Current Topics in Membranes and Transport, Volume 19: Structure, Mechanism, and Function of the Na K Pump

Advisory Board

M.P. Blaustein A. Essig R. K. H. Kinne Robert J . AdamF Norma C. Adragna Khalil Ahmed Toi Akera R. WayneAlbers L. M. Amende Beatrice M. Anner Masako Arai WilliamBall, Jr. WilliamH. Barry Gerhard Bauriedel L. Beaugt R. Berger P. Berlin Isabel Bize Rhoda Blostein H. H. Bodemann S. L. Bonting Gerda E. Breinvieser C. Bron Lindsay Brown A. M. Brown VardamM M.Buckalew, Jr. James Buggy T. J. Callahan Mitzy L. Canessa Lewis C. Cantley Cynthia T. Carilli G. CastaAeda-Hedndez J. D. Cavieres Gilbert Chin David M. Chipman A. R. ChippegWd S. P. Chock N. 0.Christiansen Randil L. Clark David Clough John H. Collins John S. Cook James B. Cooper A. Stephen Dahms M. DeLuise Paul De Weer Tamboue Deffo J. J. H. H. M.de Pont A. de Pover R. DiPolo S. Dissing F. Dittrich Philip B. Dunham Isidore Melman D. A. Eisner E. Elhanany J . Clive Ellory D. Epps Erland Erdmann Mikael Esmann Robert A. Farley Beverley E. Farquharson P. W. Flatman J. Flier Bliss Forbush III Michael Forgac Eric T. Fossel

P. A. Knauf SirH. L. Kornberg P. Llluger C.A. Pastemak Contributors L. K. Lane

h n a l d M. Foster J@ey P. Froehlich Arthur H. L. From Yarhihiro Fukushima Dwight S. Fullerton R. P. Garay La[ C. Garg P. J. Garrahan K. Geering J. Ghysel-Burton M. Girardet Carlos Gitler I. M. Glynn T. Godfraind Maurice Goeldner Kenneth A. Gruber Ingrid L. Grupp Gunter Grupp Francis J. H d y CliffordC. Hall P. Hannaert Otto Hansen YukichiHara Richard Harkins Ward E. Harris T. Hasegawa Marion Hasselberg Gamer T. Haupert Y.Hayashi Hans Hebert George R. Henderson Andrew Hiatt ChristianHirth Ann S. Hobbs Joseph F. Ho$mnn H. Homareda Stephen Huot Shoichi Iida Kenji Ikejiri H. Ishikura Peter Leth Jdrgensen WilliamP. Jencks Jdrgen Jensen Carl Johnson L Josephson James G. Kapakos Jack H. Kaplan N o m n J. Karin Steven J. D. Karlish M. Kawamura Brian G. Kennedy Eitaro Kitatsuji Roger A. Klein Iwar Klimes Irena Klodos Hermann Koepsell Juha P. Kokko K. KOpke George R. Kracke J.-P. Kraehenbllhl WorfgangKrawietz Donna L. Kropp Itsuo Kurobane Jack Kyte

W. D. Stein W. Stoeckenius K.J. Ullrich

D. E. Richards Joseph D. Robinson P. K. Lauf Henry Rodriguez Michel Lazdunski W. J. Lederer Douglas C. Rohrer Bernard Rossi Lionel G. Lelievre R. Rossi A. Lev B. C. Rossier Seymour R. Levin v. L. Lew Arnold E. Ruoho Stanley J. Russell E. Ling Russell B. Lingham D. D. Sabatini A. G. Lowe John R. Sachs Gorm Lunn J . Sakamoto Sven Mardh Friedrich A. Sauer Gerard D. Schellenberg I. G. Macara Wilhelm Schoner Alicia McDonough Amold Schwartz Julie E. M. McGeoch R. Marh Amar K. Sen Diana Marver Parimal C. Sen H. Matsui Engin H. Serpersu Arvid B. Maunsbach J. Sherman Robert W. Mercer D. G. Shoemaker Joelle E. Miara S. R. Simon J . C. Skou Manisha D. Mone T. Morimoto Elisabeth Skriver David M. Mott H. Gilbert Smith Kimberly A. Muczynski R. D. Smith Jens G. Ndrby Roderic L. Smith K. Nagano Thomas W. Smith Makoto Nakao A. K. Solomon M. Nakao Harold Solomon Toshiko Nakao D. J. Sorce D. L. Nandi Susan C. Specht C. Nazaret WilliamL. Stahl M. T. Nelson W. D. Stein J . G. Ndrby Marcia Steinberg Tomoko Ohno F. M.A. H. Schuurmans T. Ohta Stekhoven David 1. Stewart G. T. okita Doris Ollig Kuniaki Suzuki Alan C. Swann Paul Onolenghi H. G. P. Swarts Motilal Pamnani L. A. Parodi Kathleen J . Sweadner Rosemark Patzelt-Wenczler M. Taguchi Harmut Pads Kazuya Taniguchi Irene V. Pech Kyosuke Temma David Perlman C. Craig Tisher Y. Tonomura W. H. M. Peters Daniel C. Tosteson Kevin J. Petty Douglas R. Pfeiffer P. Usher M. T. Piascik G. Valet R. D. Vaughn-Jones J. F. Pincus Liselone Plesner E. T. Wallick Igor W. Plesnrr Horst Walter Gilles Ponzio Knrl Werdan Robert L Post Janice M. Whitaker Kyra R. Whitmer J . D. Potter Trevor Powell John S. Willis F. Proverbio Charles G. Winter T. Proverbio M. Yamaguchi L. A. Reeve Satoshi Yamamoto A. F. Rega Atsunobu Yoda H. Reichmann Shizuki Yoda Gerold Rempeters A. (Reeves)Zot K. R. H. Repke

Current Topics in Membranes and Transport Edited by Felix Bronner Department of Oral Biology University of Connecticut Health Center Farmington, Connecticut

Arnost Kleinzeller Depament of Physiology University of Pennsylvania School of Medicine Philadelphia, Pennsylvania

VOLUME 19 Structure, Mechanism, and Function of the Na/K Pump Guest Editors Joseph F. Hoffman Department of Physiology Yale University School of Medicine New Haven, Connecticut

Bliss Forbush 111 Depaltment of Physiology Yale University School of Medicine New Haven, Connecticut

Volume 19 is part of the series (p. xxix) from the Yale Department of Physiology under the editorial supervision of:

Joseph F. Hoffman Department of Physiology Yale University School of Medicine New Haven, Connecticut

Gerhard Giebisch Depaltment of Physiology Yale University School of Medicine New Haven, Connecticut

1983

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83 84 85 86

9 8 16 5 4 3 2 1

Contents

List of Contributors xv Preface xxv Acknowledgments xxvii Yale MembraneTransport Processes Volumes Contents of Previous Volumes xxxi

PART 1.

THERMODYNAMIC ASPECTS OF MEMBRANETRANSPORT

What Is a Coupled Vectorial Process? WILLIAM P. JENCKS

1

The Membrane Equilibrium with Chemical Reactions FRIEDRICH A. SAUER

PART 11.

xxix

21

STRUCTURAL ANALYSIS OF Na,K-ATPase

Structural Aspects of Na,K-ATPase ROBERT L. POST

53

Detergent Solubilization of Na,K-ATPase MIKAEL ESMANN

67

Methods for the Cleavage of the Large Subunit of Na,K*ATPase and the Resolution of the 83 Peptides Produced HENRY RODRIGUEZ, RICHARD HARKINS, AND JACK KYTE Selective Purification of Na,K-ATPase and Caz+,Mgz+-ATPasefrom Eel 103 Electroplax L. M. AMENDE, S. P. CHOCK, A N D R . W. ALBERS 107 High-Performance Gel Chromatography of Horse Kldney Na,K-ATPase MAKOTO NAKAO, TOSHMO NAKAO, TOMOKO OHNO, YOSHIHIRO FUKUSHIMA, YUKICHI HARA, AND MASAKO ARAI

Native Membranes from Dog Kidney Outer Medulla, Enriched in Na,K-ATPase, and 113 Vesicular In Nature BLISS FORBUSH III

CONTENTS

vi

Ultrastructureof Na,K-ATPase in Plasma Membranes Vesicles 119 ELISABETH SKRIVER, ARVID B. MAUNSBACH. AND PETER LETH J0RGENSEN Electron Microscope Analysis of Two-DimensionalCrystals of Membrane-Bound 123 Na,K-ATPase ARVID B. MAUNSBACH, ELISABETH SKRIVER, HANS HEBERT, AND PETER LETH J0RGENSEN Organizationof the Transmembrane Segmentsof Na,K-ATPase. Labellngof Lipid Embedded and Surface Domains of the a-Subunit and Its Tryptic Fragments with [1Wl lodonaphthylazide, [3*P]ATP, and Photolabeied Ouabain 127 PETER LETH J0RGENSEN, STEVEN J. D. KARLISH, AND CARLOS GITLER Structural Studies on Lamb Kidney Na,K-ATPase 131 J. H. COLLINS, BLISS FORBUSH III, L. K. LANE, E. LING, ARNOLD SCHWARTZ, AND A. (REEVES) ZOT Two Slightly Different a-Subunit Components of Kidney Na,K-ATPase Induced by Heat 135 Treatment T. OHTA, M. KAWAMURA, T. HASEGAWA, H. ISHIKURA, ANDK. NAGANO Radiation Inactivation Analysis of Na,K-ATPase 139 PAUL OTTOLENGHI, J. CLlVE ELLORY, AND ROGER A. KLEIN Stoichiometrical Binding of Ligandsto Less than 160 Kilodaltons of Na,K-ATPase 145 H. MATSUI, Y. HAYASHI, H. HOMAREDA, AND M. TAGUCHI The Active Site Structure of Na,K-ATPase: Location of a Specific Fluorescein Isothiocyanate-ReactiveSite 149 CYNTHIA T. CARILLI, ROBERT A. FARLEY, AND LEWIS C. CANTLEY Subunit Distribution of Sulfhydryl Groupsand Disulflde Bonds in 153 Renal Na,K-ATPase M. KAWAMURA, T. OHTA, AND K. NAGANO Lipid Regions of Na,K-ATPase Examined with Fluorescent Lipid Probes 157 KIMBERLY A. MUCZYNSKI, WARD E. HARRIS, AND WILLIAM L. STAHL Role of Cholesteroland Other Neutral Lipids in Na,K-ATPase 163 J. J. H. H. M. DE PONT, W. H. M. PETERS, AND S. L. BONTING

PART III.

LIGAND INTERACTIONS: CARDIAC GLYCOSIDES AND IONS

Cardiotonic Steroid Binding to Na,K-ATPase BLISS FORBUSH I11

167

203 Binding of MonovalentCations to the Na,K-ATPase M. YAMAGUCHI, J. SAKAMOTO, ANDY. TONOMURA

Half-of-the-SitesReactivity of Na,K-ATPase Examined by the Accessibility of Vanadate 219 and ATP Into Enzyme Ouabein Complexes OTTO HANSEN

-

Binding of Rb+ and ADP to a Potassium-LikeForm of Na,K-ATPase J0RGEN JENSEN AND PAUL OTTOLENGHI

223

CONTENTS

vii

Side-Dependent Ion Effects on the Rate of Ouabain Binding to Reconstituted Human Red Ceii Ghosts 229 H. H. BODEMANN, T. J. CALLAHAN, H. REICHMANN, ANDJ. F. HOFFMAN lntracellular Sodium Enhancement of Ouabain Binding to Na,K-ATPase and the Development of Glycoslde Actions 235 TAI AKERA, KYOSUKE TEMMA, AND SATOSHI YAMAMOTO Lithium-Cataiyzed Ouabain Bindingt o Canine Kidney Na,K-ATPase GEORGE R. HENDERSON

241

Ouabain Binding and Na,K-ATPase in Resealed Human Red Cell Ghosts D. G. SHOEMAKER AND P.K. LAUF

247

Stereoelectronic interaction between Cardiotonic Steroids and Na,K-ATPase: Molecular Mechanism of Digitalis Action 251 F. DITTRICH, P. BERLIN, K. KOPKE, AND K. R. H. REPKE

Use of Prophet and MMS-X Computor Graphics in the Study of the Cardiac Steroid 257 Receptor Site of Na,K-ATPase DWIGHT S. FULLERTON, DOUGLAS C. ROHRER, KHALIL AHMED, ARTHUR H. L. FROM, EITARO KITATSUJI, AND TAMBOUE DEFFO Photoafflnity Labeling of the Ouabain Binding Site of Na,K-ATPase CLIFFORD C. HALL AND ARNOLD E. RUOHO

265

New Ouabain Derivatives to Covalently Label the Digitalis Binding Site 271 BERNARD ROSSI, MAURICE GOELDNER, GILLES PONZIO, CHRISTIAN HIRTH, AND MICHEL LAZDUNSKI Ouabain Sensitivity: Diversity and Disparities JOHN S. WILLIS AND I. CLIVE ELLORY

PART IV.

277

LIGAND INTERACTIONS: NUCLEOTiDES, VANADATE, AND PHOSPHORYLATION

Ligand Interactions with the Substrate Site of Na,K-ATPase: Nucieotides, Vanadate, and Phosphotyiation 281 JENS G . N0RBY 315 Conformationai Changes of Na,K-ATPase Necessary for Transport LEWIS C. CANTLEY, CYNTHIA T. CARILLI, RODERICL. SMITH, AND DAVID PERLMAN

On the Mechanism behind the Ability of Na,K-ATPase to Discriminate between Na+ andK+ 323 JENS CHR. SKOU Characteristics of the Electric Eel Na,K-ATPase Phosphoprotein ATSUNOBU YODA AND SHIZUKO YODA

343

CONTENTS

viii

Sulfhydryl Groups of Na,K-ATPase: Effects of N-Ethytmaleimlde on Phosphorylation from ATP in the Presence of Na+ + Mg2+ 349 MIKAEL ESMANN AND IRENA KLODOS Alternatlve Pathways of Phosphorylation of Na,K-ATPase Regulated by Na+ Ions on Both 353 Sides of the Plasma Membrane HORST WALTER Structurally Different Nucleotide Binding Sites in Na,K-ATPase HERMANN KOEPSELL AND DORIS OLLIG

355

Study of Na,K-ATPase with ATP Analogs 361 WILHELM SCHONER, HARTMUT PAULS, ENGIN H. SERPERSU, GEROLD REMPETERS, ROSEMARIE PATZELT-WENCZLER, AND MARION HASSELBERG Affinity Labeling Studies of the ATP Binding Site of Canine Kldney Na,K-ATPase JAMES B. COOPER, CARL JOHNSON, AND CHARLES G. WINTER

367

31P[1aOl NMR Kinetic Analysis of ' 8 0 Exchange Reaction between P, and HzOCatalyzed by 371 Na,K-ATPase A. STEPHEN DAHMS AND JOELLE E. MIARA

PART V.

CONFORMATIONALCHANGES, STRUCTUREIFUNCTION, AND ACTIVE SITE PROBES

377

Principal Conformations of the a-Subunit and Ion Translocation PETER L. J0RGENSEN

403 Magnesium-Induced Conformational Changes In Na,K-ATPase S. L. BONTING, H. G. P. SWARTS, W. H. M. PETERS, F. M. A. H. SCHUURMANS STEKHOVEN, AND J. J. H. H. M. DE PONT

Rubidium Movements in Vesicles Reconstituted with Na,K-ATPase, Measured in the Absence of ATP and P,, in the Presence of Either Llgand, and in the Presence of Both Ligands: Role of the "Occluded State" in Allowing for the Control of the Direction of Ion Movements 425 S. J. D. KARLISH AND W. D. STEIN Eosin: A Fluorescent Probe of ATP Binding to Na,K-ATPase J . C. SKOU AND MIKAEL ESMANN

451

Interaction of Divalent Cations with Fluorescein-Labeled Na,K-ATPase MARCIA STEINBERG, JAMES G. KAPAKOS, AND PARIMAL C. SEN Cation Activation of Na,K-ATPase after Treatment with Thimerosal MANISHA D. MONE AND JACK H. KAPLAN

457 405

Alteration of Conformational Equilibria in Na,K-ATPase by Glutaraldehyde 471 Treatment DAVID M. CHIPMAN, E. ELHANANY, R. BERGER, AND A. LEV

CONTENTS

ix

Conformational Transition between ADP-Sensitive Phosphoenzyme and PotassiumSensitive Phosphoenzyme 477 KAZUYA TANIGUCHI, KUNIAKI SUZUKI, AND SHOICHI IIDA Relation between Red Cell Membrane Na,K-ATPase and Band 3 EFUC T. FOSSEL AND A. K.SOLOMON

PART V1.

481

REACTION MECHANISM AND KINETIC ANALYSIS

Kinetic Analyses and the Reaction Mechanism of the Na,K-ATPase JOSEPH D. ROBINSON

485

Evidence for Parallel Pathways of Phosphoenzyme Formation in the Mechanism of ATP Hydrolysis by Electrophorous Na,K-ATPase 513 JEFFREY P. FROEHLICH, ANN S. HOBBS, AND R. WAYNE ALBERS Evaluation of the Reaction Mechanism of the Sodium Pump by Steady-State Kinetics 537 JOHN R. SACHS Kinetic Evidence in Favor of a Consecutive Model of the Sodium Pump D. A. EISNER AND D. E. RICHARDS

547

Kinetic Models of Na-Dependent Phosphorylation of Na,K-ATPase from Rat Brain DONALD M.FOSTER, STANLEY 1. RUSSELL, AND KHALJL AHMED Reinvestigation of the Sequence of Sensitivity of Phosphoenzyme of Na,K-ATPase to ADP and K+ during the Presteady State of the Phosphorylation by ATP 557 Y. FUKUSHIMA AND M. NAKAO interaction of Na+, K+, and ATP with Na,K-ATPase P. J. GARRAHAN, R. ROSSI, AND A. F. REGA Sodium Ion Discharge from Pig Kidney Na,K-ATPase YUKICHI HARA AND MAKOTO NAKAO

561 565

ADP Sensitivity of the Native and Ollgomycin-Treated Na,K-ATPase ANN S. HOBBS, R.WAYNE ALBERS, AND JEFFREY P. FROEHLICH

569

Three (at Least) Consecutive Phosphointermediates of Na-ATPase I. KLODOS, J.G. N0RBY. AND N. 0. CHRISTIANSEN

573

Aspects of the Presteady State Hydrolysis of ATP by Na,K-ATPase A. G. LOWE AND L. A. REEVE

577

Identity of the Na Activation Sites in ATPase wlth the K Activation Sites in p-Nltrophenyiphosphatase 581 L. A. PARODI, J. F. PINCUS, L. JOSEPHSON, D. J. SORCE, AND S. R. SIMON On the Existence of Two Distinct Hydrolysis Cycles for Na,K-ATPase with Only One Active Substrate Site 587 IGOR W.PLESNER

553

CONTENTS

X

591

Kinetic Analysis of the Effects of Na+and K+ on Na,K-ATPase LISELOlTE PLESNER AND IGOR W. PLESNER DivaientCations and ConformatlonalStates of Na,K-ATPase JOSEPH D. ROBINSON

PART VII.

595

ION TRANSLOCATION AND REACTION MECHANISM

Na,K-ATPase: Reaction Mechanisms and ion TranslocatingSteps PAUL DE WEER Existence and Role of Occluded-Ion Forms of Na,K-ATPase I. M. GLYNN AND D. E. RICHARDS

599

625

Na and K Fluxes Medlated by ATP-Free and ATP-Activated Na,K-ATPase In Liposomes 639 BEATRICE M. ANNER Sidedneas of Cations and ATP interactions with the Sodium Pump L. BEAUGE AND R. DIPOLO

643

Sidednessof Sodium lnteractlons wlth the Sodlum Pump in the Absence of K + RHODA BLOSTEIN

649

MagnesiumDependence of Sodium PumpMediated Sodium Transport in Intact Human RedCeils 653 P. W.FLATMAN AND V. L. LEW K+-IndependentActlve Transport of Na+by Na,K-ATPase MICHAEL FORGAC AND GILBERT CHIN

659

-

665 ADP ATP Exchange in Internally DialyzedSquid Giant Axons PAUL DE WEER, GERDA E. BREITWIESER, BRIAN G. KENNEDY, AND H . GILBERT SMITH

-

Sodium Pumpcatalyzed ATP ADP Exchange In Red Blood Cells: The Effects of lntraceliular and ExtracellularNa and K Ions 671 JACK H.KAPLAN

-

Ouabain-SensitiveATP ADP Exchange and Na-ATPase of Resealed Red Cell Ghosts 677 J. D. CAVIERES

-

Effect of Internal Adenine Nucieotideson Sodium Pump-Catalyzed Na -Na and Na K Exchanges 683 BRIAN G. KENNEDY, GORM LUNN, AND JOSEPH F. HOFFMAN NdK Pump in InsldeQut Veslcles Utlllzlng ATP Syntheslzedat the Membrane 687 ROBERT W. MERCER, BEVERLEY E. FARQUHARSON, AND PHILIPB. DUNHAM Anion-Coupled Na Efflux Mediated by the NalK Pump in Human Red Blood Cells S. DISSINGAND J. F. HOFFMAN

Effectof Trypdn Digestlon on the Kinetlc Eiehavior of the NalK Pump In Intact 697 Erythrocytes DONNA L. KROPP

693

CONTENTS

xi

Sodium Movement and ATP Hydrolysis in Basolateral Plasma Membrane Vesicles from 703 Proximal Tubular Cells of Rat Kidney F. PROVERBIO, T. PROVERBIO, AND R. MARiN Stoichiometry of the Electrogenic Na Pump in Barnacle Muscle: Simultaneous Measurement of Na Efflux and Membrane Current 707 M. T. NELSON AND W. J. LEDERER

PART VIII.

BIOSYNTHESIS, MULTIPLEFORMS, AND IMMUNOLOGY

Regulation of Na,K-ATPase by Its Biosynthesis and Turnover NORMAN J. KARIN AND JOHN S. COOK

713

Biosynthesis of Na,K-ATPase in MDCK Cells 753 J. SHERMAN. T. MORIMOTO, AND D. D. SABATINI Possible Functional Differences between the Two Na,K-ATPases of the Brain KATHLEEN J. SWEADNER

765

Antigenic Properties of the CY,6, and Subunits of Na,K-ATPase 781 WILLIAM BALL, JR., JOHN H. COLLINS, L. K. LANE, AND ARNOLD SCHWARTZ Antibodies to Na,K-ATPese: Characterization and Use in Cell-Free Synthesis 787 Studies ALICIA McWNOUGH, ANDREW HIATT, AND ISIDORE EDELMAN lmmunoreactivity of the a-and d( +)-Subunits of Na,K-ATPase in Different Organs and 791 Species GERARD D. SCHELLENBERG, IRENE V. PECH. AND WILLIAM L. STAHL Role of Na+ and Ca2+ Fluxes in Terminal Differentiation of Murine Erythroleukemia 797 Cells I. G. MACARA, R. D. SMITH. AND LEWIS C . CANTLEY NalK Pumps and Passive K+ Transport in Large and Small Reticulocytes of Anemic 803 Low- and High-Potassium Sheep P. K. LAUF AND G . VALET Enhancement of Biosynthesis of Na,K-ATPase in the Toad Urinary Bladder by 809 Aldosterone But Not T3 K. GEERING, M.GIRARDET, c . BRON, J.-P. KRAEHENBUHL, ANDB. c . ROSSIER Na,K-ATPase Activity in Rat Nephron Segments: Effect of Low-Potassium Diet and 813 Thyroid Deficiency LAL C. GARG AND C. CRAIG TISHER Axonal Transport of Na,K-ATPase in Optic Nerve of Hamster SUSAN C. SPECHT PART IX.

819

Na,K-ATPase AND POSITIVE INOTROPY; ENDOGENOUS GLYCOSIDES

Positive lnotropic Action of Digitalis and Endogenous Factors: Na,K-ATPase and 825 Posltlve Inotropy; “Endogenous Glycosides” ARNOLD SCHWARTZ

CONTENTS

xii

EndogenousGlycoside-Like Substances GARNER T. HAUPERT, JR.

843

Monovalent Cation Transport and Mechanisms of Digltalis-Inducedlnotropy THOMAS W. SMITH AND WILLIAM H.BARRY

857

Effects of Sodium Pump Inhibition on Contraction in Sheep Cardiac Purkinje 885 Fibers D. A. EISNER, W. J. LEDERER, AND R. D. VAUGHAN-JONES Quantitative Evaluation of [3H]Ouabain Binding to Contracting Heart Muscle, Positive 891 Inotropy, Na,K-ATPase Inhibition, and W b + Uptake in Several Species ERLAND ERDMANN, LINDSAY BROWN, KARL WERDAN, AND WOLFGANG KRAWIETZ Contractile Force Effects of Low Concentrationsof Ouabain in IsolatedGuinea Pig, Rabbit, Cat, and Rat Atria and Ventricles 897 GUNTER GRUPP, INGRID L. GRUPP, J. GHYSEL-BURTON, T. GODFRAIND, A. DE POVER, AND ARNOLD SCHWARTZ Difference of Digitalis Binding to Na,K-ATPase and Sarcolemma Membranes I. KUROBANE, D. L. NANDI, ANDG. T. OKITA

903

Pharmacologicaland BiochemicalStudies on the Digitalis Receptor: A Two-Site Hypothesisfor Positive lnotropic Action 907 ARNOLD SCHWARTZ, INGRID L. GRUPP, ROBERT J. ADAMS, TREVOR POWELL, GUNTER GRUPP, AND E. T. WALLICK Hypothesis for the Mechanism of Stimulation of the NalK Pump by Cardiac Glycosides-Role of EndogenousDigitalis-Like Factor 913 T. GODFRAIND, G . CASTA~~EDA-HERNANDEZ, J. GHYSEL-BURTON, AND A. DE POVER lmmunochemical Approaches to the Isolation of an EndogenousDigoxin-like Factor 917 KENNETH A. GRUBER, JANICE M. WHITAKER, AND VARDAMAN M. BUCKALEW, JR. Demonstrationof a Humoral NalK Pump lnhlbitor in Experimental Low-Renin Hypertension 923 MOTILAL PAMNANI, STEPHEN HUOT, DAVID CLOUGH, JAMES BUGGY, AND FRANCIS J. HADDY Absence of Ouabain-LikeActivity of the Na,K-ATPase Inhibitor in Guinea Pig Brain Extract 927 GEORGE R. KRACKE Brain Na,K-ATPase: Regulationby Norepinephrlneand an EndogenousInhibitor ALAN C. SWANN

931

Inhibitory and Stimulatory Effects of Vanadate on Sodium Pump of Cultured Heart Cells from Different Species 939 KARL WERDAN, GERHARD BAURIEDEL, WOLFGANG KRAWIETZ, AND ERLAND ERDMANN EndogenousInhibitor of Na,K-ATPase: “Endodigin” 945 K. R. WHITMER, D. EPPS, AND ARNOLD SCHWARTZ

CONTENTS PART X.

xiii

PHYSIOLOGY AND PATHOPHYSIOLOGY OF THE NalK PUMP

Disorders in Molecular Assemblies for Na Transport in Essential Hypertension 951 MITZY L. CANESSA, NORMA C. ADRAGNA, ISABEL BIZE, HAROLD SOLOMON, AND DANIEL C. TOSTESON

-

The Na K Cotransport System in Essential Hypertension R. P. GARAY C. NAZARET, AND P. HANNAERT

953

Loss of Na,K-ATPase Activity during Cataract Formation in Lens

959

PARIMAL C. SEN AND DOUGLAS R. PFEIFFER Na/K Pump: Effect of Obesity and Nutritional State M. DELUISE, P. USHER, AND J. FLIER

965

Decreased Na,K-ATPase Activity in Erythrocyte Membranes and Intact Erythrocytes from 969 ObeseMan DAVID M. MOTT, IWAR KLIMES, AND RANDIL L. CLARK Functionally Abnormal NalK Pump in Erythrocytes from a Morbidly Obese 973 Subject J. FLIER, P. USHER, AND M. DELUISE Specific Insulin Binding to Purified Na,K-ATPase Associated with Rapid Activation of the 977 Enzyme JULIE E. M. McGEOCH Mechanism for Cholinergic Stimulatlon of Sodium Pump in Rat Submandibular 985 Gland DAVID J. STEWART AND AMAR K. SEN Evidence for an Aldosterone-Mediated, Na-Dependent Activation of Na,K-ATPase in the 989 Cortical Collecting Tubule KEVIN J. PETTY, JUHA P. KOKKO. AND DIANA MARVER Vanadate and Somatostatin Having Divergent Effects on Pancreatic Islet 993 Na,K-ATPase KENJI IKElIRI AND SEYMOUR R. LEVIN Phosphorylation of a Kidney Preparation of Na,K-ATPase by the Catalytic Subunit of 999 CAMP-DependentProtein Kinase SVEN MARDH Modulation of Na,K-ATPase Activity in Rat Brain by Adenosine 1005 3'3'-Monophosphate RUSSELL B. LINGHAM AND AMAR K. SEN Stimulation and Inhibition by Plasma of Ouabain-Sensitive Sodium Efflux in Human Red Blood Cells 1013 A. R. CHIPPERFIELD Inhibition of the Na Pump by CytoplasmicCalcium in Intact Red Cells A. M. BROWN AND V. L. LEW

1017

CONTENTS

xiv

involvement of Calmodulin in the Inhibition of Na,K-ATPaseby Ouabain LIONELG. LELIEVRE, M. T. PIASCIK, J. D. POTTER, E. T. WALLICK, AND ARNOLD SCHWARTZ

Index

1029

1023

List of Contributors Numbers in parentheses refer to the pages on which the authors’ contributions begin.

Robert J. Adams, Department of Pharmacology and Cell Biophysics, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267 (907) Norma C. Adragna, Department of Physiology, Harvard Medical School, Boston, Massachusetts 02115 (951) Khalil Ahmed, Veterans Administration Medical Center, Minneapolis, Minnesota (257, 553) Tal Akera, Department of Pharmacology and Toxicology, Michigan State University, East Lansing, Michigan 48824 (235) R. Wayne Albers, National Institute of Neurological and Communicative Disorders and Stroke, National Institutesof Health, Bethesde, Maryland 20205 (513,569, 103) L. M. Amendo, NINCDS, National Institutes of Health, Bethesda, Maryland 20205 (103) Beatrice M. Anner, Department of Pharmacology, Centre MMcal Universitaire, CH-1211 Geneva 4, Switzerland (639) Masako Arai, Tokyo Medical and Dental University, School of Medicine, Yushima, Bunkyo-ku,Tokyo, Japan (107) Willlam Ball, Jr., Department of Pharmacology and Cell Biophysics, University of Cincinnati, College of Medicine, Cincinnati, Ohio 45267 (781) Willlam H. Barry, Cardiovascular Division, Brigham and Women’s Hospital, and Departments of Medicine, Brigham and Women’s Hospital, and Harvard Medical School, Boston, Massachusetts 021 15 (857) Gerhard Bauriedel, Medizinische Klinik I der Universitit Miinchen, Klinikum Grosshadern, D-8000 Munchen 70, Federal Republic of Germany (939) L. B8aug6, Division de Bioffsica, Instituto de Investigaci6n Mdica Mercedes y Martin Ferreyra, C6rdoba, Argentina, and IVIC, Caracas, Venezuela (643) R. Berger, Department of Biology, Ben Gurion University, Beer Sheva, Israel (471) P. Berlin, Biomembrane Section, Central Institute of Molecular Biology, Academy of Sciences of the German Democratic Republic, Berlin, German Democratic Republic (25 1) Isabel Blze, Department of Physiology, Harvard Medical School, Boston, Massachusetts 021 15 (951) Rhoda Biostein, Departments of Biochemistry and Experimental Medicine, McGill University, Montreal, Quebec, Canada (649) H. H. Bodemann, Department of Internal Medicine, University of Freiburg, Freiburg, Federal Republic of Germany (229) S. L. Bonting, Department of Biochemistry, University of Nijmegen, 6500 HB Nijmegen, The Netherlands (163,403) Gerda E. Breltwleser, Department of Physiology and Biophysics, Washington University School of Medicine, St. Louis, Missouri 631 10 (665) C. Bron, Institut de Biochimie, Universitb de Lausanne, CH-1066, Epalinges, Switzerland (809) Lindsay Brown, Medizinische Klinik I der Universitiit Miinchen, Klinikum Grosshadern, D-8000 Munchen 70, Federal Republic of Germany (891) A. M. Brown, Department of Physiologyand Pharmacology, University of Nottingham Medical School, Nottingham, England (1017) xv

xvi

LIST OF CONTRIBUTORS

Vardaman M. Buckalew, Jr., Departments of Medicine and Physiology and Pharmacology, Bowman Gray School of Medicine, Winston-Salem, North Carolina 27103 (917) James Buggy, Department of Physiology, Uniformed Services University, Bethesda, Maryland 20814 (923) T. J. Callahan, Department of Physiology, Yale University School of Medicine, New Haven, Connecticut 06510 (229) Mitzy L. Canessa, Department of Physiology, Harvard Medical School, Boston, Massachusetts 021 15 (951) Lewis C. Cantley, Department of Biochemistry and Molecular Biology, Harvard University, Cambridge, Massachusetts02138 (149,315,797) Cynthia T. Carllli, Department of Biochemistry and Molecular Biology, Harvard University, Cambridge, Massachusetts 02138 (149,315) G. Castaiieda-Hernhdez, Laboratoire de Pharmacodynamie G6ntrale et de Pharmocologie, Universit6 Catholique de Louvain, 8-1200 Bruxelles, Belgium (913) J. D. Cavieres, Physiological Laboratory, University of Cambridge, Cambridge CB2 3EG, England (677) Gilbert Chin, Department of Biochemistry and Molecular Biology, Harvard University, Cambridge, Massachusetts 02138 (659) David M. Chlpman, Department of Biology, Ben Gurion University, Beer Sheva, Israel (471) A. R. Chlpperfleld, Department of Physiology, The University, Dundee DD14HN, United Kingdom (1013) S. P. Chock, NINCDS, National Institutes of Health, Bethesda, Maryland20205 (103) N. 0. Chrlstiansen, Institute of Biophysics, University of Aarhus, DK-8000 Aarhus, Denmark (573) Randil L. Clark, Phoenix Clinical Research Section, National Institute of Arthritis, Diabetes, Digestive and Kidney Diseases, National Institutes of Health, Phoenix, Arizona 85016 (969) David Clough, Department of Physiology, Uniformed Services University, Bethesda, Maryland 20814 (923) John H. Collins, Department of Pharmacology and Cell Biophysics, University of Cincinnati, College of Medicine, Cincinnati, Ohio45267 (131, 781) John S. Cook, The University of Tennessee-Oak Ridge Graduate School of Biomedical Sciences and The Biology Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37830 (713) James B. Cooper, Department of Biochemistry, University of Arkansas College of Medicine, Little Rock, Arkansas 72205 (367) A. Stephen Dahms, Department of Chemistry and Molecular Biology Institute, San Diego State University, San Diego, California 92182 (371) M. DeLuise, Beth Israel Hospital, Harvard Medical School, Boston, Massachusetts 02215 (965,973) Paul De Weer, Department of Physiology and Biophysics, Washington University School of Medicine, St. Louis, Missouri 631 10 (599, 665) Tamboue Deffo, School of Pharmacy, Oregon State University, Corvallis, Oregon 97330 (257) J. J. H. H. M. de Pont, Department ofBiochemistry. University of Nijmegen, 6500 HB Nijmegen, The Netherlands (163,403) A. d9 Pover, Laboratoire de Pharmacodynamie, G6n6rale et de Pharmacologie, Universit6 Catholique de Louvain, B-1200 Bruxelles, Belgium (897,913) R. DIPolo, Division de Bioffsica, Instituto de Investigaci6n Mkdica Mercedes y Martin Ferreyra, C6rdoba, Argentina, and IVIC, Caracas, Venezuela (643) S. Dissing, Department of Physiology, Yale University School of Medicine, New Haven, Connecticut 06510 (693) F. Dittrich, Biomembrane Section, Central Institute of Molecular Biology, Academy of Sciences of the German Democratic Republic, Berlin, German Democratic Republic (25 1) Phlllp B. Dunham, Department of Biology, SyracuseUniversity, Syracuse, New York 13210 (687) lsidore Edelman, Department of Biochemistry, Columbia University, New York, New York 10032 (787)

LIST OF CONTRIBUTORS

xvii

D. A. Eisner, Physiological Laboatory, University of Cambridge, Cambridge CB2 3EG, England (547, 885) E. Elhanany, Department of Biology, Ben Gurion University, Beer Sheva, Israel (471) J. Clive ElloW, Department of Physiology, University of Cambridge, Cambridge, England (139.277) D. Epps, Department of Pharmacology and Cell Biophysics, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267 (945) Erland Erdmann, Medizinische Klinik I der Universitat Miinchen, Klinikum Grosshadern, D-8000 Miinchen, Federal Republic of Germany (891,939) Mikael Esmann, Institute of Biophysics, University of Aarhus, DK-8000 Aarhus, Denmark (67, 349, 451) Robert A. Farley, Department of Biochemistry and Molecular Biology, HHarvard University, Cambridge, Massachusetts 02138 (149) Beverley E. Farquharson, Department of Biology, Syracuse University, Syracuse, New York 13210 (687) P. W. Flatman, Department of Physiology, University Medical School, Edinburgh EH8 9AG, Scotland and Physiological Laboratory, Cambridge CB2 3EG, England (653) J. Flier, Beth Israel Hospital, Harvard Medical School. Boston, Massachusetts 02215 (965,973) Bliss Forbush 111, Department of Physiology, Yale University School of Medicine, New Haven, Connecticut06510(113, 131, 167) Michael Forgac, Department of Biochemistry and Molecular Biology, Harvard University, Cambridge, Massachusetts 02138 (659) Eric T. Fossel, Biophysical Laboratory, Harvard Medical School, Boston, Massachusetts 021 15 (481) Donald M.Foster, Olin E. Teague Veterans’ Center and College of Medicine, Texas A & M University, Temple, Texas 76501 (553) Jeffrey P. Froehlich, National Institute on Aging, National Institutes of Health, Gerontology Research Center, Baltimore City Hospitals, Baltimore, Maryland 21224 (513,569) Arthur H. L. From, Veterans Administration Medical Center, Minneapolis, Minnesota 55417 (257) Yoshihiro Fukushima, Laboratory of Active Transport, National Institute for Physiological Sciences, Okazaki 444,Japan (107,557) Dwight S. Fullerton, School of Pharmacy, Oregon State University, Corvallis, Oregon 97330 (257) R. P. Garay, INSERM U 7ICNRS LA 318, Hapital Necker, 75015 Paris Cedex, France (953) La1C. Garg, Department of Pharmacology and Division of Nephrology, Department of Medicine, University of Florida, College of Medicine, Gainesville. Florida 32610 (813) P. J. Garrahan, Departamento de Quimica Biologica, Facultad de Farmacia Bioquimica, Universidad de Buenos Aires, Buenos Aires, Argentina (561) K. Geering, Institut de Pharmacologie, Universitt de Lausanne, CH-101 1 Lausanne, Switzerland (809) J. Ghysel-Burton, Laboratoire de Pharmacodynamie, GCnCrale et de Pharmacologie, Universite Catholique de Louvain, B-1200 Bruxelles, Belgium (897,913) M. Girardet, Institut de Pharmacologie, Universite de Lausanne. CH-101 I , Switzerland (809) Carlos Gitler, Department of Biochemistry and Department of Membrane Research, Weizmann Institute of Science, Rehovot, Israel (127) 1. M. Glynn, Physiological Laboratory, University of Cambridge, Cambridge CB2 3EG, England (625)

T. Godfralnd, Laboratoire de Pharmacodynamie, G6nCrale et de Pharmacolgie, Universiti Catholique de Louvain, B-1200 Bruxelles, Belgium (897,913) Maurice Goeldner, Institut de Chimie, UniversitC Louis Pasteur, 67008 Strasbourg Cedex, France (27 1) Kenneth A. Gruber, Departments of Medicine and Physiology and Pharmacology, Bowman Gray School of Medicine, Winston-Salem, North Carolina 27103 (917) Ingrid L. Grupp, Department of Pharmacology and Cell Biophysics, University of Cincinnati College of Medicine, Cincinnati, Ohio45267 (897,907) Gunter Grupp, Department of Pharmacology and Cell Biophysics, University of Cincinnati College of Medicine, Cincinnati, Ohio45267 (897,907)

xviii

LIST OF CONTRIBUTORS

Francis J. H a m , Department of Physiology, Uniformed Services University, Bethesda, Maryland 20814 (923) Clifford C. Hail, Department of Pharmacology, University of Wisconsin Medical School, Madison, Wisconsin 53706 (265) P. Hannaert, INSERM U 7/CNRS LA318, Hbpital Necker, 75015 Paris Cedex, France (953) Otto Hanwn, Institute of Physiology, University of Aarhus, DK-8000Aarhus, Denmark (219) Yukichl Hara, Tokyo Medical and Dental University School of Medicine, Yushima, Bunkyo-ku, Tokyo, Japan (107,565) Richard Harklns, Department of Chemistry, University of California, San Diego, La Jolla, California 92093 (83) Ward E. Harrls, Veterans Administration Medical Center, Seattle, Washington98108, and Department of Medicine (Neurology), University of Washington School of Medicine, Seattle,Washington 98108 (157) T. Hasegawa, Department of Chemistry, Jichi Medical School, Yakushiji, Minamikawachi-machi, Kawachi-gun, Tochigi, Japan (135) Marlon Haerelberg,Institute fiir Biochemie und Endokrinologie, Justus-Liebig-Universitiit Giessen, D-6300 Giessen, Federal Republic of Germany (361) Garner T. Haupert, Jr., Renal Unit and Cellular and Molecular Research Laboratory, Massachusetts General Hospital, Boston, Massachusetts021 14 (843) Y. Hayashi, Departmentof Biochemistry, Kyorin University School of Medicine, Mitaka, Tokyo, Japan (145) Hans Hebert, Max-Planck-Institutfiir Biochimie, Abteilung ftir Strukturforschung I, Martiensried bei Miinchen, Federal Republic of Germany (123) George R. Henderson, Department of Pharmacology, Medical College of Ohio, Toledo, Ohio 43699 (241) Andrew Hiatt, Department of Biochemistry, Columbia University, New York, New York 10032 (787) Chrlstlan Hlrth, Institut de Chimie, Universiti5Louis Pasteur, 67008 StrasbourgCedex, France (27 1) Ann S. Hobbs, National Institute of Neurological and Communicative Disorders and Stroke, National Institutesof Health, Bethesda, Maryland 20205 (513,569) Joseph F. Hoffman, Department of Physiology, Yale University School of Medicine, New Haven, Connecticut 06510 (229,683,693) H. Homareda, Department of Biochemistry, Kyorin University School of Medicine, Mitaka, Tokyo, Japan (145) Stephen Huot, Department of Physiology, Uniformed ServicesUniversity, Bethesda, Maryland 20814 (923) Sholchl Ilda, Department of Pharmacology, School of Dentistry, Hokkaido University, Sapporo, Japan (477) Kenjl Ikejlri,Wadsworth Veteran’s Administration Hospital and University of California at Los Angeles School of Medicine, Los Angeles, California90073 (993) H. Ishlkura, Department of Chemistry, Jichi Medical School, Yakushiji, Minamikawachi-machi, Kawachi-gun, Tochigi, Japan (135) Peter Leth Jbrgensen, Institute of Physiology, University of Aarhus, DK-8ooD Aarhus, Denmark (1 19, 123,127,377) Wllllam P. Jencks, Graduate Department of Biochemistry, Brandeis University, Waltham, Massachusetts (1) Jdrgen Jensen, Institute of Physiology, University of Aarhus, DK-8000 Aarhus, Denmark (223) Carl Johnson, Department of Biochemistry, University of Arkansas College of Medicine, Little Rock, Arkansas 72205 (367) L. Josephson, Department of Biochemistry, State University of New York at Stony Brook, Stony Brook, New York 11794 (581) James 0. Kapakos, Department of Pharmacology, State University of New York, Upstate Medical Center, Syracuse, new York 13210(457)

LIST OF CONTRIBUTORS

xix

Jack H. Kaplan, Department of Physiology, University of Pennsylvania, Philadelphia, Pennsylvania 19104 (465,671) Norman J. Karin, The University of Tennessee-Oak Ridge Graduate School of Biomedical Sciences and The Biology Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37830 (713) Steven J. D.Karlish, Department of Biochemistry and Department of Membrane Research, Weizmann Institute of Science, Rehovot, Israel (127,425) M. Kawamura, Department of Biology, Jichi Medical School, Yakushiji, Minamikawachi-machi, Kawachi-gun, Tochigi, Japan 329-04 (135, 153) Brian G. Kennedy, Department of Physiology and Biophysics. Washington University School of Medicine, St. Louis, Missouri 631 10 (665,683) Eitaro Kitatsujl, School of Pharmacy, Oregon State University, Corvallis, Oregon 97330 (257) Roger A. Klein, Medical Research Council, Molteno Institute, University of Cambridge, Cambridge, England (139) lwar Kllmes, Phoenix Clinical Research Section, National Institute of Arthritis, Diabetes, Digestive and Kidney Diseases, National Institutes of Health, Phoenix, Arizona 85016 (969) lrena Klodos, Institute of Biophysics, University of Aarhus, DK18OOO Aarhus, Denmark (349,573) Hermann Koepsell, Max-Planck-Institut fiir Biophysik, 6ooo Frankfurt (Main), Federal Republic of Germany (355) Juha P. Kokko, Departments of Internal Medicine and Biochemistry, University of Texas Health Science Center, Dallas, Texas (989) K. KBpke, Biomembrane Section, Central Institute of Molecular Biology, Academy of Sciences of the German Democratic Republic, Berlin, German Democratic Republic (25 1) George R. Kracke, Department of Physiology and Biophysics, Washington University School of Medicine, St. Louis, Missouri 631 10 (927) J.-P. Kraehenbuhl, Institut de Biochimie, Universite de Lausanne, CH-1066 Epalinges, Switzerland (809) Wolfgang Krawietz, Medizinische Klinik I der Universitat Miinchen, Klinikum Grosshadern, D-8000 Miinchen 70, Federal Republic of Germany (891.939) Donna L. Kropp, Department of Physiology, UMDNJ-New Jersey Medical School, Newark, New Jersey 07 103 (697) ltsuo Kurobane, Department of Pharmacology, Northwestern University Medical School, Chicago, Illinois 6061 1 (903) Jack Kyte, Department of Chemistry, University of California, San Diego, La Jolla, California 92093 (83) L. K. Lane, Department of Pharmacology and Cell Biophysics, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267 (I3 1,78 I ) P. K. Lauf, Department of Physiology, Duke University Medical Center, Durham, NorthCarolina 27710 (247,803) Michel Lazdunski, Centre de Biochimie du CNRS, Faculte des Sciences, Universitk de Nice, 06034 Nice Cedex. France (271) W. J. Lederer. Department of Physiology, University of Maryland School of Medicine, Baltimore, Maryland21201 (707,885) Lionel 0. Lelibvre, Department of Pharmacology and Cell Biophysics, College of Medicine, Cincinnati, Ohio 45267 (1023) A. Lev, Department of Biology, Ben Gurion University, Beer Sheva, Israel (471) Seymour R. Levin, Wadsworth Veterans Administration Hospital and University of California at Los Angeles School of Medicine, Los Angeles, California 90073 (993) V. L. Lew, Department of Physiology, University of Edinburgh Medical School, Edinburgh EH8 9AG, and Physiological Laboratory, Cambridge CB2 3EG, England (653, 1017) E. Ling, Department of Pharmacology and Cell Biophysics, University of Cincinnati College of Medicine, Cincinnati, Ohio45267 (131) RU88ell 6. Lingham, Department of Pharmacology, Faculty of Medicine, University of Toronto, Toronto M5S 1A8, Ontario, Canada (1005)

LIST OF CONTRIBUTORS

xx

A. 0. Lowe, Department of Biochemistry, University of Manchester, Manchester, England (577) Gorm Lunn, Department of Physiology, Yale University School of Medicine, New Haven, Connecticut 065 10 (683) Sven M%rdh,Institute of Medical and Physiological Chemistry, Biomedical Center, Uppsala University, S-75123 Uppsala, Sweden (999) 1. G. Macara, Department of Biochemistry and Molecular Biology, Harvard University, Cambridge, Massachusetts 02138 (797) Alicia McDonough, Department of Biochemistry, Columbia University, New York, New York 10032 (787) Julie E. M. McGeoch, Harvard Medical School, Department of Medicine, Beth Israel Hospital, Boston, Massachusetts 02215 (977) R. Marin, Centro de Biofisica y Bioquimica, Instituto Venezolano de Investigaciones Cientificas, IVIC, Caracas 1010A, Venezuela (703) Diana Marver, Departments of Internal Medicine and Biochemistry, University of Texas Health Science Center, Dallas, Texas (989) H. Matsui, Department of Biochemistry, Kyorin University School of Medicine, Mitaka, Tokyo, Japan (145)

Arvid 6. Maunsbach, Department of Cell Biology, The Institute'of Anatomy and Institute of Physiology, University of Aarhus, DK-8000 Aarhus, Denmark (I 19, 123) Robert W. Mercer, Department of Biology, Syracuse University, Syracuse, New York 13210 (687) Joelle E. Mlara, Department of Chemistry and Molecular Biology Institute, San Diego State University, San Diego, California 92182 (371) Manieha D. Mone, Department of Physiology, University of Pennsylvania, Philadelphia, Pennsylvania 19104 (465) T. Morimoto, Department of Cell Biology, New York University School of Medicine, New York, New York 10016 (753) David M. Mott, Phoenix Clinical Research Section, National Institute of Arthritis, Diabetes, Digestive and Kidney Diseases, National Institutes of Health, Phoenix, Arizona 85016 (969) Kimberly A. Muczynski, Veterans Administration Medical Center, Seattle, Washington 98 108, and the Department of Pathology, University of Washington School of Medicine, Seattle, Washington 98108 (157) K. Nagano, Department of Biology, Jichi Medical School, Yakushiji, Minamikawachi-machi, Kawachigun, Tochigi, Japan (135, 153) Makoto Nakao, Tokyo Medical and Dental University School of Medicine, Yushima, Bunkyo-ku, Tokyo, Japan (107,565) M. Nakao, Laboratory of Active Transport, National Institute for Physiological Sciences, Okazaki, 444 Japan (557) Toshiko Nakao, Tokyo Metropolitan Research Laboratory of Public Health, Shinjuku-ku, Tokyo, Japan (107) D. L. Nandi, Department of Pharmacology, Northwestern University Medical School, Chicago, Illinois 60611 (903) C. Nazaret, INSERM U 7/CNRS LA 318, Hbspital Necker, 75015 Paris Cedex, France (953) M. T. Nelson, Department of Physiology, University of Maryland School of Medicine, Baltimore, Maryland 21201 (707) Jens 0. Ndrby, Institute of Biophysics, University of Aarhus, DK-8000 Aarhus, Denmark (281,573) Tomoko Ohno, Tokyo Metropolitan Research Laboratory of Public Health, Shinjuku-ku, Tokyo, Japan (107) f. Ohta, Department of Biology, Jichi Medical School, Yakushiji, Minamikawachi-machi, Kawachigun, Tochigi, Japan (135, 153) G. T. Okita, Department of Pharmacology, Northwestern University Medical School, Chicago, Illinois 6061 1 (903)

LIST OF CONTRIBUTORS

xxi

Doris Ollig, Max-Planck-Institut fiir Biophysik, 6OOO Frankfurt (Main), Federal Republic of Germany (355) Paul Ottolenghi,Institute of Physiology, University of Aarhus, DK-8000 Aarhus, Denmark (139,223) Motilal Pamnani, Department of Physiology, Uniformed Services University, Bethesda, Maryland 20814 (923) L. A. Parodi, Department of Biochemistry, State University of New YOrk at Stony Brook, Stony Brook, New York 11794 (581) Rosemarie Patzelt-Wenczler, Institut fiir Biochemie und Endokrinologie, Justus-Liebig-Universitiit Giessen, D-6300 Giessen, Federal Republic of Germany (361) Hartrnut Pauls, Institut ftir Biochemie und Endokrinologie, Justus-Liebig-Universitat Giessen, D-6300 Giessen. Federal Republic of Germany (361) irene V. PMh, Veterans Administration Medical Center, and the Departments of Medicine (Neurology), and Physiology and Biophysics, University of Washington School of Medicine, Seattle, Washington (79 1) David Perlman, Department of Biochemistry and Molecular Biology, Harvard University, Cambridge, Massachusetts 02138 (315) W. H. M. Peters, Department of Biochemistry, University of Nijmegen, 6500 HB Nijmegen, The Netherlands (163,403) Kevin J. Petty, Department of Internal Medicine, University of Texas Health Science Center, Dallas, Texas (989) Douglas R. Pfeiffer, The Hormel Institute, University of Minnesota, Austin, Minnesota 55912 (959) M. 1.Piascik, Department of Pharmacology and Cell Biophysics, College of Medicine, Cincinnati, Ohio 45267 (1023) J. F. Pincus, Department of Biochemistry, State University of New York at Stony Brook, Stony Brook, New York 11794 (581) Liselotte Plesner, Institute Biophysical, Aarhus University, DK-8000 Aarhus, Denmark (59 1) lgor W. Plesner, Department of Physical Chemistry, Aarhus University, Kenisk Institute, DK-8000 Aarhus, Denmark (587, 591) Gilles Ponzio, Centre de Biochimie du CNRS, Facult6 des Sciences, Universite de Nice. 06034 Nice Cedex, France (271) Robert L. Post, Department of Physiology, Vanderbilt University Medical School, Nashville, Tennessee 37232 (53) J. D. Potter, Department of Pharmacology and Cell Biophysics, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267 (1023) Trevor Powell, Department of Physics as Applied to Medicine, Middlesex Hospital Medical School, London W 1P 7PN, England (907) F. Proverbio, Centro de Biofisica y Bioquimica, Instituto Venezolano de Investigaciones Cientificas, IVIC, Caracas lOIOA, Venezuela (703) T. Proverblo, Centro de Biofisica y Biquimica. Instituto Venezolano de Investigaciones Cientificas, IVIC, Caracas 1010A, Venezuela (703) L. A. Reeve, Department of Biochemistry, University of Manchester, Manchester, England (577) A. F. Rega, Lkpartamento de Quimica Biologica, Facultad de Farmacia Bioquimica, Universidad de

Buenos Aires, 1113 Buenos Aires, Argentina (561) H. Reichmann, Department of Internal Medicine, University of Freiburg, Freiburg, Federal Republic of Germany (229)

Geroid Rempeters, Institut fiir Biochemie und Endokrinologie, Justus-Liebig-Universitit Giessen, D6300 Giessen, Federal Republic of Germany (361) K. R. H. Repke, Biomembrane Section, Central Institute of Molecular Biology, Academy of Sciences of the German Democratic Republic, Berlin, German Democratic Republic (251)

xxii

LIST OF CONTRIBUTORS

D. E. Rlchards, Physiological Laboratory, University of Cambridge, Cambridge CB2 3EG, 'England (547,625) Joseph D. Roblnson, Department of Pharmacology, State University of New York, Upstate Medical Center, Syracuse, New York 13210(485,595) Henry Rodriguez, Department of Chemistry, University of California, San Diego, La Jolla, California 92093 (83) Douglas C. Rohrer, Medical Foundation of Buffalo, Buffalo, New York (257) Bernard Rossi, Centre de Biochimie du CNRS, Facult6 des Sciences, Universitc? de Nice, 06034 Nice Cedex, France (271) R. Rossi, Departamento de Quimica Bioldgica, Facultad de Farmacia Bioquimica, Universidad de Buenos Aires, Buenos Aires, Argentina (561) 8. C. Rossler, Institut de Pharmacologie, Universit6 de Lausanne, CH-1066 Epalinges, Switzerland (809) Arnold E. Ruoho, Department of Pharmacology, University of Wisconsin Medical School, Madison, Wisconsin 53706 (265) Stanley J. Russell, Department of Mechanical and Aerospace Engineering, Arizona State University, Tempe, Arizona 85281 (553) D. D. Sabatlni, Departmentof Cell Biology, New YorkUniversity School of Medicine, New York, New York 10016 (753) John R. Sachs, Department of Medicine, State University of New York at Stony Brook, Stony Brook, New York 11790(537) J. Sakamoto, Department of Biology, Faculty of Science, Osaka University, Toyonah, Osaka, Japan (203) FrledrichA. Sauer, Max-Planck-Institutfiir Biophysik, 6ooo Frankfurt 70, Federal Republic of Germany (21) Gerard D. Schellenberg, Veterans Administration Medical Center, and the Departments of Medicine (Neurology),and Physiology and Biophysics, University of WashingtonSchool of Medicine, Seattle, Washington (791) Wllhelm Schoner, htitut fiir Biochemie und Endokrinologie, Justus-Liebig-Universitat Giessen, D6300 Giessen, Federal Republic of Germany (361) Arnold Schwartz, Department of Pharmacologyand Cell Biophysics, University of Cincinnati College ofMedicine, Cincinnati, Ohio45267 (131,781,825,897,907,945, 1023) Amar K. Sen, Department of Pharmacology, University of Toronto, Toronto M5S 1A8, Ontario, Canada (985, 1005) Parlmal c. Sen, The Hormel Institute, University of Minnesota, Austin, Minnesota 55912 (457,959) Engln H. SerpWSu, Institute fiir Biochemie und Endokrinologie, Justus-Liebig-Universitiit Giessen, D6300 Giessen, Federal Republic of Germany (361) J. Sherman, Department of Cell Biology, New York University School of Medicine, New York, New York 10016(753) D. 0. Shoemaker, Department of Physiology, Duke University Medical Center, Durham, North Carolina 27710 (247) S. R. Simon, Department ofliochemistry, State University of New York at Stony Brook, Stony Brook, New York 11794(581) J. C. Skou, Institute of Biophysics, University of Aarhus, DK-8000 Aarhus, Denmark (323,451) Elisabeth Skrlver, Department of Cell Biology, The Institute of Anatomy and Institute of Physiology, Universityy of Aarhus, DK-8000 Aarhus, Denmark (119, 123) H. Gilbert Smlth, Department of Physiology and Biophysics, Washington University School of Medicine, St. Louis, Missouri 631 10 (665) R. 0. Smith, Department of Biochemistry and Molecular Biology, Harvard University, Cambridge, Cambridge, Massachusetts02 138 (797) Roderlc L. Smith, Department of Biochemistry and Molecular Biology, Harvard University, Cambridge, Massachusetts02138(315)

LIST OF CONTRIBUTORS

xxiii

Thomas w. Smith, CardiovascularDivision, Brigham and Women’s Hospital, and the Departments of Medicine, Brigham and Women’s Hospital, and Harvard Medical School, Boston, Massachusetts 02115 (857) A. K. Solomon, Biophysical Laboratory, Harvard Medical School, Boston, Massachusetts021 15 (481) Harold Solomon, Department of Physiology, Harvard Medical School, and Brigham and Women’s Hospital, Boston, Massachusetts021 15 (951) D. J. SOrCe, Department of Biochemistry, State University of New York at Stony Brook, Stony Brook, New York 11794 (581) Susan c. Specht, Department of Pharmacology, School of Medicine, University of Puerto Rico, San Juan, Puerto Rico (819) Willlam L. Stehl, Veterans Administration Medical Center, and the Depanments of Medicine (Neurology), and Physiology and Biophysics, University of Washington School of Medicine, Seattle, Washington (157,791) W. D. Steln, Department of Biochemistry, Hebrew University, Jerusalem, Israel (425) Marcia Steinberg, Department of Pharmacology, State University of New York, Upstate Medical Center, Syracuse, New York 13210 (457) F.M. A. H. Schuurmans Stekhoven, Department ofliochemistry, University of Nijmegen, 6500 HB Nijmegen, The Netherlands (403) David J. Stewart, Department of Pharmacology, University of Toronto, Toronto M5S 1A8, Ontario, Canada (985) Kunlaki SUZuW, Department of Pharmacology, School of Dentistry, Hokkaido University, Sapporo, Japan (477) Alan C. Swann, Department of Psychiatry, University of Texas Medical School, Houston, Texas 77025 (931) H. G. P. Swarts, Department of Biochemistry, University of Nijmegen, 6500 HB Nijmegen, The Netherlands (403) Kathleen J. Sweadner, Department of Physiology, Harvard Medical School, Boston, Massachusetts 02115 (765) M. Taguchi, DepartmentofBiochemistry, Kyorin University School of Medicine, Mitaka. Tokyo, Japan ( 145) KezUya Tanlguchi, Department of Pharmacology, School of Dentistry, Hokkaido University, Sapporo, Japan (477) Kyosuke Temma, Department of Pharmacology and Toxicology, Michigan State University, East Lansing, Michigan 48824 (235) C. Craig Tisher, Department of Pharmacology and Division of Nephrology, Department of Medicine, University of Florida, College of Medicine, Gainesville, Florida 32610 (813) Y. Tonomura, Department of Biology, Faculty of Science, Osaka University, Toyonaka, Osaka, Japan (203) Danlel C. Tosteson, Department of Physiology, Harvard Medical School, Boston, Massachusetts 02115 (951) P. Usher, Beth Israel Hospital, Harvard Medical School, Boston, Massachusetts02215 (965,973) G. Valet, Max-Planck-lnstitut fiir Biochimie, 8033 Martinsried, Federal Republic of Germany (803) R. D. Vaughan-Jones, Department of Pharmacology, Oxford OX1 3QT, England (885) E. T. Walllck, Department of Pharmacology and Cell Biophysics, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267 (907, 1023) Horst Walter Blumenweg 10, Domstadt Ulm, Federal Republic of Germany Karl Werdan, Medizinische Klinik I der UniversitAt Miinchen, Klinikum Grosshadern, D-8000 Miinchen 70, Federal Republic of Germany (891,939) Janice M. Whltaker, Departments of Medicine and Physiology and Pharmacology, Bowman Gray School of Medicine, Winston-Salem, North Carolina 27103 (917) Kym R. Whitmer, Department of Pharmacology and Cell Biophysics, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267 (945)

xxiv

LIST OF CONTRIBUTORS

John S. Willis, Department of Physiology and Biophysics, University of Illinois, Urbana, Illinois 61801 (277) Charles G. Wlnter, Department of Biochemistry, University of Arkanas College of Medicine, Little Rock,Arkansas 72205 (367) M. Yamaguchi, Department ofBiology, Faculty of Science, Osaka University, Toyonaka, Osaka, Japan (203) Satoshi Yamamoto, Department of Pharmacology and Toxicology, Michigan State University, East Lansing, Michigan 48824 (235) Atsunobu Yoda, Department of Pharmacology, University of Wisconsin Medical School, Madison, Wisconsin 53706 (343) Shizuko Yoda, Department of Pharmacology, University of WisconsinMedical School, Madison, Wisconsin 53706 (343) A. (Reeves) Zot, Department of Pharmacology and Cell Biophysics, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267 (131)

Preface

The Na/K pump present in essentially all animal cells is known to play a critical role in many cellular processes. Much work has been directed toward defining the characteristics of sodium and potassium transport by the pump and the pump's functional role in regulating ionic composition and determining cellular activity. The maintenance of high potassium and low sodium inside the cell is of fundamental importance in the control of cell volume and in the electrical excitability of nerve and muscle. The sodium gradient establishedby the Na/K pump is used by coupled transport systems to drive the cellular accumulation of solutes such as sugars and amino acids and the extrusion of others such as calcium and protons. In epithelia, the net movement of salt, water, and other solutes across the tissue results when sodium is pumped out of only one side of the cell. In heart, through changes in intracellular calcium coupled to changes in the sodium gradient, the Na/K pump may be the site of the positive inotropic action of cardiac glycosides. Since Skou's discovery that Na,K-ATPase is the enzyme in the plasma membrane responsible for coupling the hydrolysis of ATP to the outward transport of sodium and inward movement of potassium, considerable effort has been directed toward detailing the molecular and biochemical aspects of the Na,K-ATPase, i.e. the Na/K pump. This volume presents a summary and evaluation of the current status of the field. The various contributions included were either written as overviews or presented as lectures or posters at the Third International conference on the Properties and Functions of Na,K-ATPase held on August 17-21, 1981 under the auspices of the Department of Physiology at Yale University School of Medicine, New Haven, Connecticut. It is apparent that much new and important information has emerged since the previous conference on the same subject was held in Sandbjerg Castle in Denmark in 1978. It will also be clear that while considerable insight has been provided, many problems still remain to be solved before we fully understand the manner in which the Na/K pump works. We hope that this volume will serve not only as a reference source for past work but as a stimulus for seeking new approaches to the problems that remain.

JOSEPHF. HOFFMAN B. FORBUSH 111

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Acknowledgments

We particularly want to thank J. S. Cook, P. DeWeer, P. L. Jdrgensen, B. G. Kennedy, M. Milanick, J. G. Ndrby, R. L. Post, J. D. Robinson, W. F. Schmidt 111, and D. Shoemaker for their help in editing the manuscript. In addition, we are grateful to Jean Milton and Kathy Barger-Shoemaker for their dedcated organizational effort with regard to the meeting. The conference on which this volume is based would not have been possible without financial aid. We wish to express our appreciation and our thanks for the support provided to Ciba-Geigy Pharmaceutical Company, E. I. DuPont De Nemours and Company, Hoechst-Roussel Pharmaceutical Company, ICI Americas, Incorporated, Merck Sharp and Dohme, Miles Laboratories, Incorporated, National Institutes of Health, The Upjohn Company, and USV Pharmaceutical Corporation.

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Yale Membrane Transport Processes Volumes

Joseph F. Hoffman (ed.). (1978). “Membrane Transport Processes,” Vol. 1. Raven, New York. Daniel C. Tosteson, Yu. A. Ovchinnikov, and Ramon Latorre (eds.). (1978). “Membrane Transport Processes,” Vol. 2. Raven, New York. Charles F. Stevens and Richard W. Tsien (eds.). (1979). “Membrane Transport Processes,” Vol. 3: Ion Permeation through Membrane Channels. Raven, New York. Emile L. Boulpaep (ed.). (1980). “Cellular Mechanisms of Renal Tubular Ion Transport”: Volume 13of Current Topics in Membranes and Transport (F. Bronner and A. Kleinzeller, eds.). Academic Press, New York. William H. Miller (ed.). (1981). “Molecular Mechanisms of Photoreceptor Transduction”: Volume 15 of Current Topics in Membranes and Transport (F. Bronner and A. Kleinzeller, eds.). Academic Press, New York. Clifford L. Slayman (ed.). (1982). “Electrogenic Ion Pumps”: Volume 16 of Current Topics in Membranes and Transport (A. Kleinzeller and F. Bronner, eds.). Academic Press, New York. Joseph F. Hoffman and Bliss Forbush 111 (eds.). (1983). “Structure, Mechanism, and Function of the Na/K Pump”: Volume 19 of Current Topics in Membranes and Transport (F. Bronner and A. Kleinzeller, eds.). Academic Press, New York.

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Contents of Previous Volumes

Volume 1

Volume 3

Some Considerations about the Structure of Cellular Membranes MAYNARDM. DEWEY AND LLOYDBARR The Transport of Sugars across Isolated Bacterial Membranes H. R. KABACK Galactoside Permease of Escherichiu coli ADAMKEPES Sulfhydryl Groups in Membrane Structure and Function

The Na+, K+-ATPase Membrane Transport System: Importance in Cellular Function ARNOLDSCHWARTZ, GEORGE E. LINDENMAYER, AND JULIUS C. ALLEN Biochemical and Clinical Aspects of Sarcoplasmic Reticulum Function ANTHONYMARTONOS1 The Role of Periaxonal and Perineuronal Spaces in Modifying Ionic Flow across Neural Membranes w. J. ADELMAN,JR.AND Y . PALTI Properties of the Isolated Nerve Endings GEORGINA RODRiGUEZ DE LORES A R N A l Z AND EDUARDO DE ROBERTIS Transport and Discharge of Exportable Proteins in Pancreatic Exocrine Cells: I n Vitro Studies J . D. JAMIESON The Movement of Water across Vasopressin-Sensitive Epithelia RICHARD M. HAYS Active Transport of Potassium and Other Alkali Metals by the Isolated Midgut of the Silkworm WILLIAM R. HARVEY AND KARLZERAHN

ASER ROTHSTEIN Molecular Architecture of the Mitochondrion DAVID H. MACLENNAN Author Index-Subject Index

Volume 2 The Molecular Basis of Simple Diffusion within Biological Membranes w. R. LlEB AND w . D.STEIN The Transport of Water in Erythrocytes ROBERT E. FORSTER Ion-Translocation in Energy-Conserving Membrane Systems AND M. MONTAL B. CHANCE Structure and Biosynthesis of the Membrane Adenosine Triphosphatase of Mitochondria ALEXANDER TZAGOLOFF Mitochondria1Compartments: A Companson of Two Models HENRY TEDESCHI Author Index-Subject Index

Author Index-Subject Index

Volume 4 The Genetic Control of Membrane Transport CAROLYN W.SLAYMAN

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xxxii

Enzymic Hydrolysis of Various Components in Biomembranes and Related Systems MAHENDRA KUMAR JAIN Regulation of Sugar Transport in Eukaryotic Cells HOWARD E. MORGAN AND CAROL F. WHITFIELD Secretory Events in Gastric Mucosa RICHARD P. DURBIN Author Index-Subject Index

Volume 5 Cation Transport in Bacteria: K+. Na+, and H+ FRANKLIN M. HAROLD AND KARLHEINZ ALTENWRF Pro and Contra Carrier Proteins: Sugar Transport via the Periplasmic GalactoseBinding Protein WINFRIED Boos Coupling and Energy Transfer in Active Amino Acid Transport ERICHHEINZ The Means of Distinguishing between Hydrogen Secretion and Bicarbonate Reabsorption: Theory and Applications to the Reptilian Bladder and Mammalian Kidney WILLIAM A. BRODSKY AND THEODORE P. SCHILB Sodium and Chloride Transport across Iso. lated Rabbit Ileum STANLEY G . SCHULTZ AND PETERF. CURRAN A Macromolecular Approach to Nerve Excitation ICHIJITASAKI AND EUILIO CARBONE Subject Index

Volume 6 Role of Cholesterol in Biomembranes and Related Systems MAHENDRAKUMAR JAIN Ionic Activities in Cells A. A. LEVAND W. McD. ARMSTRONG Active Calcium Transport and Ca*+-Activated ATPase in Human Red Cells H. J. SCHATZMANN The Effect of Insulin on Glucose Transport in Muscle Cells TORBEN CLAUSEN

CONTENTS OF PREVIOUS VOLUMES

Recognition Sites for Material Transport and Information Transfer HALVOR N. CHRISTENSEN Subject Index

Volume 7 Ion Transport in Plant Cells E. A. C. MACROBBIE H+ Ion Transport and Energy Transduction in Chloroplasts A. DILLEY AND RICHARD ROBERT T. GIAQUINTA The Present State of the Carrier Hypothesis PAULG. LEFEVRE Ion Transport and Short-circuit Technique S. REHM WARREN Subject Index

Volume 8 Chemical and Physical Properties of Myelin Proteins M. A. MOSCARELLO The Distinction between Sequential and Simultaneous Models for Sodium and Potassium Transport P. J. GARRAHAN AND R. P. GARAY Soluble and Membrane ATPase of Mitochondria, Chloroplasts, and Bacteria: Molecular Structure, Enzymatic Properties, and Functions RIVKAPANET AND D. RAo SANADI Competition, Saturation, and InhibitionIonic Interactions Shown by Membrane Ionic Currents in Nerve, Muscle, and Bilayer Systems ROBERT J. FRENCH AND WILLIAM J. ADELMAN, JR. Properties of the Glucose Transport System in the Renal Brush Border Membrane R. KINNE Subject Index

Volume 9 The State of Water and Alkali Cations within the Intracellular Fluids: The Contribution of NMR Spectroscopy MORDECHAI SHPoRER AND MORTIMER M.CIVAN

xxxiii

CONTENTS OF PREVIOUS VOLUMES

Electrostatic Potentials at Membrane-Solution Interfaces STUART MCLAUGHLIN A Thermodynamic Treatment of Active Sodium Transport S. ROYCAPLAN A N D ALVINESSIG Anaerobic Electron Transfer and Active Transport in Bacteria AND WILN. KONINGS JOHANNES BOONSTRA Protein Kinases and Membrane Phosphorylation HOSEYA N D M. MARLENE MARIANO TAO Mechanism and Physiological Significance of Calcium Transport across Mammalian Mitochondrial Membranes LEENA MELA Thyroidal Regulation of Active Sodium Transport F. ISMAIL-BEIGI Subject Index

Volume 10 Mechanochemical Properties of Membranes E. A. EVANS A N D R. M. HOCHMUTH Receptor-Mediated Protein Transport into Cells. Entry Mechanisms for Toxins, Hormones, Antibodies, Viruses, Lysosomd Hydrolases, Asialoglycoproteins, and Carrier Proteins M. NEVILLE, JR. A N D DAVID TALMIN CHANG The Regulation of Intracellular Calcium ERNEST0 CARAFOLI AND MARTINCROMPTON Calcium Transport and the Properties of a Calcium-Sensitive Potassium Channel in Red Cell Membranes VIRGIL10 L. LEWA N D HUGOG. FERREIRA Proton-Dependent Solute Transport in Microorganisms A. A. EDDY Subject Index

Volume 11 MI Surtace Glycoprotelns: Structure, Blosynthwls, and Biological Functions

The Cell Membrane-A Short Historical Perspective ASER ROTHSTEIN The Structure and Riosynthesis of Membrane Glycoproteins JENNIFER STURGESS, MARIOMOSCARELLO, AND HARRY SCHACHTER Techniques for the Analysis of Membrane Glycoproteins R. L. JULIANO Glycoprotein Membrane Enzymes AND JOHNR. RIORDAN GORDON G. FORSTNER Membrane Glycoproteins of Enveloped Viruses RICHARD w . COMPANS A N D MAURICEC. KEMP Erythrocyte Glycoproteins MICHAELJ. A. TANNER Biochemical Determinants of Cell Adhesion LLOYD A. CULP Proteolytic Modification of Cell Surface Macromolecules: Mode of Action in Stimulating Cell Growth KENNETHD. NOONAN Glycoprotein Antigens of Murine Lymphocytes MICHELLELETARTE Subject Index

Volume 12 Carriers and Membrane Transport Proteins

Isolation of Integral Membrane Proteins and Criteria for Identifying Carrier Proteins MICHAEL J. A. TANNER The Carrier Mechanism S. B. HLADKY The Light-Driven Proton Pump of Halobacterium halobium: Mechanism and Function MICHAELEISENBACH AND S. ROY CAPLAN Erythrocyte Anion Exchange and the Band 3 Protein: Transport Kinetics and Molecular Structure ~ I L I A. P KNAUF

CONTENTSOF PREVIOUSVOLUMES

xxxiv The Use of Fusion Methods for the Microinjection of Animal Cells R. G. KULKA AND A. h Y T E R Subject Index

Volume 13 Cellular Mechanismsof Renal Tubular Ion Transporl

PART I: ION ACTIVITY AND ELEMENTAL COMPOSITION OF INTRAEPITHELIAL COMPARTMENTS Intracellular pH Regulation WALTER F. BORON Reversal of the pH,-Regulating System in a Snail Neuron R. C. THOMAS How to Make and Use Double-Barreled Ion-Selective Microelectrodes THOMAS ZUETHEN The Direct Measurement of K, CI. Na, and H Ions in Bullfrog Tubule Cells MAMORU FUJIMOTO, KUNIHIKO KOTERA, AND YUTAKAMATSUMURA Intracellular Potassium Activity Measurements in Single Proximal Tubules of Necturus Kidney TAKAHIRO KUBOTA, BRUCEBIAGI,AND GERHARD GIEBISCH Intracellular Ion Activity Measurements in Kidney Tubules RAlA N. KHURI Intracellular Chemical Activity of Potassium in Toad Urinary Bladder JOELDELONGAND MORTIMER M.CIVAN Quantitative Determination of Electrolyte Concentrations in Epithelial Tissues by Electron Microprobe. Analysis ROGERRICK,A D ~ LD F~RGE, RICHARD BAUER,FRANZBECK, JUNEMASON,CHRISTIANE ROLOFF, AND KLAUS THURAU PART 11: PROPERTIES OF INTRAEPITHELIAL MEMBRANE BARRIERS IN THE KIDNEY wih.12 .A

‘11

iiiN

Hormonal Modulation of Epithelial Structure JAMESB. WADE Changes in Cell Membrane Surfaces Associated with Alterations of Transepithelial Ion Movement MICHAELKASHGARIAN The Dimensions of Membrane Barriers in Transepithelial Flow Pathways LARRY w. WELLING AND DANJ. WELLING Electrical Analysis of Intraepithelial Barriers EMILEL. BOULPAEP AND HENRY SACKIN Membrane Selectivity and Ion Activities of Mammalian Tight ‘Epithelia SIMONA. LEWIS,NANCYK. WILLS, AND DOUGLAS C. EATON Ion Conductances and Electrochemical Potential Differences across Membranes of Gallbladder Epithelium Luis REUSS A Kinetic Model for Ion Fluxes in the Isolated Perfused Tubule BRUCEBIAGI,ERNESTO GONWLEZ, AND GERHARD GIEBISCH The Effects of Voltage Clamping on Ion Transport Pathways in Tight Epithelia ARTHUR L. FINNAND PAULA ROGENES Tubular Permeability to Buffer Components as a Determinant of Net H Ion Fluxes G. MALNIC, V. L. COSTASILVA,S. S. CAMPIGLIA, M. DE MELLOAIRES,AND G. GIEBISCH Ionic Conductance of the Cell Membranes and Shunts of Necturus Proximal Tubule GENJIRO KIMURA AND KENNETH R. SPRING Luminal Sodium Phosphate Cotransport as the Site of Regulation for Tubular Phosphate Reabsorption: Studies with Isolated Membrane Vesicles HEINIMURER,REINHARD STOLL. CARLAEVERS,ROLFKINNE, JEAN-PHILIPPE BONIOUR, AND HERBERT FLEISCH The Mechanism of Coupling between Glucose Transport and Electrical Potential in the Proximal Tubule: A Study of Potential8flO193RUY ISSlQOlO#tr On6 ,~ISWltW@OtB

CONTENTS OF PREVIOUSVOLUMES

Dependent Phlorizin Binding to Isolated Renal Microvillus Membranes PETER s. ARONSON Electrogenic and Electroneutral Na Gradient-Dependent Transport Systems in the Renal Brush Border Membrane Vesicle BERTRAM SACKTOR PART 111: INTRAMEMBRANE CARRIERS AND ENZYMES IN TRANSEPITHELIAL TRANSPORT

Sodium Cotransport Systems in the Proximal Tubule: Current Developments R. KINNE, M. BARAC,AND H.MURER ATPases and Salt Transport in the Kidney Tubule MARGARITA P E R E Z - m N G L E Z DE LA MANNA,FULCENCIO PROVERBIO, AND GUILLERMO WHITEMBURY Further Studies on the Potential Role of an Anion-Stimulated Mg-ATPase in Rat Proximal Tubule Proton Transport E. KINNE-SAFFRAN A N D R. KINNE Renal Na+- K+-ATPase: Localization and Quantitation by Means of Its K+-Dependent Phosphatase Activity RElNlER BEEUWKES 111 A N D SEYMOUR ROSEN Relationship between Localization of N+K+-ATPase, Cellular Fine Structure, and Reabsorptive and Secretory Electrolyte Transport STEPHEN A. ERNST, CLARA v . RIDDLE, AND KARLJ. KARNAKY, JR. Relevance of the Distribution of Na+ Pump Sites to Models of Fluid Transport across Epithelia JOHNW. MILLSA N D D ~ N A LR.DDIBONA Cyclic AMP in Regulation of Renal Transport: Some Basic Unsolved Questions THOMASP. DOUSA Distribution of Adenylate Cyclase Activity in the Nephron F. MOREL,D. CHABARDBS, AND M. IMBERT-TEBOUL Subject Index

xxxv

Volume 14 Carriers and Membrane Transport Proteins

Interface between Two Immiscible Liquids as a Tool for Studying Membrane Enzyme Systems L . I. BOGUSLAVSKY Criteria for the Reconstitution of Ion Transport Systems ADlL E. S H A M 0 0 AND WILLIAM F. TIvoL The Role of Lipids in the Functioning of a Membrane Protein: The Sarcoplasmic Reticulum Calcium Pump J. P. BENNETT,K. A. McGiLL, AND G. B. WARREN The Asymmetry of the Hexose Transfer System in the Human Red Cell Membrane w.F. WlDDAS Permeation of Nucleosides, Nucleic Acid Bases, and Nucleotides in Animal Cells PETERG. W. PLAGEMANN AND ROBERTM. WOHLHUETER Transmembrane Transport of Small Peptides D. M. MATTHEWS AND J. W. PAYNE Characteristics of Epithelial Transport in Insect Malpighian Tubules S. €3. P. MADDRELL Subject Index

Volume 15 Molecular Mechanisms of Photoreceptor Transduction

PART I: T H E ROD PHYSIOLOGICAL RESPONSE The Photocurrent and Dark Current of Retinal Rods G. MATTHEWS AND D. A. BAYLOR Spread of Excitation and Background Adaptation in the Rod Outer Segment K.-W. YAU,T. D. LAMB,AND P. A. MCNAUGHTON Ionic Studies of Vertebrate Rods W. GEOFFREY OWENAND VINCENT TORRE

xxxvi

Photoreceptor Coupling: Its Mechanism and Consequences GEOFFREY H. GOLD PART 11: THE CYCLIC NUCLEOTIDE ENZYMATIC CASCADE AND CALCIUM ION First Stage of Amplification in the CyclicNucleotide Cascade of Vision LUBERT STRYER, JAMESB. HURLEY, A N D BERNARD K.-K. FUNC Rod Guanylate Cyclase Located in Axonemes DARRELL FLEISCHMAN Light Control of Cyclic-Nucleotide Concentration in the Retina THOMAS G. EBREY, PAULKILBRIDE, JAMES B. HURLEY, ROGERCALHOON, AND MOTOYUKI TSUDA Cyclic-GMP Phosphodiesterase and Calmodulin in Early-Onset Inherited Retinal Degenerations G. J. CHADER, Y. P. LIU, R. T. FLETCHER, G. AGUIRRE, R. SANTOS-ANDERSON, AND M. T'SO Control of Rod Disk Membrane Phosphodiesterase and a Model for Visual Transduction P. A. LIEBMAN AND E. N. PUCH, JR. Interactions of Rod Cell Proteins with the Disk Membrane: Influence of Light, Ionic Strength, and Nucleotides HERMANN KUHN Biochemical Pathways Regulating Transduction in Frog Photoreceptor Membranes M. DERICBOWNDS The Use of Incubated Retinas in Investigating the Effects of Calcium and Other Ions on Cyclic-Nucleotide Levels in Photoreceptors ADOLPHI. COHEN Cyclic AMP Enrichment in Retinal Cones DEBORA B. FARBER Cyclic-Nucleotide Metabolism in Vertebrate Photoreceptors: A Remarkable Analogy and an Unraveling Enigma M. W. BITENSKY. G. L. WHEELER, A. YAMAZAKI, M. M. RASENICK, AND P. J. STEIN

CONTENTSOF PREVIOUSVOLUMES

Guanosine Nucleotide Metabolism in the Bovine Rod Outer Segment: Distribution of Enzymes and a Role of GTP HITOSHISHICHI Calcium Tracer Exchange in the Rods of Excised Retinas ETE z. SZUTS The Regulation of Calcium in the Intact Retinal Rod: A Study of Light-Induced Calcium Release by the Outer Segment GEOFFREY H. GOLDAND JUANI. KORENBROT Modulation of Sodium Conductance in Photoreceptor Membranes by Calcium Ions and cGMP ROBERT T. SORBI PART 111: CALCIUM, CYCLIC NUCLEOTIDES, AND THE MEMBRANE POTENTIAL Calcium and the Mechanism of Light Adaptation in Rods BRUCEL. BASTIAN AND GORDONL. FAIN Effects of Cyclic Nucleotides and Calcium Ions on Bufo Rods JOELE. BROWN AND GERALDINE WALOCA The Relation between Ca2+and Cyclic GMP in Rod Photoreceptors STUART A. LIFTON AND JOHNE. DOWLING Limits on the Role of Rhodopsin and cGMP in the Functioning of the Vertebrate Photoreceptor SANFORD E. OSTROY, EDWARD P. MEYERTHOLEN, PETERJ. STEIN, ROBERTA A. SVOBODA, A N D MEEGAN J. WILSON [Ca*+],Modulation of Membrane Sodium Conductance in Rod Outer Segments BURKSOAKLEY I1 AND LAWRENCE H. PINTO Cyclic-GMP-Induced Depolarization and Increased Response Latency of Rods: Antagonism by Light WILLIAM H. MILLERAND GRANT D. NICOL

CONTENTS OF PREVIOUS VOLUMES

PART 1V: AN EDlTORlAL OVERVIEW Ca2+and cGMP WILLIAM H. MILLER Index

Volume 16 Electrogenic Ion Pumps

PART 1. DEMONSTRATlON O F PUMP ELECTROGENlClTY IN EUKARYOTlC CELLS Electrophysiology of the Sodium Pump in a Snail Neuron R. C. THOMAS Hyperpolarization of Frog Skeletal Muscle Fibers and of Canine Purkinje Fibers during Enhanced Na+-K+ Exchange: Extracellular K+ Depletion or lncreased Pump Current? DAVID C. GADSBY The Electrogenic Pump in the Plasma Membrane of Nitella RMXR M. SPANSWICK Control of Electrogenesis by ATP, Mga+. H+, and Light in Perfused Cells of Cham MASASHI TAZAWA AND T ~ R USO HlMMtN PART 11. THE EVlDENCE IN EPlTHELl AL MEMBRANES An Electrogenic Sodium Pump in a Mammalian Tight Epithelium s. A. LEWIS A N D N. K. WILLS A Coupled Electrogenic Na+-K+ Pump for Mediating Transepithelial Sodium Transport in Frog Skin ROBERTNIELSEN Transepithelial Potassium Transport in lnsect Midgut by an Electrogenic Alkali Metal lon Pump MICHAEL G . WOLFERSBERCER, WILLIAM R. HARVEY, AND MOIRAClOFFl The ATP-Dependent Component of Gastric Acid Secretion G . SACHS, B. WALLMARK. G. SACCOMANI, E. RABON, H. B. STEWART, D. R. DIBONA,A N D T. BERGLINDH

xxxvii

PART 111. REVERSlBILlTY: ATP SYNTHESIS DRIVEN BY ELECTRlC FIELDS Effect of Electrochemical Gradients on Active H+ Transport in an Epithelium QAISAL-AWQATI A N D TROYE. DIXON Coupling between H+ Entry and ATP Synthesis in Bacteria PETERC. MALONEY Net ATP Synthesis by H+-ATPase Reconstituted into Liposomes YASUOKAGAWA Phosphorylation in Chloroplasts: ATP Synthesis Driven by A+ and by ApH of Artificial or Light-Generated Origin ~ T E GRABER R PART 1V. SOME THEORETICAL QUESTlONS Response of the Proton Motive Force to the Pulse of an Electrogenic Proton Pump ERlCH HElNZ Reaction Kinetic Analysis of CurrentVoltage Relationships for Electrogenic Pumps in Neurospora and Acetabularia DIETRICH GRADMANN, ULF-PETER HANSEN, AND CLIFFORD L . S L A Y M A N Some Physics of Ion Transport HAROLD J. MOROWITZ PART V. MOLECULAR MECHANISMS OF CHARGE SEPARATION An H+-ATP Synthetase: A Substrate Translocation Concept 1. A. KOZLOVA N D V. P. SKULACHEV Proton Translocation by Cytochrome Oxidase MARTENWIKSTROM Electrogenic Reactions of the Photochemical Reaction Center and the UbiquinoneCytochrome b / c 2Oxidoreductase P. LESLIEDU-ITON,PAULMUELLER. DANIEL P. O'KEEFE, "GEL K. PACKHAM. ROGERC. PRINCE,A N D DAVIDM. TIEDE

xxxviii Proton-Membrane Interactions in Chloroplast Bioenergetics R. A. DILLEY, L. J. PROCHASKA, G. M. BAKER,N. E. TANDY, AND A. MILLNER Photochemical Charge Separation and Active Transport in the Purple Membrane BARRY HoNlG Mitochondrial Transhydrogenase: General Principles of Functioning I. A. KOZLOV Membrane Vesicles. Electrochemical Ion Gradients, and Active Transport H. R. KABACK PART V1. BlOLOGlCAL SIGNIFICANCE OF ELECTROGENIC ION PUMPS The Role of Electrogenic Proton Translocation in Mitochondria1 Oxidative Phosphor ylation JA N N A €? WEHRLE Electrogenic Reactions and Proton Pumping in Green Plant Photosynthesis WOLFGANG JUNGE The Role of the Electrogenic Sodium Pump in Controlling Excitability in Nerve and Cardiac Fibers MARIO VASSALLE Pumps and Currents: A Biological Perspective FRANKLIN M. HAROLD Index

Volume 17 Membrane Lipids of Prokaryota Lipids of Prokaryotes-Structure and Distribution HOWARD GOLDFINE Lipids of Bacteria Living in Extreme Environments THOMAS A. LANGWORTHY Lipopolysaccharides of Gram-Negative Bacteria OTTO LUDERITZ, MARINA A. FREUDENBERG, CHRISGALANOS, VOLKER LEHMANN. ERNSTTH. RIETSCHEL, AND DEREKH. SHAW

CONTENTS OF PREVIOUS VOLUMES Prokaryotic Polyterpenes: Phylogenetic Precursors of Sterols GUY OURISSON AND MlCHEL ROHMER Sterols in Mycoplasma Membranes S H M U E L RAZlN Regulation of Bacterial Membrane Lipid Synthesis 0. RCXK A N D CHARLES JOHNE. CRONAN, JR. Transbilayer Distribution of Lipids in Microbial Membranes SHLOMO ROTTEM Lipid Phase Transitions and Regulation of Membrane Fluidity in Prokaryotes DONALD L. MELCHIOR Effects of Membrane Lipids on Transport and Enzymic Activities RONALD N. MCELHANEY Index

Volume 18

Part I. Adenylate Cyclase-Related Receptors Hormone Receptors and the Adenylate Cyclase System: Historical Overview B. RICHARDMARTIN The Elucidation of Some Aspects of Receptor Function by the Use of a Kinetic Approach A. M. TOLKOVSKY The @-Adrenergic Receptor: Ligand Binding Studies Illuminate the Mechanism of Receptor Adenylate Cyclase Coupling AND ROBERT J. JEFFREY M. STADEL LEFKOW~TZ Receptor-Mediated Stimulation And Inhibition of Adenylate Cyclase DERMOT M. F. COOPER Desensitization of the Response of Adenylate Cyclase to Catecholamines JOHNP. PERKINS Hormone-Sensitive Adenylate Cyclase: Identity, Function, and Regulation of the Protein Components ELLIOTT M. Ross, STEENE. F’EDERSEN, AND VINCENT A. FLORIO The Regulation of Adenylate Cyclase. by Glycoprotein Hormones BRIANA. COOKE

CONTENTS OF PREVIOUS VOLUMES The Activity of Adenylate Cyclase Is Regulated by the Nature of Its Lipid Environment AND LARRY M. MILESD. HOUSLAY GORDON The Analysis of Interactions between Hormone Receptors and Adenylate Cyclase by Target Size Determinations Using Irr.ah4adiation Inactivation B. RICHARD MARTIN

Part II. Receptors Not InvolvingAdenylate Cyclase Vasopressin Isoreceptors in Mammals: Relation to Cyclic AMP-Dependent and Cyclic AMP-Independent Transduction Mechanisms SERGE JARD Induction of Hormone Receptors and Responsiveness during Cellular Differentiation MICHAEL C. LINA N D SUZANNE L. BECKNER

xxxix Receptors for Lysosomal Enzymes and Glycoproteins VIRGINIA SHEPHERD, PAULSCHLESINGER. AND PHILIP STAHL The Insulin-Sensitive Hexose Transport System in Adipocytes J. GLIEMANN AND w.D. REES Epidermal Growth Factor Receptor and Mechanisms for Animal Cell Division MANlUSRl DAS The Linkage between Ligand Occupation and Response of the Nicotinic Acetylcholine Receptor PALMER TAYLOR. ROBERT DALEBROWN, AND DAVID A. JOHNSON The Interaction of Cholera Toxin with Gangliosides and the Cell Membrane SIMON VANHEYNINGEN Subject Index

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Part I

Thermodynamic Aspects of Membrane Transport

Part I Ion Activity and Elemental Composition of lntraepithelial Compartments

2

WILLIAM P. JENCKS

I t i s important t o formulate t h e s e questions as c l e a r l y a s p o s s i b l e and t o a v o i d i m p r e c i s e l y d e f i n e d terms and models t h a t may d e l a y p r o g r e s s i f t h e y are b e l i e v e d t o r e p r e s e n t a r e a l u n d e r s t a n d i n g of t h e c o u p l i n g p r o c e s s . Many models have been proposed t o " e x p l a i n " t h e a c t i v e t r a n s p o r t of i o n s and s m a l l molec u l e s and t h e s y n t h e s i s o f ATP from p r o t o n t r a n s p o r t i n o x i d a t i v e p h o s p h o r y l a t i o n and p h o t o p h o s p h o r y l a t i o n . Some o f t h e s e p r o p o s a l s have i n c l u d e d (1) mechanisms i n which a change from t i g h t b i n d i n g on one s i d e t o weak b i n d i n g on t h e o t h e r s i d e of a membrane i s respons i b l e f o r coupled t r a n s p o r t of a l i g a n d , ( 2 ) p a r t i c u l a r s t e p s i n which c o u p l i n g o r e n e r g y t r a n s f e r o c c u r s , ( 3 ) " e n e r g i z e d s t a t e s , " and ( 4 ) hydrogen-bonded o r o t h e r channels f o r ion transport. I t i s n o t always c l e a r what i s t h e r e l a t i o n s h i p t o a coupled p r o c e s s o r even I t can be t h e meaning o f t h e s e p r o p o s a l s and terms. h e l p f u l i n u n d e r s t a n d i n g t h e q u e s t i o n s t h a t need t o be answered and t h e mechanism o f t h e s e systems i f t h o s e c h a r a c t e r i s t i c s of a coupled v e c t o r i a l process t h a t a r e d i r e c t l y r e s p o n s i b l e f o r c o u p l i n g are s e p a r a t e d from t h o s e t h a t f a c i l i t a t e t h e p r o c e s s by i n c r e a s i n g t h e t u r n o v e r r a t e u n d e r p h y s i o l o g i c a l c o n d i t i o n s . These c h a r a c t e r i s t i c s may be r e l a t e d i n some s y s t e m s , b u t a c l e a r d i s t i n c t i o n can u s u a l l y be made. The sodiumand c a l c i u m - t r a n s p o r t i n g A T P a s e s a r e c o n s i d e r e d from t h i s p o i n t of view i n t h i s b r i e f a r t i c l e , which r e p r e s e n t s one s t a g e i n a n e n z y m o l o g i s t ' s a t t e m p t t o unders t a n d what i s meant by a c o u p l e d v e c t o r i a l p r o c e s s . The c o u p l i n g p r o c e s s i t s e l f c a n be d e s c r i b e d by a set o f r u l e s . Understanding c o u p l i n g t h e n r e p r e s e n t s an u n d e r s t a n d i n g o f t h e s e r u l e s and how t h e y a r e enf o r c e d . The r u l e s o f t e n r e p r e s e n t enzyme s p e c i f i c i t i e s f o r c a t a l y s i s t h a t a r e s t r i c t l y comparable t o t h e s p e c i f i c i t i e s o f o r d i n a r y enzymes. They d i f f e r from t h e s p e c i f i c i t i e s o f most enzymes, however, i n t h a t t h e y are t u r n e d on and o f f , o r change i n d i f f e r e n t s t a t e s of t h e enzymes ( J e n c k s , 1 9 8 0 ) . These s p e c i f i c i t i e s i n v o l v e t h e b i n d i n g of l i g a n d s , b u t t h e y d i f f e r from s i m p l e b i n d i n g i n t h a t v e r y l a r g e d i f f e r e n c e s i n c a t a l y t i c s p e c i f i c i t i e s a re p o s s i b l e . Binding of a l i g a n d may change by 103 between d i f f e r e n t s t a t e s , b u t enzyme s p e c i f i c i t i e s can d i s c r i m i n a t e e v e n a g a i n s t s m a l l m o l e c u l e s by f a c t o r s of 1010 (Ray and Long, 1976; Ray et a l . , 1976) o r more. Thus, t h e s p e c i f i c i t y r u l e s have t h e p o t e n t i a l t o g i v e v e r y t i g h t c o u p l i n g . To t h e e x t e n t t h a t t h e r u l e s are v i o l a t e d t h e system becomes uncoupled. T h i s o c c u r s i n some s i t u a t i o n s and may p r o v i d e a n e x p e r i m e n t a l approach t o u n d e r s t a n d i n g t h e enzymatic b a s i s f o r t h e r u l e s . The s p e c i f i c i t y r u l e s a r e

WHAT IS A COUPLED VECTORIAL PROCESS

3

o f t e n i m p l i c i t , and sometimes are e x p l i c i t i n t h e models t h a t have been proposed f o r c o u p l e d v e c t o r i a l p r o c e s s e s ( H i l l , 1969, 1977; S t e i n and Honig, 1977; de M e i s and Vianna, 1979; C a n t l e y , 1 9 8 1 ) . The p r i m a r y f u n c t i o n of t h e u t i l i z a t i o n o f b i n d i n g e n e r g y , on t h e o t h e r hand, i s t o make t h e system work a t a u s e f u l r a t e under p h y s i o l o g i c a l c o n d i t i o n s ( H i l l , 1 9 7 7 ; S t e i n and Honig, 1977; J e n c k s , 1980; H i l l and E i s e n b e r g , 1 9 8 1 ) . Binding e n e r g y may be e x p r e s s e d i n one s t a t e i n o r d e r t o p i c k up a n i o n from d i l u t e s o l u t i o n and n o t e x p r e s s e d i n a n o t h e r s t a t e when t h e i o n i s r e l e a s e d i n t o a c o n c e n t r a t e d s o l u t i o n , f o r example, Noncovalent b i n d i n g e n e r g y i s u t i l i z e d t o s t a b i l i z e high-energy chemical compounds s u c h a s ATP and a c y l p h o s p h a t e s t h a t a r e formed s p o n t a n e o u s l y from i n o r g a n i c p h o s p h a t e , so t h a t s p e c i e s c o n t a i n i n g high- and lowe n e r g y chemical bonds can c o e x i s t a t comparable concent r a t i o n s and be i n t e r c o n v e r t e d r a p i d l y . T h i s f a c i l i t a t e s t h e o v e r a l l p r o c e s s by making p r o c e s s e s r e a d i l y r e v e r s i b l e t h a t would be e f f e c t i v e l y i r r e v e r s i b l e i n d i l u t e sol u t i o n . I t h e l p s t o p r e s e r v e t h e e n e r g y of ATP f o r conv e r s i o n t o chemical o r o s m o t i c work and a v o i d s v e r y h i g h energy s p e c i e s of t h e r e a c t a n t s i n t h e c a t a l y t i c c y c l e t h a t would be p r e s e n t i n low c o n c e n t r a t i o n s and r e q u i r e l a r g e t u r n o v e r numbers, o r v e r y low e n e r g y s p e c i e s t h a t would c a u s e p i l i n g up of t h e enzyme i n o n e s t a t e . O u r p r e s e n t u n d e r s t a n d i n g of t h e s e s y s t e m s s u g g e s t s t h a t t h e p r i m a r y r o l e of b i n d i n g e n e r g y i s k i n e t i c , t o a l l o w f a s t turnover. I f t h e b i n d i n g e n e r g i e s w e r e n o t so n i c e l y b a l a n c e d , t h e system c o u l d s t i l l b e c o u p l e d , b u t it would t u r n over too slowly t o be useful. The changes i n b i n d i n g e n e r g y t o a l i g a n d when o t h e r l i g a n d s b i n d o r i n d i f f e r e n t s t a t e s o f t h e system can be described i n t e r m s of linked functions o r i n t e r a c t i o n e n e r g i e s (Wyman, 1964; Weber, 1975; H i l l , 1 9 7 7 ) . The u t i l i z a t i o n of b i n d i n g e n e r g y i n t h e s e and o t h e r systems can be summarized by t h e s t a t e m e n t t h a t i n t r i n s i c b i n d i n g energy t o a l i g a n d i s expressed d i r e c t l y i n one s t a t e of t h e s y s t e m , b u t i s n o t e x p r e s s e d ( o r i s less e x p r e s s e d ) i n a n o t h e r s t a t e ; i n s t e a d i t may be u t i l i z e d t o b r i n g a b o u t loss o f e n t r o p y and t o pay f o r s t r a i n o r o t h e r des t a b i l i z a t i o n mechanisms. T h i s c a n be i l l u s t r a t e d by e n e r g y b a r diagrams ( J e n c k s , 1975, 1 9 8 0 ) .

WILLIAM P.JENCKS

4

11.

SPECIFICITY RULES

The r u l e s t h a t d e f i n e a c o u p l e d v e c t o r i a l p r o c e s s can o f t e n be d e s c r i b e d by a s i m p l e c y c l e l s u c h as a t w o by two m a t r i x i n which d i f f e r e n t s p e c i f i c i t i e s and processes o c c u r i n two p a i r s of two s t a t e s (Scheme 1 ) . Transport No

Yes

Chemical specificity

Scheme 1 F o r example, d i f f e r e n t c h e m i c a l s p e c i f i c i t i e s f o r catal y s i s can be r e p r e s e n t e d by s t a t e s 1 and 2 and d i f f e r e n t s p e c i f i c i t i e s f o r i o n t r a n s p o r t by t h e s t a r r e d and u n s t a r r e d s t a t e s o f an enzyme. There i s no o n e s t e p i n s u c h a c y c l e a t which c o u p l i n g can be s a i d t o o c c u r . A complete r e a c t i o n c y c l e g i v e s r i s e t o t h e c o u p l e d p r o c e s s , and a f a i l u r e o f t h e c o u p l i n g mechanism i n any of s e v e r a l steps c a n d e s t r o y c o u p l i n g . The sodium-ATPase and t h e c a l c i u m - A T P a s e p r o v i d e t h e b e s t examples o f s u c h stoichiometric r e a c t i o n c y c l e s t h a t are a v a i l a b l e a t t h e present t i m e . W e would l i k e t o i d e n t i f y t h e s i m p l e s t set of s p e c i f i c i t y r u l e s f o r v e c t o r i a l coupling t h a t can be i d e n t i f i e d i n t h e models t h a t have been proposed f o r t h e s e systems and can be d i r e c t l y r e l a t e d t o t h e chemic a l p r o p e r t i e s of t h e enzymes. The c o u p l i n g i n t h e conv e r s i o n o f t h e chemical e n e r g y of ATP t o mechanical work through r e a c t i o n s o f a c t i n and myosin c a n be d e s c r i b e d by a q u i t e d i f f e r e n t s e t of s p e c i f i c i t y r u l e s ( J e n c k s , 1980 I 1982). The r e v e r s i b l e t r a n s p o r t o f c a l c i u m i n t o a s a r c o p l a s m i c r e t i c u l u m v e s i c l e w i l l be c o u p l e d t o t h e hydrol y s i s of ATP i f E l reacts r e v e r s i b l y o n l y w i t h ATP t o form enzyme-phosphate and E2 r e a c t s r e v e r s i b l y o n l y w i t h i n o r g a n i c p h o s p h a t e t o form enzyme-phosphate, w h i l e E i n t e r c o n v e r t s between o u t s i d e - and i n s i d e - e x p o s e d

WHAT IS A COUPLED VECTORIAL PROCESS

5

calcium-binding s i t e s o n l y i n t h e absence of c a l c i u m , and E* i n t e r c o n v e r t s t h e b i n d i n g s i t e s o n l y i f calcium i s bound t o them (Scheme 2 ) ( d e Meis and Vianna, 1979; Y a m a m o t o e t al., 1979; J e n c k s , 1 9 8 0 ) . E* i s t h e phosphoenzyme, E-PI i n t h i s system. 2+ 2Caout

ATP

2 /

EhP 2+ 1- C a 2

s*

-P

2 - C a 2+ 2

+

ADP

( n o t HOH)

+

HOH ( n o t ADP)

Scheme 2 S t a r t i n g w i t h t h e a c y l phosphate i n t e r m e d i a t e , t h e s p e c i f i c i t y o f t h e enzyme changes from c a t a l y s i s of t h e t r a n s f e r of p h o s p h a t e t o ADP from E l % P and t o water from E2-P. I t i s o b v i o u s t h a t f a i l u r e of t h i s s p e c i f i c i t y w i l l g i v e uncoupled ATP h y d r o l y s i s i f E1QP can a l so t r a n s f e r p h o s p h a t e t o water. F a i l u r e o f t h e s p e c i f i c i t y of E 2 w i l l a l s o g i v e u n c o u p l i n g because i f E2-P can t r a n s f e r p h o s p h a t e t o ADP t h e r e v e r s e r e a c t i o n must a l s o be c a t a l y z e d . ATP w i l l t h e n r e a c t w i t h E 2 t o g i v e Ez-P, which w i l l undergo h y d r o l y s i s w i t h o u t t r a n s p o r t of c a l cium. The r u l e s f o r i n t e r c o n v e r s i o n o f E l and E 2 w i t h and w i t h o u t bound calcium a r e r e q u i r e d i n o r d e r t o p r e v e n t f o r m a t i o n of E2-P from ElhP and h y d r o l y s i s o f ATP w i t h o u t calcium t r a n s p o r t , and t o p r e v e n t l e a k a g e of c a l c i u m w i t h o u t ATP s y n t h e s i s . T h i s c y c l e w i l l g i v e t r a n s p o r t o f t w o calcium i o n s across t h e membrane t h a t i s coupled t o t h e h y d r o l y s i s o r s y n t h e s i s of ATP e a c h t i m e it i s completed i n t h e f o r w a r d o r reverse d i r e c t i o n , r e g a r d l e s s of t h e a f f i n i t y of t h e d i f f e r e n t s t a t e s f o r calcium o r o t h e r l i g a n d s ; d i f f e r e n t amounts o f e x p r e s s i o n o f b i n d i n g e n e r g y f o r t h e l i g a n d s d e t e r m i n e how f a s t t h e reactions occur. These r u l e s are u s e f u l i f t h e y f o c u s a t t e n t i o n on t h e mechanism by which t h e c h e m i c a l p r o p e r t i e s and s p e c i f i c i t i e s o f t h e enzyme b r i n g a b o u t c o u p l i n g . A s i m p l e r s e t o f r u l e s can be f o r m u l a t e d t h a t i s based more d i r e c t l y on known c a t a l y t i c p r o p e r t i e s of t h e

6

WILLIAM P. JENCKS

Ca2+-ATPase. I t h a s been known f o r a long t i m e , b u t n o t always r e c o g n i z e d e x p l i c i t l y , t h a t t h e enzyme f o l lows a n o r d e r e d k i n e t i c mechanism i n which calcium i s n o t r e l e a s e d from El%P.Ca2 t o t h e o u t s i d e of t h e v e s i c l e u n t i l a f t e r t h e phosphate i s removed by t r a n s The o p p o s i t e sequence i s followed on t h e f e r t o ADP. i n s i d e , i n which phosphate i s n o t removed from E 2 - P - C a 2 by t r a n s f e r t o w a t e r u n t i l a f t e r calcium i s r e l e a s e d t o t h e i n s i d e of t h e v e s i c l e . The enzyme behaves a s i f t h e calcium were covered up by phosphate, so t h a t i t c a n n o t come o f f t o t h e o u t s i d e b u t i s o n l y r e l e a s e d i n s i d e a f t e r t h e c o v a l e n t a c y l phosphate bond i s formed. Cantley ( 1 9 8 1 ) h a s s u g g e s t e d a s i m i l a r c o v e r i n g of sodium by phosphate i n t h e Na-ATPase. (However, t h e r e i s no e x p e r i m e n t a l e v i d e n c e f o r e i t h e r enzyme t h a t t h i s i s t h e a c t u a l r e a s o n f o r an o r d e r e d mechanism.) The absence of s i g n i f i c a n t calcium release from E - P - C a 2 t o t h e o u t s i d e h a s been e v i d e n t from t h e o b s e r v a t i o n t h a t v e s i c l e s loaded w i t h calcium can s y n t h e s i z e ATP from ADP and phosphate i n t h e p r e s e n c e of t h e c h e l a t i n g a g e n t EGTA ( B a r l o g i e e t a l . , 1 9 7 1 ; Makinose and Hasselbach, 1 9 7 1 ) . Calcium i s r e q u i r e d f o r phosp h o r y l a t i o n of t h e enzyme by ATP and must a l s o be req u i r e d f o r t h e r e v e r s e r e a c t i o n ; i f it could d i s s o c i a t e t o t h e o u t s i d e from E - P - C a 2 , t h e r e s u l t i n g E-P could n o t S e v e r a l more r e c e n t s t u d i e s have conr e a c t w i t h ADP. f i r m e d t h a t t h e calcium i n E - P - C a 2 i s occluded (Kanazawa e t a l . , 1 9 7 1 ; Dupont, 1 9 8 0 ; Takakuwa and Kanazawa, 1 9 8 1 ; Takisawa and Makinose, 1 9 8 1 ) . The complementary o r d e r e d mechanism on t h e i n s i d e i s shown by t h e i n h i b i t i o n of t h e h y d r o l y s i s of E-P and by t h e synt h e s i s of ATP from E-P i n t h e p r e s e n c e of h i g h concent r a t i o n s of calcium (Ikemoto, 1975; Knowles and Racker, 1975; de Meis and Tume, 1 9 7 7 ) . The l a t t e r o b s e r v a t i o n and t h e s y n t h e s i s of ATP from loaded v e s i c l e s show t h a t calcium can bind t o E-P t o g i v e a s p e c i e s t h a t c a n d o n a t e phosphate t o ATP r a t h e r t h a n undergo h y d r o l y s i s . These f a c t s a r e c o n s i s t e n t w i t h t h e f o l l o w i n g s i m p l e set of r u l e s (Scheme 3 ) :

WHAT IS A COUPLED VECTORIAL PROCESS

7

2+ 2Caout E

.C a 2+ 2

%:

pi HOH

ATP ADP

2+ 2Cain

Scheme 3

1. Binding and d i s s o c i a t i o n o f c a l c i u m on t h e o u t s i d e occurs o n l y w i t h f r e e enzyme; t h e r e i s a k i n e t i c b a r r i e r so t h a t t h e b i n d i n g and d i s s o c i a t i o n o f c a l c i u m on t h i s s i d e w i t h enzyme-phosphate d o e s n o t o c c u r i n t h e coupled r e a c t i o n c y c l e . 2. Binding and d i s s o c i a t i o n o f calcium on t h e i n s i d e of t h e v e s i c l e o c c u r s o n l y w i t h enzyme-phosphate; b i n d i n g to free E does n o t l e a d t o any r e a c t i o n , i f i t o c c u r s a t a l l , and d i s s o c i a t i o n o f c a l c i u m from E - C a t o t h e i n s i d e does not occur. 3. The c a t a l y t i c s p e c i f i c i t y o f enzyme-calcium i s f o r reaction with nucleoside phosphates, not inorganic p h o s p h a t e and water. 4. The c a t a l y t i c s p e c i f i c i t y of f r e e enzyme o r enzyme-potassium i s f o r r e a c t i o n w i t h i n o r g a n i c phosp h a t e and w a t e r , n o t n u c l e o s i d e p h o s p h a t e s . I t i s a l t o g e t h e r r e a s o n a b l e t h a t t h e a c t i v i t y and s p e c i f i c i t y o f a n enzyme f o r p h o s p h a t e t r a n s f e r r e a c t i o n s s h o u l d be c o n t r o l l e d by i n o r g a n i c c a t i o n s . The v e c t o r i a l s p e c i f i c i t y f o r calcium i s c o n t r o l l e d e n t i r e l y by t h e s t a t e of p h o s p h o r y l a t i o n of t h e enzyme a c c o r d i n g t o t h i s set o f r u l e s . T h i s l i n k s t h e v e c t o r i a l s p e c i f i c i t y t o a s i m p l e chemical p r o p e r t y o f t h e system. I t a l s o a v o i d s d e s i g n a t i o n s s u c h as E l and E 2 , f o r which t h e r e are s e v e r a l d i f f e r e n t d e f i n i t i o n s ; some of these d e f i n i t i o n s a r e d i f f i c u l t t o a p p l y e x p e r i m e n t a l l y . Conf o r m a t i o n changes upon i o n b i n d i n g o r p h o s p h o r y l a t i o n a r e l i k e l y t o p l a y a r o l e i n these changes o f s p e c i f i c i ty.

WILLIAM P. JENCKS

8

The e l e g a n t e x p e r i m e n t s on t h e exchange of sodium and potassium a c r o s s t h e membrane of t h e r e d c e l l t h a t have been c a r r i e d o u t by Glynn and o t h e r s s u g g e s t t h a t e s s e n t i a l l y t h e same set of r u l e s can be used t o desc r i b e t h e Na-ATPase (Scheme 4 ) (Glynn and K a r l i s h , 1 9 7 5 ) . The c y t o p l a s m i c s i d e of t h e membrane f o r t h i s ATPase ( c y t ) c o r r e s p o n d s t o t h e o u t s i d e of t h e s a r c o plasmic r e t i c u l u m v e s i c l e f o r t h e Ca2+-ATPase, which i s a l s o exposed t o t h e cytoplasm: t h e e x t r a c e l l u l a r s i d e ( e x t ) on t h e o u t s i d e of t h e r e d c e l l c o r r e s p o n d s t o t h e i n s i d e of t h e s a r c o p l a s m i c r e t i c u l u m v e s i c l e . The requirement f o r b o t h ATP and ADP on t h e c y t o p l a s m i c

-

pi HOH

4

ATP

ADP

+ 2Kext

3Na:xt

Scheme 4 s i d e of t h e membrane f o r exchange of sodium s u g g e s t s t h a t sodium c a n n o t d i s s o c i a t e from E%P*Na3on t h i s s i d e u n t i l t h e phosphate h a s been t r a n s f e r r e d t o ADP (Glynn and Hoffman, 1 9 7 1 ) . I n t h e r e v e r s e d i r e c t i o n sodium adds t o t h e f r e e enzyme from t h e c y t o p l a s m i c s i d e b e f o r e p h o s p h o r y l a t i o n by ATP ( B l o s t e i n , 1 9 7 9 ) . The s y n t h e s i s of ATP upon t h e a d d i t i o n o f sodium and ADP t o enzyme-phosphate shows t h a t E-P*Na3 does n o t undergo h y d r o l y s i s and does r e a c t w i t h ADP t o g i v e ATP (Taniguc h i and P o s t , 1 9 7 5 ) , a s w i t h calcium and t h e calcium ATPase. T h i s r u l e , and o t h e r r u l e s , may be e n f o r c e d k i n e t i c a l l y r a t h e r t h a n by an a b s o l u t e c a t a l y t i c s p e c i f i c i t y of t h e i n i t i a l l y formed E-P-Na3. What i s r e q u i r e i s t h a t t h i s s p e c i e s m u s t d o n a t e phosphate t o ADP f a s t e r t h a n t o water under p h y s i o l o g i c a l c o n d i t i o n s (presumably a f t e r a conformation c h a n g e ) .

WHAT IS A COUPLED VECTORIAL PROCESS

9

T h e r e i s e v i d e n c e f o r a s i m i l a r o r d e r e d mechanism f o r p o t a s s i u m on t h e l e f t - h a n d s i d e o f .the c y c l e . The e v i d e n c e i s c o n s i s t e n t w i t h two a d d i t i o n a l r u l e s f o r t h e Na,K-ATPase, f o r which t h e e v i d e n c e i s s t r o n g e s t : 5 . B i n d i n g and d i s s o c i a t i o n of p o t a s s i u m on t h e e x t r a c e l l u l a r s i d e o c c u r o n l y w i t h enzyme-phosphate; t h e r e i s a k i n e t i c b a r r i e r f o r t h e d i s s o c i a t i o n and b i n d i n g of p o t a s s i u m o n t h i s s i d e w i t h t h e f r e e enzyme. 6. B i n d i n g and d i s s o c i a t i o n o f p o t a s s i u m on t h e c y t o p l a s m i c s i d e o c c u r o n l y w i t h f r e e enzyme.

B i n d i n g of p o t a s s i u m t o E-P from t h e o u t s i d e i s e v i d e n t from t h e well-known s t i m u l a t i o n of t h e h y d r o l y s i s o f E-P by e x t e r n a l p o t a s s i u m i n t h e normal r e a c t i o n c y c l e (Glynn and K a r l i s h , 1975; Drapeau and B l o s t e i n , 1980; C a n t l e y , 1 9 8 1 ) . The d i s s o c a t i o n o f p o t a s s i u m from E ' K 2 t o t h e i n s i d e is a s l o w , k i n e t i c a l l y s i g n i f i c a n t s t e p i n t h e r e a c t i o n c y c l e of t h e Na-ATPase. The observed active, s t o i c h i o m e t r i c t r a n s p o r t of potassium t o t h e i n s i d e suggests t h a t t h e r e is a k i n e t i c b a r r i e r which p r e v e n t s d i s s o c i a t i o n o f p o t a s s i u m from E - K z t o t h e outside. The r a t e of d i s s o c i a t i o n t o t h e i n s i d e i s i n c r e a s e d by ATP and ATP a n a l o g s , b u t d o e s n o t r e q u i r e p h o s p h a t e t r a n s f e r o r h y d r o l y s i s of ATP ( P o s t e t a l . , 1972; Simons, 1974; Glynn and K a r l i s h , 1975; Beau96 and Glynn, 1 9 7 9 ) . The r e q u i r e m e n t f o r i n o r g a n i c p h o s p h a t e f o r exchange o f p o t a s s i u m a c r o s s t h e r e d c e l l membrane p r o v i d e s a d d i t i o n a l e v i d e n c e c o n s i s t e n t w i t h an o r d e r e d mechanism i n which p o t a s s i u m c a n d i s s o c i a t e t o t h e o u t s i d e o n l y from E-P-K ( P o s t and Sen, 1965; Glynn e t a l . , 1 9 7 0 ) . I t i s u n l i k e f y t h a t it d i s s o c i a t e s from E.P.K2 t o t h e o u t s i d e , b e c a u s e i t b i n d s t o E-P from t h e o u t s i d e and t h e n d i s s o c i a t e s from E a K 2 t o t h e i n s i d e i n a s l o w s t e p i n t h e normal c a t a l y t i c c y c l e (Beau96 and Glynn, 1979; Drapeau and B l o s t e i n , 1 9 8 0 ) . P o t a s s i u m e x h i b i t s s i m i l a r , b u t n o t i d e n t i c a l , behavior w i t h t h e Ca2+-ATPase. P o t a s s i u m b i n d s t o t h e E-P i n t e r m e d i a t e of t h e calcium enzyme and s t i m u l a t e s i t s h y d r o l y s i s , b u t t o a smaller e x t e n t t h a n w i t h t h e sodium enzyme (Shigekawa and Akowitz, 1 9 7 9 ) . P o t a s s i u m on t h e i n s i d e of t h e v e s i c l e s t i m u l a t e s calcium t r a n s p o r t , and it h a s been s u g g e s t e d , b u t n o t proved d i r e c t l y , t h a t potassium i s t r a n s p o r t e d from t h e i n s i d e t o t h e o u t s i d e d u r i n g t h e c a t a l y t i c c y c l e (Kanazawa e t a l . , 1 9 7 1 ; Chiu and Haynes, 1 9 8 0 ) .

WILLIAM P. JENCKS

10

111.

SUBSTRATE S P E C I F I C I T Y

Some u n d e r s t a n d i n g of t h e n a t u r e of t h e s p e c i f i c i t y r u l e s t h a t a r e r e s p o n s i b l e f o r c o u p l i n g may come from an examination of t h e range of s u b s t r a t e s p e c i f i c i t y t h a t i s p e r m i t t e d by t h e r u l e s . Information regarding t h i s s p e c i f i c i t y i s a v a i l a b l e from t h e range of nonphysiolog i c a l phosphate compounds t h a t undergo uncoupled hydrol y s i s o r d r i v e i o n t r a n s p o r t w i t h t h e s e enzymes. The Na-ATPase e x h i b i t s a phosphatase a c t i v i t y t h a t i s s t i m u l a t e d by o r r e q u i r e s potassium f o r most subs t r a t e s (Judah e t a l . , 1 9 6 2 ; C a n t l e y , 1 9 8 1 ) . There i s a s i m i l a r phosphatase a c t i v i t y f o r t h e calcium enzyme t h a t h a s n o t been examined s o e x t e n s i v e l y ( d e M e i s and Vianna, 1 9 7 9 ; Yamamoto e t a l . , 1 9 7 9 ) . The s i m p l e s t exp l a n a t i o n f o r t h i s phosphatase a c t i v i t y i s t h a t i t r e p r e s e n t s t h e range of v a r i a t i o n i n t h e s t r u c t u r e of t h e l e a v i n g group, HOH o r ROH, t h a t i s t o l e r a t e d by E . K 2 i n i t s r e a c t i o n w i t h HOP032” o r ROP032’ t o form E-P.K2 (Scheme 5 ) . The normal c a t a l y t i c c y c l e i n v o l v e s c a t a l y s i s of t h e a d d i t i o n of water t o E-P-K2 i n one d i r e c t i o n and removal of water from i n o r g a n i c phosphate t o g i v e E - P - X 2 i n t h e o t h e r d i r e c t i o n (Scheme 5 , R = H) The enzyme e x h i b i t s phosphatase a c t i v i t y toward ROP032- t o t h e e x t e n t t h a t i t t o l e r a t e s t h e s u b s t i t u t i o n of ROH f o r HOH. Phosphatase a c t i v i t y t h e n i n v o l v e s t h e r e a c t i o n of ROPO32’ w i t h E - K z t o g i v e E-P*K2 followed by a r e v e r s a l of t h i s p r o c e s s w i t h w a t e r (ROH = HOH) t o g i v e phosphate and r e g e n e r a t e E.K2 (Scheme 5 ) ( P o s t e t a l . , 1 9 7 2 ; C a n t l e y , 1 9 8 1 ) . Phosphatase a c t i v i t y has been observed only w i t h u n s t a b l e phosphate compounds t h a t have an enhanced chemical r e a c t i v i t y , which can compensate f o r r e l a t i v e l y poor enzymatic c a t a l y s i s .

.

WHAT IS A COUPLED VECTORIAL PROCESS

11

+ 2Kext Scheme 5 Drapeau and B l o s t e i n (1980) have shown t h a t t h e c l e a v a g e of p - n i t r o p h e n y l p h o s p h a t e by t h e Na-ATPase r e q u i r e s t h a t p o t a s s i u m add t o t h e f r e e enzyme from t h e c y t o p l a s m i c s i d e ( t h e o u t s i d e of i n v e r t e d v e s i c l e s ) b e f o r e react i o n w i t h t h e e s t e r , a s r e q u i r e d by Scheme 5 . The app a r e n t a b s e n c e o f a r e q u i r e m e n t f o r p o t a s s i u m on t h e opp o s i t e s i d e s u g g e s t s t h a t p o t a s s i u m may be o c c l u d e d w i t h r e s p e c t t o t h i s s i d e ; i . e . , t h e d i s s o c i a t i o n o f potassium o n t h e e x t r a c e l l u l a r s i d e may a l s o be s i g n i f i c a n t l y s l o w , so t h a t E-P.K2 can undergo h y d r o l y s i s and r e c y c l e t o E.X2 w i t h o u t loss o f p o t a s s i u m (Scheme 5 , R = H). However, i n t h e p r e s e n c e o f ATP and sodium, p o t a s s i u m i s a l s o r e q u i r e d on t h e e x t r a c e l l u l a r s i d e . This i s c o n s i s t e n t w i t h t h e requirement t h a t potassium add t o t h e E-P formed from ATP o n l y from t h e e x t r a c e l l u l a r side, t o g e n e r a t e E - K 2 t h a t can react w i t h t h e ester (Scheme 5 ) . C e r t a i n high-energy a c y l p h o s p h a t e s and phenyl phosp h a t e s w i t h good l e a v i n g g r o u p s w i l l a l s o r e a c t w i t h t h e E"a3 o r E - C a 2 forms of t h e enzymes, and may r e a c t w i t h t h e s e forms i n p r e f e r e n c e t o E.K2. T h i s t e n d e n c y app e a r s t o be g r e a t e r f o r t h e C a 2 + - A T P a s e and g i v e s normal, s t o i c h i o m e t r i c a c t i v e t r a n s p o r t of c a l c i u m w i t h i n t a c t v e s i c l e s ( d e Meis, 1 9 6 9 ; Puce11 and M a r t o n o s i , 1 9 7 1 ; I n e s i , 1 9 7 1 ) . The Ca2+-ATPase shows a l a r g e p r e f e r e n c e

12

WILLIAM P. JENCKS

f o r r e a c t i o n o f t h e E*Ca2 s p e c i e s w i t h some s u b s t r a t e s of t h i s c l a s s , such as a c e t y l phosphate. With o t h e r a c y l phosphates , such a s carbamoyl phosphate , t h e r e i s l i t t l e o r no s u c h s p e c i f i c i t y (Puce11 and Martonosi, 1 9 7 1 ) . With t h e s e s u b s t r a t e s h y d r o l y s i s can occur by p h o s p h o r y l a t i o n of E-Ca2 t o g i v e t r a n s p o r t i n t h e p r e s e n c e of calcium and through t h e f r e e enzyme i n t h e absence of calcium. There a p p e a r s t o be a d e c r e a s e i n s p e c i f i c i t y w i t h i n c r e a s i n g chemical r e a c t i v i t y of subs t i t u t e d a c y l phosphates. The calcium requirement f o r h y d r o l y s i s i s d e c r e a s e d as t h e l e a v i n g a c e t y l group becomes more a c i d i c , so t h a t t h e more r e a c t i v e phosphoryla t i n g a g e n t s can r e a c t w i t h f r e e E , a s w e l l as w i t h E'Ca2 t o g i v e t r a n s p o r t (A. Tolkovsky, p e r s o n a l communic a t i o n , 1 9 8 1 ) . I t i s of i n t e r e s t t h a t t h e s p e c i f i c i t y f o r h y d r o l y s i s of f u r y l a c r y l o y l phosphate changes from p r e f e r e n t i a l r e a c t i o n w i t h E*Ca2 t o r e a c t i o n w i t h f r e e E i n t h e p r e s e n c e of dimethyl s u l f o x i d e ( I n e s i e t a l . , 1 9 8 0 ) . F u r t h e r examination of t h e r e a c t i v i t y of t h e d i f f e r e n t enzyme s p e c i e s toward s u b s t i t u t e d p h o s p h a t e s should p r o v i d e a b e t t e r u n d e r s t a n d i n g of t h e n a t u r e of t h e change i n enzymatic s p e c i f i c i t y t h a t i s m a n i f e s t e d i n r u l e s 3 and 4 . The phosphatase a c t i v i t y of t h e Na-ATPase toward most phosphate e s t e r s i s i n h i b i t e d by sodium because t h e enzyme i s c o n v e r t e d t o t h e u n r e a c t i v e E-Na3 s p e c i e s . However, h y d r o l y s i s of t h e r e a c t i v e s u b s t r a t e s 2 , 4 d i n i t r o p h e n y l phosphate and f u r y l a c r y l o y l phosphate occ u r s i n t h e p r e s e n c e of b o t h potassium and sodium a t a r a t e t h a t i s s e v e r a l times f a s t e r t h a n t h a t f o r t h e h y d r o l y s i s of ATP (Gache et al., 1977; Odom e t al., 1 9 8 1 ) . T h i s r e q u i r e s t h a t t h e h y d r o l y s i s o c c u r through a p a t h t h a t a v o i d s t h e r a t e - d e t e r m i n i n g s t e p ( s ) of ATP h y d r o l y s i s . Most of t h e h y d r o l y s i s presumably o c c u r s through t h e normal phosphatase pathway of Scheme 5 , b u t when potassium d i s s o c i a t e s and a m o l e c u l e of enzyme i s c o n v e r t e d t o E-Na3, t h i s s p e c i e s can r e a c t w i t h t h e s u b s t r a t e t o form E-P. The E-P s p e c i e s t h e n b i n d s potassium and r e g e n e r a t e s t h e r e a c t i v e E.K2 s p e c i e s r a p i d l y enough t o a v o i d i n h i b i t i o n . T h i s i s t h e same mechanism by which ATP a c t i v a t e s phosphatase a c t i v i t y i n t h e p r e s e n c e of sodium ( P o s t et al., 1 9 7 2 ) . I t i s c u r i o u s t h a t t h e Na-ATPase f o l l o w s t h e same s e q u e n t i a l , o r d e r e d mechanism f o r t h e removal of phosp h a t e and of i o n s on both s i d e s of t h e c y c l e . With b o t h sodium and potassium t h e d i s s o c i a t i o n (or b i n d i n g ) of t h e i o n on t h e c y t o p l a s m i c s i d e r e q u i r e s t h e p r i o r removal of phosphate. D i s s o c i a t i o n (and b i n d i n g ) on If the t h e o u t s i d e o c c u r s :nly w i t h enzyme-phosphate. b i n d i n g of t h e two i o n s f o l l o w s t h e same r u l e s , how t h e n

WHAT IS A COUPLED VECTORIAL PROCESS

13

a r e t h e d i r e c t i o n a l i t i e s maintained f o r t h e t r a n s p o r t of sodium and potassium a g a i n s t c o n c e n t r a t i o n g r a d i e n t s a t t h e expense of ATP h y d r o l y s i s ? The d i r e c t i o n a l i t y f o l l o w s simply from t h e change i n enzymatic s p e c i f i c i t y w i t h t h e d i f f e r e n t i o n s . Enzyme-sodium r e a c t s o n l y w i t h ATP, and enzyme-potassium o n l y w i t h i n o r g a n i c phosp h a t e , o r phosphate esters t h a t a r e a n a l o g s of i n o r g a n i c phosphate. The calcium enzyme behaves s i m i l a r l y w i t h calcium i n s t e a d of sodium. These changes i n c a t a l y t i c s p e c i f i c i t y and t h e o r d e r e d mechanisms f o r r e l e a s e of i o n s and phosphate a r e s u f f i c i e n t t o account f o r a coupled p r o c e s s t h a t t r a n s p o r t s i o n s i n a p a r t i c u l a r d i r e c t i o n . I n b o t h enzymes i t i s t h e i o n t h a t i s t r a n s p o r t e d a g a i n s t a c o n c e n t r a t i o n g r a d i e n t from t h e s i d e of t h e membrane on which ATP i s a v a i l a b l e t h a t a c t i v a t e s t h e enzyme f o r r e a c t i o n w i t h ATP; t h e s p e c i f i c i t y of t h e o t h e r h a l f - r e a c t i o n i s less d i s c r i m i n a t i n g and t h e hyd r o l y s i s of E-P o c c u r s a t a moderate r a t e i n t h e absence of potassium w i t h b o t h enzymes. These s i m p l e enzyme s p e c i f i c i t i e s may p r o v i d e a h i n t r e g a r d i n g t h e d e t a i l e d chemical mechanism f o r t h e s e coupled v e c t o r i a l p r o c e s s e s .

IV.

B I N D I N G ENERGIES AND DESTABILIZATIONS

The b a l a n c e of b i n d i n g e n e r g i e s i n t h e s e systems a p p e a r s t o be r e l a t e d o n l y i n d i r e c t l y t o t h e s p e c i f i c i t y r u l e s t h a t a r e r e s p o n s i b l e f o r c o u p l i n g . The primary r o l e of t h e e x p r e s s i o n o f d i f f e r e n t amounts of i n t r i n s i c b i n d i n g e n e r g i e s t o l i g a n d s i n d i f f e r e n t s t a t e s of t h e s e systems i s k i n e t i c ; it r e s u l t s i n t h e maintenance of comparable c o n c e n t r a t i o n s of t h e d i f f e r e n t s t a t e s under p h y s i o l o g i c a l c o n d i t i o n s so t h a t t u r n o v e r can o c c u r a t a u s e f u l r a t e , a s d e s c r i b e d above. The most i m p o r t a n t k i n e t i c r e q u i r e m e n t f o r t h e b i n d i n g of i o n s i s t h a t t h e i n t r i n s i c b i n d i n g energy f o r Na+ o r Ca2+ s h o u l d b e exp r e s s e d on t h e s i d e of t h e membrane from which t h e i o n i s normally t r a n s p o r t e d and e x p r e s s e d l e s s o r n o t a t a l l on t h e o t h e r s i d e , so t h a t t h e r e i s a s i g n i f i c a n t popul a t i o n of bound i o n s t o undergo t r a n s p o r t from one s i d e b u t t h e enzyme i s n o t t i e d up w i t h bound i o n s on t h e o t h e r s i d e . The requirement f o r r e v e r s i b l e t r a n s f e r of phosphate i s t h a t i n one s t a t e t h e i n t r i n s i c b i n d i n g energy of phosphate be f u l l y e x p r e s s e d t o s t a b i l i z e t h e a c y l phosphate i n t e r m e d i a t e , so t h a t it c a n be formed from i n o r g a n i c phosphate, and i n a n o t h e r s t a t e it i s less e x p r e s s e d so t h a t t h e phosphate i s h i g h e r i n energy -,,,,UTBs ,$w, anddcan -beAtr.ans&qr.rad,,fo,A4P L?easka,,.,P&Q)

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WILLIAM P.JENCKS

p r e s s i o n of t h e s e binding e n e r g i e s i s n i c e l y balanced i n some way such t h a t t h e b i n d i n g e n e r g i e s of t h e i o n s and of phosphate are e a c h e x p r e s s e d when t h e o t h e r i s n o t p r e s e n t and are l e s s e x p r e s s e d when b o t h are p r e s e n t t o g e t h e r i n one o r more s t a t e s . Taniguchi and P o s t (1975) have s u g g e s t e d t h a t t h i s can o c c u r by a d i r e c t mutual d e s t a b i l i z a t i o n o r i n t e r a c t i o n energy of phosphate and sodium i o n s i n t h e N a ATPase. A s i m i l a r mutual d e s t a b i l i z a t i o n p r o v i d e s t h e s i m p l e s t mechanism t o f u l f i l l t h e k i n e t i c r e q u i r e m e n t s f o r t h e Caz+-ATPase ( J e n c k s , 1 9 8 2 ) . The o r d e r e d k i n e t i c mechanism p l a y s an e s s e n t i a l r o l e i f such d e s t a b i l i z a t i o n i s t o be c o n s i s t e n t w i t h a f u n c t i o n i n g system t h a t can t u r n over rapidly. I f d i s s o c i a t i o n of N a o r C a t o t h e c y t o p l a s m i c s i d e c o u l d o c c u r r e a d i l y from E-P-Na3 or E - P - C a 2 and t h e i o n w e r e d e s t a b i l i z e d i n t h i s comp l e x , i t would immediately pop o f f and t r a n s p o r t would n o t be observed a t a u s e f u l r a t e . The k i n e t i c b a r r i e r of t h e o r d e r e d mechanism p r e v e n t s s u c h d i s s o c i a t i o n u n t i l a f t e r t h e d e s t a b i l i z e d phosphate h a s been t r a n s f e r r e d t o ADP and t h e d e s t a b i l i z a t i o n removed. Conv e r s e l y , t h e d e s t a b i l i z e d phosphate i n t h e complex would come o f f b e f o r e d i s s o c i a t i o n of t h e i o n on t h e o t h e r s i d e i f t h e r e were no o r d e r e d mechanism, s o t h a t t h e d e s t a b i l i z a t i o n of t h e i o n would be removed and i t would n o t d i s s o c i a t e i n t o t h e h i g h c o n c e n t r a t i o n of i o n s on t h i s s i d e . Again, t h e o r d e r e d k i n e t i c mechanism p r e v e n t s t r a n s f e r of t h i s phosphate u n t i l a f t e r t h e i o n h a s d i s s o c i a t e d . Honig and S t e i n (1978) have c o n s i d e r e d t h i s p o i n t i n s l i g h t l y d i f f e r e n t terms. I t i s d i f f i c u l t t o t e s t t h i s d e s t a b i l i z a t i o n mechanism e x p e r i m e n t a l l y because t h e k i n e t i c b a r r i e r s f o r t h e t r a n s f e r of i o n s and of phosphate i n t e r f e r e w i t h t h e d e t e r m i n a t i o n of e q u i l i b r i u m c o n s t a n t s and e n e r g i e s f o r t h e d i f f e r e n t s t a t e s of t h e system. An a l t e r n a t i v e way o f d e a l i n g w i t h t h i s problem i s p o s s i b l e i f t h e r e a r e t w o s t a t e s o f t h e E-Poion complex, i n one of which t h e i n t r i n s i c b i n d i n g e n e r g y of phosp h a t e i s e x p r e s s e d , b u t n o t t h a t o f t h e i o n , and i n t h e o t h e r t h e i n t r i n s i c b i n d i n g e n e r g y of t h e i o n b u t n o t t h a t of phosphate i s e x p r e s s e d . I n one s t a t e p h o s p h a t e i s s t a b i l i z e d and t h e bound i o n i s e f f e c t i v e l y d e s t a b i l ized; the reverse occurs i n t h e o t h e r state (Jencks, 1980; H i l l and E i s e n b e r g , 1981; C . Tanford, p e r s o n a l communication, 1 9 8 1 ) . T h i s mechanism a v o i d s some of t h e r e q u i r e m e n t s of t h e s i m p l e mutual d e s t a b i l i z a t i o n mechanism. The f i n d i n g t h a t low c o n c e n t r a t i o n s of calcium can a c t i v a t e i o n - f r e e E-P t h a t w a s formed from ATP f o r phosphate t r a n s f e r back t o ADP ( i n t h e a b s e n c e of magnesium) is c o n s i s t e n t w i t h t h i s h y p o t h e s i s i f i t re-

WHAT IS A COUPLED VECTORIAL PROCESS

15

s u l t s from b i n d i n g t o t h e i o n - t r a n s p o r t s i t e , n o t t h e magnesium s i t e (Takakuwa and Kanazawa, 1 9 8 1 ) . The s p e c i f i c i t y r u l e t h a t r e s t r i c t s c a l c i u m b i n d i n g and d i s s o c i a t i o n w i t h E-P i s n o t a b s o l u t e , and t h e s e p r o cesses w i l l o c c u r o v e r a p e r i o d o f t i m e . The a c t i v a t i o n by low c o n c e n t r a t i o n s o f calcium i m p l i e s t h a t c a l cium i s n o t d e s t a b i l i z e d i n t h e E-P.Ca complex, a l t h o u g h t h e phosphate is s u f f i c i e n t l y d e s t a b i l i z e d to permit i t s t r a n s f e r t o ADP. Conversion t o t h e o t h e r s t a t e r e s u l t s i n d e s t a b i l i z a t i o n o f t h e bound calcium o r sodium a s l i t t l e o r none of i t s b i n d i n g e n e r g y i s e x p r e s s e d t o g i v e a s t a b l e complex, so t h a t it pops o f f r e a d i l y from t h e o t h e r s i d e o f t h e membrane i n t o t h e i n s i d e of t h e v e s i c l e . C a n t l e y (1981) h a s s u g g e s t e d t h a t i n t h e sodium enzyme s u c h a d e s t a b i l i z a t i o n c o u l d arise from r o t a t i o n o f t h e a c y l p h o s p h a t e group i n s u c h a way as t o e n l a r g e t h e sodium-binding s i t e and open a c h a n n e l t o t h e e x t r a c e l l u l a r s i d e o f t h e membrane; such a r o t a t i o n might r e l i e v e d e s t a b i l i z a t i o n o f t h e a c y l p h o s p h a t e group. The u t i l i z a t i o n o f b i n d i n g e n e r g y and t h e s p e c i f i c i t y r u l e s are r e l a t e d a t t h i s p o i n t because l o s s of t h e i o n s and t h e c o n f o r m a t i o n change t h a t presumably accompanies t h e d i s a p p e a r a n c e o f p h o s p h a t e d e s t a b i l i z a t i o n c a u s e t h e change i n c a t a l y t i c s p e c i f i c i t y t h a t p e r m i t s p h o s p h a t e t r a n s f e r t o water. However, t h e n a t u r e of t h e i n t e r a c t i o n with calcium i n t h e state t h a t i s formed f i r s t from calcium on t h e i n s i d e of t h e v e s i c l e ( e . g . , " E 2 - P . C a 2 " , Scheme 2 ) i s n o t known. The o b s e r v e d " l o w - a f f i n i t y b i n d i n g " f o r calcium on t h e i n s i d e w i t h K~ % 1 mM p r o b a b l y d o e s n o t r e p r e s e n t b i n d i n g t o a s i t e w i t h a d i s s o c i a t i o n c o n s t a n t o f 1 mM because a p p a r e n t b i n d i n g c o n s t a n t s c o n t a i n e q u i l i b r i u m c o n s t a n t s f o r conv e r s i o n of t h e enzyme t o o t h e r s t a t e s t h a t e x i s t a t s i g n i f i c a n t c o n c e n t r a t i o n s ( S t e i n and Honig, 19771, and K~ v a l u e s are l i k e l y t o i n c l u d e k i n e t i c a s w e l l a s e q u i l i b r i u m c o n s t a n t s . O t h e r e q u i l i b r i u m c o n s t a n t s are i m p o r t a n t i n t h i s system because most o f t h e E-P.Ca2 e x i s t s i n t h e ADP-sensitive s t a t e i n t h e p r e s e n c e of h i g h c a l cium c o n c e n t r a t i o n s u n d e r c o n d i t i o n s i n which t h e hydrol y s i s o f enzyme-phosphate i s i n h i b i t e d ( P i c k a r t and J e n c k s , 1 9 8 2 ) . What can be s a i d i s t h a t t h e r e i s some d e s t a b i l i z a t i o n s u c h t h a t " E 2- P. C a2 , I' a n ADP-insensitive form t h a t i s t h e immediate p r o d u c t of c a l c i u m b i n d i n g on t h e i n s i d e , does n o t accumulate. I t i s l i k e l y t h a t t h e r e i s o n l y weak b i n d i n g of calcium i n t h e s p e c i e s t h a t i s formed i n i t i a l l y from c a l c i u m on t h e i n s i d e ( K m > 1 m M ) ; i n t h e l i m i t i n g case t h i s s p e c i e s i s a r a n dom e n c o u n t e r complex, E-P.. - C a 2 [Eq. ( l ) ] . T h i s i s n o t u n l i k e l y i f there i s a s i n g l e b i n d i n g r e g i o n f o r

WILLIAM P. JENCKS

16

%P

1 ' -CaZ

[

* Ez.caj

+

ETP .Ca 2

(1)

calcium t h a t i s n o t normally exposed t o t h e i n s i d e of t h e v e s i c l e . The s p e c i e s i n which t h e c a l c i u m b i n d i n g s i t e becomes exposed t o t h e i n s i d e may t h e n b e a highly unstable s t a t e , [EpP*Ca2]*, with a s t r u c t u r e t h a t is close or identical t o the t r a n s i t i o n s t a t e f o r t h e f o r m a t i o n of El'~PoCa2. F u r t h e r work i s needed t o d e f i n e t h e r e l a t i v e Gibbs f r e e e n e r g i e s of t h e d i f f e r e n t states of t h e s e systems and t h e i r r e l a t i o n s h i p t o t h e b i n d i n g of l i g a n d s , i n c l u d i n g potassium and magnesium.

ACKNOWLEDGMENT

T h i s i s P u b l i c a t i o n 1442 from t h e Graduate Department of Biochemistry, Brandeis U n i v e r s i t y , Waltham, Massachusetts 02254. T h i s work w a s supported i n p a r t by g r a n t s f r a n t h e N a t i o n a l Science Foundation (BMS 77-08369) and t h e N a t i o n a l I n s t i t u t e s of Health (GM 20888).

REFERENCES

B a r l o g i e , B., Hasselbach, W., and Makinose, M. (1971). A c t i v a t i o n of calcium e f f l u x by ADP and i n o r g a n i c phosphate. FEES L e t t . 12, 267-268. Beaug6, L. A., and Glynn, I . M. (1979). Occlusion of K i o n s i n t h e unphosphorylated sodium pump. Nature (London) 2 8 0 , 510-512. B l o s t e i n , R. (1979). S i d e - s p e c i f i c e f f e c t s o f sodium on ( N a , K ) ATPase. S t u d i e s w i t h i n s i d e - o u t r e d c e l l membrane v e s i c l e s . J. Biol. Chem. 254, 6673-6677. Cantley, L. C. (1981). S t r u c t u r e and mechanism o f t h e ( N a , K ) A T P a s e . Curr. Top. Bioenerg. 11, 201-231. Chiu, V. C. K., and Haynes, D. H. (1980). Rapid k i n e t i c s t u d i e s J. of a c t i v e C a 2 + t r a n s p o r t i n s a r c o p l a s m i c r e t i c u l u m . Membr. B i o l . 56, 219-239. de Meis, L. (1969). Ca2+ uptake and a c e t y l phosphatase of s k e l e t a l Li', and adenosine I n h i b i t i o n by Na', ,'K muscle microsomes. triphosphate. J. Biol Chem. 244, 3733-3739. de Meis, L., and Tume, R . K. (1977). A new mechanism by which an H+ c o n c e n t r a t i o n g r a d i e n t d r i v e s t h e s y n t h e s i s o f adenosine t r i p h o s p h a t e , pH jump , and adenosine t r i p h o s p h a t e s y n t h e s i s

.

WHAT IS A COUPLED VECTORIAL PROCESS

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2+ by t h e C a -dependent a d e n o s i n e t r i p h o s p h a t a s e of s a r c o p l a s m i c r e t i c u l u m . Biochemistry 1 6 , 4455-4463. de M e i s , L . , and Vianna, A. L. (1979). Energy i n t e r c o n v e r s i o n by t h e Ca2+-dependent ATPase of t h e s a r c o p l a s m i c r e t i c u l u m . Annu. Rev. Biochem. 48, 275-292. Drapeau, P . , and B l o s t e i n , R. (1980). I n t e r a c t i o n s o f K+ w i t h ( N a , K ) -ATPase. O r i e n t a t i o n of K+-phosphatase s i t e s s t u d i e d J. B i o l . Chem. w i t h i n s i d e - o u t r e d c e l l membrane v e s i c l e s . 255, 7827-7834. Dupont, Y. ( 1 9 8 0 ) . Occlusion of d i v a l e n t c a t i o n s i n t h e phosphorEur. J. y l a t e d calcium pump of s a r c o p l a s m i c r e t i c u l u m . Biochem. 109, 231-238. Gache, C., Rossi, B . , and Lazdunski, M. ( 1 9 7 7 ) . M e c h a n i s t i c a n a l y s i s o f t h e (Na+,K+) -ATPase u s i n g new p s e u d o s u b s t r a t e s . Biochemistry 1 6 , 2957-2965. Glynn, I. M., and Hoffman, J. F. ( 1 9 7 1 ) . N u c l e o t i d e r e q u i r e m e n t s f o r sodium-sodium exchange c a t a l y s e d by t h e sodium pump i n (London) 218 , 239-256. human r e d c e l l s . J. Physiol Glynn, I. M . , and K a r l i s h , S. J. D. (1975). The sodium pump. Annu. Rev. P h y s i o l . 3 7 , 13-55. Glynn, I . M., Lew, V. L . , and L i t h i , U. (1970). R e v e r s a l of t h e potassium e n t r y mechanism i n r e d c e l l s , w i t h and w i t h o u t J. P h y s i o l . (London) 207, r e v e r s a l of t h e e n t i r e pump c y c l e . 371-391. H i l l , T. L. (1969). A proposed common a l l o s t e r i c mechanism f o r a c t i v e t r a n s p o r t , muscle c o n t r a c t i o n , and ribosomal t r a n s l o cation. Proc. N a t l . Acad. Sci. USA 64, 267-274. H i l l , T. L. (1977). "Free Energy T r a n s d u c t i o n i n Biology." Academic Press, New York. H i l l , T. L . , and E i s e n b e r g , E. ( 1 9 8 1 ) . Can f r e e e n e r g y t r a n s d u c t i o n be l o c a l i z e d a t some c r u c i a l part. of t h e enzymatic c y c l e ? Q. Rev. Biophys. 14, 463-511. Honig, B., and S t e i n , W. D. (1978). Design p r i n c i p l e s f o r a c t i v e J. Theor. B i o l . 75, 299-305. t r a n s p o r t systems. Ikemoto, N. (1975). T r a n s p o r t and i n h i b i t o r y Ca2+ b i n d i n g s i t e s on t h e ATPase enzyme i s o l a t e d from t h e s a r c o p l a s m i c r e t i c u l u m . J. B i o l . Chem. 250, 7219-7224. I n e s i , G. (1971). p-Nitrophenyl phosphate h y d r o l y s i s and calcium i o n t r a n s p o r t i n fragmented s a r c o p l a s n i c r e t i c u l u m . S c i e n c e 171 , 901-903. I n e s i , G . , Kurmack, M . , Nakamoto, R . , de M e i s , L., and Bernhard, S. A ( 1 9 8 0 ) . Uncoupling of calcium c o n t r o l and phosphohyd r o l a s e a c t i v i t y i n s a r c o p l a s m i c r e t i c u l u m v e s i c l e s . J. B i o l . Chem. 255, 6040-6043. J e n c k s , W. P. (1975). Binding e n e r g y , s p e c i f i c i t y and enzymic Adv. Enzymol. 43, 219-410. c a t a l y s i s : The C i r c e e f f e c t . Jencks, W. P. (1980). The u t i l i z a t i o n o f b i n d i n g e n e r g y i n coupled v e c t o r i a l p r o c e s s e s . Adv. Enzymol. 51, 75-106. Jencks, W. P. (1982). Rules and t h e economics o f energy b a l a n c e i n coupled v e c t o r i a l p r o c e s s e s . I n "Membranes and T r a n s p o r t : A C r i t i c a l Review" ( A . Martonosi, e d . ) , pp. 515-520. Plenum, N e w York.

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J. D., Ahmed, K., and McLean, A. E. M. (1962). Ion t r a n s Biochim. p o r t and p h o s p h o p r o t e i n s o f human r e d cells. B i o p h y s . A c t a 6 5 , 472-480. Kanazawa, T., Yamada, S., Yamamoto, T., and Tonomura, Y. (1971). R e a c t i o n mechanism o f t h e Ca2+-dependent ATPase o f s a r c o plasmic r e t i c u l u m from s k e l e t a l muscle. V. V e c t o r i a l r e q u i r e ments f o r calcium and magnesium i o n s of t h r e e p a r t i a l react i o n s of ATPase: Formation and decomposition of a phosp h o r y l a t e d i n t e r m e d i a t e and ATP-formation from ADP and t h e i n t e r m e d i a t e . J . B i o c h e m . (Tokyo) 70, 95-123. Knowles, A. F . , and Racker, E. ( 1 9 7 5 ) . Formation o f a d e n o s i n e t r i phosphate from P i and a d e n o s i n e d i p h o s p h a t e by p u r i f i e d Ca2+-adenosine t r i p h o s p h a t a s e . J. B i o l . Chem. 250, 19491951. Makinose, M. , and H a s s e l b a c h , W. (1971). ATP s y n t h e s i s by t h e rev e r s e o f t h e s a r c o p l a s m i c calcium pump. FEBS Lett. 12, 271272. Odom, T. A . , Chipman, D. M., B e t t s , G . , and Bernhard, S. A. ( 1 9 8 1 ) . T r a n s i e n t and s t e a d y - s t a t e k i n e t i c s t u d i e s of sodium-potassium a d e n o s i n e t r i p h o s p h a t a s e u s i n g 8- ( 2 - f u r y l ) a c r y l o y l p h o s p h a t e as chromophoric s u b s t r a t e a s s a y . B i o c h e m i s t r y 20 , 480-486. P i c k a r t , C . , and J e n c k s , W P. ( 1 9 8 2 ) . J . B i o l . C h e m . 257, 53195322. P o s t , R. L., and Sen, A. K. ( 1 9 6 5 ) . An enzymatic mechanism o f act i v e sodium and potassium t r a n s p o r t . J. Histochem. C y t o c h e m . 1 3 , 105-112. P o s t , R. L., Hegyvary, C . , and Kume, S. (1972). A c t i v a t i o n by adenosine t r i p h o s p h a t e i n t h e phosphorylation k i n e t i c s o f sodium and potassium i o n t r a n s p o r t a d e n o s i n e t r i p h o s p h a t a s e . J. B i o l . C h e m . 247, 6530-6540. P u c e l l , A., and M a r t o n o s i , A. ( 1 9 7 1 ) . S a r c o p l a s m i c r e t i c u l u m . X I V . A c e t y l p h o s p h a t e and carbamylphosphate as e n e r g y s o u r c e s f o r Ca++ t r a n s p o r t . J. B i o l . Chem. 246, 3389-3397. Ray, W. J., Jr., and Long, J. W. (1976). Thermodynamics and mechanism o f t h e PO3 t r a n s f e r p r o c e s s i n t h e phosphoglucomutase r e a c t i o n . B i o c h e m i s t r y 15, 3993-4006. Ray, W. J . , Jr., Long, J. W . , a n d O w e n s , J. D. ( 1 9 7 6 ) . An a n a l y s i s o f t h e s u b s t r a t e - i n d u c e d r a t e e f f e c t i n t h e phosphoglucomutase system. B i o c h e m i s t r y 1 5 , 4006-4017. Shigekawa, M., and Akowitz, A. A. ( 1 9 7 9 ) . On t h e mechanism o f CaZ+-dependent a d e n o s i n e t r i p h o s p h a t a s e o f s a r c o p l a s m i c re ticulum. J. B i o l . Chem. 254, 4726-4730. Simons, T. J. B . (1974). Potassium:potassium exchange c a t a l y s e d by t h e sodium pump i n human r e d c e l l s . J. P h y s i o l . (London) 237 , 123-155. S t e i n , W. D . , and Honig, B. (1977). Models f o r t h e a c t i v e transp o r t of cations The s t e a d y - s t a t e a n a l y s i s . Mol. C e l l . B i o c h e m . 15, 27-44. Takakuwa, Y . , and Kanazawa, T. ( 1 9 8 1 ) . R e a c t i o n mechanism of (Ca2+,Mg2+)-ATPase o f s a r c o p l a s m i c r e t i c u l u m v e s i c l e s . I. Judah,

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WHAT IS A COUPLED VECTORIAL PROCESS

Phosphoenzyme w i t h bound Ca2+ which i s exposed t o t h e ext e r n a l medium. J . B i o l . Chem. 2 5 6 , 2691-2695. Occluded bound calcium on Takisawa, H . , and Makinose , M. (1981) t h e phosphorylated s a r c o p l a s m i c t r a n s p o r t ATPase. N a t u r e ( L o n d o n ) 2 9 0 , 271-273. Taniguchi, K. , and Post, R. L. (1975). S y n t h e s i s of adenosine t r i p h o s p h a t e and exchange between i n o r g a n i c phosphate and adenosine t r i p h o s p h a t e i n sodium and potassium i o n t r a n s p o r t adenosine t r i p h o s p h a t a s e . J. B i o l . Chem. 2 5 0 , 3010-3018. Weber, G. (1975). E n e r g e t i c s o f l i g a n d b i n d i n g to p r o t e i n s . Adv. P r o t e i n Chem. 2 9 , 1-83. Wyman, J . , Jr. (1964). Linked f u n c t i o n s and r e c i p r o c a l e f f e c t s i n hemoglobin: A second look. A d v . P r o t e i n C h e m . 1 9 , 223-286. Yamamoto, T . , Takisawa, H . , and Tonomura, Y. (1979). Reaction mechanisms f o r ATP h y d r o l y s i s and s y n t h e s i s i n t h e s a r c o p l a s C u r r . T o p . B i o e n e r g . 9 , 179-236. mic r e t i c u l u m .

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CURRENT TOPICS IN MEMBRANES AND TRANSPORT,VOLUME 19

The Membrane Equilibrium with Chemical Reactions FRIEDRICH A. SAUER Max-Planck-Imtitut filr Biophysik Frankfurt, Federal Republic of Germany

I.

INTRODUCTION

Many b i o l o g i c a l membranes show a t i g h t c o u p l i n g between f l o w and chemical r e a c t i o n s , which o c c u r i n o r a t t h e membrane. I f t h i s coupling i s complete, t h e s t o p f l o w o r s t a t i c head s t a t e of t h e membrane s y s t e m In becomes a c o n s t r a i n e d thermodynamic e q u i l i b r i u m . t h i s a r t i c l e w e w i l l a n a l y z e t h i s e q u i l i b r i u m and comp a r e it w i t h t h e r e s u l t s of n o n e q u i l i b r i u m thermodynamics. W e d i s c u s s t h e thermodynamic e q u i l i b r i u m between two p h a s e s I and 'I s e p a r a t e d by a membrane which i s r i g i d and c a n n o t move. Phases and have t h e volumes V' and v " , r e s p e c t i v e l y ( F i g . 1 ) . Without loss of g e n e r a l i t y , w e assume t h a t t h e system ( b o t h p h a s e s and t h e membrane) have t h e common t e m p e r a t u r e T. Both p h a s e s c o n t a i n t h e component water ( d e n o t e d by s u b s c r i p t w t h r o u g h o u t ) , t h e s o l v e n t f o r which t h e membrane i s permeable. There are s o l u t e conponents of t h e f o l l o w i n g kinds: components k ( k = 1, n ) , f o r which t h e

...,

21

Copyright 0 1983 by Academic Press, Inc. All nghts of reproduction in any form reserved. ISBN 0-12-1533190

22

FRIEDRICHA. SAUER

I1

V I

V

T

T

F i g . 1 . A membrane i n contact with two homogeneous phases and Selective reversible electrodes are used t o measure electrochemical potential d i f f e r e n c e s .

'

".

...,

membrane i s permeable; components j ( j = j + 1, m), f o r which t h e membrane is impermeable; and components t ( t = m + 1, r ) , f o r which t h e membrane i s i m permeable. They react a t t h e s i d e of t h e membrane f a c u ) , for i n g t h e t phase. Components s (s = r + 1, which t h e membrane i s permeable o n l y i n complete c o u p l i n g w i t h t h e chemical r e a c t i o n . According t o Gibbs ( 1 9 6 1 , pp. 83-85) o n e g e t s t h e e q u i l i b r i u m c o n d i t i o n s from t h e v a r i a t i o n s of t h e Helmholtz f r e e e n e r g y F . More p r e c i s e l y , t h e f i r s t - o r d e r v a r i a t i o n o f F = F ' + F " around e q u i l i b r i u m must be zero, when v', v " , and T a r e k e p t c o n s t a n t and t h e system a s a whole i s c l o s e € o r m a t e r i a l exchange. T h i s means t h a t

...,

...,

6F = 6F'

+

6F"

= 0

(1.1)

o b s e r v i n g t h e above-mentioned c o n s t r a i n t s . Because t h e d e f i n e d system i s t o o complex, w e d i s c u s s it s t e p by s t e p , a t f i r s t l e a v i n g o u t many o f t h e components i n t r o duced above and d i s c u s s i n g i n d e t a i l t h e d i f f e r e n c e s between e l e c t r o l y t e s and n o n e l e c t r o l y t e s . A t t h e end w e come back t o t h e g e n e r a l e q u i l i b r i u m c a s e and d i s c u s s p o s s i b l e consequences.

MEMBRANE EQUILIBRIUM

11.

23

NONELECTROLYTE SOLUTION WITHOUT CHEMICAL REACTION

The s y s t e m u n d e r d i s c u s s i o n c o n t a i n s w a t e r ( w ) a s t h e s o l v e n t , t h e p e r m e a n t component ( k ) , and t h e impermeant component ( j ) . A l l components a r e assumed t o b e nonelectrolytes. The v a r i a t i o n s o f F ' and F " a r e g i v e n by

where t h e p are c h e m i c a l p o t e n t i a l s and t h e n a r e t h e mole numbers of t h e components. I t i s assumed t h a t 6 T = 0;

6V'

(2.2)

= 6V" = 0

and f o r t h e components ( j ) w e have I

6n

j

II

= 6n

j

= 0

(2.3)

Because t h e s y s t e m as a whole i s c l o s e d , one h a s t h e conditions I

6n

W

+

1

(1

6 n w = 0;

6n

k

+

11

6nk = 0

(2.4)

I n t r o d u c i n g Eqs. ( 2 . 1 ) and ( 2 . 4 ) i n t o Eq. ( l . l ) ,o n e g e t s t h e e q u i l i b r i u m c o n d i t i o n i n t h e form

If, as w i t h A b e i n g t h e d i f f e r e n c e between I and ' I . w a s assumed, t h e components ( k ) and water a r e i n d e p e n d e n t components, one c o n c l u d e s from Eq. ( 2 . 5 ) t h a t Apk = 0

( k = 1,

...,

n);

Auw = 0

(2.6)

FRIEDRICHA. SAUER

24

The e q u i l i b r i u m c o n d i t i o n s ( 2 . 6 ) e n a b l e u s t o c a l c u l a t e p r e s s u r e and composition o f t h e 'I p h a s e , i f p r e s s u r e and c o m p o s i t i o n o f t h e I phase and t h e concen phase t r a t i o n s of t h e impermeant components ( j ) i n t h e are known. T h i s u s u a l l y l e a d s t o t h e s i t u a t i o n t h a t o n e h a s t o s o l v e a system of t r a n s c e n d e n t a l e q u a t i o n s . In case t h e s o l u t i o n i s d i l u t e , one h a s 'k

-

'kO ( T , p)

+

I n ck

RT

(2.7)

where p i s t h e p r e s s u r e and C k t h e c o n c e n t r a t i o n . Then o n e c a n f i n d a n approximate s o l u t i o n of Eqs. ( 2 . 6 ) . R e w r i t i n g Eqs. ( 2 . 6 1 , one g e t s 1

I

Pk(P'

I

Cj)

CkI

1

-

Vk(P",

I

Ck,

c

.I

J

and

II

= Pw(P",

Ck,

II

c

.I J

-

PW(PflI c

I

I

k'

c.) J

(2.9)

E q u a t i o n s ( 2 . 8 ) and ( 2 . 9 ) are of t h e form

-

(AIJ)c=c'

(2.10)

-(AP)P=pll

I n t r o d u c i n g Eq. ( 2 . 7 ) i n t o Eq. ( 2 . 8 ) and n e g l e c t i n g t h e p r e s s u r e dependence of t h e p a r t i a l molar volumes vk gives

vk

I

II

AP = - R T l n ( c k / c k )

I n a s i m i l a r way t h e l e f t - h a n d s i d e of Eq. be e x p r e s s e d and one g e t s

vw

AP =

-

(AVw)p=pll

(2.11)

( 2 . 9 ) can (2.12)

where vW i s t h e p a r t i a l molar volume o f t h e w a t e r . F o r small c o n c e n t r a t i o n d i f f e r e n c e s t h e e q u a t i o n s c a n be s i m p l i f i e d f u r t h e r . I f (2.13)

MEMBRANE EQUILIBRIUM

Eq.

25

( 2 . 1 1 ) becomes

...,

1

Vk

AP = -RT

A ck/ c k

( k = 1,

n)

(2.14)

F o r d i l u t e s o l u t i o n s w e have v k c l < < 1. T h e r e f o r e , R T I A <~ < ~ A P~ f o r t h e p e r m e a n t chmponents k . Under t h e s e c o n d i t i o n s t h e Gibbs-Duhem r e l a t i o n become s

---fLTl C

(

w

n

1

rn

Ack

1

+

Acj)

(2.15)

j=n+l

k=l

Combination o f Eq. ( 2 . 1 5 ) w i t h Eq;. gives f o r the pressure difference

(2.12)

and ( 2 . 1 4 )

rn

+

(iVw

k = l c L V A~ p = R T

j=1 n+l

AC j

(2.16a)

or rn

( 1

-

j=n+l

1

c;V\)*.

= RT j=n+l A c j

(2.16b)

F o r d i l u t e s o l u t i o n s one h a s rn

and one g e t s rn Ap = R T

c

Ac

j

(2.17)

j=n+1

which i s v a n ' t H o f f ' s l a w .

The c o n c e n t r a t i o n d i f f e r e n c e

FRIEDRICH A. SAUER

26

o f t h e p e r m e a n t components i s g i v e n by

(2.18) j=n+l

ELECTROLYTE SOLUTION WITHOUT CHEMICAL REACTIONS

111.

Homogeneous e l e c t r o l y t e s o l u t i o n s must f u l f i l l t h e e l e c t r o n e u t r a l i t y condition: n

rn

1

eknk

+

k=l

1 e ~j.n j = n +1

=

o

(3.1)

where t h e e k and e . are t h e e l e c t r i c a l c h a r g e s p e r mole of t h e i o n s . T h a t ’ c o n d i t i o n h a s c o n s e q u e n c e s f o r t h e d e f i n i t i o n a n d measurement of t h e c h e m i c a l p o t e n t i a l s of t h e ions. ( G i b b s , 1961; Guggenheim, 1 9 6 7 ) . Every v a r i a t i o n of t h e mole numbers must f u l f i l l Eq. ( 3 . 1 ) a g a i n . Because of Eq. ( 3 . 1 ) rn - 1 i n d e p e n d e n t v a r i a t i o n s of t h e m o l e numbers a r e p o s s i b l e . Choosing t h e n t h i o n a s t h e key i o n , o n e g e t s n-1 en

6nn -

- 1

m

-

6 nk

ek

k= 1

1

e

j

6n

j

(3.2)

j=n+l

F o r t h e v a r i a t i o n of t h e Helmholtz f r e e e n e r g y a t cons t a n t T I v, and nW o n e g e t s rn

n-1 6

~

1

=

k=l

( n1

vk

6nk

+

1

v j( n ) 6 n j

(3.3)

j=n+l

where p i n ’ and p j n ) a r e t h e c h e m i c a l p o t e n t i a l s of t h e i o n s . They depend on t h e c h o i c e o f t h e key i o n c o r r e s p o n d i n g t o Eq. ( 3 . 2 ) . The c h e m i c a l p o t e n t i a l s of t h e i o n s so d e f i n e d are m e a s u r a b l e q u a n t i t i e s . Choosing a n o t h e r i o n 1 # n a s t h e key i o n , o n e g e t s

MEMBRANE EQUILIBRIUM

27

m

n

(3.4)

and n

rn (3.5)

Comparing E q s . ( 3 . 2 ) and ( 3 . 3 ) w i t h E q s . ( 3 . 4 ) and ( 3 . 5 1 , one g e t s t h e r e l a t i o n s between t h e c h e m i c a l pot e n t i a l s o f i o n s d e f i n e d f o r d i f f e r e n t key i o n s n and

(3.6)

(3.7)

(3.8)

(3.9)

( j = n

+

1,

...,

rn)

(3.10)

S i m i l a r c o n s i d e r a t i o n s must be a p p l i e d f o r t h e d e f i n i t i o n of t h e p a r t i a l molar volumes of i o n s . They depend on t h e c h o i c e of t h e key i o n . One g e t s t h e t h e r m o s t a t i c r e l a t i o n f o r t h e p a r t i a l molar volume v i n ) :

FRIEDRICH A. SAUER

28

(3.11) Now w e c o n s i d e r t h e v a r i a t i o n of t h e f r e e e n e r g y a r o u n d t h e e q u i l i b r i u m s t a t e and g e t f o r e a c h p h a s e and " n-1 k=l

(3.12) n-1 k=l

for I

and w i t h t h e component n b e i n g t h e key i o n . Because t h e s y s t e m as a whole i s c l o s e d , w e have t h e c o n d i t i o n s 1

bnw

+

1

I1

6nw = 0;

6nk

I1

+

6nk = 0

(k =

1,

...,

n

-

1)

(3.14) Then t h e e q u i l i b r i u m c o n d i t i o n (1.1) h a s t h e form

(3.15)

From E q .

(3.15) we conclude t h a t

Apw = 0;

"k

(n) = 0

(k =

1,

...,

n

-

1)

(3.16)

These a r e t h e e q u i l i b r i u m c o n d i t i o n s f o r e l e c t r o l y t e sol u t i o n s , which a r e i n a c c o r d a n c e w i t h t h e e l e c t r o n e u t r a l i t y condition (3.1). To g e t t h e e l e c t r i c a l p o t e n t i a l d i f f e r e n c e s a t e q u i l i b r i u m , w e i n t r o d u c e p a i r s o f i d e n t i c a l , r e v e r si b l e e l e c t r o d e s i n t o t h e p h a s e s ' and " and p a s s a s m a l l amount of c h a r g e q a c r o s s t h e system. The e q u i l i b r i u m c o n d i t i o n (1.1) c h a n g e s t o 6F = 6F'

+

6F" = E l

69

(3.17)

MEMBRANE EQUILIBRIUM

29

f o r t h e permeant i o n s and t o

+

6 F = 6F'

6F" = E

j

(3.18)

6g

f o r t h e impermeant i o n s , Here E l and E j a r e t h e e l e c t r i c a l p o t e n t i a l d i f f e r e n c e s measured a t e q u i l i b r i u m w i t h t h e h e l p o f r e v e r s i b l e e l e c t r o d e s f o r k and j , r e s p e c t i v e l y . E q u a t i o n s ( 3 . 1 7 ) and ( 3 . 1 8 ) e x p r e s s t h e f a c t t h a t t h e change o f t h e f r e e e n e r g y must be e q u a l t o t h e r e v e r s i b l e e l e c t r i c work done o n t h e system by c h a r g e t r a n s f e r . I f we t a k e a p a i r of e l e c t r o d e s r e v e r s i b l e f o r t h e l t h p e r m e a b l e i o n and t r a n s f e r a c h a r g e 6 q , we have f o r t h e v a r i a t i o n of t h e mole numbers of t h e impermeant i o n s 6n

j

= 6n

j

= 0

( j

= n + 1,

1

+

II

(k =

6nk = 0

1,

(3.19)

For t h e p e r m e a n t i o n s w e

because t h e system i s c l o s e d . have a g a i n 6nk

..., rn)

...,

(3.20)

n)

T h i s e q u a t i o n i s v a l i d a l s o f o r t h e l t h permeant i o n . Because of t h e charge t r a n s f e r , we g e t 1

6n 1 = 6 q / e l

+ 6nl ,

II

6nl = - 6 q / e l

-

-

6n 1

(3.21)

where 6 q / e l e q u a l s t h e amount o f component 1 coming from t h e e l e c t r o d e and 6nl i s t h e exchange of 1 v i a t h e membrane. Combination of Eqs. ( 3 . 2 1 ) l e a d s t o Eq. ( 3 . 2 0 ) . The v a r i a t i o n of t h e f r e e e n e r g y becomes n-1 6F'

+

6F"

=

1

A p k( n ) tin'

k

+ ~u~

tin:

(3.22)

k= 1

Because of t h e e q u i l i b r i u m c o n d i t i o n s ( 3 . 1 6 ) , t h i s e x p r e s s i o n i s z e r o and w e c o n c l u d e from Eq. ( 3 . 1 7 ) t h a t a t equilibrium E

1

= 0

( 1 = 1,

...,

n)

or A?

:e E = 0 1 1

( 1 = 1,

...,

n)

(3.24)

30

FRIEDRICHA. SAUER

where A q l i s c a l l e d t h e e l e c t r o c h e m i c a l p o t e n t i a l d i f f e r e n c e . T h i s means t h a t a t e q u i l i b r i u m t h e e l e c t r o c h e m i c a l p o t e n t i a l d i f f e r e n c e (ECPD) of a l l p e r m e a n t i o n s i s zero. The ECPD i s measured w i t h a p a i r of i d e n t i c a l r e v e r s i b l e e l e c t r o d e s . These e q u i l i b r i u m c o n d i t i o n s a r e a g a i n i n accordance w i t h t h e e l e c t r o n e u t r a l i t y c o n d i t i o n ( 3 . 1 ) . I f w e t a k e a p a i r of i d e n t i c a l revers i b l e e l e c t r o d e s f o r a n impermeant i o n , s a y j = y I t h e c o n d i t i o n (3.19) changes i n t o I

6n

Y

II

= 6q/ey;

6n

Y

= -6q/ey

(3.25)

( 3 . 3 ) we g e t f o r t h e v a r i a t i o n of t h e

and b e c a u s e of Eq. free energy

n-1

6F'

+

6F" =

1

1

A p k( n1 6 n k

+

I

Apw

6nw

+

Apy( n ) 6 q / e

k= 1

(3.26) Under e q u i l i b r i u m (3.16) t h i s becomes

and t h e ECPD f o r a n impearmeant i o n becomes

Taking a n o t h e r impermeant i o n , s a y m , as t h e key i o n , one g e t s b e c a u s e of Eq. ( 3 . 1 0 ) (3.29) P u t t i n g j = m and o b s e r v i n g t h a t A u E ) E 0 , one g e t s (3.30) Combining Eqs.

( 3 . 2 9 ) and ( 3 . 3 0 ) y i e l d s e .

Aqj

= Apjm)

+

e m An rn

(3.31)

MEMBRANEEQUILIBRIUM

31

a r e l a t i o n between ECPDs and c h e m i c a l p o t e n t i a l d i f f e r e n c e s o f i o n s . N o t e t h a t t h i s r e l a t i o n c o n t a i n s measurable q u a n t i t i e s only. I t should n o t be mistaken f o r t h e nonthermodynamic r e l a t i o n A n = Ap

+

e A$

(3.32)

where A $ i s t h e s o - c a l l e d "membrane p o t e n t i a l " and Ap i s a s i n g l e i o n chemical p o t e n t i a l d i f f e r e n c e . F i n a l l y , Eq. ( 3 . 3 1 ) i s v a l i d f o r any c h o i c e o f t h e key i o n . So w e have a l s o e

k + e

Ank = ApLn)

An

n

(3.33)

n

I n terms o f t h e ECPDs t h e e q u i l i b r i u m c o n d i t i o n s r e a d Apw = 0;

Ank = 0

( k = 1,

f o r t h e permeant components. meable i o n s are g i v e n by

...,

n)

(3.34)

The E C P D s f o r t h e imper-

(3.35)

i f Eqs. ( 3 . 3 4 ) are f u l f i l l e d and t h e key i o n i s a p e r meable i o n . I t s h o u l d b e mentioned t h a t t h e ECPDs f o r uncharged components i w i t h e i = 0 , b e c a u s e of Eq. ( 3 . 3 3 ) , g o o v e r So w e have i n t o t h e chemical p o t e n t i a l d i f f e r e n c e s A p i . Ani

(3.36)

= Api

= Apln)

f o r ei E 0 . Coming back t o t h e e q u i l i b r i u m c o n d i t i o n s f o r t h e p e r m e a b l e i o n s ( 3 . 1 6 1 , w e found t h a t "k

(n)

= 0

(k =

1,

...,

n

-

1)

(3.37)

i f n ( t h e k e y i o n ) i s a l s o permeant. Choosing a n impermeant i o n s a y m , a s t h e key i o n , Eq. ( 3 . 3 7 ) c h a n g e s i nt o (m "k

_

- ee-

m

(3.38)

32

FRIEDRICH A. SAUER

U s e h a s been made of Eq.

3.10). O t h e r w i s e Eq. ( 3 . 3 7 ) r e m a i n s form i n v a r i a n t a s l o n g a s t h e key i o n i s a p e r meable i o n . Summarizinq t h e r e s u t s of t h e f o r e g o i n g c o n s i d e r a t i o n s , we f o u n d - t h e e q u i l i b r i u m c o n d i t i o n s (n) = 0

Avw = 0 ;

and p h a s e s

and

...,

n

-

1)

(3.39)

obey t h e e l e c t r o n e u t r a l i t y c o n d i t i o n

'I

n

rn

l e k n k +

1

e n = O

j = n +1

k=l

( k = 1,

j

(3.40)

j

A t e q u i l i b r i u m t h e s e c o n d i t i o n s are v a l i d r e g a r d l e s s o f

whether w e p u t e l e c t r o d e s i n t o t h e p h a s e s ' and I' o r n o t . A c t i v a t i n g one more d e g r e e of freedom by c h a r g e t r a n s f e r with t h e h e l p of r e v e r s i b l e e l e c t r o d e s [because of Eq. ( 3 . 4 0 ) t h i s i s i m p o s s i b l e w i t h o u t e l e c t r o d e s ] , w e f i n d f o r t h e ECPDs t h a t

Ank=o

(k=l,

..., n ) ;

A n j = A p j( n )

( j = n + l ,

..., rn) (3.41)

T o c a l c u l a t e p r e s s u r e and c o m p o s i t i o n o f t h e I' p h a s e , if p r e s s u r e and c o m p o s i t i o n of t h e I p h a s e and t h e c o n c e n t r a t i o n s of t h e impermeable components j i n t h e I' p h a s e are g i v e n , w e p r o c e e d a s w e d i d i n S e c t i o n 11. For d i l u t e e l e c t r o l y t e s o l u t i o n s w e make t h e Ansatz

From Eqs.

vW

( 3 . 3 9 ) and ( 3 . 4 2 ) w e g e t

AP = - ( A ' w ) P = p "

For small c o n c e n t r a t i o n d i f f e r e n c e s w e g e t

(3.44)

MEMBRANE EQUILIBRIUM

33

(3.45) Making u s e of Eqs. we f i n d

(3.39) and t h e Gibbs-Duhem e q u a t i o n ,

n-1

= c

I

‘kV c k1 Ap

k= 1

rn

(n)

W

- -RC TI w

1 j=n+l

( c j

-2GAc) n c

n

(3.46) and t h e r e f o r e from Eq.

(3.44)

(3.47) F o r d i l u t e s o l u t i o n s , b e c a u s e of

(3.48)

N e g l e c t i n g t e r m s o f t h e form

34

FRIEDRICH A. SAUER

and making u s e of t h e e l e c t r o n e u t r a l i t y c o n d i t i o n s , (3.48) goes over i n t o

Eq.

Ap =

n

n

rn

1

1

e k ( e k - e .)c' J k

AC

k=l

j

1

(3.49)

T h i s i s t h e g e n e r a l i z a t i o n of v a n ' t H o f f ' s l a w f o r electrolyte solutions. Taking j u s t one permeable i o n ( n = 1) and one i m p e r m e a b l e F i o n (rn = 2 1 , E q . ( 3 . 4 9 ) becomes Ap = R T ( 1

-

(3.50)

e2/el)Ac2

F o r t h e e l e c t r o c h e m i c a l p o t e n t i a l d i f f e r e n c e of t h e j t h impermeant i o n w e g e t from E q . ( 3 . 2 8 )

(j = n

+ 1,

..., rn)

(3.51)

Combining E q s . ( 3 . 4 5 ) and o b s e r v i n g t h e e l e c t r o n e u t r a l i t y c o n d i t i o n , o n e ge t s

(3.52) I n t r o d u c t i o n of E q .

(3.52) i n t o Eq.

(3.51) g i v e s

(3.53) I n t h e s p e c i a l c a s e of E q .

(3.50) one g e t s

MEMBRANE EQUILIBRIUM

35

(3.54) I f a g a i n c '2 ~ 2

< < 1, t h e n AC

IV.

2

(3.55)

NONELECTROLYTE SOLUTION W I T H CHEMICAL REACTIONS

I n a d d i t i o n t o t h e components k and j , w e i n t r o duce components t , which c a n n o t p e r m e a t e t h e membrane b u t r e a c t a t t h e s i d e of t h e membrane f a c i n g t h e ' p h a s e according t o

where t h e v t arg! t h e s t o i c h i o m e t r i c numbers. change of t h e n t i s g i v e n by 1

6n

t

= v

t

65

( t = rn

+

1,

...,

r)

Every

(4.2)

where 6 5 i s t h e change i n t h e p r o g r e s s of t h e r e a c t i o n . Because t h e t-components c a n n o t p e r m e a t e t h r o u g h t h e membrane, w e have II

An, = 0

( t = rn

+ 1,

...,

r)

(4.3)

F u r t h e r m o r e , we have components s which c a n p e r m e a t e t h e membrane o n l y i f 6 6 i s u n e q u a l t o z e r o . The s - c o m p o n e n t s do n o t undergo a c h e m i c a l change b u t a r e t r a n s f e r r e d a c r o s s t h e membrane. For a c l o s e d s y s t e m w e have 1

6ns

-

-

It

6n

S

= ts 6 5

(s = r

+

1,

...,

u)

(4.4)

36

FRIEDRICH A. SAUER

Here t h e t , a r e t h e t r a n s f e r e n c e numbers. They s h o u l d n o t be confused w i t h t h e s t o i c h i o m e t r i c numbers v t , because t h e components s a r e c o n s e r v e d ( s remains s ) . F o r t h e change of t h e f r e e e n e r g y i n b o t h p h a s e s w e get n

r

U

and

(4.5) n 6F"

U 11

=

tsuz

65

s=r+l

k=l

U s e h a s been made of E q s .

+ vw

and ( t h e system i s c l o s e d and j i s impermeant). rium w e have 6F'

+

(4.41,

n 'Pk

6 n k1

k=l

I

n j

-

II

= O

n

A t equiiib-

(4.6)

6F" = 0

and t h e r e f o r e

1

(4.31,

II

6 nw

+(

t s Aps

-

+

.)tic

Apw 6 n i =

0

s=r+l

(4.7)

F o r t h e c l o s e d system i t w a s assumed I

6n

k

+

I

II

6nk = 0;

6n

W

+

II

6nw = 0

Because 6 n k , f n w r and 6 5 a r e i n d e p e n d e n t v a r i a t i o n s , w e g e t the equilibrium conditions Apk = 0

(k = 1,

..., n ) ;

Allw=

0

U

1 s=r+l

t s Aps

-

A'

= 0

(4.9)

37

MEMBRANE EQUILIBRIUM

Here

(4.10)

i s t h e D e Donder a f f i n i t y of t h e c h e m i c a l r e a c t i o n ( 4 . 1 ) . To c a l c u l a t e t h e p r e s s u r e and t h e c o m p o s i t i o n of t h e 'I p h a s e f o r a g i v e n p r e s s u r e and c o m p o s i t i o n of t h e I phase, t h e e q u i l i b r i u m c o n d i t i o n s are incomplete i f t h e number o f s-components i s l a r g e r t h a n o n e . One g e t s t h e a d d i t i o n a l e q u a t i o n s by i n t e g r a t i o n of Eqs. ( 4 . 4 ) . T h i s i n t e g r a t i o n s t a r t s w i t h an a r b i t r a r y n o n e q u i l i b r i u m s t a t e 0 a n d g o e s t o t h e r e a l e q u i l i b r i u m s t a t e 5,. Therefore 11

II

n S - n so = - tS

-

w i t h A t = 5, 50. c o n s t a n t , one g e t s c

11

s

- c

II

SO

= - -t S V I#

A5

(s = r

+

1,

...,

A f t e r d i v i s i o n by

At

(s

= r

+

1,

(4.11)

U)

v",

which i s k e p t

..., u )

(4.12)

From Eqs. ( 4 . 1 2 ) one sees t h a t t h e e q u i l i b r i y m d i s t r i b u t i o n d e p e n d s on t h e i n i t i a l c o n c e n t r a t i o n s c s o I i f t h e number o f s-components i s l a r g e r t h a n o n e . E l i m i n a t i o n o f A 5 i n Eqs. ( 4 . 1 2 ) g i v e s II

c

s

II

= c

s o + -t u

uo

(s

= r

+

1,

...,

u

-

1)

(4.13) The s e t of e q u a t i o n s ( 4 . 1 3 ) t o g e t h e r w i t h t h e e q u i l i b rium c o n d i t i o n s ( 4 . 9 ) e n a b l e one t o c a l c u l a t e t h e e q u i l i b r i u m p r e s s u r e and c o m p o s i t i o n o f t h e I' p h a s e . In g e n e r a l t h i s l e a d s t o t h e s o l u t i o n of a system of t r a n s cendental equations. I n t h e following we g i v e an approximate s o l u t i o n for t h e s p e c i a l case of a d i l u t e s o l u t i o n . The compon e n t s w i l l b e w a t e r (w), one s-component ( s = r + 1 1 , and t h e r e a c t a n t t ( t = m + 1, , r ) Because t h e r e a c t a n t s do n o t p e r m e a t e a c r o s s t h e membrane, t h e i r c o n c e n t r a t i o n s are p r e s c r i b e d . l , T ~d e t e r m i n e t h e p r e s s u r e p " and t h e c o n c e n t r a t i o n c ~ + w ~ e , have t h e equations

. ..

.

38

FRIEDRICH A. SAUER

Under t h e assumption of a d i l u t e s o l u t i o n t h i s l e a d s t o r I

c

1

cw Vw AP = - c w ( A ~ w ) p = p = ,l R T (

Act +

A'Cr+l

t=m+l

1

(4.15)

1

If E q .

( 2 . 7 ) i s v a l i d and cWvw > > c ~ + ~ V ~ one + ~ g, e t r

1

Ap = R T

AC

+

t

t=m+l A'

A1/tr+l

r+l

(

1

Cr + l

C1

I

Acr+l

Act

+

'Ic'+')

RTtr+l

t=m+l

(4.16)

T h i s means t h e o s m o t i c p r e s s u r e d i f f e r e n c e Ap depends On A ' . I f 1 >> c r + l'r+1' w e g e t 1

(4.17) F o r more t h a n o n e s-component w e make u s e of E q s . and rewrite them i n t h e form Acs

= AcsO

+tS

(Acu

-

Acuo)

(s = r

+

1,

(4.13)

..., u )

tU

(4.18) where t h e l a s t e q u a t i o n f o r s = u i s a n i d e n t i t y . The e q u i l i b r i u m c o n d i t i o n s f o r d i l u t e s o l u t i o n s and s m a l l c o n c e n t r a t i o n d i f f e r e n c e s Acs and A c t go o v e r i n t o

MEMBRANE EQUILIBRIUM

39

(4.19) and U

U

1

t V Ap s s

+

RT

tS i-

s=r+l

s=r+l

Combination of Eqs.

C

(4.20)

A c S = A'

s

(4.18) and (4.20) leads to

U

-

1

(4.21) C

s=r+l

S

The contribution of the s-components to the osmotic pressure difference becomes then

2 -1

-

R*(

s=r+1

U

A' + -R T

2) Cs

ts

s=r+l

U

J

U

c

tstvV"

s = r + l v=r+l U

1

s=r+l v = r + l tvf
-

>)Ac CS

1

c

Here we assume that

[

U

<<

1

(4.22)

so

FRIEDRICHA. SAUER

40

V.

ELECTROLYTE SOLUTION W I T H CHEMICAL W A C T I O N S

Assuming t h a t t h e r e i s a t l e a s t one i o n of t h e t y p e k p r e s e n t which can permeate t h e membrane i n d e p e n d e n t l y of t h e r e a c t i o n , w e t a k e t h i s i o n as t h e key i o n . From t h e f o r e g o i n g p a r a g r a p h s w e f i n d t h e e q u i librium conditions

"k

(n) = 0

(k =

1,

..., n -

1 ) ; Apw = 0

s=r+l

where n i s t h e key i o n . Acs = Acso

+

tS

Together w i t h

(Ac

U

U

-

(s = r

Acuo)

+ 1,

..., u ) (5.21

Eqs. ( 5 . 1 ) allow o n e t o c a l c u l a t e t h e e q u i l i b r i u m d i s t r i b u t i o n o f t h e components and t h e p r e s s u r e i f t h e I phase i s g i v e n . For t h e ECPDs w e g e t

(s = r

+

1,

...,

u)

(5.3)

T h e r e f o r e , w e c a n w r i t e t h e g e n e r a l i z e d e q u i l i b r i u m cond i t i o n s i n t h e form

U

1

ts A n s

-

A'

= 0

(5.41

s=r+l

T h i s h a s been d e r i v e d u n d e r t h e c o n d i t i o n t h a t t h e r e e x i s t s a n i o n which can permeate i n d e p e n d e n t l y of t h e chemical r e a c t i o n . I f t h i s i s n o t t h e case, w e have t o d i s t i n g u i s h two s i t u a t i o n s .

MEMBRANE EQUILIBRIUM

41

L e t us assume t h e o n l y i o n s p r e s e n t a r e t h o s e o f W e take t h e ion w i t h t h e index u as t h e the s-type. key i o n . For t h e v a r i a t i o n o f t h e f r e e e n e r g y one g e t s u-1

1

bF =

(u)

ts

' -

6ns

A'

65

+

I

Apw

6nw

(5.5)

If t h e system a s a whole i s c l o s e d (no e l e c t r o d e s )

,

we

have I

tins = t

65

S

(s =

r

+ 1,

...,

u

-

1)

(5.6)

and b e c a u s e of t h e e l e c t r o n e u t r a l i t y c o n d i t i o n u-1 I

=

6n

eS

c

-

e ts

(5.7)

6E

s=r+l

A s on t h e o t h e r s i d e u i s o f t h e s - t y p e , 6nU = t

w e have (5.8)

65

ni

To g e t v a r i a t i o n s of t h e i n t h e closed system w i t h o u t v i o l a t i n g the e l e c t r o n e u t r a l i t y condition, U

1

e t

s s

= O

(5.9)

s=r+l

m u s t be f u l f i l l e d . l I f Eq. ( 5 . 9 ) i s n o t f u l f i l l e d , v a r i a t i o n s of t h e n s a r e p o s s i b l e o n l y by means o f e l e c t r o d e s . Assuming t h e v a l i d i t y o f E q . ( 5 . 9 ) , one g e t s the equilibrium conditions u-1

1

A v w = 0;

t s A p s( u )

-

A'

(5.10)

= 0

s=r+l

Together w i t h the material c o n d i t i o n s Ac

S

= Ac

so

tS + tU

(AcU

-

Ac

uo

)

(s =

r

+ 1,

...,

u

-2)

(5.11)

FRIEDRICH A. SAUER

42

t h e e q u i l i b r i u m d i s t r i b u t i o n i s determined. Under t h e c o n d i t i o n ( 5 . 9 ) it i s i m p o s s i b l e t o p a s s e l e c t r i c a l c h a r g e a c r o s s t h e membrane w i t h t h e h e l p of r e v e r s i b l e electrodes. L e t u s assume w e have a p a i r o f e l e c t r o d e s r e v e r s i b l e t o t h e i o n u . Then w e have (5.12) 1

6nS

--

From Eqs.

ts 65

s

#

(5.13)

u

( 5 . 1 2 ) and ( 5 . 1 3 ) w e g e t

U

U

1

es 6 n i = 6q

+

s=r+l

1

t s e s 65

(5.14)

s=r+l

Because of Eq. t i o n we g e t

( 5 . 9 ) and t h e e l e c t r o n e u t r a l i t y c o n d i (5.15)

6q = 0

T h i s means t h a t it i s i m p o s s i b l e t o p a s s e l e c t r i c a l c h a r g e a c r o s s t h e membrane. T h e r e i s no e l e c t r i c cont a c t between b o t h p h a s e s and no ECPD i s d e f i n e d . On the o t h e r side i f w e release the condition (5.91, w e have from Eq. ( 5 . 1 4 )

(5.16)

I f 6 q = 0 ( c l o s e d s y s t e m ) . I f c:=r+l t e # 0, we f i n d 6 5 = 0 b e c a u s e of t h e e l e c t r o n e u t r a l i t y c o n d i t i o n . T h i s means no v a r i a t i o n of t h e mole numbers are poss i b l e i n t h e c l o s e d system. For an open system ( w i t h e l e c t r o d e s ) one g e t s from Eq. (5.14) U

6q =

-

c s=r+l

Because of

tses 65

(5.17)

MEMBRANE EQUILIBRIUM

43

6q e

(5.18)

6F =

U

t h e e q u i l i b r i u m c o n d i t i o n becomes

(5.19) and t h e r e f o r e

(5.20) Making u s e of E q .

( 3 . 3 3 1 , one g e t s

(5.21)

E q u a t i o n s ( 5 . 2 0 ) c a n be u s e d t o c a l c u l a t e t h e p r e s s u r e d i f f e r e n c e Ap and t h e ECPD A n u , which b u i l d up a t e q u i l i b r i u m . Because C;=r+l e t # 0 and no o t h e r p e r m e a t i n g i o n s a r e p r e s e n t , t h e d i s t r i b u t i o n o f t h e comp o n e n t s w i l l n o t change. I n t h e s p e c i a l case t h a t Acs - A c s O = 0 w e g e t

(5.22) and Ans

= Anu es/eu

( s = r + 1,

.. .,

u

-

1)

(5.23)

I n t h e g e n e r a l c a s e , when a l l t h e d i f f e r e n t k i n d s o f components ( k , j , t , s ) a r e p r e s e n t , o n e g e t s t h e e q u i librium conditions

FRIEDRICH A. SAUER

44

Apw

= 0;

"k

(n) = 0

..., n

( k = 1,

-

1)

U

(5.24) s=r+l

where n i s t h e k e y i o n . T o g e t h e r w i t h Eq. ( 4 . 1 8 ) I Eqs. ( 5 . 2 4 ) d e t e r m i n e t h e e q u i l i b r i u m d i s t r i b u t i o n of p r e s s u r e and components. F o r t h e ECPDs o n e g e t s ( k = 1,

Aqk= 0

... , n ) ;

(t=m

+

1,

Aqs

- A y s( n )

..., r ) ; ( j = n

+

A

(s=r

~

1,

~

+ 1,

= (n A) V

..., rn)

..., u )

~

(5.25)

With t h e h e l p of Eqs. ( 5 . 2 5 ) t h e " c h e m i c a l " e q u i l i b r i u m c o n d i t i o n c o u l d be w r i t t e n i n t h e form U

(5.26)

I n t h e f o l l o w i n g w e compare t h i s r e s u l t of e q u i l i b r i u m thermodynamics w i t h t h e s t o p - f l o w s i t u a t i o n o f nonequil i b r i u m thermodynamics. For s i m p l i c i t y w e assume t h a t o n l y s- and t-components are p r e s e n t . Then we g e t f o r t h e s-components U II

Js =

'sh

Anh

w=r+l

+ Ls 5

A'

(s = r

+

1,

...,

u)

(5.27) and f o r t h e r e a c t i o n r a t e

J

5

U

J

5

=

1

L S h Anh

+

L

55

A'

(5.28)

h=r+l

For t h e t i m e b e i n g w e release t h e c o n d i t i o n o f c o m p l e t e coupling. Then t h e c o n d i t i o n s of u n c o n s t r a i n e d thermodynamic ' e q u i l i b r i u m are .

MEMBRANE EQUILIBRIUM

45

II

J s = 0;

J

5

= 0

(s

= r

+

...,

1,

u)

(5.29)

From t h a t w e c o n c l u d e i f t h e Onsager m a t r i x i s nons i n g u l a r (independent flows) t h a t = 0;

A'

Anh = 0

a t equilibrium.

#

J~

0;

+

(h = r

1,

...,

u)

(5.30)

I n t h e stop-flow s i t u a t i o n

J~ = 0

(s =

r

+

1,

...,

U )

(5.31)

we g e t U

(5.32)

o r i n vector notation (5.33) where Lo h a s t h e e l e m e n t s L s h and t h e v e c t o r L5 h a s t h e components ~ c = ; L, ~ assuming Onsager r e c i p r o c i t y . Here t h e s u p e r s c r i p t "sf" d e n o t e s s t o p flow. F u r t h e r Introduction more, w e assume t h a t Lo i s n o n s i n g u l a r . of t h e v e c t o r t of t h e t r a n s f e r e n c e numbers d e f i n e d by

... , n ) (5.34) leads t o

(5.35) The r e a c t i o n r a t e a t s t o p f l o w i s g i v e n by

FRIEDRICHA. SAUER

46

(5.36)

55

s=r+l

Because t h e t o t a l Onsager m a t r i x is p o s i t i v e d e f i n i t e , w e have (5.37) T h i s means t h a t t h e f a c t o r i n f r o n t of A ' i n Eq. ( 5 . 3 5 ) is positive. From t h e p o s i t i v e v a l u e o f t h e d e t e r m i n a n t of t h e t o t a l Onsager m a t r i x o n e can show t h a t t h i s f a c t o r i s less t h a n u n i t y . The p r o o f g o e s a s f o l l o w s . W e have t h e r e l a t i o n ( d e t Ltot)

=

5

)

(5.38)

[ H i n t : One g e t s t h i s r e l a t i o n by e x p a n s i o n o f ( d e t & , t o t ) a b o u t t h e e l e m e n t s o f t h e l a s t row.] Because ( d e t Ltot)

0

and ( d e t L ) are p o s i t i v e , w e h a v e

T 0-laL 0 < (L5*L

5)'L5s

< 1

(5.39)

Taking t h e a b s o l u t e v a l u e of Eq. ( 5 . 3 5 ) and making u s e of i n e q u a l i t y ( 5 . 3 9 ) , one g e t s t h e i n e q u a l i t y

(5.40)

From r e l a t i o n s ( 5 . 3 5 ) and (5.39) one c o n c l u d e s t h a t A ' and E T * A n S f have t h e same s i g n . T h e r e f o r e , w e c a n w r i t e two p o s s i h e i n e q u a l i t i e s :

(5.41)

or

MEMBRANE EQUILIBRIUM

47

(5.42) s=r+l

d e p e n d i n g on t h e s i g n of A ' . The i n e q u a l i t i e s (5.41) and (5.42) a r e d i r e c t c o n s e q u e n c e s of t h e s e c o n d law o f thermodynamics. Comparing t h i s r e s u l t w i t h Eq. (5.36) I w e come t o t h e c o n c l u s i o n t h a t €or an i n c o m p l e t e c o u p l e d s y s t e m ( a l l f l o w s i n c l u d i n g J~ a r e i n d e p e n d e n t ) a t s t o p flow i n general

#

Jsf

5

(5.43)

0

For a c o m p l e t e l y c o u p l e d s y s t e m i n n o n e q u i l i b r i u m t h e c o n d i t i o n (4.4) g o e s o v e r i n t o II

J~ = -t

J

s 5

(s = r

+

1,

. . . I

u)

(5.44)

i n e a c h s t a t e of t h e s y s t e m and n o t o n l y i n l e v e l flow. T h i s means t h e f l o w s a r e d e p e n d e n t now and t h e t o t a l Onsager m a t r i x becomes s i n g u l a r . Comparing Eqs. (5.44) w i t h Eqs. (5.27) and (5.281, one g e t s 0

(5.45)

L s h = Ls5Lh(/L5E

and

J5 = L5

5

(4'

-

is

Ans)

s=r+l

which f u l f i l l s c o n d i t i o n (5.44). F o r a c o m p l e t e l y c o u p l e d s y s t e m o n e h a s one i n d e p e n d e n t f l o w and o n e i n d e p e n d e n t d r i v i n g f o r c e o n l y . The f l o w m i g h t be t h e J 5 and t h e d r i v i n g f o r c e i s t h e n

A'

is

s=r+l

Aqs

(5.47)

FRIEDRICHA. SAUER

40

The s t o p - f l o w s i t u a t i o n c o i n c i d e s w i t h t h e c o n s t r a i n e d thermodynamic e q u i l i b r i u m . A t s t o p f l o w w e have

and t h e c o n d i t i o n f o r t h e d r i v i n g f o r c e (5.47) becomes U

A'

-

1

(5.49)

is

s=r+l

t h e same c o n d i t i o n a s w a s d e r i v e d ,at Eq. ( 5 . 4 ) . Then, and o n l y t h e n , t h e i n e q u a l i t i e s ( 5 . 4 ) and ( 5 . 4 2 ) become equalities. I f one u s e s Eq. (5.49) € o r t h e e v a l u a t i o n o f t h e a f f i n i t y A ' o f t h e d r i v i n g chemical r e a c t i o n , one must be s u r e t h a t t h e system is c o m p l e t e l y c o u p l e d . T h i s means Eqs. (5.44) must b e f u l f i l l e d i n e a c h s t a t e o f t h e system. O t h e r w i s e , Eq. (5.35) must be used. Here t h e knowledge o f t h e Onsager m a t r i x i s needed. R e w r i t i n g Eq. (5.351, one g e t s

(5.50) L

55

c s=r+l

0-1 tsthLsh h=r+t

I f o n l y some of t h e f l o w s are c o m p l e t e l y c o u p l e d , t h e stop-flow s t a t e a g a i n c o i n c i d e s w i t h t h e c o n s t r a i n t e q u i l i b r i u m . L e t u s assume w e have s-components (5.51)

...,

and k-components ( k = 1, n ) which are n o t completel y c o u p l e d . Then t h e s t o p - f l o w c o n d i t i o n s II

Js =

0

(s = r

+

1,

...,

II

u);

Jk = 0

( k = 1,

..., n ) (5.52)

because of Eqs.

(5.51) imply

MEMBRANE EQUILIBRIUM

J

5

49

= o

(5.53)

T h i s means c o n s t r a i n e d e q u i l i b r i u m w i t h t h e e q u i l i b r i u m conditions U

Ank = 0

( k = 1,

...,

n);

A'

-

1

is

Ans

= 0

s=r+l

(5.54) T h e r e w i l l be no b u i l d u p of E C P D s f o r t h e k-components. T h i s a l s o happens i n t h e c a s e of n o n v a n i s h i n g Lk It s h o u l d be mentioned t h a t s u c h a s y s t e m , n e v e r t h e i e s s , shows a l e v e l f l o w

.

I n a system f o r which p a r t s o f i t a r e c o m p l e t e l y c o u p l e d , t h e s t o p - f l o w e x p e r i m e n t g i v e s no i n d i c a t i o n f o r t h e a c t i v e t r a n s p o r t of t h e incompletely coupled components. C o n s i d e r a t i o n s b a s e d on the i n e q u a l i t i e s ( 5 . 3 7 ) a l l o w e d Kedem and Caplan ( 1 9 6 5 ) t o d e r i v e t h e i r r e s u l t s on thermodynamic e f f i c i e n c y of membrane s y s t e m s w i t h active transport.

VI.

SUMMARY

U s u a l l y components which p e r m e a t e a c r o s s a memb r a n e do n o t c o n t r i b u t e t o t h e o s m o t i c p r e s s u r e d i f f e r ence a t theryodynamic e q u i l i b r i u m i n d i l u t e s o l u t i o n s . As l o n g as ckvk i s s m a l l compared t o u n i t y , t h e i r cont r i b u t i o n c a n be n e g l e c t e d . T h a t i s n o t t r u e f o r components which are completel y c o u p l e d t o a chemical r e a c t i o n . These components c a n b u i l d up l a r g e r d i f f e r e n c e s o f t h e i r c h e m i c a l potent i a l s , which t h e n c o n t r i b u t e t o t h e o s m o t i c p r e s s u r e d i f f e r e n c e . For t h i s osmochemical e q u i l i b r i u m o n e g e t s Eq. ( 4 . 1 6 ) :

50

FRIEDRICH A. SAUER

T h i s means t h a t t h e a f f i n i t y A' of t h e chemical r e a c t i o n g i v e s an a d d i t i o n a l c o n t r i b u t i o n t o t h e osmotic p r e s The s u r e depending on t h e t r a n s f e r e n c e number t,+l. s i g n of t h e t e r m w i t h A' depends o n t h e s i g n of t h e t r a n s f e r e n c e number and t h e s i g n of A' i t s e l f . T h i s res u l t can e a s i l y be e x t e n d e d f o r membranes where t h e r e a c t i n g components p e r m e a t e t h e membrane and o t h e r i m permeant components j a r e p r e s e n t . W e g e t them t h e osm o t i c p r e s s u r e d i f f e r e n c e between d i l u t e s o l u t i o n s

By a d j u s t m e n t of t h e A c j i t i s p o s s i b l e t o v a r y t h e Ap. I f t h e r e i s more t h a n one component p r e s e n t which i s c o m p l e t e l y c o u p l e d , w e g e t memory e f f e c t s i n t h e osmot i c pressure. T h i s means t h e f i n a l osmotic p r e s s u r e d i f f e r e n c e depends on t h e i n i t i a l c o n c e n t r a t i o n d i f f e r of t h e c o m p l e t e l y coupled components. F o r ences A c t h a t comB8re Eq. (4.22). The w i d e l y used r e l a t i o n o f Eq. (5.49):

which r e l a t e s t h e e l e c t r o c h e m i c a l p o t e n t i a l d i f f e r e n c e s i n a s t o p - f l o w s t a t e w i t h t h e a f f i n i t y A ' o f t h e metab o l i c r e a c t i o n , i s r i g o r o u s l y v a l i d i f t h e components are c o m p l e t e l y c o u p l e d t o t h e chemical r e a c t i o n . Then t h e s t o p - f l o w s t a t e becomes a c o n s t r a i n e d thermodynamic e q u i l i b r i u m . For i n c o m p l e t e l y c o u p l e d systems Eq. (5.49) must be r e p l a c e d by t h e i n e q u a l i t y (5.40) :

which i s a consequence o f t h e second l a w o f thermodynamics.

MEMBRANE EQUlLlBRlUM

51

REFERENCES

Gibbs, J. W. (1961). "The S c i e n t i f i c Papers," pp. 83-85, 331-349, 4 06-4 12. Guggenheim, E. A, (1967). "Thermodynamics," Chap. 8. North Holland, Amsterdam. Kedem, O., and Caplan, S. R. (1965). T r a n s . F a r a d a y SOC. 6 1 , 1897.

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Part 11

Structural Analysis of Na,K-ATPase

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CURRENT TOPICS IN MEMBRANES AND TRANSPORT, VOLUME 19

Structural Aspects of Na,K-ATPase ROBERTL. POST Department of Physiology Vanderbilt University Medical School Nashville, Tennesse

I.

INTRODUCTION

This chapter provides an introduction t o t h e s t r u c t u r a l a s p e c t s of Na,K-ATPase d i s c u s s e d i n P a r t I of t h i s volume. I t r e l a t e s t o problems of p u r i t y , s u b u n i t a s s o c i a t i o n , and t o p o l o g i c a l mapping. I t aims t o p r o v i d e a b r o a d e r and more b a l a n c e d view t h a n m i g h t b e o b t a i n e d by r e a d i n g o n l y i n d i v i d u a l r e p o r t s . The f o l l o w i n g r e c e n t reviews g i v e f u r t h e r i n f o r m a t i o n on s t r u c t u r a l a n a l y s i s o f t h i s enzyme: C a n t l e y (19811, Grisham ( 1 9 7 9 ) , Hobbs and A l b e r s (1980) , J g k g e n s e n (1980, 1 9 8 2 ) , Kyte (1981) , Robinson and F l a s h n e r ( 1 9 7 9 ) , Schuurmans S t e k h o v e n and B o n t i n g ( 1 9 8 1 ) , and W a l l i c k et a l . ( 1 9 7 9 ) .

53

Copyright 0 1983 by Academic Press. Inc. All rights of reproduction inany form reserved.

KENO-12-1533194

ROBERT L. POST

54

11. A.

BACKGROUND DESCRIPTION

OF

THE ENZYME

Sodium and potassium ion-transport adenosinetriphosphatase, Na,K-ATPase, is an enzymatic transport system that characteristically moves 3 Na+ outward and 2 K+ inward across the plasma membrane of animal cells per terminal phosphate group of intracellular ATP split off with catalysis by intracellular Mg2+. It is characteristically identified specifically by its inhibition by, or binding of, extracellular cardioactive steroids such as ouabain. It is conveniently assayed in preparations of broken membranes as an ATPase activity that requires Mg2+, Na+, and K+ and is inhibited by ouabain. It is an intrinsic protein of plasma mernbranes. Its purity is often estimated semiquantitatively by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, SDS-PAGE. In this test a purified enzyme shows only 2 bands, one at about 100 kilodaltons and a less distinct one at about 50 kilodaltons. B.

COMPONENTS

OF

THE ENZYME

Na,K-ATPase contains at least 2 polypeptides, the The a-subunit is the "catalytic" subunit. It binds ATP or ouabain and accepts a covalent phosphate group from ATP or inorganic phosphate, Pi, as an intermediate in the reaction. Since the same molecule binds intracellular ATP or extracellular ouabain, the molecule spans the membrane. It may (Churchill et a l . , 1979; Schuurmans Stekhoven and Bonting, 1981) or may not (Freytag and Reynolds, 1981) contain a little carbohydrate. It may be inhomogeneous (Ohta et a l . , this chapter). It undergoes prominent conformational changes between an Na form and a K form. The 8-subunit is a glycoprotein showing microheterogeneity (Marshall and Hokin, 1979). It is resistant to digestion by trypsin (Churchill and Hokin, 1976 and relatively unreactive with photoaffinity or other covalent probes. Its function is obscure. I suggest here that it serves as a stable fulcrum against which the catalytic subunit executes its conformational changes more precisely than it would were it acting alone. After all, the catalytic subunit makes a sharp distinction between 3 Na+ and 2 K+. a- and 8-subunits, and phospholipid.

STRUCTURAL ASPECTS OF Na,K-ATPase

55

The mass of the a-subunit is 84-120 kilodaltons and that of the $-subunit is 40-60 kilodaltons according to various estimates. With respect to protein the mass of the $-subunit is 35-56 kilodaltons (Cantley, 1981; Hobbs and Albers, 1980). Assuming that the subunits associate stoichiometrically, 3 recent reports favor an a : $ ratio of 1:l (Craig and Kyte, 1980; Peters e t a l . , 1981a; Peterson and Hokin, 1981) and one report favors a ratio of 2:3 (Freytag and Reynolds, 1981). The enzyme requires phospholipid. It can function with only 90 moles of phospholipid per 250 kilodaltons of enzyme (Schuurmans Stekhoven and Bonting, 1981). Detergents can partially replace phospholipid. Acidic phospholipids enhance activity relative to that in the presence of other phospholipids and possibly may be replaced by acidic detergents. In a study of equilibrium binding of membrane lipids to Na,K-ATPase, the enzyme clearly preferred negatively charged lipid. About 60-65 binding or contact sites were estimated per a282 diprotomer (Brotherus e t al., 1981a). Fluorescent lipid probes indicate that there may be direct interaction between Na,K-ATPase and phospholipids (Muczynski e t a l . , Part I of this volume). Cholesterol and other neutral lipids do not seem to be essential (dePont e t a l . , and Muczynski e t a l . , in Part I of this volume). A y-subunit is a candidate for componency. It is labeled by some photoaffinity derivatives of cardioactive steroids. It is a proteolipid with a mass of 12 kilodaltons (Collins e t a l . , Part I, this volume). It may not be essential for activity (Hardwicke and Freytag, 1981).

111.

EXAMPLES AND MODELS

As guides for thinking one can look to other transport systems. As an example one membrane transport system outshines the others. This is the bacteriorhodopsin in the purple patches in the plasma membrane of Halobacterium halobium. It is a planar crystal in the native functioning state! It pumps out one proton per photon of light absorbed by its chromophore, retinal. As a crystal it is pure in the native state. By electron scattering its shadow shows seven progressively tilted alpha helices (Unwin and Henderson, 1975). By amino acid sequencing it shows clusters of hydrophobic residues, which may correspond to the helices (Khorana e t a l . , 1979).

ROBERT L. POST

56

Another admirable model is hemoglobin, the soluble globular tetrameric carrier protein for oxygen, protons, and carbon dioxide in the vascular transport system. It crystallizes and has been x-rayed more than once (Baldwin and Chothia, 1979). The native molecule actively alternates between two conformations that have two corresponding crystal habits, one for oxyhemoglobin and another for deoxyhemoglobin. These habits are so incompatible that admission of oxygen to crystalline deoxyhemoglobin dissolves it and replaces its crystals with those of oxyhemoglobin (Haurowitz, 1938). Perhaps bacteriorhodopsin functions in the crystalline state because it does not undergo large conformational rearrangements d u r i n g t s reaction cycle. As a model for half-of-the-sites reactivity, thyroxine-binding prealbumin is instructive (Jorgensen, 1981). It is a plasma protein carrier of thyroxine. It is composed of four identical subunits containing 127 amino acids each. Its shape is an ellipsoid containing one central water-filled channel along the long axis. This axis is a 2-fold axis of symmetry. The free molecule also has another 2-fold axis of symmetry through the center at right angles to the longitudinal axis. Thyroxine is an asymmetric molecule. One molecule of thyroxine enters the channel at either end and is bound asymmetrically. Binding of the asymmetric thyroxine induces an asymmetrical conformational change. This reduces the affinity of the site at the other end of the molecule 100-fold. This is strong negative cooperativity. The reaction of the molecule thus shows marked half-of-the-sites reactivity although the free molecule has two identical binding sites. Models of association of subunits may be useful. Heterogeneous subunits can associate in any pattern. But for homogeneous subunits Levitzki (1978) emphasizes two patterns: isologous and heterologous. Isologous association can be represented by the relationship between the numerals "6" and "9" in the number "69." Two subunits form a pair. Notice the 2-fold axis of symmetry at right angles to the plane of the paper. In a heterologous association the front of each molecule fits to the back of the next. This pattern can extend indefinitely leading to the development of strands. One can think of the relationship of the numerals "3" and "3" in the number " 3 3 " with a possibility of in.3333333.. to form a definite extension as in strand. The a- and 8-subunits of hemoglobin associate asymmetrically to form a protomer; these protomers now associate isologously leaving a water-filled channel along the axis of symmetry between them.

"..

."

STRUCTURAL ASPECTS OF Na,K-ATPase

57

Na,K-ATPase is clearly not crystalline in the native cell membrane (Skriver e t a l . , this chapter). It swims about in the 2-dimensional domain of the fluid mosaic -- bila er of the membrane (Singer and Nicholson, 1 9 6 t s subunits appear to associate with each other, although possibly not as completely as do those of hemoglobin. Na,K-ATPase can crystallize, but so far only in an inhibited complex with van adate and magnesium. In the process it appears to form strands. The unit cell is large enough for only an aB protomer, and there is no evidence of isologous "69" pairing (Maunsbach e t a l . , Poster 10; but see JpSrgensen, this volume).

IV.

PURITY

Accurate structural analysis requires a pure enzyme preparation. Furthermore, purity is estimated in part by structural analysis. So purification lifts itself by its own bootstraps stepping uncertainly by successive approximations. An ideally pure enzyme would have all components necessary for the maximum activity that might be possible under physiological conditions. It would have no other components. In the estimation of activity, cofactors and substrates would be provided at levels for maximal activity, and end products would not be allowed to accumulate to inhibitory levels. Any regulatory mechanisms, such as phosphorylation by a protein kinase, would be adjusted for maximal activity. Those who purify have to take account of regulatory mechanisms. Ligand binding capacity can be used to estimate purity. The following ligands bind with 1:l stoichiometry relative to each other: ATP, the intermediate phosphate group, ouabain, vanadate, and Mn2+ (Cantley, 1981). Relative to these, R b ' , a congener of K+, binds with a 2:l stoichiometry, and Na+ binds at approximately 3:l (Matsui e t a l . , Part I of this volume; Tonomura et a l . , Part 11, this volume). The binding capacity for these ligands can thus define a "functional unit." [Nevertheless, Harris e t a l . , 1973) once reported that graded thermal denaturation depressed activity more than it did ouabain binding. Jorgensen (1980) found that graded tryptic digestion of the Na form depressed activity more than ATP binding. So even this criterion may have limitations.]

ROBERT L. POST

58

I n 1 9 7 4 Jfirgensen r e p o r t e d a h i g h l y p u r i f i e d p r e p a r a t i o n with a s t o i c h i o m e t r i c r a t i o of f u n c t i o n a l u n i t t o The enzyme w a s presumed t o show h a l f a - s u b u n i t of 1 : 2 . o f - t h e - s i t e s r e a c t i v i t y and v a r i o u s models were proposed by v a r i o u s workers. I n 1 9 8 1 two l a b o r a t o r i e s r e f i n e d t h e e s t i m a t i o n of p r o t e i n and a s s i g n e d somewhat h i g h e r v a l u e s t o t h e m o l e c u l a r r a t i o s of t h e s u b u n i t s . They concluded t h a t t h e c o r r e c t s t o i c h i o m e t r y i s 1:l (Moczydlowski and F o r t e s , 1981; Peters e t a l . , 1 9 8 1 b ) . Jfirgensen (1982) h a s a p p a r e n t l y confirmed t h i s conclus i o n w i t h r e s p e c t t o t h e Rb' b i n d i n g c a p a c i t y of h i s p r e p a r a t i o n ( P a r t I V o f t h i s volume). A.

P U R I F I C A T I O N METHODS

P u r i f i c a t i o n of Na,K-ATPase s t a r t s w i t h t h e c h o i c e of a t i s s u e . A t p r e s e n t t h e most s u i t a b l e a r e : (1) t h e o u t e r r e d m e d u l l a o f mammalian kidney, ( 2 ) t h e elect r i c o r g a n o f t h e e l e c t r i c e e l , ( 3 ) t h e r e c t a l g l a n d of t h e d o g f i s h , o r ( 4 ) t h e s a l t g l a n d of t h e s a l t - f e d duck. A microsomal f r a c t i o n i s o b t a i n e d from a homog e n a t e by d i f f e r e n t i a l c e n t r i f u g a t i o n and t h e n t h e proc e d u r e d i v e r g e s . One may n e x t employ n e g a t i v e p u r i f i c a t i o n o r p o s i t i v e p u r i f i c a t i o n (Esmann, t h i s volume). I n n e g a t i v e p u r i f i c a t i o n t h e microsomal f r a c t i o n i s c a r e f u l l y t r e a t e d w i t h an a n i o n i c d e t e r g e n t , sodium d o d e c y l s u l f a t e o r d e o x y c h o l a t e , i n s u c h a way a s t o remove most o f t h e e x t r a n e o u s p r o t e i n s w i t h minimum By t r e a t m e n t w i t h sodium damage t o t h e N a , K - A T P a s e . dodecyl s u l f a t e a h i g h l y p u r i f i e d p r e p a r a t i o n can be o b t a i n e d a c c o r d i n g t o SDS-PAGE. By e l e c t r o n microscopy t h e enzyme a p p e a r s a s p a r t i c l e s embedded i n membrane f r a g m e n t s o b t a i n e d a s s h e e t s o r d i s c s 0 .1 - 0 .6 pm i n d i a m e t e r w i t h free e d g e s . Enzyme p a r t i c l e s a p p e a r t o occupy t h e f r e e edges. Free edges are u s u a l l y u n s t a b l e i n a p h o s p h o l i p i d p r e p a r a t i o n . Vesicles a r e more s t a b l e . Accordingly one might s p e c u l a t e t h a t t h e discs are s t a b i l i z e d by sodium dodecyl s u l f a t e m o l e c u l e s bound t o N a , K - A T P a s e m o l e c u l e s a t t h e e d g e s . These Na,K-ATPase m o l e c u l e s might be d i f f e r e n t from t h o s e embedded i n t h e p h o s p h o l i p i d b i l a y e r . N e g a t i v e p u r i f i c a t i o n might i n t r o d u c e a d e t e c t a b l e f u n c t i o n a l inhomog e n e i t y i n t h e Na,K-ATPase. I n p o s i t i v e p u r i f i c a t i o n t h e microsomal f r a c t i o n i s t r e a t e d w i t h a n o n i o n i c d e t e r g e n t (Esmann, P a r t I of t h i s volume). The p u r i f i e d enzyme i s s o l u b i l i z e d and t h e i n s o l u b l e r e s i d u e i s s e p a r a t e d by high-speed c e n t r i f u g a t i o n and d i s c a r d e d . With o c t a e t h y l e n e g l y c o l d o d e c y l monoether (C12E8), p r e p a r a t i o n s as a c t i v e a s

STRUCTURALASPECTS OF Na,K-ATPaoe

59

those obtained by negative purification have been obtained with good stability below 23OC. The stability at 37OC was less. The mass of the soluble particles was 265 kilodaltons or 170 kilodaltons in different laboratories with corresponding estimates of the quaternary structure as a282 or a B . By this procedure one hopes that replacing the membrane phospholipids with a nonionic detergent will not affect the characteristics of the enzyme significantly while permitting application of all the expectations, methods, experience, and interpretations available for investigating soluble enzymes. However, this hope requires substantiation in each case. The work of Amende et a l . (Part I of this volume) emphasizes the point by showing selective effects of different nonionic detergents on different membrane-bound enzymes. Thus each of these approaches to purification has limitations. The specific activity of fresh microsomal preparations of Na,K-ATPase from tissue homogenates can be increased considerably by suitable addition of a detergent. Concern has been expressed that such activation might alter the molecular properties of the enzyme. Forbush (Part I, this volume) and Skriver et a l . (Part I, this volume) conclude that detergent activation is due only to the opening of vesicles.

V.

SUBUNIT ASSOCIATIONS

I like to distinguish association from interaction. I think of association as expressing the stability of structural contact between subunits and interaction as an influence which one subunit has on the function of another subunit, with which it is in contact. Interaction implies association but association does not imply interaction. I do not aim to discuss interaction. There are four ways to look €or subunit associations: (1) cross-linking, (2) solubilization, (3) radiation inactivation, and ( 4 ) electron microscopy. (1) Cross-linking forms a stable covalent bond between subunits that are in contact at the moment of the reaction. The reaction is irreversible. So the extent of association at equilibrium cannot be estimated easily. Even estimation of changes in equilibrium from changes in the initial rate of cross-linking is uncertain since the reactivity may change more than the association. In the presence of lateral diffusion in the plane of the membrane the process of cross-linking can be in-

60

ROBERT L. POST

f l u e n c e d by many v a r i a b l e s ( K y t e , 1 9 8 1 ) . (2) Solubiliz a t i o n c h a l l e n g e s a s s o c i a t i o n of s u b u n i t s o f t h e enzyme by r e p l a c i n g t h e membrane p h o s p h o l i p i d w i t h a d e t e r gent. Presumably s u b u n i t s t h a t d i s s o c i a t e i n t h e memb r a n e would s o l u b i l i z e a s s e p a r a t e p a r t i c l e s ; and t h o s e t h a t a s s o c i a t e i n t h e membrane would remain a s s o c i a t e d a f t e r s o l u b i l i z a t i o n s i n c e t h e y w e r e n o t s e p a r a t e d from e a c h o t h e r by t h e p h o s p h o l i p i d b i l a y e r b e f o r e s o l u b i l i z a tion. Nakao e t a l . ( P a r t I , t h i s volume) a r e e x p l o r i n g h i g h performance chromatography of t h e s o l u b i l i z e d enzyme a s a means t o i n v e s t i g a t e s u b u n i t a s s o c i a t i o n s . ( 3 ) R a d i a t i o n i n a c t i v a t i o n u n d e r t a k e s t o estimate t h e s i z e of t h e t a r g e t t h a t i t i n a c t i v a t e s . ( 4 ) I n elect r o n m i c r o s c o p y a s s o c i a t i o n c a n be v i s u a l i z e d d i r e c t l y b u t t h e sample may undergo f i x a t i o n a r t i f a c t s . I would e x p e c t f r e e z e - f r a c t u r e t o show l e s s i n t e r f e r e n c e t h a n n e g a t i v e s t a i n i n g . Enzyme p a r t i c l e s c a n b e s e e n e m bedded i n t h e membrane; numbers, s i z e s , and c o n t a c t s c a n be e s t i m a t e d . T h e r e i s e v i d e n c e f o r a s s o c i a t i o n between t h e aand t h e 8 - s u b u n i t s . Cross-linking of t h e p u r i f i e d , membrane-bound Na,K-ATPase w i t h i m i d a t e d e r i v a t i v e s y i e l d s a,e-complexes i n l o w y i e l d (Hobbs and A l b e r s , 1980). S o l u b i l i z a t i o n with d i g i t o n i n y i e l d s a,Bcomplexes w i t h o u t Na,K-ATPase a c t i v i t y b u t w i t h some of t h e p a r t i a l a c t i v i t i e s of t h i s enzyme. Solubilization w i t h C 1 2 E 8 h a s y i e l d e d a282- and a,P-complexes w i t h f u l l a c t i v i t y a f t e r less t h a n 1 0 % d i l u t i o n by t h e a s s a y medium (Esmann et al., 1 9 8 0 ; B r o t h e r u s et al., 1 9 8 1 b ) . C r o s s - l i n k i n g o f s o l u b i l i z e d enzyme w i t h c u p r i c phenant h r o l i n e p r o d u c e s a,B-complexes ( C r a i g and K y t e , 1980; Hobbs and A l b e r s , 1 9 8 0 ) . I t seems r e a s o n a b l e t o t h i n k t h a t t h e s t r o n g e s t s u b u n i t a s s o c i a t i o n i s between 1 as u b u n i t and 1 8 - s u b u n i t . This thought suggests t h a t a n a,B-complex s h o u l d have a name; I w i l l u s e " p r o t o m e r " f o r t h i s purpose. To what e x t e n t do p r o t o m e r s a s s o c i a t e ? R a d i a t i o n i n a c t i v a t i o n a n a l y s i s e s t i m a t e s t h e mass o f t h e a c t i v e u n i t a t 2 6 2 k i l o d a l t o n s ( O t t o l e n g h i et a ] . , P a r t I , t h i s volume). This i s l a r g e r than t h e protomer. This s i z e s u g g e s t s n o t only t h a t protomers a s s o c i a t e b u t a l s o t h a t t h e a c t i v i t y o f e a c h one depends o n t h e i n t e g r i t y o f a p a r t n e r . But c r o s s - l i n k i n g w i t h c u p r i c p h e n a n t h r o l i n e of t h e n e g a t i v e l y p u r i f i e d enzyme d o e s n o t p r o d u c e a,B-complexes; i n s t e a d i t p r o d u c e s d i m e r , trimer, tetramer, and pentamer complexes o f t h e a - s u b u n i t a n d , a t a much s l o w e r r a t e , complexes of 8 - s u b u n i t s t o o l a r g e t o e n t e r a sodium d o d e c y l s u l f a t e - p o l y a c r y l a m i d e g e l during e l e c t r o p h o r e s i s ( G i o t t a , 1 9 7 6 ) . Furthermore, u n d e r s p e c i f i c c o n d i t i o n s t h i s c r o s s - l i n k i n g can b e con-

STRUCTURAL ASPECTS OF Na,K-ATPase

61

t r o l l e d by t h e r e a c t i v e s t a t e o f t h e enzyme. Under o n e s e t o f c o n d i t i o n s o n l y t h e phosphoenzyme c r o s s - l i n k s r e g a r d l e s s o f t h e pathway o f p h o s p h o r y l a t i o n . Under another set of c o n d i t i o n s c r o s s - l i n k i n g occurs i n t h e p r e s e n c e of a l l c o m b i n a t i o n s o f l i g a n d s e x c e p t i n t h e p r e s e n c e of a p a i r of a n t a g o n i s t i c l i g a n d s , K+ and ATP ( A s k a r i and Huang, C h a p t e r 4 ) . I have n o t f i t t e d a l l these r e s u l t s together. P e r h a p s my a s s u m p t i o n a b o u t protomers needs r e v i s i o n . E l e c t r o n microscopy shows p r o t o m e r s by n e g a t i v e s t a i n i n g , and d i - o r t e t r a p r o t o m e r s by f r e e z e - f r a c t u r e and r o t a r y shadowing (Maunsbach e t a l . , 1 9 7 9 ; Haase and K o e p s e l l , 1 9 7 9 ) . A s w a s mentioned b e f o r e , t h e v i s u a l i z e d p a r t i c l e s t e n d t o form c l u s t e r s and s t r a n d s .

VI.

TOPOLOGICAL MAPPING OF a-SUBUNIT

T o p o l o g i c a l mapping o f t h e a - s u b u n i t h a s been f u r t h e r s t u d i e d ( C a s t r o and F a r l e y , 1979; J $ r g e n s e n , 1982). T r y p s i n and c h y m o t r y p s i n s p l i t t h e c h a i n a t f o u r l o c a t i o n s g i v i n g f i v e d e f i n a b l e fragments. Starti n g from t h e a m i n o - t e r m i n a l end t h e f r a g m e n t s have a mass o f a p p r o x i m a t e l y 2 , 2 4 , 2 0 , 1 6 , and 4 2 k i l o d a l t o n s . J B r g e n s e n names t h e l o c a t i o n s t h u s : (1) l i e s between ( 2 + 2 4 + 2 0 ) and (16 + 2 4 ) ; ( 2 ) l i e s between ( 2 ) and ( 2 4 + 2 0 + 1 6 + 4 2 ) ; and ( 3 ) l i e s between ( 2 + 2 4 ) and (20 + 16 + 4 2 ) . H e c o u l d h a v e named ( 4 ) a s l y i n g between ( 2 + 24 + 2 0 + 1 6 ) and ( 4 2 ) . I have used p a r e n s ( 1 , t o i n d i c a t e t h e l i m i t s of f r a g m e n t s . L o c a t i o n (1) i s a t t a c k e d p r e f e r e n t i a l l y by t r y p s i n i n t h e p r e s e n c e of K + . L o c a t i o n ( 2 ) i s a t t a c k e d p r e f e r e n t i a l l y by t r y p s i n i n t h e p r e s e n c e of N a + . L o c a t i o n ( 3 ) i s a t t a c k e d s l o w l y by t r y p s i n i n t h e presence of N a + . L o c a t i o n ( 4 ) i s a t t a c k e d s l o w l y by c h y m o t r y p s i n i n t h e presence of ouabain. Fragment ( 2 0 ) b e a r s t h e i n t e r m e d i a t e p h o s p h a t e group. Fragment (16 + 4 2 ) a c c e p t s a f l u o r e s c e n t a d d u c t t h a t b l o c k s a l l a c t i o n s of ATP ( C a r i l l i e t a l . , t h i s chapter. Fragments ( 2 0 ) and ( 1 6 + 4 2 ) a c c e p t l a b e l i n g by a p h o t o a f f i n i t y d e r i v a t i v e of ouabain (Karlish et a l . , this chapter). Fragment ( 1 6 ) a c c e p t s from i o d o a c e t i c a c i d a l a b e l t h a t d o e s n o t modify a c t i v i t y .

ROBERT L. POST

62

B.

OTHER PROBES A N D PROSPECTS

Kyte (Part I, this volume) is developing methods for isolating and analyzing specific fragments of the primary sequence of both subunits. Collins e t a l . , (Part I, this volume) are also sequencing the enzyme. It will be interesting to learn where more active sites are located. In order to label covalently an active site in the center for ATP binding Winter (this volume) has developed a fluorosulfonyl derivative of ATP that labels covalently and specifically in the active center. Munson (1981) has labeled the same active center with a photoaffinity derivative of ATP using chromic ion in place of Mg2+ to stabilize the initial noncovalent complex. Some sulfhydryl groups are important in the function of the enzyme. Identification and characterization of them has advanced significantly (Esmann and Klodos, Part I, this volume; Kawamura e t a l . , Part I, this volume; Winslow, 1981).

ACKNCWLEDGMENT

This work w a s supported by a g r a n t 2 R 0 1 HL-01974 from t h e N a t i o n a l Heart, Lung, and Blood I n s t i t u t e of t h e N . I . H .

REFERENCES Baldwin, J. , and C h o t h i a , C. (1979). Hemoglobin: The s t r u c t u r a l changes r e l a t e d t o l i g a n d b i n d i n g and i t s a l l o s t e r i c J . Mol. B i o l . 1 2 9 , 175-220. mechanism. B r o t h e r u s , J. R . , G r i f f i t h , 0. H . , B r o t h e r u s , M. O., J o s t , P. C . , S i l v i u s , J. R . , and Hokin, L. E. (1981a). L i p i d - p r o t e i n B i o c h e m i s t r y 20, m u l t i p l e b i n d i n g e q u i l i b r i a i n membranes. 5261-5267. B r o t h e r i s , J. R . , Mdller, J. V . , and Jdrgensen, P. L. (1981b). S o l u b l e and a c t i v e Na,K-ATPase w i t h maximum p r o t e i n molecul a r mass 170,000 ? 9,000 d a l t o n s ; formation of l a r g e r u n i t s by secondary a g g r e g a t i o n . B i o c h e m . B i o p h y s . R e s . Commun. 1 0 0 , 146-154. C a n t l e y , L. C . , Jr. (1981). S t r u c t u r e and mechanism o f t h e (Na,K)ATPase. C u r r . T o p . B i o e n e r g . 11, 201-237.

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C a s t r o , J . , and F a r l e y , R. A. ( 1 9 7 9 ) . P r o t e o l y t i c f r a g m e n t a t i o n o f t h e c a t a l y t i c s u b u n i t o f t h e sodium and potassium adenos i n e t r i p h o s p h a t a s e . Alignment o f t r y p t i c and chymotryptic fragments and l o c a t i o n o f s i t e s l a b e l e d w i t h ATP and iodoa c e t a t e . J . B i o l . Chem. 2 5 4 , 2221-2228. C h u r c h i l l , L . , and Hokin, L. E . ( 1 9 7 6 ) . The s u s c e p t i b i l i t y o f t h e g l y c o p r o t e i n from t h e p u r i f i e d ( N a + , K + ) - a c t i v a t e d adenos i n e t r i p h o s p h a t a s e t o t r y p t i c and chymotryptic d e g r a d a t i o n w i t h and w i t h o u t Na+ and K+. Biochim. Biophys. A c t a 434, 258-264. C h u r c h i l l , L . , P e t e r s o n , G. L . , and Hokin, L. E. ( 1 9 7 9 ) . The l a r g e s u b u n i t of (sodium + p o t a s s i u m ) - a c t i v a t e d a d e n o s i n e t r i p h o s p h a t a s e from t h e e l e c t r o p l a x of E l e c t r o p h o r u s e l e c t r i c u s i s a g l y c o p r o t e i n . B i o c h e m . B i o p h y s . R e s . Commun. 9 0 , 488-490, C r a i g , W . S . , and Kyte, J. ( 1 9 8 0 ) . S t o i c h i o m e t r y and m o l e c u l a r w e i g h t of t h e minimum asymmetric u n i t of c a n i n e r e n a l sodium and potassium i o n - a c t i v a t e d a d e n o s i n e t r i p h o s p h a t a s e . J. B i o l . Chem. 2 5 5 , 6262-6269. Esmann, M . , C h r i s t i a n s e n , C . , K a r l s s o n , K.-A., Hansson, G. C . , and Skou, J. C . ( 1 9 8 0 ) . Hydrodynamic p r o p e r t i e s o f s o l u b i l i z e d (Na+ + K+)-ATPase from r e c t a l g l a n d s of Squalus acanthias. B i o c h i m . B i o p h y s . A c t a 6 0 3 , 1-12. F r e y t a g , J . W . , and Reynolds, J. A . ( 1 9 8 1 ) . P o l y p e p t i d e molecular w e i g h t s o f t h e (Na+,K+)-ATPase from p o r c i n e kidney medulla. B i o c h e m i s t r y 2 0 , 7211-7214. + + G i o t t a , G. J . ( 1 9 7 6 ) . Quaternary s t r u c t u r e of ( N a + K )-depend e n t adenosine t r i p h o s p h a t a s e . J . B i o l . Chem. 2 5 1 , 1 2 4 7 1252. + + Grisham, C . M. ( 1 9 7 9 ) . The s t r u c t u r e o f t h e ( N a + K )-ATPase. I m p l i c a t i o n s f o r t h e mechanism o f sodium and p o t a s s i u m t r a n s p o r t . A d v . I n o r g . B i o c h e m . , 193-218. Haase, W . , and K o e p s e l l , H . ( 1 9 7 9 ) . S u b s t r u c t u r e o f membraneP f l u e g e r s A r c h . 3 8 1 , 127-135. bound Na+-K+-ATPase. Hardwicke, P. M. D . , and F r e y t a g , J . W. ( 1 9 8 1 ) . A p r o t e o l i p i d a s s o c i a t e d w i t h Na,K-ATPase i s n o t e s s e n t i a l f o r ATPase a c t i v i t y . B i o c h e m . B i o p h y s . R e s . Commun. 1 0 2 , 250-257. H a r r i s , W. E . , Swanson, P. D . , and S t a h l , W. L. ( 1 9 7 3 ) . Ouabain b i n d i n g sites and t h e (Na+,K+)-ATPase o f b r a i n microsomal membranes. B i o c h i m . B i o p h y s . A c t a 2 9 8 , 680-689. Haurowitz, F. ( 1 9 3 8 ) . Das Gleichgewicht zwischen H k o g l o b i n und S a u e r s t o f f . H o p p e - S e y l e r ' s 2. P h y s i o l . Chem. 254, 266-274. Hobbs, A . , and A l b e r s , R . W. ( 1 9 8 0 ) . The s t r u c t u r e of p r o t e i n s i n v o l v e d i n a c t i v e membrane t r a n s p o r t . A n n u . R e v . B i o p h y s . B i o e n g . 9 , 259-291. In J o r g e n s e n , E . C. ( 1 9 8 1 ) . The d e s i g n o f t h y r o i d d r u g s . "Molecular B a s i s of Drug Action" (T. P. S i n g e r and R . N . Ondarza, e d s . ) , pp. 223-233. Elsevier/North-Holland, N e w York

.

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J$rgensen, P. L. (1974). Purification and characterization of (Na+ + K+)-ATPase IV. Estimation of the purity and of the molecular weight and polypeptide content per enzyme unit in preparations from the outer medulla of rabbit kidney. B i o c h i m . B i o p h y s . A c t a 3 5 6 , 53-67. Jgkgensen, P. L. (1982). Topology in the membrane and principal conformations of the a-subunit of Na,K-ATPase. In "Membranes and Transport," (A. Martonosi , ed. ) Vol. 1 , pp. 537-546. Plenum, New York. Khorana, H. G., Gerber, G. E., Herlihy, W. C., Gray, C. P., Anderegg, R. J., Nihei, K., and Biemann, K. (1979). Amino acid sequence of bacteriorhodopsin. Significant clustering of hydrophobic amino acids. P r o c . N a t l . A c a d . S c i . U.S.A. 7 6 , 5046-5050. Kyte, J. (1981). Molecular considerations relevant to the mechanism of active transport. Nature ( L o n d o n ) 2 9 2 , 201-204. Levitzki, A. (1978). "Quantitative Aspects of Allosteric Mechanisms." Springer-Verlag, New York. Marshall, P. J., and Hokin, L. E. (1979). Microheterogeneity of the glycoprotein subunit of the (sodium + potassium)activated adenosine triphosphatase from the electroplax of E l e c t r o p h o r u s electricus. B i o c h e m . B i o p h y s . Res. Commun. 87, 476-482. Maunsbach, A. B., Skriver, E., and &drgensen, P. L. (1979). Ultrastructure of purified Na,K-ATPase membranes. In "Na,K-ATPase. Structure and Kinetics" (J. C. Skou and J. G. Norby, eds.), pp. 1-13. Academic Press, New York. Moczydlowski, E. G., and Fortes, P. A. G. (1981). Characterization of 2',3'-0-(2,4,6-trinitrocyclohexadienylidine)adenosine 5'-triphosphate as a fluorescent probe of the ATP site of sodium and potassium transport adenosine triphosphatase. Determination of nucleotide binding stoichiometry and ioninduced changes in affinity for ATP. J. Biol. Chem. 2 5 6 , 2346-2356. Munson, K. B. (1981). Light-dependent inactivation of (Na+ + K+) ATPase with a new photoaffinity reagent, chromium arylazido8-alanyl ATP. J. B i o l . Chem. 2 5 6 , 3223-3230. Peters, W. H. M., dePont, J. J. H. H. M., Koppers, A,, and Bonting, S. L. (1981a). Studies on (Na+ + K+)-activated ATPase XLVII. Chemical composition, molecular weight and molar ratio of the subunits of the enzyme from rabbit kidney outer medulla. B i o c h i m . B i o p h y s . A c t a 6 4 1 , 55-70. Peters, W. H. M., Swarts, H. G. P., dePont, J. J. H. H. M., Schuurmans Stekhoven, F. M. A. H., and Bonting, S. L. (1981b). (Na+ + K+)ATPase has one functioning site per a subunit. N a t u r e ( L o n d o n ) 2 9 0 , 338-339. Peterson, G., and Hokin, L. E. (1981). Molecular weight and stoichiometry of the sodium- and potassium-activated adenosine triphosphatase subunits. J. B i o l . Chem. 2 5 6 , 3751-3761.

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Robinson, J. D . , and F l a s h n e r , M. S . ( 1 9 7 9 ) . The (Na' + Kf)a c t i v a t e d ATPase. Enzymatic and t r a n s p o r t p r o p e r t i e s . B i o c h i m . B i o p h y s . A c t a 549, 145-176. Schuurmans S t e k h o v e n , F . , and B o n t i n g , S . L. ( 1 9 8 1 ) . T r a n s p o r t adenosine triphosphatases: P r o p e r t i e s and f u n c t i o n s . P h y s i o l . R e v . 61, 1-76. S i n g e r , S . J . , and N i c h o l s o n , G. L. ( 1 9 7 2 ) . The f l u i d m o s a i c model o f t h e s t r u c t u r e o f c e l l membranes. Science 1 7 5 , 720-731. Unwin, P. N . T . , and Henderson, R . ( 1 9 7 5 ) . M o l e c u l a r s t r u c t u r e d e t e r m i n a t i o n by e l e c t r o n m i c r o s c o p y of u n s t a i n e d c r y s t a l l i n e s p e c i m e n s . J. Mol. B i o l . 94, 425-440. W a l l i c k , E . T . , Lane, L. K . , and S c h w a r t z , A. ( 1 9 7 9 ) . Biochemic a l mechanism of t h e sodium pump. Annu. R e v . Physiol. 41, 397-411. Winslow, J. W. ( 1 9 8 1 ) . The r e a c t i o n o f s u l f h y d r y l g r o u p s o f sodium and p o t a s s i u m i o n - a c t i v a t e d a d e n o s i n e t r i p h o s p h a t a s e w i t h N-ethylmaleimide. The r e l a t i o n s h i p between l i g a n d d e p e n d e n t a l t e r a t i o n s o f n u c l e o p h i l i c i t y and e n z y m a t i c conf o r m a t i o n a l s t a t e s . J . B i o l . Chem. 256, 9522-9531.

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CURRENT TOPICS IN MEMBRANES AND TRANSPORT. VOLUME 19

Detergent Solubilization of Na,K-ATPase MIKAEL ESMANN Institure ofBiophysics University of Aarhus Aarhus. Denmark

I.

INTRODUCTION

D e t e r g e n t s h a v e been u s e d e x t e n s i v e l y i n t h e s t u d y of t h e s t r u c t u r e and f u n c t i o n o f t h e N a , K - A T P a s e . For p r e p a r a t i v e p u r p o s e s t h e d e t e r g e n t s have been u s e d t o s e l e c t i v e l y e x t r a c t i m p u r i t i e s from membranes c o n t a i n i n g t h e Na,K-ATPase (Skou, 1 9 6 2 1 , t h i s method termed " p u r i f i c a t i o n by e x t r a c t i o n " (Hokin, 1 9 8 1 ) . By t h i s p r o c e d u r e t h e enzyme i s l e f t i n t h e membrane t h r o u g h t h e whole p r e p a r a t i o n . D e t e r g e n t s have a l s o been used t o e x t r a c t t h e N a , K - A T P a s e s e l e c t i v e l y from t h e memb r a n e , t h u s s o l u b i l i z i n g t h e enzyme i n t h e d e t e r g e n t micelles. The s o l u b i l i z e d enzyme i s t h e n p u r i f i e d by c o n v e n t i o n a l t e c h n i q u e s , and a t some l a t e s t e p i n t h e p r e p a r a t i v e p r o c e d u r e t h e enzyme i s p r e c i p i t a t e d o u t of s o l u t i o n , forming membranes w i t h t h e enzyme embedded i n t h e l i p i d b i l a y e r (Hokin e t a ] . , 1 9 7 3 ) . T h i s p r o c e d u r e h a s been termed " p u r i f i c a t i o n by s o l u b i l i z a t i o n " o r "positive p u r i f i c a t i o n " (Holin, 19811, t h e l a t t e r t e r m being obviously biased. 67

Copyright 0 1983 by Academic Press, Inc. All rights of reproductionin any form reserved ISBN 0-12-153319-0

TABLE I.

Detergents and Sources o f Enzyme Used i n the P r e p a r a t i o n o f Na,K-ATPase by t h e Method o f " P u r i f i c a t i o n by S o l u b i l i z a t i o n " a b S p e c i f i c ATPase a c t i v i t y S o l u b i l i z e d F i n a l enzyme

Source ~~~

~~

~

~~~

Red blood c e l l (SDS) Rabbit kidney (DOC) Dog kidney (DOC) Dog kidney (DOC) Dog kidney (DCC/CHO)f R e c t a l gland9 ( D K ) R e c t a l gland ( T r i t o n ) Bovine b r a i n ( L u b r o l WX) Pig b r a i n (Lubrol WX) R e c t a l gland (Lubrol WX) E e l 1 (Lubrol WX)

a

c

Purity

d Molar activity

Reference

~

0.1 24 800 494 912 2419 882 65 324 640 670

N D ~

mh ND 1552 1552 1524 NDh

ND ND >90 >95 >95 >95 76

617

=50

4650 1460 1310

>90 >95 >95

ND ND 2400 6480 6480 8190 ND 6049 12900 6300 ND

D u n h a m and Hoffman (1971) Towle and Copenhaver (1970) Kyte (1971) Lane e t a l . (1973) Lane e t a l . (1973) Skou and Esmann (1979) Marshall (1976) Uesugi et al. (1970) Nakao e t a l . (1973) Hokin e t a l . (1973) Dixon and Hokin (1978)

T h e s p e c i f i c ATPase a c t i v i t y i s g i v e n f o r the s o l u b i l i z e d e n z y m e and f o r the f i n a l p u r i f i e d membrane-bound e n z y m e . bGiven i n ~ m o l e s / m gper h o u r . C G i v e n as content o f c1 and $ i n p e r c e n t . dGiven i n min-1. e N o t determined. fCHO, C h o l a t e . 9From S q u a l u s a c a n t h i a s . hThe s o l u b i l i z e d e n z y m e w a s not p r e c i p i t a t e d . iFrom E l e c t r o p h o r u s e l e c t r i c u s .

69

DETERGENT SOLUBILIZATIONOF Na,K-ATPase

0 II

0-;

- 0'Na' S DS

0 H I

"CH,

DIGITONIN

DOC

HO

dFooQN

wo OH

OCTYL

GLUCOSIDE

0 - [CH, - CH, - 01,-

H

LUBROL

WX

F i g . 1 . S t r u c t u r e s o f d i f f e r e n t a n i o n i c (sodium d o d e c y l s u l f a t e , S D S , and d e o x y c h o l a t e , DOC) and nonionic d e t e r g e n t s u s e d i n p u r i f i c a t i o n of Na,K-ATPase ( L u b r o l W X , d i g i t o n i n , o c t y l g l u c o s i d e and o c t a e t h y l e n e g l y c o l d o d e c y l m o n o e t h e r , C 1 2 E g ) . R i n d i c a t e s a sugar moiety.

MIKAEL ESMANN

70

S o l u b i l i z a t i o n o f Na,K-ATPase i n d e t e r g e n t s h a s a l s o been used t o s t u d y t h e m o l e c u l a r w e i g h t o f t h e act i v e N a , K - A T P a s e and of t h e s e p a r a t e d , i n a c t i v e a - and 8 - s u b u n i t s , a s w e l l a s t h o s e p r o p e r t i e s of t h e enzyme which are c o n v e n i e n t l y s t u d i e d i n s o l u t i o n . The r o l e o f t h e l i p i d s h a s a l s o been s t u d i e d u s i n g d e t e r g e n t t o d e l i p i d a t e t h e enzyme i n s u c h a way t h a t t h e enzyme c o u l d be r e a c t i v a t e d by a d d i t i o n o f l i p i d s . T h i s a r t i c l e w i l l summarize some--but by no means all--of t h e procedures using detergent f o r solubilizat i o n , a s w e l l a s some o f o u r own r e s u l t s w i t h o c t a e t h y l e n e g l y c o l d o d e c y l monoether ( C 1 2 E g ) (see T a b l e I and F i g . 1 ) .

11.

A .

DETERGENTS SODIUM

DODECYL SULFATE

Although sodium d o d e c y l s u l f a t e (SDS) h a s been used e x t e n s i v e l y f o r e x t r a c t i o n o f i m p u r i t i e s from memb r a n e s c o n t a i n i n g t h e N a , K - A T P a s e ( " p u r i f i c a t i o n by ext r a c t i o n , " J d r g e n s e n and Skou, 1 9 7 1 ) , t h e r e i s o n l y one r e p o r t of a S D S - s o l u b i l i z e d e n z y m a t i c a l l y a c t i v e N a , K A T P a s e (Dunham and Hoffman, 1 9 7 0 ) . The enzyme, a l t h o u g h v e r y impure, had a s e d i m e n t a t i o n c o e f f i c i e n t of 1 4 S , e q u i v a l e n t t o a m o l e c u l a r w e i g h t around 4 0 0 , 0 0 0 , i n c l u d i n g d e t e r g e n t . T h i s i s i n t h e same r a n g e a s h a s been observed w i t h p u r i f i e d s o l u b i l i z e d N a , K - A T P a s e (Esmann e t a l . , 1979; H a s t i n g s and Reynolds, 1 9 7 9 ) . I n most o t h e r p r e p a r a t i o n s SDS i n a c t i v a t e s t h e enzyme as it i s s o l u b i l i z e d . The i n a c t i v a t i o n by SDS i s accompanied by a d e l i p i d a t i o n and a d i s s o c i a t i o n o f t h e a- and @ - s u b u n i t s (Kyte, 1 9 7 1 ) . The m o l e c u l a r w e i g h t s of t h e SDS-solubilized s u b u n i t s have been d e t e r m i n e d by polyacrylamide g e l e l e c t r o p h o r e s i s i n t h e presence of SDS, by g e l f i l t r a t i o n , and by s e d i m e n t a t i o n e q u i l i b r i u m c e n t r i f u g a t i o n , see T a b l e 11. The m o l e c u l a r w e i g h t of t h e a - s u b u n i t i s around 1 0 0 , 0 0 0 and t h a t of 8 i s around 35,000, taking t h e sedimentation equilibrium values as t h e most r e l i a b l e . The p r e c i s e m o l e c u l a r w e i g h t s w i l l be known when t h e amino a c i d sequence h a s been d e t e r mined.

TABLE 11.

Molecular w e i g h t s o f Na,K-ATPase S u b u n i t s Molecular w e i g h t Source

Method

SDS-solubilized e n z y m a t i c a l l y i n a c t i v e s u b u n i t s Shark r e c t a l g l a n d PAGE^ b Shark r e c t a l g l a n d Sed. e q u i l . Sed. e q u i l . Shark r e c t a l g l a n d PAGE Dog kidney Sed. e q u i l . Porcine kidney -1 f i l t r a t i o n Dog kidney PAGE Ee 1 PAGE B r i n e shrimps Source

Method

Higher a g g r e g a t e s of a and f3 Dog kidney ( T r i t o n ) Sed. Dog kidney ( d i g i t o n i n ) Sed. Shark r e c t a l g l a n d (Lubrol) Sed. Shark r e c t a l g l a n d (C E ) Sed. l2 Sed. P i g kidney (C12E8)

a

veloc. veloc. equil. equil. veloc.

C

a

B

R e €erence

104,200

35,800 36,600 40,000 40,200 32,300

P e t e r s o n and Hokin (1981) H a s t i n g s and Reynolds (1979) Esmann et a l . (1980) P e t e r s o n and Hokin (1981) F r e y t a g and Reynolds (1981) C r a i g and Kyte (1980) P e t e r s o n and Hokin (1981) P e t e r s o n and Hokin (1981)

106 ,400

106,400 97,000 94,000 121,000 97,700 97,800

42,000 40,100

Molecular weight

Activity

(aB)

No Partial Full Full Full

140,000 165,000 380,000 265,000 170,000

(aB)

(a,B,)

(a2821 (a6)

PAGE: polyacrylamide gel electrophoresis. bSed. equil. : sedimentation equilibrium centrifugation. CSed. veloc. : sedimentation velocity centrifugation.

Reference

C l a r k e (1975) Winter and Moss (1979) H a s t i n g s and Reynolds (1979) Esmann et a l . (1979, 1980) B r o t h e r u s et a l . (1981)

72

B.

MIKAEL ESMANN

DEOXYCHOLATE

Deoxycholate ( D O C ) , an a n i o n i c d e t e r g e n t l i k e SDS b u t less d e l e t e r i o u s , h a s a l s o been used e x t e n s i v e l y i n b o t h p u r i f i c a t i o n by e x t r a c t i o n (Skou, 1962; J d r g e n s e n and Skou, 1971; Kyte, 19711, and i n p u r i f i c a t i o n by s o l u b i l i z a t i o n (Towle and Copenhaver, 1970; Kyte, 1 9 7 1 ; Lane et al. , 1 9 7 3 ) . Because DOC h a s a m i l d e r e f f e c t t h a n SDS, DOC h a s a l s o been u s e d t o o b t a i n s o l u b l e a c t i v e Na,K-ATPase, w i t h a c t i v i t i e s r a n g i n g from 800 pmoles/mg p e r hour (Kyte, 1 9 7 1 ) t o 2 , 4 0 0 pmoles/mg p e r h o u r (Skou and E s mann, 1 9 7 9 ) . Extreme care h a s t o be t a k e n i n t h e s o l u b i l i z a t i o n s t e p a s t o t h e c o n d i t i o n s of i o n i c s t r e n g t h , t e m p e r a t u r e , and DOC c o n c e n t r a t i o n (see Skou and Esmann, 1979, f o r d e t a i l s ) . It i s a l s o important t o note t h a t the s p e c i f i c activity--but not t h e phosphorylation c a p a c i t y - - d e c r e a s e d t o a b o u t 6 0 % when t h e DOC-solubilized enzyme w a s p r e c i p i t a t e d w i t h p o l y e t h y l e n e g l y c o l (PEG) g i v i n g a membrane-bound enzyme w i t h low molar a c t i v i t y see Skou and Esmann, 1 9 7 9 ) . The mo( a b o u t 7,000 min”, l e c u l a r w e i g h t o f t h e DOC-solubilized enzyme h a s n o t been d e t e r m i n e d . Na,K-ATPase s o l u b i l i z e d i n DOC i s d e l i p i d a t e d when it i s p a s s e d o v e r a g e l f i l t r a t i o n column i n t h e p r e s e n c e o f a s u f f i c i e n t l y h i g h DOC c o n c e n t r a t i o n (Kyte, 1971; O t t o l e n g h i , 1 9 7 5 ) . The i n a c t i v e enzyme e l u t e s a t a p o s i t i o n c o r r e s p o n d i n g t o a m o l e c u l a r w e i g h t of a b o u t 500,000, which i n d i c a t e s t h a t DOC s o l u b i l i z e s t h e enzyme as a g g r e g a t e s of ( a $ ) . No d i s s o c i a t i o n between t h e two s u b u n i t s i s o b s e r v e d . The enzyme c a n be r e a c t i v a t e d a f t e r p r e c i p i t a t i o n w i t h PEG, b u t n o t t o f u l l a c t i v i t y (Ottolenghi, 1975). I n c o n c l u s i o n , i t seems as i f something i s changed i r r e v e r s i b l y i n t h e s t r u c t u r e of t h e enzyme when t h e s o l u b i l i z e d enzyme i s p r e c i p i t a t e d , l e a d i n g t o enzyme s p e c i e s w i t h lower m o l e c u l a r a c t i v i t i e s (see t h e c i t e d papers f o r f u r t h e r discussion). C.

TRITON

The n o n i o n i c p o l y o x y e t h y l e n e T r i t o n X-100 s o l u b i l i z e s t h e N a , K - A T P a s e , b u t i n a c t i v a t e s t h e enzyme i r r e v e r s i b l y , p r o b a b l y by t h e d i s r u p t i o n of a q u a r t e r n a r y a282 s t r u c t u r e i n t o a$-fragments o f m o l e c u l a r w e i g h t 1 4 0 , 0 0 0 ( C l a r k e , 1 9 7 5 ) . I t i s a l s o p o s s i b l e , however, t h a t t h e i n a c t i v a t i o n i s due t o a d e l i p i d a t i o n , b u t t h i s h a s n o t been i n v e s t i g a t e d . M a r s h a l l (1976) r e p o r t s

DETERGENT SOLUBlLlZATlON OF Na,K-ATPase

73

a s p e c i f i c a c t i v i t y o f a b o u t 8 0 0 pmoles/mg p e r h o u r i n t h e presence of T r i t o n , b u t t h e s i z e of t h e s e p a r t i c l e s was n o t d e t e r m i n e d . However, a f t e r a f f i n i t y chromatography i n c o n c a n a v a l i n A-Sepharose t h e enzyme w a s i n a c t i v e , p r o b a b l y due t o d e l i p i d a t i o n . D.

DIGITONIN

W i n t e r ( 1 9 7 2 ) h a s shown t h a t d i g i t o n i n i n a c t i v a t e s t h e Na,K-ATPase, b u t l e a v e s t h e p a r t i a l r e a c t i o n s Na+d e p e n d e n t p h o s p h o r y l a t i o n and X+-dependent p h o s p h a t a s e i n t a c t . The l o s s o f N a , K - A T P a s e a c t i v i t y i s t a k e n t o b e a r e s u l t o f t h e d i s r u p t i o n of o l i g o m e r s of an 8-0. p a r t i c l e i n t o s i n g l e a 8 - p a r t i c l e s (Winter and Moss, 1 9 7 9 ) , a s d e t e r m i n e d by t h e i r b e h a v i o r under s e d i m e n t a tion velocity centrifugation. D i g i t o n i n h a s n o t been used f o r p r e p a r a t i v e procedures. E.

OCTYL GLUCOSIDE

T h i s n o v e l d e t e r g e n t h a s been u s e d t o p r e p a r e N a , K A T P a s e from E h r l i c h a s c i t e s tumor c e l l s ( S p e c t o r e t a l . , 1 9 8 0 ) by t h i o l exchange chromatography, b u t t h e enzyme r e s u l t i n g from t h i s h a s n o t been c h a r a c t e r i z e d . An o c t y l g l u c o s i d e ( 0 G ) - s o l u b i l i z e d Na,K-ATPase w i t h f u l l a c t i v i t y would b e v e r y s u i t a b l e f o r r e c o n s t i t u t i o n p u r p o s e s b e c a u s e o f t h e h i g h c r i t i c a l micelle c o n c e n t r a t i o n of OG, b u t p r e l i m i n a r y e x p e r i m e n t s (M. Esmann, unpublished observations) i n d i c a t e t h a t t h e shark Na,KATPase i s i n a c t i v a t e d t o a l a r g e e x t e n t by s o l u b i l i z i n g c o n c e n t r a t i o n s o f OG ( a b o u t 20 m~ a t 0 . 3 mg p r o t e i n p e r milliliter)

.

F.

LUBROL

The n o n i o n i c d e t e r g e n t L u b r o l h a s been u s e d e x t e n s i v e l y by Hokin and h i s co-workers a s p a r t of p u r i f i c a t i o n schemes u s i n g " p u r i f i c a t i o n by s o l u b i l i z a t i o n " of N a , K - A T P a s e from b o v i n e b r a i n (Uesugi e t a l . , 19701, r e c t a l g l a n d s o f S q u a l u s a c a n t h i a s (Hokin e t a l . , 1 9 7 3 ; Dixon and Hokin, 1 9 7 8 ) , t h e e l e c t r i c o r g a n o f E l e c t r o p h o r e t i c u s (Dixon and Hokin, 1 9 7 8 ) , and from t h e b r i n e s h r i m p ( P e t e r s o n and Hokin, 1 9 8 0 ) . The mass of t h e L u b r o l - s o l u b i l i z e d p a r t i c l e s from t h e b r a i n enzyme w a s found t o b e a b o u t 6 0 0 , 0 0 0 d a l t o n s as j u d g e d from g e l f i l t r a t i o n i n t h e p r e s e n c e o f L u b r o l . Nakao and CO-

MIKAEL ESMANN

74

workers (1973) have a l s o used Lubrol t o s o l u b i l i z e t h e b r a i n enzyme, and found t h r e e t y p e s of p a r t i c l e s a l l i n t h e same r a n g e o f s i z e as t h a t r e p o r t e d by Uesugi e t al. ( 1 9 7 0 ) . One o f t h e p a r t i c l e s c o n s i s t e d o f as u b u n i t s a l o n e . The enzymic a c t i v i t y of t h i s p a r t i c l e w a s v e r y l a b i l e (Nakao e t a l . , 1 9 7 3 ) . The m o l e c u l a r w e i g h t of a Lubrol s o l u b i l i z e d s h a r k enzyme h a s been d e t e r m i n e d by t h e r i g o r o u s method of s e d i m e n t a t i o n e q u i l i b r i u m c e n t r i f u g a t i o n by H a s t i n g s and Reynolds ( 1 9 7 9 ) . They measured a m o l e c u l a r w e i g h t of 380,000 f o r t h e p r o t e i n p a r t o f t h e p a r t i c l e , which, i f bound d e t e r g e n t , s u g a r , and l i p i d are i n c l u d e d , i s i n agreement w i t h t h e g e l f i l t r a t i o n estimate o f Uesugi e t al. ( 1 9 7 0 ) . Care w a s t a k e n t o measure t h e e n z y m a t i c a c t i v i t y under c o n d i t i o n s where t h e enzyme w a s i n s o l u t i o n , and t h e s p e c i f i c a c t i v i t y o f t h e s o l u b i l i z e d s h a r k enzyme w a s a b o u t 1,500 pmoles/mg p e r h o u r , which i s comparable t o t h e h i g h e s t a c t i v i t i e s o b t a i n e d i n t h e membrane ( J d r g e n s e n , 1 9 7 4 ) . G.

O C T A E T H Y L E N E G L Y C O L D O D E C Y L MONOETHER

C12Eg i s chemically pure nonionic d e t e r g e n t with t h e same o v e r a l l s t r u c t u r e as L u b r o l (see T a n f o r d e t a l . , 1 9 7 7 , f o r d e t a i l s on t h e d e t e r g e n t ) C12E8 h a s been used t o s o l u b i l i z e a p u r i f i e d membrane-bound N a , K A T P a s e from r e c t a l g l a n d s o f s a c a n t h i a s (Esmann e t al., 1979, 1980) and a f u l l y a c t i v e , ( 2 , 1 0 0 pmoles/mg p e r h o u r ) homogenous s o l u t i o n o f p a r t i c l e s w a s o b t a i n e d a f ter g e l f i l t r a t i o n i n t h e presence of C12Eg. The amount o f C 1 2 E g needed t o s o l u b i l i z e t h e enzyme depends on t h e i o n i c s t r e n g t h . With no c a t i o n s added, a b o u t 2 mg C12E8/ml i s r e q u i r e d f o r a t o t a l s o l u b i l i z a t i o n of a 1 mg/ml Na,K-ATPase s u s p e n s i o n , whereas o n l y 50-60% of t h e enzyme would be i n t h e s u p e r n a t a n t a f t e r c e n t r i f u g a t i o n a t 1 0 0 , 0 0 0 g f o r 6 0 min w i t h t h i s amount o f d e t e r g e n t i f 150 m~ KC1 o r N a C l w a s i n c l u d e d i n t h e s o l u b i l i z a t i o n medium. T h i s p r o t e c t i v e e f f e c t o f cat i o n s towards s o l u b i l i z a t i o n must be an e f f e c t on t h e enzyme o r on t h e membrane i t s e l f as t h e s o l u b i l i z i n g p r o p e r t i e s o f n o n i o n i c d e t e r g e n t s are i n d e p e n d e n t o f t h e i o n i c s t r e n g t h . T i t r a t i o n c u r v e s of o p t i c a l d e n s i t y a s a f u n c t i o n o f added d e t e r g e n t a r e g i v e n i n F i g . 2 . A s can be s e e n from t h e f i g u r e t h e a d d i t i o n of 25% g l y c e r o l does n o t change t h e amount o f d e t e r g e n t needed, whereas 150 mM K C 1 ( o r N a C 1 , n o t shown) increases t h e amount o f d e t e r g e n t needed by a f a c t o r of a b o u t 1 . 5 . I n p r a c t i c e t h i s means t h a t enzyme s o l u b i l i z e d a t l o w i o n i c s t r e n g t h may p r e c i p i t a t e o u t o f s o l u t i o n when t h e

.

DETERGENT SOLUBlLlZATlON OF Na,K-ATPase

DETERGENT

75

(

UL )

F i g . 2 . E f f e c t of i n c r e a s i n g a m o u n t s o f C12Eg on o p t i c a l The o p t i c a l d e n s i t y d e n s i t y (OD) o f a s u s p e n s i o n o f Na,K-ATPase. was m e a s u r e d i n a V a r i a n - C a r y 219 a t 550 nm w i t h a I - c m l i g h t p a t h , a n d C12Eg (100 m g / m l ) was a d d e d w i t h a Hamilton s y r i n g e . T h e v a l u e s a r e g i v e n a s che d i f f e r e n c e b e t w e e n the OD a f t e r a n a d d i t i o n of C12Eg and the OD a f t e r a d d i t i o n o f 25 v l C12Eg, a n d are e x p r e s s e d i n p e r c e n t a g e o f the maximal d i f f e r e n c e ( 1 .e. , no C12Eg) a d d e d ) . T h e v o l u m e was 1 . 9 m l , and the e n z y m e ( 0 . 2 5 m q / m l ) was s u s p e n d e d i n 30 mM h i s t i d i n e , pH 7 . 4 , c o n t a i n i n g no a d d i t i o n s ( 0 ) or 1 5 0 mM KCI ( 0 ,A ) and 24% ( v / v ) g l y c e r o l ( 0 , * ) . The t e m p e r a t u r e was 23OC.

i o n i c strength is increased. Centrifugation experiments, where t h e p r o t e i n c o n t e n t i n t h e s u p e r n a t a n t i s measured, c o n f i r m s t h e f i n d i n g s i n F i g . 2 ( n o t s h o w n ) , and t h e s p e c i f i c a c t i v i t i e s of enzyme s o l u b i l i z e d w i t h h i g h o r low i o n i c s t r e n g t h are i d e n t i c a l . The C 1 2 E 8 - s o l u b i l i z e d enzyme i s s t a b l e o n s t o r a g e a t low t e m p e r a t u r e s ( 0 o r 2 3 ' C ) , w h e r e a s it i s r a p i d l y and i r r e v e r s i b l y i n a c t i v a t e d a t 37OC ( t 1 / 2 , 2-4 h r ) (see Esmann e t a l . , 1 9 8 1 ) . F i g u r e 3 shows t h e e f f e c t o f t h e i o n i c s t r e n g t h , g l y c e r o l , and d e t e r g e n t c o n c e n t r a t i o n on t h e s t a b i l i t y o f t h e e n z y m a t i c a c t i v i t y on s t o r a g e . N e i t h e r h i g h i o n i c s t r e n g t h (150 mM KC1) n o r g l y c e r o l ( 2 0 % ) i s n e c e s s a r y a t low d e t e r g e n t c o n c e n t r a t i o n s ( 0 . 5 mg/ml) a t O°C,

76

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100

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.

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72 '

F i g . 3. S t a b i l i t y of C 1 2 E g - s o l u b i l i z e d Na,K-ATPase a t 0°C ( A ) and 23 C ( B ) . Na,K-ATPase (0.24 mg p r o t e i n / m l ) i n 30 mM h i s t i d i n e , pH 7.0, w a s i n c u b a t e d a t the g i v e n t e m p e r a t u r e i n the W ) , or 2 . 5 mg C 1 2 E g / m l ) p r e s e n c e o f 0.5 mg C12Eg/ml ( 0 ,n,., ( A , ,A , 8 ) w i t h 150 mM KCI ( 0 , L ,W , 63 ) a n d 20% ( v / v ) g l y cerol ( f i l l e d s y m b o l s and a ) . Tlhe e n z y m a t i c a c t i v i t y was m e a s u r e d a t the i n d i c a t e d times a s follows: 50 l.11 of the incubation medium was w i t h d r a w n and a d d e d t o 950 l.11 o f t e s t solution (0.15 mg C 1 2 E g / m l , see l e g e n d t o F i g . 4) a t 23OC and i n c u b a t e d f o r 3 min. T h e a c t i v i t y i s g i v e n i n p e r c e n t a g e o f the a c t i v i t y before t h e incubation was s t a r t e d .

*

whereas a h i g h d e t e r g e n t c o n c e n t r a t i o n ( 2 . 5 mg/ml) l e a d s t o a slow i n a c t i v a t i o n i f g l y c e r o l and K C 1 a r e o m i t t e d . A t 23OC t h i s e f f e c t i s much more marked, and it i s s e e n t h a t o n l y a t l o w d e t e r g e n t and h i g h i o n i c s t r e n g t h i s more t h a n 80% of t h e a c t i v i t y r e t a i n e d a f t e r 70 h r . A t h i g h d e t e r g e n t c o n c e n t r a t i o n s 150 mM K C 1 can p r o t e c t a l i t t l e a g a i n s t t h e l o s s of a c t i v i t y . W e have n o t found c o n d i t i o n s where t h e a c t i v i t y c a n be r e t a i n e d i n d e t e r g e n t s o l u t i o n a t 37OC f o r more t h a n 1-2 h r . In summary, h i g h i o n i c s t r e n g t h p r o t e c t s t h e enzyme, and g l y c e r o l does n o t seem t o be i m p o r t a n t f o r p r o t e c t i o n of enzymatic a c t i v i t y . The C12E8-Solubilized Na,K-ATPase h a s a r e q u i r e m e n t f o r d e t e r g e n t under t u r n o v e r c o n d i t i o n s ( F i g . 4 1 , b u t t o o h i g h c o n c e n t r a t i o n s o f d e t e r g e n t make t h e enzyme l a b i l e i n t h e t e s t s o l u t i o n a t 23OC. A s c a n be s e e n

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77

B

A

2

MINUTES

MINUTES

C

4

D

43 i 100

Z

0 rn

l-

0 3

n 0

cc a I

a Z a

MINUTES Fig. 4. p-nitrophenol

MINUTES

P r o d u c t i o n o f i n o r g a n i c p h o s p h a t e ( A and B ) a n d (C and 0) a s a f u n c t i o n o f t i m e a t d i f f e r e n t C12E8

concentrations.

T h e s u p e r n a t a n t e n z y m e u s e d was p r e p a r e d a s p r e v i o u s l y d e s c r i b e d (Esmann e t a l . , 1 9 7 9 ) a n d the p r o t e i n c o n c e n t r a t i o n i n t h e s o l u t i o n was 14 u g / m l . T h e C12E8 c o n c e n t r a t i o n is g i v e n i n p g / m l a n d h a s been c o r r e c t e d f o r the amount o f C12E8 b o u n d t o the p r o t e i n (see Esmann e t a l . , 1 9 8 0 ) . T h e incubation took p l a c e a t 23OC, pH 7 . 1 , i n a h i s t i d i n e b u f f e r ( 3 0 mM) c o n t a i n i n g 1 3 0 mM N a C l , 20 mM KCI, 4 mM MgC12, 3 mM ATP ( T r i s s a l t ) , C12E8 a s i n d i c a t e d ( i n p g / m l ) , a n d 22% ( w / v ) g l y c e r o l i n B and D , no g l y cerol i n A and C . T h e o r d i n a t e s i n A and B a s w e l l a s i n C and D are identical.

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from F i g . 4 , t h e i n i t i a l s l o p e o f t h e Pi-produced v e r s u s t i m e c u r v e i s a b o u t t h e same a t a l l C12E8 c o n c e n t r a t i o n s b u t a t c o n c e n t r a t i o n s e x c e e d i n g 0.5 mg C12E8/rnlI t h e A T P a s e a c t i v i t y i s v e r y low a f t e r 4 min i n c u b a t i o n . The p - n i t r o p h e n y l p h o s p h a t a s e (pNPPase) i s more r e s i s t a n t t o t h e d e t e r g e n t t h a n i s t h e A T P a s e , i . e . , more d e t e r g e n t i needed t o i n a c t i v a t e it ( F i g . 4 C ) . The e f f e c t o f g l y cerol i n t h e t e s t s o l u t i o n i s a l s o shown ( F i g . 4B and D) G l y c e r o l a c t i v a t e s t h e pNPPase and i n h i b i t s t h e A T P a s e , a s w i t h t h e membrane-bound enzyme ( A l b e r s and Koval, 1972), but glycerol also p r o t e c t s against t h e inactivat i o n of t h e pNPPase. The l a b i l i t y of t h e enzyme under t u r n o v e r c o n d i t i o n s i s more pronounced a t 37OC, b u t t h e l a b i l i t y c a n be somewhat reduced by i n c l u s i o n o f albumin i n t h e t e s t s o l u t i o n as a kind of d e t e r g e n t b u f f e r (Esmann et a l . , 1 9 7 9 ) . The m o l e c u l a r w e i g h t w a s d e t e r m i n e d by s e d i m e n t a t i o e q u i l i b r i u m c e n t r i f u g a t i o n a f t e r c o r r e c t i o n f o r bound d e t e r g e n t , l i p i d , and s u g a r t o b e 265,000 f o r t h e p r o t e i n a l o n e , c o r r e s p o n d i n g t o a a 2 8 2 - p a r t i c l e (Esmann et a l . , 1 9 8 0 ) . I t w a s a s c e r t a i n e d t h a t a l l t h e m a t e r i a l used wa r e c o v e r e d i n t h e I n c v e r s u s r 2 p l o t , and t h a t t h e e q u i l i b r i u m p r o f i l e s were unchanged w i t h t i m e , i . e . , no time-dependent a g g r e g a t i o n o c c u r r e d w i t h i n 5 days a t 15OC.

111.

DISCUSSION

Although d e t e r g e n t s have been used e x t e n s i v e l y i n t h e p u r i f i c a t i o n o f t h e N a , K - A T P a s e , t h e i r e f f e c t s on t h p r o p e r t i e s o f t h e enzyme s t i l l remain u n c l e a r and deb a t e d . The a c t i v a t i n g e f f e c t o f low c o n c e n t r a t i o n s of d e t e r g e n t on microsomal p r e p a r a t i o n s h a s been a t t r i b u t e d t o a n opening o f v e s i c l e s (unmasking of l a t e n t e n z y m a t i c a c t i v i t y , see J 6 r g e n s e n and Skou, 1971) and t o e f f e c t s o t h e p r o p e r t i e s of t h e enzyme i t s e l f (Hokin e t al., 1973) T h i s a c t i v a t i n g e f f e c t i s s e e n w i t h b o t h a n i o n i c and non i o n i c d e t e r g e n t s , and t h e same d e g r e e o f a c t i v a t i o n i s obtained. This r u l e s o u t t h e p o s s i b i l i t y t h a t anionic d e t e r g e n t s g i v e a s p e c i a l a c t i v a t i o n o f t h e Na,K-ATPase (Skou and Esmann, 1 9 7 9 ) . The molar a c t i v i t i e s o f t h e d i f f e r e n t enzyme p r e p a r a t i o n s r a n e from 4,000 min-1 ( K y t e , 1 9 7 1 ) t o 1 1 , 0 0 0 13,000 min-? (Skou and Esmann, 1979; Yoda and Yoda, 1 9 8 1 ) . The d i f f e r e n c e s must somehow be r e l a t e d t o t h e p r e p a r a t i v e p r o c e d u r e , as enzyme from t h e same s o u r c e g i v e s w i d e l y d i f f e r e n t a c t i v i t i e s (Kyte, 1971; J a r g e n -

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s e n , 1 9 7 4 ) . A c l a r i f i c a t i o n o f t h e e f f e c t of d e t e r g e n t on t h e enzyme i s t h u s v e r y i m p o r t a n t . S o l u b i l i z a t i o n i n n o n i o n i c d e t e r g e n t s d o e s n o t seem t o harm t h e enzyme (Hokin e t a l . , 1973; H a s t i n g s and R e y n o l d s , 1979; Esmann e t a l . , 1979) , and a c t i v e s o l u b l e enzymes c a n b e o b t a i n e d . However, p r e c i p i t a t i o n of t h e s o l u b i l i z e d ATPase c a n l e a d t o a d e c r e a s e i n t h e molar a c t i v i t y (Skou and Esmann, 1979) which m i g h t e x p l a i n why t h e enzymes p r e p a r e d by " p u r i f i c a t i o n by s o l u b i l i z a t i o n " g e n e r a l l y have lower m o l a r a c t i v i t i e s . The m o l e c u l a r w e i g h t of t h e A T P a s e i s d e b a t e d a t t h e moment ( H a s t i n g s and Reynolds, 1979; Esmann e t a l . , 1981; B r o t h e r u s e t a l . , 1981) and no c o n c l u s i v e answer c a n b e g i v e n y e t . A f u r t h e r problem i n t h i s matter i s how t o r e l a t e t h e m o l e c u l a r w e i g h t of t h e enzyme i n t h e d e t e r g e n t s o l u t i o n under e q u i l i b r i u m c o n d i t i o n s t o t h e m o l e c u l a r w e i g h t ( a ) u n d e r t u r n o v e r c o n d i t i o n s and ( b ) i n t h e membrane. N e w t e c h n i q u e s w i l l h a v e t o b e emp l o y e d t o answer t h e s e i m p o r t a n t q u e s t i o n s .

ACKNOWLEDGMENTS

I wish t o thank t h e Danish Medical Research Council and Ingeborg and Leo Dannins Foundation f o r S c i e n t i f i c Research f o r f i n a n c i a l support.

REFERENCES

A l b e r s , R . W . , and Koval, G . J. ( 1 9 7 2 ) . Sodium-potassium adenos i n e t r i p h o s p h a t a s e . V I I . J. B i o l . C h e m . 2 4 7 , 3088-3092. B r o t h e r u s , J. R . , M$ller, J. V . , and Jpkgensen, P. L. ( 1 9 8 1 ) . S o l u b l e and a c t i v e r e n a l Na,K-ATPase w i t h maximum p r o t e i n m o l e c u l a r mass 170,000 5 9,000 d a l t o n s . B i o c h e m . B i o p h y s . R e s . Commun. 1 0 0 , 146-154. C l a r k e , S . ( 1 9 7 5 ) . The s i z e and d e t e r g e n t b i n d i n g o f membrane p r o t e i n s . J . B i o l . Chem. 2 5 0 , 5459-5469. Dixon, J. F . , and Hokin, L. E. ( 1 9 7 8 ) . A s i m p l e p r o c e d u r e f o r t h e p r e p a r a t i o n of h i g h l y p u r i f i e d (sodium + p o t a s s i u m ) adenosine-triphosphatase from t h e r e c t a l s a l t g l a n d o f S q u a l u s a c a n t h i a s and t h e e l e c t r i c o r g a n o f E l e c t r o p h o r u s electricus. A n a l . B i o c h e m . 8 6 , 378-385. Dunham, P. B. , and Hoffman, 'J. F. ( 1 9 7 0 ) . P a r t i a l p u r i f i c a t i o n of t h e ouabain-binding component and o f N a , K - A T P a s e from human r e d c e l l membranes. P r o c . N a t l . A c a d . S c i . USA 6 6 , 936-943.

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Esmann, M . , Skou, J . C . , and C h r i s t i a n s e n , C. ( 1 9 7 9 ) . S o l u b i l i z a t i o n and m o l e c u l a r w e i g h t d e t e r m i n a t i o n o f t h e (Na+ -+ K + ) ATPase from r e c t a l g l a n d s of S q u a l u s a c a n t h i a s . B i o c h i m . B i o p h y s . A c t a 5 6 7 , 410-420. Hansson, G. C . , and Esmann, M., C h r i s t i a n s e n , C . , K a r l s s o n , K.-A., Skou, J . C . ( 1 9 8 0 ) . Hydrodynamic p r o p e r t i e s o f s o l u b i l i z e d (Na+ + K+)-ATPase from Rectal g l a n d s o f S q u a l u s a c a n t h i a s . B i o c h i m . B i o p h y s . A c t a 6 0 3 , 1-12. F r e y t a g , J. W . , and Reynolds, J. A. ( 1 9 8 1 ) . P o l y p e p t i d e m o l e c u l a r w e i g h t s o f t h e Na+K+ATPase from p o r c i n e kidney medulla. Biochemistry ( i n press) H a s t i n g s , D. F., and Reynolds, J . A. ( 1 9 7 9 ) . Molecular w e i g h t o f (Na+ + K+)ATPase from s h a r k r e c t a l g l a n d . B i o c h e m i s t r y 1 8 , 817, Hokin, L. E. ( 1 9 8 1 ) . R e c o n s t i t u t i o n o f c a r r i e r s i n a r t i f i c i a l J . Membr. B i o l . 6 0 , 77-93. membranes. Hokin, L. E . , Dahl, J. L . , Deupree, J. D . , Dixon, J. F . , Hackney, J. F . , and Perdue, J. F. (1973). S t u d i e s on t h e c h a r a c t e r i z a t i o n o f t h e sodium-potassium t r a n s p o r t adenosine t r i p h o s p h a t a s e . X . P u r i f i c a t i o n of t h e enzyme from t h e r e c t a l g l a n d o f Squalus a c a n t h i a s . J . B i o l Chem. 2 4 8 , 2593-2605. J$rgensen, P. L. (1974) P u r i f i c a t i o n and c h a r a c t e r i z a t i o n o f (Na+ + K+) -ATPase. 111. B i o c h i m . B i o p h y s . A c t a 356 , 36-52. Jgkgensen, P. L . , and Skou, J. C . ( 1 9 7 1 ) . P u r i f i c a t i o n and c h a r a c t e r i z a t i o n of (Na+ + K+)-ATPase. I . The i n f l u e n c e o f d e t e r g e n t s on t h e a c t i v i t y o f ( N a + + K+)-ATPase i n p r e p a r a Biochim. t i o n s from t h e o u t e r medulla of rabbit kidney. B i o p h y s . A c t a 2 3 3 , 366-380. Kyte, J. (1971). P u r i f i c a t i o n o f t h e sodium and potassiumdependent adenosine t r i p h o s p h a t a s e from c a n i n e r e n a l medulla. J . B i o l . Chem. 2 4 6 , 4157-4165. Lane, L. K . , Copenhaver, J. H . , J r . , Lindenmayer, G. E . , and Schwartz, A. ( 1 9 7 3 ) . P u r i f i c a t i o n and C h a r a c t e r i z a t i o n o f and [ 3H]ouabain b i n d i n g of t h e t r a n s p o r t adenosine t r i p h o s p h a t a s e from o u t e r medulla o f c a n i n e kidney. J. B i o l . Chem. 2 4 8 , 7197-7200. M a r s h a l l , M. 0. ( 1 9 7 6 ) . S t u d i e s on t h e g l y c o p r o t e i n component o f (Na+ + K+)-ATPase from dog f i s h s a l t gland. B i o c h i m . B i o p h y s . A c t a 4 5 5 , 837-848. Nakao, T . , Nakao, M . , Nagai, F . , Kawai, K . , F u j i h a r a , Y . , Hara, Y . , and F u j i t a , M. (1973). P u r i f i c a t i o n and some p r o p e r t i e s o f Na,K-transport ATPase. J . B i o c h e m . ( T o k y o ) 73, 781-791. The r e v e r s i b l e d e l i p i d a t i o n o f a s o l u b i l O t t o l e n g h i , P. (1975) i z e d sodium-plus-potassium ion-dependent adenosine t r i p h o s p h a t a s e from t h e s a l t g l a n d o f t h e s p i n y d o g f i s h . B i o c h e m . J . 1 5 1 , 61-66. P e t e r s o n , G. L., and Hokin, L. E . ( 1 9 8 0 ) . Improved p u r i f i c a t i o n of b r i n e shrimp ( A r t e m i a s a l i n e ) ( N a + + K + ) - a c t i v a t e d adenos i n e t r i p h o s p h a t a s e and amino-acid and c a r b o h y d r a t e a n a l y s i s o f t h e i s o l a t e d s u b u n i t s . B i o c h e m . J . 1 9 2 , 107-118.

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P e t e r s o n , G. L . , and Hokin, L. E. ( 1 9 8 1 ) . Molecular w e i g h t and s t o i c h i o m e t r y o f t h e sodium- and p o t a s s i u m - a c t i v a t e d adenos i n e t r i p h o s p h a t a s e s u b u n i t s . J. B i o l . Chem. 256, 37513761. Skou, C. (1962). P r e p a r a t i o n from mammalian b r a i n and k i d n e y o f t h e enzyme system i n v o l v e d i n a c t i v e t r a n s p o r t o f N a + and K+. Biochim. Biophys. A c t a 58 , 314-325. Skou, J. C . , and Esmann, M. ( 1 9 7 9 ) . P r e p a r a t i o n of membranebound and o f s o l u b i l i z e d ( N a + + K+)-ATPase from r e c t a l g l a n d s o f S q u a l u s a c a n t h i a s . Biochim. Biophys. Acta 567, 436-444. S p e c t o r , M . , O ‘ N e a l , S . , and Racker, E. ( 1 9 8 0 ) . R e c o n s t i t u t i o n o f t h e Na+K+ pump o f E h r l i c h a s c i t e s tumor and enhancement o f e f f i c i e n c y by q u e r c e t i n . J. B i o l . Chem. 255, 5504-5507. Tanford, C . , Nozaki, Y., and Rohde, M. F. ( 1 9 7 7 ) . S i z e and s h a p e o f g l o b u l a r micelles formed i n aqueous s o l u t i o n by n - a l k y l J . P h y s . Chem. 81, 1555-1560. polyoxyethylene e t h e r s . Towle, D. W . , and Copenhaver, J. H . , Jr. ( 1 9 7 0 ) . P a r t i a l p u r i f i c a t i o n o f a s o l u b l e ( N a + + K+)-dependent A T P a s e from r a b b i t kidney. Biochirn. Biophys. Acta 203, 124-232. Uesugi, S . , Dulak, N . C . , Dixon, J. F . , Hexum, T. D . , Dahl, J . L . , Perdue, J . F . , and Hokin, L. E . ( 1 9 7 0 ) . S t u d i e s on t h e c h a r a c t e r i z a t i o n o f t h e sodium-potassium t r a n s p o r t a d e n o s i n e t r i p h o s p h a t a s e . J. B i o l . Chem. 246, 531-543. W i n t e r , C . G . ( 1 9 7 2 ) . D i f f e r e n t i a l e f f e c t s of d i g i t o n i n on some enzyme a c t i v i t i e s o f t h e sodium pump. Biochim. Biophys. A c t a 266, 135-142. W i n t e r , C . G . , and MOSS, A. J . , Jr. ( 1 9 7 9 ) . U l t r a c e n t r i f u g a l a n a l y s i s o f t h e e n z y m a t i c a l l y a c t i v e fragments produced by In “Na,K-ATPase: Structure d i g i t o n i n a c t i o n on Na,K-ATPase. and K i n e t i c s ” (J. C. Skou and J . G. Nglrby, e d s . ) , pp. 25-33. Academic P r e s s , New York. Yoda, A . , and Yoda, S. (1981). A new s i m p l e p r e p a r a t i o n method f o r NaK-ATPase-rich membrane fragments. Anal. Biochern. 110, 82-88.

J.

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CURRENT TOPICS IN MEMBRANES AND TRANSPORT, VOLUME 19

Methods for the Cleavage of the Large Subunit of Na,K-ATPase and the Resolution of the Peptides Produced HENRY RODRIGUEZ,' RICHARD HARKINS,' AND JACK KYTE Department of Chemistry University of Gdifornia San Diego. La Jolla, California

I.

INTRODUCTION

A t t h e p r e s e n t t i m e , t h e r e a re a v a i l a b l e a number of radiochemical r e a g e n t s t h a t are capable of l a b e l i n g covalently various positions within the t e r t i a r y struct u r e of n a t i v e Na,K-ATPase2. Examples of t h e s e are a f f i n i t y r e a g e n t s f o r t h e c a r d i a c g l y c o s i d e s i t e and t h e MgATP s i t e , as w e l l a s l i p i d - s o l u b l e c a r b e n e and n i t r e n e p r e c u r s o r s t h a t modify p o r t i o n s o f t h e p r o t e i n w i t h i n t h e p h o s p h o l i p i d b i l a y e r . To l o c a t e w i t h i n t h e sequence t h e p a r t i c u l a r amino a c i d s t h a t a r e m o d i f i e d by e a c h o f t h e s e r e a g e n t s , methods must b e d e v e l o p e d t o cleave t h e p r o t e i n i n t o fragments, i d e n t i f y those fragments t h a t a r e l a b e l e d , and d e t e r m i n e where w i t h i n t h e f r a g m e n t t h e r a d i o l a b e l i s l o c a t e d . C o n d i t i o n s are d e s -

' P r e s e n t a d d r e s s : G e n e n t e c h C o r p o r a t i o n , 460 P o i n t San B r u n o B l v d . , S o u t h San F r a n c i s c o , C a l i f o r n i a 9 4 0 8 0 .

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Copyright 0 1983 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-153319-0

HENRY RODRIGUEZ et a/.

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c r i b e d f o r t h e d i g e s t i o n of t h e l a r g e chain of Na,KA T P a s e w i t h t r y p s i n o r cyanogen bromide o r fragmentat i o n of a s p a r t y l - p r o l i n e bonds i n d i l u t e a c i d . Methods have been developed t o s e p a r a t e t h e p e p t i d e s i n t h e s e d i g e s t s . I n t h e case o f t h e cyanogen bromide f r a g m e n t s , t h e d i g e s t i o n i s performed i n s o l u t i o n s c o n t a i n i n g sodium dodecyl s u l f a t e (SDS), t h e d e t e r g e n t i s removed, and t h e f r a g m e n t s are s e p a r a t e d by g e l f i l t r a t i o n i n s o l v e n t s c o n t a i n i n g 3 M guanidinium c h l o r i d e f o l l o w e d by h i g h - p r e s s u r e l i q u i d chromatography. T r y p t i c d i g e s t i o n , on t h e o t h e r hand, c a n be performed on t h e waters o l u b l e l a r g e c h a i n , p r e v i o u s l y s t r i p p e d of d e t e r g e n t , and t h e t r y p t i c p e p t i d e s can be s e p a r a t e d by c a t i o n exchange chromatography. The Asp-Pro f r a g m e n t a t i o n proc e e d s i n SDS s o l u t i o n , and t h e p r o d u c t s can be p u r i f i e d by g e l f i l t r a t i o n . The g r e a t e s t gap i n o u r u n d e r s t a n d i n g o f a c t i v e t r a n s p o r t i n g e n e r a l and Na,K-ATPase i n p a r t i c u l a r i s t h a t o f t h e m o l e c u l a r s t r u c t u r e of t h e enzyme. While it i s c l e a r t h a t a t l e a s t f o u r d i s t i n c t conformat i o n s o f t h e enzyme e x i s t (Winslow, 1981; Glynn and R i c h a r d s , L e c t u r e 2 , S e s s i o n 61, it i s n o t known how ext e n s i v e t h e d i f f e r e n c e s are between them o r where i n t h e p r o t e i n t h e s e changes o c c u r . Although it h a s been est a b l i s h e d t h a t a t l e a s t t h e l a r g e c h a i n of Na,K-ATPase s p a n s t h e plasma membrane (Kyte, 19751, it h a s n o t been d e t e r m i n e d how much o f t h e mass o f t h e enzyme r e s i d e s on t h e o u t s i d e o f t h e c e l l , w i t h i n t h e b i l a y e r i t s e l f , and on t h e c y t o p l a s m i c s i d e o f t h e membrane. While it i s s a f e t o assume t h a t t h e c a t i o n s move a c r o s s t h e memb r a n e t h r o u g h a c h a n n e l formed by t h e p r o t e i n (Kyte, 1974) , how t h a t c h a n n e l is c o n s t r u c t e d and from which s t r a n d s of amino a c i d sequence remains a m y s t e r y . A l though it i s now known t h a t e a c h l a r g e c h a i n h a s one and o n l y one b i n d i n g s i t e f o r e a c h of t h e l i g a n d s , ATP, o u a b a i n , v a n a d a t e , and Mg2+ (Moczydlowski and F o r t e s , 1 9 8 1 ) , how t h e s e s i t e s a r e d i s t r i b u t e d o v e r t h e p r o t e i n r e l a t i v e t o t h e membrane b i l a y e r i s p r e s e n t l y a c o n j e c t u r e . Na,K-ATPase i s a v e r y l a r g e p r o t e i n , s i g n i f i c a n t l y g l y c o s y l a t e d as w e l l as membrane bound, and t h e r e f o r e it w i l l remain w e l l beyond t h e realm o f highr e s o l u t i o n c r y s t a l l o g r a p h y f o r a t least s e v e r a l decades. With t h i s i n mind, it i s c l e a r t h a t a n approach t h r o u g h p r o t e i n chemistry t o t h e important s t r u c t u r a l questions 'Abbreviations used: Na,K-ATPase, sodium and potassium ionactivated adenosinetriphosphatase; CaZf-ATPase, calcium ionactivated adenosinetriphospha tase

.

PROTEIN CHEMISTRY OF Na.K-ATPase

85

c o n c e r n i n g t h i s enzyme i s e s s e n t i a l . I f t h o s e amino a c i d s on t h e e x t e r n a l s u r f a c e o f t h e enzyme, t h o s e on t h e cytoplasmic surface, t h o s e w i t h i n t h e alkane phase of t h e b i l a y e r , t h o s e t h a t n e i g h b o r e a c h o t h e r , t h o s e found i n t h e b o u n d a r i e s between s u b u n i t s , t h o s e t h a t form t h e c a t i o n c h a n n e l , t h o s e whose r e a c t i v i t y changes d u r i n g l i g a n d - i n d u c e d c o n f o r m a t i o n a l c h a n g e s , and t h o s e t h a t s u r r o u n d t h e MgATP s i t e and t h e c a r d i a c g l y c o s i d e s i t e c a n be p o s i t i o n e d i n t h e sequence o f t h e enzyme, it s h o u l d be p o s s i b l e , w i t h i n t u i t i o n , t o c o i l t h e prot e i n i n t o i t s t h r e e - d i m e n s i o n a l s t r u c t u r e and t o unders t a n d t h e m o l e c u l a r f e a t u r e s of t h e c o n f o r m a t i o n a l changes t h a t perform a c t i v e t r a n s p o r t . To do t h i s i t w i l l be n e c e s s a r y t o modify t h e p r o t e i n a t s p e c i f i c l o c a t i o n s and t h e n i s o l a t e f r a g m e n t s o f small enough s i z e t o a s s i g n unambiguously t h e sequence p o s i t i o n s o f t h e labeled residues. I n f a c t , many o f t h e p r e s e n t a t i o n s i n c l u d e d i n t h i s symposium volume d e s c r i b e methods f o r l a b e l i n g t h e prot e i n . The r e a c t i o n between f l u o r e s c e i n i s o t h i o c y a n a t e and Na,K-ATPase, presumably o c c u r r i n g a t o n l y o n e l y s i n e i n t h e p r o t e i n , i s d e s c r i b e d by C a r i l l i e t a l . ( P a r t I , t h i s volume). The e f f e c t s o f changes i n enzyme c o n f o r m a t i o n on t h e r e a c t i v i t y o f a s m a l l number of s u l f h y d r y l r e s i d u e s i n t h e p r o t e i n toward 3H- and 1 4 C l a b e l e d N-ethylmaleimide have been d e f i n e d b o t h by Winslow (1981) and by Esmann and Klodos ( P a r t 111, t h i s volume). S e v e r a l a f f i n i t y l a b e l s f o r t h e c a r d i a c g l y c o s i d e s i t e a r e a v a i l a b l e : t h e r e d u c t i v e a m i n a t i o n add u c t s between o u a b a i n and e i t h e r [ 3 H ] - ~ - [ 2 - n i t r o - 5 azidobenzoyl]-1,2-diaminoethane (Forbush e t al., 1978; Forbush, P o s t e r 6 , S e s s i o n 1) o r [3H] -1-amino-2- [ p nitrophenyltriazenyll-ethane ( R o s s i e t al., 1980; Rossi et a 1 , P a r t 11, t h i s volume) and t h e [3H] e t h y l d i a z o malonyl d e r i v a t i v e s o f t h e d i g i t a l i s series ( H a l l and Ruoho, 1980; H a l l and Ruoho, P a r t 11, t h i s v o l u m e ) . Membrane-spanning p o r t i o n s of t h e l a r e c h a i n o f N a , K A T P a s e have been m o d i f i e d w i t h b o t h [?251] i o d o n a p h t h y l azide (Karlish et a l . , 1977; Jdrgensen et a l . , P a r t I , t h i s volume) and [3H] adamantane d i a z i r i n e ( F a r l e y et a l . , 1 9 8 0 ) . F i n a l l y , d e r i v a t i v e s of ATP t h a t i n s e r t i n t o t h e a c t i v e s i t e o f N a , K - A T P a s e , such a s [3H]-5'fluorosulfonylbenzoyladenosine ( W i n t e r , P o s t e r 1 7 , Sess i o n 1) and chromium (111) [ 3H] arylazidoadenosinetriphosp h a t e (Munson, 1 9 8 1 ) , have been developed. The number, v a r i e t y , and r e c e n t n a t u r e o f t h e e f f o r t s now under way t o l a b e l t h e enzyme are a c l e a r i n d i c a t i o n o f t h e i n t e r e s t i n t h i s endeavor. I n a l l o f t h e s e i n s t a n c e s , however, w h i l e Na,KA T P a s e l a b e l e d i n s p e c i f i c l o c a t i o n s h a s been produced,

.

HENRY RODRIGUEZ eta/.

86

t h e s e p r o d u c t s c a n n o t p r o v i d e any i n f o r m a t i o n p e r t i n e n t t o t h e s t r u c t u r e of t h e p r o t e i n u n t i l t h e modified residues a r e l o c a t e d p r e c i s e l y 3 i n t h e amino a c i d sequence. To accomplish t h i s i d e n t i f i c a t i o n , p r o c e d u r e s must be developed f o r c l e a v i n g t h e l a b e l e d l a r g e c h a i n of Na,KATPase t o produce a r e p r o d u c i b l e , dependable p a t t e r n of fragments each of which c a n be recognized r e a d i l y and i s o l a t e d f o r s e q u e n t i a l Edman d e g r a d a t i o n . I t i s t h e purpose of t h i s r e p o r t t o d e s c r i b e o u r p r o g r e s s i n dev e l o p i n g p r o c e d u r e s t h a t w i l l p e r m i t u s t o do t h i s .

11.

EXPERIMENTAL PROCEDURES MATERIALS

A.

Disodium a d e n o s i n e t r i p h o s p h a t e (vanadium-free, e q u i n e m u s c l e ) , L - h i s t i d i n e ( f r e e b a s e ) , 2-[N-morphol i n o ] e t h a n e s u l f o n i c a c i d , T r i s ( f r e e base, Trizma), T r i s - H C 1 (Trizma h y d r o c h l o r i d e ) , glucagon, m e l i t t i n , and r i b o n u c l e a s e A (Type I A ) , were o b t a i n e d from Sigma; (2-bromoethy1)trimethylammonium bromide, from A l d r i c h ; f o r m i c a c i d ( 8 8 % i n g l a s s ) , from Baker; [ l - l 4 C ] i o d o acetamide and [3H]NaBH4 , from N e w England N u c l e a r ; guanidinium c h l o r i d e , from Heico Corp.; t r y p s i n (TPCK t r e a t e d ) , from Worthington; bovine serum albumin, from M i l e s L a b o r a t o r i e s ; a p r o t i n i n , from Boehringer Mannheim; a c e t o n i t r i l e (HPLC g r a d e ) , from F i s c h e r ; U l t r a g e l ACA54 and ACA22, from LKB; and Aminex A-5 (13 5 2 p ) , from BioRad. Sodium dodecyl s u l f a t e (Sigma) was rec r y s t a l l i z e d by t h e procedure of Burgess ( 1 9 6 9 ) ; i m i d a z o l e (Matheson) was r e c r y s t a l l i z e d from benzene t h e n acetone; pyridine (Mallinckrodt) w a s r e d i s t i l l e d a f t e r r e f l u x i n g w i t h 5 % p h t h a l i c anhydride; iodoacetamide (Calbiochem) w a s r e c r y s t a l l i z e d from 9 5 % e t h a n o l ; cyanogen bromide (Matheson) was allowed t o sublime o n t o t h e w a l l s of i t s c o n t a i n e r a t 4OC, and t h e c r y s t a l s t h a t r e s u l t e d were employed; and XAD-4 r e s i n ( s o l d a s BioBeads SM-4 by BioRad) was a g i f t of t h e Rohm and Haas co

.

3An a m b i g u i t y of even 210 r e s i d u e s t r a n s l a t e s t o f15-35

1.

PROTEIN CHEMISTRY OF Na,K-ATPase

B.

87

E N 2 YME P U R I F I C A T I O N

The method h a s been d e s c r i b e d by J $ r g e n s e n ( 1 9 7 4 ) and Munson ( 1 9 8 1 ) . Microsomes from c a n i n e r e n a l m e d u l l a (Kyte, 1 9 7 1 ) a r e mixed w i t h a s o l u t i o n p r e p a r e d s u c h t h a t t h e f i n a l c o n c e n t r a t i o n s a r e 0 . 7 mg m l - 1 sodium dod e c y l s u l f a t e , 2 mg m l - 1 p r o t e i n , 25 mM i m i d a z o l i u m c h l o r i d e , H 7.5, 0 . 1 % 2 - m e r c a p t o e t h a n o l , 1 mM EDTA, and 4.5 mg m l - y ATP. The sample i s l a y e r e d o n t o d i s c o n t i n u o u s s u c r o s e g r a d i e n t s ( 3 7 . 3 % , 2 8 . 8 % , and 1 5 % s u c r o s e ) t h a t a r e t h e n c e n t r i f u g e d a t 4 0 , 0 0 0 rpm i n a T i 45 f i x e d angle rotor. The p u r e enzyme g a t h e r s a t t h e i n t e r f a c e between 28.8% and 37.3% s u c r o s e , i s c o l l e c t e d , d i l u t e d , and c o n c e n t r a t e d by f u r t h e r c e n t r i f u g a t i o n . A l l buffers are made 0 . 1 % i n 2 - m e r c a p t o e t h a n o l j u s t b e f o r e u s e . The f i n a l a c t i v i t y i s between 1 6 and 3 0 pmoles min-1 mg-l. C.

LARGE-CHAIN

PURIFICATION

(Kyte,

1972)

Na,K-ATPase (15 m l of 4-6 mg m l - 1 ) i n s t o r a g e b u f f e r (0.25 M s u c r o s e , 30 mM h i s t i d i n i u m c h l o r i d e , 0 . 1 % 2-merc a p t o e t h a n o l ) i s mixed w i t h s u f f i c i e n t 2 0 % sodium d o d e c y l s u l f a t e t o b r i n g t h e d e t e r g e n t - t o - p r o t e i n mass r a t i o t o 3. A f t e r t h e s o l u t i o n c l a r i f i e s , i t i s b r o u g h t t o 1 0 0 ° C f o r 1 m i n , c o o l e d , and a p p l i e d t o a S e p h a r o s e 4 B column ( 5 x 1 2 0 c m ) e l u t e d w i t h 40 mM T r i s s u l f a t e , 0 . 1 % sodium d o d e c y l s u l f a t e , pH 8 . 0 . Large c h a i n i s p o o l e d ( a b o u t 20-30 mg f o r e a c h r u n ) and c o n c e n t r a t e d by l y o p h i l i z a tion. D.

REDUCTION AND A L K Y L A T I O N

Large c h a i n (2-3 mg m 1 - l ) a s t h e d o d e c y l s u l f a t e complex i s d i a l y z e d u n d e r N 2 a g a i n s t 0 . 0 1 M d i t h i o t h r e i t o l , 0 . 2 M sodium b o r a t e , pH 9 . 5 , f o r 20 h r . S o l i d u r e a i s t h e n added t o 8 M and m e r c a p t o e t h a n o l t o 5 % and t h e sample i n c u b a t e d u n d e r N 2 f o r 2-3 h r a t 37OC. It i s then dialyzed a g a i n s t 8 M urea, 0 . 0 1 M d i t h i o t h r e i t o l , 0 . 2 M sodium b o r a t e , pH 9 . 5 , u n d e r N 2 f o r 20 hr. S o l i d (2-bromoethyl)trimethylammonium bromide i s added t o 1 . 0 M and t h e r e a c t i o n a l l o w e d t o p r o c e e d a t room t e m p e r a t u r e f o r 6 h r ( I t a n o and Robinson, 1 9 7 2 ) . The sample i s t h e n d i a l y z e d e x h a u s t i v e l y . The e x t e n t of r e a c t i o n i s a s s e s s e d d i r e c t l y by amino a c i d a n a l y s i s .

88

E.

CYANOGEN

BROMIDE CLEAVAGE

A l k y l a t e d l a r g e c h a i n (0.2-1.0 mg m 1 - l ) i n 0.2-1.0% sodium d o d e c y l s u l f a t e , 5 mM sodium p h o s p h a t e , pH 7 , isb r o u g h t t o 0 . 1 M i n HC1 a n d a n e q u a l volume o f 30 mg m l cyanogen bromide i n 0 . 1 M HC1, 0 . 1 % sodium d o d e c y l s u l f a t e i s added. The s o l u t i o n i s s e a l e d u n d e r A r a n d s t a n d s a t room t e m p e r a t u r e f o r 2 0 h r . The d i g e s t i s d i l u t e d w i t h water and l y o p h i l i z e d . Upon amino acid a n a l y s i s less t h a n 5% of t h e m e t h i o n i n e s h o u l d r e m a i n , and t h e e x p e c t e d amounts o f homoserine (Degen and K y t e , 1978) s h o u l d b e o b s e r v e d . F.

D I S C O N T I N U O U S S T A C K I N G S Y S T E M ON S L A B G E L FOR U R E A DODECYL S U L F A T E - P O L Y A C R Y L A M I D E G E L S ( K y t e a n d R o d r i g u e z , 1983)

The g e l s y s t e m o f Swank and Munkries (1971) w a s ext e n s i v e l y modified using t h e equations of O r n s t e i n ( 1 9 6 4 ) . Upper r e s e r v o i r b u f f e r i s 0.213 M 2 - ( ~ - m o r p h o 1 i n o ) e t h a n e s u l f o n i c a c i d , 0.269 M p y r i d i n e , 0.1% sodium dodecyl s u l f a t e ; t h e s t a c k i n g g e l i s 4% acrylamide, 0.1% b i s a c r y l a m i d e , 8 M u r e a , 0.368 M HC1, 0.951 M p y r i d i n e , 0 . 1 % sodium d o d e c y l s u l f a t e ; t h e r u n n i n g g e l i s 20% a c r y l a m i d e , 1%b i s a c r y l a m i d e , 0 . 1 % sodium d o d e c y l s u l f a t e , 0.323 T r i s C 1 , 8 M urea; and t h e lower r e s e r v o i r b u f f e r i s 0.32 M T r i s C 1 , 0.32 M T r i s f r e e b a s e . Samples are p r e p a r e d s u c h t h a t t h e f i n a l c o n c e n t r a t i o n s are 8 M u r e a , 0.368 M H C 1 , 0 . 4 5 1 M p y r i d i n e , 1-5% sodium d o d e c y l s u l f a t e , 1%2 - m e r c a p t o e t h a n o l . A s a m p l e o f up t o 70 111 can be added t o a s l o t on a 2 mm s l a b , 7 c m long. The g e l s are r u n a t 70 V f o r 16-20 h r . Staining w i t h Coomassie B r i l l i a n t Blue c a n d e t e c t p e p t i d e s as s h o r t a s 26 r e s i d u e s . G.

TRITIATION

( R i c e a n d Means,

1971)

L a r g e c h a i n , a l k y l a t e d w i t h (2-bromoethy1)trimethylammonium bromide and c l e a v e d w i t h cyanogen bromide, i s d i s s o l v e d i n 0.2 M sodium b o r a t e , p H 8.5. To t h e solut i o n , ~ 1 m0C i sodium [ 3 H ] b o r o h y d r i d e i s added f o r e a c h F r e s h l y d i s t i l l e d f o r m a l d e h y d e i s added mg of p r o t e i n . i n 5 a d d i t i o n s o v e r a 30 min p e r i o d s u c h t h a t t h e f i n a l c o n c e n t r a t i o n of f o r m a l d e h y d e a t any g i v e n a d d i t i o n i s e q u i m o l a r w i t h l y s i n e i n t h e p r o t e i n . The r e a c t i o n m i x t u r e i s allowed t o s t a n d 30 min a t which t i m e a 2 0

PROTEIN CHEMISTRY OF Na,K-ATPase

89

molar e x c e s s o f g l y c i n e i s added and t h e s o l u t i o n a l lowed t o s t a n d a t room t e m p e r a t u r e o v e r n i g h t i n a fume hood. The sample i s t h e n d i a l y z e d a g a i n s t 5 m M sodium p h o s p h a t e , 0 . 1 % sodium dodecyl s u l f a t e , pH 7 . 0 , t o remove u n r e a c t e d m a t e r i a l and l y o p h i l i z e d . An a p p r o p r i a t e amount of t h i s m a t e r i a l i s r o u t i n e l y mixed w i t h a l a r g e c h a i n cyanogen bromide d i g e s t t o a s s i s t i n f o l l o w i n g t h e fragments. H.

STRIPPING,

CONCENTRATION,

AND T R A N S F E R R I N G P R O T E I N

P r o b a b l y t h e most d i f f i c u l t problem e n c o u n t e r e d i n t h e s e e x p e r i m e n t s i s t r a n s f e r r i n g p r o t e i n among s o l u t i o n s c o n t a i n i n g t h e v a r i o u s d e n a t u r a n t s . Sodium d o d e c y l s u l f a t e and guanidinium c h l o r i d e a r e i n c o m p a t i b l e . Sodium dodecyl s u l f a t e must b e s t r i p p e d away from t h e p r o t e i n w i t h a n i o n exchange r e s i n s (Weber and K u t e r , 1 9 7 1 ) . Sol u t i o n s of p e p t i d e s i n 8 M u r e a o r 6 M guanidinium c h l o r i d e c a n n o t be c o n c e n t r a t e d r e a d i l y . There a r e s e v e r a l t e c h n i q u e s , however, which have s i m p l i f i e d t h e s e problems. D i a l y s i s t u b i n g t h a t w i l l r e t a i n peptides l a r g e r than about 1 0 residues, while p a s s i n g b u f f e r c o n s t i t u e n t s r e a d i l y , h a s become a v a i l a b l e l a t e l y ( S p e c t r a p o r e C o r p . ) , and i t h a s been employed h e a v i l y . Because many of t h e s o l u t i o n s a r e conc e n t r a t e d by l y o p h i l i z a t i o n , which works v e r y w e l l w i t h d o d e c y l s u l f a t e s o l u t i o n s , t h e e x c e s s sodium d o d e c y l s u l f a t e t h a t r e s u l t s m u s t be removed. T h i s can be accomplished by s t i r r i n g t h e sample ( 2 % i n sodium d o d e c y l By s u l f a t e ) w i t h o n e - t e n t h volume o f t h e r e s i n XAD-4. following the decrease i n detergent concentration over s e v e r a l h o u r s w i t h a methylene b l u e a s s a y (Mukerjee, 19561, t h e a d s o r p t i o n can be t e r m i n a t e d when t h e d e s i r e d t o t a l c o n c e n t r a t i o n o f d e t e r g e n t i s r e a c h e d . Because o n l y a p o r t i o n of t h e d e t e r g e n t i s removed, p r o t e i n remains i n s o l u t i o n and i t s l o s s o v e r t h e c o u r s e o f t h e a d s o r p t i o n i s minimal. T h i s p r o c e d u r e can be accomp l i s h e d i n 8 M u r e a s o l u t i o n s a s w e l l and i s a conven i e n t p r e l u d e t o Dowex 1 s t r i p p i n g (Weber and K u t e r , 1971) because i t p e r m i t s complete removal o f t h e sodium d o d e c y l s u l f a t e t o be accomplished w i t h a minimum of t h e p o l y s t y r e n e , maximizing t h e y i e l d o f p r o t e i n . Finally, d i f f i c u l t i e s i n t r a n s f e r r i n g cyanogen bromide f r a g m e n t s from t h e ACA54 p o o l s i n 3 M guanidinium c h l o r i d e i n t o a s o l v e n t compatible w i t h r e v e r s e phase high-pressure l i q u i d chromatography were e n c o u n t e r e d . I t w a s d i s c o v e r e d , however, t h a t t h e sample c o u l d be d i a l y z e d f i r s t i n t o 5% f o r m i c a c i d , l y o p h i l i z e d , and r e d i s s o l v e d i n 88% f o r m i c acid p r i o r t o i n j e c t i o n . S e v e r a l o t h e r p r o t o c o l s l e a d t o a l m o s t t o t a l l o s s of f r a g m e n t s .

HENRY RODRIGUEZ et a/.

90

I.

TRYPTIC D I G E S T I O N

Large c h a i n of Na,K-ATPase a s t h e dodecyl s u l f a t e complex i s reduced and a l k y l a t e d a s d e s c r i b e d above w i t h iodoacetamide u s i n g 5 0 mM iodoacetamide, 8 M u r e a , 0 . 2 M T r i s s u l f a t e , 5 mg m l - 1 i n l a r g e c h a i n , 50 mM 2-mercaptoethanol, pH 7.8. I t i s t h e n s t r i p p e d of t h e dodecyl s u l f a t e (Weber and K u t e r , 1 9 7 1 ) and d i a l y z e d T r y p s i n , 1 0 pg (mg l a r g e a g a i n s t 1%NH4HC03, pH 7.9. c h a i n ) ' l , i s added and t h e m i x t u r e i n c u b a t e d a t 37OC f o r s e v e r a l h o u r s , followed by a second a d d i t i o n of t r y p s i n . During t h e d i g e s t i o n t h e s o l u t i o n remains comp l e t e l y c l e a r . Completeness of d i g e s t i o n w a s judged by d e t e r m i n i n g t h e amount of l y s i n e and a r g i n i n e l i b e r a t e d by c a r b o x y p e p t i d a s e . The d i g e s t i s l y o p h i l i z e d t o g i v e a f l u f f y powder. The powder i s d i s s o l v e d i n a s m a l l amount of 50% a c e t i c a c i d and d i l u t e d t o 5 % a c e t i c a c i d .

111.

RESULTS AND DISCUSSION

Reduced and a l k y l a t e d l a r g e c h a i n from Na,K-ATPase has been d i g e s t e d w i t h e i t h e r t r y p s i n o r cyanogen bromide o r c l e a v e d a t i t s a s p a r t y l - p r o l i n e (Asp-Pro) pept i d e bonds i n d i l u t e a c i d . Each of t h e s e r e a c t i o n s produces a complicated m i x t u r e of fragments t h a t can be r e s o l v e d by g e l f i l t r a t i o n , h i g h - p r e s s u r e l i q u i d chromatography, and i o n exchange chromatography. A l l of t h e s e e f f o r t s a r e d i r e c t e d toward t h e d i s c o v e r y of s y s t e m a t i c and r e l i a b l e methods f o r i s o l a t i n g and i d e n t i f y i n g fragments of t h e l a r g e c h a i n t h a t have been Before t h e d e t a i l s a r e labeled a t defined positions. d e s c r i b e d , it i s i m p o r t a n t t o remember t h a t t h e l a r g e c h a i n i s a v e r y long p o l y p e p t i d e , which makes t h e mixt u r e s q u i t e complicated; t h a t it i s d i f f i c u l t t o p u r i f y , which r e q u i r e s t h a t s e p a r a t i o n s be performed on o n l y hundreds of moles a t a t i m e ; and t h a t i t i s a membranespanning p r o t e i n , which l e a d s t o r e f r a c t o r y b e h a v i o r from many of t h e p e p t i d e m i x t u r e s . A.

CYANOGEN BROMIDE D I G E S T I O N

S i n c e t h e l a r g e c h a i n c o n t a i n s 2.3 mole p e r c e n t methionine (Kyte, 1 9 7 2 1 , t h e mean l e n g t h of t h e f r a g ments should be a b o u t 45 r e s i d u e s , w i t h i n t h e range t h a t can be sequenced by automated p r o c e d u r e s . F u r t h e r -

PROTEIN CHEMISTRY OF Na,K-ATPase

91

more, s i n c e m e t h i o n i n e r e s i d u e s are o f t e n p r e s e n t i n membrane-spanning s e q u e n c e s (Khorana et ai., 1 9 7 9 ) , cyanogen bromide t r e a t m e n t s h o u l d b r e a k s u c h s e q u e n c e s i n t h e i r i n t e r i o r while r e t a i n i n g t h e i r connections t o more h y d r o p h i l i c p o r t i o n s o f t h e p o l y p e p t i d e c h a i n , a p o s s i b i l i t y t h a t m i g h t improve t h e s o l u b i l i t y of t h e fragments. F i n a l l y , t h i s cleavage proceeds w i t h high y i e l d and h a s been w i d e l y employed ( G r o s s , 1 9 6 7 ) . The l a r g e c h a i n of Na,K-ATPase, i n d o d e c y l s u l f a t e s o l u t i o n , i s r e d u c e d and a l k y l a t e d w i t h (2-bromoethy1)trimethylammonium bromide ( I t a n o and Robinson, 1 9 7 2 ) . This a l k y l a t i n g agent introduces a fixed, t e t r a a l k y l ammonium c a t i o n a t e v e r y c y s t e i n e r e s i d u e , i n c r e a s i n g t h e s o l u b i l i t y o f t h e f r a g m e n t s d u r i n g l a t e r manipulations. Severe c o n d i t i o n s are necessary t o a l k y l a t e t h e c y s t e i n e s q u a n t i t a t i v e l y . The problem i s n o t w i t h t h e unusual a l k y l a t i n g agent b u t w i t h i n e f f i c i e n t reduction. T h i s c o n c l u s i o n f o l l o w s from t h e f a c t t h a t i n c o m p l e t e reaction a l s o occurred with i o d o a c e t i c a c i d . Furthermore, a s i m i l a r d i f f i c u l t y had b e e n e n c o u n t e r e d w i t h C a 2 + - A T P a s e 2 ( A l l e n and Green, 1 9 7 8 ) . By m o d i f y i n g t h e r e d u c t i o n c o n d i t i o n s , however, i t i s p o s s i b l e t o p r o d u c e 2.3-2.6 mole p e r c e n t L-[2-[(2'-amino-2'-carboxye t h y l ) t h i o ] e t h y l ] trimethylammonium c a t i o n ( I t a n o and Robinson, 1 9 7 2 ) d u r i n g t h e a l k y l a t i o n , which compares f a v o r a b l y w i t h t h e t o t a l c y s t e i n e c o n t e n t o f 2 . 5 mole p e r c e n t f o r t h i s p r o t e i n (Kyte, 1 9 7 2 ) . The a l k y l a t e d l a r g e c h a i n i s c l e a v e d w i t h cyanogen bromide i n d o d e c y l s u l f a t e s o l u t i o n , and t h e n l y o p h i l i z e d . The d i g e s t , which s t i l l c o n t a i n s t h e d o d e c y l s u l f a t e , i s c o m p l e t e l y s o l u b l e i n aqueous b u f f e r s . If, on t h e o t h e r hand, t h e l a r g e c h a i n i s i n i t i a l l y s t r i p p e d o f i t s d o d e c y l s u l f a t e and c l e a v e d w i t h cyanogen bromide i n 70% formic a c i d , t h e l y o p h i l i z e d product i s completely i n s o l u b l e i n s o l v e n t s commonly employed w i t h p r o t e i n s . One o f t h e cyanogen bromide d i g e s t s w a s r e d u c t i v e l y a l k y l a t e d w i t h [3H]NaBHq ( R i c e and Means, 1 9 7 1 ) t o v e r y h i g h s p e c i f i c a c t i v i t y (10,500 cpm p g - 1 ) . This proc e d u r e m e t h y l a t e s l y s i n e r e s i d u e s and t h e amino t e r m i n a l s of t h e f r a g m e n t s , a f a c t t h a t h a s been v e r i f i e d by amino a c i d a n a l y s i s . A s m a l l amount o f t h i s r a d i o a c t i v e m a t e r i a l i s added r o u t i n e l y t o s u b s e q u e n t d i g e s t s t o p e r m i t a c c u r a t e l o c a t i o n of t h e f r a g m e n t s d u r i n g t h e s e p a r a t i o n procedures. B.

ANAZYTICAL POLYACRYLAMIDE GEL ELECTROPHORESIS

Although p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s i s a w i d e l y employed and p o w e r f u l p r o c e d u r e , t h e r e a r e v e r y few s y s t e m s t h a t have been a d a p t e d € o r p e p t i d e s of t h e

92

HENRY RODRIGUEZ et a/.

F i g . 1 . E l e c t r o p h o r e t i c s e p a r a t i o n o f the c y a n o g e n b r o m i d e f r a g m e n t s o f the l a r g e c h a i n o f Na,K-ATPase. Reduced and a l k y l a t e d l a r g e chain was d i a l y z e d i n t o 5 mM s o d i u m p h o s p h a t e , pH 8 , and then b r o u g h t t o a f i n a l c o n c e n t r a t i o n o f 0.7 mg m l - I p r o t e i n i n >0.1% s o d i u m d o d e c y l s u l f a t e , 0.1 M H C I , and 1 5 mg m l - I c y a n o g e n b r o m i d e . A f t e r 1 6 hr the s a m p l e was l y o p h i l i z e d , r e d i s s o l v e d , l y o p h i l i z e d and d i s s o l v e d i n 8 M u r e a , 50 mM T r i s C1, pH 7 , and l o a d e d d i r e c t l y onto a u r e a - d o d e c y l s u l f a t e - p o l y a c r y a m i d e s l a b g e l ( 8 x 1 4 x 0.2 cm) w i t h the s t a c k i n g s y s t e m . One r u n w a y o f the s t a i n e d g e l was s c a n n e d a t 550 nm and the trace i s p r e s e n t e d . The The asterisk d i r e c t i o n o f e l e c t r o p h o r e s i s i s from l e f t t o r i g h t . m a r k s a component w h i c h i s o n l y o c c a s i o n a l l y o b s e r v e d and i s a s sumed t o be a p a r t i a l c l e a v a g e p r o d u c t .

4

s i z e o f t h e cyanogen bromide f r a g m e n t s from t h e l a r g e chain. The most s u c c e s s f u l i n t h i s r e g a r d i s a u r e a d o d e c y l s u l f a t e s y s t e m d e v e l o p e d by Swank and Munkries (Swank and M u n k r i e s , 1 9 7 1 ) . S e v e r a l b e n e f i c i a l m o d i f i -

PROTEINCHEMISTRY OF Na,K-ATPase

93

c a t i o n s can be made t o t h i s method (Kyte and Rodriguez, 1 9 8 3 ) . By u t i l i z i n g 2 0 % p o l y a c r y l a m i d e g e l s , t h e r e s o l u t i o n of p e p t i d e s s h o r t e r t h a n 4 0 r e s i d u e s can be i n c r e a s e d s i g n i f i c a n t l y . A d i s c o n t i n u o u s b u f f e r system ( O r n s t e i n , 1964) t h a t s t a c k s t h e sample, improving o v e r a l l r e s o l u t i o n , h a s a l s o been developed. The advantage of s u c h a s t a c k i n g system i s t h a t f a i r l y wide s a m p l e s , 5-10 mm, are compressed t o e x t r e m e l y s h a r p d i s c s a t t h e o r i g i n . T h i s i s v e r y i m p o r t a n t when i t i s r e a l i z e d t h a t a sample d e r i v e d from a l a b e l i n g e x p e r i m e n t must be p r e p a r e d i n a volume t h a t can be m a n i p u l a t e d and t h e n t h e e n t i r e sample must b e l o a d e d . When t h e complete cyanogen bromide d i g e s t o f reduced and a l k y l a t e d l a r g e c h a i n i s s u b m i t t e d t o e l e c t r o p h o r e s i s i n t h i s system, very s a t i s f a c t o r y r e s o l u t i o n of t h e l a r g e r f r a g m e n t s i s accomplished. A s c a n o f such a g e l i s p r e s e n t e d i n F i g . 1, and below t h e s c a n i s a photograph o f t h e s t a i n e d g e l . The p a t t e r n i s d i v i d e d i n t o s e v e r a l regions, 1 1 1 - V I I I , f o r reasons discussed l a t e r , and t h e a p p a r e n t l e n g t h s , d e r i v e d f r o m a s t a n d a r d c u r v e , o f t h e f r a g m e n t s i n e a c h r e g i o n are p r e s e n t e d i n T a b l e I . The g e l system c a n n o t r e s o l v e p e p t i d e s whose l e n g t h i s less t h a n a b o u t 25 r e s i d u e s and t h e s e s m a l l f r a g m e n t s b l e e d from t h e g e l d u r i n g s t a i n i n g . The elect r o p h o r e s i s r e s o l v e s 11 p e p t i d e s whose combined a p p a r e n t l e n g t h i s 7 7 0 r e s i d u e s , 7 0 % of t h e t o t a l l e n g t h of t h e l a r g e c h a i n . Furthermore, some of t h e components o f t h e p a t t e r n may r e p r e s e n t o v e r l a p p i n g f r a g m e n t s . I f t h e r e a r e 26 cyanogen bromide f r a g m e n t s and 11 are l o n g e r t h a n 3 0 r e s i d u e s , t h e remaining 15 would have a mean l e n g t h

TABLE I. Apparent Lengths of t h e Cyanogen Bromide Fragments on t h e Polyacrylamide G e l Displayed i n F i g u r e 1.a Cluster

IV V VI VLI VIII a

Lengths o f fragments

116, 109, 100, 94 67, 62 54 45, 42, 41 3 7 , <30

T o three of the r u n w a y s on t h e same s l a b , RNase (n = 1 2 4 ) , a p r o t i n i n (n = 58), and g l u c a g o n ( n = 2 9 ) w e r e a p p l i e d , and t h e i r mobilities w e r e u s e d t o p o s i t i o n the s t a n d a r d c u r v e . From the mob i l i t i e s o f the v a r i o u s f r a g m e n t s d i s p l a y e d i n F i g . 1 , a p p a r e n t l e n g t h s w e r e c a l c u l a t e d . T h e y a r e p r e s e n t e d f o r the c l u s t e r s , as numbered i n F i g . 1 .

HENRY R O D R I G U U ~ ~ ~ / .

94

15

V

N

0 4 x

f

1( 5-

CI Y

I

200

600

400 VOLUME

(ML)

Fig. 2. G e l f i l t r a t i o n chromatography o f a t o t a l cyanogen bromide d i g e s t o f a l k y l a t e d l a r g e c h a i n . A l y o p h i l i z e d cyanogen b r o m i d e d i g e s t ( 9 mg) was r e d i s s o l v e d i n 8 M u r e a , 5 0 mM T r i s a c e t a t e , pH 7 . 8 . One-tenth o f a v o l u m e o f XAD-4 resin was added t o the s a m p l e , and it was s t i r r e d f o r 4 hr, f o l l o w i n g d i s a p p e a r a n c e o f the d o d e c y l s u l f a t e ( M u k e r j e e , 1 9 5 6 ) . The XAD-4 resin was removed and e n o u g h Dowex 1 added t o r e m o v e a n y r e s i d u a l s o d i u m d o d e c y l s u l f a t e (Weber and K u t e r , 1 9 7 1 ) . T h e s a m p l e was then l o a d e d onto a n U l t r a g e l ACA54 c o l u m n ( 2 x 200 c m ) t h a t was then e l u t e d w i t h 3 M g u a n i d i n i u m chloride a t 7 ml h r - l . F r a c t i o n s were a s s a y e d f o r 3 8 ( c p m ) . Pools w e r e made o f selected f r a c t i o n s a s i n d i c a t e d . T h e l o w e r c u r v e i s the e l u t i o n p r o f i l e of a m i x t u r e c o n t a i n i n g s e r u m a l b u m i n (n = 582 a a ) , RNase (n = 1 2 6 a a ) , and m e l i t t i n ( n = 26 a a ) , r u n e a r l i e r a s s t a n d a r d s . T h e s e r u m a l b u m i n r u n s i n the v o i d v o l u m e o f the c o l u m n . S t a n d a r d s w e r e d e t e c t e d b y A280.

o f 2 2 r e s i d u e s . These f r a g m e n t s presumably are o v e r l a p p e d i n t h e f i n a l peak and have d i s a p p e a r e d d u r i n g staining.

PROTEIN CHEMISTRY OF Na,K-ATPass TABLE 11.

95

C a l c u l a t e d Lengths o f t h e Cyanogen Bromide Fragments from Each Pool from t h e ACA54 Column ( F i g . 2 ) . a Pool

Length range

I11 IV V VI VII VIII IX X

120-160 90-120 60-90 50-60 35-50 25-35 15-25 10-15

a

T h e d i s t r i b u t i o n c o e f f i c i e n t s f r o m the c a l i b r a t i o n r u n o f the s t a n d a r d s ( R N a s e , n = 124; a p r o t i n i n , n = 58; and m e l i t t i n , n = 2 6 ) were p l o t t e d a s a f u n c t i o n o f the l o g a r i t h m of t h e i r l e n g t h s . From t h i s l i n e a r f u n c t i o n , the r a n g e of l e n g t h s f o r each pool was d e t e r m i n e d .

C.

P R E P A R A T I V E R E S O L U T I O N O F THE CYANOGEN BROMIDE FRAGMENTS

The cyanogen bromide d i g e s t c a n b e s t r i p p e d of i t s d o d e c y l s u l f a t e by a c o m b i n a t i o n o f a p r o c e d u r e employi n g XAD-4 r e s i n and t h e method of Weber and K u t e r ( 1 9 7 1 ) . The s t r i p p e d p r o t e i n , i n 8 M u r e a , i s i n t r o d u c e d d i r e c t l y o n t o a n ACA54 g e l f i l t r a t i o n column e q u i l i b r a t e d w i t h 3 M guanidinium c h l o r i d e . An e l u t i o n p r o f i l e from s u c h a column i s d i s p l a y e d i n F i g . 2 . A l s o i n d i c a t e d a r e t h e p o s i t i o n s and e l u t i o n w i d t h s of several s t a n d a r d pept i d e s w i t h which t h e column was c a l i b r a t e d . P o o l s are made r o u t i n e l y a s i n d i c a t e d , and t h e l e n g t h s of t h e pept i d e s t h a t e a c h p o o l s h o u l d c o n t a i n were c a l c u l a t e d ( T a b l e 11) from t h e d i s t r i b u t i o n c o e f f i c i e n t s o f t h e c a l i b r a t i n g standards. Each p o o l d i s p l a y e d p e p t i d e s of t h e e x p e c t e d l e n g t h when examined by u r e a - d o d e c y l s u l f a t e g e l e l e c t r o p h o r e s i s , and t h e Roman n u m e r a l s i n T a b l e I and F i g . 1 c o r r e s p o n d t o t h e numbered p o o l s t h a t were made i n F i g . 2 . I t c a n b e s e e n by comparing F i g s . 1 and 2 t h a t t h e b r o a d p e a k s i n t h e e l u t i o n p r o f i l e correspond w e l l t o t h e c l u s t e r s of fragments s e e n i n t h e polyacrylamide g e l . The s e c o n d d i m e n s i o n f o r f u r t h e r p r e p a r a t i v e s e p a r a t i o n i s h i g h - p r e s s u r e l i q u i d chromatography. Initiall y , s i g n i f i c a n t d i f f i c u l t i e s were e x p e r i e n c e d t r a n s f e r r i n g t h e f r a g m e n t s from t h e d i l u t e p o o l s i n 3 M

HENRY RODRIGUEZ eta/.

96

A.

B.

0

N N

V W

< z m K

v) 0

m

4

I

ELUTION VOLUME F i g . 3 . H i g h - p r e s s u r e l i q u i d c h r o m a t o g r a p h y o f pools f r o m the U l t r a g e l ACA54 c o l u m n ( F i g . 2 ) . S a m p l e s (Q30 m l ) f r o m the ACA54 c o l u m n w e r e d i a l y z e d a g a i n s t 5% f o r m i c a c i d , l y o p h i l i z e d , and r e d i s s o l v e d i n 88% f o r m i c a c i d and i n j e c t e d onto a pdondapack column (0.8 x 30 c m ) t h a t had been e q u i l i b r a t e d w i t h 0.1 M H3PO4, 0.1 M NaH2P04. The f l o w r a t e was m a i n t a i n e d a t 4 ml min-l. A s soon a s the t r a c e had r e t u r n e d t o b a s e l i n e , a f t e r the f l o w t h r o u g h had e m e r g e d , a l i n e a r g r a d i e n t o f 400 ml i n l e n g t h f r o m 0 t o 100% a c e t o n i t r i l e was i n i t i a t e d . T h e p o r t i o n s o f the e l u t i o n p r o f i l e s p r e s e n t e d commence a t a b o u t 6 0 % a c e t o n i t r i l e and end a t 90% and c o m p r i s e a b o u t 1 2 0 ml o f the t o t a l g r a d i e n t . (A) Pool IV. ( B ) Pool V .

guanidinium chloride to the concentrated solutions necessary for injection into the high pressure liquid chromatography, but these problems have been resolved. The system of O'Hare and Nice (1979) provides very satisfactory results. The elution profiles of two of the pools from the ACA54 gel filtration column (Fig. 2 ) are displayed in Fig. 3 . The individual fragments often run in pairs owing to the homoserine lactone-homoserine equilibrium. The clearest examples of this are the first two components in Fig. 3B. The amino acid compositions of the two members of the doublet are indistinguishable from each other.

PROTEIN CHEMISTRY OF Na,K-ATPase

U.

ASP-PRO

97

CLEAVAGE

I f p u r i f i e d l a r g e chain d i s s o l v e d i n dodecyl s u l f a t e s o l u t i o n i s i n c u b a t e d a t pH 2 . 5 , 37OC (Landon, 1 9 7 7 ) f o r s e v e r a l d a y s , a s i m p l e , r e p r o d u c i b l e s e t of fragments is generated (Fig. 4 A ) . The p a t t e r n i s domin a t e d by two r a t h e r l a r g e p r o d u c t s ( n r = 500 r e s i d u e s and n r = 350 r e s i d u e s ) , which may a r i s e from t h e hydrol y s i s o f a s p e c i f i c Asp-Pro p e p t i d e bond a b o u t onet h i r d of t h e way from o n e end o f t h e p r o t e i n , and s h o r t e r p r o d u c t s are a l s o o b s e r v e d . F o l l o w i n g several p r e l i m i n a r y e x p e r i m e n t s , a l a r g e r sample ( 1 0 0 nmoles) o f l a r g e c h a i n was i n c u b a t e d f o r 8 d a y s , and t h e p r o d u c t s were s e p a r a t e d by r e p e t i t i v e g e l f i l t r a t i o n on U l t r a g e l ACA22 e l u t e d w i t h 0 . 0 4 M Tris-SOq, pH 8 , 0 . 2 % sodium d o d e c y l s u l f a t e , f o l l o w i n g t h e d i s t r i b u t i o n by ~ 2 2 0 . S c a n s of p o l y a c r y l a m i d e g e l s of t h e p u r i f i e d fragments a r e displayed i n Fig. 4B. Four d i s c r e t e f r a g m e n t s can be i s o l a t e d , and t h e a l i g n ment p r e s e n t e d i n F i g . 4 i n d i c a t e s t h a t t h e y a c c o u n t f o r a l l o f t h e m a j o r components o b s e r v e d i n t h e o r i g i n a l d i gest. I f it i s assumed t h a t e a c h f r a g m e n t r e p r e s e n t s a u n i q u e p o r t i o n of t h e l a r g e c h a i n and t h a t e a c h i s a s long a s i t s m o b i l i t y i n d i c a t e s , t h e y i e l d s through t h e p u r i f i c a t i o n are only 20-30%. E.

TRYPTIC PEPTIDES

Although t h e l a r g e c h a i n c o n t a i n s a b o u t 1 1 0 t r y p t i c p e p t i d e s , it i s p o s s i b l e t h a t some of t h e l a b e l e d p o s i t i o n s w i l l b e more r e a d i l y i d e n t i f i e d by i s o l a t i n g t h e m o d i f i e d p e p t i d e s from a t r y p t i c d i g e s t . For e x a m p l e , m e t h i o n i n e r e s i d u e s a r e v e r y f r e q u e n t i n membranes p a n n i n g s e q u e n c e s (Khorana e t a l . , 19791, c a u s i n g them t o be b r o k e n up d u r i n g cyanogen bromide d i g e s t i o n . L y s i n e s and a r g i n i n e s , however, b e i n g t h e most hydrop h i l i c r e s i d u e s known ( C h o t h i a , 1 9 7 6 1 , a r e v e r y seldom found i n t h e s e r e g i o n s of a membrane-bound p r o t e i n , and e a c h membrane-spanning s e q u e n c e s h o u l d r e m a i n i n t a c t d u r i n g t r y p s i n d i g e s t i o n , e n d i n g up w i t h i n a s i n g l e , l o n g t r y p t i c p e p t i d e , a p o s s i b i l i t y t h a t would f a c i l i tate t h e i r identification. In other instances, other c o n s i d e r a t i o n s might a l s o i n d i c a t e t h a t t r y p t i c digest i o n would b e a f a v o r a b l e r o u t e t o e x p l o r e . A t r y p t i c d i g e s t of t h e l a r g e c h a i n , r e d u c e d and carboxyamidomethylated a t i t s c y s t e i n e s , w a s t h e n c a r boxyamidomethylated w i t h [ 1 4 C ] i o d o a c e t a m i d e t o r e n d e r

HENRY RODRIGUEZ eta/.

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0

m

W

z U m a K

m 0

J

m

4

RELATIVE

MIGRATION

F i g . 4 . P o l y a c r y l a m i d e g e l s o f Asp-Pro f r a g m e n t s o f the l a r g e chain. ( A ) L a r g e chain was d i a l y z e d and added t o a s o l u t i o n s u c h t h a t the f i n a l c o n c e n t r a t i o n s would be 10% a c e t i c a c i d , 0 . 2 5 % sodium d o d e c y l s u l f a t e , 0 . 1 5 mg ml-I p r o t e i n , pH 2 . 5 , w i t h p y r i d i n e . A s a m p l e was i m m e d i a t e l y w i t h d r a w n and d i a l y z e d and the rem a i n d e r i n c u b a t e d u n d e r A r a t 37OC f o r 8 d a y s . Both the i n i t i a l s a m p l e and the 8-day s a m p l e were d i a l y z e d , l y o p h i l i z e d , and r u n on 7 . 5 % s o d i u m d o d e c y l s u l f a t e - p o l y a c r y l a m i d e g e l s (Weber and Osborn, 1 9 6 9 ) . S c a n s of s t a i n e d g e l s of the t w o s a m p l e s a r e s . u p e r i m p o s e d . ( B ) A l o w pH, Asp-Pro d i g e s t o f l a r g e chain (100 nmoles) was d i a l y z e d a g a i n s t 0.04 M T r i s SO4, pH 8 , l y o p h i l i z e d and l o a d e d onto a n U l t r a g e l ACA22 column ( 2 . 5 X 200 cm) e l u t e d w i t h 0.1% SDS, 0 . 0 4 M T r i s - S O 4 , pH 8.0. Pools f r o m t h i s p r o f i l e w e r e r e r u n on the s a m e c o l u m n , and s c a n s o f p o l y a c r y l a m i d e g e l s o f the f i n a l p u r i f i e d comp o n e n t s are p r e s e n t e d , s u p e r i m p o s e d , i n the f i g u r e . The m g n i f i c a t i o n s of these s c a n s h a v e been a d j u s t e d so t h a t the r e l a t i v e m i g r a tions i n ( A ) c o i n c i d e a s c l o s e l y a s p o s s i b l e w i t h those i n ( B )

.

PROTEIN CHEMISTRY OF Na,K-ATPase

99

0

. 5

/

I W

/

4

4 I

3

I

I

I

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100

150

200

VOLUME

(ML)

Fig. 5.

E l u t i o n p r o f i l e o f a t r y p t i c d i g e s t o f the l a r g e on Aminex A-5. A t r y p t i c d i g e s t o f carboxya m i d o m e t h y l a t e d l a r g e c h a i n (10 mg) was d i s s o l v e d i n 60% a c e t i c The a c i d and m ix e d w i t h 1 2 5 V C i [14C] i o d o a c e t a m i d e ( 1 3 p C i / p m o l e ) . reaction vessel was e v a c u a t e d , s e a l e d , and i n c u b a t e d a t 57OC f o r 3 hr. T h e s a m p l e was then a l k y l a t e d w i t h u n r a d i o a c t i v e i o d o a c e t a m i d e ( 2 5 % s o l u t i o n ) u n d e r a r g o n a t 37'C. T h e s a m p l e was d e s a l t e d on a S e p h a d e x G - 1 0 c o l u m n ( 1 . 5 x 51 c m ) , e l u t e d w i t h 6 0 % a c e t i c a c i d , a n d the v o i d v o l u m e p o l was l y o p h i l i z e d . T h i s mat e r i a l was r e d i s s o l v e d i n 60% a c e t i c a c i d and pumped onto an Aminex A-5 column ( 0 . 6 x 9.6 c m ) e q u i l i b r a t e d w i t h b u f f e r A T h e c o l u m n was run a t 24 m l ( 0 . 0 5 M p y r i d i n i u m a c e t a t e , pH 2 . 4 ) . h r - I w i t h b u f f e r A u n t i l a l l f l o w t h r o u g h h a d e m e r g e d ; then a 200 m l l i n e a r g r a d i e n t c o n s t r u c t e d from 100 m l b u f f e r A a n d 100 m l b u f f e r B ( 0 . 5 M p y r i d i n i u m a c e t a t e , pH 3 . 7 5 ) ; a g r a d i e n t cons t r u c t e d f r o m 100 m l b u f f e r B and 100 m l b u f f e r C (2.0 M p y r i d i nium a c e t a t e , p H 5 . 0 ) ; and f i n a l l y b u f f e r C (Degen and K y t e , 1 9 7 8 ) . T h e f r a c t i o n s w e r e a s s a y e d f o r 14C ( c p m ) and their pH determined.

chain of Na,K-ATPase

i t s m e t h i o n i n e r e s i d u e s r a d i o a c t i v e (Degen and K y t e , 1978). The d i g e s t w a s t h e n s u b m i t t e d t o c a t i o n exchange chromatography on s u l f o n a t e d p o l y s t y r e n e ( F i g . 5). The l a r g e c h a i n c o n t a i n s 25 m e t h i o n i n e r e s i d u e s , a number t h a t compares f a v o r a b l y w i t h t h e 1 8 - 2 0 p e a k s o b s e r v e d .

HENRY RODRIGUEZ eta/.

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F.

ALTERNATIVE

CLEAVAGES

There are s e v e r a l o t h e r c l e a v a g e s t r a t e g i e s t h a t have been c o n s i d e r e d . Hydroxylamine t r e a t m e n t of t h e d e n a t u r e d p r o t e i n , i n t h e dodecyl s u l f a t e complex (Drickamer, 19761, a c l e a v a g e s p e c i f i c f o r Asn-Gly bonds ( B o r n s t e i n and B a l i a n , 19771, y i e l d e d no d i s c r e t e fragments even though t h e i n t a c t p r o t e i n s l o w l y d i s a p p e a r e d . Although it h a s a l r e a d y been d e m o n s t r a t e d t h a t t h e l a r g e c h a i n w i t h i n t h e n a t i v e enzyme can be s p l i t w i t h t r y p s i n a t a small number of p r e c i s e l y d e f i n e d pos i t i o n s (Jgirgensen, 13751, t h e y i e l d of f r a g m e n t s , e v e n on g e l e l e c t r o p h o r e s i s , i s r a t h e r poor and t h e p r o d u c t s s i g n i f i c a n t l y o v e r l a p t h e s m a l l c h a i n , a problem t h a t would a g g r a v a t e t h e d i f f i c u l t y i n p u r i f y i n g them. On t h e o t h e r hand, b o t h Green and A l l e n , s e q u e n c i n g CaZ+-ATPase ( A l l e n e t a l . , 19801, and C o l l i n s , sequencing t h e l a r g e c h a i n of fJa,K-ATPase ( C o l l i n s e t a l . , P a r t I , t h i s v o l u m e ) , have chosen t o d i g e s t s u c c i n y l a t e d p r o t e i n with trypsin. I n t h e case o f Na,K-ATPase t h i s p r o c e d u r e i s e x p e c t e d t o y i e l d a b o u t 50 p e p t i d e s , a r a t h e r formida b l e number t o s e p a r a t e a n a l y t i c a l l y , b u t t h e r e i s p e r haps one advantage t o t h i s approach. Because a r q i n i n c r e s i d u e s l i e o u t s i d e t h e b i l a y e r o n e would e x p e c t any membrane-spanning segments t o remain i n t a c t and t h e m a j o r i t y o f t h e normal sequence t o o c c u r on o t h e r p e p t i d e s . I n f a c t , b o t h Green and A l l e n , a s well a s C o l l i n s , have found t h a t a b o u t 3 0 % of t h e i r d i g e s t s , i n c l u d i n g a l l t h e t r y p t o p h a n , r u n s i n t h e v o i d volume on g e l f i l t r a t i o n and c a n be i n i t i a l l y s e p a r a t e d from t h e remaining i n c l u d e d p e p t i d e s t h a t have a normal h y d r o p h i l i c i t y and t h a t are t r a c t a b l e t o normal s e q u e n c i n g p r o c e d u r e s . Unf o r t u n a t e l y , t h e m a t e r i a l i n t h e v o i d volume of t h e o r i g i n a l g e l f i l t r a t i o n s t e p h a s proven c o m p l e t e l y r e f r a c tory t o f u r t h e r separation. F i n a l l y , c l e a v a g e a t c y s t e i n e ( O t i e n o , 1 9 7 8 ) and t r y p t o p h a n (0201s and G e r a r d , 1 9 7 7 ) should be mentioned. N e i t h e r o f t h e s e a l t e r n a t i v e s h a s any p a r t i c u l a r a p p e a l i n t h e c a s e of t h e l a r g e c h a i n because b o t h c y s t e i n e and t r y p t o p h a n a r e p r e s e n t i n a b o u t t h e same f r e q u e n c y a s m e t h i o n i n e . Furthermore, n e i t h e r of t h e s e p r o c e d u r e s i s so q u a n t i t a t i v e as cyanogen bromide because t h e y r e l y r e s p e c t i v e l y on two r e s i d u e s which a r e v u l n e r a b l e t o o x i d a t i o n d u r i n g p u r i f i c a t i o n of t h e p r o t e i n , and considerable d i f f i c u l t i e s arise with p a r t i a l cleavage. These r e s u l t s and c o n s i d e r a t i o n s , a l t h o u g h p r e l i m i n a r y , have been p r e s e n t e d i n t h e hope t h a t t h e y w i l l p r o v i d e t e c h n i q u e s t h a t c a n be employed i n t h e i s o l a t i o n of f r a g m e n t s from t h e l a b e l e d Na,K-ATPase produced i n t h e e x p e r i m e n t s r e p o r t e d a t t h i s m e e t i n g and e a r l i e r .

PROTEIN CHEMISTRY OF Na,K-ATPase

101

I f t h e y encourage h e r e t o f o r e h e s i t a n t i n v e s t i g a t o r s t o engage t h e p r o t e i n a t t h e l e v e l of i t s amino a c i d sequence t h e y w i l l have s e r v e d t h e i r p u r p o s e .

ACKNOWLEDGMENTS

T h i s work w a s s u p p o r t e d by G r a n t s HL-17879, GM-07169, and AM-07233 f r m t h e N a t i o n a l I n s t i t u t e s of H e a l t h , G r a n t PCM7824284 from t h e N a t i o n a l S c i e n c e F o u n d a t i o n , and Grant-in-Aid R.H. w a s a p o s t AHA81-1003 from t h e American Heart A s s o c i a t i o n . d o c t o r a l f e l l o w s u p p o r t e d by T r a i n i n g G r a n t AM-07233 from t h e N a t i o n a l I n s t i t u t e s o f H e a l t h and J . K . w a s a r e c i p i e n t o f R e s e a r c h Career Development Award HL-00206 f r m t h e N a t i o n a l I n s t i t u t e s o f H e a l t h . The a s s i s t a n c e o f S t e p h e n B l a t c h f o r d and James G a l l a g h e r i n t h e p r e p a r a t i o n o f t h e enzyme i s g r e a t l y a p p r e c i a t e d .

REFERENCES

A l l e n , G . , and Green, N. M. ( 1 9 7 8 ) . B i o c h e m . J. 1 7 3 , 393-402. A l l e n , G. , Trinnamen, B. J . , and Green, N . M. ( 1 9 8 0 ) . B i o c h e m . J. 1 8 7 , 591-616. B o r n s t e i n , P . , and B a l i a n , G. ( 1 9 7 7 ) . In "Methods i n Enzymology" (C. H. W. H i r s and S. N. T i m a s h e f f , e d s . ) , V o l . 4 7 , P a r t E , p p . 132-145. Academic P r e s s , N e w York. B u r g e s s , R. R. ( 1 9 6 9 ) . J. B i o l . Chem. 244, 6168-6176. C h o t h i a , C. ( 1 9 7 6 ) . J. Mol. B i o l . 1 0 5 , 1-14. Degen, J . , and Kyte, J. ( 1 9 7 8 ) . A n a l . B i o c h e m . 8 9 , 529-539. Drickamer, L. K. ( 1 9 7 6 ) . J . B i o l . C h e m . 251, 5115-5123. F a r l e y , R. A. , G o l d m a n , D. W. , and Bayley, H. ( 1 9 8 0 ) . J. B i o l . C h w . 255, 860-864. Forbush, B., Kaplan, J. H . , a n d Hoffman, J. F. ( 1 9 7 8 ) . B i o c h e m i s t r y 1 7 , 3667-3676. Gross, E. ( 1 9 6 7 ) . In "Methods i n Enzymology" (C. H. W. H i r s , e d . ) , V o l . 11, p p . 238-255. Academic P r e s s , N e w York. H a l l , C . , a n d Ruoho, A. ( 1 9 8 0 ) . Proc. N a t l . A c a d . S c i . USA 77, 4529-4533. I t a n o , H. A . , and Robinson, E. A. ( 1 9 7 2 ) . J . B i o l . Chem. 247, 4819-4824. J & r g e n s e n , P. L. ( 1 9 7 4 ) . B i o c h i m . B i o p h y s . A c t a 356, 53-67. J g k g e n s e n , P. L. ( 1 9 7 5 ) . B i o c h i m . B i o p h y s . A c t a 401, 399-415. K a r l i s h , S . J. D . , Jgkgensen, P. L . , and G i t l e r , C. ( 1 9 7 7 ) . Nature (London) 269, 715-717. Khorana, H. G., Gerber, G. E . , H e r l i h y , W. C . , Gray, C. P . , Anderegg, R. J . , N i h e i , K . , and Biemann, K. ( 1 9 7 9 ) . P r o c . N a t l . A c a d . S c i . USA 76, 5046-5050.

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1 02

Kyte, Kyte, Kyte, Kyte, Kyte,

J. (1971). J . B i o l . J. (1972). J . B i o l . J. (1974). J . B i o l . J. (1975). J . B i o l . J . , and Rodriguez, H.

Chem. 2 4 6 , 4157-4165. Chem. 2 4 7 , 7642-7649. Chem. 2 4 9 , 3652-3660. Chem. 2 5 0 , 7443-7449. (1983). Submitted for p u b l i c a t i o n i n Analytical Biochemistry. Landon, M. ( 1 9 7 7 ) . In "Methods i n Enzymology" (C. H. W. H i r s and S. N . Timasheff, e d s . ) , Vol. 47, P a r t El pp. 145-149. Academic P r e s s , New York. Moczydlowski, E. G . , and F o r t e s , P. A. G. (1981). J . B i o l . Chem. 256, 2346-2356. Mukerjee, P. (1956). Anal. Chem. 2 8 , 870-873. Munson, K. B. (1981). J . B i o l . Chem. 2 5 6 , 3223-3230. O ' H a r e , M. J . , and Nice, E . C. (1979). J . Chromatogr. 1 7 1 , 209226. O r n s t e i n , L. (1964). Ann. N.Y. Acad. Sci. 1 2 1 , 321-349. O t i e n o , S . ( 1 9 7 8 ) . Biochemistry 1 7 , 5468-5474. O z o l s , J . , and Gerard, C. (1977). J. B i o l . Chem. 2 5 2 , 5986-5989. Rice, R. H . , and Means, G. E. (1971). J . B i o l . Chem. 2 4 6 , 831-832. Rossi, B., V u i l l e u m i e r , P., Gache, C . , B a l e r n a , M., and Lazdunski, M. (1980). J . B i o l . Chem. 255, 9936-9941. Swank, R. T., and Munkries, K. D. (1971). Anal. Biochem. 39, 462-477. Weber, K., and Kuter, D. J. (1971). J . B i o l . Chem. 2 4 6 , 4504-4509. Weber, K . , and Osborn, M. ( 1 9 6 9 ) . J . B i o l . Chem. 2 4 4 , 4406-4412. Winslow, J. ( 1 9 8 1 ) . J . B i o l . Chem. 2 5 6 , 9522-9531.

CURRENT TOPICS IN MEMBRANES AND TRANSPORT, VOLUME 19

Selective Purification of Na,K-ATPase and CA2+,Mg2+-ATPasefrom Eel Electroplax L. M.AMENDE, ' S. P. CHOCK, AND R. W.ALBERS NINCDS National Institutes of Health Bethesda, Maryland

I.

Na, K-ATPase

The demand f o r o b t a i n i n g an a c t i v e and p u r e s o l u b l e f o r s t r u c t u r a l s t u d i e s h a s prompted us t o d e v e l o p an a l t e r n a t i v e method f o r s o l u b i l i z i n g E l e c t r o p h o r u s e l e c t r o p l a x microsomes. A f t e r s c r e e n i n g a number of commercially a v a i l a b l e d e t e r g e n t s , w e found t h a t t h e n o n i o n i c p o l y o x y e t h y l e n e d e t e r g e n t B r i j - 5 6 ( C 1 6 E l o ! provides a s o l u b l e Na,K-ATPase of high s p e c i f i c a c t i v i t y w i t h h i g h y i e l d and r e l a t i v e ease of p r e p a r a t i o n . T h i s method i n v o l v e s a r e p e t i t i v e e x t r a c t i o n o f t h e e e l m i crosomes w i t h 0 . 6 M K C 1 , f o l l o w e d by a d e i o n i z e d water wash b e f o r e e x p o s i n g t h e microsome t o f i r s t a l o w ( 1 : 5 ) and t h e n a h i g h (1:l) r a t i o of d e t e r g e n t t o p r o t e i n . The l a t t e r d e t e r g e n t e x t r a c t i o n s o l u b i l i z e s t h e N a , K ATPase which e x h i b i t s a h i g h d e g r e e o f homogeneity, a s shown i n F i g . 1, and h a s a s p e c i f i c a c t i v i t y a t 25OC o f 5.2 pmoles Pi/(mg x m i n ) . T h i s s o l u b l e enzyme c a n be stored i n d e f i n i t e l y i n liquid nitrogen. Na,K-ATPase

' P r e s e n t a d d r e s s : Department of B i o c h e m i s t r y , A r m e d brces R a d i o b i o l o g y R e s e a r c h I n s t i t u t e , B e t h e s d a , Y a r y l a n d 20814. 103

104

L. M. AMENDE eta/.

F i g . 1 . SDS-slab gel electrophoresis o f proteins from the various stages of the Na ,K-ATPase p u r i f i c a t i o n and solubilization process. Lane 1 : the microsome pattern before extraction. Lane 2 : the soluble proteins extracted from the microsomes b y t h e 0.6 M KCl extraction and before the B r i j - 5 6 extraction. Lane 3 : the washed microsomes a f t e r the K C l extraction and before the B r i j - 5 6 extraction. Lane 4 : the f i n a l sol ubilized Na ,K-ATPase a f t e r a b r i e f d i l u t e ( 0 . 2 % , w/w) detergent extraction and then treated with 1% B r i j (0.9:1, Brij:Protein). The soluble enzyme has a specific, a c t i v i t y of 5.2 pmole P i / ( m g x m i n ) , 25'C, pH 7.3. Lane 5: the f i n a l insoluble p e l l e t a f t e r the Brij-56 solubilization.

PURIFICATION OF Na,K-ATPaseAND Ca2 ,Mg2+-ATPase

105

+

1000 un ng /

I

' "\

F+

I

0

109

\

I

1

2

CCal, ufl

F i g . 2 . E f f e c t o f Ca 2+ on the Ca 2+ ,Mg 2+ -ATPase a c t i v i t y o f e l e c t r o p l a x m i c r o s o m s m e a s u r e d a t d i f f e r e n t Mg2+ concentrations. E n z y m e a c t i v i t y was m e a s u r e d a t 25OC i n the p r e s e n c e o f 50 mM HEPES, pH 7 . 5 , 100 mM KCI, 0.2 mM T r i s - A T P , 2 p g / m l o l i g o m y c i n , 10 mM EGTA-Ca2+ b u f f e r f o r [Ca2+] f r o m 0.17 p M t o 100 pM, a n d MgC12 a s i n d i c a t e d .

11.

Ca2+,Mg2+-ATPase

E e l e l e c t r o p l a x a l s o contain a high concentration of calmodulin (about 4 % of t h e s o l u b l e p r o t e i n f r a c t i o n ) . Calmodulin h a s been shown t o modulate t h e a c t i v i t y of c a l c i u m pumps i n o t h e r membrane s y s t e m s s u c h as t h e r e d b l o o d c e l l , b r a i n , and a d i p o c y t e s . The p r e s e n c e of s u c h a c a l c i u m pump h a s n o t been r e p o r t e d i n e l e c t r o p l a x . By u s i n g M a l a c h i t e g r e e n t o d e t e c t i n o r g a n i c phosp h a t e (Penney, 1 9 7 6 ) , w e have c h a r a c t e r i z e d a h i g h a f f i n i t y Ca2+,Mg2+-ATPase i n e e l e l e c t r o p l a x . A s shown

106

L. M.AMENDE et el.

i n F i g . 2 , a t 2 0 0 p~ Mg2+, t h e enzyme h a s a h i g h c a l c i u n High a f f i n i t y with optimal a c t i v i t y a t 0.58 V M Ca2+. c o n c e n t r a t i o n s of Ca2+ ( 1 0 0 1 . 1 ~ 1 i n h i b i t a c t i v i t y . The enzyme a c t i v i t y i s a l s o dependent on t h e Mg2+/Ca2+ r a t i o , s i n c e a t Mg2+ c o n c e n t r a t i o n s g r e a t e r t h a n 200 p M , t h e enzyme r e q u i r e s much h i g h e r c o n c e n t r a t i o n s of Ca2+ f o r a c t i v i t y . The enzyme a c t i v i t y i s n o t a f f e c t e d by 0.5 m~ o u a b a i n , o r by t h e mitochondria1 i n h i b i t o r s DCCD (50 V M ) or oligomycin ( 2 pq/ml). Other i n h i b i t o r s shown t o a f f e c t t h e r e d c e l l Ca2+,MgZ+-ATPase a l s o i n h i b i t t h e e l e c t r o p l a x Ca2+ ,Mg2+-ATPase. Vanadate i s a p o t e n t i n h i b i t o r ( K i < 0 . 2 V M ) and t r i f l u o p e r a z i n e (TFP) i n h i b i t e t h e n z m e w i t h a K i = 25 pM. TFP i n h i b i t s s e v e r a l Ca5+ ,Mgs+-ATPases by p r e v e n t i n g t h e i r i n t e r a c t i o n w i t h calmodulin ( e . g . , Levin and Weiss, 1 9 7 7 ) . S i n c e a d d i t i o of calmodulin ( 1 0 0 nM) d i d n o t markedly s t i m u l a t e elect r i c organ Ca2+,Mgz+-ATPase a c t i v i t y , it is l i k e l y t h a t t h e e l e c t r o p l a x microsomes c o n t a i n t i g h t l y bound endogenous calmodulin. Ca2+ ,Mg2+-ATPase may be s o l u b i l i z e d from e l e c t r o p l a x microsomes u s i n g T r i t o n X-100 d e t e r g e n t a t a f i n a l c o n c e n t r a t i o n of 0 . 2 % and microsomes a t 4 mg p r o t e i n / m l , u s i n g a b u f f e r a t 20 m~ HEPES, pH 7.3, 1 0 0 mM K C 1 , 0.5 m MqC12, 50 p M CaC12, and 2 m~ DTT. T h i s detergent-enzyme mixture i s k e p t on i c e f o r 1 0 min, c e n t r i f u g e d 1 0 min a t 1 0 0 , 0 0 0 g i n a Beckman A i r f u g e , and t h e s u p e r n a t a n t saved. T h i s procedure u s u a l 1 s o l u b i l i z e s about 3 7 % of t h e p r o t e i n and 43% of t h e Cas+,Mg2+-ATPase a c t i v i t y .

REFERENCES

Levin, R. B . , and Weiss, B. ( 1 9 7 7 ) . Binding of t r i f l u o p e r a z i n e t o t h e calcium-dependent a c t i v a t o r of c y c l i c n u c l e o t i d e phosphod i e s t e r a s e . Molec. Pharmacol. 13, 690-697. Penney, C . L. (1976). A simple micro-assay f o r i n o r g a n i c phosp h a t e . A n a l . Biochem. 75, 201-210.

CURRENT TOPICS IN MEMBRANES AND TRANSPORT, VOLUME 19

High-PerformanceGel Chromatography of Horse Kidney Na,K-ATPase MAKOTO NAKAO Tokyo Medical and Dental University School of Medicine, Yushimu, Bunkyo-ku, Tokyo, Japan

TOSHIKO NAKAO AND TOMOKO OHNO Tokyo Metropolitan Research Laboratory of Public Health, Shinjuku-ku, Tokyo, Japan

YUKICHI HARA, AND MASAKO ARAI Tokyo Medical and Dental University School of Medicine Yushima,Bunkyo-ku. Tokyo, Japan

YOSHIHIRO FUKUSHIMA Laboratory of Active Transport National Institutefor Physiological Sciences Okaraki, 444 Japan

I.

INTRODUCTION

T h r e e p e a k s h a v i n g N a , K - A T P a s e a c t i v i t y were det e c t e d when t h e L u b r o l o r N i k k o l (C12E8) e x t r a c t of membrane N a , K-ATPase w a s a p p l i e d t o a n a m i n o e t h y l c e l l u l o s e column (Nakao et a i . , 1 9 7 3 ) . T o e l u c i d a t e t h e m u l t i p l i c i t y , high-performance g e l chromatography was adopted

.

11.

MATERIALS AND METHODS

N a , K - A T P a s e w a s o b t a i n e d from h o r s e k i d n e y o u t e r m e d u l l a e s s e n t i a l l y a c c o r d i n g t o J # r g e n s e n ( 1 9 7 4 ) . The membrane enzyme ( 1 . 4 mg/ml) w a s s o l u b i l i z e d w i t h 1 mg/ml o f Nikkol (C12E8) ( s p e c i f i c a c t i v i t y 1100-1800, two maj o r bands i n SDS-PAGE) and a 1 0 0 p 1 a l i q u o t w a s a p p l i e d 107

Copyright Q 1983 by Academic Press, Inc. All rightsofreproduction in any form r e ~ e ~ e d . ISBN O-I2-1533190

MAKOTO NAKAO et a/.

108

t o a Toyo Soda G4000-SW or G3000-SW column (82 x 600 nm) which had p r e v i o u s l y been e q u i l i b r a t e d w i t h 1 0 0 mM T e s - T r i s , pH 6.8, c o n t a i n i n g 0 . 0 2 % o r 0 . 0 1 % C12E8. In some e x p e r i m e n t s , 20 rrw p-NPP, 2 0 m MgCl2 and 15 m K C 1 were added v i a a n o t h e r pump t o d e t e c t t h e p-NPase a c t i v i t y o n - l i n e a f t e r p r o t e i n d e t e c t i o n a t 280 nm. Lowa n g l e l i g h t s c a t t e r i n g and r e f r a c t i v e i n d e x were measured o n - l i n e (machines: LS-8 and RI-8 Toyo Soda) a c c o r d i n g t o Takagi (1981) a s f o l l o w s . (Output) Ls/(OUtput)RI = N: ( d n / d c ) K'M where N O , d n / d c , LS, R I , K ' , and M are t h e r e f r a c t i v e i n d e x o f t h e s o l v e n t a t 633 nm, t h e r e f r a c t i v e i n c r e m e n t of t h e p r o t e i n , l i g h t s c a t t e r i n g , r e f r a c t i v e i n d e x , a n i n s t r u m e n t c o n s t a n t , and m o l e c u l a r w e i g h t , r e s p e c t i v e l y . R e f r a c t i v e i n d e x i n c r e m e n t v a l u e s w e r e assumed t o be 0.190, 0.196, 0.170, and 0.160 f o r bovine serum albumin (BSA) , t h y r o g l o b u l i n , and t h e enzyme peaks B and C , respectively. P o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s was performed w i t h 3.5, 4.5, 5 , 6 , and 7% g e l s c o n t a i n i n g 0 . 0 1 % C12E8. I n some cases, Na,K-ATPase a c t i v i t y w a s d e t e c t e d on t h e g e l w i t h t r i e t h y l a m i n e molybdate.

111.

RESULTS AND DISCUSSION

The 800K B and 200K C peaks as w e l l as a v o i d volume peak A were e l u t e d w i t h e l u e n t c o n t a i n i n g K+ from a G4000-SW column, w h i l e 400K B' and 200K C peaks were o b t a i n e d w i t h e l u e n t c o n t a i n i n g N a + ( F i g . 1 ) . The appearance of t h e 400K peak i s i n a c c o r d w i t h e a r l i e r work by us (Nakao e t al., 1973) and by H a s t i n g and Skou ( 1 9 8 0 ) . These B , B ' , and C peaks showed K-pNPase and Na,K-ATPase a c t i v i t i e s which were c o m p l e t e l y i n h i b i t e d by 10-5 M o u a b a i n . On HPLC w i t h a G3000-SW column, peak A w a s e l u t e d a t t h e v o i d volume, b u t c a r e f u l exp e r i m e n t s i n d i c a t e d t h a t f o u r peaks were p r e s e n t , namel y , A, B, B ' , and C . The l a t t e r t h r e e peaks seemed t o be i n e q u i l i b r i u m w i t h e a c h o t h e r , j u d g i n g from t h e res u l t s of rechromatography w i t h N a o r K b u f f e r . Lowa n g l e l i g h t s c a t t e r i n g d a t a o b t a i n e d o n - l i n e were cons i s t e n t w i t h rough estimates o f t h e m o l e c u l a r w e i g h t s of B and C o b t a i n e d by u s i n g bovine serum albumin and human y - g l o b u l i n a s s t a n d a r d s (Table I ) . The e l u t i o n p a t t e r n s o b t a i n e d w i t h e l u e n t s cont a i n i n g d i f f e r e n t l i g a n d s are summarized i n T a b l e 11. The N a and K forms were r e a d i l y d i s t i n g u i s h e d e x c e p t

HtGH PERFORMANCE CHROMATOGMPHY OF HORSE KIDNEY

109

A

F i g . 1 . HPLC o f C 1 2 E 8 - s o l u b i l i z e d horse k i d n e y Na,K-ATPase. A T o y o Soda G4000-SW column was p r e e q u i l i b r a t e d w i t h b u f f e r conE l u t i o n rate 1 ml/min. Detect a i n i n g 2 5 mM NaCl or 10 mM K C l . tion a t 280 nm.

w i t h M g 2 + p l u s i n o r g a n i c phosphate. The r e s u l t s are s i m i l a r t o t h e d a t a r e p o r t e d by P o s t (1979) and are c o n s i s t e n t w i t h t h e view t h a t E l and E 2 have d i f f e r e n t s u r f a c e c h a r a c t e r i s t i c s . A Ferguson p l o t of t h e res u l t s o f g e l e l e c t r o p h o r e s i s i n t h e p r e s e n c e o f ATP a l s o i n d i c a t e d N a and K forms a t 2 0 0 K , 400K, and 800K. During g e l chromatography, t h e enzyme may c o n t a i n f i r m l y bound ATP as shown by K o s e p e l l (19791, and t h i s ATP may be e l i m i n a t e d by e l e c t r o p h o r e s i s . I n t h e p r e s e n c e of EDTA t h e a p p a r e n t m o l e c u l a r w e i g h t o f t h e N a form became l a r g e r t h a n t h a t of t h e K form. These d a t a show t h a t t h e 200K form o f t h e N a , K - A T P a s e ( c o n t a i n i n g l i p i d and d e t e r g e n t ) i s a c t i v e , a l t h o u g h t h e a c t i v i t y i s v e r y l a b i l e , and t h a t C 1 2 E g s o l u b i l i z a t i o n changes t h e prope r t i e s of t h e enzyme s u r f a c e depending on t h e p r e s e n c e o f N a o r K and ATP.

ACKNOWLEDGMENT

The a u t h o r s t h a n k M r . Sakane i n Toyo Soda Company f o r h e l p w i t h t h e measurement of low-angle l a s e r l i g h t s c a t t e r i n g .

110

MAKOTO NAKAO eta/.

TABLE I.

E s t i m a t i o n of Molecular Weights of M u l t i p l e Forms o f Horse Kidney Method

B

G4000-SW Kav G3000-SW Kav Low a n g l e laser l i g h t scattering Ferguson p l o t of C12E8 g e l e l e c t r o p h o r e s i s

70-80 >60

TABLE 11.

B' 40-60 45

20-27 25

15

92 >60

C

28-50

13-23

Change i n S i z e of Peaks w i t h D i f f e r e n t Ligands i n t h e E l u a n t (G3000 SW)

Ligand

-Mgi 1

M a , 25 Mg 1, M a 25 Mg 1, N a 25, K 1 0 Na-ATP 0 . 1 ATP, 0.1, N a 25 N a 25, Mg 1, ATP 1 N a 25, Mg 1, ADP 0.03 K 1 0 , Mg 1, ATP 0.04 Mg 1, P i 1 K 1 0 , Mg 1, P i 1 K 1 0 , Mg 1 K 10 ATP 0.3, K 1 0

+ + + -b

+ + + + + + + + + + +

+ + ++ ++

++ ++ + + + + +

++

++ ++ ++ ++ + + + + + ++

+ + ++ ++ ++ ++ ++ +

+ + + +

+ + + + + + + + +

+

~

a

Molecular weight.

REFERENCES

Esmann, M., C h r i s t i a n s e n , C . , K a r l s s o n , K. A . , Hansson, G. C . , and Skou, J . C . ( 1 9 8 0 ) . Hydrodynamic p r o p e r t i e s of s o l u b i l i z e d ( N a + + K+)-ATPase from r e c t a l g l a n d s o f S q u a l u s a c a n t h i a s . Biochim. Biophys A c t a 603, 1 - 1 2 . H a s t i n g s , D. , a n d Skou, J. C . (1980). Potassium b i n d i n g t o t h e ( N a + + K+)-ATPase. Biochirn. Biophys A c t a 601, 380-385.

. .

HIGH PERFORMANCE CHROMATOGRAPHY OF HORSE KIDNEY

111

+

Jgkgensen, P. L. ( 1 9 7 4 ) . I s o l a t i o n of (Na' + K )-ATPase. In "Methods i n Enzymology" (S. F l e i s c h e r and L. P a c k e r , e d s . ) , Vol. 32, P a r t B, pp. 277-290. Academic P r e s s , New York. Nakao, T . , Nakao, M . , Nagai, F . , K a w a i , K . , F u j i h i r a , Y . , Hara, Y . , and F u j i t a , M. ( 1 9 7 3 ) . P u r i f i c a t i o n and some p r o p e r t i e s of Na,K-transport ATPase. 11. P r e p a r a t i o n s w i t h h i g h s p e c i f i c a c t i v i t y o b t a i n e d through a m i n o e t h y l c e l l u l o s e chromatography. J. Biochem. ( T o k y o ) 73, 781-791. T a k a g i , T. ( 1 9 8 1 ) . C o n f i r m a t i o n of m o l e c u l a r w e i g h t of A s p e r g i l l u s o r y z a e a-amylase u s i n g t h e low a n g l e l a s e r l i g h t s c a t t e r i n g t e c h n i q u e i n combination w i t h h i g h p r e s s u r e s i l i c a g e l J. Biochem. (Tokyo) 89, 363-368. chromatography.

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CURRENT TOPICS IN MEMBRANES AND TRANSPORT. VOLUME 19

Native Membranes from Dog Kidney Outer Medulla, Enriched in Na,K-ATPase and Vesicular in Nature BLISS FORBUSH III Department of Physiology Yule UniversirySchool of Medicine New Haven,Connecticut

I.

INTRODUCTION

The o u t e r medulla of mammalian k i d n e y i s e x c e p t i o n a l l y r i c h i n Na,K-ATPase; b u t w h i l e it h a s been e x t e n s i v e l y employed a s a s o u r c e from which t o p u r i f y t h e enzyme, t h e n a t i v e microsomal membranes have n o t been w e l l c h a r a c t e r i z e d , n o r h a s t h e i r p o t e n t i a l as " s i d e d " memb r a n e v e s i c l e s been e x p l o i t e d . T h i s i n v e s t i g a t i o n w a s u n d e r t a k e n ( a ) t o examine t h e p u r i t y o f N a , K - A T P a s e i n p u r i f i e d f r a c t i o n of n a t i v e membranes from dog k i d n e y o u t e r m e d u l l a , ( b ) t o i s o l a t e a p o p u l a t i o n of " t i g h t " membrane v e s i c l e s , and ( c ) t o t e s t t h e h y p o t h e s i s t h a t d e t e r g e n t a c t i v a t i o n o f N a , K - A T P a s e i s a r e s u l t of i n c r e a s e d p e r m e a b i l i t y of membrane v e s i c l e s .

113

CopynghI 0 1983 by Academic Press, Inc. All rights of reproduction inany form reserved. lSBN 0-12-153319-0

BLISS FORBUSH 111

114

11.

METHODS AND RESULTS

I n o r d e r t o make membrane v e s i c l e s permeable, t h u s " a c t i v a t i n g " Na,K-ATPase a c t i v i t y by making l i g a n d s acc e s s i b l e t o b o t h s i d e s o f t h e membrane (see below) , memb r a n e s were p r e t r e a t e d f o r 1 0 min a t 2OoC w i t h 0.65 mg/m SDS, 1 0 mg/ml BSA, i n 25 mM imidazole (pH 7 . 5 ) , a t a m i crosomal p r o t e i n c o n c e n t r a t i o n of 0.02-0.20 mg/ml; add i t i o n a l BSA ( t o 4 mg/ml) was added i n a 5 - f o l d d i l u t i o n p r i o r t o a s s a y . The microsomal f r a c t i o n w a s p u r i f i e d from dog kidney o u t e r medulla by a c o n v e n t i o n a l proced u r e ( J d r g e n s e n and Skou, 1 9 7 1 ) , u s i n g o n l y t h e l i g h t e r p o r t i o n of t h e f i n a l p e l l e t ( P o s t and Sen, 1 9 6 7 ) . Na,K-ATPase a c t i v i t y a f t e r SDS a c t i v a t i o n w a s 5-8 pmoles Pi/mg min. P r o t e i n was determined by t h e Lowry method u n l e s s noted o t h e r w i s e . When t h e c r u d e microsomal f r a c t i o n was s e p a r a t e d on a 32-42% (w/v) s u c r o s e g r a d i e n t , a peak of Na,K-ATPase a c t i v i t y was found a t 37-40% s u c r o s e ( F i g . 1 A ) . Althoug t h e s e p a r a t i o n p r o f i l e was q u a l i t a t i v e l y s i m i l a r t o t h a t o b t a i n e d by Jdrgensen and Skou ( 1 9 7 1 , F i g s . 1 0 , 111, t h e d e t e r g e n t - a c t i v a t e d Na,K-ATPase a c t i v i t y of t h e peak f r a c t i o n s was a b o u t 4 - f o l d h i g h e r i n t h e p r e s e n t e x p e r i ments, 15-20 pmoles Pi/mg p r o t e i n min. T h i s s p e c i f i c a c t i v i t y a s w e l l a s a d e n s i t o m e t r i c scan of an SDS g e l of t h e peak f r a c t i o n shown i n F i g . 1 B i n d i c a t e s t h a t approximately h a l f of t h e p r o t e i n i n t h e plasma membranes sedimenting a t 38% s u c r o s e c o n s i s t s of Na,K-ATPase. T h i s i s comparable t o t h e p u r i t y of Na,K-ATPase i n nat i v e membranes from e e l e l e c t r i c organ (Yoda and Yoda, 1 9 8 1 ) . Note a l s o t h a t t h e o u a b a i n - i n s e n s i t i v e Mg-ATPase a c t i v i t y was found i n d i f f e r e n t membranes, i s o l a t e d a t -<32%s u c r o s e . The d e g r e e of a c t i v a t i o n by SDS v a r i e d o n l y a s m a l l amount across t h e peak of Na,K-ATPase act i v i t y , t y p i c a l l y from 5 - f o l d t o 3-fold ( n o t shown); t h i s i s c o n s i s t e n t w i t h p r e v i o u s o b s e r v a t i o n s t h a t on sucrose gradients, v e s i c l e s containing sucrose a r e not e a s i l y s e p a r a t e d from open membranes. Membrane v e s i c l e s which a r e " t i g h t " t o s m a l l molec u l e s have been found t o f l o a t a t low d e n s i t i e s i n isot o n i c dense c e n t r i f u g a t i o n media, due t o a t r a p p e d volume of less dense medium. On c e n t r i f u g a t i o n i n Hypaque (3,5-diacetamido-2,4,6-triiodobenzoate, meglumine s a l t , Winthrop L a b s . ) , t h e microsomal f r a c t i o n was s e p a r a t e d i n t o two broad peaks of p r o t e i n , a t 1 6 % and a t 2 2 % (w/v) Hypaque ( F i g . 2 ) . The l i g h t e r peak ( H 1 ) was found t o have a l m o s t no o u a b a i n - s e n s i t i v e Na,K-ATPas a c t i v i t y i n t h e absence of d e t e r g e n t ; however, a f t e r det e r g e n t a c t i v a t i o n , peak a c t i v i t y was 11-15 pmoles P i / m g

DOG KIDNEY OUTER MEDULLA IN Na,K-ATPase

I

t

32% Sucrose

115

10

Fraction No.

42% Sucrore

B

u

Sucrose Gradient Peak

I .o

c 0

-f

s

9

0.5

0.0 0

5

10

Gel Length (cm) F i g . 1 . ( A ) S u c r o s e g r a d i e n t c e n t r i f u g a t i o n o f the c r u d e m i crosomal f r a c t i o n f r o m dog k i d n e y o u t e r medulla. Twelve m i l l i g r a m s o f m i c r o s o m a l p r o t e i n w e r e c e n t r i f u g e d a t 20,000 rpm f o r 15 hr i n a Beckman SW27 rotor. F r a c t i o n s w e r e a s s a y e d d i r e c t 1 y f o r o u a b a i n sensitive (-@- and -insensitive (-.I ATPase a c t i v i t y (no d e t e r g e n t ) and p r o t e i n (-0-1 was e s t i m a t e d b y d y e b i n d i n g ( B r a d f o r d , 1976). T h e d e t e r g e n t - a c t i v a t e d s p e c i f i c a c t i v i t y o f f r a c t i o n s 1 5 and 1 6 was d e t e r m i n e d ( 1 8 pmoles Pi/mg m i n ) a f t e r p e l l e t i n g , u s i n g the Lowry p r o t e i n d e t e r m i n a t i o n . ( B ) D e n s i t o m e t r i c s c a n o f a Coomass i e B l u e - s t a i n e d p o l y a c r y l a m i d e g e l o f the p e a k f r a c t i o n a t 17% s u c r o s e f r o m a g r a d i e n t s i m i l a r t o t h a t i n A ( 6 % a c r y l a m i d e , 3.8% C t o 20% a c r y l a m i d e , 7% C, L a e m l l e b u f f e r s ) .

BLISS FORBUSH 111

10

5

0

I

t 10% Hypaque

5

Fraction No.

10

tI

22% Hypoque

F i g . 2. Hypaque g r a d i e n t c e n t r i f u g a t i o n o f the c r u d e m i c r o soma1 f r a c t i o n . 4 mg o f m i c r o s o m a l p r o t e i n was c e n t r i f u g e d a t 40,000 rpm f o r 1 5 hr i n a Beckman SW 50.1 rotor, on a 10-22% ( w / v ) Hypaque g r a d i e n t . F r a c t i o n s w e r e a s s a y e d a s i n F i g . 1 , e x c e p t t h a t a l l f r a c t i o n s w e r e a s s a y e d both w i t h and w i t h o u t a n SDS p r e t r e a t m e n t t o increase the p e r m e a b i l i t y of membrane vesicles. (-0-) estimated p r o t e i n , (-0-) o u a b a i n - s e n s i t i v e ATPase a c t i v i t y w i t h o u t d e t e r g e n t , (-A-) increment of ouabain-sensitive a c t i v i t y exposed b y d e t e r g e n t ,

m i n ) . These r e s u l t s a r e c o n s i s t e n t w i t h H 1 c o n s i s t i n g of t i g h t membrane v e s i c l e s and w i t h H2 c o n s i s t i n g of broken membranes o r l e a k y v e s i c l e s .

111.

DISCUSSION

While i n c r e a s e d p e r m e a b i l i t y of membrane v e s i c l e s (Jgkgensen and Skou, 1971; Walter, 1975; J o n e s e t al., 1977) h a s been most g e n e r a l l y g i v e n a s t h e e x p l a n a t i o n of d e t e r g e n t a c t i v a t i o n of N a , K - A T P a s e , o t h e r hypotheses have been advanced, i n c l u d i n g p e r t u r b a t i o n of

DOG KIDNEY OUTER MEDULLA IN Na,K-ATPase

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p r o t e i n - l i p i d i n t e r a c t i o n s , and a l t e r a t i o n s of s u b u n i t c o o p e r a t i v i t y . To f u r t h e r examine t h e s e h y p o t h e s e s , t r y p s i n t r e a t m e n t w a s employed; t r y p s i n , a s w e l l a s p r o n a s e , completely i n a c t i v a t e s Na,K-ATPase from t h e c y t o p l a s m i c f a c e of t h e membrane, b u t i s i n e f f e c t i v e from t h e o u t s i d e of t h e c e l l (Knauf e t ai., 1 9 7 4 ; G i o t t a , 1 9 7 5 ) . Treatment of microsomes w i t h a h i g h conc e n t r a t i o n o f p o r c i n e t r y p s i n (Sigma T 8 1 2 8 o r T0134) a l most e l i m i n a t e d Na,K-ATPase a c t i v i t y determined i n t h e absence o f SDS, i n d i c a t i n g t h i s a c t i v i t y i s due t o memb r a n e s w i t h an exposed c y t o p l a s m i c f a c e : i n s i d e - o u t v e s i c l e s , l e a k y v e s i c l e s , o r broken membranes. However when t r y p s i n exposure preceded SDS t r e a t m e n t , t h e i n crement of Na,K-ATPase a c t i v i t y exposed by SDS w a s comp l e t e l y u n a f f e c t e d by t r y p s i n , i n d i c a t i n g t h a t t h e cyt o p l a s m i c s i d e of t h e membrane i s i n a c c e s s i b l e t o t r y p s i n . This strongly supports t h e v e s i c l e hypothesis t o e x p l a i n SDS a c t i v a t i o n , and i s c o n s i s t e n t w i t h " c r y p t i c " a c t i v i t y a s b e i n g due t o r i g h t - s i d e - o u t v e s i c l e s i n t h i s p r e p a r a t i o n . Note t h a t t h i s c o n c e n t r a t i o n of t r y p s i n a b o l i s h e d a l l a c t i v i t y when it was used f o l l o w i n g SDS a c t i v a t i o n . The e f f e c t s of t r y p s i n and SDS on [3H]ouabain b i n d i n g i n t h e p r e s e n c e of NaMgATP o r MgPi a r e a l s o c o n s i s t e n t w i t h t h i s h y p o t h e s i s , a s w i l l be det a i l e d e l s e w h e r e . S i m i l a r examination of H 1 and H 2 w i t h t r y p s i n i n d i c a t e s t h a t H 1 c o n s i s t s of r i g h t - s i d e o u t membrane v e s i c l e s , w h i l e H 2 i s s t i l l a mixed populat i o n of r i g h t - s i d e - o u t and l e a k y o r i n s i d e - o u t v e s i c l e s . I n summary, i t h a s been found t h a t ( a ) some n a t i v e membranes i n dog kidney o u t e r medulla c o n s i s t of a t l e a s t 50% ( o f p r o t e i n ) N a , K - A T P a s e , ( b ) a p o p u l a t i o n o f membranes can be i s o l a t e d t h a t a r e t i g h t r i g h t - s i d e - o u t v e s i c l e s and t h a t e x h i b i t Na,K-ATPase a c t i v i t y o n l y when d e t e r g e n t a c t i v a t e d , and ( c ) r e s u l t s of t r y p s i n t r e a t ment of n a t i v e membranes a r e c o n s i s t e n t w i t h an i n c r e a s e d p e r m e a b i l i t y of membrane v e s i c l e s as an explanat i o n of detergent a c t i v a t i o n .

ACKNOWLEDGMENT

I thank M r s . Grace Jones f o r e x c e l l e n t t e c h n i c a l a s s i s t a n c e . This r e s e a r c h was supported by N I H g r a n t RO1-GM 27920 and by a PMA Foundation Research S t a r t e r Grant.

118

BLISS FORBUSH 111

REFERENCES

B r a d f o r d , M. M. ( 1 9 7 6 ) . Anal. B i o c h e m . 72, 248-254. G i o t t a , G . J. (1975). J . B i o l . C h e m . 250, 5159-5164. J o n e s , L. R., Besch, H. R . , J r . , and Watanabe, A . M. ( 1 9 7 7 ) . J . B i o l . C h e m . 252, 3315-3323. J b r g e n s e n , P. L., and Skou, J . C. (1971). B i o c h i m . B i o p h y s . A c t a 233, 366-380. Knauf, P. A . , P r o v e r b i o , F., and Hoffman, J. F. ( 1 9 7 4 ) . J . Gen. P h y s i o l . 63, 305-323. P o s t , R. L., and Sen, A . K. ( 1 9 6 7 ) . In "Methods i n Enzymology" (R. W. E s t a b r o o k and M. E. Pullman, e d s . ) , V o l . 10, pp. 762-768. Walter, H. ( 1 9 7 5 ) . Eur. J. B i o c h e m . 58, 595-601. Yoda, A . , and Yoda, S. (1981). Anal. B i o c h e m . 110, 82-88.

CURRENT TOPICS IN MEMBRANES AND TRANSPORT, VOLUME 19

Ultrastructure of Na,K-ATPase in Plasma Membranes ELISABETH SKRIVER AND ARVZD B. MAUNSBACH Depanment of Cell Biology the Institute of Anatomy University of Aarhus Aarhus. Denmark

PETER LETH J#RGENSEN Institute of Physiology University of Aarhus Aarhus, Denmark

I.

INTRODUCTION

F r e e z e - f r a c t u r e e l e c t r o n microscopy h a s v i s u a l i z e d t h e p r o t e i n s of t h e p u r i f i e d Na,K-ATPase a s intramemb r a n e p a r t i c l e s (Deguchi e t a l . , 1 9 7 7 ; Maunsbach e t a l . , 1 9 7 9 ) . The purpose of t h e p r e s e n t s t u d y was t o examine t h e o r g a n i z a t i o n of t h e Na/K pump b o t h i n i n t a c t plasma membranes of c e l l s of t h e t h i c k a s c e n d i n g limb of Henle (TALI and i n i s o l a t e d plasma membranes i s o l a t e d by d i f ferential centrifugation.

11.

MATERIALS AND METHODS

A c r u d e membrane f r a c t i o n w a s i s o l a t e d from homog e n a t e s of o u t e r r e n a l medulla of p i g kidney a s b e f o r e (J$rgensen, 1 9 7 4 ) . Mitochondria w e r e removed by cent r i f u g a t i o n a t 1 0 , 0 0 0 rpm f o r 1 5 min. The membranes 119

Copyright 0 1983 by Academic Press. Inc. All rights of reproduction in any form r ~ ~ e ~ e d . ISBN 0-12-153319-0

120

ELISABETH SKRIVER eta/.

F i g . 1 . V e s i c l e s i n i s o l a t e d membrane f r a c t i o n . M a g n i f i c a tion ~ 2 5 , 0 0 0 . F i g . 2 . Basal p a r t of c e l l s i n TAL showing l a t e r a l intereell u l a r s p a c e ( a r r o w s ) , p e r i t u b u l a r s p a c e ( P S ) , mitochondrion ( M ) , and P - f a c e and E - f a c e o f b a s o l a t e r a l membranes ( P and E ) . M a g n i f i c a t i o n ~46,000. F i g . 3 . F r e e z e - f r a c t u r e d membrane vesicles showing convex f r a c t u r e f a c e w i t h a h i g h f r e q u e n c y o f p a r t i c l e s and concave f r a c t u r e f a c e s w i t h few p a r t i c l e s . M a g n i f i c a t i o n x60,OOO. F i g . 4 . Membrane vesicles a f t e r DOC t r e a t m e n t f o l l o w e d b y f e r r i t i n i n c u b a t i o n . Notice f e r r i t i n on both membrane s u r f a c e s . M a g n i f i c a t i o n ~65,000.

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were c o l l e c t e d from t h e s u p e r n a t a n t by c e n t r i f u g a t i o n a t 2 0 , 0 0 0 rpm f o r 30 min ( S o r v a l l SS 3 4 r o t o r ) . N a , K A T P a s e a c t i v i t y w a s measured i n t h e membrane f r a c t i o n b e f o r e and a f t e r a c t i v a t i o n w i t h d e o x y c h o l a t e ( D O C ) . The i s o l a t e d membrane f r a c t i o n and t h e m e d u l l a r y t i s s u e w e r e b o t h a n a l y z e d by f r e e z e - f r a c t u r e e l e c t r o n microscopy. The membrane f r a c t i o n was a l s o s t u d i e d by t h i n s e c t i o n e l e c t r o n microscopy b e f o r e and a f t e r a c t i v a t i o n w i t h DOC and f o l l o w i n g i n c u b a t i o n w i t h c a t i o n i c f e r r i tin.

111.

RESULTS AND DISCUSSION

T h i n - s e c t i o n e l e c t r o n microscopy showed t h a t t h e membrane f r a c t i o n c o n s i s t e d o f v e s i c l e s w i t h d i a m e t e r s v a r y i n g from 1 0 0 0 t o 4 0 0 0 A ( F i g . 1 ) . F r e e z e - f r a c t u r e of b a s o l a t e r a l plasma membranes i n TAL c e l l s showed t h a t t h e P-face ( c y t o p l a s m i c l e a f l e t ) had a h i g h p a r t i c l e f r e q u e n c y w h i l e t h e E-face ( e x o p l a s m i c l e a f l e t ) w a s a l most d e v o i d of intramembrane p a r t i c l e s ( F i g . 2 ) . The membrane v e s i c l e s showed convex and concave f r a c t u r e f a c e s w i t h v a r y i n g p a r t i c l e f r e q u e n c i e s ( F i g . 3 ) . Thus, 4 5 % o f a l l convex f r a c t u r e f a c e s , b u t o n l y 3% o f t h e concave f r a c t u r e f a c e s , e x h i b i t e d a h i g h f r e q u e n c y of intramembrane p a r t i c l e s . The s i m i l a r i t y between t h e p a r t i c l e - r i c h P-face o f b a s o l a t e r a l c e l l membranes of i n t a c t TAL c e l l s and t h e p a r t i c l e - r i c h convex f r a c t u r e f a c e s of t h e membrane v e s i c l e s i d e n t i f i e s t h e m a j o r i t y of t h e plasma membrane v e s i c l e s a r e r i g h t - s i d e - o u t vesicles. The o r i e n t a t i o n o f t h e Na,K-ATPase i n t h e i s o l a t e d , u n t r e a t e d membrane v e s i c l e s i s t h u s d i s t i n c t l y asymmetric, i n c o n t r a s t t o p h o s p h o l i p i d v e s i c l e s rec o n s t i t u t e d w i t h p u r e N a , K - A T P a s e where t h e enzyme molec u l e s a r e randomly o r i e n t e d i n t h e membranes ( S k r i v e r e t a l . , 1980). The N a , K - A T P a s e a c t i v i t y i n t h e membrane f r a c t i o n w a s i n c r e a s e d 4- t o 5 - f o l d upon t r e a t m e n t w i t h DOC. T h i n - s e c t i o n e l e c t r o n microscopy showed t h a t DOC t r e a t ment w a s c o r r e l a t e d t o o p e n i n g of t h e plasma membrane v e s i c l e s and e x p o s u r e of b o t h membrane s u r f a c e s t o t h e medium. The u n t r e a t e d membrane v e s i c l e s e x c l u d e d cat i o n i c f e r r i t i n , w h i l e a f t e r DOC t r e a t m e n t c a t i o n i c f e r r i t i n w a s bound on b o t h membrane s u r f a c e s ( F i g . 4 ) . These o b s e r v a t i o n s i n d i c a t e t h a t t r e a t m e n t of t h e memb r a n e f r a c t i o n w i t h d e t e r g e n t opens t h e v e s i c l e s and res u l t s i n f r e e access o f ATP and c a t i o n s t o t h e c a t a l y t i c sites.

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ELISABETHSKRIVER eta/.

REFERENCES

Deguchi, N . , J b r g e n s e n , P. L., and Maunsbach, A. B. (1977). U1t r a s t r u c t u r e o f t h e sodium pump. Comparison of t h i n sect i o n i n g , n e g a t i v e s t a i n i n g , and f r e e z e - f r a c t u r e of p u r i f i e d , membrane-bound (Na+,K+) -ATPase. J. C e l l Biol. 75, 639-634. In J b r g e n s e n , P. L. ( 1 9 7 4 ) . I s o l a t i o n of (Na+ + K+)-ATPase. "Methods i n Enzymology" (S. F l e i s c h e r and L. Packer, e d s . ) , V o l . 32, P a r t B , pp. 277-290. Academic P r e s s , New York. Maunsbach, A. B., S k r i v e r , E . , a n d Jjzkgensen, P. L. ( 1 9 7 9 ) . U1t r a s t r u c t u r e o f p u r i f i e d Na,K-ATPase membranes. I n "Na,KATPase: S t r u c t u r e and K i n e t i c s " (J. C. Skou and J. G. Ndrby, e d s . ) , pp. 3-13. Academic P r e s s , New York. S k r i v e r , E . , Maunsbach, A. B., and Jldrgensen, P. L. (1980). U1t r a s t r u c t u r e of Na,K-transport v e s i c l e s r e c o n s t i t u t e d w i t h J. C e l l Biol. 8 6 , 746-754. p u r i f i e d r e n a l Na,K-ATPase.

CURRENT TOPICS IN MEMBRANES AND TRANSPORT, VOLUME 19

Electron Microscope Analysis of Two-Dimensional Crystals of Membrane-Bound Na,K-ATPase ARVZD B. MAUNSBACH AND EUSABETH SKRIVER Department of Cell Biology at the Institute of Anatomy University of Aarhus Aarhus. Denmark

HANS HERBERT, I

Mar-Planck-lnstitut f r Biochemie Abteilung f i r Struktwforschung I Martiensried bei Munchen M&achen,Federal Republic of Germany

PETER LETH J0RGENSEN Institute of Physiology University of Aarhus Aarhus, Denmark

I.

INTRODUCTION

Membrane-bound N a , K - A T P a s e i s o l a t e d from t h e o u t e r r e n a l m e d u l l a (Jjdrgensen, 1974) can b e o b s e r v e d by elect r o n microscopy a s 30 t o 5 0 8 s u r f a c e p a r t i c l e s p r o t r u d i n g above t h e p l a E e o f t h e membrane b i l a y e r a f t e r n e g a t i v e s t a i n i n g (Maunsbach and J$drgensen, 1 9 7 4 ) . W e have p r o p o s e d t h a t e a c h s u r f a c e p a r t i c l e c o n t a i n s one a , B u n i t ( p r o t o m e r ) of t h e enzyme p r o t e i n (Deguchi e t al., 1977; Maunsbach e t a l . , 1 9 7 9 ) and have r e c e n t l y d e m o n s t r a t e d t h a t two-dimensional c r y s t a l s c a n b e i n duced i n membrane f r a g m e n t s o f p u r i f i e d Na,K-ATPase d u r i n g i n c u b a t i o n w i t h v a n a d a t e and magnesium ( S k r i v e r e t a l . , 1981). W e r e p o r t here f o r t h e f i r s t t i m e t h a t computer-averaged images of t h e s e c r y s t a l s i n d i c a t e t h a t t h e y are composed o f p r o t o m e r s o f t h e enzyme p r o t e i n . ‘ P r e s e n t a d d r e s s : D e p a r t m e n t of V e d i c a l B i o p h y s i c s , K a r o l i n s k a I n s t i t u t e , S t o c k h o l m , Sweden.

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Copyright 0 1983 by Academic F’ress, Inc. All rights of reproductionin any form reserved. ISBN 0-12-1533194

ARVlD B. MAUNSBACH eta/.

124

F i g . 1 . T w o - d i m e n s i o n a l c r y s t a l s of membrane-bound Na,KATPase i n d u c e d by i n c u b a t i o n i n 0.25 mM NaVO3 and 1 mM MgC12 for 4 w e e k s at 4OC. F i g u r e 2 was c a l c u l a t e d f r o m p a r t o f the a r e a w i t h i n the box. x165,OOO.

11.

MATERIALS AND METHODS

Na,K-ATPase was p u r i f i e d from t h e o u t e r r e n a l medulla o f t h e p i g k i d n e y (Jfdrgensen, 1 9 7 4 ) . The enzyme was suspended i n 1 0 m M T r i s - H C 1 ( p H 7.5 a t 2OoC) and exposed t o 1 mM MgC12 and 0 . 2 5 mM NaV03. The membrane s u s p e n s i o n s were s t o r e d a t 4-6OC f o r p e r i o d s r a n g i n g from 2 h r t o 4 weeks and e l e c t r o n micrographs r e c o r d e d a f t e r n e g a t i v e s t a i n i n g w i t h u r a n y l acetate. Two-dimens i o n a l c o m p u t e r - r e c o n s t r u c t e d images were c a l c u l a t e d f o r a r e a s from vanadate-induced c r y s t a l s on a VAX 11/780 usi n g t h e e l e c t r o n micrograph p r o c e s s i n g s y s t e m "EM" (Hegerl, 1980)

.

111.

RESULTS AND DISCUSSION

Following i n c u b a t i o n o f t h e membrane-bound N a , K A T P a s e w i t h sodium monovanadate i n t h e p r e s e n c e o f magnesium, two-dimensional c r y s t a l s formed i n a r e p r o d u c i b l e way i n a l m o s t a l l membrane fragments ( F i g . 1 ) . S i n c e t h e p r e p a r a t i o n s show a h i g h p u r i t y w i t h r e s p e c t t o u l t r a s t r u c t u r e , enzymatic a s s a y s , and p r o t e i n compos i t i o n (Deguchi e t a l . , 1 9 7 7 ; J b r g e n s e n , 19741, t h e r e

TWO-DIMENSIONAL CRYSTALS OF MEMBRANE-BOUND Na,K-ATPase

125

F i g . 2 . C o m p u t e r - r e c o n s t r u c t e d image f r o m v a n a d a t e - i n d u c e d c r y s t a l . T h e l a r g e r e p e a t i n g u n i t s r e p r e s e n t enzyme p r o t e i n . T h e small, shaded r e g i o n s c o r r e s p o n d t o u r a n y l a c e t a t e - r i c h r e g i o n s and may represent d e p r e s s i o n s i n the membrane s u r f a c e . O n e u n i t c e l l i s o u t l i n e d . S c a l e : 1 nun c o r r e s p o n d s t o 2.3 1.

i s no doubt t h a t t h e c r y s t a l s a r e composed o f t h e prot e i n s o f N a , K-ATPase. The vanadate-induced two-dimensional c r y s t a l s showed a n o n o r t h o g o n a l l a t t i c e and many c r y s t a l s exh i b i t e d a h i g h d e g r e e of o r d e r . The d i f f r a c t i o n patt e r n s of t h e membrane c r y s t a l s e x t e n d e d t o 2 5 8. Twod i m e n s i o n a l c o m p u t e r - r e c o n s t r u c t e d images w e r e c a l c u l a t e d t o o b t a i n t h e averaged s t r u c t u r e of t h e N a , K A T P a s e p r o t e i n i n p r o j e c t i o n s p e r p e n d i c u l a r t o t h e memb r a n e . The u n i t c e l l d i m e n s i o n s o f t h e computera v e r a g e d image shown i n F i g . 2 are a = 69 f 4 8 , b = 5 3 k 4 8 , and y = 1 0 5 2'. The symmetry i s p l , and t h e u n i t c e l l c o n t a i n s o n l y one p r o t e i n p a r t i c l e which i s i n t e r p r e t e d as an a , $ u n i t ( p r o t o m e r ) s i n c e t h e r e i s no e v i d e n c e f o r o l i g o m e r i z a t i o n . The recons t r u c t e d images s u g g e s t t h a t t h e a , $ u n i t i n t h i s proj e c t i o n i s a r a t h e r compact s t r u c t u r e w i t h approximate dimensions 4 1 x 5 2 8 . I t s h o u l d be n o t e d t h a t t h e abs e n c e of e v i d e n c e f o r o l i g o m e r i z a t i o n w i t h i n t h e s e vanadate-induced c r y s t a l s d o e s n o t e x c l u d e t h e e x i s t ence o f o l i g o m e r s i n t h e membranes o u t s i d e t h e c r y s t a l s o r within c r y s t a l s during other experimental conditions. _+

ARVID B. MAUNSBACHeta/.

126

ACKNOWLEDGMENT

s u p p o r t e d by t h e Da i s h Medi s l Research Council.

REFERENCES

Deguchi, N . , J d r g e n s e n , P. L . , and Maunsbach, A. B. (1977). U l t r a s t r u c t u r e o f t h e sodium pump. J. C e l l B i o l . 7 5 , 619-634. Hegerl, R. (1980). New developments of t h e "EM" program system. P r o c . E u r . C o n g r . E l e c t r o n Microsc. 7 t h , 1 9 8 0 , Vol. 2 , pp. 700-701. Jdrgensen, P. L. (1974). P u r i f i c a t i o n and c h a r a c t e r i z a t i o n of (Na+ + K+)-ATPase. 111. B i o c h i m . B i o p h y s . A c t a 3 5 6 , 36-52. Maunsbach, A. B . , and Jgkgensen, P. L. (197$). U l t r a s t r u c t u r e of h i g h l y p u r i f i e d p r e p a r a t i o n s of (Na+ + K+)-ATPase from o u t e r medulla of t h e r a b b i t kidney. Proc. Int. Congr. E l e c t r o n Microsc. 8 t h , 1 9 7 4 , Vol. 2 , pp. 214-215. Maunsbach, A. B., S k r i v e r , E . , and Jgkgensen, P. L. (1979). U l t r a s t r u c t u r e of p u r i f i e d Na,K-ATPase membranes. I n "Na,K-ATPase: S t r u c t u r e and K i n e t i c s " (J. C. Skou and J. G. Ngfrby, e d s . ) , pp. 3-13. Academic P r e s s , New York. S k r i v e r , E . , Maunsbach, A. B . , and Jgkgensen, P. L. (1981). Formation of two-dimensional c r y s t a l s i n p u r e membranebound Na+,K+-ATPase. FEBS L e t t . 131, 219-222.

CURRENT TOPICS IN MEMBRANES AND TRANSPORT, VOLUME 19

Organization of the Transmembrane Segments of Na,K-ATPase. Labeling of Lipid Embedded and Surface Domains of the a-Subunit and ks Tryptic Fragments with [‘251] lodonaphthylazide, [32P]ATP, and Photolabeled Ouabain P E E R LETU J0RGENSEN Institute of Physiology University of Aarhus Aarhus, Denmark

STEPHEN J. D. W I S H Department of Biochemistry and Department of Membrane Research W e i m n n Institute of Science Rehovot. Israel

CARLOS GITLER Department of Membrane Research W e i m n n Institute of Science Rehovot, Israel

I.

INTRODUCTION

Combination of c o n t r o l l e d p r o t e o l y s i s w i t h s p e c i f i c c h e m i c a l l a b e l i n g can p r o v i d e i n f o r m a t i o n a b o u t t h e t o p o l o g y o f i n t r i n s i c membrane p r o t e i n s . In principle t h i s technique can be pursued t o high r e s o l u t i o n i f t h e p r o t e i n c a n be l a b e l e d from b o t h membrane s u r f a c e s and from w i t h i n t h e l i p i d b i l a y e r . The p u r p o s e o f t h i s work h a s been t o examine t h e o r g a n i z a t i o n o f t h e intramemb r a n o u s p o r t i o n o f t h e a - s u b u n i t i n p u r e , membrane-bound N a , K - A T P a s e ( J d r g e n s e n , 1 9 7 4 ) . C o v a l e n t l a b e l i n g of t h e a - s u b u n i t and i t s t r y p t i c f r a g m e n t s from w i t h i n t h e l i p i d b i l a y e r w i t h [ 1 2 5 I ] i o d o n a p h t h y l a z i d e ( B e r c o v i c i and G i t l e r , 1978; K a r l i s h e t a l . , 1977) w a s combined w i t h cov a l e n t l a b e l i n g w i t h 32P from [y32P]ATP a t t h e c y t o p l a s m i c s u r f a c e and w i t h [3H]NAP-ouabain (Rogers and Lazduns k i , 1 9 7 9 ) from t h e e x t r a c e l l u l a r s u r f a c e ( J d r g e n s e n e t a l . , 1982).

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128

11.

PETER L. J0RGENSEN

METHODS AND RESULTS

Control experiments confirm t h a t iodonaphthylazide l a b e l s segments of t h e p r o t e i n w i t h i n t h e l i p i d b i l a y e r . The s u s c e p t i b l e p r o t e i n r e s i d u e s a r e n o t o u t s i d e t h e l i p i d b i l a y e r , b e c a u s e l a b e l i n g o f t h e p r o t e i n i s unaff e c t e d b o t h by e x t e n s i v e p r o t e o l y s i s ( K a r l i s h e t a l . , 1977) and by t h e p r e s e n c e o f r e d u c e d g l u t a t h i o n e , a n i t r e n e s c a v e n g e r (Bayley and Knowles, 1980) i n t h e aqueous phase. I n a d d i t i o n , t h e 1 2 K segments o f t h e a - s u b u n i t produced by e x t e n s i v e p r o t e o l y s i s are select i v e l y e x t r a c t e d by a c i d chloroform/methanol o r by a c i d butanol. Both a t a low (0.25 mole i o d o n a p h t h y l a z i d e p e r mole a - s u b u n i t ) and a t a h i g h c o n c e n t r a t i o n o f iodonapht h y l a z i d e ( 1 0 moles i o d o n a p h t h y l a z i d e p e r mole as u b u n i t ) , a b o u t 50% o f i o d o n a p h t h y l a z i d e added t o t h e medium was c o v a l e n t l y a t t a c h e d t o p r o t e i n and l i p i d . I n b o t h c o n d i t i o n s , more t h a n 9 0 % of p r o t e i n l a b e l w a s recovered i n 1 2 K polypeptides a f t e r e x t e n s i v e p r o t e o l y s i s . A t t h e l o w i o d o n a p h t h y l a z i d e c o n c e n t r a t i o n , t h e NH2t e r m i n a l 46K fragment of t h e a - s u b u n i t was p r e f e r e n t i a l 1 labeled, while a t t h e higher iodonaphthylazide concentra t i o n , b o t h t h e 46K fragment and t h e COOH-terminal 58K fragment were l a b e l e d . Phosphorus-32 from [ y 3 2 P ] -ATP i s i n c o r p o r a t e d exc l u s i v e l y i n t o t h e 46K fragment from t h e c y t o p l a s m i c mem b r a n e s u r f a c e . P h o t o l a b e l i n g w i t h [3H]-NAP-ouabain p r e sumably from t h e e x t r a c e l l u l a r s u r f a c e l a b e l s a l l t h e major f r a g m e n t s , 7 8 K , 58K, and 4 6 K . A model f o r p o s i t i o n i n g of t h e 1 2 K segments r e l a t i v t o t h e primary t r y p t i c s p l i t s and t o t h e s i t e s f o r l a b e l i n g i s shown i n F i g . 1. The segment o f t h e a - s u b u n i t between bond 1 and bond 3 i s l a b e l e d w i t h b o t h o u a b a i n and 32P from ATP. S i n c e b o t h t r y p t i c s p l i t s o c c u r a t t h e c y t o p l a s m i c s u r f a c e , t h i s segment o f t h e a - s u b u n i t i s proposed t o t r a v e r s e t h e b i l a y e r t w i c e . The neighb o r i n g segment o f t h e a - s u b u n i t between bonds 2 and 3 i n c o r p o r a t e s up t o two m o l e c u l e s of i o d o n a p h t h y l a z i d e p e r mole a - s u b u n i t and is assumed t o c o n s i s t of two 12K segments which t r a v e r s e t h e b i l a y e r . The p a r t of t h e a - s u b u n i t between bond 1 and t h e COOH-terminal i s a l s o assumed t o t r a v e r s e t h e b i l a y e r s i n c e it i n t e r a c t s w i t h o u a b a i n a t t h e e x t r a c e l l u l a r s u r f a c e , and i t s NH2t e r m i n a l i s exposed a t t h e c y t o p l a s m i c s u r f a c e . The s i d e d n e s s of t h e COOH-terminal of t h e a - s u b u n i t i s unknown. T o g e t h e r , t h i s i n f o r m a t i o n s u g g e s t s t h e model i n which f o u r segments of t h e a - s u b u n i t t r a v e r s e t h e b i l a y e r w i t h t h e NH2-terminals and t h e t h r e e t r y p s i n s e n s i t i v e bonds exposed a t t h e c y t o p l a s m i c s u r f a c e .

IGANIZATIONOF THE TRANSMEMBRANE SEGMENTS

Q

129

Q l L E

78K

F i g . 1 . Model f o r ( a ) a r r a n g e m e n t of a - s u b u n i t i n membrane-bound Na ,K-ATPase and (b) t r y p t i c c l e a v a g e of a - s u b u n i t . The numbered a r r o w s mark the s i t e s of p r i m a r y t r y p t i c c l e a v a g e i n p r e s e n c e o f KCI or NaCl. ( ) i n d i c a t e s i n c o r p o r a t i o n of [I2511 i o d o n a p h t h y l a z i d e . Position of t r y p t i c s p l i t s and NHzand COOH-terminals w e r e d e t e r m i n e d i n p r e v i o u s work (J$drgensen, 1975; Farley e t a l . , 1980).

111.

DISCUSSION

3 A f t e r i n c o r p o r a t i o n of [ Hladamantane d i a z a r i n e i n t o membrane-bound N a , K - A T P a s e , a l l l a b e l i n g s i t e s are l o c a t e d w i t h i n t h e 5 8 K segment of t h e a - s u b u n i t , w h i l e no l a b e l i n g i s d e t e c t e d w i t h i n t h e 4 1 K (46K) segment forming t h e NH2-terminal of t h e a - s u b u n i t ( F a r l e y e t a l . , 1 9 8 0 ) . These a u t h o r s proposed t h a t t h e a - s u b u n i t con-

s i s t s of two s t r u c t u r a l domains, a NH2-termina1, cytop l a s m i c domain and a COOH-terminal membrane-associated domain. The r e a s o n f o r t h i s d i s c r e p a n c y w i t h o u r res u l t s i s n o t clear a t p r e s e n t , b u t n o t a b l e d i f f e r e n c e s i n t h e b e h a v i o r of i o d o n a p h t h y l a z i d e and adamantane d i a z a r i n e a r e a p p a r e n t . Adamantane d i a z a r i n e h a s a p a r t i t i o n c o e f f i c i e n t of only 1 , 7 5 0 f o r e r y t h r o c y t e ghosts while t h e p a r t i t i o n c o e f f i c i e n t f o r iodonaphtyl-

PETER L. JORGENSEN

130

azide is as high as 163,000 (Bayley and Knowles, 1980). Adamantane diazarine may also be more selective than iodonaphthylazide because diazirines photoisomerize to diazo compounds that are powerful electrophiles and may label proteins by paths not related to carbene reactions. A very important point in comparing results obtained with the two probes is that no data of exhaustive proteolysis are available for preparations labeled with adamantane diazirine. The resistance of protein-bound iodonaphthylazide to extensive proteolysis of the membranes by trypsin or thermolysin (Karlish et al., 1977; Jqkgensen e t a l . , 1982) constitutes a major argument for the expected behavior of iodonaphthylazide as an apolar phase affinity label.

REFERENCES

Bayley, H., and Knowles, J. (1980). Photogenerated r e a g e n t s for membranes: S e l e c t i v e l a b e l i n g of i n t r i n s i c membrane p r o t e i n s i n the human e r y t h r o c y t e membrane. B i o c h e m i s t r y 1 9 , 3883-3892. 5 [1251]i o d o n a p h t h y l a z i d e , B e r c o v i c i , T. , and G i t l e r , C. (1978) a r e a g e n t t o determine t h e p e n e t r a t i o n of p r o t e i n s i n t o the l i p i d b i l a y e r of b i o l o g i c a l membranes. B i o c h e m i s t r y 1 7 , 1484-1489. F a r l e y , R. A., Goldman, D. W . , and Bayley, H. C. (1980). I d e n t i f i c a t i o n of r e g i o n s o f t h e c a t a l y t i c subunit o f Na,K-ATPase embedded w i t h i n t h e c e l l membrane. J . B i o l . Chem. 2 5 5 , 860-864. P u r i f i c a t i o n of Na,K-ATPase. Biochim, Jgkgensen, P. L. (1974) B i o p h y s . A c t a 356, 36-52. Jgkgensen, P. L. (1975). Conformational changes i n Na,K-ATPase. B i o c h i m . B i o p h y s . A c t a 401 , 399-415. Jgkgensen, P. I,., K a r l i s h , S. J. D., and G i t l e r , C. (1982). Evidence f o r t h e o r g a n i z a t i o n of t h e transmembrane segments of Na,K-ATPase based on l a b e l i n g lipid-embedded and s u r f a c e doJ . Biol. Chem. 257, 7435-7442. mains of t h e a-subunit. K a r l i s h , S. J . D., Jdrgensen, P. L . , and G i t l e r , C. ( 1 9 7 7 ) . I d e n t i f i c a t i o n o f a membrane-embedded segment of t h e l a r g e p o l y p e p t i d e c h a i n of Na,K-ATPase. Nature (Landon) 269, 715-717. Rogers, T., and Lazdunski, M. (1979). P h o t o a f f i n i t y l a b e l i n g of Biochemistry t h e d i g i t a l i s r e c e p t o r i n t h e Na,K-ATPase. 1 8 , 135-140.

.

.

CURRENT TOPICS IN MEMBRANES AND TRANSPORT. VOLUME 19

Structural Studies on Lamb Kidney Na,K-ATPase J . H. COLLINS, L. K.LANE, E. LING,' A. S C W A R l Z , AND A. (REEVES) ZOT Department of Pharmacology and Cell Biophysics Universiw of Cincinnati College of Medicine Cincinnati, Ohio

B. FORBUSH 111 Department of Physiology Yale University School of Medicine New Haven, Connecticut

INTRODUCTION

I.

I n t h i s a r t i c l e we r e p o r t the r e s u l t s o f s t r u c t u r a l We also report the s t u d i e s on lamb k i d n e y Na,K-ATPase. i s o l a t i o n of p r o t e o l i p i d components from p u r i f i e d enzyme l a b e l e d t.$Sith a p h o t o a f f i n i t y d e r i v a t i v e of o u a b a i n . 2

11.

METHODS

F o r o u r s e q u e n c e s t u d i e s w e have p u r i f i e d gram quanfrom t h e o u t e r m e d u l l a o f lamb k i d ney (Lane e t a l . , 1 9 7 9 ) . The h i g h l y p u r i f i e d enzyme w a s s o l u b i l i z e d i n 1%SDS, and chromatographed on a S e p h a r o s e

t i t i e s of N a , K - A T P a s e

'Present a d d r e s s : D e p a r t m e n t of B i o c h e m i s t r y , E . K . for Y e n t a l R e t a r d a t i o n , Wal t h a m , Y a s s a c h u s e t t s .

Shriver Center

n

L A b b r e v i a t i o n s : NAB-ouabain, 2-nitro-5-azidobenzoyl d e r i v a t i v e of o u a b a i n ; S D S , s o d i u m d o d e c y l s u l f a t e . 131

Copyright 0 1983 by Academic Press, Inc. All rights of reprchction in m y form ~ ~ e ~ e d . ISBN O-I2-153319-0

132

J. H. COLLINS eta/.

CL-6B column r u n i n 0 . 1 % SDS. T h i s p r o c e d u r e s e p a r a t e s the three proteins present i n the preparation: a (catal y t i c s u b u n i t , Mr 2 1 0 0 , 0 0 0 ) , B ( g l y c o p r o t e i n , M r E 5 0 , 0 0 0 ) , and y ( p r o t e o l i p i d , M y = 1 2 , 0 0 0 ) . The y compon e n t i s s e p a r a t e d from s a l t s , l i p i d s , and o t h e r l o w mol e c u l a r w e i g h t m a t e r i a l by chromatography on Sephadex LH-60 i n a n o r g a n i c s o l v e n t . (Using t h e same method, a, 8 , and y w e r e p u r i f i e d from r a t k i d n e y N a , K - A T P a s e , and found t o be s i m i l a r i n s i z e and amino a c i d composit i o n t o t h e c o r r e s p o n d i n g lamb k i d n e y p r o t e i n s . ) The a - s u b u n i t of t h e lamb k i d n e y enzyme was r e d u c e d and c a r b o x y m e t h y l a t e d a t SH g r o u p s w i t h i o d o a c e t i c a c i d , and i t s N - t e r m i n a l s e q u e n c e w a s d e t e r m i n e d t o be GlyArg-Asp-Lys-Tyr-Gluw i t h t h e u s e of t h e Beckman Sequencer. The c a r b o x y m e t h y l a t e d a - s u b u n i t was p a s s e d t h r o u g h a n AG1-X2 column i n o r d e r t o remove SDS, and t h e n d i gested with trypsin. The d i g e s t i o n was v e r y i n c o m p l e t e , presumably due t o t h e i n a c c e s s i b i l i t y o f t h e l y s i n e and arginine residues. A r a p i d and c o m p l e t e c l e a v a g e a t t h e 4 0 a r g i n i n e s o f c1 c o u l d be o b t a i n e d , however, i f t h e p r o t e i n were s u c c i n y l a t e d p r i o r t o t r y p t i c d i g e s t i o n . When a t r y p t i c d i g e s t o f c a r b o x y m e t h y l a t e d , s u c c i n y l a t e d c1 i s chromatographed on a Sephadex G-50 column, f o u r m a j o r , w e l l - d e f i n e d f r a c t i o n s ( A t o D ) a r e r e p r o d u c i b l y obt a i n e d . F r a c t i o n A , which e l u t e s a t t h e v o i d volume of t h e column, c o n t a i n s a g g r e g a t e d , v e r y h y d r o p h o b i c pept i d e s presumably d e r i v e d from r e g i o n s of a t h a t are b u r i e d w i t h i n t h e membrane l i p i d b i l a y e r i n t h e n a t i v e enzyme. F r a c t i o n s B and D c o n t a i n w a t e r - s o l u b l e p e p t i d e : and t o g e t h e r a c c o u n t € o r a b o u t 7 5 % of t h e a p o l y p e p t i d e c h a i n . S e v e n t e e n s m a l l ( 2 - 1 5 r e s i d u e s ) p e p t i d e s have been i s o l a t e d from f r a c t i o n D by c o n v e n t i o n a l chromatog r a p h i c p r o c e d u r e s , and t h e s e a r e b e i n g s e q u e n c e d . The p e p t i d e s of f r a c t i o n s A t o C a r e b e i n g s e p a r a t e d by HPLC.

111.

DISCUSSION

W e have d e v e l o p e d t h e f i r s t p r e p a r a t i v e - s c a l e p r o cedure f o r t h e i s o l a t i o n of t h e p r o t e o l i p i d a s s o c i a t e d w i t h t h e Na,K-ATPase (Reeves e t al., 1980) and have c a l l e d t h i s v e r y h y d r o p h o b i c p r o t e i n t h e y component of t h e enzyme. T h i s procedure h a s been modified r e c e n t l y t o g i v e i n c r e a s e d y i e l d s ( C o l l i n s e t a l . , 1 9 8 2 ) . The l y o p h i l i z e d y f r a c t i o n from t h e S e p h a r o s e CL-6B column (see above) was d i s s o l v e d i n a minimum volume of 88% f o r m i c a c i d and chromatographed o n a Sephadex LH-60 colw.r? i n a 3 : l ( v / v ) m i x t u r e o f 95% e t h a n o l and 88%

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formic a c i d . Two v e r y s i m i l a r p r o t e o l i p i d f r a c t i o n s were i s o l a t e d : y l , which emerges a t t h e v o i d volume of t h e column, and y2. When rechromatographed on t h e same LH-60 column, y l and y 2 a r e i n t e r c h a n g e a b l e , s u g g e s t i n g t h a t y l i s an a g g r e g a t e d form of y2. S t a t i s t i c a l comparisons of t h e amino a c i d composit i o n s of p r o t e o l i p i d s from v a r i o u s s o u r c e s i n d i c a t e t h a t y is s i m i l a r t o pr oteolipids t h a t a r e p resen t i n sarcop l a s m i c r e t i c u l u m from c a r d i a c ( C o l l i n s e t a l . , 1 9 8 1 ) and s k e l e t a l muscle ( J . H C o l l i n s , A . S. Zot, and E . G. K r a n i a s , u n p u b l i s h e d ) , b u t v e r y d i f f e r e n t from t h e DCCDb i n d i n g p r o t e o l i p i d of t h e m i t o c h o n d r i a 1 ATPase ( B l o n d i n , 1 9 7 9 1 , t h e p r o t e o l i p i d a s s o c i a t e d w i t h t h e Ca-ATPase (MacLennan e t a l . , 1 9 7 2 ) and phospholamban, t h e phosp h o r y l a t a b l e a c t i v a t o r of t h e Ca-ATPase of c a r d i a c s a r coplasmic r e t i c u l u m ( C o l l i n s e t al., 1 9 8 1 ) . S t r u c t u r a l s t u d i e s on y a r e i n p r o g r e s s and i t w i l l be of i n t e r e s t t o compare t h e sequence of y w i t h t h e sequences of various other proteolipids. Highly p u r i f i e d lamb kidney Na,K-ATPase was photoa f f i n i t y l a b e l e d w i t h t r i t i a t e d NAB-ouabain ( C o l l i n s e t al., 1 9 8 3 ) . The enzyme was l a b e l e d on t h e a and y comp o n e n t s , a s determined by polyacrylamide g e l e l e c t r o phoresis, This l a b e l i n g p a t t e r n i s q u a l i t a t i v e l y the same a s t h a t o b t a i n e d p r e v i o u s l y by Forbush et a l . ( 1 9 7 8 ) w i t h l a b e l e d p i g kidney N a , K ATPase. The l a b e l e d lamb kidney Na,K-ATPase was mixed w i t h u n l a b e l e d c a r r i e r enzyme, s o l u b i l i z e d i n S D S , and chromatographed on Sepharose CL-6B and Sephadex LH-60 (see a b o v e ) . The l a b e l e d p r o t e o l i p i d s c o p u r i f i e d w i t h t h e u n l a b e l e d prot e o l i p i d components ( y l and y 2 ) i s o l a t e d p r e v i o u s l y . The c o p u r i f i c a t i o n of l a b e l e d and u n l a b e l e d m a t e r i a l p r o v i d e s e v i d e n c e t h a t t h e p r o t e o l i p i d i s o l a t e d by Reeves e t al. (1980) i s i d e n t i c a l t o t h a t l a b e l e d by Forbush e t a l . ( 1 9 7 8 ) .

ACKNOWLEDGMENTS

T h i s work w a s s u p p o r t e d by a g r a n t f r o m t h e M u s c u l a r Dyst r o p h y A s s o c i a t i o n a n d b y N I H g r a n t s R01-AM-20875, R01-GM-27920, and 5-T32-HL-07382. K04-HL-00555, Pol-HL-22619,

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REFERENCES

Blondin, G . A. (1979). Resolution of t h e mitochondria1 N,N-dicyclohexylcarbodiimide binding p r o t e o l i p i d f r a c t i o n i n t o t h r e e s i m i l a r s i z e d p r o t e i n s . B i o c h e m . B i o p h y s . R e s . Commun. 8 7 , 1087-1094. C o l l i n s , J. H., Kranias, E. G . , Reeves, A. S., B i l e z i k j i a n , L. M., and Schwartz, A. (1981). I s o l a t i o n of phospholamban and a second p r o t e o l i p i d component from canine c a r d i a c sarcoplasmic reticulum. B i o c h e m . B i o p h y s . R e s . Commun. 9 9 , 796-803. C o l l i n s , J. H., Fo-rbush, B., Lane, L. K . , Ling, E . , Schwartz, A., and Zot, A. S. (1982). P u r i f i c a t i o n and c h a r a c t e r i z a t i o n of a p r o t e o l i p i d component o f Na,K-ATPase l a b e l e d with a p h o t o a f f i n i t y d e r i v a t i v e of ouabain. B i o c h i m . B i o p h y s . Acta 6 8 6 , 7-12. Forbush, B., Kaplan, J. H . , and Hoffman, J. F. (1978). C h a r a c t e r i z a t i o n of a new p h o t o a f f i n i t y d e r i v a t i v e of ouabain: Labeling of t h e l a r g e polypeptide and of a p r o t e o l i p i d component B i o c h e m i s t r y 1 7 , 3667-3676. of t h e Na,K-ATPase. Lane, L. K., P o t t e r , J. D., and C o l l i n s , J. H. (1979). Large-scale p u r i f i c a t i o n of Na,K-ATPase and i t s s u b u n i t s from lamb kidney medulla. P r e p . B i o c h e m . 9 , 157-190. MacLennan, D. H . , Yip, C. C . , Iles, G. H . , and Seeman, P. (1972). . I s o l a t i o n of sarcoplasmic reticulum p r o t e i n s . C o l d S p r i n g Harbor S y m p . Q u a n t . Biol. 37, 469-477. Reeves, A . S., C o l l i n s , J. H . , and Schwartz, A. (1980). I s o l a t i o n and c h a r a c t e r i z a t i o n of Na,K-ATPase p r o t e o l i p i d . Biochern. B i o p h y s . R e s . C o m u n . 9 5 , 1591-1598.

CURRENT TOPICS IN MEMBRANES AND TRANSPORT, VOLUME 19

Two Slightly Different a-Subunit Components of Kidney Na,K-ATPase Induced by Heat Treatment T. OHTA Depanmem of Biology and Chemistry Jichi Medical School Yakushiji,Minamikawachi-machi Kawachi-gun, Tochigi. Japan

M.K A WAMURA,' T. HASEGA WA, H. ISHIKURA, AND K.NAGANO Department of Biology Jichi Medical School Yakushiji,Mlnamikawachi-machi,Kawachi-gun Tochigi, Japan

I.

INTRODUCTION

C h e m i c a l m u l t i p l i c i t y of t h e a - s u b u n i t of N a , K A T P a s e i s i n d i c a t e d i n several v e r t e b r a t e n e r v o u s t i s s u e enzymes (Sweadner, 1 9 7 9 ) a s w e l l as i n t h e a - s u b u n i t o f t h e b r i n e s h r i m p Na,K-ATPase ( P e t e r s o n a n d Hokin, 1 9 8 1 ) . T h e s e d a t a s u g g e s t a closer r e e x a m i n a t i o n o f t h e s u b u n i t c o m p o s i t i o n of p u r i f i e d Na,K-ATPase p r e p a r a t i o n s

11.

MATERIALS AND METHODS

Na,K-ATPase w a s p u r i f i e d from dog k i d n e y by t h e method of Jjdrgensen. SDS-gel e l e c t r o p h o r e s i s w a s carried o u t by t h e method of Weber and Osborn. U n l e s s o t h e r w i s e s t a t e d , s a m p l e s were h e a t e d i n a b o i l i n g water b a t h f o r 5 min. [3H]NAP-ouabain w a s p r e p a r e d a s desc r i b e d by R o g e r s and L a z d u n s k i ( 1 9 7 9 ) . ' P r e s e n t a d d r e s s : D e p a r t m e n t of D e p a r t m e n t of B i o l o g y , W c u l t y of Science C h i b a M i v e r s i t y Y a y o i - c h o C h i b a , J a p a n . 135

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For p h o t o a f f i n i t y l a b e l i n g , a sample of Na,K-ATPase was i n c u b a t e d i n a medium c o n t a i n i n g 5 m~ MgC12, 5 m Tris-Phosphate, and 5 0 m i m i d a z o l e b u f f e r ( p H 7 . 4 ) i n t h e p r e s e n c e o f 5 U M [3H]NAP-ouabain a t 37OC f o r 30 min. A c o n t r o l sample was p r e i n c u b a t e d i n t h e same medium, b u t i n t h e p r e s e n c e of 1 mM c o l d ouabain i n s t e a d of [3H]NAP-ouabain, washed by c e n t r i f u g a t i o n , and t h e r e a f ter l a b e l e d w i t h [3H]NAP-ouabain a s above. A f t e r b e i n g i l l u m i n a t e d f o r 5 min under an u l t r a v i o l e t lamp, samples were d i l u t e d and c e n t r i f u g e d . The p r e c i p i t a t e s were suspended i n 5% t r i c h l o r o a c e t i c a c i d (TCA) t o a l l o w nonc o v a l e n t l y bound [ 3H]NAP-ouabain t o d i s s o c i a t e . Both samples were t h e n r e c o n c e n t r a t e d i n t h e s t a r t i n g volume of t h e imidazole b u f f e r by c e n t r i f u g a t i o n .

111.

RESULTS

When Na,K-ATPase was analyzed by SDS-polyacrylamidc g e l e l e c t r o p h o r e s i s a f t e r b o i l i n g i n t h e SDS medium (1% SDS, 1%2-mercaptoethanol, 1 0 mbf sodium phosphate, and 4 M u r e a ) , two c l o s e l y spaced bands appeared i n s t e a d of t h e s i n g l e a band o f t h e unheated c o n t r o l sample. The

F i g . 1 . SDS-gel e l e c t r o p h o r e s i s s h o w i n g the d o u b l e t bax of the a - s u b u n i t . (A) P u r i f i e d a-subunit. ( B ) Standard proti p l u s p u r i f i e d a-subunit. ( C ) S t a n d a r d p r o t e i n s (RNA p o l y m e r a and bovine s e r u m a l b u m i n ) .

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SUBUNIT COMPONENTS INDUCED BY HEAT

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cm)

SDS-gel electrophoresis of Na ,K-ATPase labeled with (A) Radioactivity profiles of labeled ( 0 ) and control ( 0 ) sample. (B) Densitogram of the stained gel. Fig. 2.

[ 3H]NAP-ouabain.

two bands were d e s i g n a t e d as a 1 ( t h e s l o w e r moving band) and a 1 1 ( t h e s l i g h t l y f a s t e r b a n d ) . M o l e c u l a r w e i g h t s of a 1 and a 1 1 w e r e d e t e r m i n e d t o b e 1 0 1 , 0 0 0 and 9 3 , 0 0 0 , respectively (Fig. 1). To check t h e t e m p e r a t u r e dependence of t h e band s p l i t t i n g , t h e enzyme p r o t e i n s o l u b i l i z e d i n SDS medium w a s t r e a t e d a t v a r i o u s t e m p e r a t u r e s f o r 1 0 min. Enzyme p r e p a r a t i o n s t r e a t e d a t 50 o r 6 0 ° C gave o n l y t h e o r i g i n a l s i n g l e t band, w h i l e t h e d o u b l e t s t r u c t u r e a p p e a r e d above 70°C. The amount of t h e a 1 1 band i n c r e a s e d up t o a c e r t a i n l e v e l w i t h i n c r e a s i n g t e m p e r a t u r e above 70°C.

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The d o u b l e t bands were w e l l s e p a r a t e d on 5.6% acrylamide g e l s and w e r e e s s e n t i a l l y u n a f f e c t e d by t h e p r e s e n c e o r absence of r e d u c i n g a g e n t s . TCA-resistant b i n d i n g , which probably re r e s e n t e d t h e c o v a l e n t l y p h o t o l a b e l e d f r a c t i o n of t h e [ H I N A P ouabain (Forbush e t al., 1978), was s m a l l i n q u a n t i t y b u t r e l i a b l y p o s i t i v e . Enzyme t r e a t e d w i t h TCA w a s analyzed by g e l e l e c t r o p h o r e s i s ( F i g . 2 ) . A s e x p e c t e d , all of t h e TCA-resistant l a b e l c o n c e n t r a t e d i n t h e two a bands, i n d i c a t i n g t h a t b o t h of them were d e r i v e d from a - s u b u n i t of Na,K-ATPase. When t h e enzyme was p r e t r e a t e d w i t h c o l d ouabain b e f o r e l a b e l i n g w i t h [3H]NAPouabain, no s i g n i f i c a n t r a d i o a c t i v i t y peak was observed on t h e g e l .

3

REFERENCES

.

CharacForbush, B. , Kaplan, J. H. , and Hoffman, J . F. (1978) t e r i z a t i o n o f a p h o t o a f f i n i t y d e r i v a t i v e o f ouabain: L a b e l i n g of t h e l a r g e p o l y p e p t i d e and o f a p r o t e o l i p i d B i o c h e m i s t r y 1 7 , 3667-3676. component o f t h e Na,K-ATPase. P e t e r s o n , G. L . , and Hokin, E. (1981). Molecular w e i g h t and s t o i c h i o m e t r y o f t h e sodium and p o t a s s i u m - a c t i v a t e d adenos i n e t r i p h o s p h a t a s e s u b u n i t s . J . B i o l . C h e m . 2 5 6 , 37513761. Rogers, T. B., and Lazdunski, M. (1979). P h o t o a f f i n i t y l a b e l i n g of t h e d i g i t a l i s receptor i n t h e ( N a + K ) - a c t i v a t e d adenos i n e t r i p h o s p h a t a s e . B i o c h e m i s t r y 18, 135-140. Sweadner, K. J. ( 1 9 7 9 ) . Two m o l e c u l a r forms o f ( N a + + K+)s t i m u l a t e d ATPase i n b r a i n . S e p a r a t i o n , and d i f f e r e n c e i n a f f i n i t y f o r s t r o p h a n t h i d i n . J. B i o l . C h e m . 2 5 4 , 60606067.

CURRENT TOPICS IN MEMBRANES AND TRANSPORT, VOLUME 19

Radiation Inactivation Analysis of Na,K-ATPase PAUL OVOLENGHI Institute of Physiology University ofAarhus Aarhus, Denmark

J. CLIVE ELLORY Department of Physiology University of Cambridge Cambridge, England

ROGER A. KLEIN Medical Research Council, Molten0 Institute University of Cambridge Cambridge, England

I.

INTRODUCTION

Radiation i n a c t i v a t i o n is a well-established technique f o r determining t h e molecular weights of both s o l u b l e and membrane-bound enzymes (Kepner and Macey, 1968; Kempner and S c h l e g e l , 1 9 7 9 ; E l l o r y , 1 9 7 9 ) . The method i n v o l v e s i r r a d i a t i o n o f s a m p l e s w i t h s p a r s e l y i o n i z i n g r a d i a t i o n ( e . g . , h i g h - e n e r g y e l e c t r o n s ) and works b e s t f o r enzymes o f known m o l e c u l a r w e i g h t . Q u a n t i t a t i o n of r e s u l t s normally involves e m p i r i c a l c a l i b r a t i o n by i r r a d i a t i n g enzymes of e s t a b l i s h e d molecular sizes. The u n i v e r s a l l y a p p l i e d f o r m u l a , u s i n g d a t a s e l e c t e d by Kepner and Macey ( 1 9 6 8 1 , i s MW = 6 . 4 x 1O1l/D37 where 037 i s t h e d o s e i n r a d s necessary t o reduce t h e a c t i v i t y t o l / e ( i . e . , 37%) of i t s o r i g i n a l value. There a r e s e v e r a l d i f f i c u l t i e s and a s s u m p t i o n s i n a p p l y i n g t h i s t e c h n i q u e t o membranebound p e p t i d e s . I r r a d i a t i o n a r t i f a c t s can involve t e m p e r a t u r e , f r e e r a d i c a l , a n d s c a v e n g e r e f f e c t s , and s a m p l e s a r e u s u a l l y l y o p h i l i z e d and i r r a d i a t e d i n V ~ C U 139

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PAUL OTTOLENGHI eta/.

10” 1

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Dose (Mrads) F i g . 1 . R a d i a t i o n i n a c t i v a t i o n of p i g k i d n e y Na,K-ATPase a c t i v i t y . Enzyme ( S D S - a c t i v a t e d p i g k i d n e y m i c r o s o m e s , see Hansen e t a l . , 1 9 7 9 ) s u s p e n d e d i n 250 mM sucrose, 30 mM h i s t i d i n e , pH 7 . 4 , was l y o p h i l i z e d a n d i r r a d i a t e d i n V ~ C U Ow i t h 1 6 MeV electrons a t 2 - 3 Mrad min-1 a t a t e m p e r a t u r e of 1Oo-6O0C. P e r s p e x H X d o s i m e t r y was u s e d . Enzyme w a s a s s a y e d i n 1 3 5 mM N a , 1 5 mM K , 4 mM Mg, 3 mM ATP, 30 mM h i s t i d i n e , w i t h and w i t h o u t 1 mM o u a b a i n , Control a c t i v i t y was 550 pmoles ATP a t 37OC and pH 7 . 4 . s p l i t .hr-l ‘mg p r o t e i n - 1

.

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a t 0'-40°C. A f u r t h e r consideration is t h a t calibrat i o n i s u s u a l l y c a r r i e d o u t w i t h s o l u b l e enzymes, and so may n o t n e c e s s a r i l y a p p l y f o r membrane-bound s y s t e m s . Nevertheless, s i n c e t h e molecular weight of Na,K-ATPase i s s t i l l t h e s u b j e c t o f c o n s i d e r a b l e debate, i t i s worthwhile a p p l y i n g t h i s i n d i r e c t t e c h n i q u e t o t h i s s y s t e m . P r e v i o u s estimates f o r N a , K - A T P a s e mol e c u l a r w e i g h t s by t h i s method r a n g e between 2 0 0 , 0 0 0 and 606,000, w h i l e r e s u l t s f o r K-dependent pNPPase act i v i t y a p p e a r t o be 250% o f t h e v a l u e f o r t o t a l A T P a s e (Kepner and Macey, 1968; E l l o r y e t a l . , 1979; K a n i i k e et a l . , 1 9 7 6 ) .

11.

RESULTS AND DISCUSSION

Our r e s u l t s from i r r a d i a t i o n of k i d n e y microsome are p r e s e n t e d i n F i g . 1. The d a t a g i v e an e x c e l l e n t f i t t o a s t r a i g h t l i n e down t o below 0 . 0 1 % r e s i d u a l a c t i v i t y , w i t h a 037 o f 2 . 4 2 Mrads, c o r r e s p o n d i n g t o a m o l e c u l a r w e i g h t o f 262,000 u s i n g Kepner and Macey's f o r m u l a . A p i g b r a i n enzyme p r e p a r a t i o n gave an i d e n t i c a l v a l u e . R e s u l t s f o r t h e two p a r t i a l r e a c t i o n s , K-pNPPase and N a - A T P a s e , are shown i n F i g . 2 . The two f u n c t i o n s behave i d e n t i c a l l y , and d i f f e r e n t l y from N a , K - a c t i v a t e d ATPase. Over t h e r a n g e 1-6 Mrad a s t r a i g h t l i n e c a n b e drawn t h r o u g h t h e d a t a p o i n t s , g i v i n g a 037 of 4 . 0 Mrad (MW 1 6 0 , 0 0 0 ) f o r k i d n e y enzyme, and a 037 of 3.43 Mrad (MW 1 8 6 , 0 0 0 ) f o r b r a i n enzyme. This i s not half t h e v a l u e f o r Na,K-activated ATPase a c t i v i t y 7 W h e n t h e s e p a r t i a l r e a c t i o n s are a s s a y e d a f t e r h i g h e r d o s e s o f r a d i a t i o n t h e f i t d e v i a t e s from l i n e a r i t y and may i n d i c a t e multi-hit o r p a r t i a l i n a c t i v a t i o n behavior. Other e x p e r i m e n t s on o u a b a i n b i n d i n g and Na,K-pNPPase i n d i c a t e s i m i l a r complex b e h a v i o r and lower i n i t i a l s l o p e s f o r decay o f a c t i v i t y . I r r a d i a t i o n i n t h e p r e s e n c e o f l i g a n d s (Na, K , A T P , Mg) d i d n o t a f f e c t t h e t a r g e t s i z e . Measurement o f k i n e t i c s ( t h e a p p a r e n t K m f o r K+ of pNPPase) on s a m p l e s i r r a d i a t e d w i t h 6 Mrad d i d n o t show any d i f f e r e n c e s from the control. W e conclude t h a t i r r a d i a t i o n i n a c t i v a t i o n a n a l y s i s of Na,K-activated ATPase a c t i v i t y g i v e s simple r e s u l t s c o n s i s t e n t w i t h an apparent molecular weight around 260,000. T h i s i s i n good agreement w i t h some u l t r a c e n t r i f u g a l d a t a (Esmann e t a l . , 1 9 8 1 ) , and a l t h o u g h subj e c t t o some t h e o r e t i c a l c r i t i c i s m , i s c e r t a i n l y t o o Na,K-ATPase

142

PAUL OlTOLENGHI eta/.

10"

I

0

I

I

I

1

I

5

10

15

20

25

I

30

Dose (Mrads) F i g . 2. R a d i a t i o n i n a c t i v a t i o n of K-pNNPase a n d Na-ATPase Enzyme a s i n F i g . 1 . A s s a y c o n d i t i o n s : either 150 mM Na, 4 mM Mg, 3 mM ATP, 30 mM h i s t i d i n e , w i t h a n d w i t h o u t 1 mM o u a b a i n (= Na-ATPase, A ); o r 150 mM K , 20 mM Mg, 10 mM pNPP, 30 mM h i s t i d i n e , w i t h a n d w i t h o u t 1 mM ouabain (= K-pNPPase, 0 ) T h e l i n e c o r r e s p o n d i n g t o the e x p e r i m e n t i n F i g . 3 i s a l s o s h o w n .

activities.

.

RADIATION INACTIVATION ANALYSIS OF Na,K-ATPase

143

l a r g e t o be accommodated w i t h i n c u r r e n t v a l u e s f o r a n a , $ - s u b u n i t , e v e n t a k i n g a c c o u n t of bound c a r b o h y d r a t e . The m o s t p r o b a b l e estimates of t h e m o l e c u l a r w e i g h t s of a and $ p e p t i d e s and f o r c a r b o h y d r a t e are i n t h e r a n g e 92,000-120,000, 32,000-40,000, a n d 9,000-20,000, res p e c t i v e l y (see P e t e r s o n and Hokin, 1 9 8 1 ) . The b e h a v i o r of t h e measured p a r t i a l r e a c t i o n s (pNPPase, Na-ATPase, o u a b a i n - b i n d i n g ) i s complex, b u t d o e s n o t f i t w i t h a s i m p l e model where pNPPase r e p r e s e n t s h a l f t h e s i z e of N a , K - a c t i v a t e d ATPase.

REFERENCES

E l l o r y , C. (1979). R a d i a t i o n i n a c t i v a t i o n f o r molecular s i z e determinations. T I B S , pp. N99-Nl00. E l l o r y , J. C . , Green, J. P., J a r v i s , S. M . , and Young, J. D. (1979). Measurement of t h e a p p a r e n t molecular volume of membrane-bound t r a n s p o r t systems by r a d i a t i o n i n a c t i v a t i o n . J. P h y s i o l . ( L o n d o n ) 2 9 5 , 1OP-UP. Esmann, M . , C h r i s t i a n s e n , C . , K a r l s s o n , K.-A., Hansson, G. C., and Skou, J. C . (1981). Hydrodynamic p r o p e r t i e s of s o l u b i l i z e d ( N a + + K+)-ATPase from rectal g l a n d s o f S q u a l us a c a n t h i a s . B i o c h i m . B i o p h y s . A c t a 603, 1-12. Hansen, O . , J e n s e n , J . , NZrby, J. G . , and O t t o l e n g h i , P. (1979). A new p r o p o s a l r e g a r d i n g t h e s u b u n i t composition of (Na+ + K+) -ATPase. Nature ( L o n d o n ) 280, 410-412. Kaniike, K . , Miyamoto, H . , and Kurogochi, Y. (1976). Molecular weights of Na+,K+-dependent ATPase and K+-dependent phosp h a t a s e . A c t a Med. K i n k i Univ. 1 , 23-29. Kempner, E. S . , and S c h l e g e l , W. (1979). S i z e d e t e r m i n a t i o n of enzymes by r a d i a t i o n i n a c t i v a t i o n . A n a l . Biochern. 92, 2-10. Kepner, G. R., and Macey, R. I. (1968). Membrane enzyme systems: Molecular s i z e d e t e r m i n a t i o n s by r a d i a t i o n i n a c t i v a t i o n . B i o p h y s . A c t a 1 6 3 , 188-203. P e t e r s o n , G. L . , and Hokin, L. E . (1981). Molecular weight and s t o i c h i o m e t r y of t h e sodium- and p o t a s s i u m - a c t i v a t e d adenosine t r i p h o s p h a t a s e s u b u n i t s . J. B i o l . Chern. 2 5 6 , 3751-3761.

This Page Intentionally Left Blank

CURRENT TOPICS IN MEMBRANES AND TRANSPORT, VOLUME 19

Stoichiometrical Binding of Ligands to Less than 160 Kilodattons of Na,K-ATPase H. MATSUI, Y. HAYASHI, H. HOMAREDA AND M . TAGUCHI Department of Biochemistry Kyorin University School of Medicine Mitaka. Tokyo,Japan

I.

METHODS

N a , K - A T P a s e w a s p u r i f i e d from o u t e r medulla of dog kidney by t h e method of JBrgensen (1974) m o d i f i e d by Y . Hayashi and R. L. P o s t ( p e r s o n a l communication). P r o t e i n w a s estimated a c c o r d i n g t o Hayashi and P o s t ( 1 9 8 0 ) . About 1 5 mg of enzyme p r o t e i n w i t h s p e c i f i c act i v i t y of more t h a n 4 0 umoles Pi/mg p r o t e i n / m i n a t 37OC w a s o b t a i n e d p e r one z o n a l c e n t r i f u g a t i o n w i t h Beckman T i 15 r o t o r . The h i g h e s t a c t i v e f r a c t i o n r e a c h e d a s p e c i f i c a c t i v i t y o f 45. S i n c e t h e a c t i v i t y o f t h e p r e s e n t p r e p a r a t i o n w a s a l m o s t twice a s much as t h a t o f o u r p r e v i o u s p r e p a r a t i o n t o which o u a b a i n b i n d i n g w a s 3 nmoles/mg p r o t e i n ( M a t s u i et a l . , 1 9 7 7 1 , i t was examined whether t h i s p r e p a r a t i o n c o u l d b i n d 6 nmoles ouabain/mg p r o t e i n i n p r o p o r t i o n t o t h e i n c r e a s e d specific activity.

145

Copyright 0 1983 by Academic Press, Lnc. All rights of reproduction in any form reserved. ISBN 0-12-1533190

H. MATSUI et el.

146

0

u

S a t u r a t i o n Binding = 5 . 5 nmol/mg

.-'(Unbound

)

0

2

I

I

4

6

1

100

T o t a l Ouabain (uM) Fig. 1 .

T i t r a t i o n o f o u a b a i n b i n d i n g t o p u r i f i e d Na,K-ATPast

The r e a c t i o n mixture i n a f i n a l volume of 1 ml c o n t a i n e d 0.5 mq pu. r i f i e d e n z y m e , 5 mM MgCl2, 4 mM P i , 1 mM EDTA, 50 mM imidazole-HC1 (pH 7.0 a t 37OC), and v a r i o u s amounts of [ 3 H ] o u a b a i n ; the mixture was i n c u b a t e d for 30 m i n a t 37OC. A f t e r c e n t r i f u g a t i o n a t 40,000 rpm f o r 20 m i n , the r a d i o a c t i v i t y o f [ 3 H ] o u a b a i n i n the p e l l e t w a s determined. ( 0 ) , ( A ) r e p r e s e n t d i f f e r e n t enzyme p r e p a r a t i o n s .

11.

RESULTS AND DISCUSSION

F i g u r e 1 shows t h e t i t r a t i o n p r o f i l e of [ 3Hlouabair b i n d i n g t o t h e p u r i f i e d Na,K-ATPase. S i n c e t h e enzyme c o n c e n t r a t i o n used (%3 P M ) w a s t w o o r d e r s of magnitude g r e a t e r t h a n t h e d i s s o c i a t i o n c o n s t a n t of t h e enzymeo u a b a i n complex (%30 nM), t h e ouabain added t o t h e react i o n m i x t u r e w a s c o m p l e t e l y t r a p p e d by t h e enzyme u n t i l t h e ouabain c o n c e n t r a t i o n exceeded t h e enzyme c o n c e n t r a t i o n . The maximal v a l u e of o u a b a i n b i n d i n g w a s 6.3 nmoles/mg rotein. I n a d d i t i o n t o [ 3H] o u a b a i n b i n d i n g , [14C]ATP, $2K+, and 22Na+ b i n d i n g were measured t o see t h e s t o i c h i o m e t r y of l i g a n d b i n d i n g ( T a b l e I ) . The amounts of o u a b a i n , ATP, K+, and N a + s p e c i f i c a l l y bound t o t h e same enzyme p r e p a r a t i o n were 6.1, 5 . 5 , 1 1 . 3 , and 14.7 nmoles/mg p r o t e i n , r e s p e c t i v e l y . S i n c e t h e amounts

STOICHIOMETRICAL BINDING OF LIGANDS TO Na,K-ATPase

3 S t o i c h i o m e t r i c a l B i n d i n g of [ HIOuabain, and 22Na+ t o Na,K-ATPasea

TABLE I.

L i g a n d s added 3

5 pM [ H]Ouabaind 10 pM [3H]Ouabaind 100 pM [%I]Ouabain

14

1 0 pM [14C]ATP 20 pM [ CIATP 20 pM 42K+ 50 pM 42K+

500 pM

147

[14C]ATP, 42K+,

L i g a n d s i n p e l l e t (nmol/mg) Nonspecific specific T o t a l Unboundb b i n d i n g binding'

Water space (pl/mg)

--

6.06 6.04 6.10

13.ge 12.2f

5.45 5.57

17.2 17.3

0.78 1.96

9.87 11.26

14.7 15.7

5.52 12.96

14.29 14.68

15.3 15.7

0

6.10 6.16 7.48

0.04 0.12 1.38

5.59 5.88

0.14 0.31

0 0

10.91 13.95

0.26 0.73

22.80 35.34

2.99 7.70

0

<0.02

a

The reaction m i x t u r e (1 m l ) c o n t a i n e d 0 . 5 mg enzyme, 5 0 mM imidazole-HC1 (pH 7.8 a t OOC), 1 mM EDTA, 1 0 mM [3H]sucrose, and i n d i c a t e d l i g a n d w a s i n c u b a t e d a t O°C f o r 10 min. A f t e r c e n t r i f u g a t i o n a t 40,000 r p m f o r 20 min, t h e r a d i o a c t i v i t i e s i n t h e p e l l e t w e r e d e t e r m i n e d . The w a t e r space i n t h e p e l l e t w a s measured w i t h [ 3 H ] s u c r o s e . bunbound = water s p a c e x free l i g a n d c o n c e n t r a t i o n . CSpecific b i n d i n g of [3H]ouabain and [14C]ATP = t o t a l - unSpecific b i n d i n g of 42K a n d 22Na = T o t a l - t o t a l w i t h bound. o u a b a i n = t o t a l - [unbound + n o n s p e c i f i c b i n d i n g ] . d I n c u b a t e d w i t h 5 mM MgC12, 4 mM P . and n o n r a d i o a c t i v e 1 sucrose a t 37OC f o r 30 min. eMeasured w i t h c o n t r o l t u b e s c o n t a i n i n g [ 3 H ] s u c r o s e a n d nonradioactive ouabain. 5 mM MgC12, 0.1 M N a C l , and fMeasured w i t h [14C]sucrose. 0.1 M KCI were added a f t e r 1 h r i n c u b a t i o n w i t h [3H]ouabain. 9 A t o t a l of 5 p g o l i g o m y c i n was added t o i n c r e a s e t h e a f f i n i t y f o r Na'.

of bound l i g a n d s a r e s a t u r a t i o n l e v e l s ( M a t s u i and Homareda, 1982), t h e o b s e r v e d r a t i o of bound l i g a n d s i n d i c a t e s t h a t t h e s t o i c h i o m e t r y of o u a b a i n , ATP, ,'K and N a + b i n d i n g t o t h e enzyme i s 1 : l : 2 : 3. The maxim a l v a l u e of o u a b a i n b i n d i n g , 6 . 3 nmoles/mg p r o t e i n , c o r r e s p o n d s t o one m o l e c u l e of o u a b a i n bound p e r 1 6 0 k i l o - d a l t o n s of p r o t e i n . Consequently, t h e s e r e s u l t s

148

H. MATSUI etal.

s u g g e s t t h a t t h e main p r o t e i n components € o r t h e funct i o n a l u n i t of t h e enzyme c o n s i s t of one a and one B chain.

ACKNOWLEDGMENT

Supported by g r a n t s from t h e M i n i s t r y of Education, S c i e n c e , and C u l t u r e of Japan.

REFERENCES Hayashi, Y . , and Post, R. L. (1980). Fed. Proc., Fed. Am. SOC. E x p . B i o l . 39, 1704. Jgkgensen, P. L. (1974). B i o c h i m . B i o p h y s . A c t a 3 5 6 , 36. Matsui, H . , Hayashi, Y . , Homareda, H . , and Kimimura, M. (1977). B i o c h e m . B i o p h y s . Res. Commun. 7 5 , 373. Matsui, H. , and Homareda, H. (1982). J. B i o c h e m . ( T o k y o ) 9 2 , 193.

CURRENT TOPICS IN MEMBRANES AND TRANSPORT, VOLUME 19

The Active Site Structure of Na,K-ATPase: Location of a Specific Fluorescein Isothiocyanate-Reactive Site CMVTHIA T. C A R I . , ROBERTA. FARLEY, AND LEWIS CANTLEY Department of Biochemiswy and Moleculur Biology Harvard Universiry Cambridge. Massachusetts

I t h a s r e c e n t l y been d e m o n s t r a t e d t h a t f l u o r e s c e i n i s o t h i o c y a n a t e ( F I T C ) c o v a l e n t l y reacts w i t h p u r i f i e d N a , K - A T P a s e and c o m p l e t e l y i n h i b i t s t h e A T P a s e a c t i v i t y when added a t a s l i g h t molar e x c e s s ( K a r l i s h , 1979; K a r l i s h et a l . , 1 9 7 9 ) . T h i s i n h i b i t i o n w a s p r e v e n t e d by ATP, s u g g e s t i n g m o d i f i c a t i o n a t t h e a c t i v e s i t e o f t h e enzyme. I n t h i s r e p o r t w e d e s c r i b e f u r t h e r charact e r i s t i c s o f t h e f l u o r e s c e i n - l a b e l e d Na,K-ATPase. FITC reacts w i t h a s t o i c h i o m e t r y e q u a l t o t h e s t o i c h i o m e t r y of o u a b a i n o r v a n a d a t e b i n d i n g t o t h e p u r i f i e d enzyme. The l a b e l e d s i t e i s l o c a t e d a b o u t 1 4 from t h e s i t e o f d i v a l e n t c a t i o n b i n d i n g d u r i n g c a t a l y s i s b u t i s found on a d i f f e r e n t t r y p t i c fragment o f t h e A T P a s e t h a n t h e a s p a r t a t e residue t h a t i s phosphorylated during turn3ver.

149

Copyright 0 1983 by Academlc Press. Inc. All rights of reprcduclion in any form reserved. ISBN 0-12-153319-0

CYNTHIA T. CARlLLl eta/.

150

I.

LOCATION OF THE FLUORESCEIN-LABELED SITE ON THE PROTEIN

The p u r i f i e d Na,K-ATPase from r e n a l medulla was l a b e l e d w i t h 1 0 p M FITC i n 50 m M T r i s - H C 1 , 1 0 0 mM N a C 1 , 5 mM EDTA, pH 9 . 2 , f o r 30 min a't 25OC. P r o t e i n w a s s e p a r a t e d from u n r e a c t e d F I T C by g e l f i l t r a t i o n . F l u o r e s c e i n - l a b e l e d A T P a s e was t r e a t e d w i t h t r y p s i n o r chymotrypsin a c c o r d i n g t o p r e v i o u s l y p u b l i s h e d proc e d u r e s ( F a r l e y e t al., 1980) t o g e n e r a t e p r o t e o l y t i c f r a g m e n t s whose l o c a t i o n s w i t h i n t h e a - s u b u n i t o f t h e A T P a s e are known ( C a s t r o and F a r l e y , 1 9 7 9 ) . A f t e r separ a t i o n o f t h e p r o t e o l y t i c f r a g m e n t s by SDS-polyacrylamide g e l e l e c t r o p h o r e s i s t h e l o c a t i o n o f t h e f l u o r e s c e i n l a b e l e d f r a g m e n t s w a s d e t e r m i n e d by f l u o r e s c e n c e a f t e r i l l u m i n a t i o n w i t h l o n g wavelength W l i g h t . F l u o r e s c e i n was found t o be a s s o c i a t e d w i t h t h e 77,000-dalton chymot r y p t i c fragment and t h e 58 I 000-dalton t r y p t i c f r a g m e n t , i n d i c a t i n g t h a t t h e r e a c t i v e s i t e s are l o c a t e d w i t h i n t h e c a r b o x y - t e r m i n a l h a l f of t h e a - p o l y p e p t i d e . No f l u o r e s c e n c e w a s a s s o c i a t e d w i t h t h e NH2-terminal 41,000-dalton t r y p t i c fragment p r e v i o u s l y shown t o cont a i n t h e a s p a r t a t e r e s i d u e t h a t i s p h o s p h o r y l a t e d by ATP d u r i n g t u r n o v e r ( C a s t r o and F a r l e y , 1 9 7 9 ) . Although t h e f l u o r e s c e i n - l a b e l e d A T P a s e e x h i b i t s Na+-dependent and K+-dependent f l u o r e s c e n c e changes ( K a r l i s h , 1979; K a r l i s h e t al., 1 9 7 9 ) , w i t h t r y p s i n c l e a v a g e t h e l a b e l e d A T P a s e i s no l o n g e r s e n s i t i v e t o t h e s e l i g a n d s . 11.

STOICHIOMETRY O F FITC LABELING

A f t e r m o d i f i c a t i o n by F I T C t h e p u r i f i e d Na,K-ATPase was d e n a t u r e d i n 1%SDS, and t h e a b s o r p t i o n of t h e fluorescein-ATPase complex w a s measured a t 495 nm, t h e a b s o r p t i o n maximum. P r o t e i n d e t e r m i n a t i o n s were made e i t h e r by amino a c i d a n a l y s i s a f t e r a c i d h y d r o l y s i s o r by t h e method o f Lowry. Using a n e x t i n c t i o n c o e f f i c i e n t f o r t h e f luorescein-ATPase complex of 75 mM-1crn-l a s t o i c h i o m e t r y of 2.7 nmoles/mg w a s measured f o r an enzyme p r e p a r a t i o n t h a t had a n ouabain-binding s t o i c h i o m e t r y of 2.5 nmoles/mg. P u r i f i c a t i o n of t h e f l u o r e s c e i n l a b e l e d a - s u b u n i t s by g e l f i l t r a t i o n i n SDS y i e l d e d a p r e p a r a t i o n of a-subunits with a f l u o r e s c t i n stoichiomet r y of 4 . 2 nmoles/mg. No f l u o r e s c e n c e was found assoc i a t e d w i t h t h e 8-subunit o f t h e A T P a s e . These r e s u l t s i n d i c a t e t h a t F I T C r e a c t s w i t h a p p r o x i m a t e l y one s i t e pel o u a b a i n ( o r n u c l e o t i d e ) b i n d i n g s i t e t o c a u s e complete i n h i b i t i o n of N a , K - A T P a s e a c t i v i t y . The s i t e of m o d i f i c a t i o n i s on t h e a - p o l y p e p t i d e and t h e r e a p p e a r s t o be a p p r o x i m a t e l y one s i t e p e r two a - p o l y p e p t i d e s .

FlTC SITE ON Na.K-ATPase

111.

151

FLUORESCENCE ENERGY TRANSFER FROM FLUORESCEIN TO A DIVALENT CATION Although t h e f l u o r e s c e i n - l a b e l e d ATPase h a s no

A T P a s e a c t i v i t y , it w i l l s t i l l h y d r o l y z e p - n i t r o p h e n y l -

p h o s p h a t e and b i n d v a n a d a t e i n t h e p r e s e n c e o f a d i valent cation. S i t e s of b i n d i n g o f t h e s e l i g a n d s , t h e r e f o r e , must s t i l l e x i s t on t h e enzyme. P r e v i o u s work h a s shown t h a t i t i s p o s s i b l e t o t r a p o n e mole o f a d i v a l e n t c a t i o n p e r mole o f v a n a d a t e a t t h e a c t i v e s i t e , and t h a t t h i s t r a p p e d c a t i o n d i s s o c i a t e s v e r y s l o w l y , a s v a n a d a t e d i s s o c i a t e s from t h e enzyme, o v e r s e v e r a l h o u r s (Smith e t a ] . , 1 9 8 0 ) . The d i v a l e n t cat i o n s Co2+ and Cu2+ w i l l s u b s t i t u t e f o r Mg2+ i n t h e norm a l o p e r a t i o n of t h e N a , K - A T P a s e , and w i l l a l s o a b s o r b l i g h t i n t h e s e c t r a l r e g i o n where f l u o r e s c e i n w i l l f l u o r e s c e . Co$+ o r Cu2+ w a s t r a p p e d on f l u o r e s c e i n l a b e l e d Na,K-ATPase, and t h e quenching of f l u o r e s c e i n f l u o r e s c e n c e w a s measured. Quenching w a s found t o depend on t h e p r e s e n c e o f enzyme-bound d i v a l e n t c a t i o n , and n o t t o be a f f e c t e d by unbound c a t i o n . A n a l y s i s o f t h e r e s u l t s of t h e s e experiments i n d i c a t e s t h a t t h e from t h e b i n d i n g s i t e f l u o r e s c e i n moiety i s 14 ? 2 f o r t h e d i v a l e n t c a t i o n , and i s bound v e r y r i g i d l y .

ACKNOWLEDGMENT

T h i s work w a s supported by g r a n t #GM 26199 from the N a t i o n a l I n s t i t u t e s of H e a l t h , and by a p o s t d o c t o r a l f e l l o w s h i p from t h e C y s t i c F i b r o s i s Foundation (R.A.F.)

.

REFERENCES

C a s t r o , J . , and F a r l e y , R. A. ( 1 9 7 9 ) . J. Biol. Chern. 254, 22212228. F a r l e y , R. A . , Goldman, D. W., and Bayley, H. ( 1 9 8 0 ) . J. Biol. C h e m . 255, 860-864. K a r l i s h , S. J. D. (1979). In "Na-K-ATPase: S t r u c t u r e and K i n e t i c s " (J. C. Skou and J. G. Nhrby, e d s . ) , pp. 115-128. Academic P r e s s , N e w York. K a r l i s h , S. J. D., Beaug6, L. A. , and Glynn, I. M. (1979). N a t u r e (London) 282 , 333-335. Smith, R. L., Zinn, K . , and C a n t l e y , L. C . (1980). J. Biol. C h e m . 255, 9852-9859.

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CURRENT TOPICS IN MEMBRANEF AND TRANSPORT, VOLUME 19

Subunit Distribution of Sulfhydryl Groups and Disulfide Bonds in Renal Na,K-ATPase M.K A WAMURA,' T.OHTA, AND K. NAGANO Depanmeni of Biology Jichi Medical School Yakushiji, Minnmiknwachi-machi Kawachi-gun, Tochigi. Japan

I.

INTRODUCTION

S u l f h y d r y l g r o u p s and d i s u l f i d e bonds h a v e been s u g g e s t e d t o h a v e e s s e n t i a l r o l e s i n Na,K-ATPase (see S c h o o t e t a l . , 1980; Kawamura e t a l . , 1 9 8 0 ) . Here w e r e p o r t t h e s u b u n i t d i s t r i b u t i o n of t h e s e t w o k i n d s of g r o u p s o n a p u r i f i e d Na,K-ATPase.

11.

MATERIALS AND METHODS

P u r i f i e d Na,K-ATPase w a s o b t a i n e d from dog k i d n e y by t h e method o f J B r g e n s e n . T i t r a t i o n s o f f r e e s u l f h y d r y l g r o u p s were p e r formed i n a medium c o n t a i n i n g 1%SDS w i t h 1 mM N - [ e t h y l l-14C]maleimide ([14C]NSM). The number of d i s u l f i d e ' P r e s e n t a d d r e s s : D e p a r t m e n t of B i o l o g y , Chiba l h i v e r s i t y , Yayoi-cho, Chiba, Japan. 153

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TABLE I.

Subunit Distribution of Free Sulfhydryl Groups and Disulfide Bonds of Na,K-ATPase

Sulfur group

SH

ss

SH +- SS a

Sul f h y d r y l groups titrated Na,K-ATPase Subunit (mole/mole)a (Y B 21.2 15.5 39.4

14.5 2.8 17.8

5.0 9.8 14.8

Y

1.5 2.7 6.0

M o l e c u l a r w e i g h t was assumed t o be 2 5 0 , 0 0 0 .

bonds w a s determined from t h e d i f f e r e n c e i n q u a n t i t y of s u l f h y d r y l groups t i t r a t e d b e f o r e and a f t e r r e d u c t i o n of t h e enzyme. The d i s t r i b u t i o n of t h e 1 4 C l a b e l over t h e s u b u n i t s of N a , K - A T P a s e a f t e r t r e a t m e n t w i t h [14C]NEM w a s d e t e r mined by Sepharose CL-4B column chromatography.

111.

RESULTS

The t o t a l number of f r e e s u l f h y d r y l groups of N a , K ATPase t i t r a t a b l e i n t h e p r e s e n c e of 1%SDS was found t o be 2 3 . 3 k 2 . 0 ( n = 4 ) w i t h [14C]NEM. The m o l e c u l a r w e i g h t of t h e u n i t o f Na,K-ATPase was assumed t o be 250,000. A s k a r i e t a l . ( 1 9 7 9 ) and Schoot et a l . (1978) e s t i m a t e d t o t a l s u l f h y d r y l groups i n one Na,K-ATPase molecule t o be 2 4 and 3 6 , r e s p e c t i v e l y . O u r r e s u l t was n e a r l y ident i c a l t o t h a t of Askari. The s u b u n i t d i s t r i b u t i o n of s u l f h y d r y l groups determined w i t h [14C]NEM is shown i n Table I. The number of s u l f h y d r y l groups i n each subu n i t was c a l c u l a t e d from t h e p e r c e n t a g e i n e a c h f r a c t i o n of t o t a l r a d i o a c t i v i t y r e c o v e r e d from t h e column of Sepharose 4B. Reduction of d i s u l f i d e bonds of Na,K-ATPase w i t h 1 m~ d i t h i o t h r e i t o l i n t h e p r e s e n c e of 1%SDS followed by a l k y l a t i o n w i t h [14C]NEM i n c r e a s e d d e t e c t a b l e s u l f h y d r y l groups up t o 38.4 f 2.0 ( n = 3 ) , s u g g e s t i n g t h e p r e s e n c e of 8-9 d i s u l f i d e bonds i n t h e u n i t of Na,KA T P a s e (Table I). T h i s w a s a l s o confirmed by t h e d i f f e r e n t i a l l a b e l i n g experiment, where t h e enzyme p r e a l k y l a t e d w i t h c o l d NEM was reduced w i t h d i t h i o t h r e i t o l and r e a l k y l a t e d w i t h [14C]NEM, as above. I n t h i s ex-

SUBUNIT DISTRIBUTION IN RENAL Na,K-ATPase

155

p e r i m e n t , 16.2 f 1 . 5 ( n = 3 ) s u l f h y d r y l g r o u p s w e r e found. From t h e s e d a t a , w e c o n c l u d e d t h a t t h e r e were e i g h t d i s u l f i d e bonds i n t h e u n i t o f Na,K-ATPase. The number of d i s u 1 f i . d e bonds i n e a c h s u b u n i t c a l c u l a t e d w a s b a s e d on t h e d i s t r i b u t i o n of r a d i o a c t i v i t y ( T a b l e I ) .

REFERENCES

A s k a r i , A . , Huang, W., and Henderson, G. R. ( 1 9 7 9 ) . Na,K-ATPase: F u n c t i o n a l and s t r u c t u r a l m o d i f i c a t i o n induced by m e r c u r i a l s . In "Na,K-ATPase: S t r u c t u r e and K i n e t i c s " (J. C. Skou and J. G. N$rby, e d s . ) , pp. 205-215. Academic P r e s s , New York. Kawamura, M. , Ohta, T. , a n d Nagano, K. ( 1 9 8 0 ) . E f f e c t o f r e d u c i n g a g e n t s on t h e s o l u b i l i z a t i o n o f r e n a l sodium and potassium dependent A T P a s e w i t h d e t e r g e n t . J. Biochem. (Tokyo) 8 7 , 132 7-1333. S c h o o t , B. M . , d e P o n t , J . J. H. H. M . , and Bonting, S. L. ( 1 9 7 8 ) . S t u d i e s on ( N a + K)-ATPase. X L I I . Evidence f o r two classes o f e s s e n t i a l s u l f h y d r y l groups. B i o c h i m . B i o p h y s . A c t a 522, 602-613. Schoot, B. M . , Van Emst-de V r i e s , S. E . , Van Haard, P. M. M., de P o n t , J. J. H. H. M . , and Bonting, S . L. ( 1 9 8 0 ) . S t u d i e s on ( N a + K)-ATPase. XLVI E f f e c t o f c a t i o n induced c o n f o r m a t i o n a l Biochim. Biophys. changes on s u l f h y d r y l group m o d i f i c a t i o n . Acta 602, 144-154.

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CURRENT TOPICS IN MEMBRANES AND TRANSPORT, VOLUME 19

Lipid Regions of Na,K-ATPase Examined with Fluorescent Lipid Probes KIMBERLY A. MUCZMVSKI VeteransAdministration Medical Center Seattle, Washington Department of Pathology University of Washington School of Medicine Seattle, Washington

WARD E. HARRIS Veterans AdministrationMedical Center Seattle. Washington Department of Medicine (Neurology) University of Washington School of Medicine Seattle, Washington

WILLIAML. STAHL VeteransAdministration Medical Center Seattle. Washington Department of Physiology and Biophysics University of Washington School of Medicine Seattle, Washington Department of Medicine (Neurology) University of Washington School of Medicine Seattle. Washington

I.

INTRODUCTION

Lipid i s e s s e n t i a l f o r Na,K-ATPase a c t i v i t y b u t i t s p r e c i s e f u n c t i o n remains unknown. I n c o n t r a s t t o "del i p i d a t i o n - r e l i p i d a t i o n " approaches t o t h i s problem, t h i s s t u d y a t t e m p t s t o examine i n t e r a c t i o n s between l i p i d s and N a , K - A T P a s e w i t h o u t d i s r u p t i o n of t h e n a t i v e membrane l i p i d . F l u o r e s c e n t d e r i v a t i v e s of c h o l e s t e r o l ( d e h y d r o e r g o s t e r o l , DHE) and p h o s p h o l i p i d s ( [ 3 H ] d a n s y l phosphatidylethanolamine, DNS-PE; and [ 3 H ] d a n s y l p h o s p h a t i d y l s e r i n e , DNS-PS) w e r e s y n t h e s i z e d and i n c o r p o r a t e d i n t o E . e l e c t r i c u s Na,K-ATPase ( C a n t l e y e t a l . , 1978) u s i n g a p h o s p h o l i p i d exchange p r o t e i n (PLEP) p r e p a r a t i o n ( C r a i n and Z i l v e r s m i t , 1 9 8 0 ) . From d a t a g a t h e r e d on f l u o r e s c e n t l i p i d p r o b e s , t h i s i n v e s t i g a t i o n (1) desc r i b e s i n t e r a c t i o n s between membrane l i p i d and N a , K A T P a s e , and ( 2 ) compares Na,K-ATPase w i t h E . e l e c t r i c u s membranes e n r i c h e d i n a c e t y l c h o l i n e s t e r a s e (AChE) and with erythrocyte ghosts t o determine i f these interact i o n s are s p e c i f i c f o r t h e Na,K-ATPase o r i f t h e y can be g e n e r a l i z e d t o o t h e r membrane p r o t e i n s . 157

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The d e t a i l e d methodology f o r u s i n g t h e PLEP prepar a t i o n t o i n c o r p o r a t e f l u o r e s c e n t l i p i d p r o b e s i n t o memb r a n e s i s d e s c r i b e d e l s e w h e r e (Muczynski e t a l . , 1 9 8 1 ) . B r i e f l y , s o n i c a t e d mixed l i p i d v e s i c l e s c o n t a i n i n g DNS-PE, DNS-PS , o r DHE and [14C] t r i o l e i n ( a s a t r a c e nonexchangeable l a b e l ) w e r e formed. L i p i d p r o b e s were t r a n s f e r r e d t o Na,K-ATPase by i n c u b a t i n g t h e l i p i d v e s i c l e s and Na,K-ATPase w i t h PLEP (40 min a t 32OC). Na,K-ATPase was p e l l e t e d by c e n t r i f u g a t i o n . N o n s p e c i f i c "adherence" of l i p i d s u b s t r a t e t o Na,K-ATPase membranes was c a l c u l a t e d from t h e l o s s of [14C] t r i o l e i n i n t h e s u p e r n a t a n t . G r e a t e r t h a n 8 0 % of t h e Na,K-ATPase act i v i t y was r e c o v e r e d .

11.

RESULTS AND DISCUSSION

Na,K-ATPase had d r a m a t i c e f f e c t s on t h e f l u o r e s c e n t p r o p e r t i e s of DNS-PE and DNS-PS. (1) The e m i s s i o n maxima (Aemmax) of t h e DNS-phospholipids (DNS-PLs) were b l u e - s h i f t e d i n Na,K-ATPase membrane fragments: AemmaX of DNS-PE o r DNS-PS i n mixed l i p i d v e s i c l e s = 5 2 2 nm; Aesax of DNS-PE i n Na,K-ATPase = 513 nm, w h i l e hemmax of DNS-PS i n Na,K-ATPase = 500-504 nm. These s h i f t s a r e c o n s i s t e n t w i t h t h e DNS-PLs b e i n g i n a l e s s p o l a r environment i n Na,K-ATPase membrane fragments t h a n i n mixed l i p i d v e s i c l e s (Waggoner and S t r y e r , 1 9 7 0 ) . The hemmax of t h e DNS-PLs i n v e s i c l e s formed from ext r a c t e d Na,K-ATPase l i p i d s r e t u r n e d t o t h e i n i t i a l v a l u e of t h e p r o b e s i n donor mixed l i p i d v e s i c l e s . ( 2 ) Energy t r a n s f e r was demonstrated from t h e t r y p t o p h a n r e s i d u e s of t h e Na,K-ATPase t o b o t h DNS-PE and DNS-PS; however, no changes i n t h e f l u o r e s c e n t p r o p e r t i e s of t h e s e probes could be induced w i t h l i g a n d s r e p o r t e d t o i n d u c e c o n f o r m a t i o n a l changes i n Na,K-ATPase ( H a r r i s and S t a h l , 1 9 7 7 ) . ( 3 ) F l u o r e s c e n c e p o l a r i z a t i o n of DNS-PLs i n t h e N a , K A T P a s e w a s e l e v a t e d beyond a l e v e l a t t r i b u t a b l e t o probe d i l u t i o n (Fig. 1 ) . DNS-PS had a h i g h e r p o l a r i z a t i o n t h a n DNS-PE (Table I ) , i n d i c a t i n g t h a t t h e c a r b o x y l group and n e g a t i v e charge on DNS-PS made i t more r i g i d i n a b i l a y e r t h a n DNS-PE. ( 4 ) I n mixed l i p i d v e s i c l e s , DNS-PLs had a homogeneous l i f e t i m e of approximately 1 2 nsec; however, i n t h e Na,K-ATPase t h e l i f e t i m e of t h e DNS-PLs became heterogeneous. The f l u o r e s c e n t d a t a from t h e DNS-PLs i n AChE and e r y t h r o c y t e g h o s t s were similar t o t h a t o b t a i n e d from t h e probe i n Na,K-ATPase. This suggests t h a t t h e observations characterize general protein-lipid interactions.

Na,K-ATPaseEXAMINED WITH LIPID PROBES

159

0.32

P c

0.30

?'! a

0.28

2

0.24 0.22

0.26

W Y 0.20

8"

0.18

g 3

0.16 0.14 0.12

u.

0.10

#

UA: K* -AT?ase

AChE

0.341 0.32

'

RBC GHOSTS

1

0.26

W 0.24

5%0.20

3 5 3 Y

0.18 0.16

0.14 0.12 0.10

Ua*, K+ -ATPace

AChE

RBC GHOSTS

F i g . 1 . Polarization o f the l i p i d probes i n a membrane could be due t o membrane protein or t o probe concentration. T o make t h i s d i s t i n c t i o n , v e s i c l e s were formed from chloroform: methanol (2:1, v o l / v o l ) extracted membrane l i p i d and polarization o f the fluorescent probes determined. The polarization o f DNS-PLs i n membranes was due t o the presence o f protein, while the increased polarization o f DHE was a concentration e f f e c t . DNS-PS gave a pattern identical t o DNS-PE. Fluorescence polarization was determined i n an SLM 4800 nanosecond spectrofluorometer Samples were maintained i n a nitrogen atmosphere a t 19O-22OC. Measurements were corrected f o r l i g h t s c a t t e r . F o r DNS-PLs exciting wavelength = 3 5 6 nm, emission wavelength c u t o f f = 470 nm; f o r DHE exciting wavelength = 330 nm, emission wavelength c u t o f f = 370 nm.

.

However, t h e a c t u a l p o l a r i z a t i o n v a l u e s of DNS-PE o r DNS-PS i n membranes c o n t a i n i n g Na,K-ATPase, AChE, and e r y t h r o c y t e plasma membrane p r o t e i n s were d i f f e r e n t (Table I ) , s u g g e s t i n g t h a t t h e r e s t r i c t i o n i n t h e motion of DNS-PE and DNS-PS i s p r o t e i n s p e c i f i c .

KIMBERLY A. MUCZYNSKI et el.

160

TABLE I.

Fluorescence P o l a r i z a t i o n ( P ) of DNS-PE and DNS-PS i n Na,K-ATPase, AChE, and Erythrocyte Ghostsa P

preparation

DNS-PE

Na,K-ATPase AChE Ghosts

0.213 f 0.003 0.199 +- 0.004 0.243

DNS-PS 0.278 0.257 0.294

f 0.004 k 0.002

a

Values f o r Na,K-ATPase and AChE represent t h e mean f standard d e v i a t i o n of 3 o r 4 p o l a r i z a t i o n s determined on elect r i c organ membrane fragments i s o l a t e d independently and incorporated with d i f f e r e n t s t o c k s o f DNS-PL v e s i c l e s using d i f f e r e n t p r e p a r a t i o n s o f PLEP.

The f l u o r e s c e n t p r o p e r t i e s of DHE were n o t a f f e c t e d by Na,K-ATPase, AChE, o r e r y t h r o c y t e g h o s t p r o t e i n s . S p e c i f i c a l l y , (1) Na,K-ATPase l i g a n d s d i d n o t induce changes i n t h e f l u o r e s c e n t p a r a m e t e r s of DHE; ( 2 ) t h e i n c r e a s e d f l u o r e s c e n c e p o l a r i z a t i o n of DHE'adsociated w i t h i t s i n c o r p o r a t i o n i n t o a membrane ( F i g . 1) can be accounted f o r by d i l u t i o n ; and ( 3 ) t h e l i f e t i m e of DHE i n a membrane was homogeneous and i d e n t i c a l t o t h e l i f e t i m e of DHE i n P L - s t e r o l v e s i c l e s . T h e r e f o r e , it seems t h a t t h e s e membrane p r o t e i n s have no e f f e c t on t h e o r g a n i z a t i o n of s t e r o l i n a b i l a y e r t h a t can be d e t e c t e d by f l u o r e s c e n c e . Assuming t h a t t h e f l u o r e s c e n t probes used i n t h i s study represent t h e i r nonfluorescent analogs, i n t e r a c t i o n s of E . e l e c t r i c u s Na,K-ATPase w i t h membrane l i p i d s can be d e s c r i b e d . The o r g a n i z a t i o n of c h o l e s t e r o l w i t h i n Na,K-ATPase membrane fragments i s homogeneous and u n a f f e c t e d by t h e p r e s e n c e of t h e enzyme. I n cont r a s t , t h e Na,K-ATPase c r e a t e s a heterogeneous e n v i r o n ment (based on l i f e t i m e h e t e r o g e n e i t y d a t a , Klausner e t al., 1 9 8 0 ) f o r phosphatidylethanolamine (PE) and phosp h a t i d y l s e r i n e (PS) t h a t i s less p o l a r (hemmax s h i f t s ) and more r i g i d ( p o l a r i z a t i o n d a t a ) t h a n t h a t of a l i p i d b i l a y e r w i t h o u t p r o t e i n . The c a r b o x y l group and negat i v e charge of PS make i t s environment less p o l a r and i t s motion more r e s t r i c t e d t h a n PE. The d e g r e e t o which t h e r o t a t i o n a l motion of t h e PLs i s r e s t r i c t e d i s s p e c i f i c a l l y determined by t h e Na,K-ATPase. How t h e Na,KA T P a s e e x e r t s t h e s e e f f e c t s i s unknown, b u t t h r e e p o s s i b i l i t i e s a r e o f f e r e d . F i r s t , s i n c e PE and PS a r e i n c l o s e p r o x i m i t y t o Na,K-ATPase (energy t r a n s f e r d a t a ) ,

Na,K-ATPase EXAMINED WITH LIPID PROBES

161

t h e r e may be a d i r e c t i n t e r a c t i o n between enzyme and PL. Second, t h e Na,K-ATPase may s e t up l i p i d domains w i t h i n t h e membrane (which would a c c o u n t f o r t h e s i t e h e t e r o g e n e i t y o f PE and PSI T h i r d , t h e Na,K-ATPase may occupy more area a t t h e g l y c e r o l r e g i o n of t h e membrane t h a n i n t h e h y d r o p h o b i c c o r e , t h e r e b y f o r c i n g t h e headg r o u p s of P L s t o g e t h e r w h i l e l e a v i n g c h o l e s t e r o l u n a f fected

.

.

ACKNOWLEDGMENTS

T h i s work w a s s u p p o r t e d by t h e U S. V e t e r a n s A d m i n i s t r a t i o n and by N I H G r a n t NS 15790. K. A. Muczynski w a s s u p p o r t e d i n p a r t by a Medical S c i e n t i s t T r a i n i n g Program F e l l o w s h i p , N I H g r a n t #GM-07266.

REFERENCES

C a n t l e y , L. C . , Jr., Gelles, J . , and Josephson, L. ( 1 9 7 8 ) . React i o n o f (Na-K)ATPase w i t h 7-chloro-4-nitrobenzo-2-oxa-lI3d i a z o l e : Evidence f o r an e s s e n t i a l t y r o s i n e a t t h e a c t i v e s i t e . B i o c h e m i s t r y 1 7 , 418-425. C r a i n , R C . , and Z i l v e r s m i t , D. ( 1 9 8 0 ) . TWO n o n s p e c i f i c phosphol i p i d exchange p r o t e i n s from beef l i v e r . I. P u r i f i c a t i o n and B i o c h e m i s t r y 1 9 , 1433-1439. characterization. Harris, W. E . , and S t a h l , W. L. (1977). Conformational changes o f p u r i f i e d (Na+ + K+)-ATPase d e t e c t e d by a s u l f h y d r y l B i o c h i m . B i o p h y s . A c t a 4 8 5 , 203-214. f l u o r e s c e n c e probe. K l a u s n e r , R. D., K l e i n f e l d , A. M . , Hoover, R. L . , and Karnovsky, M. J. ( 1 9 8 0 ) . L i p i d domains i n membranes: Evidence d e r i v e d from s t r u c t u r a l p e r t u r b a t i o n s i n d u c e d by free f a t t y a c i d s J. B i o l . Chem. 255, and l i f e t i m e h e t e r o g e n e i t y a n a l y s i s . 1286-1295. Muczynski, K. A., Harris, W. E . , and S t a h l , W. L. ( 1 9 8 1 ) . A l t e r i n g e r y t h r o c y t e membrane composition w i t h p h o s p h o l i p i d exchange I n t . J. B i o c h e m . 1 3 , 959-962. protein. Waggoner, A. S . , and S t r y e r , L. (1970). F l u o r e s c e n t p r o b e s of P r o c . N a t l . A c a d . Sci. USA 6 7 , 579b i o l o g i c a l membranes. 589.

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CURRENT TOPICS M MeMBRANES AND TRANSPORT. VOLUME 19

Role of Cholesterol and Other Neutral Lipids in Na, K-ATPase J. J. H. H. M.DEPONT, W. H. M.PETERS, ANDS. L. BONTING Depanmenr of Biochemistry University of Nijmegen Nijmegen, 7he Netherlands

The highly purified Na,K-ATPase preparation from kidney outer medulla as described by J#rgensen (1974) is now widely used for studies of this enzyme system. This preparation contains, in addition to Na,K-ATPase (ca. 90% of all protein present) a large complement of lipids in bilayer arrangement. While the composition and role of the phospholipids have been investigated in detail (de Pont et al., 19781, not much attention has so far been paid to the neutral lipids. Of these neutral lipids cholesterol is the most interesting one, since there are indications that modification of the cholesterol content in crude membrane preparations might influence the activity of the enzyme. We report here the detailed lipid analysis of the purified Na,K-ATPase preparation, and show that removal or conversion of cholesterol does not significantly affect the activity of the enzyme. 163

Copyright 0 1983 by Academic Press, Inc. All rightsof reproduction in any form reserved. ISBN 0-12-1533190

164

J. J. H. H. M. DEPONTetal.

TABLE I.

L i p i d Composition and P r o p e r t i e s of Bound and F r e e F a t t y Acids i n Na,K-ATPase From R a b b i t Kidney Outer Medulla

Compound

Lipid content (mole/mole enzyme)

Sphingomyelin Phosphatidylcholine Phosphatidyl-

serine Phosphatidylinositol Phosphatidylethanolamine Free f a t t y a c i d s Monoglycerides Diglycerides Triglycerides Cholesterol Free E s t e r if i e d

Fatty acids Average c h a i n U n s a t u r a t i o n length indexa

68

18.2

136

136

17.1

83

50

18.0

113

21

18.2

158

107

17.6

158

17.1

76

17.6 17.5

124 188

67 9 16 12 249

19

--

--

17.3

175

a Sum of p e r c e n t a g e o f each f a t t y a c i d , m u l t i p l i e d b y i t s number o f d o u b l e b o n d s .

11.

L I P I D ANALYSIS

I n a d d i t i o n t o 382 moles of p h o s p h o l i p i d s , t h e p r e p a r a t i o n c o n t a i n s 249 moles f r e e c h o l e s t e r o l , 1 9 moles c h o l e s t e r o l , 6 7 moles f r e e f a t t y a c i d s and minor amounts of mono-, d i - , and t r i g l y c e r i d e s . Two p o i n t s need t o be emphasized: (1) The h i g h molar c h o l e s t e r o l / p h o s p h o l i p i d r a t i o (0.71, t o g e t h e r w i t h t h e h i g h sphingomyelin c o n t e n t , i s c h a r a c t e r i s t i c f o r mammalian (2) There i s a s t r i k i n g l y h i g h conplasma membranes. t e n t of f r e e f a t t y a c i d s , which may have i n c r e a s e d d u r i n g t h e p u r i f i c a t i o n procedure (Table I ) . I n any c a s e , t h e presence of t h i s n e g a t i v e l y charged l i p i d class m u s t be t a k e n i n t o a c c o u n t i n t h e d i s c u s s i o n of a poss i b l e r o l e of n e g a t i v e l y charged ( p h o s p h o ) l i p i d s f o r t h e a c t i v i t y of t h e enzyme. The f a t t y a c i d a n a l y s i s of t h e v a r i o u s l i p i d s i s a l s o p r e s e n t e d i n Table I . The a v e r a g e c h a i n l e n g t h v a r i e s between 1 7 . 1 and 18.2 carbon atoms p e r f a t t y

ROLE OF CHOLESTEROL AND OTHER NEUTRAL LIPIDS

165

*---__I-----_+

f-

( N a * + K ' ) A T P a s e activity

20

00

I

I

2

I

I

4

I

8

6 TIME (h)

F i g . 1 . R e l a t i o n s h i p b e t w e e n cholesterol o x i d a t i o n ( 0 ) and Na,K-ATPase a c t i v i t y ( 0 ) upon i n c u b a t i o n of Na,K-ATPase w i t h cholesterol e s t e r a s e and cholesterol o x i d a s e a t 37'C. Averages from three e x p e r i m e n t s . From Peters e t a l . ( 1 9 8 1 ) .

a c i d ; t h e u n s a t u r a t i o n i n d e x v a r i e s between 76 and 1 8 8 . Most u n s a t u r a t e d a r e t h e f a t t y a c i d s of p h o s p h a t i d y l i n o s i t o l I phosphatidylethanolamine , t r i g l y c e r i d e s , and c h o l e s t e r o l e s t e r s , whereas t h o s e of p h o s p h a t i d y l c h o l i n e and of t h e f r e e f a t t y acids and monoglycerides a r e r e l a t i v e l y more s a t u r a t e d . I n g e n e r a l , t h e more s a t u r a t e d f a t t y a c i d s have a somewhat s h o r t e r c h a i n l e n g t h .

111.

EFFECT O F CHOLESTEROL REMOVAL AND O X I D A T I O N

Hexane e x t r a c t i o n of a l y o p h i l i z e d Na,K-ATPase p r e p a r a t i o n removes a l l c h o l e s t e r o l and o t h e r n e u t r a l l i p i d s , and a l s o 20-25% of t h e p h o s p h o l i p i d s . The comp o s i t i o n of t h e e x t r a c t e d p h o s p h o l i p i d s was n o t s i g n i f i c a n t l y d i f f e r e n t from t h a t of t h e n a t i v e enzyme p r e p a r a t i o n , i n d i c a t i n g t h a t n o s p e c i f i c c l a s s of phosp h o l i p i d s i s e x t r a c t e d . T h e r e s i d u a l Na,K-ATPase act i v i t y i n t h e p e l l e t was 6 6 % (SE 1 0 ; n = 3 ) of t h a t i n t h e l y o p h i l i z e d p r e p a r a t i o n . No a c t i v i t y was found i n t h e s u p e r n a t a n t . T h i s s u g g e s t s t h a t removal of n e u t r a l l i p i d s h a s o n l y a minor e f f e c t on t h e enzyme a c t i v i t y .

166

J. J. H. H. M. DE PONTetsl.

F i g u r e 1 shows t h a t t o t a l o x i d a t i o n of c h o l e s t e r o l t o 4-cholesten-3-one i s o b t a i n e d a f t e r i n c u b a t i o n f o r 8 h r a t 37OC w i t h c h o l e s t e r o l e s t e r a s e and c h o l e s t e r o l o x i d a s e . I n t h a t t i m e t h e a c t i v i t y of t h e c o n t r o l p r e p a r a t i o n i s h a r d l y d e c r e a s e d , whereas t h e t r e a t e d p r e p a r a t i o n h a s l o s t o n l y 1 5 % of t h e Na,K-ATPase a c t i v i t y . T h i s means t h a t m o d i f i c a t i o n of t h e hydroxyl group of a l l c h o l e s t e r o l does n o t g r e a t l y a f f e c t t h e Na,KATPase a c t i v i t y of t h e p r e p a r a t i o n . I n summary, c h o l e s t e r o l and o t h e r n e u t r a l l i p i d s do n o t seem t o be e s s e n t i a l f o r t h e enzyme a c t i v i t y i n a h i g h l y p u r i f i e d Na,K-ATPase p r e p a r a t i o n .

REFERENCES

de Pont, J. J. H. H. M . , Van Prooyen-van Eeden, A., and Bonting, S. L. (1978). S t u d i e s on (Na+ + K + ) - a c t i v a t e d ATPase. XXXIX. Role o f n e g a t i v e l y charged p h o s p h o l i p i d s i n h i g h l y p u r i f i e d ( N a + + K+)-ATPase from r a b b i t kidney o u t e r medulla. B i o c h i r n . B i o p h y s . A c t a 5 0 8 , 464-477. J$rgensen, P. L. (1974). P u r i f i c a t i o n and c h a r a c t e r i z a t i o n of ( N a + + K+)-ATPase. 111. P u r i f i c a t i o n from t h e o u t e r medulla of mammalian kidney a f t e r s e l e c t i v e removal o f membrane components by sodium dodecyl s u l p h a t e . B i o c h i r n . B i o p h y s . A c t a 356, 35-52. Peters, W. H. M . , Fleuren-Jakobs, A. M. M . , de Pont, J. J. H. H. M. , and Bonting, S . L. (1981). S t u d i e s on ( N a + + K+)a c t i v a t e d ATPase. XLIX. Content and r o l e o f c h o l e s t e r o l and o t h e r n e u t r a l l i p i d s i n h i g h l y p u r i f i e d r a b b i t kidney enzyme p r e p a r a t i o n s . B i o c h i r n . B i o p h y s . A c t a 6 4 9 , 541-549.

Part I11

Ligand Interactions: Cardiac Glycosides and Ions

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CURRENT TOPICS IN MEMBRANES AND TRANSPORT, VOLUME I9

Cardiotonic Steroid Binding to Na,K-ATPase BLISS FORBUSH 111 Yale University School of Medicine New Haven, Connecticut

I n t h i s o v e r v i e w , I c o n s i d e r v a r i o u s a s p e c t s of t h e i n t e r a c t i o n between c a r d i a c g l y c o s i d e s and N a , K - A T P a s e . T h i s i s n o t i n t e n d e d t o be an e x h a u s t i v e r e v i e w , and r e f e r e n c e s were chosen p r i m a r i l y f o r i l l u s t r a t i v e purp o s e s . I n some c a s e s I have a t t e m p t e d t o d i s t i l l a l a r g e number of o b s e r v a t i o n s , w i t h t h e hope t h a t t h e t r u t h h a s n o t been l e f t behind i n t h e p o t . C e r t a i n m a j o r areas, such as t h e i n o t r o p i c a c t i o n of t h e c a r d i a c g l y c o s i d e s and t h e u s e o f f l u o r e s c e n t c a r d i a c g l y c o s i d e s , have been a d e q u a t e l y c o n s i d e r e d e l s e w h e r e i n t h i s volume, and a r e n o t reviewed h e r e ; l i k e w i s e , " i o n bindi n g t o N a , K - A T P a s e , " i s t h o r o u g h l y t r e a t e d by Tonomura e t a l . ( t h i s volume) and Glynn (Volume 4 ) , and w i l l n o t be d i s c u s s e d here.

167

Copyright 0 1983 by Academic Press. Inc. All rights ofreproductionin any form reserved ISBN 0-12-153319-0

BLISS FORBUSH 111

168

I.

FACTORS AFFECTING OUABAIN B I N D I N G

The i n t e r a c t i o n between c a r d i a c g l y c o s i d e s and i s of high a f f i n i t y ( t y p i c a l d i s s o c i a t i o n c o n s t a n t Kd = 2 x 1 0 - 9 M , under o p t i m a l c o n d i t i o n s ) and r a t h e r slow ( t y p i c a l a s s o c i a t i o n r a t e c o n s t a n t k a = 3 X 1 0 4 M-lsec-1). Since t h e dissociation rate c o n s t a n t i s v e r y s m a l l and i s f a i r l y c o n s t a n t i n t h e p r e s e n c e of v a r i o u s b i n d i n g l i g a n d s , i t i s p r i m a r i l y t h e a s s o c i a t i o n r a t e c o n s t a n t t h a t d e c r e a s e s under cond i t i o n s of nonoptimal b i n d i n g ; t h u s e q u i l i b r i u m b i n d i n g u n d e r t h e s e c o n d i t i o n s may r e q u i r e many h o u r s . Most i n v e s t i g a t o r s , r e c o g n i z i n g t h i s , have c h o s e n t o measure a s s o c i a t i o n and/or d i s s o c i a t i o n rate c o n s t a n t s r a t h e r t h a n t o measure t h e e q u i l i b r i u m c o n s t a n t ( K d ) from S c a t c h a r d p l o t s ( c f . I n a g a k i e t a l . , 1974; Yoda, 1973; Bodemann and Hoffman, 1976a; W a l l i c k et a l . , 1 9 7 7 , 1 9 8 0 ) ; Kd may t h e n be o b t a i n e d d i r e c t l y from t h e r a t i o of t h e two r a t e c o n s t a n t s i f t h e s e v a l u e s have been obt a i n e d under t h e same c o n d i t i o n s . T h i s approach h a s t h e advantage of g i v i n g a d d i t i o n a l i n f o r m a t i o n , s i n c e d e t e r m i n a n t s o f b i n d i n g and d i s s o c i a t i o n are q u i t e d i f f e r e n t , as w i l l be s e e n below. Ouabain b i n d i n g o c c u r s w i t h h i g h e s t r a t e and g r e a t e s t a f f i n i t y t o p h o s p h o r y l a t e d forms o f N a , K A T P a s e , t h o s e produced i n t h e p r e s e n c e of N a + , Mg2+, ATP, o r Mg2+ + P i (Matsui and Schwartz, 1968; A l b e r s e t a l . , 1 9 6 8 ) ; t h e c h a r a c t e r i s t i c s of t h i s b i n d i n g have been s t u d i e d i n g r e a t e s t d e t a i l and w i l l be d i s c u s s e d below. Ouabain a l s o b i n d s under c o n d i t i o n s i n which p h o s p h o r y l a t i o n seems u n l i k e l y o r i m p o s s i b l e , and it may be s a f e s t t o assume t h a t o u a b a i n can i n t e r a c t w i t h any enzyme c o n f o r m a t i o n a t a f i n i t e , i f s m a l l , r a t e . I t s h o u l d be n o t e d however, t h a t i t i s d i f f i c u l t t o p r o v e t h a t a g i v e n enzyme c o n f o r m a t i o n i s i n v o l v e d i n nonoptimal o u a b a i n b i n d i n g , s i n c e i n t h e c o u r s e of t h e ouabain-binding e x p e r i m e n t , a number of enzyme c o n f o r m a t i o n s w i l l be i n r a p i d e q u i l i b r i u m and o u a b a i n w i l l " t r a p " t h o s e which r e a c t most r e a d i l y . Very slow ouab a i n b i n d i n g h a s been r e p o r t e d i n t h e absence of l i g a n d s (Sen et a l . , 1969; Mandel et a l . , 1 9 7 7 ) , much h i g h e r r a t e s are found i n t h e p r e s e n c e o f Mg2+ a l o n e (Sen et al., 1969; A l l e n et a l . , 1 9 7 1 ) ; and i n t h i s volume, Henderson r e p o r t s t h a t L i + a p p e a r s t o be a b l e t o subI n t h e p r e s e n c e of Mg2++ ATP, t h e r a t s t i t u t e f o r Mg2+. o f o u a b a i n b i n d i n can be a s much a s 25-80% t h a t of t h e r a t e w i t h N a + + Mg9+ + ATP (Wallick et al. , 1977; M a t s u i and Schwartz, 1 9 6 8 ; Hoffman, 1969; Skou et al., 1 9 7 1 ) . While " t r a p p i n g " of a low l e v e l of p h o s p h o r y l a t e d i n t e r Na,K-ATPase

CARDIOTONIC STEROID BINDING TO Na,K-ATPase

169

m e d i a t e formed from r e s i d u a l P i o r from ATP i n t h e abs e n c e o f N a h a s been proposed t o e x p l a i n t h e s e o b s e r v a t i o n s , t h e q u a n t i t a t i v e a s p e c t s make t h i s u n l i k e l y . A.

+

Na + Mg2+ + ATP-STIMULATED TYPE I O U A B A I N B I N D I N G

OUABAIN BINDING:

I n t h e p r e s e n c e of N a + and Mg2+, ATP a c t s a t a h i g h - a f f i n i t y s i t e t o s t i m u l a t e o u a b a i n b i n d i n g , cons i s t e n t w i t h p h o s p h o r y l a t i o n of Na,K-ATPase (Matsui and Schwartz, 1968; Skou e t a l . , 1 9 7 1 ; Shoemaker and L a u f , t h i s volume). Whether under t h e s e c o n d i t i o n s phosphory l a t i o n i s r e q u i r e d f o r t h e s t i m u l a t i o n o f o u a b a i n bindi n g h a s n o t been c l e a r , s i n c e n u c l e o t i d e s which are hydrolyzed poorly support optimal rates of binding even a t low c o n c e n t r a t i o n s (Matsui and Schwartz, 1968; Hoffman, 1 9 6 9 ) ; b u t n o n h y d r o l y z a b l e a n a l o g s do n o t (Tobin e t a l . , 1 9 7 4 ) . I n a r a t b r a i n microsomal p r e p a r a t i o n Tobin et a l . (1971) found t h a t t h e s t i m u l a t i o n by UTP, I T P , and CTP c o u l d be e x p l a i n e d by slow phosp h o r y l a t i o n ( e i t h e r d i r e c t o r i n d i r e c t ) by t h e s e n u c l e o t i d e s . Some o f t h e e a r l i e r r e s u l t s may have been comp l i c a t e d by t h e p r e s e n c e o f t r a n s p h o s p h o r y l a t i n g enzymes i n t h e membrane p r e p a r a t i o n s ; Shoemaker and Lauf r e p o r t i n t h i s volume t h a t UTP and ADP f a i l t o s u p p o r t b i n d i n g i n r e d c e l l g h o s t s i f n u c l e o s i d e d i p h o s p h o k i n a s e and adenylate kinase i n h i b i t o r s a r e present t o prevent slow f o r m a t i o n o f ATP. On t h e o t h e r hand, t h e h i g h r a t e of o u a b a i n b i n d i n g s e e n i n t h e p r e s e n c e of Mg2+ + ATP, b u t a b s e n c e o f N a + , would n o t be e x p e c t e d t o i n v o l v e phosp h o r y l a t i o n , and might be s u p p o r t e d by n o n h y d r o l y z a b l e n u c l e o t i d e s ; t h e e f f e c t i v e n e s s of n u c l e o t i d e s o t h e r t h a n ATP h a s n o t been r e p o r t e d under t h e s e c o n d i t i o n s . While E2-P i s p r o b a b l y t h e r e l e v a n t o u a b a i n - b i n d i n g enzyme c o n f o r m a t i o n i n most t i s s u e s , it i s worth n o t i n g t h a t E l - P i s t h e predominant form of t h e N a , K - A T P a s e from e e l e l e c t r i c o r g a n i n t h e p r e s e n c e of 1 0 0 mM N a + , M q 2 + + ATP (Yoda, t h i s v o l u m e ) . I n t h i s r e g a r d , Hegyvary (1976) and Wallick e t a i . (19781, have shown t h a t o u a b a i n b i n d s t o E1P b l o c k e d from t r a n s i t i o n t o E2P by N-ethylmaleimide; t h e r a t e c o n s t a n t f o r b i n d i n g i s somewhat less than w i t h E2P. I t i s a l s o i n t e r e s t i n g t h a t t h e r a t e of Mg2+ + ATP-supported b i n d i n g i s n o t altered by NEM (Wallick e t a ] . , 1978). + I n t h e p r e s e n c e of Mg2+ + ATP (and a b s e n c e of K 1 , N a + s t i m u l a t e s ouabain binding with an apparent a f f i n i t y of 0.6 m M ( I n a g a k i e t a l . , 1 9 7 4 ) , c o n s i s t e n t w i t h N a + a c t i n g a t t h e s i t e involved i n phosphorylation [ P o s t e t a l . , 1965; k + ( N a ) = 1 . 6 mM]. The s i t e promoting phos-

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p h o r y l a t i o n i s a t t h e i n t r a c e l l u l a r f a c e of t h e N a pump ( B l o s t e i n and Chu, 1 9 7 7 ) ; b u t t h e r e l e v a n t o u a b a i n b i n d i n g e x p e r i m e n t s have n o t been performed i n " s i d e d " membrane p r e p a r a t i o n s . F u r t h e r e v i d e n c e s u p p o r t i n g a n i n t r a c e l l u l a r o r i e n t a t i o n of t h e h i g h - a f f i n i t y N a + s i t e i s t h e a f f i n i t y f o r N a i i n uncoupled N a + e f f l u x ( k + i 1 m ; K a r l i s h and Glynn, 1974) and N a / K pumping a t l o w K i ( 0 . 2 m ~ Garay ; and Garrahan, 1973; see a l s o Garrahan et al., 1 9 7 9 ) . A t h i g h e r c o n c e n t r a t i o n s of N a , t h e r a t e of o u a b a i n b i n d i n g c o n t i n u e s t o i n c r e a s e , w i t h an a p p a r e n t a f f i n i t y f o r N a + of a b o u t 1 5 mM (Lindemayer and Schwartz, 1973; I n a g a k i e t al., 1 9 7 4 ) . S i n c e t h i s e f f e c t c a n n o t be e x p l a i n e d by an i n c r e a s e i n E 2 P , which i s maximal a t N a = 5 m , i t s u g g e s t s t h a t a n o t h e r conf o r m a t i o n of Na,K-ATPase, w i t h bound N a , b i n d s o u a b a i n more r a p i d l y t h a n E 2P. Swann and A l b e r s (1980) have p r e s e n t e d e v i d e n c e i n f a v o r of a n E 2 conformation w i t h N a bound t o a l o w - a f f i n i t y s i t e , from d a t a d e s c r i b i n g N a + + ATP s t i m u l a t i o n of K+ p-nitrophenylphosphatase a c t i v i t y . A p a r a l l e l i s m w i t h a low a f f i n i t y N a s i t e f o r s t i m u l a t i o n o f Na-ATPase a c t i v i t y (Mardh and P o s t , 1977; Garrahan e t al., 1979) on t h e o u t s i d e o f t h e c e l l (Glynn and K a r l i s h , 1976) i s a l s o a p p e a l i n g . Hobbs and Dunham (1978) have found t h a t N a o s t i m u l a t e s o u a b a i n b i n d i n g a t a v e r y low a f f i n i t y N a s i t e ("a]+ > 50 mM; a l t h o u g h Sachs, 1 9 7 4 , and Bodeman and Hoffman, 1976a, found no e f f e c t of N a o i n t h e a b s e n c e of KO; i n e a r l i e r s t u d i e s [ K I o w a s p r o b a b l y r a i s e d by l e a k a g e o f K from the cells. I t thus appears l i k e l y t h a t ouabain binding t o N a o * E 2 - P , i s more r a p i d t h a n t o t h e l i g a n d - f r e e form of t h e phosphoenzyme (see d i s c u s s i o n of S a c h s , 1 9 7 4 ; Hobbs and Dunham, 1 9 7 8 ) . I n t h e p r e s e n c e of K , t h e dependence of ouabain b i n d i n g on N a + c o n c e n t r a t i o n i s more complex. Although t h e i n t e r a c t i o n w i t h N a , K - A T P a s e i n fragmented membranes can be a d e q u a t e l y modeled by N a + s t i m u l a t i o n of E2-P f o r m a t i o n and K+ i n i t i a t i o n o f E 2 P h y d r o l y s i s ( B a r n e t t , 1 9 7 0 ) o r by c o m p e t i t i o n o f N a and K f o r a s i n g l e "modul a t i n g s i t e " (Lindenmayer and Schwartz, 19731, s t u d i e s w i t h " s i d e d " membrane p r e p a r a t i o n s show t h a t s i t e s f o r b o t h i o n s p e c i e s are i n v o l v e d on b o t h sides o f t h e memb r a n e . E x t e r n a l K+ i s known t o a n t a g o n i z e o u a b a i n b i n d i n g as it s t i m u l a t e s N a / K pumping (Schatzmann, 1953; Glynn, 1957; S a c h s , 1974; Bodemann and Hoffman, 1976a; Hobbs and Dunham, 1978) by a c c e l e r a t i n g h y d r o l y s i s o f E2-P. E x t e r n a l N a competes w i t h K and i n h i b i t s N a / K pump a c t i v i t y ( P o s t e t a l . , 1 9 6 0 ; Garrahan and Glynn, 1 9 6 7 ) i n a k i n e t i c a l l y complex manner ( S a c h s , 1 9 7 7 ) ; i t a p p e a r s t h a t t h e accompanying s t i m u l a t i o n o f ouabain +

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b i n d i n g (Beau96 and Adragna, 1 9 7 1 ; Baker and W i l l i s , 1972; S a c h s , 1974; Hobbs and Dunham, 1 9 7 8 ) o c c u r s b o t h by p r e v e n t i o n o f K+ b i n d i n g and by f o r m a t i o n o f a Naos t a b i l i z e d form of E2-P w i t h enhanced o u a b a i n b i n d i n g (see Hobbs and Dunham, 1 9 7 8 ) . When t h e N a pump i s a c t i v a t e d by e x t r a c e l l u l a r K + , t h e r a t e of o u a b a i n b i n d i n g i s f u r t h e r r e d u c e d by t h e p r e s e n c e o f i n t r a c e l l u l a r K+ a t low N a i (Bodemann and Hoffman, 1 9 7 6 a ) . T h i s a c t i o n c o r r e l a t e s r e a s o n a b l y w e l l w i t h i n h i b i t i o n of N a / K pump t u r n o v e r (Hoffman, 1962; Garay and G a r r a h a n , 1973; Knight and W e l t , 1 9 7 4 ; Bodemann and Hoffman, 1976a1, p o s s i b l y r e f l e c t i n g t h e s t a b i l i t y of t h e K . E 1 form o f t h e pump, l e a d i n g t o a r e d u c e d s t e a d y s t a t e l e v e l of E2-P. In t h i s situation, i n c r e a s e d N a i i s r e q u i r e d t o compete f o r E l and promote p h o s p h o r y l a t i o n , as e v i d e n c e d by b o t h i n c r e a s e d N a / K pump a c t i v i t y and ouabadn b i n d i n g ( J o i n e r and L a u f , 1978; Bodemann e t a l . , t h i s volume; Akera e t a l . , t h i s volume). I n d e c r e a s i n g t h e a p p a r e n t a f f i n i t y f o r N a i from 0. 4 t o 3.5 mM f o r s t i m u l a t i o n of Na pumping, K i a c t s a t a s i t e w i t h an a p p a r e n t a f f i n i t y of 9 mM (Garay and G a r r a h a n , 1973; [K], = 0 o r 1 0 mM; [ N a I o = 1 4 0 m M ) . Since K-Na i n t e r a c t i o n s i n ouabain binding t o N a , K A T P a s e i n b r o k e n membrane p r e p a r a t i o n s have been s t u d i e d c h i e f l y a t K c o n c e n t r a t i o n s below 5 m M , i t i s l i k e l y t h a t those i n t e r a c t i o n s took p l a c e a t t h e e x t e r n a l f a c e of t h e pump, as p r o p o s e d by Lindenmayer and Schwartz (1973). When [ K ] i i s below 1 0 mM and [K], i s 5-10 m M , t h e N a / K pump i s maximally s t i m u l a t e d by N a i c o n c e n t r a t i o n s less t h a n a b o u t 5 m M (Garay and G a r r a h a n , 1973; p u b l i s h e d d a t a are n o t a v a i l a b l e f o r [ N a ] i > 5 m ~ ) ,c o n s i s t e n t w i t h t h e N a i r e q u i r e m e n t f o r uncoupled N a e f f l u x and f o r N a + + Mg2+ + ATP-stimulated p h o s p h o r y l a t i o n , d i s c u s s e d above. Bodemann and Hoffman ( 1 9 7 6 a ) have found t h a t when K i i s low, N a i h a s a n u n e x p e c t e d i n h i b i t o r y e f f e c t on ouabain binding. T h i s behavior does n o t f i t any a v a i l a b l e model o f o u a b a i n b i n d i n g t o E2-P. Lindenmayer and Schwartz (1973) made a p a r a l l e l o b s e r v a t i o n w i t h f r a g m e n t e d membranes from beef b r a i n : o u a b a i n i n h i b i t i o n i n t h e p r e s e n c e of 1 0 mM K + , 2.5 mM ATP, 2 . 5 mM Mg2+ w a s d e c r e a s e d a s t h e N a + c o n c e n t r a t i o n w a s i n c r e a s e d from 1 t o 1 0 mM. I have c o n f i r m e d t h i s f i n d i n g and a l s o d e t e r m i n e d t h a t t h e r a t e of [3H]ouabain b i n d i n g i s dec r e a s e d when N a + i s added a t low c o n c e n t r a t i o n s t o a b i n d i n g medium c o n t a i n i n g K + , Mg2+, and ATP ( F i g . 1). 1 l T h e e f f e c t i s not d u e t o N a i i n h i b i t i o n of (Mg2+ + P i ) s u p p o r t e d ouabain b i n d i n g , w i t h P i r e l e a s e d b y h y d r o l y s i s of ATP. In r e d c e l l s , the r a t e of (Mg2+ + P i ) - s u p p o r t e d b i n d i n g is l o w e r than the rates c o n s i d e r e d here, even w i t h 2 mM P i (Bodemann a n d

BLISS FORBUSH 111

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F i g . 1 . Dependence on Na o f o u a b a i n b i n d i n g t o beef b r a i n ( A ) Inhibition of m i c r o s o m e s i n the p r e s e n c e of Mg2+ + ATP + K+. Na,K-ATPase a c t i v i t y b y o u a b a i n . B e e f b r a i n m i c r o s o m e s (0.11 mg/ m l ) w e r e i n c u b a t e d f o r 10 m i n a t 37'C i n the p r e s e n c e o f 4 mM T r i s ATP, 0 . 3 mM EDTA, 4 mM K+, 6 . 3 mM Mg2+, 10 mM i m i d a z o l e , pH 7 . 2 , and the i n d i c a t e d c o n c e n t r a t i o n o f Na+, a f t e r w h i c h r e l e a s e d P i was d e t e r m i n e d . For e a c h Na c o n c e n t r a t i o n , o u a b a i n - i n s e n s i t i v e a c t i v i t y was d e t e r m i n e d i n the p r e s e n c e o f 10-3 M o u a b a i n and s u b t r a c t e d : (- 0 -) control a c t i v i t y ; ( - A -) a c t i v i t y i n p r e s e n c e o f 10-6 M o u a b a i n ; ( - 0 -) % i n h i b i t i o n b y 10-6 M o u a b a i n . ( B ) Ouabain b i n d i n g . Microsomes ( 0 . 3 m g / m l ) w e r e i n c u b a t e d f o r 4 5 sec (37'C) i n the p r e s e n c e o f the above c o n c e n t r a t i o n s o f l i g a n d s and 10-6 M [3H]oua[3H]Ouabain b i n d i n g was d e t e r m i n e d b y f i l t r a t i o b a i n ( i n 0.12 ml). (Gelman GN-6) a f t e r d i l u t i o n i n t o 5 ml of 10-4 M o u a b a i n , 100 mM KCI, 25 mM i m i d a z o l e a t O'C. The mean o f 6 d e t e r m i n a t i o n s (2SEM) i p r e s e n t e d . S i m i l a r r e s u l t s w e r e o b t a i n e d w i t h p u r i f i e d dog k i d n e y Na ,K-ATPase

.

.

'(Cont ' a ) ( H o f f m a n ,

197613) Control e x p e r i m e n t s a l s o d e m o n s t r a t e t h a t the r e s u l t s shown i n P i g . 1 cannot be d u e t o the small amount o f P i r e l e a s e d i n the t i m e c o u r s e o f the e x p e r i m e n t ( B . F o r b u s h I I l unpublished r e s u l t s )

.

CARDIOTONIC STEROID BINDING TO Na,K-ATPase

173

A s Lindenmayer and Schwartz (1973) s u g g e s t e d , i t seems l i k e l y t h a t i n t h e a b s e n c e of N a , o u a b a i n b i n d i n g i s

s u p p o r t e d by Mg2+ + ATP, p r o b a b l y w i t h o u t phosphorylat i o n . The i n h i b i t i o n a t low N a would t h e n be exp l a i n e d by t h e d i s a p p e a r a n c e o f t h e E - A T P i n t e r m e d i a t e a s t h e enzyme was p h o s p h o r y l a t e d ; i n t h e p r e s e n c e o f K + , E2-P would a l s o be r a p i d l y broken down. Stimulation o f o u a b a i n b i n d i n g by h i g h e r N a + c o n c e n t r a t i o n s i s cons i s t e n t w i t h an e x t r a c e l l u l a r a c t i o n of N a + i n competit i o n w i t h KO, as d i s c u s s e d above. A t h i g h N a i , K i s t i m u l a t e s t h e N a / K pump, and ouab a i n b i n d i n g a s w e l l (Garay and Garrahan, 1973; J o i n e r and Lauf, 1978; Bodemann e t a l . , t h i s volume. Sachs (1981) h a s e x p l a i n e d t h i s by a n e f f e c t o f on metabolic p r o c e s s e s u n r e l a t e d t o t h e pump, which a l t e r t h e l e v e l o f ATP i n t h e e x p e r i m e n t s . T h a t o u a b a i n - b i n d i n g and pump r a t e are b o t h a f f e c t e d is c o n s i s t e n t w i t h a change i n t h e s t e a d y s t a t e l e v e l o f E2-P. The s i t u a t i o n d e s c r i b e d above p e r t a i n s a t moderate l e v e l s of f r e e Mg2+, 0.1-2 mM, t h e r a n g e i n which most s t u d i e s have been u n d e r t a k e n . A t v e r y low Mg2+ concent r a t i o n s t h e a f f i n i t y f o r N a + (presumably N a i l i s dec r e a s e d (Skou et al., 1 9 7 1 ) , and t h e i n h i b i t i o n o f ouab a i n b i n d i n g by N a i ( a t low K i ) i s l o s t (Bodemann and Hoffman, 1 9 7 6 ~ ) . I n v e s t i g a t i o n s a t h i g h e r t h a n normal Mg?+ a r e d i s c u s s e d by Bodemann e t a l . i n t h i s volume. The mechanism u n d e r l y i n g t h e e f f e c t s o f Mg2+ i s n o t known, a l t h o u g h a l t e r a t i o n i n t h e E 1 P + E 2P e q u i l i b r i u m h a s been d i s c u s s e d i n t h e above r e f e r e n c e s .

KI

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2+ (Mg + Pi)-SUPPORTED OUABAIN B I N D I N G

OUABAIN

A second mode o f o u a b a i n N a K-ATPase i s p h o s p h o r y l a t e d Mgl+. A s w i t h Type I b i n d i n g , proposed t o be E z - P , however, N a , K - A T P a s e i n t e r m e d i a t e s can

BINDING:

T Y P E 11

b i n d i n g t a k e s p l a c e when from P i i n t h e p r e s e n c e o f the species involved i s t h e two t y p e s of ouabainbe d i s t i n g u i s h e d by t h e i r d i s s o c i a t i o n r a t e s , as d i s c u s s e d l a t e r . For Mg2+ + P i promoted o u a b a i n b i n d i n g t o o c c u r , P i i s r e q u i r e d on t h e i n s i d e f a c e o f t h e membrane (Lishko e t a l . , 1 9 7 2 ) . P i b i n d s w i t h a n a f f i n i t y of a b o u t 0.1-0.6 m M (Hansen and Skou, 1973; Bodemann and Hoffman, 197623) which i s cons i s t e n t w i t h a requirement f o r phosphorylation ( P o s t et a l . , 1975). A v e r y l a r g e i n c r e a s e i n t h e a f f i n i t y of P i ,x! f o r p h o s p h o r y l a t i o n ( t o = 6 V M ; Schuurmans Stekhoven e t a l . , 1976) o c c u r s when o u a b a i n i s bound.

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Sodium s t r o n g l y i n h i b i t s (Mg2+ + P . ) -promoted ouab a i n b i n d i n g ( A l b e r s et a l . , 1968; “ a ] $ 2i 4 r n M , Hansen and Skou, 1 9 7 3 ) ; t h e a c t i o n of N a t a k e s p l a c e on t h e i n s i d e of t h e membrane o n l y (Bodemann and Hoffman, 197613). T h i s can be e x p l a i n e d by f o r m a t i o n of NaE which p r e v e n t s p h o s p h o r y l a t i o n from P i ( P o s t et al. , 1 9 7 4 ) . P o t a s s i u m a l s o i n h i b i t s ( A l b e r s e t ai., 1968; Hansen and Skou, 1 9 7 3 ) , b u t from b o t h s i d e s of t h e membrane (Lishko et a l . , 1 9 7 2 ) . The K+ i n h i b i t i o n i s n o t complete; t h e poss i b i l i t y t h a t t h i s r e f l e c t s f o r m a t i o n o f a low l e v e l o f K-E-P ( P o s t e t a l . , 1 9 7 5 ) , and t h a t K*E-P b i n d s o u a b a i n , h a s been r a i s e d by Hansen and Skou ( 1 9 7 3 ) . C.

D I S S O C I A T I O N OF O U A B A I N FROM N a , K - A T P a s e

3 The r a t e of d i s s o c i a t i o n of [ Hlouabain from N a , K A T P a s e depends on t h e c o n d i t i o n u n d e r which it w a s bound, as w e l l a s on t h e composition o f t h e d i s s o c i a t i o n medium. The p r o c e s s i s most r e a d i l y measured by a d d i t i o n o f n o n r a d i o a c t i v e o u a b a i n t o p r e v e n t r e b i n d i n g of t h e l a b e l e d compound, and i n t h i s way t h e d i s s o c i a t i o n r a t e can be measured u n d e r t h e same c o n d i t i o n a s t h e a s s o c i a t i o n r a t e . I t h a s been n o t e d i n many l a b o r a t o r i e s t h a t when t h e l a t t e r e x p e r i m e n t i s performed, d i s s o c i a t i o n t a k e s p l a c e a t a b o u t t h e same r a t e , f o r an o f he b i n d i n g c o n d i t i o n s , i . e . , Mg2+ + ATP, Mgs+ + N a + + ATP, Mg2+ + N a + + K + , Mg2+, Mg2+ + P i ; f o r many t i s s u e s and s p e c i e s , t h e h a l f t i m e i s a b o u t 150 min ( c f . Wallick et al., 1 9 8 0 ) . When ouabain-Na,K-ATPase complex i s c e n t r i f u g e d and r e s u s p e n d e d , or more simply j u s t d i l u t e d i n t o b u f f e r cont a i n i n g no monovalent c a t i o n , Type I and I1 complexes are markedly d i f f e r e n t i n t h e i r d i s s o c i a t i o n r a t e s : t h e h a l f - t i m e o f Type I: d i s s o c i a t i o n i s a b o u t 1 0 min, w h i l e t h a t o f Type I1 i s o n l y s l i g h t l y changed from t h e h a l f time i n t h e b i n d i n g medium ( e . g . , 1 5 0 min; A l l e n et a l . , 1971; Akera e t a l . , 1 9 7 4 ; Tobin e t a l . , 1 9 7 4 ) . The d i f f e r e n c e i n d i s s o c i a t i o n r a t e between Type I and Type I1 complex i s n o t d u e t o t h e n a t u r e of t h e p h o s p h o r y l a t e d i n t e r m e d i a t e , s i n c e i n e a c h case, d e p h o s p h o r y l a t i o n i s v e r y r a p i d r e l a t i v e t o [3H] o u a b a i n d i s s o c i a t i o n (Sen e t a l . , 1969; Hansen, 1979) The Type I enzyme-ouabain complex can be s t a b i l i z e d % (t+ = 150 min) by a d d i t i o n of a s m a l l amount of K+ ( 2 1 mM) t o t h e d i s s o c i a t i o n medium. While Akera e t a l . ( 1 9 7 4 ) r e p o r t e d t h a t t h e K+ e f f e c t w a s r e v e r s i b l e on removal of K + , Yoda and Yoda (1974) found it t o be irrev e r s i b l e . R e c e n t l y I have found t h a t t h e K+ ( o r Rb+) i o n s r e s p o n s i b l e f o r t h e e f f e c t a r e t i g h t l y bound

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3 F i g . 2 . 86Rb and [ H l o u a b a i n d i s s o c i a t i o n f r o m Na,K-ATPase. P a r t i a l 1 y p u r i f i e d Na,K-ATPase f r o m d o g k i d n e y (method o f J 4 r g e n s e n ; 0 . 8 mg i n 0.4 ml) was i n c u b a t e d w i t h M [ 3 H ] o u a b a i n i n the p r e s e n c e o f 10 mM Na', 2 . 5 mM Mg2+, and 5 mM ATP f o r 1 m i n a t 37OC, d i l u t e d t o 27 m l i n 25 mM i m i d a z o l e , 2 mM EDTA (OOC), and centrif u g e d ( 2 5 , 0 0 0 9 , 30 m i n ) . T h e p e l l e t was r e s u s p e n d e d i n 100 p 1 8 mM 86Rb+, 25 mM i m i d a z o l e , 2 mM EDTA (pH 7 . 5 ) and i n c u b a t e d a t 37OC f o r 5 min. T h e enzyme was p e l l e t e d i n a n a i r f u g e , r e s u s p e n d e d i n 1 m l i m i d a z o l e / E D T A b u f f e r ( O O C ) and q u i c k l y d i l u t e d i n t o 335 m l i m i d a zole/EDTA b u f f e r a t 37OC, pH 7 . 5 ( t = 0 ) . A d d i t i o n s (0.5-3 m l ) w e r e made t o 55 mZ p o r t i o n s o f the cont r o l (-'D-) b a t c h t o r e s u l t i n f i n a l c o n c e n t r a t i o n s o f ( - * - ) 1 0 mM K, ( - A - ) 100 mM Na, and (-v-) 100 mM Na, 2 mM ATP. (These a d d i t i o n s were made a t t = 1 , 2 , and 3 m i n , r e s p e c t i v e l y , and the d a t a h a v e been p l o t t e d t o r e f l e c t t i m e a f t e r a d d i t i o n . ) A t a p p r o p r i a t e t i m e s , 5-ml a l i q u o t s w e r e f i l t e r e d (Gelman GN-6, 5 m l w a s h , O°C, imidazole/ED!l'A + 1 0 mM K ) a n d bound 86Rb ( A ) and [ 3 H ] o u a b a i n ( B ) w e r e d e t e r m i n e d on the same f i l t e r b y t w o - c h a n n e l s c i n t i l l a t i o n c o u n t i n g . N o n s p e c i f i c b i n d i n g was n e g l i g i b l e , and no correction h a s been made. T h e amount o f [ 3 H ] o u a b a i n a t t = 0 i s o n l y a b o u t 25% of the maximal b i n d i n g level f o r t h i s p r e p a r a t i o n , p r e s u m a b l y d u e t o dissociation i n s t e p s prior to t = 0 (also, [protein] i s referenced t o the o r i g i n a l s a m p l e , and i s not c o r r e c t e d for p o s s i b l e losses i n c e n t r i f u g a t i o n ) . The d a s h e d l i n e i n B i n d i c a t e s the r a t e of [ 3 H ] o u a b a i n d i s s o c i a t i o n i n the control i f the 86Rb i n c u b a t i o n i s omitted

.

o r " o c c l u d e d " by t h e enzyme bound 86Rb: 3H o u a b a i n 2 2:1), and o n d i l u t i o n are r e l e a s e d w i t h a h a l f - t i m e of a b o u t 3 0 min: F i g u r e 2 shows t h e r e s u l t s of a n e x p e r i m e n t i n which T y p e I [3H]ouabain-Na,K-ATPase was i n c u -

176

BLISS FORBUSH 111

b a t e d f o r 1 0 min w i t h 8 mM 86Rb (and no o t h e r l i g a n d s ) A s assayed by and t h e n d i l u t e d more t h a n 10,000-fold. f i l t r a t i o n , b o t h t h e amount of [3H]ouabain and of 86% bound t o t h e p r o t e i n d e c r e a s e d w i t h a h a l f - t i m e of about 30-40 min. K+ i n t h e d i l u t i o n medium d i d n o t a f f e c t 86Rb+ d i s s o c i a t i o n , b u t i t d i d slow o u a b a i n d i s s o c i a t i o n , presumably by b i n d i n g t o t h e s i t e s v a c a t e d by t h e d i s s o c i a t i n g 86Rb. These and o t h e r d a t a a r e cons i s t e n t w i t h t h e h y p o t h e s i s t h a t upon d i l u t i o n of t h e K + - s t a b i l i z e d Type I i n t e r m e d i a t e , d i s s o c i a t i o n of bound K+ l i m i t s t h e r a t e of o u a b a i n d i s s o c i a t i o n . Na+ a l s o i n f l u e n c e s t h e r a t e of Type I ouabain-Na, K-ATPase complex d i s s o c i a t i o n , r e l a t i v e t o d i l u t i o n i n t o a medium c o n t a i n i n g no monovalent c a t i o n s . Akera et al. ( 1 9 7 4 ; a l s o B. Forbush 111, unpublished o b s e r v a t i o n s ) have found t h a t Na+ a t h i g h c o n c e n t r a t i o n s (100 m M ) s t a b i l i z e s t h e complex l i k e K+, a l t h o u g h t h e a c t i o n of N a + i s completely r e v e r s i b l e . While Yoda and Yoda ( 1 9 7 4 ) have r e p o r t e d a d e s t a b i l i z i n g e f f e c t of Na, t h e d i f f e r e n c e i s probably a t t r i b u t a b l e t o a s i g n i f i c a n t amount of ATP ( 0 . 1 m M ) p r e s e n t d u r i n g d i s s o c i a t i o n i n t h e l a t t e r work. When Na+ i s added t o t h e R b ( o r K ) s t a b i l i z e d form of t h e t y p e I complex, i t a c c e l e r a t e s r e l e a s e of 86Rb and s t a b i l i z e s t h e bound o u a b a i n ( F i g . 2); t h i s i n d i c a t e s t h a t t h e K+ s i t e s and Na+ s i t e s a r e s e p a r a t e , and t h a t b o t h can be occupied a t t h e same time The Type I1 complex d i s s o c i a t e s a t a l o w r a t e under a l l c o n d i t i o n s t e s t e d , e x c e p t t h a t i n t h e p r e s e n c e of Na and absence of Mg2+, ATP can a c t a t a low a f f i n i t y s i t e t o a c c e l e r a t e t h e p r o c e s s , p r o b a b l y w i t h o u t phosphorylat i o n ( t + = 1 0 mini Tobin e t al., 1 9 7 4 ; Yoda and Yoda, 1 9 7 4 ) . The d e s t a b i l i z a t i o n i s r e v e r s i b l e on f u r t h e r d i l u t i o n , i n d i c a t i n g t h a t t h e Type I1 complex has n o t been " c o n v e r t e d i n t o " t h e Type I complex. Type I complex d i s s o c i a t i o n c a n a l s o be a c c e l e r a t e d by Na+ + ATP relat i v e t o t h e r a t e i n t h e p r e s e n c e of b u f f e r a l o n e (Yoda and Yoda, 1 9 7 4 ; Huang and A s k a r i , 1 9 7 5 ) . When a h i g h c o n c e n t r a t i o n of K+ o r Na+ i s added t o t h e (Mg2+ + P i ) b i n d i n g medium, t h e ouabain-enzyme comp l e x e s t h a t a r e formed ( a t a low l e v e l i n t h e l a t t e r c a s e ) a r e less s t a b l e t h a n t h e u s u a l Type I1 complex. Hansen ( 1 9 7 9 ) has i n t e r p r e t e d t h e s e r e s u l t s , and s i m i l a r r e s u l t s from experiments i n which ouabain b i n d i n g was s u p p o r t e d by low energy phosphate d o n o r s , i n terms of a model i n which "Na-form" and "K-form" conformations a r e c o n s i d e r e d . To d a t e , t h e l a c k of e x t e n s i v e d a t a on t h e e f f e c t of v a r i o u s l i g a n d s p r e s e n t d u r i n g a s s o c i a t i o n ( p a r t i c u l a r l y i n " s i d e d " p r e p a r a t i o n s ) on t h e r a t e of ouabain d i s s o c i a t i o n a f t e r d i l u t i o n hampers t h e f i l l i n g - i n of such models.

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The most i m p o r t a n t s t r u c t u r a l f e a t u r e s o f t h e card i o t o n i c s t e r o i d s can be d i s c u s s e d i n t e r m s o f t h e t h r e e t o p o g r a p h i c a l r e g i o n s of t h e m o l e c u l e (see F i g . 3 ) : (1) A s t e r o i d moiety w i t h u n u s u a l s t e r e o c h e m i s t r y i s req u i r e d ; C/D c i s , B/C t r a n s a re f u n c t i o n s needed, and t h e A/B c i s c o n f i g u r a t i o n a l s o e n h a n c e s p o t e n c y . The lact o n e r i n g i s t h e most i m p o r t a n t s u b s t i t u e n t , w i t h t h e C - 1 4 ( 8 ) hydroxyl n e x t m o s t i m p o r t a n t . ( 2 ) An u n s a t u r a t e d s i d e group a t t h e C-17(8) p o s i t i o n i s an a b s o l u t e requirement. I n n a t u r a l p r o d u c t s , t h i s can be t h e 5membered l a c t o n e r i n g i n t h e c a r d e n o l i d e s , o r t h e 6membered r i n g of t h e b u t e n o l i d e s . Thomas e t ai. ( 1 9 7 4 ) have p r e p a r e d a number of o t h e r a c t i v e s i d e g r o u p s and have p o i n t e d o u t t h a t t h e y a l l have i n common a c o p l a n a r

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-R, where A i s =O, = N H , o r E N , and arrangement (217-C-CR i s p r e f e r a b l y smaller t h a n t h e e t h o x y group. The E r y t h r o p h l o e u m alkaloid cassaine f i t s t h i s requirement, a l t h o u g h i t l a c k s t h e D r i n g of t h e s t e r o i d . ( 3 ) A su-

g a r group a t t a c h e d t o t h e s t e r o i d a t t h e C-3(8) p o s i t i o n , w h i l e n o t n e c e s s a r y f o r a c t i v i t y , h a s a profound a f f e c t on t h e d i s s o c i a t i o n r a t e from N a , K - A T P a s e (see b e l o w ) . The l i t e r a t u r e r e g a r d i n g s t r u c t u r e - a c t i v i t y r e l a t i o n s h i p s for s e m i s y n t h e t i c compounds h a s been reviewed by Thomas e t ai. ( 1 9 7 4 1 , and f o r n a t u r a l p r o d u c t s , by Chen ( 1 9 6 2 ) ; w h i l e t h e l a t t e r work i s concerned w i t h c a r d i o t o n i c and c a r d i o t o x i c e f f e c t s , t h e weight of r e c e n t p h y s i o l o g i c a l d a t a c o n n e c t i n g Na,K-ATPase and c a r d i a c e f f e c t s makes these data equally relevant. Q u a n t i t a t i v e e v a l u a t i o n s o f c a r d i a c steroid-Na,KA T P a s e s t r u c t u r a l i n t e r a c t i o n s w i l l be g r e a t l y a i d e d by p r e c i s e s t r u c t u r a l d a t a f o r t h e s t e r o i d s . F u l l e r t o n et a1 (1979, and t h i , s volume) have d e t e r m i n e d c r y s t a l conf o r m a t i o n s o f a number o f compounds from x - r a y d a t a and have used p o t e n t i a l e n e r g y c a l c u l a t i o n s t o d e t e r m i n e t h e r o t a t i o n a l freedom o f t h e C-17 s i d e group. Applying t h e s e c a l c u l a t i o n s , t h e p o s i t i o n of t h e l a c t o n e c a r b o n y l , r e l a t i v e t o t h e p o s i t i o n found f o r d i g i t o x i g e n i n , shows an e x c e l l e n t c o r r e l a t i o n w i t h i n h i b i t o r y p o t e n c y , f o r a l m o s t 2 0 a g l y c o n e s ( F u l l e r t o n e t a l . , t h i s volume). Repke and P o r t i u s (1965) have p o i n t e d o u t t h a t t h e i n h i b i t o r y p o t e n c y o f t h e c a r d i o t o n i c s t e r o i d s i s rel a t e d t o t h e d e g r e e t o which t h e l a c t o n e r i n g i s e l e c t r i c a l l y p o l a r i z e d , as i f i t i n t e r a c t e d w i t h a n e g a t i v e l y c h a r g e d group on t h e p r o t e i n . Such an i n t e r a c t i o n i s s u p p o r t e d by t h e r e l a t i v e p o t e n c i e s of s t e r o i d a l quanyl-

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hydrazones, which can c a r r y a p o s i t i v e c h a r g e (Thomas et a l . , 1974). More r e c e n t l y Repke et a l . (1974) and Repke and D i t t r i c h (1980) have developed a t h e o r y which cons i d e r s t h e e n e r g y of i n t e r a c t i o n o f t h e d i p o l e moment of t h e e n t i r e cardiac s t e r o i d w i t h a h y p o t h e t i c a l e l e c t r i c d i p o l e i n t h e b i n d i n g s i t e . Given t h e freedom t o c h o o s e one of two r o t a t i o n a l o r i e n t a t i o n s of t h e l a c t o n e r i n g , t h e r e i s q u i t e good c o r r e l a t i o n f o r 20 d e r i v a t i v e s of d i g i t o x i g e n i n ( D i t t r i c h et a l . , t h i s volume). And, a l though t h e r e i s no o t h e r e v i d e n c e f o r a n e l e c t r i c f i e l d w i t h i n t h e o u a b a i n - b i n d i n g s i t e , t h e magnitude of t h e d i p o l e moment of p e p t i d e u n i t s o r i e n t e d i n a n a - h e l i x and the possible r e s u l t a n t electrical p o t e n t i a l gradient near a - h e l i c a l segments o f p e p t i d e s are v e r y l a r g e , as c a l c u l a t e d by Hol et a l . (1978) I t r e m a i n s t o b e s e e n t o what d e g r e e l o c a l i n t e r a c t i o n s w i l l be a n e c e s s a r y m o d i f i c a t i o n o f t h e stereoe l e c t r o n i c t h e o r y of Repke et a l . ( 1 9 7 4 ) . S i m i l a r l y , s p e c i f i c i n t e r a c t i o n s between t h e c a r d i o t o n i c s t e r o i d and N a , K - A T P a s e o b v i o u s l y t a k e p l a c e a t o t h e r p o s i t i o n s t h a n t h e l a c t o n e c a r b o n y l , s o t h e f i t of F u l l e r t o n e t a l . ( t h i s volume) c a n n o t be a s g e n e r a l a s i t h a s a p p e a r e d so f a r . I t i s i n t e r e s t i n g t o n o t e t h a t i t a p p e a r s t h a t i n order t o reasonably f i t the binding d a t a f o r the series o f a n a l o g s s t u d i e d by D i t t r i c h et a l . ( t h i s v o l ume), b o t h t h e o r i e s would need t o assume t h a t i n t h e p o o r l y b i n d i n g compounds t h e l a c t o n e r i n g is 180' r o t a t e d r e l a t i v e t o t h e lowest energy c o n f i g u r a t i o n . I n t h e one t h e o r y t h i s p r o v i d e s a d i e l e c t r i c component opp o s i t e go t h e p r i n c i p a l v e c t o r , and i n t h e o t h e r t h e o r y t h e s2 A d i s p l a c e m e n t o f t h e c a r b o n y l group would e x p l a i n t h e lowered A G O . Yoda h a s found t h a t t h e d i s s o c i a t i o n r a t e of card i a c g l y c o s i d e s from b o t h Type I ( f o r d i s s o c i a t i o n i n t h e p r e s e n c e o f K+) and Type I1 complexes w i t h beef b r a i n Na,K-ATPase i s u n a f f e c t e d by t h e n a t u r e of t h e s t e r o i d moiety and i s determined s o l e l y by t h e n a t u r e and number o f s u g a r g r o u p s (Yoda, 1973; Yoda et a l . , 1975; Yoda and Yoda, 1 9 7 5 ) . For i n s t a n c e , 3 ' - and 5 ' h y d r o x y l s are p a r t i c u l a r l y i m p o r t a n t i n t h a t s u b s t i t u t i o n a t t h e s e p o s i t i o n s on t h e f i r s t s u g a r l e a d s t o more r a p i d d i s s o c i a t i o n . D i s s o c i a t i o n i s slowed by a d d i t i o n a l sugar groups a f t e r t h e f i r s t , c o n s i s t e n t w i t h f u r t h e r int e r a c t i o n s w i t h t h e s i t e . Even t h e r a t e of d i s s o c i a t i o n of t h e c a r d i a c a g l y c o n e s w a s found t o be i n d e p e n d e n t of t h e s t e r o i d s t r u c t u r e ; a l l of t h e s e (ouabagenin, d i g o x i g e n i n , s t r o p h a n t h i d i n , d i g i t o x i g e n i n ) were found t o have t h e same h i g h d i s s o c i a t i o n r a t e : 0.63 min-1, a t 3OOC. T h i s s t r o n g l y s u g g e s t s t h a t t h e d i s s o c i a t i o n of g l y c o s i d e s and a g l y c o n e s p r o c e e d s t h r o u g h a common r a t e -

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CARDIOTONICSTEROID BINDING TO k.K-ATPase

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l i m i t i n g s t e p , which i s independent of s t e r o i d s t r u c t u r e , a l t h o u g h t h e a b s o l u t e rates a r e v e r y d i f f e r e n t f o r t h e two c l a s s e s of compounds (see model b e l o w ) . With Type I complexes i n t h e absence of K + , d i s s o c i a t i o n of t h e c a r d i a c g l y c o s i d e s i s more r a p i d (see a b o v e ) , and t h e s t e r o i d p l a y s some r o l e i n d e t e r m i n i n g d i s s o c i a t i o n r a t e (Yoda and Yoda, 1 9 7 4 ) . A s s o c i a t i o n r a t e c o n s t a n t s f o r b o t h Type I and Type I1 complexes of t h e c a r d i a c g l y c o s i d e s and t h e i r corresponding aglycones depend markedly on t h e n a t u r e of t h e s t e r o i d moiety, i n c r e a s i n g i n t h e o r d e r o u a b a i n , d i g o x i n , s t r o p h a n t h i d i n g l y c o s i d e , d i g i t o x i n (Yoda e t a]., 1973; Yoda and Yoda, 1 9 7 7 ) . The n ---a t u r e of a s u g a r group i s unimportant, i m p l y i n g , a s t h e a u t h o r s have sugg e s t e d , t h a t t h e r a t e - d e t e r m i n i n g s t e p i s b i n d i n g of t h e s t e r o i d , and t h a t a s s o c i a t i o n of t h e s u g a r group w i t h t h e s u g a r - s p e c i f i c s i t e i s slower. On t h e o t h e r hand, t h e number of s u g a r groups h a s a s i g n i f i c a n t e f f e c t which may be m u l t i p l i e d t i m e s t h e c o n t r i b u t i o n of t h e steroid t o give the association rates: r e l a t i v e t o the a g l y c o n e s , t h e mono-, b i s - , and t r i - s a c c h a r i d e d e r i v a t i v e s have r a t e s of 0 . 3 , 0 . 2 , and 0.15, r e s p e c t i v e l y . The i n v e r s e dependence on " q u a n t i t y b u t n o t q u a l i t y " suggests a s t e r i c hindrance i n t h e i n i t i a l i n t e r a c t i o n of t h e i n h i b i t o r w i t h t h e s i t e , i n which t h e p r e s e n c e of t h e bulky s u g a r groups d e c r e a s e s t h e p r o b a b i l i t y of the s t e r o i d entering the s i t e within a given t i m e . A minimal model t o encompass Yoda's s t r u c t u r e a c t i v i t y r e s u l t s f o r Type I1 c a r d i a c g l y c o s i d e b i n d i n g i s g i v e n i n Scheme 1, where E r e p r e s e n t s Na,K-ATPaSe, and G r e p r e s e n t s t h e g e n i n p o r t i o n , and S t h e s u g a r port i o n of the glycoside.

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BLISS FORBUSH 111

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w i t h r e g a r d t o s t e r o i d - s i t e i n t e r a c t i o n , and

G-E

t h e r e f o r e s t e p l a b u t n o t s t e p l b depends on t h e s t r u c t u r e of t h e i n h i b i t o r . ( 2 ) k-2 i s r a p i d r e l a t i v e t o k-1. Although a t f i r s t c o u n t e r i n t u i t i v e , t h i s i s r e a s o n a b l e i n view o f t h e low r a t e of s t e p 1. Thus, v e r y slow cardiac glycoside dissociation r e l a t i v e t o dissociation of t h e corresponding a g l y c o n e s i s due t o t h e low e q u i l i b r i u m c o n c e n t r a t i o n of S , t h e s p e c i e s which can d i s -

I

G-E

sociate. Yoda has r e a c h e d t h i s c o n c l u s i o n , t h a t t h e r a t e - l i m i t i n g s t e p (k-l i n Scheme 1) i n d i s s o c i a t i o n i s a c o n f o r m a t i o n a l change f o l l o w i n d i s s o c i a t i o n of t h e s u g a r groups ( k- 2 i n Scheme the finding t h a t t h e a c t i v a t i o n energy f o r d i s s o c i a t i o n i s t h e same f o r a l l of t h e g l y c o s i d e s t e s t e d ( 2 20 kcal/mole; Yoda e t al., 1 9 7 5 ) . The simple model above, w i t h assumptions 1 and 2, f i t s t h e f o l l o w i n g o b s e r v a t i o n s of Yoda and others: ( a ) Association rate, b u t n o t d i s s o c i a t i o n rate of b o t h g l y c o s i d e s and a g l y c o n e s depends on t h e s t e r o i d s t r u c t u r e (from assumption 1 ) . (b) The d i s s o c i a t i o n ( c ) The d i s s o c i a r a t e of a l l aglycones i s t h e same. t i o n r a t e of t h e g l y c o s i d e s depends o n l y on t h e n a t u r e of t h e s u g a r group: t h e a c t i v a t i o n energy f o r d i s s o c i a t i o n i s t h e same f o r a l l of t h e g l y c o s i d e s . ( d ) The a s s o c i a t i o n rates of g l y c o s i d e s and aglycones a r e s i m i l a r , t h e 3-fold d i f f e r e n c e representing a reasonable c o n t r i b u t i o n t o k from hindrance by a s i n g l e s u g a r . ( e ) For Na,K-ATPahe from d i f f e r e n t t i s s u e s and s p e c i e s , w i t h widely d i f f e r i n g a f f i n i t i e s f o r c a r d i a c g l y c o s i d e s , t h e a f f i n i t i e s f o r t h e aglycones v a r y i n t h e same way; t h e d i f f e r e n c e s are p r i m a r i l y i n t h e d i s s o c i a t i o n r a t e , c o n s i s t e n t w i t h s p e c i e s t o s p e c i e s v a r i a t i o n i n k-1 (see below). For Type I enzyme-ouabain complexes, o u a b a i n bindi n g and d i s s o c i a t i o n p r o c e e d s through a c y c l e of phosp h o r y l a t i o n by ATP, o u a b a i n b i n d i n g , d e p h o s p h o r y l a t i o n , and ouabain d i s s o c i a t i o n ; t h i s i s a s t e a d y s t a t e , r a t h e r than e q u i l i b r i u m s i t u a t i o n and r e q u i r e s a more complex, c y c l i c model. Yoda and Yoda ( 1 9 7 7 ) have developed s u c h a model t o e x p l a i n t h e i r unusual o b s e r v a t i o n t h a t v a r i a t i o n s i n l i g a n d c o n c e n t r a t i o n s ( N a + , Mg2+, P i , ATP) change t h e a p p a r e n t number of s i t e s , b u t n o t t h e a f f i n i t y f o r [3H]d i g o x i g e n i n : e s s e n t i a l l y , b i n d i n g of l i g a n d s a t c e r t a i n s t e p s i n t h e c y c l e "removes" a f r a c t i o n of t h e s i t e s from t h e a p p a r e n t e q u i l i b r i u m . I t s h o u l d be n o t e d t h a t one c a n n o t a p p l y , a s t h e y have, t h e same reasoning to s i m i l a r observations i n t h e t r u e e q u i l i b r i u m s i t u a t i o n of (Mg2+ + P i ) - s t i m u l a t e d b i n d i n g .

*ram

CARDIOTONIC STEROID BINDING TO Na,K-ATPase

A.

183

S P E C I E S AND T I S S U E DIFFERENCES

Very l a r g e d i f f e r e n c e s i n t h e s e n s i t i v i t y of Na,KATPase from v a r i o u s t i s s u e s and s p e c i e s t o i n h i b i t i o n by ouabain c o n s t i t u t e one of t h e remarkable f e a t u r e s of c a r d i a c glycoside-Na,K-ATPase i n t e r a c t i o n s : t h e a f f i n i t y f o r o u a b a i n r a n g e s from a b o u t 2 x 1 0 - 9 M i n sens i t i v e t i s s u e s such a s dog kidney t o g r e a t e r t h a n 10-5 M i n r a t h e a r t ( c f . Tobin and Brody, 1 9 7 2 ; Erdmann and Schoner, 1 9 7 3 ) . An e x p l a n a t i o n h a s been s o u g h t i n t h e d e g r e e of i n t e r a c t i o n between t h e Na,K-ATPase and v a r i ous p o r t i o n s of t h e i n h i b i t o r molecule. Comparing t h e r e l a t i v e i n h i b i t o r y p o t e n c i e s of v a r i o u s c a r d i o t o n i c d r u g s having a l t e r e d l a c t o n e r i n g s t r u c t u r e , Akera e t a l . ( 1 9 7 9 ) r u l e d o u t t h e l a c t o n e r i n g r e g i o n of t h e b i n d i n g s i t e . S i m i l a r l y , Wallick e t a i . (1980) found t h a t t h e r e l a t i v e e f f e c t i v e n e s s of ouabain and t h e c o r r e s p o n d i n g aglycone, ouabagenin, ( 1 0 - t o 20-fold r a t i o ) w a s t h e same f o r most t i s s u e s and s p e c i e s , so t h a t o n l y a s m a l l p a r t of t h e s p e c i e s d i f f e r e n c e c o u l d be due t o i n t e r a c t i o n a t t h e sugar-binding s i t e . Akera e t a l . ( 1 9 7 4 ) , have proposed a " l i p i d b a r r i e r " t o c a r d i a c glycoside d i s s o c i a t i o n t h a t i s lacking i n the ouabain-insensitive species; there is l i t t l e d i r e c t I t is t h e s s o e v i d e n c e f o r such a barrier, however. c i a t i o n rate (Tobin and Brody, 1 9 7 2 ; Wallick e t al., 1 9 8 0 1 , and n o t t h e a s s o c i a t i o n r a t e (Erdmann and Schoner, 1973; Wallick et a l . , 1 9 8 0 ) , t h a t i s markedly d i f f e r e n t i n d i f f e r e n t s p e c i e s , presumably f o r t h e aglycones a s w e l l a s t h e g l y c o s i d e s ( s i n c e t h e 150's a r e a l t e r e d f o r b o t h types of i n h i b i t o r ) . T h i s is cons i s t e n t w i t h an a l t e r a t i o n i n k - 1 i n Scheme 1, which acc o r d i n g t o t h e proposed model i s n o t r e l a t e d t o i n t e r a c t i o n s a t e i t h e r t h e s t e r o i d o r s u g a r r e g i o n s of t h e b i n d i n g s i t e , b u t t o a c o n f o r m a t i o n a l change e l s e w h e r e i n t h e molecule.

111.

COVALENT LABELING O F THE OUABAIN B I N D I N G SITE

Four g o a l s of s p e c i f i c c o v a l e n t l a b e l i n g of t h e (1) I d e n t i f i c a ouabain b i n d i n g s i t e may be i d e n t i f i e d : t i o n of t h e p e p t i d e s making up t h e b i n d i n g s i t e . T h i s 2Abbreviations u s e d : AD-, aryl diazonium-; CM-, ethylchloromalonyl-; DAM-, ethyl diazomlonyl-; NAB-, 2-nitro-5-azidobenzoyl-; NAP, 2-nitro-l-azidophenyl-; NPT, p-nitrophenyltriazene.

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h a s been accomplished, a s w i l l be reviewed below. ( 2 ) I d e n t i f i c a t i o n w i t h i n a g i v e n s u b u n i t of p e p t i d e domains t h a t a r e i n v o l v e d i n t h e s i t e . Only p r e l i m i n a r y e f f o r t s have been made i n t h i s d i r e c t i o n . (3) U s e of t h e c o v a l e n t l y a t t a c h e d molecule t o i s o l a t e o n l y those peptides t h a t are covalently labeled. This i s a u s e f u l t e c h n i q u e (Forbush and Hoffman, 1 9 7 9 a ) , b u t n o t a n e n d i n i t s e l f , and it w i l l n o t be d i s c u s s e d f u r t h e r here. ( 4 ) Mapping t h e e n t i r e ouabain-binding s i t e i n v a r i o u s c o n f o r m a t i o n a l s t a t e s , u s i n g a l a r g e number of d e r i v a t i v e s w i t h r e a c t i v e groups a t d i f f e r e n t l o c a t i o n s ; t h i s i s an a m b i t i o u s g o a l , which w i l l r e q u i r e c a u t i o u s i n t e r p r e t a t i o n of d a t a . Aspects of c o v a l e n t l a b e l i n g t e c h n i q u e s are c o n s i d e r e d below i n r e l a t i o n t o t h i s g o a l , and p r o g r e s s t o d a t e i s reviewed. S i n c e i t i s des i r a b l e t o generate very r e a c t i v e species within t h e b i n d i n g s i t e , t h e d i s c u s s i o n below centers around photoa f f i n i t y l a b e l i n g , a t e c h n i q u e which h a s been r e c e n t l y reviewed (Tometsko and R i c h a r d s , 1980; Chowdry and Westheimer, 1979; J o r i and S p i k e s , 1978; Bayley and Knowles, 1 9 7 6 ) . C e r t a i n a f f i n i t y l a b e l s h a v i n g reactive g r o u p s which a r e g e n e r a t e d w i t h i n t h e i n h i b i t o r y s i t e may a l s o be u s e f u l (e.9. , NPT-ouabain) , and are cons i d e r e d i n t h e same c o n t e x t . A.

SPECIFICITY

a l l of t h e I t s h o u l d be s t r e s s e d a t t h e o u t s e t t h a t s p e c i f i c i t y i n p h o t o a f f i n i t y - l a b e l i n g experiments arises from t h e s p e c i f i c i t y of b i n d i n g of t h e p a r e n t c a r d i o t o n i c s t e r o i d m o l e c u l e . The r e a c t i v e s p e c i e s i n p h o t o a f f i n i t y l a b e l i n g r e a g e n t s are i n d i s c r i m i n a t e and w i l l l a b e l most p r o t e i n s i n t h e r e a c t i o n m i x t u r e under c o n d i t i o n s where t h e r e a g e n t i s p r e s e n t i n e x c e s s . Phot o a f f i n i t y d e r i v a t i v e s of o u a b a i n , f o r i n s t a n c e , are u s u a l l y a m p h i p a t h i c m o l e c u l e s , w i t h a h y d r o p h i l i c ouab a i n p o r t i o n and a hydrophobic p h o t o a f f i n i t y group; t h e l a t t e r moiety i s l i k e l y t o p a r t i t i o n i n t o membranes, and t o p r e f e r e n t i a l l y l a b e l hydrophobic r e g i o n s of p r o t e i n s . The o c c u r r e n c e of n o n s p e c i f i c p h o t o l a b e l i n g , which may show p r e f e r e n c e s f o r o n e p e p t i d e ( e . g . , hydrophobic pept i d e s ) o v e r a n o t h e r , i s t h u s n e i t h e r s u r p r i s i n g nor v e r y informative r e l a t i v e t o c a r d i a c glycoside binding, but it c a n b e e l i m i n a t e d by p r o p e r e x p e r i m e n t a l d e s i g n . I n t h e case o f c a r d i a c g l y c o s i d e b i n d i n g t o Na,K-ATPase from nonrodent s p e c i e s it i s a simple matter t o e x p l o i t t h e low r a t e o f c a r d i a c g l y c o s i d e d i s s o c i a t i o n t o a c h i e v e v e r y l o w l e v e l s o f n o n s p e c i f i c l a b e l i n g , by ext e n s i v e washing of t h e enzyme-glycoside complex p r i o r t o

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p h o t o l y s i s , u s i n g e i t h e r f i l t r a t i o n or c e n t r i f u g a t i o n . When a p a r t i c u l a r e x p e r i m e n t a l o b j e c t i v e d o e s n o t p e r m i t t h e washing s t e p , n o n s p e c i f i c b i n d i n g must be minimized by p e r f o r m i n g p h o t o l y s i s under c o n d i t i o n s where most o f t h e d e r i v a t i v e i s bound a t t h e i n h i b i t o r y s i t e . I n a d d i t i o n , s o l u b l e p r o t e i n s o r chemical s c a v e n g e r s c a n be added t o d e c r e a s e n o n s p e c i f i c l a b e l i n g . R o s s i e t a l . d e s c r i b e ( t h i s volume) a compound, ADo u a b a i n , which o f f e r s some t h e o r e t i c a l a d v a n t a g e , i n t h a t it i s a c t i v a t e d by e n e r g y t r a n s f e r from t r y p t o p h a n s n e a r t h e b i n d i n g s i t e ; however, w h i l e t h e compound would n o t be e x c i t e d i n free s o l u t i o n , i t would p r o b a b l y l a b e l t r y p t o p h a n - c o n t a i n i n g p r o t e i n s t o which it w a s n o n s p e c i f i c a l l y adsorbed. B.

SELECTIVITY

An i m p o r t a n t p a r t o f t h e r a t i o n a l e behind p h o t o a f f i n i t y l a b e l i n g i s t h a t t h e p h o t o r e a c t i v e g r o u p be ext r e m e l y r e a c t i v e , and t h u s s h o r t - l i v e d and n o n s e l e c t i v e . There are two r e a s o n s f o r t h i s . (1) I f t h e photogenera t e d s p e c i e s i s l o n g - l i v e d , t h e molecule may l e a v e t h e b i n d i n g s i t e and n o n s p e c i f i c a l l y l a b e l o t h e r p r o t e i n s . T h i s i s of l i t t l e c o n c e r n w i t h r e g a r d t o c a r d i a c g l y c o s i d e s and t h e Na,K-ATPase because of t h e low r a t e s of d i s s o c i a t i o n ; even n o n r e a c t e d , p h o t o l y z e d m o l e c u l e s remain bound a t t h e s i t e (Forbush e t al., 1 9 7 8 b ) . ( 2 ) The p h o t o a f f i n i t y group may be a t t a c h e d t o t h e p a r e n t molec u l e by a l o n g and f l e x i b l e s p a c e r a r m , and may be f r e e t o move a b o u t w i t h i n and around t h e b i n d i n g s i t e . A l t e r n a t i v e l y , movement of amino-acid s i d e c h a i n s may b r i n g d i f f e r e n t groups i n t o proximity w i t h t h e p h o t o l a b e l o v e r p e r i o d s of many nanoseconds. I f , a f t e r p h o t o l y s i s , t h e r e a c t i o n i s immediate ( n a n o s e c o n d s ) , t h e d i s t r i b u t i o n of l a b e l w i l l c o r r e c t l y r e f l e c t t h e s p a t i a l d i s t r i b u t i o n o f t h e group b e f o r e p h o t o l y s i s . If reaction i s slow ( h u n d r e d s of nanoseconds) t h e group may move around u n t i l i t f i n d s t h e most r e a c t i v e p a r t n e r ( f o r i n s t a n c e , a n amino g r o u p ) ; i n which case l a b e l i n g may be due t o a n o r i e n t a t i o n o c c u r r i n g o n l y a s m a l l f r a c t i o n of t h e t i m e . W e l a c k a c o h e r e n t body of d a t a r e g a r d i n g t h e r a t e s o f r e a c t i o n and s e l e c t i v i t i e s o f p h o t o l a b e l i n g by any o f t h e p h o t o a f f i n i t y l a b e l s , b u t a number of import a n t o b s e r v a t i o n s have been made r e c e n t l y . Bayley and Knowles (1978a) and Gupta e t a l . ( 1 9 7 9 ) have found t h a t n i t r e n e s formed on p h o t o l y s i s o f p h e n y l a z i d e s and n i t r o p h e n y l a z i d e s ( u s e d i n NAP-ouabain, NAB-ouabain) do n o t r e a c t t o a s i g n i f i c a n t e x t e n t by i n s e r t i o n i n t o C-H bonds, e v e n when more r e a c t i v e bonds a r e a b s e n t . T h u s

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n i t r e n e s p r o b a b l y select f o r C=C bonds i n aromatic a m i no a c i d s , and f o r n u c l e o p h i l i c g r o u p s (-OH, -NH2, -SH). On t h e o t h e r hand, c a r b e n e s , which a r e known t o be s h o r t e r - l i v e d , are found t o i n s e r t i n t o -CH bonds w i t h good y i e l d , and are b e t t e r c a n d i d a t e s € o r n o n s e l e c t i v e p r o b e s . These t o o , however, p r e f e r C=C bonds and nuc l e o p h i l e s when t h e y a r e i n c l o s e p r o x i m i t y (Brunner and Richards, 1 9 8 0 ) . I n a d d i t i o n , rearrangement of a c y l c a r b e n e s , e . g . , from DAM-digitoxin, can y i e l d l o n g - l i v e d r e a c t i v e s p e c i e s . The carbonium i o n produced by t h e i n situ decomposition of NPT-ouabain, a l t h o u g h h i g h l y r e a c t i v e , i s s e l e c t i v e f o r nucleophilic groups; f u r t h e r s e l e c t i o n may r e s u l t from t h e p r o x i m i t y of groups on t h e p r o t e i n t h a t c a t a l y z e t h e decomposition o f t h e p a r e n t t r i a z e n e . Thus an i d e a l l y n o n s e l e c t i v e p h o t o a f f i n i t y group i s n o t known. The b e s t c a n d i d a t e s t o d a t e a r e p r e c u r s o r s such as a r y l d i a z i r a n e s and c e r t a i n d i a z o compounds which a r e p r o t e c t e d from rearrangement (Brunner and R i c h a r d s , 1980; Gupta e t al., 1 9 7 9 ; Bayley and Knowles, 197833; Chowdry et a l . , 1 9 7 6 ) ; none of t h e s e has y e t been used t o d e r i v a t i z e c a r d i o t o n i c s t e r o i d s . C.

LOCALIZATION

Because of t h e s i z e of t h e a r y l n i t r e n e and a r y l carbene p r e c u r s o r s , and t h e l e n g t h of t h e s p a c e r arms w i t h which t h e y a r e most e a s i l y a t t a c h e d t o c a r d i o t o n i c s t e r o i d s , t h e r e i s i n e v i t a b l e ambiguity w i t h r e g a r d t o t h e r e l a t i o n s h i p between t h e s i t e of l a b e l i n g and t h e b i n d i n g s i t e . For i n s t a n c e i n NAP-strophanthidin, which has been r e p o r t e d a s a probe of t h e "primary" r e g i o n of t h e b i n d i n g s i t e (Rogers and Lazdunski, 1 9 7 9 a ) , t h e n i t r o p h e n y l a z i d e can be extended 1 2 d fgom t h e s t e r o i d , and as f a r from t h e l a c t o n e r i n g ( 2 0 A) as t h e rhamnose moiety i n o u a b a i n (see F i g . 3 ) . D e r i v a t i v e s d e s i g n e d t o l a b e l t h e " s u g a r b i n d i n g region'' appear t o have an advantage i n t h i s r e g a r d i n t h a t t h e y can be t h e same l e n g t h a s o t h e r c a r d i a c g l y c o s i d e s ; however, it seems u n l i k e l y t h a t t h e hydrophobik p h o t o a f f i n i t y groups would be bound i n t h e same way, or even i n t h e same p l a c e , as s u g a r groups. I t would be r e a s s u r i n g i f t h e a d d i t i o n o f a p h o t o a f f i n i t y group a t t h e 3 - p o s i t i o n would c o n f e r on a c a r d i a c aglycone t h e marked d e c r e a s e i n d i s s o c i a t i o n r a t e constant seen with t h e glycosides. Unfortunately, t h e r a t e of d i s s o c i a t i o n from Na,K-ATPase of NAP-glycyld i g i t o x i g e n i n (Ruoho and H a l l , 1 9 8 0 ) has n o t y e t been r e p o r t e d . The second and t h i r d s u g a r g r o u p s have less e f f e c t on b i n d i n g , and i t i s t h u s h a r d e r t o assess changes a t t h e s e p o s i t i o n s . G e n e r a l l y , t h e r e does n o t

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a p p e a r t o be an e a s y r e s o l u t i o n of p o s i t i o n a l ambiguity w i t h t h e p r o b e s c u r r e n t l y a v a i l a b l e . However, by p r e p a r i n g s t r i c t l y analogous f l u o r e s c e n t (and/or s p i n l a b e l ) d e r i v a t i v e s , and s t u d y i n g p o l a r i z a t i o n l i f e t i m e s , i t may be p o s s i b l e t o e s t i m a t e t h e d e g r e e of m o b i l i t y of t h e p r o b e s w i t h i n t h e b i n d i n g s i t e . Acyldiazo groups ( e . g . , DAM-) a r e advantageous from t h e p o i n t of view of l o c a l i z a t i o n , s i n c e n o t o n l y a r e t h e y less bulky and s h o r t e r t h a n a r y l g r o u p s , t h e y may a l s o be c o n s i d e r a b l y less hydrophobic. " D i r e c t " photoaffinity labeling exploits the sensit i v i t y of t h e n a t i v e c a r d i o t o n i c s t e r o i d and/or t h e Na,K-ATPase t o short-wave W l i g h t t o produce c o v a l e n t l i n k a g e s between t h e two molecules. Although t h e r e i s no u n c e r t a i n t y r e g a r d i n g t h e p o s i t i o n of a d a n g l i n g p r o b e , t h e mechanism of a t t a c h m e n t and t h e l o c a t i o n of c o v a l e n t bonds a r e unknown. I n p r a c t i c e (Forbush and Hoffman, 1 9 7 9 b ) , t h e method produces s p e c i f i c l a b e l i n g of low e f f i c i e n c y , b u t i s s e v e r e l y l i m i t e d by photoc r o s s - l i n k i n g o f t h e p r o t e i n . The f i r s t r e p o r t e d photol a b e l i n g of Na,K-ATPase (Ruoho and K y t e , 1 9 7 4 , 1 9 7 7 ) i n volved " d i r e c t " p h o t o a f f i n i t y l a b e l i n g , s i n c e CM-cymarin (which was t w i c e a s e f f e c t i v e a s DAM-cymarin) does n o t have a known p h o t o a f f i n i t y l a b e l i n g group. D.

PHOTOLYSIS

Two p r a c t i c a l a s p e c t s of c o n s i d e r a b l e importance i n p h o t o a f f i n i t y l a b e l i n g a r e t h e c h a r a c t e r i s t i c s of l i g h t a b s o r p t i o n and t h e o v e r a l l e f f i c i e n c y of c o v a l e n t i n c o r p o r a t i o n of p h o t o l a b e l . To be g e n e r a l l y u s e f u l , t h e compound should be a c t i v a t e d e f f e c t i v e l y by l i g h t g r e a t e r t h a n 310 nm, because i f l i g h t <300 nm h a s t o be u s e d , p h o t o i n h i b i t i o n of N a , K-ATPase and e x t e n s i v e photoc r o s s - l i n k i n g w i l l r e s u l t (Forbush and Hoffman, 1 9 7 9 b ) . Of t h e compounds d i s c u s s e d h e r e , o n l y t h e diazomalonyl (DAM-) group i s d e f i c i e n t i n t h i s r e g a r d . I t i s a l s o h e l p f u l i f t h e molar absorbance and quantum e f f i c i e n c y of t h e compound a r e a s h i g h a s p o s s i b l e , t o p e r m i t photolysis i n a short t i m e . With t h e n i t r o a r y l a z i d e s ( N A P , N A B ) , p h o t o l y s i s can be complete i n less t h a n 0 . 5 s e c if l i g h t from a 1 KW mercury-zenon a r c lamp ( w e l l - f i l t e r e d t o 310-380 nm f o r NAB) i s focused on a 5 mm2 sample a r e a . T h i s i s r a p i d enough t o p e r m i t s t u d y of r e l a t i v e l a b e l i n g of d i f f e r e n t c a r d i O t o n i c steroid-Na,K-ATPase c o n f o r m a t i o n s , e . g . , E I g u a b a l n v s . E-Ouabain when t h e enzyme d e p h o s p h o r y l a t e s a f t e r o u a b a i n b i n d i n g ( B . Forbush 111, unpublished r e s u l t s ) . I t s h o u l d a l s o be p o s s i b l e t o

BLISS FORBUSH 111

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p h o t o l a b e l r o d e n t Na,K-ATPase a f t e r r a p i d f i l t r a t i o n and washing, b e f o r e t h e p h o t o a f f i n i t y d e r i v a t i v e has d i s s o c i a t e d from t h e enzyme. A high e f f i c i e n c y of p h o t o i n c o r p o r a t i o n - - t h e r a t i o of molecules c o v a l e n t l y i n c o r p o r a t e d t o t h o s e r e v e r s i b l y bound b e f o r e p h o t o l y s i s - - i s c l e a r l y d e s i r a b l e , i f only f o r ease of d e t e c t i o n . When p e p t i d e mapping s t u d i e s a r e contemplated, t h i s becomes much more i m p o r t a n t , e s p e c i a l l y s i n c e it i s l i k e l y t h a t l a b e l i n g w i l l t a k e p l a c e a t more t h a n one r e s i d u e . U n f o r t u n a t e l y , t h e photochemical l i t e r a t u r e i s o f v i r t u a l l y no h e l p i n p r e d i c t i n g t h e comp a r a t i v e p h o t o l a b e l i n g e f f i c i e n c i e s of d i f f e r e n t p r o b e s even i n s i m p l e model systems: as a r e s u l t i t i s n o t poss i b l e t o p r e d i c t t h e r e s u l t when t h e e f f i c i e n c y of l a b e l I f a g i v e n probe i s i n g a p r o t e i n i s t o be determined. known t o be f r e e of i n t r a m o l e c u l a r r e a c t i o n s , t h e n low l a b e l i n g e f f i c i e n c y m u s t be due t o e a s y a c c e s s of aqueous medium i n t o t h e r e g i o n of t h e r e a c t i v e g r o u p , and/or a high a b s o l u t e r a t e of r e a c t i o n of t h e probe w i t h water compared w i t h t h e r a t e of r e a c t i o n w i t h t h e p r o t e i n moiety. I n p r a c t i c e , 20% or g r e a t e r photoincorp o r a t i o n i s u n u s u a l l y good, w h i l e amounts l e s s t h a n 2 % p r e s e n t problems of s e p a r a t i o n of p r o d u c t s , n o n s p e c i f i c l a b e l i n g , and i n some c a s e s doubt a s t o t h e mechanism of covalent attachment. I n summary, none of t h e a v a i l a b l e p h o t o l a b e l s i s o p t i m a l i n e v e r y r e g a r d . NAB- and NPT-derivatives l a b e l t h e Na,K-ATPase w i t h h i g h e f f i c i e n c y (see b e l o w ) , b u t t h e r e a c t i v e groups may p a r t i t i o n i n t o hydrophobic reg i o n s n e a r t h e b i n d i n g s i t e and l a b e l s e l e c t i v e l y . The diazomalonyl (DAM) group i s more l i k e l y t o remain i n t h e sugar-binding s i t e , b u t i t r e q u i r e s b i o l o g i c a l l y damaging amounts of s h o r t wavelength UV l i g h t f o r p h o t o l y s i s , and t h e photoproduct i n s e r t s w i t h low e f f i c i e n c y (see b e l o w ) . I t may be more s e l e c t i v e t h a n c e r t a i n o t h e r c a r b e n e s : t h e l a t t e r , however, have n o t y e t been used t o l a b e l t h e ouabain-binding s i t e . E.

P R O G R E S S I N LABELING SITE

THE CARDIAC GLYCOSIDE-BINDING

D e r i v a t i v e s of c a r d i a c s t e r o i d s t h a t have been developed i n f o u r l a b o r a t o r i e s i n t h e l a s t seven y e a r s t o l a b e l t h e Na,K-ATPase a r e l i s t e d i n Table I , t o g e t h e r w i t h an a b b r e v i a t e d s t a t e m e n t of t h e r e s u l t s o b t a i n e d . Included a r e one d e r i v a t i v e and two n a t i v e compounds t h a t have been found t o c o v a l e n t l y i n c o r p o r a t e i n t o t h e Na,K-ATPase i n short-wave UV l i g h t by " d i r e c t " (mechanism unknown) p h o t o a f f i n i t y l a b e l i n g . The compounds

TABLE I.

Summary o f Covalent L a b e l i n g R e s u l t s w i t h C a r d i o t o n i c S t e r o i d D e r i v a t i v e sa 0

Steroid 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16.

Digitoxigenin Ouabain CM-cymarin DAM-cymarin Oxidized ouabain AD-ouabain NPT-ouabain 4'-DAM-monodigitoxoside NAP-strophanthidin NAP-ouabain

NAP-glycyl-digitoxigenin NAP-ouabain I NAB-ouabain (11) 4"-DAM-bisdigitoxoside 3"' -DAM-digitoxin 4 I" -DAM-digitoxin

D i s t a n c e (A) a of r e a c t i v e group Frm From c e n t e r s t e r o i d of l a c t o n e 0 0- 6 0-14 0-14 4 4-8 7 9 12* 13 13 13 13 14 17 18

0-11 0-17 0-25 0-25

15 15-18 18 20 20" 23 24 24 24 24 28 29

E f f i c i e n c y of photoincorporation

Labeling of other peptides as f r a c t i o n of l a b e l i n g of a

? "Direct" ? "Direct " ? "Direct"

" D i r e c t " o r carbene Aldehyde Carbonim ion Carbonium i o n Acyl c a r b e n e Aryl n i t r e n e Aryl n i t r e n e Aryl n i t r e n e A r y l nitrene A r y l nitrene A r y l carbene A r y l carbene Aryl carbene

Y

B

Reactive species 0.5 1 <2 <1

0 0 0 0

20 20

0

-

0.5

It <5 20 35

-

<5 <5

t

0 0

-

-

0 0

0 0 0

0

0

0

0.7

0 <0.1 <0.06 <0.1 0.4

-

Ql

0

2.2 0.8 0

-

a D i s t a n c e s were e s t i m a t e d f r o m m o l e c u l a r m o d e l s . I*) N A P - h a s a C-19 a t t a c h m e n t t o the s t e r o i d ; a l l o t h e r d e r i v a t i v e s a r e a t C-3. " E f f i c i e n c y o f i n c o r p o r a t i o n " i s the f r a c t i o n o f r e v e r s i b l y bound l a b e l t h a t becomes i r r e v e r s i b l y bound on p h o t o l y s i s , when t h i s i s a v a i l a b l e i n the l i t e r a t u r e ; i n t w o c a s e s (t) a n a t t e m p t i s made t o c a l c u l a t e this v a l u e f r o m d a t a i n f i g u r e l e g e n d s o f the a p p r o p r i a t e references. R e f e r e n c e s f o r the u s e o f these compounds i n c l u d e : compounds 1 , 2 , F o r b u s h and Hoffman, 1979b; 3 , 4 , Ruoho and K y t e , 1 9 7 4 , 1 9 7 7 ; 5 , H e g y v a r y , 1 9 7 5 ; 6 , R o s s i e t a l . , t h i s v o l u m e ; 7 , R o s s i e t a l . , 1 9 8 0 ; 8 , 14, H a l l and Ruoho, t h i s v o l u m e ; 9 , 1 0 , R o g e r s and L a z d u n s k i , 1 9 7 9 a , b ; 11, Ruoho and H a l l , 1 9 8 0 ; 1 2 , 1 3 , Forbush e t a l . , 1978b; 1 5 , 1 6 , H a l l and Ruoho, 1 9 8 0 .

BLISS FORBUSH 111

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a r e l i s t e d i n o r d e r of i n c r e a s i n g d i s t a n c e between t h e l a c t o n e r i n g and t h e r e a c t i v e atom, as e s t i m a t e d from m o l e c u l a r models. Although t h i s d i s t a n c e i s d i s c u s s e d h e r e , a s it h a s been i n t h e l i t e r a t u r e , it should be reemphasized t h a t d i f f e r e n t r e a c t i v i t i e s and p a r t i t i o n i n g b e h a v i o r of t h e p r o b e s may t u r n o u t t o be more i m p o r t a n t i n d e t e r m i n i n g t h e i r l a b e l i n g p a t t e r n . Repres e n t a t i v e examples of t h e s e compounds a r e shown i n F i g . 3. The a - s u b u n i t of Na,K-ATPase i s s p e c i f i c a l l y l a b e l e d by a l l of t h e o u a b a i n s i t e - s p e c i f i c r e a g e n t s t h a t have been r e p o r t e d , and it c l e a r l y c o n s t i t u t e s t h e major p o r t i o n of t h e ouabain-binding s i t e , e x t e n d i n g o v e r 20-30 b . Furthermore, t h e a - s u b u n i t i s t h e o n l y s u b u n i t l a b e l e d by any of t h e r e a g e n t s s h o r t e r t h a n 23 b , s u g g e s t i n g t h a t it a l o n e makes up t h e primary reg i o n of t h e b i n d i n g s i t e (Ruoho and Kyte, 1974, 1 9 7 7 ; Forbush e t al., 1978b; Rogers and Lazdunski, 1979b) and p o s s i b l y t h e f i r s t p a r t of t h e s u g a r s p e c i f i c p o r t i o n of t h e s i t e a s w e l l . Only t h e N-terminal 36K-dalton t r y p t i c fragment of t h e a p o l y p e p t i d e i s l a b e l e d by NPT-ouabain ( R o s s i et al., t h i s volume), b u t b o t h 57Kand 45K-dalton fragments a r e about e q u a l l y l a b e l e d by NAB-ouabain (Forbush e t al., 1 9 7 8 a ) . I t remains t o be determined i f t h i s d i f f e r e n c e i s due t o t h e 6 b d i f f e r ence i n p o s i t i o n of t h e r e a c t i v e group of t h e t w o p r o b e s and t h e r e f o r e i s i t s e l f an i m p o r t a n t p i e c e of "mapping" i n f o r m a t i o n , o r i f t h e d i f f e r e n c e c o u l d be a t t r i b u t e d t o d i f f e r e n t chemical r e a c t i v i t i e s of t h e p h o t o l a b e l s . The 8-subunit i s l a b e l e d only by 3 " ' - and 4"' -DAMd i g i t o x i n s i n which t h e p h o t o l a b e l i s s l i g h t l y beyond t h e t h i r d s u g a r moiety i n d i g i t o x i n ( H a l l and Ruoho, 1980, and t h i s volume) The 8-subunit i s n o t s i g n i f i c a n t l y l a b e l e d above background by DAM-, NAP-, o r NABd e r i v a t i v e s which could r e a c h j u s t t o t h e p o s i t i o n of t h e t h i r d s u g a r i n t h e b i n d i n g s i t e . [A s m a l l amount of l a b e l i n g by NAB-ouabain of a 55,000-dalton peak w a s rep o r t e d (Forbush et al., 197833) a s a t y p i c a l (see Fig. 4 f o r t h e u s u a l p a t t e r n ) , and may have been due t o p r o t e o l y s i s . ] Thus, t h e a v a i l a b l e e v i d e n c e s u g g e s t s t h a t t h e 8-subunit and t h e a - s u b u n i t may be i n c l o s e p r o x i m i t y j u s t beyond t h e s i t e f o r t h e t h i r d s u g a r group i n d i g i toxin. A s m a l l r o l e i n car diac glycoside binding, i f any, i s i m p l i e d f o r t h e @ - s u b u n i t . A low-molecular-weight hydrophobic p e p t i d e , termed y ( o r p r o t e o l i p i d ) , i s s p e c i f i c a l l y l a b e l e d by NABouabains and NAP-ouabain (Forbush et a l . , 1978a,b; Rogers and Lazdunski, 1979b) , a l l of which produce a r e a c t i v e n i t r e n e 23-24 from t h e c e n t e r of t h e l a c t o n e r i n g . T h i s p e p t i d e h a s an a p p a r e n t m o l e c u l a r w e i g h t of

a

CARDIOTONIC STEROID BINDING TO Na,K-ATPase

191

COOYASSIE BLUE SCAN

0.6

1

b

4

i

T.D.

w

0 IS

a

0 0

2

4 6 Mlgration Diatancr (cm)

8

10

12

Fig. 4 . P h o t o a f f i n i t y l a b e l i n g o f crude p i g k i d n e y microsomes F r e s h l y p r e p a r e d m i c r o s o m e s ( 4 . 3 m g / m l ) were b y [ 3 H ] NAB-ouabain. i n c u b a t e d f o r 2 m i n i n the p r e s e n c e of 5 mM Mg, 3 mM P i and 1.3 W M [3H]NAB-ouabain, f i l t e r e d , w a s h e d , a n d p h o t o l y z e d on the s u r f a c e o f a N u c l e o p o r e f i l t e r (see F o r b u s h e t a l . , 1 9 7 8 b ) . T h e m i c r o s o m e s were s o l u b i l i z e d i n SDS and rbn on S D S - p o l y a c r y l a m i d e g e l s . ( A ) One (B) A d u p l i c a t e gel g e l track was s t a i n e d , d e s t a i n e d , and s c a n n e d . t r a c k was s l i c e d a n d c o u n t e d for 3 8 w i t h o u t s t a i n i n g . A r r o w s i n 3A ( b ) B c h a i n o f Na,Kmark the p o s i t i o n of ( a ) a c h a i n of Na,K-ATPase, A T P a s e , ( c ) y c o m p o n e n t , ( d ) f r e e [3H]NAB-ouabain (TD) t r a c k i n g d y e .

1 2 , 0 0 0 on SDS g r a d i e n t g e l s ( F o r b u s h e t a l . , 1978b) and 9,500 on SDS-urea g e l s ( B . F o r b u s h I11 a n d M. Sussman, u n p u b l i s h e d r e s u 1 , t s ; g e l s y s t e m of R o d r i g u e z e t a l . , t h i s v o l u m e ) . S i n c e t h e p h o t o l a b e l i n g e x p e r i m e n t s cons t i t u t e t h e o n l y s t r o n g e v i d e n c e t o date t h a t t h e s m a l l

0

0

rn

W

3 c

-

1.0

0

U

W

c

80

E

0

0

e L

c

t?

a"

8

rn

:a

8

0

?a

0

.-c

0 'CI

0

0

2

0.5

3 0

B 0

n

rn Reversibly bound

c .-

0 Label on lorge

0

n

choin

0 Label on proteolipid

0 3 0

m U

z I m -L

0.0 0

I

I 1.0

0.5

FH] - N A B ouaboin (BM) 3

F i g . 5 . R e v e r s i b l e b i n d i n g and c o v a l e n t l a b e l i n g o f t w o p e p t i d e s b y [ HINAB-ouabain. Purified p i g k i d n e y Na,K-ATPase ( 0 . 5 mg/ml) was i n c u b a t e d w i t h [ 3 H ] NAB-ouabain a t the i n d i c a t e d c o n c e n t r a t i o n s f o r 1 hr a t 37OC w i t h the Mg2+ + P i b i n d i n g medium. S a m p l e s were f i l t e r e d , p h o t o l y z e d , and r u n on S D S g e l s as d e s c r i b e d i n the l e g e n d t o F i g . 4 . ( W ) [jHINAB-ouabain r e v e r s i b l y b o u n d , d e t e r m i n e d on N u c l e o p o r e f i l t e r s , and p l o t t e g as f r a c t i o n of 2.1 n m o l e s / m g . ( 0 ) [3H]NAB-ouabain i n 9 5 K - d a l t o n peak (01 [3H]NAB-ouabain i n 12Kon S D S g e l s , p l o t t e d as f r a c t i o n of 0.21 nmoles/mg o r i g i n a l p r o t e i n . d a l t o n p e a k on SDS g e l s , p l o t t e d as f r a c t i o n o f 0 . 2 3 nmole/mg o r i g i n a l p r o t e i n .

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hydrophobic p e p t i d e i s a s s o c i a t e d with Na,K-ATPase, t h e r e l e v a n t o b s e r v a t i o n s w i l l be reviewed h e r e . The h i g h d e g r e e o f c o v a l e n t l a b e l i n g of y (15-20% o f r e v e r s i b l y bound i n h i b i t o r ) r e m a i n s t h e most p e r s u a s i v e p i e c e o f d a t a t h a t t h e r e i s a s e c i f i c a s s o c i a t i o n between y and t h e Na,K-ATPase. Roug l y t e same f r a c t i o n o f l a b e l i n g i s s e e n i n a c r u d e microsomal f r a c t i o n from p i g k i d n e y ( F i g . 4 ) a s i n a h i g h l y p u r i f i e d p r e p a r a t i o n from t h e same s o u r c e ( F o r b u s h e t al., 197833). Even i n t h e p u r i f i e d p r e p a r a t i o n , where y c o u l d c o n s t i t u t e a s much a s 5-10% of t h e p r o t e i n , it d o e s n o t seem t h a t random mot i o n o f d i s s o c i a t e d p e p t i d e s w i t h i n t h e b i l a y e r would l e a d t o a 20% p r o b a b i l i t y t h a t t h e r e a c t i v e g r o u p of NAB-ouabain would f i n d and r e a c t w i t h t h e s m a l l p e p t i d e among t h e o t h e r p r o t e i n and l i p i d m o l e c u l e s . The y p e p t i d e i s l a b e l e d by t h e same p o p u l a t i o n o f bound NAB-ouabain m o l e c u l e s as i s a , s i n c e t h e a p p a r e n t a f f i n i t i e s o f r e v e r s i b l e NAB-ouabain b i n d i n g , a - s u b u n i t l a b e l i n g , and of l a b e l i n g of y are i d e n t i c a l w i t h i n e x perimental e r r o r (Fig. 5 ) . The small p o l y p e p t i d e from lamb k i d n e y Na,K-ATPase h a s been p u r i f i e d by HPLC, and i t s amino a c i d composit i o n d e t e r m i n e d (Reeves e t al., 1 9 8 0 ) . I n c o r p o r a t e d NAB-ouabain cochromatographs w i t h t h e p e p t i d e ( C o l l i n s e t al., t h i s v o l u m e ) . R e c e n t l y , Hardwicke and F r e y t a g (1981) r e p o r t e d t h e amino a c i d c o m p o s i t i o n o f a s m a l l h y d r o p h o b i c p e p t i d e ( M =~ 1 1 , 1 0 0 ) i n Na,K-ATPase p u r i f i e d from s h a r k r e c t a l g l a n d t o b e v e r y s i m i l a r t o t h a t from lamb k i d n e y , and p o i n t e d o u t t h a t t h i s a r g u e s f o r c o n s e r v a t i o n of s e q u e n c e and hence f o r some r o l e f o r the peptide. The mass r a t i o of y / a s u p p o r t s a s t o i c h i o m e t r y n e a r 1 y p e p t i d e p e r a - s u b u n i t i n enzyme p u r i f i e d from b o t h s p e c i e s (Reeves et a l . , 1980; Hardwicke and F r e y t a g , 1981; C o l l i n s e t a l . , t h i s v o l u m e ) . A l t h o u g h t h e p o s s i b i l i t y of p r o t e o l y s i s i s a l w a y s v e r y h a r d t o r u l e o u t , t h e r e i s no e v i d e n c e s u p p o r t i n g i t , and t h e s m a l l p e p t i d e i s s t i l l found when t h e enzyme p r e p a r a t i o n i s performed i n t h e p r e s e n c e of p r o t e a s e i n h i b i t o r s (Hardwicke and F r e y t a g , 1 9 8 1 ) . The NAB-ouabainl a b e l e d p e p t i d e i s c l e a r l y d i f f e r e n t from t h e . ~ 1 2 , 0 0 0 d a l t o n t r y p t i c f r a g m e n t o f Na,K-ATPase t h a t i s l a b e l e d w i t h [ 1 2 5 I ] i o d o n a p h t h y l a z i d e ( K a r l i s h e t a l . , 19771, s i n c e t h e two a r e w e l l s e p a r a t e d i n e i t h e r o n e of two d i f f e r e n t SDS-gel e l e c t r o p h o r e s i s s y s t e m s ( S . J . D. K a r l i s h and B. Forbush 111, u n p u b l i s h e d r e s u l t s ) . I t i s q u i t e p o s s i b l e t h a t t h e y p e p t i d e i s n o t req u i r e d f o r t h e complete f u n c t i o n o f Na,K-ATPase, i . e . , The b e s t f o r t h e ATP-fueled movement o f N a + and K'. e v i d e n c e f o r t h i s i s i n f e r e n t i a l , by a n a l o g y t o C a 2 + A T P a s e from s a r c o p l a s m i c r e t i c u l u m , which when h i g h l y

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p u r i f i e d c o n t a i n s a p r o t e o l i p i d component s i m i l a r t o y . (1980) have been a b l e t o remove a l l d e t e c t a b l e p r o t e o l i p i d from a p u r i f i e d p r e p a r a t i o n o f C a 2 + - A T P a s e , r e c o n s t i t u t e t h e enzyme i n t o p h o s p h o l i p i d v e s i c l e s , and measure t h e same r a t e and s t o i c h i o m e t r y of ATP-driven C a 2 + t r a n s p o r t . Further support is given by an e x p e r i m e n t o f Hardwicke and F r e y t a g (1981) i n which a- and B-subunits o f N a , K - A T P a s e w e r e c o s o l u b i l i z e d i n B r i j 58 and were found t o e x h i b i t f u l l ATPase a c t i v i t y , w h i l e most o f t h e s m a l l p e p t i d e remained i n t h e s o l i d r e s i d u e . When t h e B r i j - s o l u b i l i z e d enzyme can b e shown t o be f r e e o f t h e y p e p t i d e , and r e c o n s t i t u t e d t o g i v e N a + K t r a n s p o r t , t h e l a c k of a f u n c t i o n a l r e q u i r e m e n t f o r t h e s m a l l p e p t i d e w i l l have been d e m o n s t r a t e d . I n any case, f u r t h e r e v i d e n c e r e g a r d i n g i n t e r a c t i o n o f t h e peptide a t t h e ouabain-binding s i t e w i l l a i d i n c l a r i f y i n g t h e " s p e c i f i c i t y " o f i n t e r a c t i o n of y w i t h t h e N a , K ATPase and may h e l p d e t e r m i n e i f t h e p e p t i d e p l a y s a r e g u l a t o r y r o l e o r s u b s t a n t i a l l y a f f e c t s ouabain binding parameters. I f a s y s t e m a t i c mapping of t h e o u a b a i n b i n d i n g s i t e i s t o be u n d e r t a k e n , a number of v a r i a b l e s i n a d d i t i o n t o t h o s e d i s c u s s e d above w i l l need t o be c o n t r o l l e d , and perhaps exploited. ( a ) A s r e g a r d s d e t e r g e n t s and p u r i f i c a t i o n , w e have o b s e r v e d q u a n t i t a t i v e d i f f e r e n c e s i n l a b e l i n g of N a , K - A T P a s e p u r i f i e d from t h e same s p e c i e s by d i f f e r e n t methods. With Na,K-ATPase p u r i f i e d from p i g k i d n e y by t h e method of Lane e t a l . (1973) t h e p e r c e n t p h o t o i n c o r p o r a t i o n o f r e v e r s i b l y bound NAB-ouabain i n t o t h e a c h a i n w a s 11-13% compared t o 1 7 - 2 0 % w i t h p r e p a r a t i o n s p u r i f i e d by t h e method of J d r g e n s e n ( 1 9 7 4 ) , and 13-16% i n t h e c r u d e microsomes ( F i g . 1 ) . ( b ) Even among s p e c i e s w i t h a h i g h a f f i n i t y f o r o u a b a i n , w e have n o t e d l a r g e d i f f e r e n c e s i n l a b e l i n g w i t h NAB-ouabain. With Na,K-ATPase p u r i f i e d from p i g k i d n e y , dog k i d n e y , and s h a r k r e c t a l g l a n d t h e e f f i c i e n c y of i n c o r p o r a t i o n i n t o a w a s 17-20%, 10-13%, and 8 % ; and i n t o t h e y pept i d e it was 1 5 % , 5 % , and 20.5%, r e s p e c t i v e l y (Type I i n termediate; unpublished r e s u l t s ) ( c ) The l a b e l i n g p a t t e r n i s s u b s t a n t i a l l y a f f e c t e d by t h e c o n f o r m a t i o n a l s t a t e of t h e N a , K - A T P a s e ; t h a t i s , by t h e l i g a n d s p r e s e n t d u r i n g b i n d i n g of t h e c a r d i o t o n i c s t e r o i d d e r i v a t i v e , a s w e l l a s by t h e l i g a n d s p r e s e n t d u r i n g p h o t o l y s i s ( F o r b u s h et a l . , 197833; Ruoho and H a l l , 1980; t h i s v o l u m e ) . If a s y s t e m a t i c mapping e f f o r t i s u n d e r t a k e n , t h e s e d i f f e r e n c e s may g i v e a g r e a t d e a l of i n s i g h t i n t o t h e n a t u r e of t h e conformational changes.

MacLennan e t a l .

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ACKNOWLEDGMENTS

T h i s work w a s supported by g r a n t s from t h e American H e a r t A s s o c i a t i o n (AHA 81-998) and from N I H (R01-GM27920). O r i g i n a l experiments r e p o r t e d h e r e i n were performed w i t h t h e e x c e l l e n t t e c h n i c a l a s s i s t a n c e o f M r s . Grace Jones.

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sample c o n t a i n i n g an unknown amount o f bound c a r d i a c glycos i d e . L i f e S c i . 16, 1253-1262. I n a g a k i , C., Lindenmayer, G. E., and Schwartz, A. (1974). E f f e c t s of sodium and potassium on b i n d i n g of ouabain t o t h e t r a n s port adenosine t r i p h o s p h a t a s e . J . B i O l . Chem. 249, 52355140. J o i n e r , C. H . , and Lauf, P. K. (1978). Modulation o f ouabain binding and potassium pump f l u x e s by c e l l u l a r sodium and Potassium i n human and sheep e r y t h r o c y t e s . J. P h y s i o l . ( L o n d o n ) 283, 177-196. J$rgensen, P. L. (1974). P u r i f i c a t i o n and c h a r a c t e r i z a t i o n of Na,K-ATPase. 111. P u r i f i c a t i o n from o u t e r medulla of m m m a l i a n kidney a f t e r s e l e c t i v e remove1 o f membrane components by sodium d o d e c y l s u l f a t e . B i o c h i m . B i o p h y s . A c t a 356, 36-52. J o r i , G. , and S p i k e s , J. D. (1978). Mapping 3-dimensional s t r u c t u r e of p r o t e i n s by photo-chemical t e c h n i q u e s . P h o t o c h e m . P h o t o b i o l . R e v . 3, 193-275. K a r l i s h , S. J. D . , and Glynn, I. M. (1974). An uncoupled e f f l u x of N a from human r e d c e l l s probably a s s o c i a t e d w i t h N a dependent ATPase a c t i v i t y . Ann. N.Y. A c a d . S c i . 242, 461470. K a r l i s h , S. J. D . , J$rgensen, P. L., and G i t l e r , C. (1977). I d e n t i f i c a t i o n o f a membrane embedded segment o f t h e l a r g e Nature (London) p o l y p e p t i d e c h a i n o f ( N a + + K+) -ATPase. 269, 715-717. Knight, A. B., and W e l t , L. G. (1974). I n t r a c e l l u l a r potassium. A d e t e r m i n a n t of t h e sodium-potassium pump rate. J. G e n . P h y s i o l . 63, 351-373. Lane, L. K., Copenhaver, J. H . , Jr., Lindenmayer, G. E . , and Schwartz, A. (1973). P u r i f i c a t i o n and c h a r a c t e r i z a t i o n o f and [3H]ouabain b i n d i n g t o t h e t r a n s p o r t adenosine t r i p h o s p h a t a s e from o u t e r medulla of c a n i n e kidney. J. B i o l . Chem. 248, 7197-7200. Lindenmayer, G . E . , and Schwartz, A. (1973). Nature o f t h e t r a n s port ATPase g l y c o s i d e complex. I V . Evidence t h a t sodium and potassium c o m p e t i t i o n f o r a common s i t e modulates t h e r a t e of g l y c o s i d e i n t e r a c t i o n . J. B i o l . Chem. 248, 1291-1300. Lishko, V. K., Malysheva, M , K., and G r e v i z i r s k a y a , T. I. (1972). The i n t e r a c t i o n o f t h e ( N a + , K + ) -ATPase of e r y t h r o c y t e g h o s t s w i t h ouabain. B i o c h i m . B i o p h y s . A c t a 288, 103-106. MacLennan, D. H . , Reithmeier, R. A. F., Shoshan, V., Campbell, K. P . , L e B e l , D., H e r m a n , T. R . , and Shamoo, A. E. (1980). Ann. N.Y. A c a d . S c i . 358, 138-148. Mandel, F , Wallick, E. T., and Schwartz, A. (1977). Mg-stimulated ouabain b i n d i n g t o p u r i f i e d Na,K-ATPase from l a m b kidney. F e d . Proc., F e d . Am. SOC. E x p . B i o l . 36(3), 274 (abstr.). Mirdh, S., and P o s t , R. L. (1977). Phosphorylation from adenosine t r i p h o s p h a t a s e of sodium and p o t a s s i u m - a c t i v a t e d adenosine t r i p h o s p h a t a s e . J. B i o l . Chem. 252, 633-638.

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Matsui, H . , and Schwartz, A. (1968). Mechanism of c a r d i a c g l y c o s i d e i n h i b i t i o n of t h e ( N a + + K+)-dependent ATPase from c a r d i a c t i s s u e . B i o c h i m . B i o p h y s . A c t a 1 5 1 , 655-663. P o s t , R. L., Merritt, C. R., Kinsolving, C. R., and A l b r i g h t , C . D. Membrane a d e n o s i n e t r i p h o s p h a t a s e a s a p a r t i c i p a n t (1960) i n t h e a c t i v e t r a n s p o r t o f sodium and potassium i n human J. Biol Chem. 2 3 5 , 1796-1802. erythrocyte. P o s t , R. L., Sen, A. K., and Rosenthal, A. S. (1965). A phosp h o r y l a t e d i n t e r m e d i a t e i n t h e ATP-dependent sodium and poJ . B i o l . Chem. tassium t r a n s p o r t a c r o s s kidney membranes. 2 4 0 , 1437-1445. P o s t , R. L., Toda, G., and R o g e r s , F. N. (1975). P h o s p h o r y l a t i o n by i n o r g a n i c phosphate o f sodium p l u s potassium i o n t r a n s J . B i o l . Chem. 2 5 0 , 691-701. p o r t adenosine t r i p h o s p h a t e . Reeves, A. S . , C o l l i n s , J. H . , and Schwartz, A. (1980). I s o l a t i o n and c h a r a c t e r i z a t i o n of ( N a , K ) -ATPase p r o t e o l i p i d 95, B i o c h e m . B i o p h y s . R e s . C o m u n . 9 5 , 1591-1598. Repke, K . , and P o r t i u s , H. J. ( 1 9 6 5 ) . A n a l y s i s of s t r u c t u r e a c t i v i t y r e l a t i o n s h i p s i n c a r d i o a c t i v e c a p o u n d s on t h e molecular l e v e l . S c i . P h a r m . P r o c . 2 5 , 39-57. Repke, K. R . H . , and D i t t r i c h , F . (1980). Thermodynamics of i n f o r m a t i o n t r a n s f e r from c a r d i o t o n i c s t e r o i d s t o r e c e p t o r t r a n s p o r t ATPase. T r e n d s Pharmacol S c i 1 , 398-402. Repke, K. R. H . , D i t t r i c h , F., B e r l i n , P . , and P o r t i u s , H. J. (1974). On p h y s i c a l f o r c e s governing c a r d i a c g l y c o s i d e a c t i v i t y . A n n . N.Y. A c a d . S c i . 2 4 2 , 737-739. Rogers, T. B . , and Lazdunski, M. (1979a). P h o t o a f f i n i t y l a b e l i n g of t h e d i g i t a l i s r e c e p t o r i n t h e (sodium + p o t a s s i u m ) B i o c h e m i s t r y 1 8 , 135a c t i v a t e d adenosinetriphosphatase 140. Rogers, T . B . , and Lazdunski, M. (1979b). P h o t o a f f i n i t y l a b e l i n g of a small p r o t e i n component of a p u r i f i e d ( N a + + K+)-ATPase FEBS L e t t . 98, 373-376. R o s s i , B . , V u i l l e u m i e r , P., Gache, C . , B a l e r n a , M., and Lazdunski, M. (1980). A f f i n i t y l a b e l i n g of t h e d i g i t a l i s r e c e p t o r w i t h p-nitrophenyltriazene-ouabain, a h i g h l y s p e c i f i c a l k a l a t i n g a g e n t . J . B i o l . Chem. 2 5 5 , 9936-9941. Ruoho, A. E . , and H a l l , C. C. ( 1 9 8 0 ) . The u s e of p h o t o l a b e l s t o Ann. probe t h e ouabain b i n d i n g s i t e o f t h e (Na,K-ATPase. N.Y. A c a d . S c i . 3 4 6 , 90-103. Ruoho, A . , and Kyte, J. (1974). P h o t o a f f i n i t y l a b e l i n g of t h e ouabain-binding s i t e on (Na+ + K+) a d e n o s i n e t r i p h o s p h a t a s e P r o c . N a t l . A c a d . S c i . USA 7 1 , 2352-2356. Ruoho, A . , and Kyte, J. (1977). The ouabain-binding s i t e on ( N a + + K+) -adenosinetriphosphatase. In "Methods i n Enzymology" (W. B. Jakoby and M. Wilchek, e d s . ) , Vol. 46, 523-531. Sachs, J. R . (1974). I n t e r a c t i o n of e x t e r n a l K, N a and c a r d i o a c t i v e s t e r o i d s w i t h t h e Na-K pump o f t h e human r e d blood c e l l . J . Gen. P h y s i o l . 6 3 , 123-143.

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Sachs, J. R. (1977). K i n e t i c s o f t h e i n h i b i t i o n o f t h e Na-K pump by e x t e r n a l sodium. J . P h y s i o l . ( L o n d o n ) 264, 449-470. Sachs, J. R. (1981). I n t e r n a l potassium s t i m u l a t e s t h e sodiumJ. potassium pump by i n c r e a s i n g c e l l ATP c o n c e n t r a t i o n . P h y s i o l . ( L o n d o n ) 319, 515-528. Schatunann, H. J. (1953). Herzglycoside a l s Hemmstoffe f u r den a k t i v e n K a l i u m and N a t r i u m T r a n s p o r t d u r e b d i e Erythrocytenmembran. Helv. P h y s i o l . P h a r m a c o l . A c t a 11, 346-354. Schuunnans Stekhoven, F. M. A. H . , VanHesmijk, M. P. E., de Pont, J. H. H. M . , and Bonting, S. L. (1976). S t u d i e s on (Na+K+)-activated ATPase. X X X V I I I . A 100,000 molecular weight p r o t e i n a s t h e 1 w energy phosphorylated i n t e r m e d i a t e of t h e enzyme. B i o c h i m . B i o p h y s . A c t a 422, 210-224. Sen, A. K . , Tobin, T., and P o s t , R. L. (1969). A c y c l e f o r ouab a i n i n h i b i t o n of sodium- and potassium-dependent adenosine t r i p h o s p h a t a s e . J . B i o l . Chem. 244, 6596-6604. Skou, J. C . , B u t l e r , K. W., and Hansen, 0. (1971). The e f f e c t of magnesium ATP, P i and sodium on t h e i n h i b i t i o n of t h e Na+,K+ATPase. B i o c h i m . B i o p h y s . A c t a 241 , 443-461. Swann, A. C., and Albers, R. W. (1980). (Na+,K+)-ATPase of mamm a l i a n b r a i n : D i f f e r e n t i a l e f f e c t s on c a t i o n a f f i n i t i e s o f p h o s p h o r y l a t i o n by ATP and a c e t y l p h o s p h a t e . A r c h . B i o c h e m . B i o p h y s . 203, 422-427. Thomas, R., Boutagy, J., and G e l b a r t , A. (1974). S y n t h e s i s and b i o l o g i c a l a c t i v i t y o f s e m i s y n t h e t i c d i g i t a l i s analogs. J . Pharm. S c i . 63, 1649-1683. Tobin, T. , and Brody, T. M. (1972). R a t e s of d i s s o c i a t i o n of enzyme-ouabain complexes and K0.5 v a l u e s i n ( N a + + K+) adenosine t r i p h o s p h a t a s e from d i f f e r e n t species. B i o c h e m . P h a r m a c o l . 2 1 , 1553-1560. Tobin, T., Baskin, S. I . , Akera, T., and Brody, T. M. (1971). Nuc l e o t i d e s p e c i f i c i t y of t h e N a + s t i m u l a t e d p h o s p h o r y l a t i o n Mol. P h a r m a c o l . and [3H]ouabain binding r e a c t i o n s o f N a + . 8, 256-263. Tobin, T . , Akera, T., Lee, C. Y . , and Brody, T. M. (1974). Ef ects of nucleoOuabain b i n d i n g t o (Na+ + K+) -ATPase. t i d e analogues and e t h a c r y n i c a c i d . B i o c h i m . B i o p h y s . A c t a 345, 102-117. Tometsko, A. M., and Richards, F. M., e d s . (1980). A p p l i c a t i o n s of photochemistry i n probing b i o l o g i c a l t a r g e t s . Ann. N. Y . A c a d . S c i . 346, 1-502. Wallick, E. T . , Lindenmayer, G. E., Lane, L. K., A l l e n , J. C . , P i t t s , B. J . R . , and Schwartz, A. (1977). Recent advances i n c a r d i a c glycoside-Na', K+-ATPase i n t e r a c t i o n . F e d . P r o c . , F e d . Am. S O C . Exp. B i o l . 36, 2214-2218. Wallick, E. T . , Anner, B. M . , Ray, M. V., and Schwartz, h. (1978). E f f e c t of temperature on phosphorylation and ouabain binding J. Biol. t o N-ethylmaleimide-treated ( N a + + K+) -ATPase. Chem. 253, 8778-8786. Wallick, E . T . , P i t t s , B. J. R . , Lane, L. K., and Schwartz, A. (1980). A k i n e t i c comparison of c a r d i a c g l y c o s i d e i n t e r a c -

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CARDIOTONIC STEROID BINDING TO Na,K-ATPase

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t i o n w i t h Na+,K+-ATPase f r o m s k e l e t a l and c a r d i a c muscle and from kidney. A r c h . B i o c h e m . B i o p h y s . 202, 442-449. A. (1973) S t r u c t u r e - a c t i v i t y r e l a t i o n s h i p s of c a r d i o t o n i c s t e r o i d s f o r t h e i n h i b i t i o n of sodium- and potassiumdependent adenosine t r i p h o s p h a t a s e . I. D i s s o c i a t i o n r a t e c o n s t a n t s of v a r i o u s enzyme c a r d i a c glycoside complexes formed i n t h e presence of magnesium and phosphate. Mol. Pharmacol. 9, 51-60. A., and Yoda, S. (1974). S t r u c t u r e - a c t i v i t y r e l a t i o n s h i p s of c a r d i o t o n i c s t e r o i d s f o r t h e i n h i b i t i o n of sodium- and potassium-dependent adenosine t r i p h o s p h a t a s e . 111. Dissoc i a t i o n r a t e c o n s t a n t s of v a r i o u s enzyme-cardiac g l y c o s i d e complexes formed i n t h e presence of sodium, magnesium and adenosine t r i p h o s p h a t e . Mol. P h a r m a c o l . 1 0 , 494-500. A . , and Yoda, S. (1975). S t r u c t u r e - a c t i v i t y r e l a t i o n s h i p s of c a r d i o t o n i c s t e r o i d s f o r i n h i b i t i o n o f sodium- and po t a ssium-dependent adenosine t r i p ho sphata se V. D i s s o c i a t i o n r a t e c o n s t a n t s of d i g i t o x i n a c e t a t e s . Mol P h a r m a c o l . 1 1 , 653-662. A. , and Yoda, S. (1977). Association and d i s s o c i a t i o n r a t e c o n s t a n t s of t h e complexes between v a r i o u s c a r d i a c aglycones and sodium- and potassium-dependent adenosine t r i p h o s p h a t a s e formed i n t h e presence of magnesium and phosphate. Mol. P h a r m a c o l . 1 3 , 352-361. A . , Yoda, S . , and S a r r i f , A. M. (1973). S t r u c t u r e - a c t i v i t y r e l a t i o n s h i p s of c a r d i o t o n i c s t e r o i d s f o r t h e i n h i b i t i o n of sodium- and po t a s s ium-depende nt adeno s i n e t r i p ho spha t a s e 11. Association r a t e c o n s t a n t s of v a r i o u s enzyme-cardiac g l y c o s i d e complexes. Mol. P h a r m a c o l . 9, 766-776. S . , S a r r i f , A. M., and Yoda, A. (1975). S t r u c t u r e - a c t i v i t y r e l a t i o n s h i p s of c a r d i o t o n i c s t e r o i d s f o r t h e i n h i b i t i o n of sodium- and potassium-dependent adenosine t r i p h o s p h a t a s e . IV. D i s s o c i a t i o n r a t e c o n s t a n t s f o r complexes of t h e enzyme with c a r d i a c o l i g o d i g i t o x i d e s . Mol. Pharmacol. 11, 647-652.

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CURRENT TOPICS IN MEMBRANES AND TRANSPORT, VOLUME 19

Binding of Monovalent Cations to the Na,K-ATPase' M. YAMAGUCHI, J. SAKAMOTO, AND Y. TONOMURA Department of Biology Faculty of Science Osaka University Toyo&, Osaka,Japan

I.

INTRODUCTION

It is well established from physiological studies performed by Sen and Post (1964) and Garrahan and Glynn (1967) that 3 Na+ and 2 K+ are actively transported through the plasma membrane, coupled to the hydrolysis of 1 ATP molecule which is catalyzed by Na,K-ATPase. A simplified reaction scheme of Na,K-ATPase is expressed as the following:

+ .

&y,

ADP, K -insensitive EP with bound 'Abbreviations: ADP; EIP, ADP-sensitive and @-insensitive EP; EzP, ADP-insensitive and e - s e n s i tive EP; AMP-PNP, adenyl-S'-yl-iddodiphosphate; DCCD, dicyclohexylcarbodiimide. 203

Copyright 0 1983 by Academic Press. Inc. All rights of repduction in any form reserved. ISBN 0-12-153319-0

204

M. YAMAGUCHI eta/.

Acceleration

Inhibition

Scheme 1

ADP

I n t h i s r e a c t i o n scheme, E;P , E I P , and E2P a r e ADP, K + - i n s e n s i t i v e , ADP-sensitive, and K + - s e n s i t i v e E P , r e s p e c t i v e l y . The o u t e r c y c l e of t h e r e a c t i o n sequence h a s been e s t a b l i s h e d by P o s t e t a l . ( 1 9 6 9 ) . On t h e o t h e r hand, w e have o b t a i n e d s e v e r a l l i n e s o f e v i d e n c e f o r E$DP o r EADP i n t e r m e d i a t e s d u r i n g ATP h y d r o l y s i s (Tonomura and Fukushima, 1974; Yamaguchi and Tonomura, 1978; Sakamoto and Tonomura, 1 9 8 0 ) . There a r e two i m p o r t a n t a s p e c t s t o t h e above r e a c t i o n scheme. One asp e c t i s t h a t t h e Na,K-ATPase c a n t a k e a t l e a s t two d i f f e r e n t c o n f o r m a t i o n s , a Na+-bound form, E l , and a K+bound form, E The o t h e r a s p e c t i s t h a t t h e a f f i n i t i e s o f t h e A4Pase € o r N a + and K+ change w i t h t h e conf o r m a t i o n a l change accompanying t h e p h o s p h o r y l a t i o n c y c l e of t h e A T P a s e r e a c t i o n . The mechanism of t h e A T P a s e r e a c t i o n h a s been i n v e s t i g a t e d mainly by k i n e t i c methods. C o n s e q u e n t l y , c o n c l u s i o n s o b t a i n e d are u s u a l l y n o t c o n c l u s i v e , and many i m p o r t a n t p o i n t s remain c o n t r o v e r s i a l . One must measure t h e amounts of N a + and K+ bound t o t h e A T P a s e a t v a r i o u s enzymatic s t a t e s t o know which s t e p i n t h e A T P a s e r e a c t i o n a c t u a l l y i n d u c e s t h e a f f i n i t y change, and whether o r n o t N a + and K+ b i n d t o t h e enzyme s i m u l t a n e o u s l y . The f i r s t measurement of b i n d i n g of monovalent cat i o n s t o t h e Na,K-ATPase w a s made by K a n i i k e e t a l . ( 1 9 7 6 ) , u s i n g t h e u l t r a c e n t r i f u g a l s e p a r a t i o n method. They showed t h a t 3 moles o f N a + b i n d t o 1 mole of ouabain-binding s i t e . M a t s u i e t a l . (1977) l a t e r showed t h a t 2 moles o f K+ b i n d t o 1 mole of ouabain-binding s i t e . H a s t i n g s (1977) and H a s t i n g s and Skou (1980) a l s o measured K+ b i n d i n g , u s i n g a K + - s e l e c t i v e e l e c t r o d e .

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BINDING OF MONOVALENT CATIONS TO THE Na,K-ATPase

205

However, t h e y have n o t measured t h e c a t i o n b i n d i n g i n t h e c o u r s e of t h e enzyme t u r n o v e r . F u r t h e r m o r e , i n p r e c i p i t a t e s o b t a i n e d by u l t r a c e n t r i f u g a t i o n , a c t i v e s i t e s do n o t d i s t r i b u t e randomly, s i n c e a l a y e r s t r u c t u r e of membrane i s formed. T h e r e f o r e , w e have d e v i s e d a new membrane f i l t r a t i o n method which does n o t d i s t u r b e q u i l i b r i u m and e n a b l e s u s t o measure t h e c a t i o n bindi n g d u r i n g t h e A T P a s e r e a c t i o n (Yamaguchi and Tonomura, 1979, 1 9 8 0 a , b , c ) .

11.

METHODS

+

The amounts of N a , K + , Rb', and ATP bound t o t h e enzyme were measured by a m o d i f i e d membrane f i l t r a t i o n method. The r e a c t i o n m i x t u r e c o n t a i n e d t h e enzyme, r a d i o a c t i v e monovalent c a t i o n s o r ATP, and [3H]glucose. About 5 0 u 1 of t h e r e a c t i o n m i x t u r e w a s p l a c e d o n t o a s e t o f 2 membrane f i l t e r s ; t h e u p p e r , a Nucleopore f i l t e r , and t h e lower, a M i l l i p o r e f i l t e r . About 3 vl of t h e r e a c t i o n m i x t u r e w a s t r a p p e d i n t h e M i l l i p o r e f i l t e r by s u c t i o n f o r 5 sec. The volume o f t h e f i l t r a t e t r a p p e d i n t h e M i l l i p o r e f i l t e r w a s d e t e r m i n e d by t h e r a d i o a c t i v i t y o f [3H]glucose. The t i m e r e q u i r e d f o r measurement of t h e c a t i o n b i n d i n g w a s less t h a n 1 0 sec. The amount o f a c t i v e s i t e w a s determined by measuring t h e maximal amount o f E P .

111.

RESULTS AND DISCUSSION

I n t h e following, w e w i l l describe t h e p r o p e r t i e s o f monovalent c a t i o n b i n d i n g t o t h e A T P a s e i n t h e o r d e r of t h e r e a c t i o n i n t e r m e d i a t e s a p p e a r i n g i n t h e r e a c t i o n scheme shown i n S e c t i o n I . A.

B I N D I N G OF M O N O V A L E N T C A T I O N S T O T H E E N Z Y M E I N A B S E N C E OF A T P ( E )

THE

F i g u r e 1 shows t h e N a + c o n c e n t r a t i o n dependence o f N a + b i n d i n g i n t h e p r e s e n c e of EDTA. I n t h e absence of K C 1 , t h e amount o f N a + bound i n c r e a s e d markedly w i t h a n i n c r e a s e i n t h e N a + c o n c e n t r a t i o n up t o a b o u t 0 . 4 mM, t h e n i n c r e a s e d g r a d u a l l y and l i n e a r l y . On t h e o t h e r hand, t h e amount o f Na+ b i n d i n g i n t h e p r e s e n c e o f a

M. YAMAGUCHI eta/.

206

0

.E 4 al

> 4-

0

m

-zE" Q

2-

n

z

I

m

0

0.2

0.4 [Na+lfrce (mM1

F i g . 1 . Dependence on amount o f Na+ b i n d i n g i n the of 30 mM KCI. T h e amount o f or 1 mM ( A ,A ) T r i s - A T P a n d

0.6

+

the c o n c e n t r a t i o n of f r e e Na o f the p r e s e n c e ( 0 ,A ) and a b s e n c e ( 0 , A ) Na+ b i n d i n g was measured i n 0 ( 0 , 0 ) 5 mM EDTA a t pH 7.5 and O°C.

high c o n c e n t r a t i o n of KC1 i n c r e a s e d g r a d u a l l y and l i n e a r l y w i t h i n c r e a s e i n t h e Na+ c o n c e n t r a t i o n . W e found t h a t t h e Na+ b i n d i n g i n t h e p r e s e n c e of a h i g h concent r a t i o n of K C 1 i s n o t r e l a t e d t o a c t i v e t r a n s p o r t , and t h a t K + - s e n s i t i v e Na+ b i n d i n g o c c u r s on Na,K-ATPase. The maximum amount of K + - s e n s i t i v e N a + b i n d i n g w a s about 3 moles p e r mole of a c t i v e s i t e . The H i l l c o e f f i c i e n t of t h e K + - s e n s i t i v e Na+ b i n d i n g was about 3 , and t h e a p p a r e n t d i s s o c i a t i o n c o n s t a n t w a s found t o b e a b o u t 0 . 3 m. + W e a l s o found t h a t t h e amount of N a + - s e n s i t i v e K b i n d i n g w i t h h i g h a f f i n i t y was 3 moles p e r 1 mole of a c t i v e s i t e . F i g u r e 2 shows t h e d i s p l a c e m e n t by N a + of Of t h e 3 moles of N a + - s e n s i t i v e K+ bound t o t h e enzyme. K+ b i n d i n g w i t h h i g h a f f i n i t y , 1 mole of K+ w a s d i s p l a c e d by adding a low c o n c e n t r a t i o n of NaC1. T h i s 1 mole of bound K+ i s t h a t bound t o t h e Na+-binding s i t e . On t h e o t h e r hand, 2 moles o f K+ w e r e d i s p l a c e d by add i n g a high c o n c e n t r a t i o n of NaCl which r e s u l t s i n a H i l l c o e f f i c i e n t of about 2. The a f f i n i t y of t h e 2 moles of K+ f o r t h e s i t e s was a b o u t 60 t i m e s h i g h e r t h a n t h a t of Na+, and t h e s e two s i t e s were named t h e K+-binding s i t e s .

BINDING OF MONOVALENT CATIONS TO THE Na,K-ATPase

z

3

2

- LOGLNaCI

+

207

e

1

'

ADDED] (MI

+

D i s p l a c e m e n t of I( bound t o the ATPase b y Na . T h e amount of K' b i n d i n g was measured i n 13.1 ( 0 ) or 17.1 ( A ) mg/ml e n z y m e , 0 . 2 4 mM 4 2 K C l , 0.5-0.7 mM EDTA, and v a r i o u s conc e n t r a t i o n s of NaCl a t pH 7.5 and O°C. Fig. 2 .

+

W e measured Rb b i n d i n g t o t h e enzyme, s i n c e it i s w e l l known t h a t Rb' f u n c t i o n s as a K+ congener. I n t h e p r e s e n c e of a h i g h c o n c e n t r a t i o n of KC1, t h e amount of

Rb+ b i n d i n g i n c r e a s e d g r a d u a l l y and l i n e a r l y w i t h i n crease i n t h e Rb+ c o n c e n t r a t i o n ( F i g . 3 ) . However, t h e K + - i n s e n s i t i v e Rb+ b i n d i n i s n o t r e l a t e d t o a c t i v e t r a n s p o r t , and o n l y t h e K q - s e n s i t i v e Rb+ b i n d i n g i s i n volved i n t r a n s p o r t . Thus, 2 moles of R b + bound s p e c i f i c a l l y t o t h e enzyme w i t h an a p p a r e n t d i s s o c i a t i o n c o n s t a n t of a b o u t 20 phf and a H i l l c o e f f i c i e n t of a b o u t 2 . Glynn and Richards ( t h i s volume) r e p o r t e d t h a t t h e amounts of b i n d i n g n o t o n l y of K+ b u t a l s o of R b + w e r e 3 moles p e r mole of a c t i v e s i t e . The r e a s o n f o r t h e d i s c r e p a n c y is n o t known. One of t h e m o s t c o n t r o v e r s i a l i s s u e s of t h e react i o n of N a + and K+ w i t h t h e N a , K - A T P a s e i s whether N a + and K+ can b i n d t o t h e enzyme s i m u l t a n e o u s l y . W e found t h a t i n t h e absence of ATP, 2.5 moles of N a + and 2 moles of Rb+ bound s i m u l t a n e o u s l y t o 1 mole of a c t i v e s i t e i n t h e p r e s e n c e of 0 . 8 m~ NaCl and 0 . 1 m~ RbC1. 3 N a + and

208

M. YAMAGUCHI eta/.

3

+

F i g . 3 . Dependence on the c o n c e n t r a t i o n o f f r e e Rb of the amount of ' b R b i n d i n g i n the p r e s e n c e ( 0 ) and a b s e n c e ( 0 ) o f 100 mM K C I . C o n d i t i o n s a r e a s i n F i g . 2 .

2 K+ were a l s o found t o b i n d s i m u l t a n e o u s l y t o t h e enzyme i n t h e p r e s e n c e o f 1 . 8 m~ N a C l and 0.13 rn KC1. I t i s w e l l known t h a t t h e Na,K-ATPase c o n t a i n s a- and B-subunits and t h a t t h e a - s u b u n i t i s phosphory l a t e d by ATP. Q u i t e r e c e n t l y , it h a s been shown t h a t t h e Na,K-ATPase p r o t e i n h a s 1 mole o f p h o s p h o r y l a t i o n s i t e o r h i g h - a f f i n i t y n u c l e o t i d e - b i n d i n g s i t e p e r mole of a - s u b u n i t ( P e t e r s e t a l . , 1981; Moczydlowski and F o r t e s , 1981a; F o r t e s and Moczydlowski, t h i s volume; Matsui e t a l . , t h i s volume). T h e r e f o r e , w e can conc l u d e t h a t t h e r e are 3 moles o f Na+-binding s i t e s and 2 moles of K+-binding s i t e s p e r mole of a - s u b u n i t i n t h e Na,K-ATPase. I t s h o u l d b e added t h a t o u r f i n d i n g s do n o t mean t h a t 3 moles of N a + and 2 moles o f K+ b i n d s i m u l t a n e o u s l y d u r i n g t h e u s u a l enzyme t u r n o v e r ( c f . S a c h s , 1981, a l s o t h i s volume). T h i s i s b e c a u s e t h e enzyme t a k e s c o n f o r m a t i o n s 1 and 2 a l t e r n a t e l y , and a t s t a t e 1 Na+ b i n d s p r e f e r e n t i a l l y , w h i l e a t s t a t e 2 K+ b i n d s p r e f e r e n t i a l l y . Our f i n d i n g s i n d i c a t e o n l y t h a t N a + b i n d i n g s i t e s and K+-binding s i t e s are p h y s i c a l l y separated.

BINDING OF MONOVALENT CATIONS TO THE Na,K-ATPase

TABLE I.

209

P r o p e r t i e s of t h e Two Kinds of Cation-Binding S i t e s o f t h e ATPase

Binding site

Ion

+ N a -binding

+ Na

sites ( 3 moles/mole)

+

K -binding

site ( 2 moles/mole)

Dissociation constant

K+

a+ K+ Rb+ Na+

(P M )

Hill

coefficient

200-320 24 >>300

2.5-3

44 24 2200

1.5 1.5-2 2

KO. 5, (pM)

2 30

1

-22 72

aKo - 5 represents the monovalent cation concentration when the protecting effect of monovalent cation against DCCD inhibition is a half of the maximum.

Table I summarizes t h e a f f i n i t i e s of t h e s p e c i f i c c a t i o n - b i n d i n g s i t e s . The v a l u e of t h e a f f i n i t y o f Na+-binding s i t e s f o r K+ w a s o b t a i n e d from t h e d a t a o f d i s p l a c e m e n t o f N a + by K+. Those of Na+-binding s i t e s f o r Rb+ and o f K+-binding s i t e s f o r N a + and K+ w e r e a l s o o b t a i n e d from d i s p l a c e m e n t e x p e r i m e n t s . B.

B I N D I N G OF MONOVALENT C A T I O N S T O T H E E N Z Y M E I N P R E S E N C E OF A T P A N D A B S E N C E OF Mg2+ ( E - A T P )

THE

I n t h e absence of Mg2+1 t h e enzyme-substrate comp l e x ( E - A T P ) i s formed by adding ATP t o t h e A T P a s e . K+ b i n d i n g w a s i n h i b i t e d by a d d i t i o n of ATP, w h i l e Na+ b i n d i n g w a s u n a f f e c t e d by ATP i n t h e absence of Mg2+ ( F i g . 1 ) . I t w a s a l s o found t h a t ATP b i n d i n g w a s i n h i b i t e d by K + , b u t n o t by N a + , a s thermodynamically expected. C.

B I N D I N G OF M O N O V A L E N T C A T I O N S T O T H E N E M - M O D I F I E D E N Z Y M E I N T H E P R E S E N C E OF A T P A N D M g 2 + ( E l P )

The n e x t problem i s t h e e f f e c t of ATP on c a t i o n b i n d i n g i n t h e p r e s e n c e of Mg2+1 where t h e enzyme i s p h o s p h o r y l a t e d w i t h ATP. S e v e r a l k i n d s of phosphory l a t e d enzyme a r e formed by t h e r e a c t i o n of t h e N a , K A T P a s e w i t h ATP ( c f . S e c t i o n I ) . One form i s t h e ADPs e n s i t i v e and K + - i n s e n s i t i v e E P , E 1 P ; a n o t h e r i s t h e ADP-insensitive and K + - s e n s i t i v e E P , E 2 P . When ATP w a s

210

M. YAMAGUCHI eta/.

added t o t h e NEM-treated enzyme i n t h e p r e s e n c e of low c o n c e n t r a t i o n s of Na+ and f r e e Mg2+, only E 1 P was produced, b u t no N a + was r e l e a s e d from t h e enzyme by t h e a d d i t i o n of ATP. W e a l s o found t h a t when R b + was added a f t e r t h e f o r m a t i o n of E1P on t h e NEM-treated enzyme, t h e amount of EP d e c r e a s e d o n l y s l i g h t l y , and t h e amount of Rb+ b i n d i n g i n t h e p r e s e n c e of ATP was o n l y s l i g h t l y l a r g e r t h a n t h a t i n t h e absence of ATP. D.

B I N D I N G O F M O N O V A L E N T C A T I O N S TO T H E E N Z Y M E IN P R E S E N C E OF A T P A N D M g 2 + ( E 2 P )

THE

I n t h e p r e s e n c e of Mg2+, a l m o s t a l l phosphoenz m e formed a t t h e s t e a d y s t a t e i s ADP-insensitive and KY s e n s i t i v e , t h a t i s , E2P. W e found t h a t t h e f o r m a t i o n of E 2 P i n d u c e s marked changes i n a f f i n i t i e s of t h e A T P a s e f o r 3 Na+ and 2 K+ o r Rb'. Na+ bound s p e c i f i c a l l y t o t h e Na+-binding s i t e s i n t h e p r e s e n c e of Mg2+ was r e l e a s e d r a p i d l y and completely by t h e f o r m a t i o n of E 2 P , t h e n rebound t o t h e enzyme g r a d u a l l y as t h e amount of E2P was reduced. As shown i n F i g . 4 , on a d d i t i o n of ATP, a b o u t 2 moles of Rb+ bound r a p i d l y p e r mole of a c t i v e s i t e , w i t h t h e f o r m a t i o n of E 2 P . S i m i l a r r e s u l t s were a l s o Thus, Na+ i s r e l e a s e d observed f o r t h e b i n d i n g of K+. completely from t h e Na+-binding s i t e and K+ o r Rb+ b i n d s almost c o m p l e t e l y t o t h e K+-binding s i t e upon t h e I t s h o u l d be n o t e d t h a t 5 min a f t e r formation of E2P. t h e a d d i t i o n of ATP , E2P d i s a p p e a r e d a l m o s t c o m p l e t e l y , w h i l e t h e enzyme s t i l l bound Rb+ ( F i g . 4 ) . P o s t et a 2 . ( 1 9 7 2 ) p r e v i o u s l y s u g g e s t e d t h a t t h e enzyme o c c l u d e s K+ j u s t a f t e r t h e d e p h o s p h o r y l a t i o n . R e c e n t l y , Beauge and Glynn ( 1 9 7 9 ) p r e s e n t e d s t r o n g e v i d e n c e f o r t h e occluded-K+ form i n t h e c o u r s e o f t h e enzyme t u r n over. The dependency of Rb+ b i n d i n g on Rb+ c o n c e n t r a t i o n i n t h e p r e s e n c e of ATP, Mg2+, and 1 0 0 mM NaCl was exp r e s s e d by a s i m p l e second-order d i s s o c i a t i o n e q u a t i o n w i t h a s i n g l e " a p p a r e n t " d i s s o c i a t i o n c o n s t a n t of 4 1 p M , a l t h o u g h t h e amount of E2P d e c r e a s e d upon i n c r e a s i n g t h e Rb+ c o n c e n t r a t i o n . T h i s i n d i c a t e s t h a t t h e a f f i n i t y o f Rb+ f o r E 2 P i s e q u a l t o t h a t f o r E 2 . The d i s s o c i a t i o n c o n s t a n t f o r E2P and E 2 i n t h e p r e s e n c e of 1 0 0 m~ N a C l was 1/30 of t h a t f o r E l .

BINDING OF MONOVALENT CATIONS TO THE Na,K-ATPase

21 1

REACTION TIME ( m i d

+

F i g . 4 . T i m e c o u r s e of R b b i n d i n g t o the e n z y m e a f t e r the s t a r t of the ATPase reaction. T h e amount o f R b f b i n d i n g was m e a s u r e d a f t e r the ATPase reaction w a s s t a r t e d b y the a d d i t i o n of 0.5 mM ATP ( 0 ) t o the e n z y m e i n the p r e s e n c e of 4 2 V M 86RbC1, 2 mM M g C 1 2 , and 0.5 mM EDTA. Open s q u a r e s ( 0 ) i n d i c a t e the amounts of Rb' b i n d i n g b e f o r e the a d d i t i o n of ATP. When 10 mM KCI was a d d e d t o the reaction m i x t u r e 2 min a f t e r the s t a r t of the ATPase reaction ( + ) , the amount o f Rb+ b i n d i n g d e c r e a s e d T h e numbers m a r k e d l y ( a ) . Other c o n d i t i o n s a r e a s i n F i g . 2 . i n p a r e n t h e s e s i n d i c a t e t h e c o n c e n t r a t i o n s o f f r e e Rb+ i o n s O J M ) .

E.

A T P B I N D I N G TO C A T A L Y T I C AND REGULATORY S I T E S

N e u f e l d and Levy (1969) and Kanazawa e t a l . ( 1 9 7 0 ) found t h a t t h e a c t i v i t y o f t h e N a , K - A T P a s e i s accelera t e d markedly by h i g h c o n c e n t r a t i o n s of ATP. P o s t e t a l . (1972) i n d i c a t e d from t h e i r k i n e t i c s t u d i e s t h a t t h e a c c e l e r a t i o n i s due t o t h a t of t h e c o n v e r s i o n o f a K+-bound form, E 2 , i n t o a Na+-bound form, E , by h i g h c o n c e n t r a t i o n s of ATP. BeaugC and Glynn (1480) showed t h a t n o t o n l y ATP b u t a l s o u n h y d r o l y z a b l e ATP a n a l o g u e s , such a s AMP-PNP, s h i f t t h e e q u i l i b r i u m between E l form and E 2 form t o E l . More r e c e n t l y , Moczydlowski and F o r t e s ( 1 9 8 1 a , b ) showed t h a t a s i n g l e ATP m o l e c u l e bindi n g t o a c a t a l y t i c s i t e can produce t h e a c c e l e r a t i o n . W e found t h a t 1 mole of ATP b i n d s t o t h e enzyme w i t h a d i s s o c i a t i o n c o n s t a n t of 1 I J M , and s e v e r a l moles

M.YAMAGUCHI eta/.

212

zm z

2

0

TIME AFTER ADDITION OF 0.5 mM NaCl (mi

'0

10 20 TIME AFTER ADDITION OF 0.5 mM NaCl

30

(s)

+

F i g . 5 . E f f e c t of ATP a n d AMP-PNP on the time course of Na b i n d i n g t o the A T P a s e p r e i n c u b a t e d w i t h XCl. U p p e r : T h e e n z y m e was i n c u b a t e d f o r 30 m i n w i t h 50 p M ( 0 ,0 ,A ) or 30 mM KCl ( A ) T h e reaction i n the p r e s e n c e of 10-20 mM EDTA a t pH 7 . 5 a n d O°C. was s t a r t e d b y the a d d i t i o n o f 0 . 5 mM 22NaC1 w i t h 1 mM ATP ( @ ) or AMP-PNP ( A ) or w i t h o u t n u c l e o t i d e ( 0 ,h ) . Lower: T h e a m o u n t s o f Na+ b i n d i n g were m e a s u r e d a f t e r the a d d i t i o n o f 0 . 5 mM 22NaC1 t o t h e reaction m i x t u r e w i t h 30 pM (A), 1 mM ( 0 ) A T P , or w i t h o u t ATP ( 0 )

.

of ATP b i n d w i t h a l o w a f f i n i t y . The former s i t e i s t h e c a t a l y t i c s i t e . The a f f i n i t y of ATP for t h e catal y t i c s i t e w a s d e c r e a s e d by t h e a d d i t i o n of K+, as mentioned b e f o r e . However, we found t h a t 50 V M K C 1 d o e s n o t a f f e c t t h e ATP b i n d i n g t o t h e c a t a l y t i c s i t e .

BINDING OF MONOVALENT CATIONS TO THE Na,K-ATPase

I

213

SLICE NUMBER

20 40 INCUBATION TIME ( m i d

0

60

14 F i g . 6 . T i m e courses o f [ CIDCCD b i n d i n g a n d o f i n a c t i v a T h e e n z y m e was i n c u b a t e d w i t h 80 p M t i o n of Na,K-ATPase. [14C]DCCD ( A , 0 ) or c o l d DCCD ( A ,0 ) i n 2% e t h a n o l and 30 mM M E S - i m i d a z o l e (pH 6 . 0 ) a t 25OC i n t h e p r e s e n c e ( A ,A ) or a b s e n c e For the DCCD-binding, a l i q u o t s were t a k e n o f 1 mM K C l ( 0 , 0 ) o f f , d i l u t e d 10 t i m e s w i t h 0.1 mM c o l d DCCD and 2% ethanol a t pH 7 . 2 and O°C, a n d l a y e r e d on the M i l l i p o r e f i l t e r u n d e r s u c t i o n . T h e f i l t e r was washed 6 t i m e s w i t h 5 m l o f 50% ethanol a t pH 7 . 5 , f o l l o w e d b y 2 washes w i t h 5 m l o f d e i o n i z e d w a t e r . A f t e r d r y i n g , the r a d i o a c t i v i t y was m e a s u r e d . Inset : SDS-gel e l e c t r o p h o r e g r a m o f [ 1 4 C ] DCCD-modified Na, K-ATPasr.

.

F i g u r e 5 shows t h e i n i t i a l phase of t h e t h e enzyme p r e i n c u b a t e d w i t h 50 p M KC1, a f f e c t t h e ATP b i n d i n g t o t h e c a t a l y t i c absence of ATP, t h e s p e c i f i c b i n d i n g of

Na+ b i n d i n g t o which does n o t site. In the Na+ t o t h e

M. YAMAGUCHI eta/.

214

enzyme, t h a t i s , t h e c o n v e r s i o n of t h e enzyme from a K+-bound form, E , i n t o a Na+-bound form, El, o c c u r r e d very slowly a s szown i n t h e upper f i g u r e . The r a t e of b i n d i n g w a s markedly a c c e l e r a t e d by t h e a d d i t i o n of ATP o r AMP-PNP. Furthermore, t h e r a t e was enhanced by i n c r e a s i n g t h e ATP c o n c e n t r a t i o n from 30 t o 1 0 0 0 V M , a s shown i n t h e lower f i g u r e . T h i s f i n d i n g i n d i c a t e s t h a t t h e b i n d i n g of ATP t o t h e l o w - a f f i n i t y r e g u l a t o r y s i t e ( s ) i s a l s o f u n c t i o n i n g i n t h e a c c e l e r a t i o n of t h e conversion. F.

M O D I F I C A T I O N OF T H E E N Z Y M E B Y D C C D

Robinson ( 1 9 7 4 ) p r e v i o u s l y showed t h a t DCCD i n h i b i t s t h e Na,K-ATPase r e a c t i o n and t h a t t h e DCCD eff e c t i s p r o t e c t e d by t h e p r e s e n c e of Na+. W e found t h a t n o t o n l y Na+ b u t a l s o K+ o r Rb+ p r o t e c t t h e i n The c a t i o n concena c t i v a t i o n of t h e ATPase by DCCD. t r a t i o n which g i v e s t h e half-maximum e f f e c t i s i n good agreement w i t h t h e a f f i n i t y o b t a i n e d by t h e measurement of monovalent c a t i o n b i n d i n g t o t h e A T P a s e (Table I ) . Furthermore, w e found t h a t t h e amount of Rb+ bound t o t h e ATPase d e c r e a s e s i n p r o p o r t i o n t o t h e e x t e n t of i n h i b i t i o n of t h e Na,K-ATPase a c t i v i t y by t h e DCCD t r e a t ment. The b i n d i n g of DCCD t o t h e ATPase w a s p a r t i a l l y i n h i b i t e d by K+ ( F i g . 6). However, t h e c o v a l e n t bond o f DCCD was u n s t a b l e , and t h e amount o f bound DCCD obt a i n e d was n o t q u a n t i t a t i v e . The i n s e t shows t h a t t h e main peak of bound DCCD i s l o c a t e d on t h e a - s u b u n i t w i t h a molecular weight of about 1 0 0 , 0 0 0 . T h e r e f o r e , it may be concluded t h a t t h e c a t i o n b i n d i n g s i t e s of t h e ATPase a r e l o c a t e d on t h e a - s u b u n i t .

IV.

SUMMARY

Two k i n d s of c a t i o n - b i n d i n g s i t e s e x i s t on t h e Three Na+-binding s i t e s and 2 K+- or Rb+-binding s i t e s e x i s t s i m u l t a n e o u s l y p e r each asubunit. 2. When E - A T P i s formed by t h e b i n d i n g of ATP t o t h e c a t a l y t i c s i t e i n t h e absence of Mg2+, t h e K+ bindi n g i s i n h i b i t e d , w h i l e t h e N a + b i n d i n g is u n a f f e c t e d . 3. When t h e enzyme i s modified w i t h NEM, E 1 P i s formed. I n t h i s c a s e , no change occurs i n t h e a f f i n i t y f o r N a + o r Rb+ upon p h o s p h o r y l a t i o n . 4. I n t h e p r e s e n c e of ATP and Mg2+, t h a t i s i n t h e E2P s t a t e , t h e a f f i n i t y of t h e Na+-binding s i t e s f o r 1.

A T P a s e molecule.

BINDING OF MONOVALENTCATIONS TO THE Na,K-ATPase

215

+

decreases m a r k e d l y , w h i l e t h a t o f t h e K+-binding s i t e s f o r K+ o r Rb+ i n c r e a s e s markedly. The s t a t e of changed a f f i n i t i e s f o r c a t i o n s i s m a i n t a i n e d e v e n a f t e r

Na

t h e dephosphorylation. 5. The c o n v e r s i o n of a K+ bound form i n t o an Na+bound form i s markedly a c c e l e r a t e d by b i n d i n g o f ATP o r AMP-PNP t o t h e c a t a l y t i c and r e g u l a t o r y s i t e s . 6 . The A T P a s e a c t i v i t y i s i n h i b i t e d by t h e b i n d i n g of DCCD t o t h e monovalent c a t i o n - b i n d i n g s i t e s l o c a t e d on t h e a - s u b u n i t .

REFERENCES

Beaug6, L. A., and Glynn, I. M. ( 1 9 7 9 ) . Occlusion o f K i o n s i n t h e unphosphorylated sodium pump. N a t u r e (London) 280, 510-512. Beaug6, L. A , , and Glynn, I. M. (1980). The e q u i l i b r i u m between d i f f e r e n t conformations of t h e unphosphorylated sodium pump: E f f e c t s o f ATP and o f potassium i o n s , and t h e i r J. P h y s i o l (London) r e l e v a n c e t o potassium t r a n s p o r t . 299, 367-383. Garrahan, P. J . , and Glynn, I . M. (1967). The s t o i c h i o m e t r y o f t h e sodium pump. J. P h y s i o l . (London) 192, 217-235. H a s t i n g s , D. F. (1977). D i f f e r e n t i a l t i t r a t i o n of potassium b i n d i n g t o membrane p r o t e i n s using i o n s e l e c t i v e e l e c t r o d e s . A n a l . B i o c h e m . 83, 416-432. H a s t i n g s , D. F . , and Skou, J. C. ( 1 9 8 0 ) . Potassium b i n d i n g t o B i o c h i m . B i o p h y s A c t a 6 0 1 , 380t h e (Na+ + K+) -ATPase. 385. Kanazawa, T., S a i t o , M., and Tonomura, Y. (1970). Formation and decomposition o f a phosphorylated i n t e r m e d i a t e i n t h e r e a c t i o n o f N a + - K+ dependent ATPase. J . B i o c h e m . ( T o k y o ) 6 7 , 693-711. Kaniike, K . , Lindenmayer, G. E . , W a l l i c k , E . T . , Lane, L. K . , and Schwartz, A. (1976). S p e c i f i c sodium-22 b i n d i n g t o a p u r i f i e d sodium + potassium adenosine t r i p h o s p h a t a s e . Inh i b i t i o n by ouabain. J. B i o l . Chem. 251, 4794-4795. Matsui, H . , Hayashi, Y . , Homareda, H . , and Kimimura, M ( 1 9 7 7 ) . O u a b a i n - s e n s i t i v e 42K b i n d i n g t o Na+,K+-ATPase p u r i f i e d Biochem. Biophys. R e s . from c a n i n e kidney o u t e r medulla. Commun. 7 5 , 373-380. Moczydlowski, E. G . , and F o r t e s , P. A. G. (1981a). C h a r a c t e r i z a t i o n of 2t,3'-0-(2,4,6-trinitrocyclohexadienylidine)adenosine 5 ' - t r i p h o s p h a t e a s a f l u o r e s c e n t probe o f t h e ATP s i t e o f sodium and potassium t r a n s p o r t adenosine t r i p h o s p h a t a s e . Determination of n u c l e o t i d e b i n d i n g s t o i c h i o m e t r y J. B i o l . Chem. and ion-induced changes i n a f f i n i t y f o r ATP. 2 5 6 , 2346-2356.

.

.

216

M. YAMAGUCHI eta/.

Moczydlowski, E G . , and F o r t e s , P. A. G. (1981b). I n h i b i t i o n of sodium and potassium adenosine t r i p h o s p h a t a s e by 2 ’ , 3 ’ - 0 (2,4,6-trinitrocyclohexadienylidene)adenine n u c l e o t i d e s . I m p l i c a t i o n s f o r t h e s t r u c t u r e and mechanism of t h e Na:K pump. J. B i o l . C h e m . 256, 2357-2366. Neufeld, A. H . , and Levy, H. M. (1969). A second ouabains e n s i t i v e sodium-dependent adenosine t r i p h o s p h a t a s e i n b r a i n microsomes. J. B i o l . C h e m . 244, 6493-6497. P e t e r s , W. H. M., Swarts, H . G. P., de Pont, J J. H. H. M., Schuurmans Stekhoven, F. M. A. H . , and Bonting, S . L. (1981). (Na+ + K+)ATPase h a s one f u n c t i o n i n g p h o s p h o r y l a t i o n s i t e p e r s u b u n i t . N a t u r e ( L o n d o n ) 290, 338-339. P o s t , R. L . , Kume, S., Tobin, T., O r c u t t , B., and Sen, A. K . (1969). F l e x i b i l i t y of a n a c t i v e c e n t e r i n sodium-pluspotassium adenosine t r i p h o s p h a t a s e . J . Gen. P h y s i o l . 54 , 3065-325s. P o s t , R. L . , Hegyvary, C., and K u m e , S. (1972). A c t i v a t i o n by adenosine t r i p h o s p h a t e i n t h e p h o s p h o r y l a t i o n k i n e t i c s o f sodium and potassium i o n t r a n s p o r t adenosine t r i p h o s p h a t a s e . J. B i o l . C h e m . 247, 6530-6540. + + Robinson, J. D. (1974). A f f i n i t y o f t h e ( N a + K )-dependent ATPase f o r Na+ measured by Na+-modified enzyme i n a c t i v a t i o n . FEBS L e t t . 38, 325-328. Sachs, J . R. (1981). Mechanistic i m p l i c a t i o n s o f t h e potassiumpotassium exchange c a r r i e d o u t by t h e sodium-potassium pump. J. P h y s i o l . ( L o n d o n ) 316, 263-277. Sakamoto, J . , and Tonomura, Y. (1980). Order of release of ADP and P i from phosphoenzyme w i t h bound ADP of CaZ+-dependent ATPase from s a r c o p l a s m i c r e t i c u l u m and of Na+,K+-dependent ATPase s t u d i e d by ADP-inhibition p a t t e r n s . J. B i o c h e m . ( T o k y o ) 87, 1721-1727. Sen, A. K . , and P o s t , R. L. (1964). S t o i c h i o m e t r y and l o c a l i z a t i o n of ATP dependent Na and K t r a n s p o r t i n t h e e r y t h r o c y t e . J. B i o l . C h e m . 2 3 9 , 345-352. Tonomura, Y . , and Fukushima, Y. (1974). K i n e t i c p r o p e r t i e s of p h o s p h o r y l a t e d i n t e r m e d i a t e s i n t h e r e a c t i o n o f Na+,K+ATPase. A n n . N . Y . A c a d . S c i . 242, 92-105. Yamaguchi, M., and Tonomura, Y. (1978). Binding of adenosine diphosphate t o r e a c t i o n i n t e r m e d i a t e s i n t h e N a + ,K+dependent ATPase from p o r c i n e kidney. J. B i o c h e m . ( T o k y o ) 83, 977-987. Yamaguchi, M . , and Tonomura, Y. (1979). Simultaneous b i n d i n g of t h r e e N a + and two K+ i o n s t o Na+,K+-dependent ATPase and changes i n i t s a f f i n i t i e s f o r t h e i o n s induced by t h e f o r J. B i o c h e m . mation o f a phosphorylated i n t e r m e d i a t e . ( T o k y o ) 86, 509-523. Yamaguchi, M., and Tonomura, Y. (1980a). Binding o f monovalent c a t i o n s t o Na+,K+-dependent ATPase p u r i f i e d from p o r c i n e kidney. I. Simultaneous b i n d i n g of t h r e e sodium and two potassium or rubidium i o n s t o t h e enzyme. J. B i o c h e m . ( T o k y o ) 88, 1365-1375.

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Yamaguchi, M . , and Tonomura, Y . (1980b). Binding o f monovalent c a t i o n s t o Na+,K+-dependent ATPase p u r i f i e d from p o r c i n e kidney. 11. A c c e l e r a t i o n o f t r a n s i t i o n from a K+-bound form t o a Na+-bound form by b i n d i n g o f ATP t o a r e g u l a t o r y s i t e o f t h e enzyme. J. B i o c h e m . (Tokyo) 8 8 , 1377-1385. Yamaguchi, M . , and Tonomura, Y . ( 1 9 8 0 ~ ) . Binding o f monovalent c a t i o n s t o Na+,K+-dependent ATPase p u r i f i e d from p o r c i n e kidney. 111. Marked changes i n a f f i n i t i e s f o r monovalent c a t i o n s induced by f o r m a t i o n o f an ADP-insensitive b u t n o t a n ADP-sensitive phosphoenzyme. J. B i o c h e m . (Tokyo) 8 8 , 1387-1397.

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CURRENT TOPICS IN MEMBRANES AND TRANSPORT, VOLUME 19

Half-of-the-Sites Reactivity of Na,K-ATPase Examined by the Accessibility of Vanadate and ATP into Enzyme-Ouabain Complexes O n 0 HANSEN Institute of Physiology Universiry of Aarhus Aarhus, Denmark

I.

INTRODUCTION

Biphasic substrate-velocity p l o t s f o r t h e Na,KA T P a s e r e a c t i o n and t h e k i n e t i c d i f f e r e n c e s of t h e i n h i b i t i o n of Na,K-ATPase and pNPPase a c t i v i t i e s by a number o f s u b s t a n c e s have been i n t e r p r e t e d a s an i n d i c a t i o n of h i g h - a f f i n i t y and l o w - a f f i n i t y s u b s t r a t e s i t e s o f t h e enzyme. Models t h a t have been proposed t o d e s c r i b e t h e r e a c t i o n c a t a l y z e d by N a , K - A T P a s e i n clude a half-of-the-sites r e a c t i v i t y o r a l t e r n a t i n g s i t e mechanism i n v o l v i n g two s u b s t r a t e s i t e s and a f l i p - f l o p mechanism. A model i n v o l v i n g two s i m u l t a n e o u s l y working subs t r a t e s i t e s p r e d i c t s b i n d i n g of ATP and P i a t one and t h e same t i m e ( S t e i n , 1 9 7 9 ) . I t h a s been s u g g e s t e d

219

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OTTO HANSEN

t h a t n u c l e o t i d e s can r e a c t w i t h phosphoenzyme s i n c e ATP a s w e l l a s nonphosphorylating ATP a n a l o g s a c c e l e r a t e t h e r e l e a s e of ouabain from an enzyme-ouabain complex t h e f o r m a t i o n of which was promoted by Mg2+ + P i (Glynn and K a r l i s h , 1 9 7 5 ) . The enzyme-ouabain complex obt a i n e d w i t h (Mg2+ + P i ) is r a t h e r s t a b l e a f t e r it h a s been washed f r e e of unbound l i g a n d s , b u t r e l e a s e o f ouabain i s a c c e l e r a t e d c o n s i d e r a b l y by ATP, e s p e c i a l l y i f N a + and EDTA a r e a l s o p r e s e n t , i . e . , under condit i o n s where p h o s p h o r y l a t i o n c a n n o t t a k e p l a c e . I f P i i s s t i l l p a r t of t h e enzyme-ouabain complex a t t h i s t i m e , t h e simultaneous b i n d i n g of P i and ATP would be a fact.

11.

RESULTS AND DISCUSSION

S i n c e Na,K-ATPase b i n d s P i w i t h low a f f i n i t y and, t o some d e g r e e , a l s o u n s p e c i f i c a l l y , ''Pi b i n d i n g measurements a r e u n r e l i a b l e f o r e s t a b l i s h i n g s p e c i f i c P i b i n d i n g t o enzyme-ouabain complexes d u r i n g c h a s e s w i t h ATP. A very similar enzyme-ouabain complex i s o b t a i n e d by s u b s t i t u t i n g very low c o n c e n t r a t i o n s of vanadate f o r P i (Hansen, 1 9 7 9 ) . Vanadate behaves a s a t r a n s i t i o n s t a t e analog of phosphate and h a s an a f f i n i t y f o r ATPase t h a t i s s e v e r a l o r d e r s of magnitude h i g h e r t h a n t h a t of P i . The enzyme-ouabain complex formed w i t h vanadate i s v e r y s i m i l a r t o t h a t formed w i t h P i and [48V]vanadate i s known t o be r e t a i n e d i n an enzymevanadate-ouabain complex from which i t i s r e l e a s e d more slowly t h a n ouabain (Hansen, 1 9 8 0 ) . A f t e r washing t h e (Mg2+ + V ) - f a c i l i t a t e d enzyme-ouabain complex f r e e of unbound l i g a n d s and r e s u s p e n d i n g i n d i l u t e T r i s b u f f e r o r i n b u f f e r + Na+ + ATP, i t appeared t h a t n e i t h e r ouabain nor vanadate r e l e a s e was a c c e l e r a t e d by (Na+ + A T P ) . I t can be concluded t h a t t h e r e i s no e v i d e n c e of ATP b i n d i n g t o an enzyme-ouabain complex under condit i o n s where t h e phosphate a n a l o g , v a n a d a t e , i s r e t a i n e d as p a r t of t h e complex. F i g u r e 1 shows t h e t i m e c o u r s e of [48V]vanadate b i n d i n g t o Na,K-ATPase and t o enzymeouabain complexes o b t a i n e d w i t h Mg2+, (Mg2+ + Na+ + ATP) , (Mg2+ + P i ) t o r (Mg2+ + V ) i n t h e p r e s e n c e ( A ) o r abs e n c e (B) of t h e l i g a n d s t h a t promoted ouabain b i n d i n g . The c o n c e n t r a t i o n of u n l a b e l e d vanadate t h a t promoted ouabain b i n d i n g was s e l e c t e d so t h a t n e a r l y a l l vanadate was bound. I t i s seen t h a t i n t h e f i r s t t h r e e c a s e s , [48V]vanadate i s bound somewhat more s l o w l y , b u t ap-

A

0

A

6

+ 100

50

Min

Min

F i g . 1 . T i m e c o u r s e of [ 4 8 V ] v a n a d a t e b i n d i n g to 118 nM Na,K-ATPase f r o m p i g k i d n e y o u t e r m e d u l l a ( A ) and t o e n z y m e - o u a b a i n c o m p l e x e s o b t a i n e d w i t h M g 2 + ( 0 ) , ( M y 2 + + Nai + ATP) ( + ) , (M++ + P i ) ( V ) or ( M y 2 + + 100 nM V ) ( 0 ) i n the p r e s e n c e ( A ) or a b s e n c e ( B ) of the l i g a n d s t h a t f a c i l i t a t e d ouabain binding. ( R e p r o d u c e d w i t h p e r m i s s i o n f r o m B i o c h i m . B i o p h y s . A c t a 6 9 2 , 187.)

O n 0 HANSEN

222

p a r e n t l y w i t h a h i g h e r a f f i n i t y t o enzyme-ouabain comp l e x e s t h a n t o t h e uncomplexed enzyme, provided compet i t i o n by P i and ATP i s avoided. Hence t h e s e t h r e e enzyme-ouabain complexes have a s i t e a v a i l a b l e f o r vanadate o r P i b i n d i n g (and probably a l s o ATP b i n d i n g l e a d i n g t o p h o s p h o r y l a t i o n ) so t h a t t h e p h o s p h o r y l a t i o n t h a t preceded ouabain b i n d i n g i n two of t h e complexes m u s t have been l o s t . I n c o n t r a s t , t h e vanadatef a c i l i t a t e d enzyme-ouabain complex i s i n a c c e s s i b l e t o f u r t h e r b i n d i n g of 48V, presumably u n t i l ouabain and u n l a b e l e d vanadate a r e r e l e a s e d .

111.

CONCLUSIONS

The experiments t h u s show (1) t h a t s i m u l t a n e o u s vanadate and ATP b i n d i n g does n o t t a k e p l a c e (and t h u s , most p r o b a b l y , n e i t h e r P i and ATP b i n d i n g ) , ( 2 ) t h a t p h o s p h o r y l a t i o n i s l o s t from enzyme-ouabain complexes o b t a i n e d w i t h P i as w e l l a s ATP, whereas v a n a d y l a t i o n i s r e t a i n e d , and ( 3 ) t h a t t h e complex o b t a i n e d w i t h Mg2+ b u t w i t h o u t added P i o r ATP cannot have been f a c i l i t a t e d by t r a c e s of vanadate.

ACKNOWLEDGMENT

T h i s s t u d y w a s s u p p o r t e d by t h e Danish Medical Research Council.

REFERENCES

Glynn, I. M., and K a r l i s h , S . J. D. (1975). The sodium pump. A n n u . R e v . P h y s i o l . 3 7 , 13-55. Hansen, 0. (1979). F a c i l i t a t i o n o f o w b a i n b i n d i n g t o ( N a + + K + ) ATP as e by v a n a d a t e a t i n vivo c o n c e n t r a t i o n s . B i o c h i r n . B i o p h y s A c t a 5 6 8 , 265-269. Hansen, 0. (1980). Vanadate i n t e r a c t i o n w i t h t h e Na,K-ATPase. B a s i c R e s . C a r d i o l . 75, 455-459. S t e i n , W. D. ( 1 9 7 9 ) . H a l f - o f - t h e - s i t e s r e a c t i v i t y and t h e N a ,K-ATPase In " N a , K-ATPase: S t r u c t u r e and K i n e t i c s " ( J . C. Skou and J. G. NZrby, e d s . ) , pp. 487-500. Academic Press, New York,

.

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CURRENTTOPICS W MEMBRANES AND TRANSPORT, VOLUME 19

Binding of Rb+ and ADP to a Potassium-Like Form of Na,K-ATPase JgRGEN JENSEN AND PAUL OlTOLENGHI Institute of Physiology University of Anrhus Anrhus, Denmark

I N TROD UC T I ON

T h e e s t i m a t i o n of t h e b i n d i n g c a p a c i t y f o r p o t a s ium t o Na,K-ATPase i s d i f f i c u l t , a s e v i d e n c e d from ? p o r t s i n t h e r e c e n t l i t e r a t u r e (Matsui e t a l . , 1977; i n t l e y et a l . , 1978; Yamaguchi and Tonomura, 1979; \ s t i n g s and Skou, 1 9 8 0 ) , where one f i n d s t h a t 2 , 4 , d 5 K+ i o n s are bound p e r o u a b a i n - b i n d i n g s i t e . W e have r e i n v e s t i g a t e d t h e problem w h i l e t r y i n g optimize t h e conditions f o r c a t i o n binding. W e v e used rubidium a s a n a n a l o g f o r p o t a s s i u m and have ?d h i g h enzyme c o n c e n t r a t i o n , l o w t e m p e r a t u r e , l o w iic s t r e n g t h , and l o w monovalent c a t i o n c o n c e n t r a m. W e have a l s o i n v e s t i g a t e d whether n u c l e o t i d e and o r Rb+ b i n d s i m u l t a n e o u s l y t o t h e enzyme. The amount of p r o t e i n c o r r e s p o n d i n g t o one nucleoe - b i n d i n g s i t e o r one o u a b a i n - b i n d i n g s i t e i s used d e f i n e a u n i t o f Na,K-ATPase.

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11.

METHODS

The rubidium- and ADP-binding s t u d i e s w e r e c a r r i e d o u t a t 0-2OC w i t h crude microsomal p r e p a r a t i o n s of p i g kidney o u t e r medulla, a c t i v a t e d w i t h SDS, t h o r o u g h l y washed, and suspended i n 200 m s u c r o s e , 0 . 5 mM EDTA, 1 4 m&f i m i d a z o l e , and 0-1.3 mM T r i s , p H 7.5 ( a t OOC). The s p e c i f i c a c t i v i t i e s (pmoles s p l i t p e r hour p e r m i l l i g r a m p r o t e i n ) were: K-pNPPase, 1 0 0 ; Na,K-ATPase, 550. The p r o t e i n c o n c e n t r a t i o n i n t h e b i n d i n g a s s a y s w a s app r o x i m a t e l y 4 mg p e r m i l l i l i t e r . The number of Rb-binding s i t e s w a s measured by cent r i f u g a t i o n u s i n g 86RbCl. ADP-binding c a p a c i t was det e r m i n e d by a r a p i d d i a l y s i s t e c h n i q u e u s i n g [I4C]ADP (Ndrby and J e n s e n , 1 9 7 1 ) . Ouabain-binding c a p a c i t y w a s measured by f i l t r a t i o n a c c o r d i n g t o Hansen and Skou (1973) u s i n g [3H]ouabain. The h i g h - a f f i n i t y ( s p e c i f i c ) b i n d i n g of rubidium i o n s a t any g i v e n c o n c e n t r a t i o n of f r e e l i g a n d was obt a i n e d as t h e d i f f e r e n c e between two c u r v e s showing (1) t o t a l rubidium bound v e r s u s f r e e rubidium concent r a t i o n ; ( 2 ) rubidium bound t o h e a t - d e n a t u r e d enzyme v e r s u s f r e e rubidium c o n c e n t r a t i o n . Assays were p e r formed on i d e n t i c a l a l i q u o t s b e f o r e and a f t e r h e a t i n g f o r 5 o r 1 0 min a t 65OC.

111.

RESULTS AND DISCUSSION

F i g u r e 1 shows two c u r v e s i n a S c a t c h a r d - t y p e p l o t f o r t h e s p e c i f i c b i n d i n g of rubidium t o Na,K-ATPase. The r e s u l t s were o b t a i n e d w i t h t h r e e d i f f e r e n t enzyme preparations: two i n t h e absence of n u c l e o t i d e and one i n t h e p r e s e n c e of 2 m~ ATP. Although t h e c o r r e c t i o n f o r nonspecific binding t h a t w a s associated with t h e t h r e e p r e p a r a t i o n s v a r i e d by a t l e a s t 300%, s p e c i f i c b i n d i n g c a p a c i t y p e r ouabain-binding s i t e showed no v a r i a t i o n , and t h e two e x p e r i m e n t s i n t h e absence of n u c l e o t i d e gave i d e n t i c a l r e s u l t s . There are t h r e e h i g h - a f f i n i t y b i n d i n g s i t e s f o r rubidium f o r e v e r y ouabain-binding s i t e on t h e enzyme, b o t h i n t h e absenct o f and i n t h e p r e s e n c e of ATP c o n c e n t r a t i o n s t h a t w e have found t o b e 5 - f o l d g r e a t e r t h a n t h o s e n e c e s s a r y t s a t u r a t e any e f f e c t upon rubidium b i n d i n g . ATP decreases t h e a f f i n i t y w i t h which rubidium b i n d s t o i t s s i t e s on t h e enzyme w i t h o u t changing t h e rubidiumbinding capacity.

BINDING OF Rb+ AND ADP TO Na,K-ATPase

225

F i g . 1 . S c a t c h a r d - l i k e p l o t of R b b i n d i n g w i t h o u t ATP ( o p e n s y m b o l s ) and i n the p r e s e n c e of 2 mM ATP ( c l o s e d symbols).

N u c l e o t i d e b i n d i n g t o t h e enzyme w a s s t u d i e d u n d e r t h e same e x p e r i m e n t a l c o n d i t i o n s a s used above. A s shown i n F i g . t h e a f f i n i t y f o r ADP w a s much l o w e r t h a n t h e 5 pM-3'found i n s t u d i e s w i t h o u t N a o r K a d d e d , b u t w i t h h i g h e r T r i s and EDTA c o n c e n t r a t i o n s . Under t h e latter conditions fluorescence spectra i n d i c a t e t h a t t h e enzyme i s i n a s o d i u m - l i k e form (Skou and Esmann, 1 9 8 1 ) . Adding sodium t o enzyme u n d e r c o n d i t i o n s u s e d i n t h i s s t u d y i n c r e a s e s t h e enzyme's a f f i n i t y f o r ADP toward v a l u e s of 5 U M - ~ . T h i s e f f e c t o f sodium c o u l d b e due t o i t s c o m p e t i t i o n w i t h p o t a s s i u m c o n t a m i n a t i n g t h e enzyme p r e p a r a t i o n o r t o a s p e c i f i c e f f e c t on t h e e q u i l i b r i u m between K forms and N a forms of t h e enzyme.

226

J0RGEN JENSEN AND PAUL OTOLENGHI

rl n

I

a

d

3

Bound/f ree F i g . 2. S c a t c h a r d - l i k e p l o t o f b i n d i n g o f ADP t o K - l i k e f o r m of the e n z y m e a t constant t o t a l ADP concentration; (*), i n a b s e n c e o f a d d e d Na or K ; ( a ) , a f t e r a d d i t i o n of 5 mM K; ( 0 ) , a f t e r a d d i t i o n o f u p t o 10 mM Na. (----- ) , Isotherm s h o w i n g the b i n d i n g o f ADP t o N a - l i k e f o r m o f the e n z y m e i n the absence of a d d e d Na or K .

Adding potassium t o enzyme d e c r e a s e s t h e enzyme's a f f i n i t y f o r nucleotide. A t high potassium concentrat i o n s t h e e x p e r i m e n t s do n o t a l l o w one t o d e c i d e whether t h i s a f f i n i t y i s d i f f e r e n t from z e r o , i . e . , whether t h e K-enzyme complex c a n ' b i n d n u c l e o t i d e . The q u e s t i o n i s s e t t l e d by t h e rubidium-binding e x p e r i m e n t s which showed t h a t rubidium and n u c l e o t i d e c o u l d b i n d s i m u l t a n e o u s l y . The a f f i n i t y of K-enzyme complex f o r ADP and ATP must b e 1 0 0 P M o r more f o r it t o be i n v i s i b l e i n t h e nucleotide-binding experiments. F l u o r e s c e n c e s p e c t r a o f e o s i n w i t h t h e enzyme as used h e r e i n d i c a t e d t h a t t h e enzyme w a s i n a potassiuml i k e form. I t i s n o t p o s s i b l e t o d e c i d e whether t h e

BINDING OF Rb+ AND ADP TO Na,K-ATPase

227

enzyme i s i n t h i s p o t a s s i u m - l i k e form b e c a u s e of t h e 5-10 U M p o t a s s i u m i o n c o n t a m i n a t i o n of t h e s t o c k s o l u t i o n s o f well-washed enzyme o r w h e t h e r it would s t i l l be i n t h i s potassium-like s t a t e i n t h e t o t a l absence of potassium. The p r e s e n t e x p e r i m e n t s show t h a t e a c h p r o t e i n u n i t h a v i n g one ouabain-binding s i t e and one n u c l e o t i d e b i n d i n g s i t e i s c a p a b l e of b i n d i n g 3 Rb' i o n s and h e n c e , presumably a l s o 3 K+ i o n s . Simultaneous b i n d i n g of n u c l e o t i d e and rubidium i s p o s s i b l e ; n u c l e o t i d e s r e d u c e t h e a f f i n i t y o f enzyme f o r rubidium a n d , t h e r e f o r e , rubidium d e c r e a s e s t h e enzyme's a f f i n i t y € o r n u c l e o t i d e .

ACKNOWLEDGMENT

T h i s s t u d y w a s s u p p o r t e d i n p a r t by t h e Danish Medical R e s e a r c h Council.

REFERENCES

C a n t l e y , L. C . , C a n t l e y , L. G . , and J o s e p h s o n , L. ( 1 9 7 8 ) . A c h a r a c t e r i z a t i o n o f vanadate i n t e r a c t i o n s w i t h t h e ( N a , K ) ATPase. J. B i o l . Chem. 2 5 3 , 7361-7360. Hansen, O . , a n d Skou, J. C . ( 1 9 7 3 ) . A s t u d y on t h e i n f l u e n c e o f t h e c o n c e n t r a t i o n o f Mg2+, P i , K+, N a + and T r i s o n (Mg2+ + Pi)-supported g-strophanthin binding t o (Na+ + K+)-inactiv a t e d ATP from ox b r a i n . B i o c h i m . B i o p h y s . A c t a 311, 5166. H a s t i n g s , D . , and Skou, J . C. ( 1 9 8 0 ) . Potassium b i n d i n g t o t h e ( N a + + K+) -ATPase. B i o c h i m . B i o p h y s . A c t a 601, 380-385. M a t s u i , H . , Hayashi, Y . , Homareda, H . , and Kimimura, M. ( 1 9 7 7 ) . O u a b a i n - s e n s i t i v e 42K-binding t o Na+,K+-ATPase p u r i f i e d Biochem. Biophys. R e s . from c a n i n e k i d n e y o u t e r medulla. commun. 75, 373-379. Nbrby, J. G . , and J e n s e n , J. ( 1 9 7 1 ) . Binding o f ATP t o b r a i n B i o c h i m . B i o p h y s . A c t a 2 3 3 , 104-116. microsomal ATPase. A f l u o r e s c e n t probe Skou, J . C . , and Esmann, M. ( 1 9 8 1 ) . Eosin. of ATP b i n d i n g t o t h e ( N a + + K+)-ATPase. Biochim. Biophys. A c t a 647, 232-240. Yamaguchi, M . , and Tonomura, Y. ( 1 9 7 9 ) . Simultaneous b i n d i n g o f t h r e e N a + and two K+ i o n s t o Na+,K+-dependent ATPase and changes i n i t s a f f i n i t i e s f o r t h e i o n s induced by t h e f o r mation o f a p h o s p h o r y l a t e d i n t e r m e d i a t e . J. B i o c h e m . ( T o k y o ) 8 6 , 509-523.

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CURRENT TOPICS IN MEMBRANES AND TRANSPORT,VOLUME 19

Side-Dependent Ion Effects on the Rate of Ouabain Binding to Reconstituted Human Red Cell Ghosts H. H. BODEMANN AND H. REICHMA" Department of Internal Medicine University of Freiburg Freiburg, Federal Republic of Germany

T. J. C'HANANDJ.

F. HOFFMAN

Department of Physiology Yale University School of Medicine New Haven, Connecticut

T h i s a r t i c l e summarizes r e c e n t work concerned w i t h t h e e f f e c t s v a r i o u s i o n s have on t h e r a t e of o u a b a i n b i n d i n g t o t h e r e d c e l l membrane. These types o f e f fects h e l p t o d e f i n e t h e determinants of ouabain i n t e r a c t i o n w i t h i t s r e c e p t o r and t h e r e l a t i o n t h i s b i n d i n g b e a r s t o t h e t r a n s p o r t f u n c t i o n o f t h e pump. W e w i l l c o n s i d e r t h e i n f l u e n c e s t h a t e x t e r n a l N a and e x t e r n a l C a have on o u a b a i n b i n d i n g as c o n t r o l l e d by i n t e r n a l Mg. I n a d d i t i o n , w e a n a l y z e t h e p a r a d o x i c a l r e l a t i o n between t h e o u a b a i n b i n d i n g r a t e and pump a c t i v a t i o n by i n t e r n a l N a as i n f l u e n c e d by i n t e r n a l K . P r e v i o u s work h a s shown t h a t o u a b a i n b i n d i n g t o t h e o u t s i d e of t h e membrane i s a n t a g o n i z e d by KO; N a o c a n r e d u c e t h e a f f i n i t y of t h e pump f o r KO, r e s u l t i n g i n a s t i m u l a t i o n o f t h e b i n d i n g r a t e o f o u a b a i n (Bodemann and Hoffman, 1976a; S a c h s , 1 9 7 4 ) . The i n t e r a c t i o n of Nao w i t h t h e o u t s i d e o f t h e pump i s complex, e v i d e n t l y req u i r i n g a t l e a s t two Na s i t e s ( C a v i e r e s and E l l o r y , 1975; Hobbs and Dunham, 1 9 7 6 ) . I n a d d i t i o n , o u a b a i n b i n d i n g i s a l s o s t i m u l a t e d by Cao and Mgo b u t t h e s e i o n s do n o t a c t 229

Copynght 0 1983 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-153319-0

230

H. H. BODEMANN eta/.

by changing t h e pump's a f f i n i t y f o r KO (Gardner and F r a n t z , 1974; Hobbs and Dunham, 1 9 7 8 ) .

I.

INTERACTION BETWEEN Mgi AND N a o

The e f f e c t o f N a o on t h e o u a b a i n - b i n d i n g r a t e depends upon t h e c o n c e n t r a t i o n of Mgi. I t w a s known from s t u d i e s on ATP promoted o u a b a i n b i n d i n g i n r e c o n s t i t u t e d g h o s t s (Bodemann and Hoffman, 197633) t h a t N a O , i n t h e p r e s e n c e of h i g h KO, i n c r e a s e d t h e r a t e o f o u a b a i n bindThis e f i n g p r o v i d e d t h a t Mgi was low ( e . g . , 0 . 5 m M ) . f e c t of N a o w a s maximal a t 1 2 m M and was l o s t when Mgi was i n c r e a s e d . Thus, a t h i g h Mgi ( e . g . , 4 m h f ) , conc e n t r a t i o n s of N a o up t o 1 5 0 mM w i t h KO p r e s e n t were w i t h o u t e f f e c t on t h e o u a b a i n - b i n d i n g r a t e (Bodemann and Hoffman, 1 9 7 6 a ) . I n c o n t r a s t , o u a b a i n b i n d i n g t o i n t a c t c e l l s w a s found t o be s t i m u l a t e d by N a o up t o 150 mM i n d e p e n d e n t o f t h e p r e s e n c e o f a h i g h KO (Hobbs and Dunham, 1 9 7 8 ) . These a p p a r e n t l y d i v e r g e n t e f f e c t s were r e s o l v e d by s y s t e m a t i c a l l y v a r y i n g M g i from 0.5 t o 1 2 mM i n t h e I t was found t h a t i n c r e a s p r e s e n c e and absence o f Na,. i n g Mgi i n c r e a s e d t h e o u a b a i n - b i n d i n g r a t e i n t h e abS i n c e N a o o n l y s t i m u l a t e d o u a b a i n bindsence of Nao. i n g a t low Mgi, t h e f a i l u r e of N a o t o a c t a t h i g h Mgi was because t h e ouabain-binding r a t e w a s a l r e a d y s t i m u l a t e d . Hence, t h e d i f f e r e n c e between t h e r e s u l t s obt a i n e d on i n t a c t c e l l s and g h o s t s e v i d e n t l y r e f l e c t s d i f f e r e n c e s i n Mgi. On t h e o t h e r hand, i n t h e absence of KO, we found t h a t N a c o u l d s t i m u l a t e t h e r a t e of o u a b a i n b i n d i n g i n d e p e n 8 e n t of t h e c o n c e n t r a t i o n o f Mgi These r e s u l t s p r o v i d e e v i d e n c e f o r a t l e a s t two N a s i t e s on t h e o u t s i d e of t h e pump, one o f which i s a v a i l a b l e o n l y i n t h e absence of KO (and t h e r e f o r e p r o b a b l y a K s i t e ) ; t h e o t h e r i s a c c e s s i b l e i n t h e p r e s e n c e of KO and i s c o n t r o l l e d by Mgi.

.

11.

INTERACTION BETWEEN M g i AND C a o

P r e v i o u s work on porous g h o s t s (when a l l added subs t a n c e s have e q u a l access t o b o t h s i d e s of t h e membrane) i n d i c a t e d t h a t w h i l e C a c o u l d n o t s u b s t i t u t e f o r Mg i n ATP-promoted o u a b a i n b i n d i n g (Hoffman, 19691, C a added i n t h e p r e s e n c e of Mg a l w a y s reduced t h e b i n d i n g r a t e i n

SIDE-DEPENDENT ION EFFECTS ON OUABAIN BINDING

231

t h e p r e s e n c e o r absence of N a and/or K (Bodemann and Hoffman, 1 9 7 6 b ) . The ouabain-binding r a t e was s e n s i t i v e , of c o u r s e , t o K whether o r n o t N a w a s p r e s e n t , b u t t h e most marked i n h i b i t o r y e f f e c t of C a w a s o b s e r v e d when K and N a were p r e s e n t t o g e t h e r (Bodemann and Hoffman, 1 9 7 6 b ) . These r e s u l t s c o n t r a s t w i t h t h e e f f e c t s of C a on i n t a c t r e d c e l l s o r on r e c o n s t i t u t e d g h o s t s , where membrane s i d e d n e s s i s p r e s e r v e d . For i n s t a n c e , i n i n t a c t r e d c e l l s , Cao i n t h e absence of KO was found t o s t i m u l a t e o u a b a i n b i n d i n g (Gardner and F r a n t z , 1 9 7 4 ; Hobbs and Dunham, 1 9 7 8 ) . With r e c o n s t i t u t e d g h o s t s , w e found t h a t t h e e f f e c t s of Cao were dependent upon t h e c o n c e n t r a t i o n of Mgi, i n a manner similar t o t h a t d e s c r i b e d above f o r Na,. Thus a t low Mgi, Cao w a s found t o markedly s t i m u l a t e t h e ouabain-binding r a t e , and t h i s e f f e c t w a s reduced by i n c r e a s i n g t h e c o n c e n t r a t i o n o f Mgi. These e f f e c t s of Cao c o u l d be observed i n t h e p r e s e n c e of N a o and KO. The c o n c e n t r a t i o n o f Cao which gave half-maximal s t i m u l a t i o n w a s l e s s t h a n 3 mM. These r e s u l t s i n d i c a t e t h a t t h e r e i s a C a s i t e on t h e o u t s i d e of t h e pump (modulating ouab a i n b i n d i n g ) , t h e a c c e s s i b i l i t y of which depends on t h e c o n c e n t r a t i o n of Mgi. A t h i g h Mgi, t h i s s p e c i f i c i t y changes and r e s e m b l e s t h e c o n t r o l t h a t Mgi h a s on t h e e f f e c t s of Na,.

111.

INTERACTION BETWEEN Nai AND Ki

The ouabain-binding r a t e c a n be markedly a l t e r e d by v a r i a t i o n s i n N a i and K i , a t normal and/or c o n s t a n t v a l u e s of Mgi, N a O , and KO. When K i i s low, i n c r e a s i n g N a i ( c h o l i n e added t o keep i n s i d e i s o t o n i c w i t h plasma) d e c r e a s e s t h e r a t e of ATP-promoted o u a b a i n b i n d i n g t o r e c o n s t i t u t e d g h o s t s (Bodemann and Hoffman, 1 9 7 6 a ) . I n c o n t r a s t , t h e ouabain-binding r a t e i s i n c r e a s e d by i n c r e a s i n g N a i when K i i s h i g h ( J o i n e r and Lauf, 1 9 7 8 ) . I n b o t h i n s t a n c e s , i t c o u l d be shown t h a t t h e pump r a t e i s s t i m u l a t e d by i n c r e a s i n g N a i and t h a t t h e half-maximal c o n c e n t r a t i o n of N a i f o r a c t i v a t i o n i n c r e a s e s as K i i s i n c r e a s e d . These r e s u l t s imply t h a t t h e r e must be some v a l u e of K i where t h e ouabain-binding r a t e would be cons t a n t and i n d e p e n d e n t of v a r i a t i o n s i n N a i . Thus w e found t h a t t h e ouabain-binding r a t e i s u n a f f e c t e d by v a r y i n g N a i when K i i s a b o u t 45 m M / l i t e r c e l l s . A l t e r n a t i v e l y , when N a i w a s a p p r o x i m a t e l y 35-40 m M / l i t e r c e l l s , t h e ouabain-binding r a t e w a s u n a f f e c t e d by varying K i .

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With a s i m i l a r e x p e r i m e n t a l d e s i g n , t h e same t y p e of i n t e r a c t i o n between N a i and Ki was s t u d i e d on o r t h o phosphate-promoted ouabain b i n d i n g t o r e c o n s t i t u t e d g h o s t s . W e found t h a t t h e ouabain-binding r a t e was una f f e c t e d by v a r y i n g N a i a t h i g h ( 1 1 0 mM) K i , o r by v a r y i n g K i when Nai was low (6 m M ) I t would a p p e a r from t h e s e r e s u l t s t h a t t h e r e a r e a t l e a s t two t y p e s of b i n d i n g s i t e s f o r Na and K on t h e i n n e r f a c e of t h e pump which i n t e r a c t t o c o n t r o l t h e a c c e s s i b i l i t y of t h e ouabain-binding s i t e a t t h e o u t e r a s p e c t of t h e pump.

.

ACKNOWLEDGMENT

This work w a s supported by N I H , USPHS Grants HL09906 and AM05644, and by t h e Deutsche Forschungsgemeinschaft.

REFERENCES

Bodmann, H. H . ,

and Hoffman, J. F.

(1976a).

Side-dependent e f -

fects of i n t e r n a l v e r s u s e x t e r n a l N a and K on ouabain bindi n g to r e c o n s t i t u t e d human r e d blood c e l l g h o s t s . J . Gen. P h y s i o l . 67, 497-525. Bodemann, H. H . , and Hoffman, J. F . (197633). E f f e c t s of Mg and C a on t h e s i d e dependencies o f N a and K on ouabain b i n d i n g to r e d blood c e l l g h o s t s and t h e c o n t r o l of N a t r a n s p o r t by i n t e r n a l Mg. J. Gen. P h y s i o l . 67, 547-561. C a v i e r e s , J. D . , and E l l o r y , J. C. (1975). A l l o s t e r i c i n h i b i t i o n of t h e sodium pump by e x t e r n a l sodium. Nature (London) 255, 338-340. Gardner, J. D . , and F r a n t z , C . (1974). E f f e c t s o f c a t i o n s on J . Membr. ouabain b i n d i n g by i n t a c t human e r y t h r o c y t e s . B i o l . 1 6 , 43-64. Hobbs, A. S., and Dunham, P. B. (1976). Evidence f o r two sodium s i t e s on t h e e x t e r n a l a s p e c t o f Na-K pump i n human e r y t h r o c y t e s . Nature (London) 260, 651-652. Hobbs, A. S., and Dunham, P. B. (1978). I n t e r a c t i o n of e x t e r n a l a l k a l i metal i o n s w i t h t h e Na-K pump of human e r y t h r o c y t e s . J . Gen. P h y s i o l . 72, 381-402. Hoffman, J. F. (1969). The i n t e r a c t i o n between t r i t i a t e d ouabain and t h e Na-K pump i n r e d blood c e l l s . J. Gen. P h y s i o l . 5 4 , 343s-350s. J o i n e r , C. H . , and Lauf, I?. K. (1978). Modulation of ouabain b i n d i n g and potassium pump f l u x e s by c e l l u l a r sodium and

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potassium i n human and sheep e r y t h r o c y t e s . J. P h y s i o l . (London) 283, 177-196. Sachs, J. R. (1974). I n t e r a c t i o n of e x t e r n a l K , Na, and cardioa c t i v e s t e r o i d s with t h e Na-K pump of t h e human r e d blood c e l l . J. Gen. P h y s i o l . 63, 123-143.

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CURReNT TOPICS IN MEMBRANES AND TRANSPORT. VOLUME 19

lntracellular Sodium Enhancementof Ouabain Binding to Na,K-ATPase and the Development of Glycoside TAI AKERA, KYOSUKE TEMMA, AND SATOSHI YAMAMOTO’ Depament of Pharmacology and Toxicology Michigan State University East Lansing, Michigan

I.

INTRODUCTION

The b i n d i n g of c a r d i a c g l y c o s i d e s t o Na,K-ATPase, observed i n v i t r o i n t h e p r e s e n c e of Mg2+ and ATP, i s enhanced by N a + (Matsui.and Schwartz, 1 9 6 8 ) . S i n c e Na+ a c t i v a t e s t h e enzyme a t t h e c y t o p l a s m i c s i d e of t h e c e l l membrane, it seems t o be i n t r a c e l l u l a r N a + ( N a + i ) t h a t s t i m u l a t e s t h e g l y c o s i d e b i n d i n g . Bodemann and Hoffman ( 1 9 7 6 1 , however, r e p o r t e d t h a t Na+i f a i l s t o s t i m u l a t e ouabain b i n d i n g i n r e s e a l e d e r y t h r o c y t e g h o s t s under c e r t a i n ionic conditions. I n myocardium, t h e p o s i t i v e i n o t r o p i c and t o x i c a c t i o n s of t h e g l y c o s i d e s develop f a s t e r when t h e muscle i s e l e c t r i c a l l y s t i m u l a t e d a t higher frequencies. Since s t i m u l a t i o n , and r e s u l t i n g membrane d e p o l a r i z a t i o n , i n c r e a s e s t h e amount of Na+i a v a i l a b l e t o Na,K-ATPase o r t h e sodium pump, t h e above f i n d i n g f a v o r s t h e c o n c e p t t h a t Na+i s t i m u l a t e s g l y c o s i d e b i n d i n g . Clausen and Hansen ( 1 9 7 7 ) r e p o r t e d t h a t glycoside-induced sodium pump i n h i b i t i o n i n s k e l e t a l muscle o r a d i p o c y t e s i s aug‘ P r e s e n t a d d r e s s : Department of Pharmacology, Keio L h i v e r s i t y School of M e d i c i n e , T o k y o , J a p a n . 235

Copynght 0 1983 by Academic Press. Inc All nghts of reproduction in any form ISBN 0-12-153319-0

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mented under t h e c o n d i t i o n s which enhance enzyme t u r n over. Since N a + i i s a f a c t o r stimulating t h e turnover, t h e s e f i n d i n g s a l s o s u p p o r t t h e above c o n c e p t . I f N a + i regulates t h e glycoside binding i n i n t a c t c e l l s , p o t e n c i e s of t h e g l y c o s i d e s which i n h i b i t i s o lated Na,K-ATPase, observed i n t h e presence of a r b i t r a r y N a + and K+ c o n c e n t r a t i o n s , c a n n o t be compared w i t h t h o s e which produce i n o t r o p i c e f f e c t s i n i n t a c t c a r d i a c c e l l s i n an a t t e m p t t o examine t h e r e l a t i o n s h i p between t h e s e two e v e n t s . S i n c e r e l e v a n t (asymmetric) l i g a n d c o n d i t i o n s c a n n o t be reproduced i n i s o l a t e d enzyme s t u d i e s , i n h i b i t o r y and i n o t r o p i c a c t i o n s might o n l y be compared i n i n t a c t c e l l s . T h e r e f o r e it i s i m p o r t a n t t o u n d e r s t a n d t h e e f f e c t s of N a + i on sodium pump a c t i v i t y ( t u r n o v e r o f Na,K-ATPase i n i n t a c t c e l l s ) , g l y c o s i d e s e n s i t i v i t y of t h e sodium pump, and t h e g l y c o s i d e b i n d i n g t o Na,K-ATPase i n b e a t i n g m y o c a r d i a l c e l l s . The p u r p o s e of t h i s a r t i c l e i s t o examine t h e abovementioned e f f e c t s o f N a + i and t o r e e v a l u a t e t h e o u a b a i n s e n s i t i v e 86Rb+- o r 42K+-uptake as a means o f e s t i m a t i n g sodium pump a c t i v i t y .

11.

RESULTS AND DISCUSSION

A r e d u c t i o n o r a u m e n t a t i o n o f t h e ouabains e n s i t i v e ( s p e c i f i c ) 8iRb+- o r 42K+-uptake h a s been f r e q u e n t l y i n t e r p r e t e d t o i n d i c a t e i n h i b i t i o n and s t i m u l a t i o n , r e s p e c t i v e l y , of t h e sodium pump. A d d i t i o n a l l y , c o n c e n t r a t i o n s of o u a b a i n which c a u s e a 5 0 % i n h i b i t i o n of t h e u p t a k e are used t o r e p r e s e n t t h e g l y c o s i d e s e n s i t i v i t y of t h e sodium pump. The f o l l o w i n g c o n s i d e r a t i o n s however, s u g g e s t t h a t t h e s e might n o t b e t h e case. Under normal c o n d i t i o n s , t h e m y o c a r d i a l sodium pump seems t o have r e s e r v e c a p a c i t y because n e i t h e r a modera t e enhancement o f t h e sodium i n f l u x , caused by a n i n crease i n h e a r t r a t e o r by a g e n t s which i n c r e a s e sodium i n f l u x ( e . g . , monensin, v e r a t r i d i n e , o r g r a y a n o t o x i n ) , nor a p a r t i a l i n h i b i t i o n o f t h e sodium pump by i o n o t r o p i c c o n c e n t r a t i o n s of c a r d i a c g l y c o s i d e s markedly e l e v a t e s m y o c a r d i a l sodium. T h e r e f o r e , when t h e s p e c i f i c Rb+ or K+ u p t a k e i s e s t i m a t e d i n q u i e s c e n t p r e p a r a t i o n s w i t h o u t a Na+ p r e l o a d i n g , t h e o b s e r v e d u p t a k e r e p r e s e n t s ongoing sodium pump a c t i v i t y , which i s determined by, and e q u i v a l e n t t o , t h e r a t e o f sodium i n f l u x ( F i g . 1 ) . Thus, t h e Rb+ o r K+ u p t a k e may be a l t e r e d by changes i n t h e N a + i n f l u x . Moreover, i f t h e sodium pump h a s res e r v e c a p a c i t y , pump i n h i b i t i o n o f a d e g r e e less t h a n t h e r e s e r v e c a p a c i t y may n o t r e s u l t i n a r e d u c t i o n o f

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sodium pump

F i g . 1 . R e s e r v c c a p a c i t y of the s o d i u m pump and ouabain s e n s i t i v i t y of 86Rb+ u p t a k e .

t h e o b s e r v e d Rb+ o r K+ u p t a k e : t h e u p t a k e may be i n h i b i t e d o n l y when t h e reserve c a p a c i t y i s e x h a u s t e d . Thus, t h e s e n s i t i v i t y of t h e sodium pump t o an i n h i b i t o r may be u n d e r e s t i m a t e d ( F i g . 1 ) . The f o l l o w i n g o b s e r v a t i o n s s u p p o r t t h e above concept: (1) N a + l o a d i n g o f l e f t a t r i a l muscle p r e p a r a t i o n s o f g u i n e a - p i g h e a r t ( e l e v a t e d N a + i due t o a 30-min i n c u b a t i o n a t O°C i n a K+- and Ca2+-free medium) enhanced t h e s p e c i f i c Rb+ u p t a k e o b s e r v e d w i t h a 7-min i n c u b a t i o n a t 37'C. A f t e r a 35-min i n c u b a t i o n , however, t h e u p t a k e was n o t s i g n i f i c a n t l y a f f e c t e d by t h e N a + loading, indicating t h a t t h e e x t r a N a + i is a v a i l a b l e t o t h e pump o n l y d u r i n g t h e f i r s t s e v e r a l m i n u t e s . ( 2 ) a - D i h y d r o g r a y a n o t o x i n (0.5-5 L I M ) , monensin (1-10 L I M ) , o r 1.5-3 Hz e l e c t r i c a l s t i m u l a t i o n enhanced t h e s p e c i f i c Rb+ u p t a k e i n p r e p a r a t i o n s which were n o t N a + l o a d e d . ( 3 ) The s t i m u l a t e d p o r t i o n w a s i n h i b i t e d by h i g h concent r a t i o n s ( 1 0 - 1 0 0 U M ) o f v e r a p a m i l , which i s known t o i n h i b i t Na+ influx. ( 4 ) R e l a t i v e l y h i g h s p e c i f i c Rb+ upt a k e of e l e c t r i c a l l y s t i m u l a t e d p r e p a r a t i o n s was n o t a f -

238

TAI AKERA eta/.

f e c t e d by monensin a t 2 . 5 U M and w a s r a t h e r i n h i b i t e d a t 1 0 VM. ( 5 ) Monensin o r g r a y a n o t o x i n d i d n o t a f f e c t Na,K-ATPase a c t i v i t y i n v i t r o , i n d i c a t i n g t h a t t h e obs e r v e d e f f e c t s o f t h e s e a g e n t s are n o t due t o a d i r e c t s t i m u l a t i o n of t h e sodium pump. (6) Concentrations of ouabain r e q u i r e d t o cause a 50% i n h i b i t i o n of s p e c i f i c Rb+ u p t a k e were markedly lowered by 2 . 5 V M monensin o r 1.5-3 Hz e l e c t r i c a l s t i m u l a t i o n . E l e c t r i c a l s t i m u l a t i o n a l s o i n c r e a s e d o u a b a i n F s e n s i t i v i t y of t h e s p e c i f i c 42K+ uptake. Exposure o f q u i e s c e n t l e f t a t r i a l muscle p r e p a r a t i o n s o f g u i n e a p i g h e a r t t o 0 . 5 V M o u a b a i n f o r 60 min a t 3 O o C r e s u l t e d i n a 1 3 . 4 % occupancy o f t h e g l y c o s i d e Monensin ( 1 0 v M ) enhanced b i n d i n g s i t e s on Na,K-ATPase. t h e o u a b a i n b i n d i n g , r e s u l t i n g i n a 34.3% occupancy. E l e c t r i c a l s t i m u l a t i o n a t 2 Hz caused a f a s t e r and g r e a t e r o u a b a i n b i n d i n g t o Na,K-ATPase t h a n t h a t obs e r v e d w i t h 0.5-Hz s t i m u l a t i o n . Concomitantly, t h e pos i t i v e i n o t r o p i c e f f e c t o f o u a b a i n developed more r a p i d l y when p r e p a r a t i o n s w e r e s t i m u l a t e d a t , h i g h e r f r e q u e n c i e s , o r i n t h e p r e s e n c e of e i t h e r g r a y a n o t o x i n o r monensin. Monensin ( 1 0 U M ) enhanced t h e development o f c o n t r a c t u r e due t o 5 P M o u a b a i n i n q u i e s c e n t muscle, i n d i c a t i n g t h a t t h e development o f t h e t o x i c e f f e c t of o u a b a i n i s a l s o enhanced by Na'i.

111.

SUMMARY

These r e s u l t s i n d i c a t e t h a t Na'i available t o the sodium pump i s t h e d e t e r m i n a n t of t h e s p e c i f i c 86Rb+o r 42K+-uptake i n c a r d i a c muscle p r e p a r a t i o n s which a r e not preloaded with Na+. The s p e c i f i c u p t a k e , t h e r e f o r e , may r e p r e s e n t t h e r a t e of N a + i n f l u x which i s e q u i v a l e n t t o N a + e f f l u x i n s t e a d y - s t a t e p r e p a r a t i o n s . The condit i o n s which i n c r e a s e N a + i a v a i l a b l e t o t h e sodium pump a f f e c t t h e c o n c e n t r a t i o n of o u a b a i n r e q u i r e d t o i n h i b i t t h e s p e c i f i c Rb+- or K+-uptake by enhancing t h e g l y c o s i d e b i v d i n g and a l s o by r e d u c i n g t h e r e s e r v e c a p a c i t y . In Na,-loaded p r e p a r a t i o n s , ouabain s e n s i t i v i t y i s probably overestimated. I n t r a c e l l u l a r N a + enhances Na,K-ATPase i n h i b i t i o n by o u a b a i n and i t s i n o t r o p i c and t o x i c actions.

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ACKNOWLEDGMENTS

T h i s work w a s s u p p o r t e d by a USPHS g r a n t , HL 16052 from t h e N a t i o n a l H e a r t , Lung, and Blood I n s t i t u t e .

REFERENCES

BOdemann, H. H . , and Hoffman, J. F. ( 1 9 7 6 ) . Comparison of t h e side-dependent e f f e c t s o f N a and K on o r t h o p h o s p h a t e - , UTP-, a n d ATP-promoted o u a b a i n b i n d i n g t o r e c o n s t i t u t e d human r e d blood c e l l g h o s t s . J. Gen. P h y s i o l . 6 7 , 527-545. Clausen, T . , and Hansen, 0. (1977). A c t i v e Na-K t r a n s p o r t and t h e r a t e o f o u a b a i n b i n d i n g . The e f f e c t o f i n s u l i n and o t h e r s t i m u l i on s k e l e t a l muscle and a d i p o c y t e s . J. P h y s i o l . (London) 2 7 0 , 415-430. Matsui, H., and Schwartz, A. ( 1 9 6 8 ) . Mechanism of c a r d i a c g l y c o s i d e i n h i b i t i o n of t h e (Na+-K+)-dependent A T P a s e from c a r d i a c t i s s u e . B i o c h i m . B i o p h y s . A c t a ( A m s t e r d a m ) 1 5 1 , 655-663.

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CURRENT TOPICS IN MEMBRANES AND TRANSPORT. VOLUME 19

Lithium-Catalyzed Ouabain Binding to Canine Kidney Na,K-ATPase GEORGE R. HENDERSON Department of Pharmacology Medical College of Ohio Toledo, Ohio

I.

INTRODUCTION

The i n t e r a c t i o n s o f m e t a l i o n s w i t h Na,K-ATPase are e x t r e m e l y complex, i n v o l v i n g t h e r e g u l a t i o n o f v a r i o u s c o n f o r m a t i o n a l t r a n s i t i o n s a s w e l l a s enzymes u b s t r a t e complex f o r m a t i o n . S i n c e t h e d i v a l e n t c a t i o n Mg2+ i s e s s e n t i a l f o r c a t a l y t i c a c t i v i t y and a l s o modul a t e s t h e s p e c i f i c i n h i b i t i o n o f t h e enzyme by cardiac g l y c o s i d e s , it seems p o s s i b l e t h a t t h e r e may be m u l t i p l e r e g u l a t o r y s i t e s f o r Mg2+ on t h e enzyme. With t h e poss i b i l i t y t h a t L i + may be c a p a b l e of s u b s t i t u t i n g f o r Mg2+, as i n d i c a t e d by p h y s i c o c h e m i c a l p r o p e r t i e s , s t u d i e s w e r e u n d e r t a k e n t o compare t h e a b i l i t y o f t h e s e two c a t i o n s t o modulate o u a b a i n i n t e r a c t i o n s w i t h t h e enzyme

.

24 1

Copyright 0 1983 by Academic Press, Inc. All rights of reproductionin any form reserved. ISBN 0-12-1533190

GEORGE R. HENDERSON

242

- LOG IOUABAIN], M F i g . 1 . Concentration d e p e n d e n c e o f o u a b a i n b i n d i n g t o Na K-ATPase U s i n g a p u r i f i e d p r e p a r a t i o n of Na K-ATPase f r o m canine renal m e d u l l a ( L a n e e t a l . , 1 9 7 3 ) o u a b a i n b i n d i n g was d e t e r m i n e d b y a m o d i f i c a t i o n of the m e t h o d o f M a t s u i and S c h w a r t z ( 1 9 6 8 ) . Incubations were c a r r i e d o u t f o r 2 hr a t 37OC w i t h 50 v g / m l o f e n z y m e i n 20 mM T r i s - H C 1 , pH 7 . 4 , a t the d e s i g n a t e d ouabain concentrations i n the p r e s e n c e of 20 mM L i C l and 2 0 mM EDTA (-); 2 mM MgC12 (-); or 2 mM MgC12 and 2 mM Pi

.

(

m-

11.

rn). RESULTS AND DISCUSSION

Using a p u r i f i e d p r e p a r a t i o n o f Na,K-ATPase from c a n i n e r e n a l m e d u l l a , L i + w a s found t o be c a p a b l e of s u p p o r t i n g t h e i n t e r a c t i o n o f o u a b a i n w i t h t h e enzyme. T h i s r e p r e s e n t s a p r o p e r t y of L i + t o t a l l y d i f f e r e n t from e i t h e r N a + o r K+ s i n c e t h e s e monovalent c a t i o n s a l o n e are i n e f f e c t i v e i n sup o r t i n g o u a b a i n b i n d i n g . I n t h e p r e s e n c e o f e i t h e r Mg5+ o r L i + , t h e amount o f o u a b a i n bound t o t h e enzyme w a s dependent on t h e concen t r a t i o n o f o u a b a i n a s d e p i c t e d i n F i g . 1. A t h i g h e r o u a b a i n c o n c e n t r a t i o n s , t h e b i n d i n maximum f o r L i + approached t h a t s e e n f o r Mg2+ and Mgj+ + Pi. However, t h e a p p a r e n t a f f i n i t y o f t h e Li+-enzyme complex f o r o u a b a i n w a s c o n s i d e r a b l y less t h a n t h e a f f i n i t y of ouab a i n f o r t h e MgZ+-associated enzyme.

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Fig. 2 . L i t h i u m a c t i v a t i o n c u r v e for o u a b a i n b i n d i n g t o Na,K-ATPase. B i n d i n g was c a r r i e d o u t for 2 hr a t the d e s i g n a t e d l i t h i u m c o n c e n t r a t i o n s i n the p r e s e n c e of 20 mM EDTA, 20 mM T r i s H C I , pH 7 . 4 , a n d 10-6 M o u a b a i n .

The L i + a c t i v a t i o n c u r v e f o r o u a b a i n b i n d i n g i s shown i n F i g . 2. The b i n d i n g was dependent on t h e L i + c o n c e n t r a t i o n and w a s a s a t u r a b l e p r o c e s s . The appar e n t Km € o r L i + a c t i v a t i o n w a s a p p r o x i m a t e l y 8 m M . The Li+-dependent o u a b a i n b i n d i n g was a l s o c a r r i e d o u t i n t h e p r e s e n c e of EDTA ( 2 0 m M ) , e l i m i n a t i n g t h e p o s s i b i l i t y o f any c o n t a m i n a t i o n by Mg2+. I t s h o u l d be n o t e d t h a t a t a s i m i l a r EDTA c o n c e n t r a t i o n , o u a b a i n b i n d i n g s t i m u l a t e d by 2 mM Mg2+ was c o m p l e t e l y b l o c k e d by t h e c h e l a t o r . These s t u d i e s p o i n t t o a d i r e c t e f f e c t o f L i + on t h e enzyme, w i t h a c a p a c i t y t o s t i m u l a t e o u a b a i n b i n d i n g i n d e p e n d e n t l y of Mg2+. A f i n a l area o f c o n s i d e r a t i o n i n v o l v e d t h e e f f e c t of L i + on Mg2+-dependent o u a b a i n b i n d i n g ( F i g . 3 ) . Ouabain b i n d i n g w a s s t u d i e d as a f u n c t i o n o f t h e Mg2+ c o n c e n t r a t i o n w i t h i n c r e a s i n g c o n c e n t r a t i o n s of L i + added t o t h e b i n d i n g r e a c t i o n . L i + w a s found t o i n h i b i t t h e MgZ+-dependent b i n d i n g i n a dose-dependent manner. However, no c o n c e n t r a t i o n o f L i + w a s found t h a t e n t i r e l y i n h i b i t e d a l l of t h e Mg2+-dependent bindi n g , a f i n d i n g s i m i l a r t o t h e r e s u l t s of Krishnan and A l b e r s ( 1 9 8 0 ) . The k i n e t i c s o f t h e L i + - i n h i b i t i o n ,

GEORGE R. HENDERSON

244

. 0

Oa6

t

E

n Z

3

0

m

5 a m U

3

0

500

F i g . 3 . E f f e c t o f l i t h i u m on magnesium-dependent o u a b a i n b i n d i n g t o Na,K-ATPase. B i n d i n g was c a r r i e d o u t f o r 2 hr a t the d e s i g n a t e d magnesium c o n c e n t r a t i o n s w i t h 10-7 M o u a b a i n i n the absence @ ( )and i n the p r e s e n c e of 5 mM ( O w ) , 10 mM -.f D ) , and 20 mM (00) L i C l .

w h i l e q u i t e complex, had some c h a r a c t e r i s t i c s of a comp e t i t i v e e f f e c t f o r Mg2+. The d a t a summarized and p r e s e n t e d i n t h i s a r t i c l e are c o n s i s t e n t w i t h t h e i n t e r p r e t a t i o n t h a t L i + p a r t i c i p a t e s i n a s p e c i f i c Mg2+-dependent r e a c t i o n o f Na,K-ATPase. Although t h e complex n a t u r e of L i + and i t s e f f e c t s on t h e enzyme i n t h e p a s t have been i n t e r p r e t e d a s N a + - o r K+-like (Robinson, 1975; BeaugB, 1978; Swann and A l b e r s , 1 9 7 9 ) , o u r d a t a c a n n o t be exp l a i n e d by a t t r i b u t i n g s u c h a h y b r i d n a t u r e t o L i + . The i n t e r p r e t a t i o n of l i t h i u m ' s e f f e c t s on o u a b a i n b i n d i n g can b e s t be e n v i s i o n e d i n terms of a p a r t i a l a g o n i s t f o r Mg2+. I n t h e p r e s e n c e o f Mg2+, L i + b i n d s t o t h e enzyme and d i s p l a c e s some of t h e d i v a l e n t c a t i o n , t h u s r e d u c i n g t h e amount o f E-Mg2+, t h e h i g h a f f i n i t y form f o r o u a b a i n . However, i n t h e absence of Mg2+, E - L i + i s more r e a d i l y formed and a l s o h a s a c a p a c i t y t o b i n d g l y c o s i d e b u t w i t h a lower a f f i n i t y . These s t u d i e s s u g g e s t t h a t i n t e r m s o f enzyme-ouabain i n t e r a c t i o n , E - L i + r e s e m b l e s E-Mg2+ t o a g r e a t e r d e g r e e t h a n E-Na+ o r E-K+.

LITHIUM-DEPENDENTOUABAIN BINDING

245

ACKNOWLEDGMENTS

T h i s s t u d y h a s b e e n s u p p o r t e d by a g r a n t from t h e American Heart A s s o c i a t i o n , N o r t h w e s t e r n Ohio C h a p t e r , I n c . , and by a B i o medical R e s e a r c h S u p p o r t G r a n t from t h e Medical C o l l e g e of Ohio 5 SO7 RR 05700 10.

REFERENCES

Beaug6, L.

(1978).

A c t i v a t i o n by l i t h i u m i o n s o f the i n s i d e sodium B i o c h i m . B i o p h y s . A c t a 5 2 7 , 472-

s i t e s i n Na+,K+-ATPase.

484. K r i s h n a n , N . , and A l b e r s , R. W. ( 1 9 8 0 ) . M o d i f i c a t i o n o f t h e r a t e o f o u a b a i n b i n d i n g t o Na+,K+-ATPase by l i t h i u m i o n s . J. Neurochem. 3 5 , 753-755. Lane, L. K., Copenhaver, J. H . , Lindenmayer, G. E., and S c h w a r t z , A. ( 1 9 7 3 ) . P u r i f i c a t i o n and c h a r a c t e r i z a t i o n of and [3H]ouabain b i n d i n g t o t h e t r a n s p o r t a d e n o s i n e t r i p h o s p h a t a s e J. B i o l . Chern. 2 1 8 , from o u t e r m e d u l l a of c a n i n e k i d n e y . 7197-7200. M a t s u i , H . , and S c h w a r t z , A, ( 1 9 6 8 ) . Mechanism o f c a r d i a c g l y c o s i d e i n h i b i t i o n o f t h e Na+,K+-dependent ATPase from c a r d i a c t i s s u e . B i o c h i r n . B i o p h y s . A c t a 1 5 1 , 655-663. Robinson, J. D. ( 1 9 7 5 ) . Mechanisms by which L i + s t i m u l a t e s t h e Na+,K+-dependent ATPase. B i o c h i m . B i o p h y s . A c t a 4 1 3 , 459471. Swann, A. C., and A l b e r s , R . W. ( 1 9 7 9 ) . Na+,K+-adenosine t r i p h o s p h a t a s e o f mammalian b r a i n c a t a l y t i c and r e g u l a t o r y K+ s i t e s d i s t i n g u i s h a b l e by s e l e c t i v i t y f o r L i + . J. B i o l . Chern. 2 5 4 , 4540-4544.

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CURRENT TOPICSIN MEMBRANES AND TRANSPORT, VOLUME 19

Ouabain Binding and Na,K-ATPase in Released Human Red Cell Ghosts D.G. SHOEMAKER' AND P . K. LAUF Depanment of Physiology Duke University Medical Center Durham, North Carolina

I.

INTRODUCTION

The s t u d y of t h e i n t e r a c t i o n of c a r d i a c g l y c o s i d e s w i t h N a , K - A T P a s e h a s proved t o be a n i n d i s p e n s a b l e t o o l i n d e f i n i n g and c h a r a c t e r i z i n g numerous p r o p e r t i e s of t h e N a , K - A T P a s e t r a n s p o r t complex. W e have e x t e n d e d o u r p r e v i o u s i n v e s t i g a t i o n s on t h e s u p p o r t of [3H]ouab a i n b i n d i n g by a number of l i g a n d s a c t i n g a t t h e i n t e r n a l face of t h e membrane of a r e s e a l e d e r y t h r o c y t e g h o s t preparation. Resealed g h o s t s a r e p a r t i c u l a r l y s u i t a b l e e x p e r i m e n t a l models a s one i s a b l e t o p r e c i s e l y c o n t r o l t h e c o n c e n t r a t i o n of d e s i r e d l i g a n d s on b o t h s i d e s o f t h e membrane.

'Present address: Department of Physiology, Yale l h i v e r s i t y School of V e d i c i n e , New Haven, Connecticut. 247

Copyright 0 1983 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-1533190

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11.

METHODS AND DISCUSSION

Resealed g h o s t s w e r e p r e p a r e d by a m o d i f i c a t i o n o f t h e method of Bodemann and Passow ( 1 9 7 2 ) t o c o n t a i n a h i g h i n t r a c e l l u l a r sodium c o n c e n t r a t i o n (150-165 mM) and v a r i o u s c o n c e n t r a t i o n s of N a / K pump l i g a n d s . [3H]Ouabain b i n d i n g a s s a y s were performed i n a h i g h sodium medium (165 mM) w i t h t h e c o n c e n t r a t i o n of l a b e l e d c a r d i a c g l y c o s i d e r a n g i n g from 10-7 t o 10-8 M. The b i n d i n g p r o c e s s was found t o be e s s e n t i a l l y i r r e v e r s i b l e o v e r t h e 3-hr t i m e c o u r s e examined, as h a s been p r e v i o u s l y r e p o r t e d f o r i n t a c t human r e d c e l l s (Hoffman, 1966; 1969; S a c h s , 1974; J o i n e r and Lauf , 1978a) By e x p o s i n g r e s e a l e d g h o s t s t o [3H]ouabain f o r v a r i ous t i m e p e r i o d s , p r e p a r i n g h y p o t o n i c a l l y l y s e d membranes from t h e s e t r e a t e d g h o s t s , and a s s a y i n g t h e f r a c t i o n a l i n h i b i t i o n o f t h e Na,K-ATPase a c t i v i t y , t h e r e l a t i o n s h i p between t h e number o f o u a b a i n m o l e c u l e s bound p e r g h o s t and t h e f r a c t i o n a l i n h i b i t i o n of N a , K - A T P a s e w a s obt a i n e d . I t w a s found t h a t o n e molecule of [3H]ouabain w a s s u f f i c i e n t t o i n h i b i t one Na,K-ATPase complex. Support o f o u a b a i n b i n d i n g was examined a s a funct i o n o f t h e v a r i o u s N a / K pump l i g a n d s : Mg2+, ATP, ADP, and i n o r g a n i c p h o s p h a t e ( P i ) . The e x p e r i m e n t a l system w a s d e s i g n e d t o examine t h e N a + - , Mg2+-, and ATP-supp o r t e d enzyme-ouabain complex. The h i g h i n t r a c e l l u l a r sodium c o n c e n t r a t i o n employed h a s been shown p r e v i o u s l y t o p r e v e n t Mg2+- and P i - s u p p o r t e d o u a b a i n b i n d i n g (Skou e t al., 1 9 7 1 ) and p h o s p h o r y l a t i o n o f t h e N a , K - A T P a s e ( P o s t et a l . , 1 9 7 5 ) . I n t r a c e l l u l a r magnesium ( 4 m ~ o ) r magnesium p l u s i n o r g a n i c p h o s p h a t e (1 mM) produced t h e l o w e s t r a t e of o u a b a i n b i n d i n g . ADP (1 mM) s u p p o r t e d n e g l i g i b l e r a t e s of o u a b a i n b i n d i n g p r o v i d e d s y n t h e s i s o f ATP through t h e r e s i d u a l a d e n y l a t e k i n a s e a c t i v i t y w a s p r e v e n t e d by t h e a d e n y l a t e k i n a s e i n h i b i t o r , p l , p 5 , d i a d e n o s i n e p e n t a p h o s p h a t e (Ap5A). The n u c l e o t i d e bTP, shown p r e v i o u s l y t o s u p p o r t s i g n i f i c a n t i n i t i a l rates o f o u a b a i n b i n d i n g i n r e s e a l e d g h o s t s (Hoffman, 1969) and p u r i f i e d N a , K - A T P a s e (Tobin e t al., 1 9 7 2 1 , w a s a l s o examined and found t o be i n c a p a b l e o f s u p p o r t i n g o u a b a i n b i n d i n g p r o v i d e d t h a t s y n t h e s i s o f ATP t h r o u g h t h e nuc l e o s i d e d i p h o s p h o k i n a s e r e a c t i o n was i n h i b i t e d w i t h Trypan Blue (Kaplan and H o l l i s , 1980) and t h a t r e s i d u a l ATP s t o r e s w e r e d e p l e t e d by i n c o r p o r a t i o n o f h e x o k i n a s e and g l u c o s e i n t h e g h o s t s . By p r e p a r i n g more d i l u t e r e s e a l e d g h o s t s and employi n g t h e c r e a t i n e p h o s p h a t e - c r e a t i n e k i n a s e ATP-regenerati n g system, t h e r a t e o f o u a b a i n b i n d i n g w a s examined a t It v a r i o u s ATP c o n c e n t r a t i o n s i n t h e r a n g e 10-6-10-7 M .

.

OUABAIN BINDING AND Na.K-ATPase IN HUMAN RED CELL

249

w a s found t h a t 1 P M i n t r a c e l l u l a r ATP s u p p o r t e d maximal r a t e s of o u a b a i n b i n d i n g , i n d i c a t i n g t h a t i t i s t h e h i g h - a f f i n i t y ATP b i n d i n g s i t e o f t h e Na,K-ATPase which i s r e s p o n s i b l e f o r promoting f o r m a t i o n of t h e N a + - , Mg2+-, and ATP-supported enzyme-ouabain complex. I n o t h e r e x p e r i m e n t s , t h e n o n h y d r o l y z a b l e ATP a n a l o g , AMPPNP, f a i l e d t o e f f e c t s i g n i f i c a n t r a t e s o f [3H]ouabain b i n d i n g , d e s p i t e t h e f a c t t h a t t h i s compound i s known t o compete w i t h ATP a t i t s b i n d i n g s i t e s on t h e enzyme ( R o b i n s o n , 1976) and t o s u p p o r t K:K exchange i n r e s e a l e d g h o s t s by b i n d i n g t o t h e l o w - a f f i n i t y ATP b i n d i n g s i t e (Simons , 1 9 7 5 ) .

111.

CONCLUSION

We conclude from t h e s e s t u d i e s t h a t t h e o u a b a i n binding process i n resealed ghosts i s a process s p e c i f i c a l l y s u p p o r t e d by ATP i n t h e p r e s e n c e o f h i g h sodium c o n c e n t r a t i o n s and magnesium. F u r t h e r m o r e , t h e r e s u l t s i n d i c a t e t h a t ATP s u p p o r t s o u a b a i n b i n d i n g by phosphor y l a t i n g t h e enzyme a t t h e h i g h - a f f i n i t y ATP s i t e .

ACKNOWLEDGMENT

T h i s work w a s s u p p o r t e d i n p a r t by N I H g r a n t AM 28236/HEM.

REFERENCES

Bodemann, H. H . , and Hoffman, J F. ( 1 9 7 6 ) . Side-dependent e f f e c t s of i n t e r n a l v e r s u s e x t e r n a l N a and K on ouabain b i n d i n g t o r e c o n s t i t u t e d human r e d blood c e l l g h o s t s . J . Gen. P h y s i o l . 67, 497-525. Bodemann, H . , and Passow, H. ( 1 9 7 2 ) . F a c t o r s c o n t r o l l i n g t h e res e a l i n g o f t h e membrane o f human e r y t h r o c y t e g h o s t s a f t e r J . Membr. Biol. 8 , 1-26. h y p o t o n i c hemolysis. Hoffman, J. F. ( 1 9 6 9 ) . The i n t e r a c t i o n between t r i t i a t e d o u a b a i n and t h e Na-K pump i n r e d b l o o d c e l l s . J . Gen. P h y s i o l . 5 4 , 343s-350s. J o i n e r , C . H . , and Lauf, P. K. ( 1 9 7 8 a ) . The c o r r e l a t i o n between ouabain b i n d i n g and p o t a s s i u m pump i n h i b i t i o n i n human and J. P h y s i o l (London) 2 8 3 , 155-175. sheep erythrocytes.

.

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D. G. SHOEMAKER AND P. K. L A W

J o i n e r , C. H . , and Lauf, P. K. (197833). Modulation o f ouabain b i n d i n g and p o t a s s i u m pump f l u x e s by c e l l u l a r sodium and potassium i n human and sheep e r y t h r o c y t e s . J. P h y s i o l . (London) 283, 177-196. Kaplan, J. H . , and H o l l i s , R. J. (1980). E x t e r n a l N a dependence o f o u a b a i n - s e n s i t i v e ATP:ADP exchange i n i t i a t e d by photol y s i s o f i n t r a c e l l u l a r caged-ATP i n human r e d c e l l g h o s t s . N a t u r e 288, 587-589. P o s t , R. L . , Toda, G . , and Rogers, F. N . (1975). P h o s p h o r y l a t i o n by i n o r g a n i c phosphate o f sodium p l u s potassium i o n t r a n s p o r t a d e n o s i n e t r i p h o s p h a t e . J. B i o l . Chem. 250, 691-701. Robinson, J. D. ( 1 9 7 6 ) . S u b s t r a t e s i t e s o f t h e (Na' + K+)B i o c h i m . B i o p h y s . Acta 429, 1006-1019. dependent ATPase. S a c h s , J. R. ( 1 9 7 4 ) . I n t e r a c t i o n o f e x t e r n a l K, N a , and c a r d i o a c t i v e s t e r o i d s w i t h t h e Na-K pump o f t h e human r e d b l o o d c e l l . J. Gen. P h y s i o l . 6 3 , 123-143. Skou, J. C . , B u t l e r , K. W. , and Hansen, 0. ( 1 9 7 1 ) . The e f f e c t o f magnesium, ATP, P i , and sodium on t h e i n h i b i t i o n o f t h e ( N a + + K+) - a c t i v a t e d enzyme system by y - s t r o p h a n t h i n . B i o c h i m . B i o p h y s . A c t a 2 4 1 , 443-461. Tobin, T . , Baskin, S. I . , Akera, T . , and Brody, T. M. ( 1 9 7 2 ) . N u c l e o t i d e s p e c i f i c i t y o f t h e Na+-stimulated p h o s p h o r y l a t i o n and [3H]ouabain-binding r e a c t i o n s o f t h e ( N a + + K+)dependent a d e n o s i n e t r i p h o s p h a t e . Mol. P h a r m a c o l . 8, 256-263.

CURRENT TOPICS IN MEMBRANES AND TRANSPORT. VOLUME 19

Stereoelectronic Interaction between Cardiotonic Steroids and Na, K-ATPase: Molecular Mechanism of Digitalis Action F. DITTRICH, P. BERLJN, K. KOPKE, AND K. R. H. REPKE Biomembrane Section in the Central Institute of Molecular Biology Academy of Sciences of the German Democratic Republic Berlin, German Democratic Republic

I.

INTRODUCTION

The d i s c o v e r y o f c a r d i a c N a , K - A T P a s e

a s t h e molecu-

l a r p o i n t of a t t a c k of c a r d i o t o n i c s t e r o i d s ( r e v i e w e d by Repke and D i t t r i c h , 1 9 8 0 ) r e n d e r e d p o s s i b l e t h e det e r m i n a t i o n of e x a c t i n p u t d a t a f o r o u r e n d e a v o r t o dec i p h e r t h e c o d i f i c a t i o n of a c t i v i t y o f t h e d i v e r s e compounds i n p h y s i c a l terms (Repke e t a l . , 1 9 7 4 ) . T h i s should a l l o w us t o r a t i o n a l i z e t h e a c t i v i t y o f s t u d i e d r e p r e s e n t a t i v e s and t o d e s i g n new d e r i v a t i v e s o f d e s i r e d a c t i v i t y . Our s t r a t e g y f o r d e c i p h e r i n g t h i s c o d i f i c a t i o n o f a c t i v i t y stemmed from t h e knowledge t h a t N a , K A T P a s e i s s t r u c t u r e d from a r a t h e r h i g h c o n t e n t of a - h e l i c a l p e p t i d e c h a i n s ( B r a z h n i k o v et a l . , 1978) and t h a t t h e backbone of a p r o t e i n m o l e c u l e , t h e h e l i c e s i n p a r t i c u l a r , g e n e r a t e s an e x t e n d e d e l e c t r i c a l f i e l d i n t h e form o f a d i p o l e moment v e c t o r which i s w e l l f i t t e d f o r l o n g - r a n g e a t t r a c t i o n and o r i e n t a t i o n o f o p p o s i t e l y c h a r g e d e f f e c t o r m o l e c u l e s (Hol e t a l . , 1 9 7 8 ) . T h i s 251

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252

F. DITTRICHeta/.

Fig. 1. Correlation between observed and calculated standard Gibbs energies, AGGbs or ,bG,!&lc, found for the interaction between congeneric digitalis derivatives and Na ,K-ATPase from guinea-pig cardiac muscle e; The regression equation is is comAGgalc = 0.461pCleff - 6.73(n = 20, rxy = 0.968). puted for the low-energy conformations with C-14 to C-22 opposition ( 0 , A ) or C-14 to C-21 opposition (0). The studied derivatives differ with respect to 3-deoxy-digitoxigenin in showing the following substituents: (1) 38,16B-(ON02)2; (2) 3$-0H, 168-OCOCH3; (3) 38,16@:(0COCH3)2; (4) 3$-OCOCH3; (5) 38-OH; ( 6 ) 38,11a-(OH)2; (7) 38,12B-(OH)2; (8) 3a,12a-(OCOCH3)2; (9) 3a-OCOCH3, l l = o ; (10) 38-OCOCH3, 168-0H; (11) 38,16@-(0H)2; (12) 38,16a-(OCH3)2; (13) 38,12a-(OH)2; (14) 3$-0H, 16a-OCH3; (15) 38,128-(ON02)2; (16) 3@,118-(OH)2; (17) 3a-OH; (18) 38-0Hf 128-oCocH3; (19) 38,168-(OCOCH3)2, 17a-OH; (20) 38,128-(OCOCH3)2.

\$Ief

appeared t o us t o be a n e c e s s a r y t o o l f o r e f f e c t i v e b i n d i n g o f c a r d i o t o n i c s t e r o i d s , s i n c e t h e s t e r o i d molec u l e s o u t s i d e t h e e l e c t r i c a l i n t e r a c t i o n f o r c e s show random t r a n s l a t i o n a l and r o t a t i o n a l m o b i l i t i e s , and s i n c e t h e mouth o f t h e b i n d i n g - s i t e c l e f t may amount t o only a s m a l l percentage of t h e e x t e r n a l p r o t e i n surface. The emerging c o n c l u s i o n - - t h a t t h e geometry and d i p o l e v e c t o r of t h e b i n d i n g - s i t e c l e f t i n N a , K - A T P a s e p r o t e i n impose s t e r e o e l e c t r o n i c p r e f e r e n c e s o r even i m p e r a t i v e s upon i n t e r a c t i o n w i t h c a r d i o t o n i c s t e r o i d s - - i s shown t o be e s s e n t i a l l y t r u e i n t h e p r e s e n t c h a p t e r .

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STEREOELECTRONIC INTERACTIONS

The c a r d i o t o n i c s t e r o i d s d i s p l a y a d i p o l e moment v e c t o r t h e q u a n t i t y and d i r e c t i o n of which i s v e r y o f t e n h i g h l y s e n s i t i v e t o t h e n a t u r e , d i s p o s i t i o n , and c h i r a l i t y o f s u b s t i t u e n t s a s t h e s t e r o i d n u c l e u s and t h e b u t e n o l i d e r i n g as w e l l a s t o t h e geometry and c o n f o r m a t i o n o f t h e v a r i o u s d e r i v a t i v e s ( f o r computat i o n of t h e d i p o l e moment v e c t o r and c o n f o r m a t i o n a l f l e x i b i l i t y see Repke e t a ] . , 1 9 7 4 , and B e r l i n , 1977, r e s p e c t i v e l y ) . The s t e r e o e l e c t r o n i c d e s c r i p t o r i n t e g r a t e s t h e i n f o r m a t i o n c o d i f i e d i n m o l e c u l a r composit i o n , c o n s t i t u t i o n , c o n f i g u r a t i o n , c o n f o r m a t i o n and e l e c t r o n d i s t r i b u t i o n and i s t h u s q u a l i f i e d f o r key functionality. As shown i n F i g . 1, t h e v a r i a b i l i t y of t h e e f f e c t i v e d i p o l e moment v e c t o r i n t h e v a r i o u s c a r d i a c steroids significantly contributes t o t h e substituentd e p e n d e n t v a r i a b i l i t y of t h e m o l e c u l a r a c t i v i t y i n d e x o f t h e v a r i o u s compounds, i . e . , t h e s t a n d a r d Gibbs e n e r g y ( A G O ) o f t h e i r i n t e r a c t i o n w i t h Na,K-ATPase ( f o r c a l c u l a t i o n , c f . Repke and D i t t r i c h , 1 9 8 0 ) . A s demons t r a t e d i n t h a t paper, t h e A G O value i s l i n e a r l y corr e l a t e d w i t h t h e v a l u e of Gibbs a c t i v a t i o n e n e r g y , and t h u s i n c l u d e s information a l s o about i n t e r a c t i o n kinetics. The A G O v a l u e i s d e t e r m i n e d by t h e a t t r a c t i o n and o r i e n t a t i o n f o r c e which d e r i v e s from i n t e r a c t i n g e l e c t r i c a l f i e l d s . They a r i s e from b o t h t h e l i n e d i p o l e of a - h e l i c e s i n t h e r e c e p t i v e enzyme s t a t e (unl o c k e d b i n d i n g - s i t e c l e f t ) and from t h e i n t e g r a t e d permanent d i p o l e s of c a r d i o t o n i c s t e r o i d s d i f f e r i n g i n t y p e , p o s i t i o n , and c h i r a l i t y o f s u b s t i t u e n t s . S i n c e t h e amount and d i r e c t i o n o f t h e enzyme d i p o l e moment can b e presumed t o be i n v a r i a n t i n t h e rec e p t i v e s t a t e , t h e a t t r a c t i o n and o r i e n t a t i o n f o r c e i s s o l e l y d e p e n d e n t on t h e dipole-moment component o f a g i v e n d i g i t a l i s compound, which d e t e r m i n e s t h e e n t r a n c e i n t o and t h e b i n d i n g t o one l o b e of b i n d i n g - s i t e c l e f t . w a s found by means T h i s e f f e c t i v e component, of m u l t i p l e l i n e a r r e g r e s s i o n a n a l y s i s t o correspond w i t h t h e e l e c t r i c a l f i e l d which r u n s p a r a l l e l t o a l i n e between C-6 and C-9 and e x t e n d s i n t h a t d i r e c t i o n o v e r t h e whole d i g i t a l i s m o l e c u l e ( F i g . 2 ) . T h i s f i e l d encompasses t h e c a r b o n y l and b r i d g e oxygen i n t h e b u t e n o l i d e r i n g and t h u s a c c o u n t s f o r t h e v a r i a t i o n i n t h e act i v i t y of compounds which d i f f e r i n t h e p o s i t i o n o f t h e s e oxygen f u n c t i o n s r e l a t i v e t o t h e s t e r o i d n u c l e u s ( P o r t i u s and Repke, 1964; F u l l e r t o n e t a l . , 1 9 7 9 ) .

IClleff,

254

F. DITTRICH eta/.

F i g . 2 . S c h e m a t i c r e p r e s e n t a t i o n of the e l e c t r i c a l f i e l d of c a r d i o t o n i c s t e r o i d s ( a r r o w s ) w h i c h i n t e r a c t s w i t h the o p p o s i t e 1 y d i r e c t e d e l e c t r i c a l f i e l d o f Na,K-ATPase i n a r e c e p t i v e s t a t e , t h u s a t t r a c t i n g and o r i e n t i n g the d i g i t a l i s m o l e c u l e t o the b i n d i n g - s i t e c l e f t . The b u t e n o l i d e r i n g i s d e p i c t e d i n the l o w - e n e r g y conform a t i o n s w i t h C-14 t o C-22 o p p o s i t i o n ( s o l i d l i n e ) or w i t h C-14 t o C-21 o p p o s i t i o n (broken l i n e ) .

-+

F o r t h e c a l c u l a t i o n of I P C l e f f , it had t o be assumed t h a t t h e v a r i o u s d i g i t a l i s d e r i v a t i v e s e n t e r t h e b i n d i n g s i t e c l e f t i n t h e low-energy c o n f o r m a t i o n s w i t h e i t h e r C - 1 4 t o C-22 o p p o s i t i o n o r C-14 t o C-21 opposit i o n because o n l y t h i s c o n d i t i o n y i e l d s an e x c e l l e n t c o r r e l a t i o n between o b s e r v e d and computed A G O v a l u e s , as shown i n F i g . 1. A p p a r e n t l y , d u r i n g t h e r e c o g n i t i o n phase t h e b i n d i n g - s i t e c l e f t s o r t s o u t one o r t h e o t h e r conformation. The extreme s p e c i f i c i t y o f d i g i t a l i s a c t i o n c o d i f i e d i n t h e almost i n f l e x i b l e s t e r o i d n u c l e u s r e q u i r e s a c l o s e f i t t o t h e s t r u c t u r a l f e a t u r e s of t h e bindings i t e c l e f t , presumably l o c k e d by d i p o l e - d i p o l e i n t e r a c t i o n . From c l o s e p a c k i n g , i n t e r a t o m i c d i s p e r s i o n e n e r g i e s a r i s e which i n c r e a s e w i t h b o t h i n c r e a s i n g p o l a r i z a b i l i t y and i n c r e a s i n g number o f atoms i n v o l v e d . While t h e diverse substituents present i n the s t e r o i d s display l o c a l i n t e r a c t i o n e n e r g i e s which do n o t c o n t r i b u t e d i f f e r e n t l y t o t h e A G O v a l u e s , c e r t a i n s u b s t i t u e n t s a t carbon atoms i n r i n g s C, D , and E may do s o a s j u s t mentioned here. The d i p o l e - d i p o l e i n t e r a c t i o n mechanism as deduced a l s o a p p l i e s t o s t e r o i d s i n which t h e l a c t o n e r i n g , c h a r a c t e r i s t i c f o r d i g i t a l i s compounds, i s r e p l a c e d ‘by other structural features.

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CONCLUSIONS

The r e g r e s s i o n e q u a t i o n o f t h e t y p e shown i n F i g . 1 was found t o g e r m i t t h e e s t i m a t e of t h e e x p e c t e d a c t i v i t y i n d e x A G o f d e s i g n e d d e r i v a t i v e s p r i o r t o synt h e s i s . The c o r r e l a t i o n between t h e A G O v a l u e and t h e i n o t r o p i c e f f i c i e n c y ( c f . Repke and D i t t r i c h , 1 9 8 0 ) r e n d e r s p o s s i b l e t h e p r e d i c t i o n of t h e p h a r m a c o l o g i c a l a c t i v i t y . C o n s e q u e n t l y , t h e e x p l o i t a t i o n of t h e molecul a r i n t e r a c t i o n mechanism p r o m i s e s t o economize t h e not o r i o u s l y d i f f i c u l t synthesizing process.

REFERENCES

.

Konformationsberechnungen von E f f e k t o r e n d e r B e r l i n , P. (1977) (Na,K)-ATPase. E r g e b . E x p . Med. 24, 181-183. B r a z h n i k o v , E . V . , C h e t v e r i n , A . B . , and C h i r g a d z e , Yu.N. ( 1 9 7 8 ) . Secondary s t r u c t u r e o f Na', K+-dependent a d e n o s i n e t r i p h o s FEBS L e t t . 93, 125-128. phatase. F u l l e r t o n , D. S . , Yoshioka, K . , R o h r e r , D. C . , From, A. H. L . , Sideand Ahmed, K. ( 1 9 7 9 ) . D i g i t a l i s g e n i n a c t i v i t y : g r o u p c a r b o n y l oxygen p o s i t i o n i s a major d e t e r m i n a n t . S c i e n c e 205, 917-915. H o l , W. G . J . , van D u i j n e n , P. T . , and Berendsen, H . J . C. ( 1 9 7 8 ) . Nature The a - h e l i x dipole and t h e p r o p e r t i e s o f p r o t e i n s . ( L o n d o n ) 273, 443-446. P o r t i u s , H. J . , and Repke, K. ( 1 9 6 4 ) . Versuch e i n e r Analyse der Beziehungen s w i s c h e n chemischer S t r u k t u r und D i g i t a l i s s h n l i c h e r Wirksamkeit a u f d e r R e z e p t o r e b e n e . A r z n e i m . Forsch. 1 4 , 1073-1077. Repke, K. R. H . , and D i t t r i c h , F. ( 1 9 8 0 ) . Thermodynamics o f i n f o r m a t i o n t r a n s f e r from c a r d i o t o n i c s t e r o i d s t o r e c e p t o r T r e n d s Pharmacol. S c i . 1 , 398-402. t r a n s p o r t ATPase. Repke, K. R. H . , D i t t r i c h , F . , B e r l i n , P . , and P o r t i u s , H. J . ( 1 9 7 4 ) . On p h y s i c a l f o r c e s g o v e r n i n g cardiac g l y c o s i d e act i v i t y . A n n . N.Y. A c a d . S c i . 242, 737-739.

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CURRENT TOPICS IN MEMBRANES AND TRANSPORT. VOLUME 19

Use of Prophet and MMS-X Computer Graphics in the Study of the Cardiac Steroid Receptor Site of Na,K-ATPase DWIGHT S. FULLERTON, EITARO KITA TS UJI, AND TAMBOUE DEFFO School of Pharmacy Oregon State Universiw Corvallis, Oregon

DOUGLAS C. ROHRER Medical Foundation of Buffalo Buffalo, New York

KHALIL AHMED AND ARTHUR H. L. FROM Cardiovascular Section Department of Medicine Universiryof Minnesota and VeteransAdministrationMedical Center Minneapolis, Minnesota

I.

INTRODUCTION

Many o f t e n c o n f l i c t i n g models have been p r o p o s e d t o d e s c r i b e t h e b i o l o g i c a l r o l e s of d i g i t a l i s s t r u c t u r e and c o n f o r m a t i o n . Our s t u d i e s ( F u l l e r t o n e t al., 1979, 1980; Rohrer e t al., 1979; Rohrer and F u l l e r t o n , 1980; K . Ahmed and A. H . L. From, u n p u b l i s h e d d a t a , 1981; A. H. L. From and K . Ahmed, u n p u b l i s h e d d a t a , 1 9 8 1 ) beg i n w i t h x-ray c r y s t a l l o g r a p h y t o provide p r e c i s e atomic c o o r d i n a t e s f o r s u b s e q u e n t a n a l y s i s o f t h e N I H PROPHET and MMS-X computer s y s t e m s . The c o n f o r m a t i o n a l f l e x i b i l i t y of t h e m o l e c u l e s i s examined w i t h p o t e n t i a l e n e r gy c a l c u l a t i o n s f o r r o t a t i o n s of t h e bonds t o t h e C-17 s i d e g r o u p and t o t h e f i r s t s u g a r ( e . g . , d i g o x i n , F i g . 1A). The s t r u c t u r e s are s u p e r i m p o s e d , and d i s t a n c e s bet w e e n c o r r e s p o n d i n g atoms are c a l c u l a t e d ( F i g . 1 B ) t o p r o v i d e a d i r e c t measure of t h e g e o m e t r i c d i f f e r e n c e s . 'Present address:Department of Industrial Chemistry, Toyama Technical College, Toyama, Japan. 257

Copynght 0 1983 by Academic Ress, Inc All rights of reproduction in any form reserved ISBN 0-12-1533194

258

DWIGHT S. FULLERTON

I

Fig. 1. ( A ) C-17 s i d e g r o u p and f i r s t s u g a r o f d i g o x i n Arrows show c o n f o r m a t i o n a l c h a n g e s under s t u d y . ( B ) A , B , and r i n g s of g i t o x i n ( G o and K a r t h a , 1980b) and o f d i g o x i n ( G o and K a r t h a , 1 9 8 0 a ) s u p e r i m p o s e d ( t o p and s i d e v i e w s ) . C a r b o n y l o x y gen d i s t a n c e i s shown.

COMPUTER GRAPHICS IN THE STUDY OF Na,K-ATPase

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GENINS

W e p r e v i o u s l y r e p o r t e d ( R o h r e r e t ai., 1979; F u l l e r t o n et a l . , 1 9 7 9 ) t h a t t h e r e l a t i v e p o s i t i o n o f t h e C-17 s i d e - g r o u p c a r b o n y l oxygen had a n e a r l y p e r f e c t (1-2 = 0 . 9 8 ) c o r r e l a t i o n w i t h r a t b r a i n Na,K-ATPase i n h i b i t i o n . F i g u r e 2 A shows a few o f t h e a n a l o g u e s examined, i n c l u d i n g t h e t o a d p o i s o n b u f a l i n and t h e p r o g e s t i n chlormadinone a c e t a t e . S i x o f t h e s e were a l s o s t u d i e d (K. Ahmed and A. H . L. From, u n p u b l i s h e d d a t a , 1981) u s i n g c a t h e a r t Na,K-ATPase. Again t h e r e l a t i v e c a r b o n y l oxygen p o s i t i o n s were f o u n d t o c o r r e l a t e w i t h i n h i b i t i o n ( F i g . 2B)--too g r e a t a d i s t a n c e ( o v e r 7 d) t o b e e x p l a i n e d by d i f f e r e n c e s i n H-bonding. [ C r y s t a l l o g r a p h i c a l l y observed conformations w e r e lowest i n e n e r g y , and u s e d f o r F i g . 2B. The e x c e p t i o n w a s ( 2 0 s ) - d i h y d r o d i g i t o x i g e n i n , whose n e x t - t o - lowes t ( I' a l t e r n a t e " ) e n e r g y c o n f o r m a t i o n p r o v i d e d t h e b e s t " f i t . "1 W e have a l s o found t h a t t h e r e i s a s t r o n g c o r r e l a t i o n of l o g T 5 0 ( c o n c e n t r a t i o n which i n c r e a s e s t h e isometric c o n t r a c t i l e t e n s i o n by 5 0 % ) w i t h t h e c a r b o n y l oxygen p o s i t i o n (A. H. L. From and K . Ahmed, u n p u b l i s h e d data, 1981). I n 1974, Repke and co-workers found a r e l a t i o n s h i p between t h e t o t a l d i p o l e moment p E of g e n i n s and t h e i r Na,K-ATPase 1 5 0 ' s . T h i s work w a s expanded i n t h e accompanying p a p e r . The r e l a t i o n s h i p i s a most i n t e r e s t i n g o n e , b u t f o r s e v e r a l compounds i t depends upon g r o s s l y i n a c c u 5 a t e C-17 s i d e group p o s i t i o n s [e.g., 0 . 0 7 and 0 . 4 0 A c a l c u l a t e d ( D i t t r i c h e t a l . , 1981) r e l a t i v e c a r b o n y l p o s i t i o n s f o r g i t o x i n and a g i t o x i g e n i n v s . 2 . 4 d a c t u a l C 1 7 s i d e g r o u p p o s i t i o n s from s t r u c t u r a l d a t a ( G o and K a r t h a , 1980b; P a r z y b y l i s k a and Ahmed, 1 9 7 9 ) l . I n t h e Repke work, t h e Na,K-ATPase i s viewed as p r e f e r r i n g t h e l o w e s t e n e r g y c o n f o r m a t i o n f o r t h e f i r s t 1 0 g e n i n s ; and an a l t e r n a t e minimum € o r t h e next 10. I n c o n t r a s t , t h e c o r r e l a t i o n i n Fig. 2B uses t h e lowest energy conformation f o r a l l b u t dihydrodigitoxigenin. F u r t h e r work s h o u l d c l a r i f y t h e s e differences.

111.

GLYCOSIDES

E l e g a n t s t u d i e s by Yoda (1973; Yoda and Yoda, 1 9 7 5 ) and o u r own ( F u l l e r t o n e t a l . , 1980) have shown t h a t t h e f i r s t s u g a r of t h e s e g l y c o s i d e s h a s t h e g r e a t e s t e f f e c t

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DWIGHT S. FULLERTON

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263

COMPUTER GRAPHICS IN THE STUDY OF Na,K-ATPase

on N a , K - A T P a s e . The second and t h i r d s u g a r s o f g l y c o s i d e s , l i k e d i g o x i n , have less. Genins c a n be v e r y act i v e ( F i g . 2 B ) b u t some s u g a r s (Rohrer and F u l l e r t o n , 1980; Yoda, 1973; Brown e t a l . , 1981) can i n c r e a s e bindi n g and a c t i v i t y by 1 0 - f o l d o r more. Yoda proposed t h a t a n a x i a l c o n f o r m a t i o n f o r t h e C-3'-OH, a s i n 8-U-digit o x o s e ( K a n t e r s e t a l . , 1978) ( F i g . 3A) i s i m p o r t a n t f o r o p t i m a l b i n d i n g . T h i s was based i n p a r t on a conformat i o n f o r a-l-rhamnose which p l a c e s t h e C-3I-OH i n an a x i a l p o s i t i o n . However, t h i s -OH i s e q u a t i o n a l b o t h as t h e f r e e s u g a r ( K a n t e r s e t a l . , 1 9 7 8 ) ( F i g . 3A) and i n o u a b a i n (Messerschmedt, 1 9 8 0 ) ; so o u a b a i n i t s e l f may n o t f i t t h i s p a r t o f t h e Yoda model. Preliminary p o t e n t i a l energy c a l c u l a t i o n s f o r r o t a t i o n of t h e f i r s t s u g a r are a l s o v e r y r e v e a l i n g . F i g u r e 3B c l e a r l y shows t h a t i n o u a b a i n , o v e r h a l f t h e p o s s i b l e c o n f o r m a t i o n s of t h e a-1-rhamnose have e n e r g i e s g r e a t e r t h a n 5 0 kcal/mole and are t h e r e f o r e e f f e c t i v e l y f o r b i d d e n . The c a l c u l a t i o n s f o r d i g i t o x i g e n i n $-Rd i g i t o x o s i d e and f o r $-U-glucoside a l s o i n d i c a t e v e r y l i m i t e d c o n f o r m a t i o n a l freedom. F i n a l l y , w e are a l s o u s i n g MMS-X t o g e n e r a t e van d e r Waals e n c l o s u r e maps t o r e p r e s e n t a p p a r e n t volumes r e q u i r e d f o r t h e s e compounds a t t h e "ouabain-binding s i t e . 'I

ACKNOWLEDGMENT

Supported by t h e N a t i o n a l Heart, Lung (HL21457).

and Blood I n s t i t u t e

REFERENCES

Brownl L. I Boutagy, J. d i a c glycosides.

and Thomas, R. (1981). S y n t h e s i s of carA r z n e i m . -Forsch 3 1 , 1059-1064.

F i g . 3 . ( A ) T o p and s i d e v i e w s o f $ - D - d i g i t o x o s e a n d a-1-rhamnose ( G o a n d K a r t h a , 1 9 8 0 a ; K a n t e r s e t a l . , 1 9 7 8 ) . ( B ) C o n f o r m a t i o n a l e n e r g y map f o r r o t a t i o n of the a-1-rhamnose o f o u a b a i n . E a c h d a r k l i n e r e p r e s e n t s 10 k c a l h i g h e r e n e r g y . X i s t h e c r y s t a l l o g r a p h i c a l l y observed c o n f o r m a t i o n . M i s the c a l c u l a t e d e n e r g y minimum.

264

DWIGHT S . FULLERTON

Brown, L . , B o u t a q y , J . , a n d Thomas, R. ( 1 9 8 1 ) . S y n t h e s i s o f cardiac glycosides. A r z n e i m . Forsch. 3 1 , 1059-1064. D i t t r i c h , F . , Repke, K . , e t al. ( 1 9 8 1 ) . Data from t h e pos t e r f o r t h e preceding paper. From, A . H . L . , and Ahmed, K. (19811, u n p u b l i s h e d d a t a . o h r e r , D. C . , From, A . , a n d F u l l e r t o n , D. S . , Y o s h i o k a , K . , Ahmed, K. ( 1 9 7 9 ) . D i g i t a l i s q e n i n a c t i v i t y . Science 205, 917-919. F u l l e r t o n , D . S . , Y o s h i o k a , K . , R o h r e r , D. C . , From, A . , and Ahmed, K . ( 1 9 8 0 ) . A c t o d i g i n a n d i t s q e n i n . Mol. P h a r m a c o l . 1 7 , 43-51. Go, K . , a n d K a r t h a , G. ( 1 9 8 0 ) . Digoxin. A c t a C r y s t a l l o g r . S e c t . B 3 6 , 1811-1819. G o , K . , and Kartha, G . ( 1 9 8 0 ) . Gitoxin. Acta C r y s t a l l o q r . Sect. B 3 6 , 3034-3040. K a n t e r s , J . , B a t e n b u r q , L . , Gaykema, W . , and R o e l o f s e n , G . ( 1 9 7 8 ) . A c t a C r y s t a l l o q r . S e c t . B 3 4 , 3049-3053. 6-D-digitoxose. K i l l e a n , R . , Lawrence, J . , a n d Sharma, V . ( 1 9 7 1 ) . a-L-rhamnose .H2O. A c t a C r y s t a l l o g r . S e c t . B 2 6 , 1707-1710. M e s s e r s c h m e d t , A. ( 1 9 8 0 ) . Ouabain.8H2O. C r y s t . S t r u c t . Commun. 9 , 1185-1194. P a r z y b y l i s k a , M . , and Ahmed, F. R. ( 1 9 7 9 ) . 5 B - h y d r o x y g i t o x i g e n i n . A c t a C r y s t a l l o g r . S e c t . B 35, 2436-2440. Repke, K. R . , D i t t r i c h , F . , B e r l i n , P . , a n d P o r t i u s , H . J . ( 1 9 7 4 ) . On p h y s i c a l f o r c e s q o v e r n i n q c a r d i a c g l y c o s i d e a c t i v i t y . Ann. N . Y . Acad. S c i . , 737-739. R o h r e r , D. C . , a n d F u l l e r t o n , D . S. ( 1 9 8 0 ) . D i g o x i g e n i n d i h y d r a t e . A c t a C r y s t a l l o g r . S e c t . B 3 6 , 1565-1568. R o h r e r , D. C . , F u l l e r t o n , D. S . , Y o s h i o k a , K., From, A , a n d Ahmed, K . ( 1 9 7 9 ) . F u n c t i o n a l receptor mapping f o r m o d i f i e d I n Computer A s s i s t e d Drug D e s i g n " (E. C . cardenolides. O l s o n and D. D. C h r i s t o f e r s e n , e d s . ) , p p . 259-279. W a s h i n q t o n , D.C. R o h r e r , D . C . , F u l l e r t o n , D. S . , e t a l . S t r u c t u r e s o f m o d i f i e d Acta Crystalloqr. ( i n press) . cardenolides. IV. Yoda, A . ( 1 9 7 3 ) . S t r u c t u r e - a c t i v i t y r e l a t i o n s h i p s . Mol. PharmaC O ~ . 9 , 51-60. Yoda, A . , and Yoda, S . ( 1 9 7 5 ) . D i s s o c i a t i o n r a t e c o n s t a n t s of Mol . P h a r m a c o l . 1 1 , 653-662. diqoxin acetates.

CURRENT TOPICS IN MEMBRANES AND TRANSPORT, VOLUME 19

Photoaffinity Labeling of the Ouabain Binding Site of Na,K-ATPase CLIFFORD C. HALL' AND ARNOLD E. RUOHO Department of Pharmacology University of Wisconsin Medical School Madison, Wisconsin

I.

INTRODUCTION

A f f i n i t y and p h o t o a f f i n i t y l a b e l i n g o f t h e o u a b a i n b i n d i n g s i t e o f t h e Na,K-ATPase h a s been r e p o r t e d (Ruoho and Kyte, 1 9 7 4 ; Forbush e t al., 1978; Rogers and L a z d u n s k i , 1 9 7 9 ; Ruoho and H a l l , 1980; H a l l and Ruoho, 1 9 8 0 ) . I n a l l cases, s p e c i f i c c o v a l e n t l a b e l i n g of t h e a - s u b u n i t ( M r = 95,000-100,000) h a s been f o u n d , i n d i c a t i n g t h a t t h i s s u b u n i t of t h e Na,K-ATPase c o n t a i n s t h e ouabain binding site.

11.

METHODS AND DISCUSSION

I n a n a t t e m p t t o probe s y s t e m a t i c a l l y t h e "sugars p e c i f i c " r e g i o n o f t h i s s i t e , w e have p r e p a r e d h i g h a f f i n i t y , t r i t i a t e d d i a z o m a l o n y l d e r i v a t i v e s of d i g i t o x i n , b i s d i g i t o x o s i d e , and m o n o d i g i t o x o s i d e i n which ' P r e s e n t a d d r e s s : D e p a r t m e n t of C h e m i s t r y , U l i v e r s i t y of Pennsylvania, Philadelphia, Pennsylvania. 265

Copyright 0 1981 by Academic Press, Inc All rights of reproduction in any form reserved ISBN 0-12-153319-0

266

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Pp

P

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268

SDS-PAGE results o f p h o t o l y s i s o f Na,K-ATPase w i t h I conditions P u r i f i e d e l e c t r i c eel Na ,K-ATPase ( 7 5 pg) was i n c u b a t e d w i t h s a t u r a t i n g concentrations o f 4 '- [ 3H] DAM-mono, 4"- [ 3 H ] DAM-bis, or 4'" - [ 3H] DAM-digi t o x i n i n the p r e s e n c e or absence o f 1 x 10-5 M o u a b a i n . C o m p a r a b l e amounts o f [3H]DAM-glycoside-enzyme c o m p l e x were p h o t o l y z e d f o r 10 sec a s a s u s p e n s i o n and t h e e n z y m e - l i g a n d c o m p l e x e s s e d i m e n t e d b y centrif u g a t i o n a t 100,000 g f o r 6 0 m i n . T h e r e s u l t i n g p e l l e t s were s o l u b i l i z e d i n SDS f o r g e l e l e c t r o p h o r e s i s . T h e t o p s o f the g e l s are a t the l e f t . T h e a - s u b u n i t m i g r a t e d a t s l i c e s 7-10 and t h e 8-subunit a t slices 1 3 - 1 6 . In these e x p e r i m e n t s , o n l y t h e p o r t i o n o f the g e l e n c o m p a s s i n g the a - and 8-subunits was a n a l y z e d f o r radioactivity. ( A ) 4 ' - 3 8 DAM-mono: s o l i d circles, 8.11 X 10-7 M 4'-[3H]DAM-mono ( 3 . 7 6 C i / m m o l e ) ; o p e n circles, 8.11 x 10-7 M 4 ' - [ 3 H ] DAM-mono ( 3 . 7 6 Ci/mmole) + 1 x 10-5 M o u a b a i n ( B ) 4"-[3H]DAM-bis: s o l i d circles, 8 . 5 7 X 10-7 M 4"-[3H]DAM-bis M 4"-[3H]DAM-bis ( 3 . 9 4 ( 3 . 9 4 C i / m m o l e ) ; open circles, 8 . 5 7 X C i / m m o l e ) + 1 X 10-5 M ouabain. ( C ) 4 " ' - 38 D A M - d i g i t o x i n ( 7 . 4 C i / m m o l e ) ; open c i r c l e s , 1 . 7 8 x 10-6 M 4 " ' [ - [ ' H ] D A M - d i g i t o x i n (7.4 C i / m m o l e ) + 1 x 10-5 M ouabain. Fig. 1.

[ 3H] DAM-gl y c o s i d e s u n d e r T y p e

.

.

SDS-PAGE results o f p h o t o l y s i s o f Na,K-ATPase w i t h P u r i f i e d electric eel Na,K-ATPase (75 p g ) was i n c u b a t e d w i t h s a t u r a t i n g concentrat i o n s o f 4 ' - [ 3 H ] D A M - m n o , 4"-[3H]-DAM-bis, or 4"' - [ 3 H ] D A M - d i g i t o x i n i n the p r e s e n c e or absence o f 1 x M ouabain. Comparable amounts o f [3H]DAM-glycoside-enzyme c o m p l e x were p h o t o l y z e d f o r 7 sec a s a s u s p e n s i o n and the e n z y m e - l i g a n d c o m p l e x e s s e d i m e n t e d b y c e n t r i f u g a t i o n a t 100,000 g f o r 6 0 m i n . T h e r e s u l t i n g p e l l e t s were s o l u b i l i z e d i n SDS for g e l e l e c t r o p h o r e s i s . T h e t o p s o f the g e l s are a t the l e f t . T h e a - s u b u n i t m i g r a t e d a t slices 7-10 and the 8-subunit a t slices 1 3 - 1 6 . In these e x p e r i m e n t s , o n l y the p o r t i o n o f the g e l e n c o m p a s s i n g the a- and B-subunits was a n a l y z e d for radioactivity. ( A ) 4'-[3H]DAM-mono: s o l i d circles, 1 . 6 3 x lom6 M 4'-[3H]DAM-mono (2.01 C i / m m o l e ) ; open circles, 1 . 6 3 x 10-6 M 4 ' [3H]DAM-mono (2.01 C i / m o l e ) + 1 X 1 0 - 5 M o u a b a i n . ( B ) 4'f-[3H]DAM-bis: s o l i d circles, 8 . 6 2 x M 4"-[3H]DAM-bis ( 3 . 9 7 C i / m o l e ) ; o p e n circles, 8 . 6 2 x M 4"-[3H]DAM-bis ( 3 . 9 7 C i / m m o l e ) + 1 x lom5 M o u a b a i n . ( C ) 4"' - [ 3 H ] D A M - d i g i t o x i n : s o l i d c i r c l e s , M 4"' [ 3 H ] D A M - d i g i t o x i n ( 4 . 6 6 C i / n n n o l e ) ; o p e n c i r c l e s , 1.48 X 1.48 X lom6 M 4"' - [ 3 H ] D d M - d i g i t o x i n ( 4 . 6 6 C i / m m o l e ) i1 X lo-' M ouabain. Fig. 2.

[ 3 H ] DAM-gl y c o s i d e s u n d e r T y p e 11 c o n d i t i o n s .

PHOTOAFFINITY LABELING OF OUABAIN BINDING SITE

269

t h e p h o t o a c t i v e group i s e s t e r i f i e d t o t h e 4"'-, 4"-, and 4 ' - h y d r o x y l s , r e s p e c t i v e l y , o f t h e t e r m i n a l s u g a r s . When e q u a l amounts o f L u b r o l - p u f i f i e d e l e c t r i c e e l N a , K - A T P a s e w e r e p h o t o l y z e d a t l 0 C w i t h s a t u r a t i n g conc e n t r a t i o n s o f e a c h p h o t o l a b e l i n b o t h t h e Type I (ATP, N a + , Mg2+) and Type I1 (Mg2+, P i ) complexes and t h e a - and 8 - s u b u n i t s s e p a r a t e d by sodium d o d e c y l s u l f a t e (SDSI-polyacrylamide g e l e l e c t r o p h o r e s i s (SDS-PAGE), t h e r e s u l t s d e p i c t e d i n F i g s . 1 and 2 were o b t a i n e d . The a - s u b u n i t w a s e x c l u s i v e l y l a b e l e d i n b o t h t h e Type I and Type I1 complexes w i t h t h e 4'-[3H]ethyldiazomalonyl d i g i t o x i g e n i n m o n o d i g i t o x o s i d e (3'-[3H]DAM-mono). This i s i n a g r e e m e n t w i t h t h e c o n c l u s i o n t h a t t h e a - s u b u n i t cont a i n s t h e c a r d i o t o n i c s t e r o i d b i n d i n g s i t e (Ruoho and K y t e , 1974; Forbush e t a l . , 1978; Rogers and L a z d u n s k i , 1979; Ruoho and H a l l , 1980; H a l l and Ruoho, 1 9 8 0 ) . Howe v e r , as t h e photoactive moiety w a s p o s i t i o n e d a t inc r e a s i n g d i s t a n c e s from t h e s t e r o i d n u c l e u s [,"- [3H] e t h y l d i a z o m a l o n y l d i g i t o x i g e n i n b i s d i g i t o x o s i d e (4"-[3H]DAM-bis) and 4"' [3H] e t h y l d i a z o m a l o n y l d i g i t o x i n ( 4 " ' - [3H] DAM-digitoxin) ] , two e v e n t s were o b s e r v e d t o o c c u r f o r b o t h t h e Type I and Type I1 complexes ( F i g s . 1 and 2 ) : ( a ) l a b e l i n g of t h e a-subunit decreased relat i v e t o t h e 4'-DAM-mono l a b e l i n g a s t h e d i s t a n c e of t h e p h o t o a c t i v e g r o u p from t h e s t e r o i d i n c r e a s e d , and ( b l s p e c i f i c l a b e l i n g o f t h e 8 - s u b u n i t c o u l d now b e det e c t e d r e a d i l y w i t h 4 " ' -DAM-digitoxin i n which t h e p h o t o a c t i v e g r o u p w a s p l a c e d on t h e t h i r d s u g a r . In replicate experiments, t h e s p e c i f i c a c t i v i t y of t h e a-subunit d e c r e a s e d 5- t o 2 0 - f o l d a s t h e p h o t o a c t i v e g r o u p w a s ext e n d e d f u r t h e r away from t h e s t e r o i d n u c l e u s , w h i l e t h a t of t h e @ - s u b u n i t i n c r e a s e d 4- t o 1 0 - f o l d . Covalent l a b e l i n g of a 1 2 , 0 0 0 - d a l t o n p r o t e o l i p i d component (Forbush et a l . , 1 9 7 8 ) c o u l d n o t be d e t e c t e d by t h e s e p h o t o l a b e l s i n t h e Type I complex. These d a t a are c o n s i s t e n t w i t h a model o f t h e ouabain binding s i t e of t h e d e t e r g e n t - p u r i f i e d eel N a , K ATPase i n which p o r t i o n s o f t h e 8 - s u b u n i t o c c u r i n c l o s e proximity t o t h e a-subunit i n t h e "sugar" r e g i o n of t h e c a r d i a c g l y c o s i d e binding s i t e . F u r t h e r experiments are necessary t o e s t a b l i s h whether t h i s i s a u n i v e r s a l s t r u c t u r a l f e a t u r e of a l l N a , K - A T P a s e s , e s p e c i a l l y t h e n a t i v e , membrane-bound form o f t h e enzyme which h a s n o t been exposed t o d e t e r g e n t .

-

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REFERENCES

Forbush, B . , Kaplan, J. H . , and Hoffman, J. F. ( 1 9 7 8 ) . Charact e r i z a t i o n o f a new p h o t o a f f i n i t y d e r i v a t i v e o f ouabain: Labeling of t h e l a r g e p o l y p e p t i d e and o f a p r o t e o l i p i d component o f the Na,K-ATPase. Biochemistry 1 7 , 3667-3675. H a l l , C. C . , and Ruoho, A. E. (1980). Ouabain-binding-site photoa f f i n i t y p r o b e s t h a t label b o t h s u b u n i t s o f Na,K-ATPase. Proc. Natl. Acad. Sci. USA 77, 4529-4533. Rogers, T. B . , and Lazdunski, M. (1979). P h o t o a f f i n i t y l a b e l i n g of t h e d i g i t a l i s r e c e p t o r i n t h e (sodium + p o t a s s i u m ) a c t i v a t e d adenosinetriphosphatase. Biochemistry 18, 135140. Ruoho, A. E . , and H a l l , C . C . (1980). The u s e of p h o t o l a b e l s t o probe t h e ouabain b i n d i n g s i t e o f t h e (Na,K)-ATPase. Ann. N.Y. Acad. Sci. 346, 90-103. Ruoho, A. E . , and Kyte, J. (1974). P h o t o a f f i n i t y l a b e l i n g of t h e ouabain-binding s i t e on ( N a + + K+) adenosinetriphosphatase. Proc. Natl. Acad. Sci. USA 71, 2352-2356.

CURRENT TOPICS IN MEMBRANES AND TRANSPORT, VOLUME 19

New Ouabain Derivatives to Covalently Label the Digitalis Binding Site BERNARD ROSSI, GIUES PONZIO, AND MICHEL LAZDUNSKI Centre de Biochimie du CNRS Fucultt? des Sciences UniversitC de Nice Nice, France

MAURICE GOELDNER AND CHRISTIAN HIRTH Institut de Chimie UniversitP Louis Pusteur Strasbourg , France

I.

INTRODUCTION

The p u r p o s e o f t h i s c h a p t e r i s t o d e s c r i b e t h e p r o p e r t i e s o f two new o u a b a i n d e r i v a t i v e s ( F i g . 1) t h a t c a n be u s e d t o c o v a l e n t l y l a b e l t h e d i g i t a l i s b i n d i n g s i t e . One o f them, p - n i t r o p h e n y l t r i a z e n e o u a b a i n (NPT-ouabain), i s an a l k y l a t i n g a g e n t ( R o s s i e t ai., 1 9 8 0 ) . The a l k y l a t i o n r e a c t i o n i n v o l v e s a p r o t o n t r a n s f e r from t h e r e c e p t o r p r o t e i n ( S i n n o t t and S m i t h , 1 9 7 8 ) . The s e c o n d m o l e c u l e , a r y l d i a z o n i u m o u a b a i n (AD-ouabain), i s a p h o t o a c t i v a b l e r e a g e n t . It d i f f e r s from o t h e r p h o t o a c t i v a b l e d e r i v a t i v e s of o u a b a i n o r o t h e r d i g i t a l i s - l i k e compounds ( F o r b u s h e t a l . , 1978; H a l l and Ruoho, 1980; Rogers and L a z d u n s k i , 1979a) by t h e f a c t t h a t it i s p o s s i b l e w i t h t h a t d e r i v a t i v e t o p h o t o a c t i v a t e s e l e c t i v e l y o n l y t h e m o l e c u l e s o f ADo u a b a i n t h a t a r e s p e c i f i c a l l y bound t o t h e d i g i t a l i s s i t e o f t h e Na,K-ATPase m o l e c u l e . T h i s a c t i v a t i o n i n v o l v e s e n e r g y t r a n s f e r t h r o u g h t h e e x c i t a t i o n of 1

P r e s e n t a d d r e s s : CNRS, C e n t r e d e N e u r o c h i m i e , S t r a s b o u r g ,

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15ance.

Copyright 0 1983 by Academic Press. Inc. All rights 01reproduction in any form reserved. ISBN 0-12-153319-01

272

BERNARD ROSS1 eta/.

CH

IN/ I CH2 CH2

Fig. 1. Chemical structures of (I) NPT-ouabain and (II) ar y l d i azoni um ouabain

.

t r y p t o p h a n r e s i d u e s of t h e r e c e p t o r p r o t e i n , a s i n d i c a t e d s c h e m a t i c a l l y i n F i g . 2 . Under these c o n d i t i o n s f r e e AD-ouabain m o l e c u l e s are n o t p h o t o a c t i v a t e d .

11.

PROPERTIES

The main p r o p e r t i e s o f t h e c o v a l e n t l a b e l i n g o f t h e d i g i t a l i s r e c e p t o r by NPT-ouabain and AD-ouabain are t h e following: (1) The p e r c e n t a g e o f c o v a l e n t i n c o r p o r a t i o n i s h i g h e r t h a n 2 0 % . ( 2 ) [3H]NPT-ouabain and [3H]AD-ouabain s p e c i f i c a l l y l a b e l t h e a - s u b u n i t o f p u r i f i e d N a , K - A T P a s e from r a b b i t kidney and from t h e e l e c t r i c o r g a n of E . e l e c t r i c u s ( F i g . 3 ) . N e i t h e r t h e

OUABAIN DERIVATIVES TO LABEL DIGITALIS BINDING SITE

Receptor R

Ligand

TrP

hY 3 2 0 n m photoact ivation

R

273

TrP

+

9

Lr

t

non specific covalent

‘1

l ~ T r 6 -

““p’t

energy transfert

L+ 1

specific labeling

association

F i g . 2 . Direct (A = 320 nm) and e n e r g y t r a n s f e r (A i r r a d i a t i o n s t r a t e g i e s f o r t h e a c t i v a t i o n of AD-ouabain.

= 290

nm)

p r o t e o l i p i d t h a t w a s a l s o l a b e l e d w i t h NAP-ouabain (Rogers and L a z d u n s k i , 197913) and NAB-ouabain ( F o r b u s h et al. 19781, n o r t h e 8 - s u b u n i t t h a t was a l s o l a b e l e d w i t h [ 1HIDAM-digitoxin ( H a l l and Ruoho, 1980) c o v a l e n t l y i n c o r p o r a t e t h e two t r i t i a t e d d e r i v a t i v e s p r e s e n t e d i n F i g . 1. D i f f e r e n t s o u r c e s g f d i g i t a l i s r e c e p t o r s h a v e been u s e d w i t h [ 3H] NPT-ouabain: t h e c r u d e microsomal f r a c t i o n o f e l e c t r i c o r g a n , crab a x o n a l membranes, and card i a c plasma membranes from c h i c k embryo ( R o s s i e t a l . , 1 9 8 0 ) . F o r a l l t h e s e p r e p a r a t i o n s t h a t d i f f e r from e a c h o t h e r i n t h e i r a f f i n i t y f o r o u a b a i n and a l s o i n t h e i r c o n t e n t of Na,K-ATPase, only t h e a-subunit of t h e enzyme w a s l a b e l e d ( R o s s i e t a l . , 1 9 8 0 ) . R e s u l t s obt a i n e d w i t h c a r d i a c c e l l membranes s t r o n g l y s u g g e s t t h a t t h e o n l y d i g i t a l i s r e c e p t o r i n t h e s e membranes i s t h e Na,K-ATPase a - s u b u n i t . The a - s u b u n i t of t h e N a , K - A T P a s e s p a n s t h e phosphol i p i d b i l a y e r exposing t h e c a r d i a c g l y c o s i d e s i t e a t t h e e x t e r n a l s i d e o f t h e plasma membrane and t h e a c t i v e s i t e f o r ATP h y d r o l y s i s on t h e c y t o p l a s m i c s i d e . T r y p t i c and c h y m o t r y p t i c d i g e s t i o n o f t h e a - s u b u n i t have a l r e a d y p e r m i t t e d i d e n t i f i c a t i o n o f t h e p o l y p e p t i d e f r a g m e n t s i n v o l v e d i n t h e p h o s p h o r y l a t i o n by [32P]yATP and i n t h e l a b e l i n g o f i n t r a m e m b r a n a l s e q u e n c e s by [3H]adamantane d i a z i r i n e and [ 1 2 5 I ] i o d o n a p h t h y l a z i d e ( F a r l e y e t a l . , 1980; K a r l i s h e t a ] . , 1 9 7 7 ) . The t r y p t i c d i g e s t i o n p a t t e r n of t h e a - s u b u n i t i s p r e s e n t e d i n F i g . 4 .

274

BERNARD ROSS1eta/.

600 0

labeling with 1 r M [t?INPTO

500

o protection with unlabeled oua bai n

400 Q) .-0

300

v)

\

E 200 n 0

100

9eK

58K

F i g . 3 . L a b e l i n g o f i n t a c t Na,K-ATPase. P u r i f i e d Na,KATPase f r o m rabbit k i d n e y ( 0 . 5 mg/ml) was i n c u b a t e d w i t h 5 pM [3H]NPT-ouabain f o r 2 hr at room t e m p e r a t u r e i n a medium cont a i n i n g 50 mM triethanolamine, 2 mM ATP, 2 mM M q C l 2 , 100 mM N a C l a t pH 7 . 4 . W i t h [3H]AD-ouabain the p h o t o a c t i v a t i o n p r o t o c o l was: ( 1 ) incubation o f Na,K-ATPase (0.5 m g / m l ) w i t h 1 p M [3H]AD-ouabain i n the d a r k f o r 5 m i n , and ( 2 ) i r r a d i a t i o n w i t h a monochromatic l i g h t (A = 290 nm) f o r 20 m i n a t 4OC. N o n s p e c i f i c l a b e l i n g was d e t e r m i n e d b y a d d i t i o n of 50 pM o f u n l a b e l e d ouabain b e f o r e the a d d i t i o n o f both t r i t i a t e d l i g a n d s .

The e x t e r n a l p e p t i d e fragment b e a r ' n g t h e d i g i t a l i s The b i n d i n g s i t e w a s l o c a l i z e d u s i n g [ HINPT-ouabain. r a d i o a c t i v i t y p r o f i l e p r e s e n t e d i n F i g . 5 shows t h a t no l a b e l i n g o c c u r r e d on t h e l a r g e C-terminal t r y p t i c T r i t i u m l a b e l i n g i s found i n fragment of MW 5 8 , 0 0 0 . Therefore t h i s t h e N-terminal p e p t i d e o f MW 3 6 , 0 0 0 . p e p t i d e domain s p a n s t h e plasma membrane and i s i n volved b o t h i n ATP h y d r o l y s i s a t t h e c y t o p l a s m i c f a c e and i n o u a b a i n b i n d i n g a t t h e e x t e r n a l f a c e .

3

OUABAIN DERIVATIVESTO LABEL DIGITALIS BINDING SITE

Trypsin(N2)

275

Trypsin(K+)

NH2 94 K

77 K 36 K

II

58 K

U phosphorylatlon s i t e domain ( i n t e r n a l )

d i g i t a l i s s i t e domain ( e x t e r n a l ) F i g . 4 . Summary of t h e t r y p t i c f r a g m e n t a t i o n o b t a i n e d i n t h e p r e s e n c e of 150 mM KCI or N a C l . 600, 0 1

500.

58K 7

direct labeling p r o t e c t e d I abeling

37K I

I F i g . 5. P r o t e o l y t i c f r a g m e n t a t i o n o f Na,K-ATPase p u r i f i e d e n z y m e l a b e l e d a s d e s c r i b e d u n d e r F i g . 3 was d i g e s t e d i n the p r e s e n c e o f 1 5 0 mM K C I , 25 mM i r n i d a z o l e , 1 mM EDTA, pH 7.5, b y TPCK t r y p s i n a t a t r y p s i n / A T P a s e w e i g h t r a t i o of 1 : l O a t 37OC for 5 min. T h e r e a c t i o n was s t o p p e d b y a d d i n g a 2 - f o l d w e i g h t excess o f s o y b e a n t r y p s i n i n h i b i t o r . Two m a j o r p o l y p e p t i d e s a p p e a r e d , w i t h a MW 58,000 and 37,000 r e s p e c t i v e l y . T h e minor p o l y p e p t i d e (MW 26 ,0 0 0 ) w h i c h a p p e a r e d u n d e r these c o n d i t i o n s was p r o b a b l y g e n e r a t e d b y a s e c o n d a r y c l e a v a g e o f t h e MW - 3 7 , 0 0 0 .

276

BERNARD ROSS1etel.

REFERENCES F a r l e y , R. A, , Goldman, D. W. , and Bayley, H. (1980). J. B i o l . C h e m . 2 5 5 , 860-864. Forbush, B. , Kaplan, J. H . , and Hoffman, J. F. (1978). B i o c h e m i s t r y 1 7 , 3667-3675. H a l l , C., and Ruoho, A. (1980). P r o c . Natl. A c a d . S c i . USA 77, 4529-4533. K a r l i s h , S. J. D., Jgfrgensen, P. L . , and G i t l e r , C. (1977). Nature (London) 2 6 9 , 715-717. Rogers, T. B., and Lazdunski, M. (1979a). B i o c h e m i s t r y 18, 135140. Rogers, T. B. , and Lazdunski, M. (197933). FEBS L e t t . 98, 373376. R o s s i , B., V u i l l e u m i e r , P . , Gache, C . , Balerna, M., and Lazdunski, M. (1980). J. B i o l . C h e m . 2 5 5 , 9936-9941. S i n n o t t , M. L., and Smith, P. L. (1978). B i o c h e m . J. 1 7 5 , 525538.

CURRENT TOPICS IN MEMBRANES AND TRANSPORT, VOLUME 19

Ouabain Sensitiwty: Diversity and Disparities JOHNS. WILLISAND J . C W E EUORY Departmenr of Physiology and Biophysics University of Illinois Urbana, Illinois

I.

SPECIES DIFFERENCES

The almost u n i v e r s a l u s e of ouabain o r o t h e r c a r d i a c g l y c o s i d e s t o d e f i n e and measure Na pump-related a c t i v i t i e s i n i n t a c t c e l l s and membrane p r e p a r a t i o n s i s based on t h e c o n s i s t e n c y and potency of i t s e f f e c t . N e v e r t h e l e s s , i t is widely r e c o g n i z e d t h a t l a r g e d i f f e r e n c e s i n s e n s i t i v i t y t o o u a b a i n do e x i s t among v a r i ous s p e c i e s and, w i t h i n a n organism, among t i s s u e s . Ouabain i n h i b i t i o n i n most t i s s u e s of r a t s and h a m s t e r s r e q u i r e s n o t o r i o u s l y high c o n c e n t r a t i o n s ( m i l l i m o l a r r a n g e ) and i s r a p i d l y r e v e r s i b l e . Furthermore, t h e ouabain dose-response of Na,K-ATPase of r a t and hamster kidney membrane p r e p a r a t i o n s f a i l s t o show t h e u s u a l s h i f t t o h i g h e r c o n c e n t r a t i o n s a t low t e m p e r a t u r e s , and t h e antagonism of o u a b a i n b i n d i n g by K may a l s o be l a c k i n g ( A l l e n and Schwartz, 1 9 6 9 ; L i , 1 9 7 1 ) . On t h e o t h e r hand, some r o d e n t t i s s u e s (ground s q u i r r e l , g u i n e a p i g ) have been r e p o r t e d t o have ouabain s e n s i t i v i t i e s i n t h e micromolar range even though r e v e r 277

Copyright 0 1983 by Academc Press, Inc All nghu of reproductionin any form rew,rvcd ISBN 0-12-153319-0

JOHN S. WlLLlS AND J. CLIVE ELLORY

278

s a l i s r a p i d i n one of t h e s e ( W i l l i s , 1 9 6 9 ; Akera e t I n a d d i t i o n , l a c k of t e m p e r a t u r e and K e f f e c t s on ouabain b i n d i n g t o Na,K-ATPase does n o t a c c o r d w i t h o b s e r v a t i o n s based on i n t a c t c e l l i o n f l u x e s and c a r d i o t o n i c e f f e c t s ( W i l l i s , 1 9 6 9 ; Akera et al., 1 9 7 0 ) . So, t h e q u e s t i o n s a r i s e a s t o whether r e v e r s i b i l i t y of b i n d i n g and i t s i n s e n s i t i v i t y t o K and t e m p e r a t u r e a r e n e c e s s a r y c o r r e l a t e s of o u a b a i n i n e f f e c t i v e n e s s and whether r o d e n t s a s a group have low s e n s i t i v i t y . W e know of no s t u d y of d i f f e r e n t i a l s e n s i t i v i t y t o c a r d i a c g l y c o s i d e s based on a c t u a l Na/K pump f l u x e s i n i n t a c t c e l l s , nor of any s i n g l e a t t e m p t s i n c e Dunham and Glynn ( 1 9 6 1 ) t o compare ouabain e f f e c t s on pumping i n i n t a c t c e l l s w i t h t h o s e on Na,K-ATPase from c e l l s of t h e same type W e measured ouabain s e n s i t i v i t v of 42K i n f l u x a t 37OC a t 1 0 mM KO i n e r y t h r o c y t e s o f - s e v e r a l s p e c i e s of r o d e n t s , and o u a b a i n r e v e r s i b i l i t y by measuring f l u x e s f o l l o w i n g t h r e e r a p i d washes a t 5 O C i n o u a b a i n - f r e e sol u t i o n . C e l l s from f o u r s p e c i e s of t h e marmotine t r i b e of s q u i r r e l s (woodchucks and t h r e e s p e c i e s of ground s q u i r r e l s , genus S p e r m o p h i l u s ) e x h i b i t e d s e n s i t i v i t y comparable t o t h a t of o t h e r h i g h l y s e n s i t i v e forms (human, r a b b i t , s h e e p ) w i t h a ~ 0 . 5 between 0 . 1 and 1 U M . Red c e l l s of t w o o t h e r s p e c i e s of s q u i r r e l ( f l y i n g and g r a y ) and o f g u i n e a p i g and coypu were o n l y a l i t t l e less s e n s i t i v e (guinea p i g Ko.5 i s 3 L I M ) , y e t t h e y exh i b i t e d almost complete r e v e r s a l of i n h i b i t i o n a f t e r washing. F i n a l l y , r e d c e l l s of h a m s t e r s , r a t s , l e m mings, and house mice a l l had low s e n s i t i v i t y t o ouabain ( ~ 0 . 5> 100 v M ) and v i r t u a l l y complete r e v e r s a l w i t h washing. a l . , 1970).

11.

DOSE-WSPONSE CURVE

W e n e x t compared t h e e f f e c t s of X c o n c e n t r a t i o n and low t e m p e r a t u r e on t h e o u a b a i n dose-response curve of K i n f l u x i n i n t a c t c e l l s and Na,K-ATPase of freeze-thawed e r y t h r o c y t e g h o s t s i n r e p r e s e n t a t i v e s o f e a c h of t h e t h r e e groups i d e n t i f i e d above: h i g h l y s e n s i t i v e (group I , human and t h i r t e e n - l i n e d ground s q u i r r e l ) , s e n s i t i v e b u t r e v e r s i b l e (group 11, g u i n e a p i g ) , and i n s e n s i t i v e and r e v e r s i b l e (group 111, r a t and h a m s t e r ) . I n c e l l s of group I , lowering t h e t e m p e r a t u r e from 37' t o 25OC s h i f t e d t h e dose-response of b o t h f l u x e s and ATPase t o h i g h e r v a l u e s by about a h a l f of an e x p o n e n t i a l u n i t ( i . e . , h a l f a d e c a d e ) . The magnitude of s h i f t produced

OUABAIN SENSITIVITY: DIVERSITY AND DISPARITIES

': OI 0I

-

279

AT Pase

.c

-

0 1

,

oK20 e, K 5

a/$

p (Ouabain ) F i g . 1 . D o s e - r e s p o n s e curves f o r o u a b a i n i n h i b i t i o n of K i n f l u x or Na,K-ATPase a c t i v i t y i n g u i n e a - p i g r e d c e l l s or broken ghosts, respectively. 42K i n f l u x was m e a s u r e d i n a medium cont a i n i n g Na 1 3 0 or 1 4 5 mM, K 5 or 20 mM, g l u c o s e 5 mM, T r i s 10 mM pH 7.5, a t 37OC f o r 3 0 m i n b y a r a p i d c e n t r i f u g a t i o n w a s h i n g m e t h o d . ATPase was m e a s u r e d i n Na 150 mM, K 5 or 20 mM, Mg 2 mM ATP 2 mM, T r i s 1 5 mM pH 7 . 5 , at 37'C f o r 1 hr.

by l o w e r i n g [K], from 1 0 t o 1 mM was s i m i l a r t o t h a t f o r l o w e r i n g t e m p e r a t u r e . I n groups I1 and I11 c e l l s , t h e e f f e c t s o f t e m p e r a t u r e and K were s i m i l a r t o t h o s e i n group I f o r dose-response o f o u a b a i n on K f l u x e s , b u t w e r e a b s e n t o r g r e a t l y d i m i n i s h e d f o r Na,K-ATPase of broken membranes ( F i g . 1 ) . Thus, n o t a l l s p e c i e s o f r o d e n t are i n s e n s i t i v e t o c a r d i a c g l y c o s i d e s ( e x c e p t i o n s : ground s q u i r r e l s , guinea p i g s ) ; high r e v e r s i b i l i t y i s n o t an i n v a r i a n t c o r r e l a t e of low s e n s i t i v i t y ( e x c e p t i o n : g u i n e a p i g s ) ; and w h i l e i n s e n q i t i v i t y o f o u a b a i n b i n d i n g t o low t e m p e r a t u r e o r h i g h K may be a f e a t u r e of N a , K - A T P a s e i n s p e c i e s w i t h r a p i d l y r e v e r s i b l e b i n d i n g , it i s n o t observed i n i n t a c t cells. R e d c e l l s o f g u i n e a p i g s may be a u s e f u l model f o r studying r a p i d l y reversing preparations, s i n c e t h e i r s e n s i t i v i t y i s s u f f i c i e n t l y h i g h t o a l l o w e a s y and meaningful measurements o f s t e a d y s t a t e b i n d i n g w i t h a low, n o n s p e c i f i c background. One f u r t h e r resemblance between g r o u p s I1 and I11 w a s found i n t h e r a n k o r d e r of e f f e c t i v e n e s s o f v a r i o u s c a r d i a c g l y c o s i d e s . In Group I (ground s q u i r r e l s ) , t h i s w a s s c i l l a r e n > o u a b a i n > s t r o p h a n t h i d i n > d i g o x i n , whereas i n group I1 ( g u i n e a p i g ) and group I11 ( h a m s t e r ) , it w a s s c i l l a r e n > o u a b a i n > digoxin >> strophanthidin.

JOHN S. WlLLlS AND J. CLIVE ELLORY

280

111.

CONCLUSIONS

Recent evidence h a s i n d i c a t e d t h a t low o u a b a i n sens i t i v i t y may be a r e f l e c t i o n of e i t h e r a c h a r a c t e r i s t i c a l l y d i f f e r e n t Na,K-ATPase (Sweadnor, 19791, o r of an impinging a c c e s s o r y molecule ( L e l i e v r e e t al., 1 9 7 9 1 , o r both. Our o b s e r v a t i o n s of an a l t e r a t i o n i n seconda r y p r o p e r t i e s w i t h membrane r u p t u r e a c c o r d s w i t h t h e n o t i o n of i n t e r a c t i o n w i t h an a c c e s s o r y molecule, b u t t h e f a c t t h a t t h e same e f f e c t s o c c u r i n a s e n s i t i v e s y s t e m ( i . e . , group 11) i s a c o m p l i c a t i o n .

ACKNOWLEDGMENT

T h i s work w a s supported by N I H Grant GM 11494.

REFERENCES

Akera, T., Larsen, F S . , and Brody, T. M. (1970). C o r r e l a t i o n o f c a r d i a c sodium- and p o t a s s i u m - a c t i v a t e d a d e n o s i n e triphosphatase a c t i v i t i e s w i t h ouabain induced i n o t r o p i c stimul a t i o n . J. Pharmacol. Exp. Ther. 1 7 3 , 145-151. A l l e n , J. C. , and Schwartz, A. (1969). A p o s s i b l e biochemical exp l a n a t i o n f o r t h e i n s e n s i t i v i t y o f t h e r a t t o c a r d i a c glycos i d e s . J. Pharrnacol. Exp. Ther. 1 6 8 , 42-46. Dunham, E. T. , and Glynn, X. M. (1961). Adenosinetriphosphatase a c t i v i t y and t h e a c t i v e movements o f a l k a l i metal i o n s . J. P h y s i o l . (London) 1 5 6 , 274-293. L e l i e v r e , L. , Zachowski, A. , Charlemagne, D. , Laget, P. , and P a r a f , A. (1979). I n h i b i t i o n of (Na -k K)-ATPase by ouabain: Biochim. involvement of calcium and membrane p r o t e i n s . Biophys. Acta 5 5 7 , 399-408. L i , N. M. (1971). Temperature s e n s i t i v i t y o f Na-K-ATPase and mechanism of ouabain a c t i o n on c a t i o n t r a n s p o r t and on NaK-ATPase. Ph.D. T h e s i s , Texas A.M U n i v e r s i t y , College S t a t ion. Sweadnor, K. J. (1979). Two molecular forms o f ( N a + K ) - s t h u l a t e d ATPase i n b r a i n . S e p a r a t i o n and d i f f e r e n c e i n a f f i n i t y f o r s t r o p h a n t h i d i n . J. E i o l . Chem. 2 5 4 , 6060-6067. W i l l i s , J. S. (1969). Ouabain i n h i b i t i o n o f i o n t r a n s p o r t and r e s p i r a t i o n i n r e n a l cortical slices o f ground squirrels and hamsters. Biochim. Biophys. A c t a 1 6 3 , 506-515.

Part IV

Ligand Interactions: Nucleotides, Vanadate, and Phosphorylation

This Page Intentionally Left Blank

CURRENT TOPICS IN MEMBRANES AND TRANSPORT. VOLUME 19

Ligand Interactions with the Substrate Site of Na,K-ATPase: Nucleotides, Vanadate, and Phosphorylation JENS G. N0RBY Institute of Biophysics University ofAarhus Aarhus, Denmark

I.

INTRODUCTION

I n t h e p r e s e n t a r t i c l e w e s h a l l be d e a l i n g w i t h t h e i n t e r a c t i o n s of c e r t a i n compounds w i t h t h e s u b s t r a t e s i t e o f Na,K-ATPase, i . e . , t h e s i t e on which T i s h y d r o l y z e d t o ADP and i n o r g a n i c p h o s p h a t e ( P i ) . More s p e c i f i c a l l y , t h i s a r t i c l e w i l l b e c o n c e r n e d w i t h t h e i n t e r a c t i o n of t h i s s i t e w i t h some n a t u r a l l y o c c u r r i n g n u c l e o t i d e s and n u c l e o t i d e a n a l o g s , w i t h v a n a d a t e (which may be c o n s i d e r e d a s a t r a n s i t i o n - s t a t e a n a l o g of p h o s p h a t e ) , and w i t h t h e p h o s p h o r y l a t e d i n t e r m e d i a t e s of Na,K-ATPase which a r e formed when ATP "del i v e r s " i t s t e r m i n a l p h o s p h a t e t o form a c o v a l e n t bond a t the substrate site. I t i s n o t i n t e n d e d t h a t a c o m p l e t e r e v i e w of t h e p r o p e r t i e s of t h e s u b s t r a t e s i t e and t h e c o n s e q u e n c e s of i t s i n t e r a c t i o n s w i t h l i q a n d s b e p r e s e n t e d h e r e . The r e a d e r who would l i k e a t h o r o u g h (and more b a l a n c e d ? ) s u r v e y o f t h i s f i e l d i s u r g e d t o c o n s u l t some of t h e many r e v i e w s , n o t a b l y t h o s e by Robinson and F l a s h n e r 281

Copyright 0 1983 by Academic Press, Inc. All rights of reproduction in any form resewed ISBN O-lZ-lS33l94

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( 1 9 7 9 a ) and by Jfdrgensen (19801, and t h e more d i r e c t l y r e l a t e d and e x c e l l e n t review by Cantley ( 1 9 8 1 ) , as w e l l a s t h e o t h e r c o n t r i b u t i o n s t o t h i s volume. R a t h e r , i n f o r m a t i o n h a s been s e l e c t e d which i s most r e l e v a n t t o t h e c u r r e n t d i s c u s s i o n concerning t h e s u b s t r a t e s i t e of t h e Na,K-ATPase and t h e r o l e and p r o p e r t i e s of t h e phosphorylated intermediates. Before t u r n i n g t o a more d e t a i l e d d i s c u s s i o n of t h e above-mentioned s u b j e c t s , i t should be emphasized t h a t t h e working h y p o t h e s i s o f t h i s a r t i c l e i s t h a t t h e r e i s one ATP-binding s i t e p e r enzyme molecule, t h e l a t t e r b e i n g d e f i n e d as t h e s m a l l e s t u n i t c a p a b l e of performing a l l t h e r e a c t i o n s of Na,K-ATPase. I t seems r e a l i s t i c t o assume t h a t t h i s s i t e cons i s t s of d i f f e r e n t r e g i o n s , o r s u b s i t e s , t h a t react w i t h t h e adenine r i n g , t h e r i b o s e , and a t l e a s t two of t h e t h r e e phosphates i n ATP. Such a view i s s u p p o r t e d by s t u d i e s on n u c l e o t i d e s p e c i f i c i t y (Njdrby and J e n s e n , 1 9 7 4 ) and i s a t t r a c t i v e i n r e l a t i o n t o o b s e r v a t i o n s on t h e i n t e r a c t i o n of Na,K-ATPase w i t h c e r t a i n s i t e - s p e c i f i c r e a g e n t s , a s d i s c u s s e d by Smith e t a l . (1980). The conc e p t of s u b s i t e s i m p l i e s t h a t a l i g a n d o t h e r t h a n ATP may bind t o t h e s u b s t r a t e s i t e of Na,K-ATPase w i t h o u t necess a r i l y occupying t h e e n t i r e s i t e , t h u s p o s s i b l y l e a v i n g room f o r t h e s i m u l t a n e o u s b i n d i n g of a n o t h e r l i g a n d t o the substrate site. I n t h e c o n t e x t of t h i s overview it s h o u l d a l s o be noted t h a t i n p u r i f i e d enzyme p r e p a r a t i o n s t h e r e i s an e q u a l number of s i t e s f o r h i g h - a f f i n i t y b i n d i n g of nuc l e o t i d e s , o u a b a i n , and v a n a d a t e , and f o r p h o s p h o r y l a t i o n by ATP o r P i (see Hansen e t al., 1979; C a n t l e y , 1 9 8 1 , pp. 206-209). I t i s l i k e w i s e i m p o r t a n t t h a t a s a consequence of c o n f o r m a t i o n a l change i n t h e enzyme p r o t e i n , t h e subs t r a t e s i t e c a n e x i s t i n a t l e a s t two forms d i f f e r i n g i n p r o p e r t i e s and t h e r e f o r e probably a l s o i n s t r u c t u r e .

11.

INTERACTION W I T H NUCLEOTIDES

The i n t e r a c t i o n of Na,K-ATPase w i t h n u c l e o t i d e s h a s been i n v e s t i g a t e d by a l a r g e v a r i e t y of t e c h n i q u e s and under many d i f f e r e n t e x p e r i m e n t a l c o n d i t i o n s . Roughly, however, t h e experiments may b e d i v i d e d i n t o t h r e e groups of i n c r e a s i n g complexity, namely, (1) e q u i l i b r i u m e x p e r i m e n t s , i n which t h e A T P a s e i s n o t t u r n i n g o v e r ; ( 2 ) d e n a t u r a t i o n o r i n h i b i t i o n e x p e r i m e n t s , i n which t h e e f f e c t of l i g a n d s f o r t h e s u b s t r a t e s i t e on t h e r a t e and e x t e n t of i n a c t i v a t i o n of t h e enzyme i s measured: and

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( 3 ) t r a n s i e n t o r s t e a d y - s t a t e e x p e r i m e n t s , i n which t h e A T P a s e i s t u r n i n g o v e r , u s u a l l y h y d r o l y z i n g ATP.

I n e a c h o f t h e s e t y p e s of e x p e r i m e n t s t h e i n t e r p l a y between o t h e r l i g a n d s , e . g . , N a + , K+, and Mg2+, and l i g a n d s f o r t h e s u b s t r a t e s i t e have been c h a r a c t e r i z e d , and t h e r e i s t h u s a v a s t amount o f i n f o r m a t i o n a v a i l a b l e on t h e s u b j e c t t o be d i s c u s s e d i n t h i s s e c t i o n . The d i s c u s s i o n s h a l l f o c u s on t h o s e r e s u l t s t h a t i l l u s t r a t e most d i r e c t l y t h e i n t e r a c t i o n s w i t h and t h e p r o p e r t i e s of t h e s u b s t r a t e s i t e . The more complete, and complic a t e d , p i c t u r e o f t h e r o l e of n u c l e o t i d e i n t e r a c t i o n i n t h e r e a c t i o n mechanism of Na,K-ATPase i s t h e s u b j e c t o f o t h e r a r t i c l e s of t h i s volume. A.

EQUILIBRIUM

BINDING STUDIES

The f i r s t d i r e c t d e m o n s t r a t i o n o f e q u i l i b r i u m bindi n g of ATP t o a h i g h - a f f i n i t y s i t e (Kd = 0 . 1 - 0 . 2 P M ) on Na,K-ATPase w a s p u b l i s h e d s i m u l t a n e o u s l y by Hegyvary and P o s t (1971) and by Ndrby and J e n s e n ( 1 9 7 1 ) . They u s e d r a d i o a c t i v e l y l a b e l e d ATP and a d i a l y s i s r a t e t e c h n i q u e t o measure t h e c o n c e n t r a t i o n o f f r e e ATP i n s o l u t i o n s c o n t a i n i n g enzyme and ATP. Bound ATP w a s t h e n c a l c u l a t e d as t h e d i f f e r e n c e between t o t a l and f r e e ATP. Also ADP w a s found t o b i n d w i t h h i g h a f f i n i t y , K d = 0.5-2 I.IM (Hegyvary and P o s t , 1971; J e n s e n and N&rby, 1971; Kaniike e t a l . , 1 9 7 3 ) . H i g h - a f f i n i t y b i n d i n g o f ATP t o a v a r i e t y o f p u r i f i e d o r p u r e enzyme p r e p a r a t i o n s , measured by d i a l y s i s o r c e n t r i f u g a t i o n t e c h n i q u e s , h a s been r e p o r t e d by o t h e r workers ( J d r g e n s e n , 1 9 7 4 ; K a n i i k e e t al., 1974; Schoner e t a l . , 1977; J e n s e n and N&rby, 1 9 7 9 ) . R e c e n t l y Yamaguchi and Tonomura (1980) and Schuurmans Stekhoven e t a l . (1981) have u s e d a f i l t r a t i o n t e c h n i q u e , which measures bound n u c l e o t i d e d i r e c t l y , t o o b t a i n b i n d i n g i s o t h e r m s of ATP and AMPP(NH)P t o p u r i f i e d p r e p a r a t i o n s o f kidney N a , K - A T P a s e a t room t e m p e r a t u r e . An e a r l i e r review on the p r o p e r t i e s of t h e h i g h - a f f i n i t y s i t e i s g i v e n by NZrby and J e n s e n ( 1 9 7 4 ) . The f u n c t i o n a l s i g n i f i c a n c e of t h e s i t e i s c l e a r l y i l l u s t r a t e d by t h e f a c t t h a t t h e e q u i l i b r i u m c o n s t a n t f o r h i g h - a f f i n i t y ATP b i n d i n g i s o f t h e same o r d e r o f magnitude a t t h e a p p a r e n t Km ( o r K0.5) v a l u e s f o r ATP f o r (1) h y d r o l y s i s a t low ATP c o n c e n t r a t i o n s (and i n t h e a b s e n c e of K+, s o - c a l l e d N a A T P a s e ) , ( 2 ) f o r m a t i o n of p h o s p h o r y l a t e d i n t e r m e d i a t e s , and ( 3 ) ATP-supported b i n d i n g of o u a b a i n ( f o r r e f e r e n c e s and d i s c u s s i o n , see NZrby and J e n s e n , 1971, 1 9 7 4 ) . The above s t u d i e s were performed i n t h e a b s e n c e o f Mg2+ ( 1 0 mM o r more o f a c h e l a t o r s u c h a s EDTA w a s added) t o a v o i d h y d r o l y s i s o f ATP and/or c o n v e r s i o n o f ADP t o

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B/ F i g . 1 . S c a t c h a r d p l o t o f I 4 C ADP b i n d i n g t o Na,K-ATPase f r o m ox b r a i n i n t h e a b s e n c e a n d p r e s e n c e of a d d e d K+ a s i n d i c a t e d . Enzyme w a s p r e p a r e d a s d e s c r i b e d b y K l o d o s e t a l . ( 1 9 7 5 1 , a n d b i n d i n g m e a s u r e d b y the d i a l y s i s - r a t e m e t h o d ( N b r b y a n d J e n s e n , 1 9 7 1 ) a t O°C w i t h 10 mM EDTA, 1 5 mM T r i s - C 1 , 88 mM i m i d a z o l e b u f f e r , 225 mM sucrose, pH 7 . 4 , v o l u m e 1 . 5 ml. KCl w a s s u b s t i t u t e d for T r i s - C 1 on a n e q u i m o l a r b a s i s . ( J . J e n s e n and J . G . N d r b y , unpublished.)

AMP + ATP by c o n t a m i n a t i n g a d e n y l a t e k i n a s e . Furthermore, it i s i m p o r t a n t t o n o t e t h a t K + h a s a d r a m a t i c i n f l u e n c e on t h e b i n d i n g e q u i l i b r i u m of t h e E-ATP complex (see S e c t i o n II,C), so t h a t h i g h - a f f i n i t y b i n d i n g can o n l y b e o b s e r v e d i n t h e a b s e n c e of added ( o r c o n t a m i n a t i n g ) K+. E q u i l i b r i u m b i n d i n g of ATP a n a l o g s t h a t change f l u o r e s c e n c e upon b i n d i n g r e p r e s e n t s a n o t h e r a p p r o a c h t o t h e s t u d y o f t h e ATP-binding s i t e of Na,K-ATPase. Part i c u l a r l y w e l l s u i t e d i s f o r m y c i n t r i p h o s p h a t e (FTP) and t h e d i p h o s p h a t e ( F D P ) , b o t h o f which showed h i g h - a f f i n i t y b i n d i n g ( K d = 1.1 and 4.8 p M , r e s p e c t i v e l y , w i t h p i g k i d n e y enzyme 2 O o C , 5 0 mM N a + ) . By c o m p e t i t i o n w i t h ATP o r AMPP(NH)P u n d e r t h e same c o n d i t i o n s , Kd f o r ATP and AMPP(NH)P were d e t e r m i n e d t o be 0 . 1 5 and 1.1 V M , r e s p e c t i v e l y ( K a r l i s h e t a l . , 1 9 7 6 , 1 9 7 8 a ) . TNP-ATP, a n o t h e r f l u o r e s c e n t n u c l e o t i d e , shows even h i g h e r a f f i n i t y f o r Na,K-ATPase t h a n ATP. Using f l u o r e s c e n c e t i t r a t i o n w i t h TNP-AT?, o r [3H]TNP-ATPI o r a d i a l y s i s t e c h n i q u e w i t h [3H]TNP-ATP, Moczydlowski and F o r t e s (1981a) showed t h a t

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h i g h l y p u r i f i e d e l e c t r o p l a x N a , K - A T P a s e b i n d s TNP-ATP w i t h a Kd = 0 . 0 4 - 0 . 0 9 p~ a t 3OC and 0 . 2 - 0 . 7 V M a t room t e m p e r a t u r e . The number o f s i t e s , found t o be e q u a l t o t h e number o f o u a b a i n - b i n d i n g s i t e s , w a s i n d e p e n d e n t of t h e method u s e d . The b i n d i n g showed s i m p l e c o m p e t i t i o n w i t h ATP, and t h e b i n d i n g s i t e had t h e same n u c l e o t i d e s p e c i f i c i t y as p r e v i o u s l y d e s c r i b e d f o r t h e h i g h - a f f i n i t y ATP s i t e (Ndrby and J e n s e n , 1 9 7 4 ) . I n a l l t h e s t u d i e s mentioned t h u s f a r (see a l s o F i g . 1) t h e b i n d i n g d a t a c a n be f i t t e d t o a b i n d i n g i s o therm i n t h e form of a r e c t a n g u l a r h y p e r b o l a (bound versus f r e e ligand) o r a s t r a i g h t l i n e i n a Scatchard p l o t (bound v e r s u s b o u n d / f r e e ) c o n s i s t e n t w i t h a homogeneous p o p u l a t i o n o f h i g h - a f f i n i t y b i n d i n g s i t e s f o r ATP

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AND NUCLEOTIDE B I N D I N G

The h y d r o l y s i s of ATP i n t h e p r e s e n c e of N a + o r K + i s o p t i m a l o n l y a t Mg c o n c e n t r a t i o n s h i g h e r t h a n t h e ATP c o n c e n t r a t i o n (Skou, 197433; Robinson and F l a s h n e r , 1979a; C a n t l e y , 1 9 8 1 ) . I n c o n t r a s t , h i g h a f f i n i t y n u c l e o t i d e b i n d i n g d o e s n o t r e q u i r e Mg, a l t h o u g h a number o f e x p e r i m e n t s seem t o i n d i c a t e t h a t Mg h a s some i n f l u e n c e on t h e a p p a r e n t a f f i n i t y f o r t h e n u c l e o t i d e . 1 The ATP a n a l o g AMPP(NH)P, which i s n o t h y d r o l y z e d by Na,K-ATPase and b i n d s w i t h r a t h e r h i g h a f f i n i t y (Kd = 1 - 4 V M i n t h e a b s e n c e o f added c a t i o n s , h a s been u s e d t o s t u d y t h e e f f e c t of Mg. A d d i t i o n of Mg a t low concentrations s l i g h t l y increases the a f f i n i t y f o r the a n a l o g (Robinson and F l a s h n e r , 197913) and t h e n d e c r e a s e s i t t o a p l a t e a u v a l u e a t Mg2+ > 2 mM of Kd = 4-6 V M (Robinson and F l a s h n e r , 1979b; Schuurmans Stekhoven e t a ] . , 1 9 8 1 ) . Q u a l i t a t i v e l y s i m i l a r r e s u l t s have been obt a i n e d i n o u r l a b o r a t o r y f o r ADP b i n d i n g i n t h e p r e s e n c e of t h e a d e n y l a t e k i n a s e i n h i b i t o r ~1,~ 5 - d i a d e n o s i n e 5 I Na+

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concentration i n the binding experiments discussed 'The Mg i n Section I I , A i s so low t h a t the nucleotides i n s o l u t i o n are a l l uncomplexed. Comparison o f the e f f e c t o f Mg2+ on nucleotide binding with k i n e t i c experiments makes i t l i k e l y t h a t Mg complexes with ATP and EATP with about the same s t a b i l i t y constant (Plesner and Plesner, 1 9 8 1 ; Plesner e t a l . , 1981), so that bound ATP i s a l s o unI f Mg i s required for binding, i t complexed i n these experiments. m u s t a c t a t an Mg s i t e with extremely high a f f i n i t y (Ndrby and Jensen, 1 9 7 1 ) .

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Fig. 2. T h e e f f e c t o f Mg on ADP b i n d i n g . T h e ADP b i n d i n g t o p u r i f i e d p i g k i d n e y outer m e d u l l a e n z y m e was m e a s u r e d e s s e n t i a l l y a s d e s c r i b e d i n F i g . 1 , b u t w i t h 5 mM EDTA i n s t e a d o f 10 mM MgCl, was s u b s t i t u t e d for T r i s - C 1 on an equimolar b a s i s and 0.1 mM Pl,P5d i a d e n o s i n e 5 ’ - p e n t a p h o s p h a t e was added t o i n h i b i t a d e n y l a t e k i n a s e . T h e ADP a f f i n i t y a t Mgt = 0 i s somewhat l o w e r than the u s u a l c a . 5 p M - 1 d u e t o c o m p e t i t i o n between the inhibitor a n d ADP f o r the b i n d i n g s i t e . ( J . J e n s e n and J . G . N d r b y , u n p u b l i s h e d . )

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pentaphosphate (Fig. 2 ) A t h i g h e r Mg2+ c o n c e n t r a t i o n s , almost a l l ADP and AMPP(NH)P o c c u r a s t h e complexes MgADP and MgAMPP “ H I P ; i n f a c t , AMPP ( N H ) P complexes Mg2+ even b e t t e r t h a n ATP (see Yount, 1 9 7 5 ) . K a r l i s h e t a l . (1978a) have a l s o r e p o r t e d t h a t Mg has o n l y a s m a l l i n f l u e n c e on t h e a f f i n i t y of t h e enzyme f o r FDP. Moczydlowski and F o r t e s (1981a) have t a k e n advantage of t h e f a c t t h a t K+ i n h i b i t s ATP h y d r o l y s i s t o s t u d y t h e e f f e c t of Mg2+ ( i n t h e p r e s e n c e of K + ) on t h e d i s p l a c e m e n t of TNP-ATP from i t s b i n d i n g s i t e by ATP. They show t h a t MgATP a s w e l l a s ATP e x h i b i t s s i m p l e c o m p e t i t i o n toward TNP-ATP, t h e a p p a r e n t a f f i n i t y f o r ElgATP b e i n g 1/5-1/10 t h a t f o r ATP. I n t h e p r e s e n c e of Mg, t h e Mg-nucleotide complexes a p p a r e n t l y b i n d almost a s w e l l a s t h e uncomplexed nucleoand t h e d i f f e r e n c e might w e l l t i d e s i n t h e absence of M be due t o an e f f e c t of Mgj; a c t i n g a t a s e p a r a t e s i t e of t h e enzyme ( C a n t l e y , 1 9 8 1 ; Schoner e t a l . , t h i s volume). There i s t h u s some i n d i c a t i o n from e q u i l i b r i u m s t u d i e s t h a t ATP and MgATP may b i n d t o t h e same s i t e ,

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and t h i s h y p o t h e s i s i s s u p p o r t e d by t h e f o l l o w i n g nonequilibrium observations. In a rather detailed investig a t i o n of t h e s t e a d y - s t a t e k i n e t i c s of Na,K-ATPase, P l e s n e r and P l e s n e r (1981) have shown t h a t t h e c a t a l y t i c complex i n t h e p r e s e n c e of b o t h Na+ and Na+ + K+ i s EATPMg. T h i s complex may be formed by t h e r e a c t i o n of E w i t h ATP t o form EATP and E A T P ' s r e a c t i o n w i t h Mg2+, o r by t h e more d i r e c t r e a c t i o n of E w i t h MgATP. T h i s i s i n accordance w i t h p h o s p h o r y l a t i o n e x p e r i m e n t s (Mflrdh and P o s t , 1 9 7 7 ; I . Klodos, t h i s l a b o r a t o r y , u n p u b l i s h e d ) i n which t h e enzyme i s p r e i n c u b a t e d w i t h [ Y - ~ ~ P ] A Ti P n t h e absence of Mg2+. m e n Mg2+ and u n l a b e l e d ATP are s u b s e q u e n t l y added s i m u l t a n e o u s l y a t r a n s i e n t phosphor y l a t i o n w i t h t h e f o r m a t i o n of E-12P of h i g h s p e c i f i c a c t i v i t y i s o b s e r v e d , i n d i c a t i n g t h a t prebound ATP i s t h e phosphoryl donor. I t s h o u l d be mentioned t h a t Skou, i n s t u d y i n g t h e o v e r a l l h y d r o l y s i s r e a c t i o n ( 1 9 7 4 b ) , w a s a b l e t o conc l u d e t h a t ATP and MgATP b i n d t o Na,K-ATPase w i t h a b o u t t h e same a f f i n i t y , s i n c e t h e c o n c e n t r a t i o n of t o t a l ATP, r a t h e r t h a n f r e e ATP o r MgATP, determined t h e a p p a r e n t r e l a t i v e a f f i n i t y of Na,K-ATPase f o r Na+ and K+ (see also Section I 1 , D ) . Munson (1981) has t a k e n a r a t h e r d i r e c t approach i n s t u d y i n g t h e i n t e r a c t i o n of c h e l a t e d n u c l e o t i d e s w i t h Na,K-ATPase, u s i n g a MgATP a n a l o g , t h e chromium-I11 coo r d i n a t i o n complex of t h e p h o t o a f f i n i t y a n a l o g a r y l a z i d o B-alanyl-ATP ( c a l l e d CrATPa), a s a marker of t h e subs t r a t e s i t e of enzyme from c a n i n e kidney o u t e r medulla. T h i s compound i s i n e r t t o h y d r o l y s i s , and i n t h e d a r k it showed r e v e r s i b l e , c o m p e t i t i v e i n h i b i t i o n of Na,K-ATPase, w i t h ~i= 0.32 mM, s i m i l a r t o t h e a p p a r e n t Km f o r MgATP. Also, CrATPa d i s p l a c e d TNP-ATP from t h e enzyme. The d a t a conformed t o a " s i n g l e c l a s s of s i t e s " model, w i t h Kd = 9 V M f o r C r A T P a (no i o n s a d d e d ) . Furthermore, t h e l i g h t - d e p e n d e n t i n a c t i v a t i o n of N a , K - A T P a s e by t h i s anal o g followed an e q u a l l y simple model and was a b l e t o be p r e v e n t e d by ATP. These r e s u l t s s u p p o r t t h e e v i d e n c e t h a t ATP and MgATP r e a c t a t t h e same s i t e whose a f f i n i t y i s dependent on t h e o t h e r l i g a n d s p r e s e n t (see a l s o S e c t i o n s I 1 , C and D ) I n summary, b o t h MgATP and ATP b i n d t o Na,K-ATPase d i r e c t l y (and n o t v i a M g ) and Mg can bind t o t h e p r e formed EATP complex, i n a l l p r o b a b i l i t y t o t h a t p a r t of t h e t r i p h o s p h a t e c h a i n of ATP t h a t i s a c c e s s i b l e from t h e s u r r o u n d i n g medium. The ( s e m a n t i c ? ) d i s c u s s i o n of whether ATP o r MgATP i s t h e " t r u e " s u b s t r a t e may be abandoned s i n c e t h e c a t a l y t i c complex EATPMg seems t o a r i s e from r e a c t i o n of t h e enzyme w i t h b o t h , Mg2+ b e i n g a " s u b s t r a t e " f o r EATP.

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C.

MONOVALENT C A T I O N S , NUCLEOTIDE B I N D I N G

ENZYME CONFORMATION,

AND

Among t h e e a r l y e v i d e n c e f o r a s t r u c t u r a l and f u n c t i o n a l r e l a t i o n s h i p between t h e h i g h - a f f i n i t y ATPb i n d i n g s i t e and t h e N a , K - A T P a s e m o l e c u l e w a s t h e obs e r v a t i o n t h a t K+ (and i t s congeners T 1 + , Rb+, CS+, and NH4') i n c o n c e n t r a t i o n s w e l l below 1 mM r e d u c e s t h e a f f i n i t y f o r ATP w i t h o u t changing t h e number o f b i n d i n g s i t e s (Hegyvary and P o s t , 1 9 7 1 ; Ng5rby and J e n s e n , 1971, 1 9 7 4 1 . 2 N a + c o u n t e r a c t s t h e e f f e c t o f K+ b u t h a s l i t t l e o r no e f f e c t i n i t s e l f ( a small e f f e c t of N a + sometimes s e e n i s p r o b a b l y c o m p e t i t i o n w i t h c o n t a m i n a t i o n K+) (Hegyvary and P o s t , 1 9 7 1 ; Ng5rby and J e n s e n , 1973, 1 9 7 4 ) . The e f f e c t of K+ h a s been confirmed i n d i r e c t b i n d i n g s t u d i e s r e p o r t e d i n s e v e r a l p a p e r s , b o t h w i t h ADP ( F i g . 1) (Kaniike e t a l . , 1 9 7 4 ; J e n s e n and O t t o l e n g h i , 1976) and ATP (Schoner e t a l . , 1 9 7 7 ; J e n s e n , Ng5rby, O t t o l e n g h i u n p u b l i s h e d ) , and w i t h t h e a n a l o g TNP-ATP (Moczydlowski and F o r t e s , 1 9 8 1 a ) . S i m i l a r l y , t h e f l u o r e s c e n t nucleot i d e s FTP and FDP bound t o N a , K - A T P a s e i n Na+-containing media can be d i s p l a c e d by T 1 + , K + , Rb+, N H 4 + , and Cs+ ( K a r l i s h e t a l . , 197813). A s w i l l b e s e e n below, t h e e f f e c t s of N a + and K+ on ATP b i n d i n g r e f l e c t t h e e x i s t e n c e of t w o major c o n f o r m a t i o n s o f t h e enzyme, o f t e n c a l l e d t h e N a + form, o r E l , w i t h a h i g h a f f i n i t y f o r ATP, and t h e K+ form, o r E2, w i t h a much lower a f f i n i t y f o r ATP. B e f o r e w e p r o c e e d , two i m p o r t a n t p o i n t s r e g a r d i n g t h e b i n d i n g r e s u l t s must be emphasized. F i r s t , t h e obs e r v a t i o n o f h i g h - a f f i n i t y b i n d i n g i n t h e a b s e n c e of added (or c o n t a m i n a t i n g ) monovalent c a t i o n s seems t o i n d i c a t e t h a t i n t h e absence of s p e c i f i c l i g a n d s , t h e E l form is predominant. T h i s , however, i s n o t n e c e s s a r i l y t h e case: J e n s e n and O t t o l e n g h i ( t h i s volume) have shown t h a t t h e a f f i n i t y f o r ADP i n t h e a b s e n c e o f added Na+ o r K+ i s d e p e n d e n t upon T r i s c o n c e n t r a t i o n . With 75 mM T r i s ( w i t h o u t N a + ) Kd f o r ATP w a s 0 . 2 v M , whereas a t 0 - 1 . 3 mM T r i s , K d was a b o u t two o r d e r s o f magnitude h i g h e r . T h i s a p p a r e n t l y unexpected o b s e r v a t i o n i s i n

2The o f t e n - q u o t e d s u g g e s t i o n o f H e g y v a r y and Post (1971) + t h a t K o p e n s u p a n e x t r a l o w - a f f i n i t y s i t e for ATP h a s not been c o n f i r m e d ( C . H e g y v a r y , p e r s o n a l c o m m u n i c a t i o n ) . It m u s t be emp h a s i z e d t h a t t e c h n i q u e s t h a t m e a s u r e bound ATP a s the d i f f e r e n c e b e t w e e n t o t a l and f r e e ATP may r e s u l t i n l a r g e e x p e r i m e n t a l errors i n the r e g i o n , w h e r e bound ATP i s o n l y a m i n u t e f r a c t i o n o f t o t a l ATP. For a d i s c u s s i o n on n o n s p e c i f i c TNP-ATP b i n d i n g , see M o c z y d l o w s k i and Fortes ( 2 9 8 1 a ) .

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a c c o r d a n c e w i t h t h e r e s u l t s o b t a i n e d by Skou and Esmann (1980) w i t h i n t r i n s i c f l u o r e s c e n c e of t h e enzyme, showi n g t h a t p r o t o n a t e d T r i 6 had an Na+-like e f f e c t on t h e c o n f o r m a t i o n o f t h e enzyme. Whether o r n o t t h i s means t h a t T r i s + b i n d s t o Na+ s i t e s i s n o t known. Second, whereas t h e b i n d i n g i s o t h e r m s f o r t h e h i g h a f f i n i t y b i n d i n g of n u c l e o t i d e s i n t h e a b s e n c e of K+ a r e s t r a i g h t l i n e s i n a S c a t c h a r d p l o t (see S e c t i o n I I , A ) , t h e y g e n e r a l l y become c u r v e d i n t h e p r e s e n c e o f K+ (see, e . g . , F i g . 1, which i s r e p r e s e n t a t i v e of a l a r g e number of b i n d i n g s t u d i e s i n o u r l a b o r a t o r y ) . TNP-ATP, t h e b i n d i n g o f which i s n o t v e r y s e n s i t i v e t o K+ (Moczydl o w s k i and F o r t e s I 1 9 8 1 a ) , may b e an e x c e p t i o n . The obs e r v a t i o n of s u c h c u r v a t u r e r e q u i r e s v e r y a c c u r a t e exp e r i m e n t s , and t h e p o s s i b l e " i n h o m o g e n e i t y " o f t h e b i n d i n g - s i t e p o p u l a t i o n t h a t i t may r e f l e c t i s e a s i l y m i s s e d i n more i n d i r e c t e x p e r i m e n t s . The above c u r v a t u r e d o e s n o t s e e m t o r e s u l t from a s y s t e m a t i c e x p e r i m e n t a l e r r o r r e l a t e d t o t h e d e c r e a s e i n a f f i n i t y f o r ATP s i n c e e x p e r i m e n t s w i t h f u r o s e m i d e and b u m e t a n i d e , b o t h of which d e c r e a s e a p p a r e n t a f f i n i t y of t h e enzyme f o r ATP, l e a d t o s t r a i g h t - l i n e S c a t c h a r d p l o t s ( J e n s e n and Ndrby, 1 9 7 9 ) . P o s s i b l e e x p l a n a t i o n s f o r t h e c u r v a t u r e i n t h e Scatchard p l o t s w i l l be discussed i n Section I I , D , b u t i t i s c l e a r t h a t t h e model f o r t h e K+ e f f e c t c a n n o t be a s s i m p l e a s t h a t o r i g i n a l l y p r o p o s e d by Ndrby and Jensen ( 1 9 7 1 ) . A t t h i s p o i n t o n e may a s k w h e t h e r t h e d e c r e a s e i n a p p a r e n t a f f i n i t y b r o u g h t a b o u t by K+ r e f l e c t s t h e absence o f ATP b i n d i n g t o EK. Ndrby and J e n s e n (1971) o r i g i n a l l y provided evidence f o r t h e e x i s t e n c e of K-E-ATP, and b a s e d on a s i m p l e model t h e y c a l c u l a t e d a d i s s o c i a t i o n c o n s t a n t f o r ATP from t h i s complex t o 0.69 PM. B u t , a s mentioned above, t h e more r e c e n t obs e r v a t i o n s t h a t S c a t c h a r d p l o t s i n t h e p r e s e n c e o f K+ are c u r v e d r e q u i r e more r e f i n e d models. T h e r e a r e a t l e a s t two o t h e r e q u i l i b r i u m - b i n d i n g e x p e r i m e n t s t h a t s p e a k i n f a v o r of t h e e x i s t e n c e of a K-E-ATP complex: f i r s t , t h e o b s e r v a t i o n by J e n s e n and O t t o l e n g h i ( t h i s volume) t h a t Rb+ ( a n d t h e r e f o r e i n a l l p r o b a b i l i t y a l s o K + ) b i n d s t o EATP; and s e c o n d , t h e e x p e r i m e n t s by Moczydlowski and F o r t e s + ( l 9 8 l a ) , showing t h a t e v e n i n t h e p r e s e n c e o f 20 mM K , ATP can d i s p l a c e TNP-ATP from t h e b i n d i n g s i t e . T h e r e seems t o b e no d o u b t , however, t h a t K-E-ATP h a s a Kd f o r ATP t h a t i s s e v e r a l o r d e r s of magnitude h i g h e r t h a n t h a t of EATP. Of e q u a l s i g n i f i c a n c e i s t h e work o f K a r l i s h e t a l . ( 1 9 7 8 b ) , K a r l i s h and Yates ( 1 9 7 8 ) , and Beauge and Glynn ( 1 9 8 0 ) which i s c o n c e r n e d w i t h t h e e f f e c t of ATP on t h e t r a n s f o r m a t i o n of t h e enzyme from E 2 (K+ form) t o E l

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( N a + f o r m ) . From t h e f i r s t - m e n t i o n e d a r t i c l e i t a p p e a r s t h a t a d d i t i o n of K + t o a m i x t u r e o f E l N a + , and FDP ( o r FTP) produced t h e r e l e a s e o f n u c l e o t i d e from t h e enzyme, and t h a t t h e r e w a s c o m p e t i t i o n between Na+ and K+ f o r t h i s e f f e c t . C o n v e r s e l y , e x p e r i m e n t s i n which N a + f FTP w a s added t o t h e enzyme p r e i n c u b a t e d w i t h K+ ( t h e K form) showed t h a t t h e n u c l e o t i d e a c c e l e r a t e d t h e c o n v e r s i o n of t h e K form t o t h e Na form, and i t i s n a t u r a l t o assume t h a t i t d i d s o by b i n d i n g t o EK. For t h e o v e r a l l mechanism i t i s i m p o r t a n t t h a t h e r e t h e n u c l e o t i d e s a c t e d w i t h o u t p h o s p h o r y l a t i n g t h e enzyme and w i t h o u t b e i n g hyd r o l y z e d . I n t h e s u b s e q u e n t a r t i c l e K a r l i s h and Yates (1978) m o n i t o r e d t h e e f f e c t of ATP on t h e a p p a r e n t r a t e c o n s t a n t f o r t h e E2 -t E l t r a n s f o r m a t i o n by measuring changes i n t h e i n t r i n s i c t r y p t o p h a n f l u o r e s c e n c e of t h e enzyme. The work by Beauge and Glynn (1980) a l s o used t h e enzyme's i n t r i n s i c t r y p t o p h a n f l u o r e s c e n c e t o d e t e r mine t h e e f f e c t of K+ and n u c l e o t i d e s on t h e b a l a n c e between t h e El and E 2 forms of t h e enzyme. S i n c e ATP had no e f f e c t on t h e i n t r i n s i c f l u o r e s c e n c e and s i n c e t h i s d i f f e r e d f o r t h e N a and t h e K f o r m s , i t c o u l d r e a d i l y b e d e m o n s t r a t e d t h a t ATP c a t a l y z e s t h e c o n v e r s i o n o f t h e K form of t h e enzyme t o t h e N a form. Although t h e r e s u l t s showed some s c a t t e r , t h e y were c o m p a t i b l e w i t h a s i m p l e model i n v o l v i n g o n l y one ATP s i t e , t h e a p p a r e n t a f f i n i t y Phosphorylao f which w a s dependent on K+ c o n c e n t r a t i o n . t i o n was n o t i n v o l v e d s i n c e ANPP(C)P and AMPP(NH)P behaved l i k e ATP. The d e t a i l e d t h e o r e t i c a l a n a l y s i s i n t h e i r a r t i c l e p o i n t s o u t t h e d i f f i c u l t i e s i n determining t h e K-E-ATP d i s s o c i a t i o n c o n s t a n t . Also, Jfdrgensen and K a r l i s h (1980) s t u d i e d t h e e f f e c t o f K+ and ATP on t h e t r y p t o p h a n f l u o r e s c e n c e o f t h e enzyme and, i n p r i n c i p l e , a r r i v e d a t t h e same r e s u l t s . The f o u r i n v e s t i g a t i o n s j u s t c i t e d were performed a t room t e m p e r a t u r e , s u g g e s t i n g t h a t t h e c o n c l u s i o n a r r i v e d a t i n e q u i l i b r i u m b i n d i n g exp e r i m e n t s a t 0-3OC may w e l l be v a l i d a t h i g h e r temperatures also. Although t h e i n t e r p r e t a t i o n of e x p e r i m e n t s i n which t h e o v e r a l l A T P a s e o r pNPPase r e a c t i o n i s s t u d i e d i s obv i o u s l y more d i f f i c u l t t h a n t h e i n t e r p r e t a t i o n o f e q u i librium experiments, the general trends i n the i n t e r p l a y between c a t i o n s and n u c l e o t i d e i n t e r a c t i o n w i t h N a , K ATPase o b s e r v e d i n t h e l a t t e r seems t o be r e f l e c t e d i n t h e more complete r e a c t i o n system. I n a series o f a r t i c l e s , Skou ( 1 9 7 4 a , b , c , 1979, a l s o t h i s volume) and Skou and Esmann (1980) have r e p o r t e d on " t h e e f f e c t of ATP on t h e . . ( N a + + K+) dependent enzyme system." C h a r a c t e r i z i n g t h e e f f e c t of l i g a n d s on t h e i n h i b i t i o n by NEM, Skou (1974a) proposed t h a t ATP and ADP ( i n a c o n c e n t r a t i o n range r a t h e r h i g h e r

.

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t h a n t h a t c h a r a c t e r i s t i c of h i g h - a f f i n i t y b i n d i n g ) i n e x p e r i m e n t s w i t h o u t h y d r o l y s i s , s h i f t t h e conformation of t h e enzyme from t h e K+ form toward t h e Na+ form. The n u c l e o t i d e e f f e c t on c o n f o r m a t i o n i s a l s o s e e n i n h y d r o l y s i s e x p e r i m e n t s (Skou, 1974b, 1979) a n d , as mentioned e a r l i e r , seems t o be t h e same f o r ATP and MgATP. I n r e c e n t a r t i c l e s by Skou ( 1 9 7 9 , a l s o t h i s volume) and by Skou and Esmann ( t h i s v o l u m e ) , t h e s e s t u d i e s a r e c o n t i n u e d t o i n c l u d e a l s o t h e r o l e of H+. Without g o i n g i n t o d e t a i l , t h e e f f e c t of ATP ill u s t r a t e d by t h e s e s t u d i e s may be summarized a s f o l l o w s : (1) Under t u r n o v e r c o n d i t i o n s , ATP i n c r e a s e s t h e a f f i n i t y of t h e enzyme f o r N a + r e l a t i v e t o t h a t f o r K+ (poss i b l y a t a n i n s i d e c a t i o n s i t e ) . The K O 5 f o r t h i s ATP e f f e c t i s l o w e r t h a n t h e KO. f o r ATP h y d r o l y s i s . ( 2 ) The i n t e r p l a y between N a , K+, H+, and ATP seems t o i n d i c a t e t h a t t h e N a + form i s f a v o r e d by low H+ concent r a t i o n and t h a t b i n d i n g of ATP ( a t low ATP c o n c e n t r a t i o n s ) p r o d u c e s a s i m i l a r e f f e c t by d e c r e a s i n g t h e p~ of t h e g r o u p ( s ) i n v o l v e d , t h e r e b y s t i m u l a t i n g d e p r o t o n a t i o n and c o n v e r s i o n t o t h e N a + form. ( 3 ) A t even h i g h e r pH v a l u e s , however, t h e e f f e c t of ATP becomes r a t h e r c o m p l i c a t e d i n t h a t low c o n c e n t r a t i o n s of ATP i n crease t h e a p p a r e n t a f f i n i t y f o r N a + , whereas h i g h e r ATP concentrations decrease the apparent a f f i n i t y There i s no u n e q u i v o c a l i n t e r p r e t a t i o n o f t h e s e exp e r i m e n t s , b u t t h e y s u p p o r t and c o n f i r m t h e w i d e s p r e a d view t h a t ATP may p l a y a c r u c i a l r o l e a t more t h a n one s t e p i n t h e h y d r o l y s i s c y c l e . Whether t h e s e i n t e r a c t i o n s of t h e enzyme w i t h ATP t a k e p l a c e a t d i f f e r e n t s i t e s o r a t a s i n g l e s i t e ( b i n d i n g r e g i o n ) t h a t changes a f f i n i t y f o r ATP depending on enzyme c o n f o r m a t i o n i s S t i l l open t o q u e s t i o n , a l t h o u g h a s i n g l e - s i t e mechanism seems t o be a b l e t o e x p l a i n most o f t h e i n f o r m a t i o n a v a i l a b l e .

5

.

D.

I S T H E R E MORE THAN ONE A T P - B I N D I N G MOLECULE?

S I T E PER ENZYME

I t h a s been c o n s i s t e n t l y o b s e r v e d t h a t N a , K - A T P a s e from a l l s o u r c e s i n v e s t i g a t e d e x h i b i t s a complex k i n e t i c b e h a v i o r when t h e r a t e o f ATP h y d r o l y s i s ( v ) i s measured a t f i x e d N a + and K+ c o n c e n t r a t i o n s as a f u n c t i o n o f MgATP c o n c e n t r a t i o n ( o r ATP c o n c e n t r a t i o n a t o p t i m a l [Mg2+]). Lineweaver-Burk p l o t s of l / v v e r s u s l/[ATP] show downward c u r v a t u r e , s u g g e s t i v e of e i t h e r inhomogeneity o f t h e enzyme p r e p a r a t i o n , homotropic n e g a t i v e c o o p e r a t i v i t y between two s u b s t r a t e s i t e s , o r a l l o s t e r i c a c t i v a t i o n o f ATP h y d r o l y s i s by ATP a t a l o w - a f f i n i t y s i t e . These obs e r v a t i o n s have s p u r r e d t h e p r o p o s a l o f models f o r

292

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Na,K-ATPase-catalyzed ATP h y d r o l y s i s i n v o l v i n g t h e s i multaneous e x i s t e n c e of high- and l o w - a f f i n i t y s i t e s f o r ATP, opening t h e p o s s i b i l i t y t h a t Na,K-ATPase h a s “ h a l f o f - t h e - s i t e s ’ ’ k i n e t i c s ( f o r r e f e r e n c e s and d i s c u s s i o n , see Robinson, 1976; Glynn and K a r l i s h , 1976; Robinson and F l a s h n e r , 1 9 7 9 a ; C a n t l e y , 1 9 8 1 ) . R e c e n t l y , however, s e v e r a l l a b o r a t o r i e s have i n d e p e n d e n t l y d e m o n s t r a t e d t h a t t h e complex s u b s t r a t e k i n e t i c s of Na,K-ATPase can be e x p l a i n e d by s i m p l e r models i n v o l v i n g o n l y o n e ATP-binding s i t e p e r enzyme m o l e c u l e . Thus, two g r u p s have shown t h a t t h e by now c l a s s i c a l and w i d e l y a c c e p t e d ( b u t see S e c t i o n IV) P o s t A l b e r s model f o r Na,K-ATPase w i l l y i e l d n o n l i n e a r l/v v e r s u s l/[ATP] p l o t s t h a t f i t t h e d a t a v e r y w e l l i f s u i t a b l e r a t e c o n s t a n t s are chosen (Smith e t a l . , 1980; Moczydlowski and F o r t e s , 1981b; see a l s o Kyte, 1 9 8 1 ; C a n t l e y e t a l . , t h i s volume). L i k e w i s e , t h e a l t e r n a t i v e model i n v o l v i n g two h y d r o l y t i c c y c l e s proposed from o u r l a b o r a t o r y ( P l e s n e r e t a l . , 1 9 8 1 ; see a l s o S e c t i o n I V ) o p e r a t e s w i t h o n l y one s i t e f o r ATP, and P l e s n e r ( t h i s volume) h a s shown t h a t t h i s model w i l l a l s o g i v e nonl i n e a r , downward-curved Lineweaver-Burk p l o t s . Before l e a v i n g t h e k i n e t i c s t u d i e s , it s h o u l d be p o i n t e d o u t t h a t t h e p u z z l i n g e f f e c t s of ATP on t h e pNPPase reaction--where micromolar c o n c e n t r a t i o n s o f ATP ( i n t h e p r e s e n c e o f N a + and K+) s t i m u l a t e pNPPase wherea s h i g h e r c o n c e n t r a t i o n s o f ATP w i l l i n h i b i t i t (Skou, 1 9 7 4 c ; Robinson, 1 9 7 6 ; Gache e t a l . , 1976)--can a l s o be e x p l a i n e d by a o n e - s i t e model as d i s c u s s e d by Robinson and F l a s h n e r (1979a) and C a n t l e y ( 1 9 8 1 ) . O f paramount importance f o r t h e complete u n d e r s t a n d i n g of the mechanism o f Na,K-ATPase i s t h e s o l u t i o n t o the following questions: W h a t i s t h e s i z e a n d s u b u n i t s t r u c t u r e o f t h e s m a l l e s t a c t i v e u n i t of e n z y m e a n d , r e l e v a n t €or t h i s o v e r v i e w , how many b i n d i n g s i t e s a r e there p e r u n i t ? There i s a t p r e s e n t no u n e q u i v o c a l

answer t o t h e s e q u e s t i o n s . U n t i l r e c e n t l y i t looked as i f t h e s m a l l e s t a c t i v e u n i t was an a 2 8 2 m o l e c u l e h a v i n g one h i g h - a f f i n i t y s i t e f o r e v e r y two a - c h a i n s , b u t s e v e r a l r e p o r t s have been p u b l i s h e d t h a t r e v i s e t h i s p i c t u r e by c l a i m i n g t h e o c c u r r e n c e of one ATP s i t e p e r one a-chain. One o f t h e key problems i n e s t a b l i s h i n g t h i s s t o i c h i o m e t r y i s t h e d e t e r m i n a t i o n of t h e p r o t e i n mass c o r r e s p o n d i n g t o 1 mole o f ATP s i t e . S e v e r a l g r o u p s now i n d e p e n d e n t l y c l a i m t h a t one of t h e h i t h e r t o a c c e p t e d p r o t e i n d e t e r m i n a t i o n s (Lowry method) o v e r e s t i m a t e s t h e amount of Na,K-ATPase p r o t e i n by up t o 3 5 % i n p u r e ATPase p r e p a r a t i o n s . A c o r r e c t i o n f o r t h i s l e a d s , of c o u r s e , immediately t o a n o t h e r ATP s i t e / a - c h a i n s t o i c h i o m e t r y Since t h e w i t h o u t any change i n t h e b i n d i n g of ATP.

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problem i s n o t q u i t e s e t t l e d , it s h a l l n o t be d i s c u s s e d f u r t h e r . The i n t e r e s t e d r e a d e r w i l l f i n d v a l u a b l e cont r i b u t i o n s t o t h i s problem i n t h e a r t i c l e s o f C r a i g and Kyte (1980) , Powell and C a n t l e y (1980) , Moczydlowski and F o r t e s (1981a1, Peters e t a l . ( 1 9 8 1 ) , B r o t h e r u s e t a l . (1981) , Kyte (1981) , Schuurmans Stekhoven et al. ( 1 9 8 1 ) , C a n t l e y et a l . ( t h i s volume) , i n C a n t l e y ' s r e v i e w ( 1 9 8 1 ) , and p r o b a b l y i n a number o f c o n t r i b u t i o n s t o t h i s volume. I n s t e a d , i n t h e r e m a i n d e r of t h i s s e c t i o n and i n S e c t i o n 111, I s h a l l d i s c u s s t h e f o l l o w i n g two q u e s t i o n s : (1) A r e t h e r e more s p e c i f i c ATP s i t e s p e r m o l e c u l e t h a n t h o s e t h a t c a n b e measured by h i g h - a f f i n i t y b i n d i n g , as d e s c r i b e d i n S e c t i o n I I , A ? and ( 2 ) D o t h e h i g h - a f f i n i t y s i t e s , a l t h o u g h c a p a b l e of c h a n g i n g c o n f o r m a t i o n t o l o w a f f i n i t y s i t e s ( S e c t i o n I I , C ) , c o n s t i t u t e a homogeneous p o p u l a t i o n o f i n d e p e n d e n t ATP s i t e s ? With r e g a r d t o t h e f i r s t q u e s t i o n , Schuurmans Stekhoven e t a 1 . (1981) performed e q u i l i b r i u m b i n d i n g e x p e r i m e n t s by means o f a f i l t r a t i o n t e c h n i q u e t h a t m e a s u r e s bound ATP d i r e c t l y and i s t h e r e f o r e b e t t e r s u i t e d t h a n o t h e r methods f o r m e a s u r i n g r e l a t i v e l y lowa f f i n i t y binding. I n t h e a b s e n c e of s p e c i f i c mono- and d i v a l e n t c a t i o n s t h e y found e x c l u s i v e l y homogeneous h i g h - a f f i n i t y b i n d i n of ATP and AMPP(NH)P, b u t i n t h e p r e s e n c e of 5 mM Mg2?, t h e AMPP(NH)P e x p e r i m e n t s d i s c l o s e d a l o w - a f f i n i t y b i n d i n g a l s o (ATP c o u l d n o t b e u s e d b e c a u s e o f h y d r o l y s i s ) . Although i t i s t e m p t i n g from t h e b i n d i n g s t o i c h i o m e t r y ( o n e h i g h - and one lowa f f i n i t y s i t e a t 5 mM Mg2+) t o a s s i g n f u n c t i o n a l i m p o r t a n c e t o t h i s "new" s i t e , t h e r e m i g h t b e o t h e r e x p l a n a t i o n s and t h u s more e x p e r i m e n t s a r e re u i r e d . First, t h e b i n d i n g c a p a c i t y i s a f u n c t i o n of Mg2+ c o n c e n t r a t i o n ( i t more t h a n d o u b l e d when Mg2+ w a s i n c r e a s e d from 0 . 5 t o 5 m M ) , which i s d i f f i c u l t t o e x p l a i n from e q u i l i b r i u m k i n e t i c s . An e q u i l i b r i u m model p r e d i c t s t h a t t h e b i n d i n g c a p a c i t y , d e t e r m i n e d by e x t r a p o l a t i o n t o a n i n f i n i t e l y h i g h f r e e ATP ( o r MgATP) c o n c e n t r a t i o n , would be c o n s t a n t € o r a l l c o n c e n t r a t i o n s of Mg h i g h e r t h a n t h e enzyme c o n c e n t r a t i o n . Second, Moczydlowski and F o r t e s (1981a) n o t e t h a t u n s p e c i f i c b i n d i n g i s sometimes s e e n w i t h t h e ATP a n a l o g u e TNP-ATP, p a r t i c u l a r l y i n t h e p r e s e n c e o f N a + o r Mg2+. One m i g h t a l s o s p e c u l a t e t h a t t h e i n c r e a s e d b i n d i n g (low a f f i n i t y ) i n t h e p r e s e n c e of Mg might r e f l e c t f o r m a t i o n o f E-AMPP ( N H ) P-Mg-AMPP ( N H ) P r a t h e r t h a n b i n d i n g o f AMPP(NH)P t o a new s i t e . I n t h i s c o n n e c t i o n one s h o u l d n o t e t h a t t h e c o m p l e x i t y cons t a n t f o r AMPP(NH)P-Mg i s c o n s i d e r a b l y h i g h e r t h a n t h a t of MgATP (Yount, 1 9 7 5 ) . S u p p o r t f o r t h e o b s e r v a t i o n t h a t Mg somehow i n c r e a s e s t h e b i n d i n g c a p a c i t y f o r cer-

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t a i n l i g a n d s comes from Skou and Esmann ( t h i s volume), In who s t u d i e d t h e b i n d i n g of e o s i n t o N a , K - A T P a s e . t h e p r e s e n c e o f N a + , e o s i n b i n d s c o m p e t i t i v e l y w i t h ATP t o a h i g h - a f f i n i t y s i t e , and when Mg i s p r e s e n t , t h e binding capacity f o r " s p e c i f i c " eos i n binding is i n c r e a s e d . The curved S c a t c h a r d p l o t s o b t a i n e d can be res o l v e d i n t o two s t r a i g h t l i n e s e a c h having t h e same o r d i n a t e ( B ~ i~n t e~ r c) e p t , i n d i c a t i v e of a "Mg-created" site f o r eosin. One more e q u i l i b r i u m b i n d i n g s t u d y d e m o n s t r a t e s ATP b i n d i n g beyond t h e high-af f i n i t y b i n d i n g . Yamaguchi and Tonomura (1980) used a f i l t r a t i o n t e c h n i q u e t o measure ATP b i n d i n g i n t h e abeence of c a t i o n l i g a n d s a t ATP conTheir r e s u l t s c e n t r a t i o n s r a n g i n g from 0 . 1 I I M t o 1 mM. i n d i c a t e d t h e e x i s t e n c e of t h e "normal" high-af f i n i t y s i t e w i t h some e x t r a l o w - a f f i n i t y b i n d i n g . T h i s b i n d i n g had reached a value of a b o u t 2 moles/mole h i g h - a f f i n i t y s i t e a t 1 mM ATP, w i t h s t i l l no s i g n s t h a t s a t u r a t i o n was b e i n g approached. Smith et a l . (1980) and Moczydlowski and F o r t e s (1981a) a l s o r e p o r t e d l o w - a f f i n i t y , n o n s a t u r a b l e b i n d i n g of ATP o r TNP-ATP which t h e y claim t o be u n s p e c i f i c , whereas Schuurmans Stekhoven e t a l . (1981) d i d n o t o b s e r v e l o w - a f f i n i t y b i n d i n g ( i n t h e absence of Mg), a l t h o u g h t h e y used t h e same e x p e r i m e n t a l approach a s Yamaguchi and Tonomura ( 1 9 8 0 ) . I t i s c l e a r t h a t f u r t h e r e x p e r i m e n t s a r e needed b e f o r e f u n c t i o n a l s i g n i f i c a n c e ( i f any) can be a s s i g n e d t o t h e l o w - a f f i n i t b i n d i n g s i t e ( s ) . One might a l s o p o i n t o u t t h a t t h e i n t e r p l a y between monovalent c a t i o n s and ATP, i n t e r p r e t e d by Yamaguchi a n d Tonomura (1980) a s o c c u r r i n g a t b o t h high- and l o w - a f f i n i t y ATP s i t e s , can e q u a l l y w e l l be e x p l a i n e d by a o n e - s i t e model where t h e s i t e may change affinity. A few o t h e r s t u d i e s m u s t be mentioned i n c o n n e c t i o n w i t h t h e f i r s t of t h e two above q u e s t i o n s . PatzeltWenczler and Schoner (1981) found t h a t t h e i n a c t i v a t i o n o f N a , K - A T P a s e w i t h t h e d i s u l f i d e of t h i o i n o s i n e t r i phosphate (SnoPPP) 2 proceeded w i t h two d i f f e r e n t v e l o c i t i e s , i n d i c a t i n g r e a c t i o n w i t h t w o d i f f e r e n t t y p e s of SH-groups. ATP p r o t e c t e d a g a i n s t t h e i n a c t i v a t i o n of b o t h t y p e s i n a manner s u g g e s t i v e of two ATP b i n d i n g s i t e s w i t h Kd = 3 and 7 7 P M , r e s p e c t i v e l y . The number of s u l f h y d r y l g r o u p s t h a t r e a c t e d w a s f o u r times t h e number of p h o s p h o r y l a t a b l e s i t e s . I n a similarl,y\comp l i c a t e d s t u d y , Koepsell and O l l i g ( t h i s volume) determined t h e e f f e c t of ATP, ADP, and AMPP(NH)P on t h e pNPPase a c t i v i t y and on t h e i n h i b i t i o n o f Na,K-ATPase by t h e SH-group r e a c t a n t s NbsGITP and DTNB. Although the r e s u l t s s e e m d i f f i c u l t t o i n t e r p r e t , they f i n d t h a t t h e "data p r o v i d e arguments f o r t h e e x i s t e n c e of a t

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l e a s t two s i m u l t a n e o u s l y p r e s e n t n u c l e o t i d e b i n d i n g s i t e s i n t h e (Na+ + K+)-ATPase." F i n a l l y , b e a r i n g on t h e f i r s t q u e s t i o n are t h e two i n v e s t i g a t i o n s on t h e i n a c t i v a t i o n of t h e A T P a s e a c t i v i t y (Grosse e t a 2 . , 1 9 7 8 , 1 9 7 9 ) and t h e A T P a s e and pNPPase a c t i v i t y ( C a n t l e y e t al., 1978a, 1 9 7 9 ; Smith e t al., 1 9 8 0 ) by NBD-Cl, and t h e a l r e a d y mentioned photoi n a c t i v a t i o n s t u d y by Munson ( 1 9 8 1 ; S e c t i o n I 1 , B ) . Based on t h e k i n e t i c s f o r t h e p r o t e c t i v e e f f e c t of ATP toward i n a c t i v a t i o n , Repke's l a b o r a t o r y p r e s e n t s a model f o r Na,K-ATPase w i t h two c a t a l y t i c c e n t e r s , p r i m a r i l y e q u i v a l e n t , b u t e x h i b i t i n g homotropic n e g a t i v e c o o p e r a t i v i t y of ATP b i n d i n g . Here t h e NBD-C1 w a s used a s a t h i o l r e a g e n t . C a n t l e y ' s group, on t h e o t h e r hand, f i n d s t h a t NBD-C1 under t h e i r c o n d i t i o n s reacts w i t h two t y r o s i n e r e s i d u e s p e r mole of enzyme (as judged from p r o t e i n s p e c t r a and [14C]NBD-C1 i n c o r p o r a t i o n ) . A t f i r s t t h e s e l a t t e r r e s u l t s were c o n s i d e r e d by t h e a u t h o r s t o be s u p p o r t i v e of a two-ATP-site model i n which t h e d e s t r u c t i o n of one s i t e l e d t o l o s s of ATPase a c t i v i t y , whereas two s i t e s had t o be blocked b e f o r e pNPPase a c t i v i t y d i s a p p e a r e d , s i n c e t h e A T P a s e a c t i v i t y showed a s i n g l e e x p o n e n t i a l and t h e pNPPase a c t i v i t y a double e x p o n e n t i a l decay i n t h e p r e s e n c e of NBD-C1. R e c e n t l y , however, an a l t e r n a t i v e e x p l a n a t i o n was o f f e r e d (Smith e t a l . , 1 9 8 0 ) i n v o l v i n g two t y r o s i n e residues a t o n e ATP s i t e . Although b o t h r e s i d u e s m u s t be i n t a c t f o r t h e A T P a s e r e a c t i o n , t h e r e would s t i l l be room a t t h e s u b s t r a t e s i t e f o r pNPP when o n l y one t y r o s i n e r e s i d u e i s blocked. The r a t h e r e x t e n s i v e s t u d y by Munson (1981) u s i n g CrATPa (see S e c t i o n I 1 , B ) t o i n h i b i t ATPase a c t i v i t y , t o d i s p l a c e TNP-ATP i n b i n d i n g e x p e r i m e n t s , o r t o photoi n a c t i v a t e t h e enzyme i s i n l i n e w i t h o n e - s i t e t h e o r y . Munson's concludes: "these r e s u l t s provide evidence € o r a s i n g l e , homogeneous p o p u l a t i o n of ATP-binding s i t e s on ( N a + + K+)-ATPase whose a f f i n i t y can be s i g n i f i c a n t l y a l t e r e d depending on t h e p a r t i c u l a r l i g a n d c o n d i t i o n s under which b i n d i n g i s determined." The second q u e s t i o n , whether t h e h i g h - a f f i n i t y ATP s i t e s c o n s t i t u t e a homogeneous p o p u l a t i o n of independent s i t e s , i s n o t e a s i l y answered e i t h e r . Under some e q u i l i b r i u m b i n d i n g c o n d i t i o n s , t h e enzyme d e f i n i t e l y behaves a s i f t h e r e w a s o n l y a s i n g l e c l a s s of independent n u c l e o t i d e s i t e s and, a s w e have s e e n , t h e m a j o r i t y of d a t a from o t h e r t y p e s of experiments a r e s u f f i c i e n t l y e x p l a i n e d by a model i n v o l v i n g one ATP s i t e t h a t changes a f f i n i t y a c c o r d i n g t o i t s l i g a n d e d s t a t e . On t h e o t h e r hand, d e v i a t i o n s from t h i s s i m p l e p i c t u r e become appar e n t i n e q u i l i b r i u m b i n d i n g s t u d i e s under s p e c i a l condi-

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t i o n s ( a s a l r e a d y mentioned i n S e c t i o n I 1 , C ) where t h e r e s u l t s always l e a d t o upward-curved S c a t c h a r d p l o t s (e.g., Fig. 1). There are a number o f p o s s i b l e models t h a t w i l l conform t o upward-curved S c a t c h a r d p l o t s ( f o r d i s c u s s i o n of t h e problems and p i t f a l l s e n c o u n t e r e d i n t h e i n t e r p r e t a t i o n of such d a t a , see N@rby e t a l . , 1 9 8 0 ) . Among (1) two o r more p o p u l a t i o n s o f them are t h e f o l l o w i n g : independent s i t e s w i t h d i f f e r e n t a f f i n i t i e s f o r t h e ( l a ) inhomogeneous enzyme p r e p a r a t i o n , p a r t i a l ligand: d e n a t u r a t i o n , and ( l b ) p r e e x i s t i n g d i f f e r e n t s i t e s on t h e molecule , ( 2 ) homotropic n e g a t i v e c o o p e r a t i v i t y between two o r more s i t e s on t h e enzyme; and ( 3 ) h e t e r o t r o p i c a l l y induced d i f f e r e n c e s i n t h e a f f i n i t y o f t h e l i g a n d f o r two o r more o t h e r w i s e i d e n t i c a l s i t e s on t h e enzyme. The n a t u r e of t h e e x p e r i m e n t s and t h e a c c u r a c y o f t h e d a t a do n o t o f t e n j u s t i f y t h e r e s o l u t i o n o f c u r v e d S c a t c h a r d p l o t s i n t o more t h a n two s t r a i g h t - l i n e d compon e n t s (see NBrby e t a 1 ., 1 9 8 0 ) . I n t h e case of models ( l b ) , (2), and ( 3 ) t h e s e l i n e s s h o u l d have i d e n t i c a l i n t e r c e p t s on t h e a x i s r e p r e s e n t i n g bound l i g a n d , whereas i n t h e case o f model ( l a ) , t h e i n t e r c e p t s , which c o r respond t o t h e s i t e c o n c e n t r a t i o n s , need n o t be e q u a l . I n t h e a b s e n c e o f added monovalent c a t i o n s , nucleot i d e b i n d i n g d a t a f o r p u r i f i e d ( d e t e r g e n t - a c t i v a t e d ) enzyme p r e p a r a t i o n s g i v e l i n e a r S c a t c h a r d p l o t s (see Section 1 1 , A ) . However, two r e p o r t s show curved b i n d i n g i s o t h e r m s w i t h c r u d e , n o t d e t e r g e n t - t r e a t e d , microsomal p r e p a r a t i o n s u n d e r such c o n d i t i o n s : ADP b i n d i n g t o Na,K-ATPase from t h e s p i n y d o g f i s h ( J e n s e n and O t t o l e n g h i , 1976) and ATP b i n d i n g t o k i d n e y enzyme from p i g (Hansen e t a l . , 1 9 7 9 ) . I n t h e l a t t e r case, c u r v e d S c a t c h a r d p l o t s were a l s o found f o r v a n a d a t e b i n d i n g and o u a b a i n b i n d i n g s u p p o r t e d by ATP o r v a n a d a t e , and a model i n v o l v i n g n e g a t i v e c o o p e r a t i v i t y between a g g r e It g a t e s of s u b u n i t s was proposed t o e x p l a i n t h e d a t a . w a s s u g g e s t e d t h a t d e t e r g e n t t r e a t m e n t more o r less abolished subunit i n t e r a c t i o n . By f a r t h e m a j o r i t y of b i n d i n g d a t a come from d e t e r g e n t a c t i v a t e d N a , K - A T P a s e , and w i t h t h e s e p r e p a r a t i o n s , c u r v a t u r e of t h e S c a t c h a r d i s o t h e r m s a l m o s t i n v a r i a b l y o c c u r s when n u c l e o t i d e b i n d i n g i s measured i n t h e p r e s e n c e o f K+ (Ngkby and J e n s e n , 1974; Schoner e t a l . , 1 9 7 7 ; t h i s a r t i c l e , F i g . 1; and s e v e r a l u n p u b l i s h e d experiments, also with q u i t e , p u r e p r e p a r a t i o n s ) . Since t h e i r d a t a f i t a t w o - s i t e model of t y p e (3) w e l l , Schoner e t a l . (1977) c o n s i d e r s u c h c u r v a t u r e t o be due t o t h e f a c t " t h a t K+ i o n s i n d u c e a n e g a t i v e c o o p e r a t i v i t y of t h e ATP b i n d i n g s i t e s . " The p o s s i b l e i n t e r p r e t a -

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t i o n s o f o u r work a r e p r e s e n t l y u n d e r c o n s i d e r a t i o n . 3 I n t h e a n a l y s i s o f t h e s e phenomena some c h a r a c t e r i s t i c s o f o u a b a i n b i n d i n g s h o u l d be t a k e n i n t o a c c o u n t . I n p u r i f i e d enzymes t h e o u a b a i n and n u c l e o t i d e b i n d i n g cap a c i t i e s are e q u a l . But, i n t h e absence of added monov a l e n t c a t i o n s , (Mg + P i ) - s u p p o r t e d o u a b a i n b i n d i n g ( i n c o n t r a s t t o n u c l e o t i d e binding) g i v e s curved Scatchard p l o t s w i t h enzyme from ox b r a i n and a d d i t i o n o f K t e n d s t o s t r a i g h t e n t h e l i n e s (Hansen, 1976; Schoner e t a l . , 1 9 7 7 ) . Both a u t h o r s found t h e i r d a t a t o be c o m p a t i b l e w i t h two p o p u l a t i o n s o f s i t e s p r e s e n t i n t h e p r o p o r t i o n 6 0 : 4 0 (Hansen, 1976) and 5 0 : 5 0 (Schoner e t a l . , 1 9 7 7 ) . F i n a l l y , it i s of s i g n i f i c a n c e t h a t Forbush and Hoffman (1979) have shown t h a t t h e h i g h - a f f i n i t y s u b s t r a t e s i t e f o r ATP and t h e o u a b a i n s i t e a r e on t h e same a-chain-an o b s e r v a t i o n which p o s e s some r e s t r i c t i o n on t h e i n t e r p r e t a t i o n s of t h e phenomena mentioned above. E.

C O N C L U D I N G REMARKS

I t a p p e a r s from t h e f o r e g o i n g t h a t t h e m a j o r i t y o f t h e d a t a on n u c l e o t i d e i n t e r a c t i o n w i t h N a , K - A T P a s e conform t o a model w i t h o n l y one ATP s i t e p e r enzyme molec u l e . On t h e o t h e r hand, i t seems o b v i o u s t h a t u n d e r c e r t a i n e x p e r i m e n t a l c o n d i t i o n s s u c h a model i s n o t ent i r e l y s a t i s f a c t o r y . C a n t l e y e t a l . ( t h i s volume) sugg e s t s t h a t a l t h o u g h s u b u n i t i n t e r a c t i o n may n o t be ess e n t i a l f o r the c a t a l y t i c p r o p e r t i e s o f t h e enzyme (and therefore n o t apparent), s t r u c t u r a l f e a t u r e s involving more t h a n one c a t a l y t i c p e p t i d e may b e i m p o r t a n t ( o r expressed) under o t h e r c o n d i t i o n s .

31t m u s t be e m p h a s i z e d t h a t a l t h o u g h the a f f i n i t y of E a n d EK for ATP a r e q u i t e d i f f e r e n t , b i n d i n g i s o t h e r m s for ATP b i n d i n g

i n the p r e s e n c e o f f i x e d [Kt] w o u l d s t i l l g i v e s t r a i g h t l i n e s i n the S c a t c h a r d p l o t i f there was o n l y one A T P - b i n d i n g s i t e and a l l e n z y m e s p e c i e s w e r e i n e q u i l i b r i u m u n d e r the c o n d i t i o n s o f b i n d i n g : EATP- E- EK- EKATP

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INTERACTION

WITH VANADATE

During t h e f e w y e a r s t h a t have e l a p s e d s i n c e t h e d i s c o v e r y of v a n a d a t e a s a p o t e n t Na,K-ATPase i n h i b i t o r c o n t a m i n a t i n g commercial ATP from e q u i n e m u s c l e (Cantley e t a l . , 1 9 7 7 1 , many a s p e c t s of t h e i n t e r a c t i o n of t h i s compound w i t h Na,K-ATPase have been s t u d i e d . The main c h a r a c t e r i s t i c s of t h i s i n t e r a c t i o n have been reviewed by Simons ( 1 9 7 9 1 , Robinson and F l a s h n e r ( 1 9 7 9 a ) , J6rgensen ( 1 9 8 0 1 , and Schuurmans Stekhoven and Bonting ( 1 9 8 1 1 , and t h e r e a d e r i s r e f e r r e d t o t h e s e reviews i f n o t o t h e r w i s e i n d i c a t e d i n t h e f o l l o w i n g . Valuable o b s e r v a t i o n s and r e f e r e n c e s a r e a l s o found i n t h e a r t i c l e of Beauge e t a l . ( 1 9 8 0 ) on t h e e f f e c t of vanadate on t h e sodium pump. There i s v e r y convincing e v i d e n c e ( a l t h o u g h circums t a n t i a l ) t h a t vanadate b i n d s n o n c o v a l e n t l y a t t h e subs t r a t e s i t e of Na,K-ATPase ( C a n t l e y e t a l . , 1 9 7 9 ; C a n t l e y , 1981, pp. 2 2 0 - 2 2 2 ) : 1. Vanadate i n v e r y l o w c o n c e n t r a t i o n s i n h i b i t s s e v e r a l ATPases. 2. I t i n h i b i t s Na,K-ATPase from t h e c y t o p l a s m i c s i d e of t h e membrane, where t h e ATP s i t e i s l o c a t e d . 3 . The i n h i b i t i o n i s c o m p e t i t i v e l y c o u n t e r a c t e d by ATP ( C a n t l e y et al., 1978b, 1 9 7 9 ; Smith e t a l . , 1980) 4. S t r u c t u r a l l y , vanadate may be c o n s i d e r e d as a t r a n s i t i o n s t a t e a n a l o g of i n o r g a n i c phosphate (see d i s c u s s i o n i n C a n t l e y et a l . , 1978b) and a s such it may be expected t o f i t e x t r e m e l y w e l l i n t o a phosphate s u b s i t e of ( c e r t a i n c o n f i g u r a t i o n s o f ) t h e ATP-binding s i t e . 5. Mg2+ + v a n a d a t e c a t a l y z e s t h e f o r m a t i o n of a n enzyme-ouabain corn l e x v e r y s i m i l a r t o t h a t o b t a i n e d i n t h e p r e s e n c e of Mg3+ and P i (Hansen, 1 9 7 9 ; Hansen et a l . 1979). 6. Vanadate b i n d i n g , l i k e p h o s p h o r y l a t i o n , t r a p s a d i v a l e n t c a t i o n on t h e enzyme (Smith et a l . , 1 9 8 0 ) . E s p e c i a l l y p e r t i n e n t t o t h e t h e s i s of t h i s overview (see I n t r o d u c t i o n ) - - t h a t t h e r e i s o n l y one ATP s i t e p e r a c t i v e enzyme u n i t - - a r e t h e s t u d i e s on vanadate b i n d i n g and t h e ATP-vanadate c o m p e t i t i o n . The f i r s t two e q u i l i b r i u m s t u d i e s on b i n d i n g of vanadate t o p u r i f i e d Na,K-ATPase seemed t o be i n c o n f l i c l w i t h one a n o t h e r . C a n t l e y et a l . (197833, 1979) found t w c vanadate s i t e s , one high- and one l o w - a f f i n i t y , p e r ouabain-binding s i t e , whereas Hansen e t a l . ( 1 9 7 9 ) d i d n o t see t h e l o w - a f f i n i t y s i t e . Both on t h e b a s i s of t h e b i n d i n g r e s u l t s and on s i m u l t a n e o u s k i n e t i c s t u d i e s on vanadate i n h i b i t i o n , C a n t l e y ' s group claimed proof of

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t h e s i m u l t a n e o u s e x i s t e n c e o f two d i f f e r e n t ATP s i t e s , b o t h a b l e t o b i n d v a n a d a t e , on t h e a c t i v e u n i t . Recentl y , however, t h i s group h a s e x t e n d e d and r e e v a l u a t e d t h e i r f i r s t e x p e r i m e n t s and on t h i s b a s i s t h e y c o n c l u d e " t h a t only a s i n g l e nucleotide binding s i t e e x i s t s p e r f u n c t i o n a l u n i t of enzyme" (Smith et al., 1980; C a n t l e y , 1981, p . 2 2 8 ) . I n t h i s d i s c u s s i o n t h e y s t a t e t h a t t h e i r s t u d i e s a r e c o n s i s t e n t w i t h t h e absence o f a h i g h a f f i n i t y ATP s i t e o n t h e v a n a d a t e - t r a p p e d enzyme, and t h e y f u r t h e r a r g u e t h a t "no low a f f i n i t y ATP s i t e e x i s t s (1) v a n a d a t e b i n d i n g i n t h e presence of vanadate s i n c e : i s c o m p e t i t i v e w i t h low a f f i n i t y ATP b i n d i n g i n k i n e t i c experiments and ( 2 ) 1 t o 4 m~ ATP had no e f f e c t on v a n a d a t e release from t h e enzyme." They v e n t u r e t h e p o s s i b i l i t y t h a t t h e l o w - a f f i n i t y v a n a d a t e b i n d i n g obs e r v e d i n t h e i r f i r s t s t u d y may be due t o o c c u p a t i o n of a second p h o s p h a t e p o s i t i o n i n t h e same n u c l e o t i d e s i t e where t h e h i g h - a f f i n i t y v a n a d a t e b i n d i n g o c c u r s . The f a i r l y complex i n t e r p l a y between v a n a d a t e and ATP s e e n i n k i n e t i c i n h i b i t i o n s t u d i e s i s t h u s no l o n g e r cons i d e r e d e v i d e n c e of more t h a n one b i n d i n g s i t e f o r vanad a t e ( o r ATP) p e r a c t i v e enzyme u n i t , s i n c e it i s j u s t a s w e l l e x p l a i n e d by t h e model mentioned i n S e c t i o n II,D i n which one ATP s i t e changes a f f i n i t y f o r ATP d u r i n g t h e hydrolytic cycle. Another approach t o t h e problem r e g a r d i n g t h e numb e r of s i t e s h a s c o n s i s t e d o f s t u d i e s on t h e e f f e c t o f ATP and v a n a d a t e on c e r t a i n E-ouabain complexes. When t h e E-ouabain complex i s formed i n t h e p r e s e n c e o f Mg + P i , i t i s g e n e r a l l y assumed t h a t P i p h o s p h o r y l a t e s a s p e c i f i c s i t e on t h e enzyme. The o b s e r v a t i o n t h a t ATP and c e r t a i n a n a l o g s s t i m u l a t e d t h e release of o u a b a i n from s u c h complexes (Tobin et a l . , 1974) w a s t h e r e f o r e t a k e n as e v i d e n c e f o r t h e s i m u l t a n e o u s e x i s t e n c e of a p h o s p h o r y l a t e d s i t e and of an ATP-binding s i t e ( e . g . , Glynn and K a r l i s h , 1 9 7 5 b ) . T h i s e x p l a n a t i o n o f t h e ATP e f f e c t d o e s n o t seem t o h o l d , however, s i n c e it h a s now been shown t h a t once t h e P-E-ouabain complex i s formed, The unphosphorylP i l e a v e s i t much f a s t e r t h a n o u a b a i n . a t e d E-ouabain complex presumably has a n open ATP s i t e s i n c e v a n a d a t e b i n d s t o it w i t h h i g h a f f i n i t y (Hansen, t h i s v o l u m e ) . These o b s e r v a t i o n s w e r e e x t e n d e d by t h e f i n d i n g t h a t ATP i s u n a b l e t o a c c e l e r a t e o u a b a i n release from t h e vanadate-E-ouabain complex a p p a r e n t l y bec a u s e v a n a d a t e and o u a b a i n always a r e p r e s e n t o n t h e enzyme s i m u l t a n e o u s l y under t h e s e c o n d i t i o n s . The complex formed from o u a b a i n , Mg, and v a n a d a t e i s a l s o u n a b l e t o b i n d any e x t r a v a n a d a t e ( u n l i k e t h a t formed from Mg and P i , see above) (Hansen, t h i s volume). A o n e - s i t e model i s thus able t o a l s o explain t h e s e r e s u l t s .

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The c o n f o r m a t i o n o f t h e enzyme f a v o r e d by v a n a d a t e Whereas ATP, a s m e n t i o n e d , b i n d s with high a f f i n i t y t o t h e s u b s t r a t e s i t e i n the conform a t i o n c o r r e s p o n d i n g t o t h e N a + form and w i t h a n a f f i n i t y s e v e r a l o r d e r s of magnitude lower t o t h e K+ form, t h e c o n v e r s e seems t o be t h e case f o r v a n a d a t e . The r o l e o f monovalent c a t i o n s and enzyme c o n f o r m a t i o n i s d i s c u s s e d e x t e n s i v e l y by C a n t l e y et a l . ( 1 9 7 8 b ) , Beau96 (1979) , Beauge et a l . (1980) , Smith et a l . (1980) , and K a r l i s h and P i c k ( 1 9 8 1 ) . The v a n a d a t e i n t e r a c t i o n w i t h t h e enzyme may t h u s , l i k e t h e nucleotide i n t e r a c t i o n , i l l u s t r a t e t h e concept of a s u b s t r a t e s i t e t h a t c a n e x i s t i n a t l e a s t two c h a r a c t e r i s t i c a l l y d i f f e r e n t c o n f o r m a t i o n s , and t h e r e i s , w i t h t h e p r e s e n t e v i d e n c e , no need f o r a model w i t h more t h a n o n e v a n a d a t e ( s u b s t r a t e ) s i t e a t a t i m e .

i s t h e K+ form, E 2 .

IV.

PHOSPHORYLATION

When a Na,K-ATPase p r e p a r a t i o n i s i n c u b a t e d w i t h + Na+ + [y-32P]ATPI a v e r y r a p i d i n c o r p o r a t i o n , even a t O°C, o f 32P i n t o t h e p r o t e i n o c c u r s . The phospho-enzyme bond i s a c i d - s t a b l e which makes it poss i b l e t o d e t e r m i n e p r o t e i n - b o u n d 35P q u a n t i t a t i v e l y i n a n a c i d p r e c i p i t a t e from t h e r e a c t i o n m i x t u r e . Under o p t i m a l c o n d i t i o n s f o r t h e E-32P f o r m a t i o n a t O°C ( e . g . , 1 mM Mg2+, 1 0 - 2 0 mM N a + , 1 0 p~ [y-32P]ATP), t h e t u r n o v e r of t h e A T P a s e i s low and a s t e a d y - s t a t e l e v e l of E-32P i s o b t a i n e d . T h i s l e v e l c o r r e s p o n d s t o t h e number of p h o s p h o r y l a t a b l e s i t e s , which, a s mentioned i n S e c t i o n I , i s e q u a l t o t h e number o f o u a b a i n - , v a n a d a t e - , and h i g h a f f i n i t y n u c l e o t i d e - b i n d i n g s i t e s . The enzyme can a l s o be p h o s p h o r y l a t e d by Mg2+ + K+ + P i , and w i t h o p t i m a l c o n c e n t r a t i o n s of t h e s e l i g a n d s 1 mole of p h o s p h a t e b i n d s c o v a l e n t l y p e r mole of a c t i v e s i t e , t h e l a t t e r b e i n g d e t e r m i n e d by 3 2 P i n c o r p o r a t i o n from [y-32P]ATP ( P o s t et a l . , 1975; T a n i g u c h i and P o s t , 1975; Schuurmans Stekhoven et al., 1 9 8 0 ) . The p h o s p h a t e i s bound t o a n a s a r t y 1 g r o u p , and t h e c o v a l e n t E-P bond formed w i t h Mg + + K+ + P i i s c h e m i c a l l y , b u t n o t k i n e t i c a l l y (see below) , i d e n t i c a l t o t h a t formed from Mg2+ + N a + + ATP ( e . g . , S i e g e 1 et a l . , 1 9 6 9 ; Schuurmans Stekhoven et a ] . , 1 9 8 0 ) . T h e r e i s c o n v i n c i n g e v i d e n c e t h a t t h e s i t e on which t h e enzyme i s p h o s p h o r y l a t e d i s i d e n t i c a l t o ( p a r t o f ) t h e high-af f i n i t y ( s u b s t r a t e ) s i t e f o r ATP F i r s t , KO. 5 f o r ATP i n t h e p h o s p h o r y l a t i o n r e a c t i o n i s c l o s e t o t h e d i s s o c i a t i o n c o n s t a n t f o r t h e h i g h - a f f i n i t y E-ATP complex (Kanazawa et a l . , 1970; see a l s o NBrby and J e n s e n , 1971, Mg2+

3

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301

THE SUBSTRATE SITE OF Na,K-ATPase

and S e c t i o n I I , A of t h i s a r t i c l e ) . Second, enzyme p r e incubated with [ Y - ~ ~ P I A T P o ,r w i t h [ Y - ~ ~ P I A T+P N a + o r Mg2+ w i l l i n i t i a l l y form E-32P w i t h t h e same s p e c i f i c r a d i o a c t i v i t y as [ Y - ~ ~ P ] A Ta P f t e r t h e missing c a t i o n s a r e added t o g e t h e r w i t h u n l a b e l e d ATP. This i n d i c a t e s t h a t i t i s t h e prebound ATP t h a t p h o s p h o r y l a t e s t h e enzyme ( P o s t e t a l . , 1 9 6 9 ; Mdrdh and P o s t , 1 9 7 7 ) . T h i r d , ATP can b e s y n t h e s i z e d from ADP and P i when E-P i s f i r s t formed from Mg2+ + K+ + P i , and N a + + ADP a r e t h e n added ( e . g . , T a n i g u c h i and P o s t , 1 9 7 5 ) . T h i s p r o c e s s i s p r o b a b l y t h e e n z y m a t i c e q u i v a l e n t o f t h e backward r u n n i n g o f t h e pump t h a t can be d e m o n s t r a t e d w i t h i n t a c t c e l l s ( G a r r a h a n and Glynn, 1 9 6 7 ; Glynn and Lew, 1 9 7 0 ) . A s i s w e l l known, t h e p h o s p h o r y l a t e d enzyme can e x i s t i n a t l e a s t two c o n f o r m a t i o n s , u s u a l l y c a l l e d E l S P and E2-P. The f o r m e r i s t r a d i t i o n a l l y c a l l e d "ADPs e n s i t i v e EP" s i n c e i t reacts w i t h ADP t o form ATP, and t h e l a t t e r i s c a l l e d "K+-sensitive EP" because a d d i t i o n o f K+ accelerates i t s s l o w , s p o n t a n e o u s h y d r o l y s i s t o E and P i . These p r o p e r t i e s formed t h e b a s i s f o r t h e o r i g i n a l p r o p o s a l by A l b e r s ( 1 9 6 7 ) and P o s t e t a l . ( 1 9 7 2 ) of a r e a c t i o n s e q u e n c e f o r t h e (Na+ + K + ) - s t i m u l a t e d ATP h y d r o l y s i s , which i n c l u d e d t h e two phosphoenzymes i n t e r m e d i a t e s . The c r u c i a l r o l e o f t h e p h o s p h o i n t e r m e d i a t e s i s now w i d e l y a c c e p t e d , and t h e P o s t - A l b e r s scheme o r expanded v e r s i o n s t h e r e o f i s a l m o s t u n i v e r s a l l y u s e d as a frame o f r e f e r e n c e i n ATPase k i n e t i c s ( K a r l i s h e t a l . , 1978b; Robinson and F l a s h n e r , 1979a; C a n t l e y , 19811, a l though Skou ( 1 9 7 5 ) , Whittam and C h i p p e r f i e l d (19751, and Klodos and N&rby ( 1 9 7 9 ) have d i s c u s s e d o t h e r p o s s i b l e mechanisms (see a l s o b e l o w ) . A r e c e n t a r t i c l e from t h i s l a b o r a t o r y (Klodos e t a l . , 1 9 8 1 ) c h a r a c t e r i z e d t h e k i n e t i c s of t h e r e a c t i o n s o f t h e s e phosphoenzymes ( i n t h e a b s e n c e of K + ) and g a v e s e v e r a l r e f e r e n c e s t o e a r l y work i n t h i s f i e l d . The app r o a c h u s e d w a s t o s t u d y t h e t i m e c o u r s e of d e p h o s p h o r y l a t i o n a t O°C o f E-32P formed by i n c u b a t i o n of enzyme w i t h 1 mM Mg2+, 150 m M N a + , and 25 LILY [y-32P]ATP a s a f u n c t i o n o f ADP c o n c e n t r a t i o n . The s i m p l e s t model t h a t c o u l d e x p l a i n t h e dephosphorylation k i n e t i c s w a s t h e following:

E, + ATP

ADP *----

Elf

e E 2 P .-AE,

Scheme I

- - - +

+ Pi

E 2 + Pi

302

JENS G. N0RBY

and a n a l y s i s o f t h e r e s u l t s by t h e mathematical model c o r r e s p o n d i n g t o Scheme 1 provided e s t i m a t e s of a l l t h e c o n s t a n t s i n v o l v e d . One of t h e c o n c l u s i o n s of t h i s a n a l y s i s w a s t h a t t h e r e s u l t s were i n c o m p a t i b l e w i t h t h e Post-Albers scheme. I n a subsequent a r t i c l e ( P l e s n e r et al., 1 9 8 1 ) w e debated some of t h e e a r l i e r k i n e t i c experiments t h a t form t h e b a s i s of t h e Post-Albers scheme and a new model f o r Na,K-ATPase w a s proposed, i n which t h e a c i d - s t a b l e p h o s p h o i n t e r m e d i a t e s a r e p a r t of t h e r e a c t i o n sequence f o r Na-ATPase b u t n o t f o r N a , K - A T P a s e . I t should be p o i n t e d o u t t h a t t h e model proposed by P l e s n e r et al. ( 1 9 8 1 ) , l i k e t h e Post-Albers model, i n v o l v e s o n l y one s i t e for ATP on t h e enzyme. I n b o t h , t h i s s i t e may change a f f i n i t y f o r ATP d u r i n g t h e hydrol y s i s c y c l e ( s ) , and t h e r e i s more t h a n one s t e p i n t h e c y c l e ( s 1 where ATP i s a p o s s i b l e r e a c t a n t . The complete r e a c t i o n mechanism of Na,K-ATPase w i l l n o t be d i s c u s s e d f u r t h e r i n t h i s s e c t i o n . I n s t e a d t h i s p a r t of t h e overview s h a l l be l i m i t e d t o a d i s c u s s i o n of t h e r o l e of Mg2+, Na+, and K + i n t h e r e a c t i o n s of t h e phosphointermediates. A.

THE ROLE OF M g

I n t h i s s e c t i o n a few a s p e c t s of t h e r o l e of Mg i n t h e s y n t h e s i s and r e a c t i o n s of t h e p h o s p h o i n t e r m e d i a t e s w i l l be d i s c u s s e d . Magnesium i s r e q u i r e d f o r p h o s p h o r y l a t i o n b o t h from In the f i r s t case the a f f i n i t y Na+ + ATP and from P i . of t h e system f o r Mg seems t o be h i g h a l t h o u g h i t i s d i f f i c u l t t o g i v e a p r e c i s e f i g u r e . O p e r a t i o n a l l y , phosp h o r y l a t i o n i s o b t a i n e d w i t h micromolar c o n c e n t r a t i o n s of Mg2+ (Klodos and Skou, 1975) and, w i t h 125 mM Na+ and 25 U M ATP, t h e half-maximal s t e a d y - s t a t e l e v e l of EP i s o b t a i n e d w i t h less t h a n 1 0 V M Mg2+. I n c o n t r a s t , t h e d i s s o c i a t i o n c o n s t a n t s f o r EMg ( a b o u t 1 mM) and EPi-Mg, when E P i i s formed from E + P i (Kd a 9 mM f o r EPi-Mg) p o i n t s towards a r e l a t i v e l y low Mg a f f i n i t y i n t h e P i p h o s p h o r y l a t i o n r e a c t i o n ( K u r i k i e t al., 1 9 7 6 ) . The p i c t u r e t h a t emerges from t h 3 above a f f i n i t y c o n s i d e r a t i o n s and t h e s t u d i e s of Fukushima and P o s t ( 1 9 7 8 ) and of Hegyvary and JBrgensen ( 1 9 8 1 ) on t h e bindi n g of d i v a l e n t c a t i o n s t o t h e phosphoenzymes, i s shown i n Scheme I1 (monovalent c a t i o n s n o t shown):

THE SUBSTRATE SITE OF Na,K-ATPase

E I

- EATP -EATPMg -;EI

303

- E,PMg I

1

Scheme I1 I n t h i s s c h e m a t i c (Na-ATPase?) c y c l e , which r u n s c l o c k w i s e , Mg e n t e r s t h e enzyme a f t e r ( o r w i t h ) ATP and s t a y s I n c o n c l u s i o n , mention on u n t i l t h e c y c l e i s complete. should be made of t h r e e o b s e r v a t i o n s r e l e v a n t t o and s u p p o r t i n g Scheme 11. F i r s t , Smith e t a l . ( 1 9 8 0 ) could show t h a t t h e P i analog v a n a d a t e , when bound t o t h e enzyme, t r a p s d i v a l e n t c a t i o n s , s u g g e s t i n g t h a t i n t h e o f f - r e a c t i o n , P i l e a v e s b e f o r e Mg. Second, e v i d e n c e obt a i n e d by Beaugg and Glynn ( 1 9 7 9 1 , t h a t ADP r a t h e r t h a n MgADP i s t h e s u b s t r a t e f o r ADP/ATP exchange, may be exp l a i n e d by t h e scheme i f one assumes t h a t t h e Mg i n ElPMg p r e v e n t s MgADP, b u t n o t ADP, from e n t e r i n g t h e s u b s t r a t e site. Final1 a l t h o u g h an e a r l i e r h y p o t h e s i s a s s i g n e d a r o l e f o r MgY; i n t h e E 1 P t o E2P c o n v e r s i o n , r e c e n t i n dependent e v i d e n c e , e s p e c i a l l y from experiments w i t h t h e e f f e c t of Mg and c h e l a t o r s (Klodos and Skou, 1975, 1 9 7 7 ) , seems t o r u l e t h a t o u t , which i s i n accordance w i t h Scheme 11. B.

T H E ROLE O F Na+ A N D K +

The i n t e r a c t i o n of Na,K-ATPase w i t h i t s a c t i v a t i n g monovalent c a t i o n s and t h e i m p l i c a t i o n s t h e r e o f i s t h e t o p i c of o t h e r c h a p t e r s i n t h i s volume. Furthermore, t h e numerous q u a l i t a t i v e and q u a n t i t a t i v e a s p e c t s of t h i s i n t e r a c t i o n , both i n r e l a t i o n t o t h e d i f f e r e n t t r a n s p o r t models of t h e sodium pump and t h e k i n e t i c s of t h e many d i f f e r e n t r e a c t i o n s of Na,K-ATPase, have been d e a l t w i t h e x t e n s i v e l y i n a number of reviews and c o n f e r e n c e p r o c e e d i n g s (Skou, 1975; Whittam and C h i p p e r f i e l d , 1975; Glynn and K a r l i s h , 1975a; C a v i e r e s , 1 9 7 7 ; Robinson and F l a s h n e r , 1 9 7 9 a ; Skou and Ngh-by, 1 9 7 9 ; C a n t l e y , 1 9 8 1 ) . The r e a d e r i s a l s o r e f e r r e d t o t h e d i s c u s s i o n s e c t i o n i n Klodos e t a l . ( 1 9 8 1 ) . I s h a l l u s e t h i s m a t e r i a l t o g e t h e r w i t h a few o r i g i n a l a r t i c l e s t o d i s c u s s a f e w rec e n t developments i n o u r and o t h e r l a b o r a t o r i e s concerni n g t h e r o l e of Na+ and K+ i n t h e r e a c t i o n s of t h e phosphointermediates.

JENS G. N0RBY

304

I n t h e r e a c t i o n ( s 1 l e a d i n g t o f o r m a t i o n of phosphoenzyme, 1~0.5 f o r N a + i s around 1 m M , and t h e r e i s ample e v i d e n c e t h a t N a + h e r e i s a c t i n g a t an i n s i d e , c y t o p l a s m i c s i t e ( s ) of t h e enzyme ( t h e ~ 0 . 5f o r s a t u r a t i o n of t h e s e s i t e s i n t r a n s p o r t s t u d i e s i s around o r less t h a n 1 mM) Maximal o b t a i n a b l e s t e a d y - s t a t e p h o s p h o r y l a t i o n i s r e a c h e d w i t h a b o u t 1 0 m M N a + when ATP and Mg concent r a t i o n s a r e o p t i m a l . A few i n v e s t i g a t i o n s w i t h i n s i d e o u t membrane v e s i c l e s from s h e e p k i d n e y o u t e r medulla (Walter and Bader, 1978; Walter, t h i s volume) o r r e d blood c e l l s ( B l o s t e i n , 1 9 7 9 ) seemed t o i n d i c a t e t h a t t h e p r e s e n c e o f N a + on l o w - a f f i n i t y e x t r a c e l l u l a r s i t e s , t h a t are f a r from s a t u r a t e d a t 1 0 mM N a + , i n c r e a s e d t h e s t e a d y - s t a t e l e v e l of E P . These r e s u l t s are t h u s i n app a r e n t c o n t r a s t t o t h e experiments w i t h p u r i f i e d N a , K A T P a s e p r e p a r a t i o n s , and t h e i r i n t e r p r e t a t i o n may be d i f ficult. I t s h o u l d be borne i n mind i n t h i s c o n n e c t i o n t h a t even under o p t i m a l c o n d i t i o n s f o r p h o s p h o r y l a t i o n ( e . g . , 1 mM Mg, 150 mM N a + , 25 pM A T P ) , small c o n c e n t r a t i o n s o f K+ (0.05-0.2 m M ) w i l l r e d u c e t h e s t e a d y - s t a t e l e v e l of EP c o n s i d e r a b l y (Klodos and Nbrby, 1 9 7 9 ) . T h i s i s a n a c t i o n on an e x t r a c e l l u l a r s i t e t h a t w i l l be more pronounced a t lower ATP c o n c e n t r a t i o n s ( B l o s t e i n h a s l e s s t h a n 0 . 1 P M A T P ) , and r a t h e r h i g h N a + concent r a t i o n s ( e v i d e n t l y h i g h e r t h a n 150 mM, see above) w i l l be needed f o r c o m p e t i t i v e o c c u p a t i o n o f t h i s K+ s i t e . There i s no d o u b t , however, t h a t N a + , by b i n d i n g t o s i t e s (low a f f i n i t y ) o t h e r t h a n t h o s e i n v o l v e d i n t h e p h o s p h o r y l a t i o n , can i n f l u e n c e t h e k i n e t i c s even when s t e a d y - s t a t e p h o s p h o r y l a t i o n i s maximal. One e x p r e s s i o n of t h i s i s t h e i n c r e a s e i n t h e E l P / E z P r a t i o b r o u g h t a b o u t by h i g h Na+ c o n c e n t r a t i o n s ( ~ 0 . 5f o r t h e e f f e c t i s 100-200 m M ) , a s d e m o n s t r a t e d by K u r i k i and Racker (19761, J 6 r g e n s e n and K a r l i s h (1980) , Hara and Nakao (1981, t h i s v o l u m e ) , Yoda and Yoda ( t h i s v o l u m e ) , and Klodos et a l . ( t h i s volume). Concomitantly, Na+ i n t h i s c o n c e n t r a t i o n range (and h i g h e r ) i n c r e a s e s t h e r a t e o f ADP/ATP exchange and a l s o t h e Na-ATPase a c t i v i t y ( e . g . , Beauge and Glynn, 1979). + These e f f e c t s o f N a , a l l s e e n a t a c o n s t a n t , maxim a l s t e a d y - s t a t e l e v e l of p h o s p h o r y l a t i o n , c a l l f o r a model i n v o l v i n g s e v e r a l l i g a n d e d forms of EP ( K a r l i s h e t a l . , 197833; Garrahan e t a l . , 1979; Hara and Nakao, 1981; Klodos et a l . , t h i s volume). The N a / N a exchange s e e n i n i n t a c t c e l l s y s t e m s must a l s o be e x p l a i n e d by t h e model. Furthermore, i f it i s a c c e p t e d t h a t i n c h e m i c a l and enzymatic r e a c t i o n s o n l y one " e v e n t " c a n t a k e p l a c e a t e a c h s t e p , t h e f o l l o w i n g minimal model f o r t h e react i o n s w i t h which w e a r e concerned emerges (Mg i s n o t shown, see Scheme 11):

.

THE SUBSTRATE SITE OF Na,K-ATPase

305

E

Scheme I11 The s u b s c r i p t s i and o s i g n i f y t h e s i d e d n e s s of t h e s y s t e m meaning " i n s i d e " and " o u t s i d e , r e s p e c t i v e l y . L e f t o u t i n Scheme I11 a r e a l l t h e "spontaneous" dephosphoryla t i o n s of t h e El-phosphoenzymes ( c f . t h e condensed models of Klodos e t a l . , 1981, t h i s volume), and f o r t h e sake of s i m p l i c i t y o n l y one o u t s i d e and one i n s i d e c a t i o n s i t e a r e shown. The d i f f e r e n t f e a t u r e s of t h i s model a r e c u r r e n t l y b e i n g i n v e s t i g a t e d e x p e r i m e n t a l l y and m a t h e m a t i c a l l y i n o u r l a b o r a t o r y . A t t h e p r e s e n t s t a g e of t h i s a n a l y s i s a few i n t e r e s t i n g p r o p e r t i e s have been r e v e a l e d (see a l s o Klodos e t a 1 , t h i s volume) : (1) The amount of EP t h a t d i s a p p e a r s r a p i d l y upon a d d i t i o n of ADP i s dependent upon "a+], and a t h i g h "a+] i t may encompass a l l t h e E l P s ( c o n v e r s i o n of E 2 P t o E 1 P i s p r o b a b l y slow, Klodos e t a 1 1981). El-P might be c a l l e d " N a - s e n s i t i v e E P , " and Na/Na exchange would i n v o l v e t h i s s p e c i e s b u t n o t E 2 P . ( 2 ) There a r e i n d i c a t i o n s t h a t " K + - s e n s i t i v e EP" c o n s i s t s of E 2 P , E l - P and maybe a l s o NaoE1-P. El-P, which has an empty o u t s i d e s i t e may r e a c t w i t h KO and d e p h o s p h o r y l a t e r a p i d l y so t h a t t h e n e t f l u x of NaoE1-P t o E -P could i n c r e a s e . P r o p e r t i e s (1) and ( 2 ) t o g e t h e r c o u l i e x p l a i n t h e o b s e r v a t i o n by Yoda and Yoda ( t h i s volume) and Klodos e t a l . ( t h i s volume) t h a t a t h i g h "a+] t h e sum of ADP- and K + - s e n s i t i v e E P s t e n d s t o be l a r g e r than 1 0 0 % . I'

.

.,

4E(P)ADP i s c o n s i d e r e d e i t h e r t o c o n s t i t u t e o n l y a v e r y s m a l l f r a c t i o n o f t h e e n z y m e a n d / o r t o have a n a c i d - l a b i l e E-P b o n d . I f neither o f these t w o r e q u i r e m e n t s w e r e f u l f i l l e d , i t w o u l d not be p o s s i b l e t o see t h e r a p i d d i s a p p e a r a n c e o f EP u p o n ADP a d d i t i o n . For the same reason t h e s a m e requirements a r e i m p o s e d u p o n EATP.

306

JENS G.N0RBY

100

CEPI

I

OIO

60 40

20

“a+]

300

10

150

20

t 2

t +

F i g . 3 . D e p h o s p h o r y l a t i o n of EP b y K ( K l o d o s e t a l . , t h i s v o l u m e ) , B r a i n Na,K-ATPase was p h o s p h o r y l a t e d a t O°C f o r 60 sec i n the p r e s e n c e of 1 mM Mg2+, 25 pM [ y - 3 2 P ] A T P , and w i t h the Na’ concentrations shown. A t t i m e 0 , 2 0 mM Kf + 1 mM u n l a b e l e d ATP was added and the d e c a y of EP as a f u n c t i o n of t i m e d e t e r m i n e d as d e s c r i b e d (Klodos e t a l . , 1 9 8 1 ) .

( 3 ) The appearance of a slow p h a s e of t h e b i ( m u l t i - ? ) p h a s i c K+-dephosphorylation c u r v e s ( e . g . , F i g . 3 ) i s n o t e a s i l y e x p l a i n T d and c o n t r a s t s w i t h t h e widely accepted n o t i o n t h a t K i n c r e a s e s t h e turnover of t h e c y c l e ( b u t see b e l o w ) . One i n t e r e s t i n g p o s s i b i l i t y i s t h a t K+ reacts w i t h N a i E 1 - P on a n o u t s i d e s i t e and t h e r e b y h i n d r e s t h e movement of N a i t o t h e o u t s i d e ( b l o c k s t h e r e a c t i o n N a i E 1 - P t o NaoE1-PI. Schemes I1 and I11 c l e a r l y i l l u s t r a t e t h e r a t h e r h i g h d e g r e e of s p e c i f i c i t y r e q u i r e d o f t h e many d i f f e r e n t i n termediates with regard t o ligand i n t e r a c t i o n . But t h e f l e x i b i l i t y of t h e system may w e l l b e g r e a t e r t h a n t h a t i l l u s t r a t e d i n t h e s e schemes. I n a r e c e n t a r t i c l e ( P l e s n e r e t a l . , 1 9 8 1 ) w e have proposed t h a t t h e s e schemes may o n l y be r e p r e s e n t a t i v e of Na-ATPase (and t h e p a r t i a l r e a c t i o n s c o r r e s p o n d i n g t o t h i s enzyme a c t i v i t y ) and t h a t t h e N a , K - A T P a s e a c t i v i t y i s d i s p l a y e d by a n o t h e r c y c l e of r e a c t i o n s . Among t h e arguments for t h i s hypo-

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t h e s i s were t h e f o l l o w i n g : ( 1 ) t h e d i s c r e p a n c y between t h e measured and t h e c a l c u l a t e d r a t e of ATP h y d r o l y s i s i n e x p e r i m e n t s where K + , and e s p e c i a l l y L i + , were shown t o s t i m u l a t e ATP h y d r o l y s i s more t h a n e x p e c t e d from t h e e f f e c t of t h e s e ions on dephosphorylation; ( 2 ) k i n e t i c c o n s i d e r a t i o n s t h a t K+ a d d i t i o n would n o t be a b l e t o i n crease t h e r a t e o f a l l t h e s t e p s i n t h e c y c l e ; ( 3 ) t h i s l i m i t a t i o n i n t h e K+ e f f e c t seemingly s u b s t a n t i a t e d by t h e a p p e a r a n c e of t h e slow d e p h o s p h o r y l a t i o n p h a s e i n t h e e x p e r i m e n t s shown i n F i g . 3 . C.

C O N C L U D I N G REMARKS

T h e o b s e r v a t i o n s on t h e p r o p e r t i e s and r e a c t i o n s of t h e phosphoenzymes and t h e i r r o l e a s i n t e r m e d i a t e s i n t h e Na-ATPase, Na,K-ATPase, and t h e p a r t i a l r e a c t i o n s o f t h e s e a c t i v i t i e s may a l l be e x p l a i n e d , a s f a r a s t h e p r o p e r t i e s of t h e s u b s t r a t e s i t e i s c o n c e r n e d , by assumi n g t h e e x i s t e n c e of o n e f l e x i b l e s i t e p e r m o l e c u l e .

V.

GENERAL CONCLUSION

The i n t e r a c t i o n s of Na,K-ATPase w i t h n u c l e o t i d e s and v a n a d a t e , and t h e p r o p e r t i e s and r e a c t i o n s o f i t s p h o s p h o r y l a t e d forms i l l u s t r a t e t h e c o m p l e x i t y and f l e x i b i l i t y o f t h e enzyme. I n s u c h a s i t u a t i o n it i s tempti n g f o r an experimenter o r a t h e o r e t i c i a n t o d e v i s e models of s u c h a d e g r e e o f c o m p l e x i t y t h a t w i l l s a t i s f y h i s o r h e r a m o u r p r o p r e . I have i n s t e a d t r i e d t o s i m p l i f y t h e p i c t u r e by i n t e r p r e t i n g the a v a i l a b l e d a t a i n s u c h a way t h a t t h e premise o f one s u b s t r a t e s i t e p e r m o l e c u l e o f N a , K - A T P a s e seems t o b e j u s t i f i e d . "But absence of e v i d e n c e i s n o t e v i d e n c e o f a b s e n c e " (Sagan, 1 9 7 7 ) .

ACKNOWLEDGMENTS

I am v e r y g r a t e f u l t o D r . P a u l O t t o l e n g h i f o r h i s i n v a l u a b l e h e l p i n t h e f i n a l s t a g e s of t h i s a r t i c l e . Thanks are due a l s o t o The work was G r e t e Ngirby f o r h e r c o o p e r a t i o n w i t h t h e m a n u s c r i p t . s u p p o r t e d by Grant No. 12-1938 from t h e Danish Research Council.

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p l i c a t i o n s f o r t h e s t r u c t u r e and mechanisn of t h e Na:K pump. J. B i o l . Chem. 2 5 6 , 2357-2366. M U ~ S O Q , K. B. (1981) Light-dependent i n a c t i v a t i o n o f (Na+ + K+) ATPase w i t h a new p h o t o a f f i n i t y r e a g e n t , chromium a r y l a z i d o 6-alanyl ATP. J. B i o l . Chem. 256, 3223-3230. Nfdrby, J. G . , and Jensen, J. ( 1 9 7 1 ) . Binding of ATP t o b r a i n m i crosomal ATPase. Determination of t h e ATP-binding c a p a c i t y and t h e d i s s o c i a t i o n c o n s t a n t of t h e enzyme-ATP complex a s a f u n c t i o n of K+ c o n c e n t r a t i o n . B i o c h i m . B i o p h y s . A c t a 233, 104-116. Nbrby, J. G . , and J e n s e n , J . (1973). Binding of ATP t o ( N a + + K + ) a c t i v a t e d ATPase. N u m b e r of b i n d i n g sites and enzyme-ATP d i s s o c i a t i o n c o n s t a n t . I n "Reaction Mechanisms and C o n t r o l P r o p e r t i e s of Phosphotranspherases, I' pp. 199-204. AkademieVerlag, B e r l i n . Nfdrby, J . G . , and Jensen, J. (1974). Binding of ATP t o Na,K-ATPase. Ann. N.Y. A c a d . S c i . 2 4 2 , 158-167. N#rby, J. G., O t t o l e n g h i , P . , and Jensen, J. (1980). S c a t c h a r d p l o t : Common m i s i n t e r p r e t a t i o n s o f b i n d i n g experiments. Anal. B i o c h e m . 102, 318-320. Patzelt-Wenczler, R., and Schoner, W. (1981). Evidence f o r two d i f f e r e n t r e a c t i v e s u l f h y d r y l groups i n t h e ATP-binding s i t e s o f (Na+ + K+)-ATPase. Eur. J. B i o c h e m . 1 1 4 , 79-87. P e t e r s , W. H. M., S w a r t s , H . G. P., de Pont, J. J. H . H. M., Schuurmans Stekhoven, F. M. A. H., and Bonting, S. L. (1981). (Na+ + K+) -ATPase h a s one f u n c t i o n i n g p h o s p h o r y l a t i o n s i t e p e r subunit. Nature ( L o n d o n ) 2 9 0 , 338-339. P l e s n e r , I. W., P l e s n e r , L., Nbrby, J. G., and Klodos, I. (1981). The s t e a d y - s t a t e k i n e t i c mechanism o f ATP h y d r o l y s i s catal y z e d by membrane-bound (Na+ + K+)-ATPase from ox b r a i n . 111. A minimal model. B i o c h i m . B i o p h y s . A c t a 6 4 3 , 483-494. P l e s n e r , L., and P l e s n e r , I. W. (1981). The s t e a d y - s t a t e k i n e t i c mechanism of ATP h y d r o l y s i s c a t a l y z e d by membrane-bound (Na+ + K+)-ATPase from ox b r a i n . I. S u b s t r a t e i d e n t i t y . B i o c h i m . B i o p h y s . A c t a 643, 449-462. P o s t , R. L. , K u m e , S . , Tobin, T. , O r c u t t , B. , and Sen, A. K. ( 1 9 6 9 ) . F l e x i b i l i t y o f an a c t i v e c e n t e r i n sodium-plus-potassium adenosine t r i p h o s p h a t a s e . J . Gen. P h y s i o l 5 4 , 306s-326s. P o s t , R. L., Hegyvary, C . , and Kume, S. (1972). A c t i v a t i o n by adenosine t r i p h o s p h a t e i n t h e p h o s p h o r y l a t i o n k i n e t i c s of sodium and potassium i o n t r a n s p o r t adenosine t r i p h o s p h a t a s e . J. B i o l . Chem. 2 4 7 , 6530-6540. P o s t , R. L . , Toda, G . , and Rogers, F. N. (1975). P h o s p h o r y l a t i o n by i n o r g a n i c phosphate of sodium p l u s potassium i o n t r a n s p o r t J. B i o l . Chem. 2 5 0 , 691-710. adenosine t r i p h o s p h a t a s e . Powell, L. D . , a n d Cantley, L. C. (1980). S t r u c t u r a l changes i n (Na+ + K+) -ATPase accompanying d e t e r g e n t i n a c t i v a t i o n . B i o c h i m . B i o p h y s . A c t a 5 9 9 , 436-447. Robinson, J. D. (1976). S u b s t r a t e s i t e s o f t h e ( N a + + K+)-depend e n t ATPase B i o c h i m . B i o p h y s . A c t a 4 2 9 , 1006-1019.

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Robinson, J . D., and F l a s h n e r , M. S . ( 1 9 7 9 a ) . The ( N a + + K + ) a c t i v a t e d ATPase. Enzymatic and t r a n s p o r t p r o p e r t i e s . Biochim. Biophys. A c t a 5 4 9 , 145-176. C a t i o n and nucleoRobinson, J. D., and F l a s h n e r , M. S. (1979b) In "Na,K-ATPase: t i d e i n t e r a c t i o n s w i t h t h e Na,K-ATPase. S t r u c t u r e and K i n e t i c s " (J. C. Skou and J. G. N#rby, e d s . ) , pp. 275-285. Academic P r e s s , N e w York. Sagan, C. ( 1 9 7 7 ) . "The Dragons o f Eden. S p e c u l a t i o n s on t h e E v o l u t i o n o f Human I n t e l l i g e n c e . " B a l l a n t i n e Books, N e w York. Schoner, W., P a u l s , H. , and P a t z e l t - W e n c z l e r , R. ( 1 9 7 7 ) . Bioc h e m i c a l c h a r a c t e r i s t i c s o f t h e sodium pump: Indications In of a h a l f - o f - s i t e s r e a c t i v i t y o f ( N a + + K+)-ATPase. "Myocardial F a i l u r e " (G. R i e c k e r , A Weber, and J. Goodwin, S p r i n g e r - V e r l a g , B e r l i n and N e w York. e d s . ) , pp. 104-119. Schuunnans Stekhoven, F. M. A. H . , and Bonting, S. L. ( 1 9 8 1 ) . T r a n s p o r t a d e n o s i n e t r i p h o s p h a t a s e s : P r o p e r t i e s and funcP h y s i o l . R e v . 61, 1-76. tions. Schuurmans Stekhoven, F. M. A. H . , S w a r t s , H. G. P., d e P o n t , J. J. H. H. M., and Bonting, S. L. ( 1 9 8 0 ) . S t u d i e s o n ( N a + 4- K + ) - a c t i v a t e d ATPase. X L I V . S i n g l e phosphate i n c o r p o r a t i o n d u r i n g d u a l p h o s p h o r y l a t i o n by i n o r g a n i c p h o s p h a t e and a d e n o s i n e t r i p h o s p h a t e . Biochim. Biophys. A c t a 5 9 7 , 100-111. Schuurmans Stekhoven, F. M. A. H . , S w a r t s , H. G. P . , d e Pont, J. J. H. H. M., a n d B o n t i n g , S. L. (1981). S t u d i e s on ( N a + + K + ) - a c t i v a t e d ATPase. XLV. Magnesium i n d u c e s two l o w - a f f i n i t y non-phosphorylating n u c l e o t i d e b i n d i n g sites p e r molecule. Biochim. Biophys. Acta 6 4 9 , 533-540. S i e g e l , G . J . , Koval, G. J., and A l b e r s , R. W. ( 1 9 6 9 ) . Sodiumpotassium-activated adenosine t r i p h o s p h a t a s e . IV. C h a r a c t e r i z a t i o n o f t h e p h o s p h o p r o t e i n formed from o r t o p h o s p h a t e i n J. B i o l . Chem. 2 4 4 , 3264-3269. t h e p r e s e n c e o f ouabain. Simons, T. J . B. ( 1 9 7 9 ) . Vanadate--a new t o o l f o r b i o l o g i s t s . N a t u r e (London) 281, 337-338. Skou, J. C. ( 1 9 7 4 a ) . E f f e c t o f ATP on t h e i n t e r m e d i a r y steps o f t h e r e a c t i o n o f t h e ( N a + + K+)-dependent enzyme system. I. S t u d i e d by t h e use o f N-ethylmaleimide i n h i b i t i o n a s a tool. Biochim. Biophys. A c t a 3 3 9 , 234-245. Skou, J. C. ( 1 9 7 4 b ) . E f f e c t o f ATP on t h e i n t e r m e d i a r y s t e p s o f t h e r e a c t i o n o f t h e ( N a + + K+)-dependent enzyme system. 11. Biochim. E f f e c t o f a v a r i a t i o n i n t h e ATP/Mg2+ r a t i o . Biophys. A c t a 3 3 9 , 246-257. Skou, J. C. ( 1 9 7 4 ~ ) . E f f e c t o f ATP on t h e i n t e r m e d i a r y steps of t h e r e a c t i o n o f t h e ( N a + + K+)-dependent enzyme system. 111. E f f e c t on t h e p-nitrophenylphosphatase a c t i v i t y of t h e system. Biochim. Biophys. A c t a 3 3 9 , 258-273. Skou, J. C ( 1 9 7 5 ) . The ( N a + + K + ) a c t i v a t e d enzyme system and i t s r e l a t i o n s h i p t o t r a n s p o r t o f sodium and potassium. Q. Rev. Biophys. 7, 401-434.

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Skou, J. C. (1979). E f f e c t o f ATP on t h e intermediary s t e p s of t h e r e a c t i o n of t h e (Na' + K+)-dependent enzyme system. IV. E f f e c t o f ATP on K0.5 f o r Na+ and on h y d r o l y s i s a t d i f f e r e n t pH and temperature. B i o c h i m . B i o p h y s . A c t a 567 , 421-435. Skou, J. C . , and Esmann, M. (1980). E f f e c t s of ATP and p r o t o n s on t h e Na:K s e l e c t i v i t y of t h e ( N a + + K+)-ATPase s t u d i e d by l i g a n d e f f e c t s on i n t r i n s i c and e x t r i n s i c fluorescence. B i o c h i m . B i o p h y s . A c t a 6 0 1 , 386-402. Skou, C . , and Nglrby, J. G . , eds. (1979). "Na,K-ATPase: S t r u c t u r e and Kinetics. " Academic Press, New York. Smith, R. L., Zinn, K . , and Cantley, L. C. (1980). A study of t h e vanadate-trapped s t a t e of t h e Na,K-ATPase. J . B i o l . Chem. 255, 9852-9859. Taniguchi, K . , and Post, R. L. (1975). Synthesis of ATP and exchange between inorganic phosphate and ATP i n sodium and potassium t r a n s p o r t ATPase. J. B i o l . Chem. 2 5 0 , 3010-3018. Tobin, T., Akera, T . , L e e , C. Y . , and Brody, T. M. (1974). Ouabain binding t o (Na+ + K+)-ATPase. E f f e c t s of n u c l e o t i d e analogues and e t h a c r y n i c a c i d . B i o c h i m . B i o p h y s . A c t a 345, 102-117. Walter, H . , and Bader, H. (1978). E f f e c t of i n t r a v e s i c u l a r monov a l e n t c a t i o n s on t h e steady s t a t e of t h e phosphoenzyme of adenosine t r i p h o s p h a t a s e dependent on sodium and potassium i o n s i n i n s i d e - o u t plasma membrane v e s i c l e s . E u r . J . B i o c h e m . 83 , 125-130. Whittam, R., and C h i p p e r f i e l d , A. R. (1975). The r e a c t i o n mechani s m o f t h e sodium pump. B i o c h i m . B i o p h y s . A c t a 415, 149-171. Binding of monovalent caYamaguchi, M. , and Tonomura, Y. (1980) t i o n s t o Na+, K+-dependent ATPase p u r i f i e d from p o r c i n e kidney. 11. Acceleration of t r a n s i t i o n from a K+-bound form t o a Na+-bound form by binding of ATP t o a r e g u l a t o r y s i t e of t h e enzyme. J . B i o c h e m . ( T o k y o ) 88, 1377-1385. Yount, R. G. (1975). ATP analogs. A d v . E n z y m o l . 43, 1-56.

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CURRENTTOPICS IN MEMBRANES AND TRANSWRT. VOLUME 19

Conformational Changes of Na,K-ATPase Necessary for Transport LEWIS C. CANZEY, CYNTHIA T. CARILLI, RODERIC L. SMITH, AND DAVID PEIUMAN Depanment of Biochemistry and Molecular Biology Hurvard University Cambridge, Massachusetts

I.

INTRODUCTION

Two major q u e s t i o n s c o n c e r n i n g t h e s t r u c t u r e and mechanism o f t h e Na,K-ATPase have been d e b a t e d f o r t h e p a s t s e v e r a l years: (1) Does ATP-driven N a / K exchange a c r o s s t h e membrane r e q u i r e a n i n t e r a c t i n g dimeric p r o t e i n s t r u c t u r e ? (2) D o Na+ and K+ i o n s occupy t h e same s e t o f b i n d i n g s i t e s i n a c o n s e c u t i v e f a s h i o n , o r do two sets o f b i n d i n g s i t e s s i m u l t a n e o u s l y e x i s t on t h e two s i d e s of t h e membrane? T e c h n i c a l problems make i t d i f f i c u l t t o d e s i g n e x p e r i m e n t s t o answer t h e s e quest i o n s unambiguously. However, a number of r e l a t e d quest i o n s have been answered, and t h e s e answers s u g g e s t t h a t what one m i g h t ( p e r h a p s n a i v e l y ) have c o n s i d e r e d a s t h e s i m p l e s t s t r u c t u r e f o r t h e p r o t e i n ( i . e . , no i n t e r a c t i n g d i m e r s and o n l y one s e t of c a t i o n s i t e s ) is p r o b a b l y true. I n f a c t , i f w e e x t e n d a b a s i c p r i n c i p l e used i n d e v e l o p i n g k i n e t i c models t o s t r u c t u r a l c o n s i d e r a t i o n s , t h e n w e s h o u l d assume t h a t t h e p r o t e i n h a s no i n t e r a c t i n g 315

Copyright 0 1983 by Academic Press. h c . All rights of reproductionin any form reserved. ISBN 0-12-1533194

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s i t e s ( i . e . , i s a f u n c t i o n a l monomer) and h a s o n l y one s e t o f monovalent c a t i o n b i n d i n g s i t e s , u n t i l s t r u c t u r a l or k i n e t i c evidence proves otherwise.

11.

STRUCTURE

The r e a s o n s f o r p r o p o s i n g t h a t a n i n t e r a c t i n g d i meric s t r u c t u r e i s needed f o r t r a n s p o r t were b a s e d p r i (I) A l l t i g h t l y b i n d i n g m a r i l y on two o b s e r v a t i o n s : l i g a n d s ( o u a b a i n , ATP, p h o s p h a t e , and v a n a d a t e ) were found t o b i n d t o o n l y one s i t e p e r ~ ~ 2 5 0 , 0 0d0a l t o n s of p u r i f i e d p r o t e i n (apparently an a 2 , 8 2 s t r u c t u r e ) . (2) I n a d d i t i o n t o t h e h i g h - a f f i n i t y ATP s i t e o b s e r v e d i n b i n d i n g e x p e r i m e n t s , a l o w - a f f i n i t y ATP s i t e was obs e r v e d k i n e t i c a l l y ( f o r r e v i e w , see C a n t l e y , 1 9 8 1 ) . Competition f o r ATP h y d r o l y s i s by v a n a d a t e w a s a l s o more c o m p l i c a t e d t h a n would have been assumed f o r a s i n g l e - s i t e model ( C a n t l e y et al. , 1 9 7 8 ) . However, t h e stoichiometry of t h e h i g h - a f f i n i t y n u c l e o t i d e binding s i t e on t h e Na,X-ATPase h a s been r e i n v e s t i g a t e d by a number o f g r o u p s , and t h e r e v i s e d estimates s u g g e s t t h a t one n u c l e o t i d e s i t e e x i s t s p e r c a t a l y t i c s u b u n i t ( o r p e r a,B-protomer; Plodzydlowski and Fortes, 1 9 8 1 a ) . Although m o l e c u l a r w e i g h t and s t o i c h i o m e t r y measurements on i n t r i n s i c membrane p r o t e i n s are s u b j e c t t o c o n s i d e r a b l e e r r o r , t h e s e r e s u l t s have n e u t r a l i z e d h a l f of t h e a r g u ment f o r a n e s s e n t i a l d i m e r i c s t r u c t u r e . For r e a s o n s e n t i r e l y i n d e p e n d e n t of s t o i c h i o m e t r y measurements, w e have concluded t h a t t h e high- and lowa f f i n i t y ATP s i t e s do n o t s i m u l t a n e o u s l y e x i s t on t h e en. zyme b u t r e p r e s e n t t h e same s i t e a t d i f f e r e n t t i m e s i n t h e t u r n o v e r c y c l e (Smith et a l . , 1 9 8 0 ) . T h i s c o n c l u s i o n n e u t r a l i z e s t h e o t h e r argument f o r i n t e r a c t i n g d i mers. The p r i m a r y r e a s o n f o r t h i s c o n c l u s i o n i s t h e obs e r v a t i o n t h a t ATP h a s no e f f e c t on t h e r a t e o f v a n a d a t e release from t h e Na,K-ATPase. This r e s u l t , along with t h e observations t h a t vanadate binding a t one s i t e per ouabain-binding s i t e i s c o m p e t i t i v e w i t h ATP b i n d i n g t o t h e l o w - a f f i n i t y s i t e i n k i n e t i c experiments (Cantley e t al., 1978) and c o m p l e t e l y b l o c k s ATP b i n d i n g t o t h e h i g h - a f f i n i t y s i t e i n e q u i l i b r i u m binding experiments (Smith et a l . , 1 9 8 0 ) , implies t h a t o n l y o n e ATP s i t e c a n S i n c e v a n a d a t e a p p a r e n t l y a c t s as a exist at a time. t r a n s i t i o n - s t a t e analog f o r phosphate h y d r o l y s i s , i t s release from t h e enzyme s h o u l d be q u i t e s e n s i t i v e t o any a l l o s t e r i c r e g u l a t o r s . T h u s , N a + and K+ h a v e d r a m a t i c e f f e c t s on t h e r a t e of v a n a d a t e release, b u t ATP h a s no effect

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(F) F i g . 1 . A k i n e t i c mechanism f o r ATP-driven NaT and K' t r a n s port. L i g a n d s on t h e o u t s i d e o f t h e c y c l e enter or l e a v e f r o m t h e o u t s i d e o f t h e c e l l and those on the i n s i d e o f the c y c l e enter or l e a v e from the i n s i d e o f the c e l l . El and E2 d e s i g n a t e c o n f o r m a t i o n s o f t h e tr7nsport s i t e s w h i c h f a v o r b i n d i n g c y t o p l a s m i c Na+ or e x t r a c e l l u l a r K , r e s p e c t i v e l y ; ( K ) i n d i c a t e s a " t r a p p e d " I@ i n -, a c c e s s i b l e f r o m either s i d e o f the membrane. D a s h e s i n d i c a t e cov a l e n t bonds and d o t s i n d i c a t e n o n c o v a l e n t bonds. T h e stoichiom e t r i e s o f Na+ and K+ s i t e s a r e o m i t t e d . A t low ATP c o n c e n t r a t i o n s , t r a n s p o r t i s a c c o m p l i s h e d b y a c l o c k w i s e movement t h r o u g h the c y c l e f r o m i n t e r m e d i a t e s A t h r o u g h F . T h e r a t e l i m i t i n g s t e p i s F +. A . A t h i g h e r ATP c o n c e n t r a t i o n s , the inner c y c l e ( B t o G ) i s f o l l o w e d since ATP c a n b i n d t o (K)E2 a n d a c c e l e r a t e K+ r e l e a s e t o the c y t o p l a s m . T h i s model i s a s l i g h t m o d i f i c a t i o n o f the model p r o p o s e d b y Post e t a l . ( 1 9 7 2 ) . R e p r i n t e d w i t h p e r z n i s s i o n o f C u r r e n t T o p i c s i n Bioenergetics from Cantley (1981).

111.

KINETICS

In order t o verify t h a t a l l k i n e t i c data thus f a r r e p o r t e d can be e x p l a i n e d by a s i n g l e ATP s i t e whose a f f i n i t y changes w i t h t i m e , w e determined t h e s t e a d y - s t a t e e q u a t i o n f o r t h e mechanism i n F i g . 1. T h i s mechanism i s a s l i g h t m o d i f i c a t i o n of one proposed by P o s t and coworkers ( 1 9 7 2 ) . The c r i t i c a l p o i n t i s t h a t ATP c a n e i t h e r b i n d t o t h e h y d r o l y s i s s i t e b e f o r e t h e E 2 -+ El c o n f o r m a t i o n a l s t e p o r a f t e r t h e E 2 +. T1 conforma-

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t i o n a l s t e p . Using t h e s o l u t i o n t o t h i s model, w e were a b l e t o g e n e r a t e n o n l i n e a r Lineweaver-Burk p l o t s which q u i t e a c c u r a t e l y f i t o u r e x p e r i m e n t a l d a t a (Smith et a l . , 1 9 8 0 ) . W e were a l s o a b l e t o e x p l a i n t h e complex c o m p e t i t i o n o b s e r v e d f o r v a n a d a t e i n h i b i t i o n of ATP hyd r o l y s i s . The f e a t u r e of t h i s model which i s e s s e n t i a l f o r f i t t i n g t h e experimental data i s t h a t t h e slowest A ) and t h a t s t e p i n t u r n o v e r i s t h e E 2 -+ E l s t e p (F a l t h o u g h ATP b i n d s p o o r l y t o E 2 ( F .+ G ) , t h i s b i n d i n g c o n s i d e r a b l y accelerates t h e E 2 + E l c o n f o r m a t i o n a l s t e p (G + B ) . A s i m i l a r c o n c l u s i o n h a s a l s o been r e a c h e d by Modzydlowski and F o r t e s (1981a,b) u s i n g somewhat d i f f e r e n t a s s u m p t i o n s f o r s o l v i n g t h e k i n e t i c model. These r e s u l t s make i t u n l i k e l y t h a t a n i n t e r a c t i n g dimer i s e s s e n t i a l f o r t h e c a t a l y t i c p r o p e r t i e s of t h e Na,K-ATPase. However, it may s t i l l be p o s s i b l e t h a t a n oligomeric s t r u c t u r e i s e s s e n t i a l f o r t h e transport p r o p e r t i e s o f t h i s p r o t e i n . For example, work on t h e r e d - c e l l anion-exchange p r o t e i n , a p r o t e i n known t o e x i s t a s a dimer b o t h i n s o l u t i o n and i n t h e membrane, i n d i c a t e s t h a t a l t h o u g h t h e two h a l v e s o f t h e dimer t r a n s p o r t anions independently, t h e dimeric s t r u c t u r e may be e s s e n t i a l f o r forming a common c a v i t y f o r access of h y d r o p h i l i c i o n s i n t o i n d e p e n d e n t t r a n s p o r t s i t e s (Macara and C a n t l e y , 1 9 8 1 a , b ) . The q u e s t i o n c o n c e r n i n g t h e number and l o c a t i o n of monovalent c a t i o n - b i n d i n g s i t e s on t h e Na,K-ATPase i s c o m p l i c a t e d by t h e low a f f i n i t y of t h e p r o t e i n f o r b o t h N a + and K+ i o n s , which h a s p r e c l u d e d a c c u r a t e b i n d i n g measurements. K i n e t i c a n a l y s e s o f t r a n s p o r t a r e cons i s t e n t with e i t h e r simultaneous o r consecutive binding schemes ( S a c h s , 1 9 7 9 ) . I n t h e i n t e r e s t of s i m p l i c i t y , w e have p r e s e n t e d a scheme which s u g g e s t s how t r a n s p o r t might o c c u r w i t h o n l y one s e t o f monovalent c a t i o n b i n d i n g s i t e s and minimal p r o t e i n movement ( F i g . 2 ) . The l e t t e r e d i n t e r m e d i a t e s i n t h i s scheme c o r r e s p o n d t o t h e r e s p e c t i v e k i n e t i c i n t e r m e d i a t e s i n F i g . 1 (A has been o m i t t e d ) . The e x p l a n a t i o n of v a r i o u s p a r t i a l react i o n s u s i n g t h i s scheme i s p r e s e n t e d i n a review ( C a n t l e y , 1 9 8 1 ) . The main p o i n t of t h e model i s t h a t one c a n c o n c e i v e o f how t r a n s p o r t c o u l d be d r i v e n w i t h o n l y l o c a l c o n f o r m a t i o n a l changes on t h e p r o t e i n and w i t h o u t t h e n e c e s s i t y o f s e p a r a t e " e x t e r n a l " and " i n t e r n a l " monovalent c a t i o n - b i n d i n g s i t e s . -+

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F i g . 2 . A h y p o t h e t i c a l a s p a r t a t e g a t e m d e l t o e x p l a i n the m o l e c u l a r mechanism o f ATP-driven Na+ and K+ t r a n s p o r t . T h e drawi n g s r e p r e s e n t cut-away v i e w s o f the c y t o p l a s m i c p o r t i o n o f the a c t i v e s i t e . A c h a n n e l t o the o u t s i d e o f t h i s cell i s i n d i c a t e d a t the bottom of e a c h f i g u r e . T h e i n t e r m e d i a t e s B t h r o u g h G a r e s t r u c t u r a l r e p r e s e n t a t i o n s o f i n t e r m e d i a t e s B t h r o u g h G i n F i g . 1 . Int e r m e d i a t e A h a s been o m i t t e d b u t would be i d e n t i c a l t o the s t r u c t u r e i n B w i t h no ATP b o u n d . T h e c i r c l e d P r e p r e s e n t s a p h o s p h a t e m o i e t y ; the s m a l l s o l i d circle i s a Mg2+ i o n ; the c i r c l e d K i s a K+ i o n ; the o p e n circle i s a Na+ i o n ; and the c i r c l e d 0 i s a c a r b o x y l i c o x y g e n o f the a s p a r t a t e r e s i d u e . T h e r e l a t i v e r a d i i o f the circles a r e a p p r o x i m a t e l y p r o p o r t i o n a l t o the i o n i c r a d i i o f the r e s p e c t i v e i o n s . T h e model p r o p o s e s t h a t the a c t i v e - s i t e a s p a r t a t e r e s i d u e blocks a c c e s s t o the m o n o v a l e n t c a t i o n s i t e s f r o m the e x t r a c e l l u l a r s i d e when i n a r e l a x e d c o n f i g u r a t i o n . P h o s p h o r y l a t i o n o f the a s p a r t a t e c a u s e s a s t r a i n e d p o s i t i o n ( i n t e r m e d i a t e C , or Na-El-P-Mg) w h i c h is r e l i e v e d b y a r o t a t i o n a b o u t the a-B c a r b o n bond o f the a s p a r t a t e . T h i s r o t a t i o n a l l o w s a c c e s s t o the c a t i o n s i t e s f r o m the o u t s i d e o f the c e l l and makes the s i t e s f a v o r a b l e t o a l a r g e r c a t i o n . D e p h o s p h o r y l a t i o n a l l o w s the a s p a r t a t e t o r e l a x t o i t s o r i g i n a l p o s i t i o n . T h e q u e s t i o n mark i n i n t e r m e d i a t e D i n d i c a t e s a n u n c e r t a i n t y a s t o w h e t h e r a t h i r d s i t e c a n accommodate a K+ i o n , a Na+ i o n , or neither. R e p r i n t e d w i t h p e r m i s s i o n of Current Topics i n Bioenergetics from Cantley (1981).

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F i g . 3 . T h e r e l a t i v e d i s t a n c e s b e t w e e n the a n t h r o y l o u a b a i n b i n d i n g s i t e and t h e f l u o r e s c e i n 5'-isothiocyanate-binding s i t e on F l u o r e s c e i n 5 ' -i so thiocyana t e i s p r e s u m a b l y b i n d the Na ,K-A TPase i n g a t the A T P - h y d r o l y s i s s i t e and i s n e a r the d i v a l e n t c a t i o n binding site ( C a r i l l i et a l . , 1981, 1982). The width o f a t y p i c a l b i l a y e r is shown f o r c o m p a r i s o n .

.

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DISCUSSION

T h i s model f o r c e s u s t o q u e s t i o n more d i r e c t l y how e x t e n s i v e a r e t h e p r o t e i n c o n f o r m a t i o n a l changes i n v o l v e d i n transport. The a b i l i t y of ATP, b i n d i n g t o t h e c y t o p l a s m i c s i d e o f t h e membrane, t o i n h i b i t ouabain b i n d i n g from t h e e x t r a c e l l u l a r s i d e (Hansen e t a l . , 1971) sugg e s t s t h a t a c o n f o r m a t i o n a l change e x t e n d s a c r o s s t h e bi-

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layer. However, i t i s p o s s i b l e t h a t t h e s e two l i g a n d s b i n d i n c l o s e p r o x i m i t y and a p p r o a c h t h e i r r e s p e c t i v e b i n d i n g s i t e s n e a r t h e c e n t e r of t h e b i l a y e r from oppos i t e s i d e s o f t h e membrane. I n o r d e r t o answer t h i s q u e s t i o n , w e have u s e d t h e t e c h n i q u e of f l u o r e s c e n c e r e s o n a n c e e n e r g y t r a n s f e r t o measure t h e d i s t a n c e from t h e o u a b a i n - b i n d i n g s i t e t o t h e ATP-binding s i t e ( C a r i l l i e t a l . , 1981, 1 9 8 2 ) . [3H]Anthroylouabain, s y n t h e s i z e d by t h e p r o c e d u r e o f F o r t e s (19771, was u s e d a s a d o n o r and f l u o r e s c e i n 5 ' - i s o t h i o c y a n a t e (FITC), r e a c t i n g a t t h e ATP-binding s i t e ( K a r l i s h e t al., 19791, was u s e d a s a n a c c e p t o r . The two p r o b e s c o u l d be shown t o b i n d s i m u l t a n e o u s l y and s t o i c h i o m e t r i c a l l y t o t h e N a , K - A T P a s e . The e f f i c i e n c y of e n e r g y t r a n s f e r w a s a p p r o x i m a t e l y 6 - 7 % a s judged by b o t h donor q u e n c h i n g and a c c e p t o r enhancement. These measurements s u g g e s t a most p r o b a b l e d i s t a n c e between t h e c e n t e r o f t h e t r a n s i t i o n d i p o l e s of t h e chromophores of a p p r o x i m a t e l y 74 f l . Taking i n t o account the uncertainty i n the t r a n s i t i o n dipole orientat i o n s , t h e minimum d i s t a n c e between t h e s e p r o b e s i s s t i l l 62 A . A s i m i l a r d i s t a n c e h a s been e s t i m a t e d by F o r t e s ( 1 9 8 1 ) . Thus, as i n d i c a t e d i n F i g . 3 , w e must c o n c l u d e t h a t t h e d i s t a n c e between t h e ATP- and o u a b a i n - b i n d i n g s i t e s i s q u i t e l a r g e and t h a t a r a t h e r e x t e n s i v e c o n f o r m a t i o n a l change o c c u r s o n t h e p r o t e i n d u r i n g t h e t r a n s p o r t cycle.

ACKNOWLEDGMENT

T h i s research w a s s u p p o r t e d by g r a n t number GM26199 f r o m t h e N a t i o n a l I n s t i t u t e s of H e a l t h .

REFERENCES

C a n t l e y , L. C . ( 1 9 8 1 ) . Curr. Top. B i o e n e r g . 1 1 , 201-237. C a n t l e y , L. C . , C a n t l e y , L. G . , a n d Josephson, L. ( 1 9 7 8 ) . J. Biol. C h e m . 2 5 2 , 7421-7423. C a r i l l i , C. T., F a r l e y , R A . , a n d C a n t l e y , L. C. ( 1 9 8 1 ) . (Yale Symposium t h i s i s s u e . ) C a r i l l i , C. T . , F a r l e y , R. A . , Perlman, D., and C a n t l e y , L. C . ( 1 9 8 2 ) . J. Biol. C h e m . 2 5 7 , 5601-5606. F o r t e s , P. A. G. ( 1 9 7 7 ) . B i o c h e m i s t r y 1 6 , 531-540. F o r t e s , P. A. G. ( 1 9 8 1 ) . (Yale Symposium t h i s i s s u e . ) Hansen, O . , J e n s e n , J . , and N4rby, J. G. ( 1 9 7 1 ) . N a t u r e ( L o n d o n ) , N e w B i o l . 2 3 4 , 122-123.

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Karlish, S. J. D . , Beauge, L. A . , and Glynn, I . M. ( 1 9 7 9 ) . N a t u r e (London) 282, 333-335. Macara, I. G . , and C a n t l e y , L. C. ( 1 9 8 1 a ) . B i o c h e m i s t r y 20, 5095-5105. Macara, I . G . , and C a n t l e y , L. C. ( 1 9 8 1 b ) . B i o c h e m i s t r y 20, 5695-5701. Moczydlowski, E. G., and F o r t e s , P. A. G. ( 1 9 8 1 a ) . J. B i o l . C h e m . 256, 2346-2356. Moczydlowski, E. G . , and F o r t e s , P. A. G. ( 1 9 8 1 b ) . J. B i o l . Chem. 256, 2357-2366. P o s t , R . L . , Hegyvary, C., and K u m e , S. ( 1 9 7 2 ) . J . Biol. C h e m . 247, 6530-6540. Sachs, J . R . ( 1 9 7 9 ) . I n "Na,K-ATPase: S t r u c t u r e and K i n e t i c s " ( J . C. Skou and J. G. Nbrby, e d s . ) , pp. 463-473. Academic P r e s s , N e w York. S m i t h , R. L . , Z i n n , K . , and C a n t l e y , L. C. ( 1 9 8 0 ) . J. Biol. C h e m . 255, 9852-9859.

CURRENT TOPICS IN MEMBRANES AND TRANSPORT,VOLUME 19

On the Mechanism Behind the Ability of Na,K-ATPase to Discriminate between Na+and K+ JENS CHR.SKOU Institute of Biophysics University of Aarhus Aarhus, Denmark

I.

INTRODUCTION

I t i s c h a r a c t e r i s t i c of Na,K-ATPase t h a t it can d i s c r i m i n a t e between K+ and N a + (Skou, 1957) and t h a t i n t h e p r e s e n c e of K+ it e x i s t s i n a c o n f o r m a t i o n which i s d i f f e r e n t from t h e c o n f o r m a t i o n i n t h e p r e s e n c e of Na+ (Nfdrby and J e n s e n , 1971; Hegyvary and P o s t , 1 9 7 1 ; Jfdrgensen, 1975; K a r l i s h e t a l . , 1978; K a r l i s h and Yates, 1978; K a r l i s h , 1 9 8 0 ; Skou and Esmann, 1 9 8 0 ) . I n o t h e r words, t h e system c a n s e n s e t h e d i f f e r e n c e between K+ and N a + , and t h e m o l e c u l a r s t r u c t u r e h a s t h e a b i l i t y t o a d a p t t o t h e d i f f e r e n c e . T h i s i s a p p a r e n t l y t h e basis €or the a b i l i t y t o discriminate. The a d a p t a t i o n of t h e m o l e c u l a r s t r u c t u r e t o K+ and N a + , r e s p e c t i v e l y , i s i n t i m a t e l y c o n n e c t e d t o a change i n t h e a f f i n i t y f o r ATP (Ndrby and J e n s e n , 1971; Hegyvary and P o s t , 1 9 7 1 ; K a r l i s h e t a l . , 1978; Skou and Esmann, 1 9 8 1 ) . When t h e s t r u c t u r e i s a d a p t e d t o N a + , a h i g h - a f f i n i t y s i t e f o r ATP is opened, w h e r e a s when a d a p t e d t o K + , t h e a f f i n i t y f o r ATP i s d e c r e a s e d ( o r t h e 323

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F i g . 1. A c t i v a t i o n b y (Na + K = 150 mM) o f the h y d r o l y s i s o f ATP w i t h 0.1, 1 , 1 0 , a n d 100 pM A T P , r e s p e c t i v e l y ; the Mg2+ concentration was 50, 1 0 0 , 1 0 0 , a n d 500 pM r e s p e c t i v e l y ; 30 mM h i s t i d i n e - H C 1 b u f f e r , pH 7.4, 37OC. The a c t i v i t y i s g i v e n a s p e r c e n t o f t h e maximum obtainable w i t h the g i v e n ATP concentrat i o n . T h e Na,K-ATPase was p r e p a r e d f r o m rectal g l a n d s f r o m Squalus a c a n t h i a s ( S k o u a n d E s m a n n , 1 9 7 9 ) . T h e s p e c i f i c a c t i v i t y was 1320 moles P i / m g p r o t e i n / h r . (From S k o u a n d E s m a n n , 1 9 8 0 . ) 4 R e p r o d u c e d b y permission f r o m B i o c h i m . B i o p h y s . A c t a .

s i t e i s c l o s e d ? ) . T h i s means t h a t ATP w i l l t e n d t o change t h e s t r u c t u r e from t h e K form t o t h e N a form. The s t r u c t u r a l a d a p t a t i o n i s observed a s an i n c r e a s e i n t h e r a t e of t h e t r a n s f o r m a t i o n of t h e K form t o t h e N a form by ATP a t a g i v e n N a : K r a t i o ( K a r l i s h e t a l . , 1 9 7 8 ; K a r l i s h and Yates, 1 9 7 8 ) . Under s t e a d y - s t a t e c o n d i t i o n s t h e m o l e c u l a r adapt a t i o n i s observed a s a d e c r e a s e i n t h e Na+ c o n c e n t r a t i o n n e c e s s a r y f o r half-maximum Na+ a c t i v a t i o n i n t h e p r e s e n c e of K+ (Na+ + K+ = 1 5 0 m M ) when t h e ATP concent r a t i o n i s i n c r e a s e d ( F i g . 1 ) . T h i s i s due t o an e f f e c t of ATP a s such and n o t t o t h e h y d r o l y s i s of ATP (Skou, 1 9 7 4 ) . B u t what i s t h e mechanism behind t h e a d a p t a t i o n of t h e molecular s t r u c t u r e t o t h e c a t i o n s ?

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Fig. 2. ( A ) Na versus K t i t r a t i o n c u r v e s o f the eosin f l u o r e s c e n c e a t d i f f e r e n t v a l u e s o f pH a n d w i t h Na+ + = 1 5 0 mM. 100 p g Na,K-ATPase w i t h the s p e c i f i c a c t i v i t y g i v e n i n F i g . 1 : a t pH 7 . 2 a n d l o w e r , 30 mM h i s t i d i n e - H C 1 , 2 mM CDTA; a t pH 7 . 2 a n d l o w e r , 30 mM h i s t i d i n e - H C 1 , 2 mM CDTA; a t pH 7 . 8 a n d h i g h e r , 30 mM ( B ) T h e e f f e c t o f pH on the eosin T r i s - H C 1 a n d 2 mM CDTA, 22OC. f l u o r e s c e n c e a t d i f f e r e n t Na:K r a t i o s a n d w i t h Na+ + K+ = 1 5 0 mM. B u f f e r s a n d e n z y m e a s i n F i g . 2 A , 22'C. ( F r o m Skou a n d E s m a n n , 1 9 8 0 Reproduced b y p e r m i s s i o n f r o m Biochim. Biophys. A c t a .

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+

An i n c r e a s e i n pH s h i f t s t h e N a + v s K ( N a + K+ = 1 5 0 mM) t i t r a t i o n c u r v e toward a lower N a + c o n c e n t r a t i o n f o r t h e t r a n s f o r m a t i o n from t h e K form (low f l u o r e s c e n c e ) t o t h e Na form ( h i g h f l u o r e s c e n c e ) (see F i g . 2 A ) . Or-what i s t h e same--an i n c r e a s e i n t h e N a : K r a t i o s h i f t s

t h e pH t i t r a t i o n c u r v e toward a l o w e r pH v a l u e f o r t h e transformation (Fig. 2 B ) . I t means t h a t a n i n c r e a s e i n t h e N a : K r a t i o d e c r e a s e s pK v a l u e s , l e a d i n g t o a deprot o n a t i o n o f t h e s y s t e m ; t h e e f f e c t i s v e r y pronounced. I t i n d i c a t e s t h a t p r o t o n s a r e t a k e n up when K+ i s bound and r e l e a s e d when Na+ i s bound--a Bohr e f f e c t . The K form i s a p r o t o n a t e d and t h e N a form a d e p r o t o n a t e d form More N a + r e l a t i v e t o K+ i s needed f o r half-maximum e f f e c t on t h e e q u i l i b r i u m d i s t r i b u t i o n between t h e K form and t h e N a form when t h e sum o f t h e two c a t i o n s i n t h e t i t r a t i o n e x p e r i m e n t s i s d e c r e a s e d t o 1 0 mM i n s t e a d of 1 5 0 mM (Skou and Esmann, 1 9 8 0 ) . A n i n c r e a s e i n t h e sum of t h e c a t i o n s ( i o n i c s t r e n g t h e f f e c t ? ) f a c i l i t a t e s t h e t r a n s f o r m a t i o n from t h e K form t o t h e Na form and f a c i l i t a t e s t h e d e p r o t o n a t i o n , i . e . , t h e d e c r e a s e i n pK. T h e r e a r e t y o K v a l u e s i n v o l v e d . T h i s i s s e e n from a p l o t of t h e N a : K r a t i o ( N a + + K+ = 1 5 0 m M ) which g i v e s a n e q u a l d i s t r i b u t i o n between t h e K form and t h e N a form a s a f u n c t i o n of pH ( F i g . 3 ) . One m o i e t y w i t h a PK between 5 and 7 may be h i s t i d i n e g r o u p s , and a n o t h e r w i t h a PK of 9 o r h i g h e r may b e €-amino g r o u p s o f l y s i n e .

.

?

B.

STEADY STATE

The e f f e c t on t h e Na+ v s K+ t i t r a t i o n c u r v e of a n i n c r e a s e i n pH i s a l s o s e e n u n d e r s t e a d y - s t a t e c o n d i t i o n s when t h e h y d r o l y s i s of n o n s a t u r a t i n g c o n c e n t r a t i o n s of ATP i s u s e d t o m o n i t o r t h e e f f e c t . An i n c r e a s e i n pH d e c r e a s e s t h e Na:K r a t i o ( N a + + K+ = 1 5 0 mM) f o r h a l f maximum N a + a c t i v a t i o n * ( F i g . 4 ) . O r , what i s t h e same, a d e c r e a s e i n t h e N a : K r a t i o s h i f t s t h e pH t i t r a t i o n c u r v e t o w a r d s a h i g h e r pH v a l u e , i n c r e a s e s pK ( F i g . 5 ) . The a c t i v i t y a t a g i v e n s u b o p t i m a l Na:K r a t i o i n F i g . 5 i s g i v e n i n p e r c e n t of t h e a c t i v i t y w i t h t h e optimum N a : K r a t i o . A change i n t h e f r a c t i o n a l *In t h e f o l l o w i n g t h e Na+ concentration f o r h a l f maximum Na+ e f f e c t w i t h Na+ + fi = 1 5 0 mM is denoted K0.5 for Na+.

327

Na,K-ATPase DISCRIMINATION BETWEEN Na+AND K +

5

6

7

PH

8

9

10

F i g . 3 . T h e e f f e c t of pH on the Na:K r a t i o f o r h a l f - m a x i m u m c h a n g e i n eosin f l u o r e s c e n c e w i t h Na’ + K+ = 1 5 0 mM, 22’C; e n z y m e a s i n F i g . 1 (Skou a n d Esmann, 1 9 8 0 ) . R e p r o d u c e d b y p e r m i s s i o n from B i o c h i m . B i o p h y s . A c t a .

a c t i v i t y a t a given suboptimal N a : K r a t i o r e f l e c t s a change i n a b i l i t y o f N a + t o compete w i t h K+ f o r N a + act i v a t i o n o f h y d r o l y s i s . The a c t i v i t y i s measured w i t h 0 . 1 p M ATP ( F i g s . 4 and 5 ) a n d , f o r t h e N a : K r a t i o of 9 0 : 6 0 , a l s o w i t h 1 0 p M ATP ( F i g . 5 ) . A c o m p a r i s o n between F i g . 4 and F i g . 1 shows t h a t a n i n c r e a s e i n pH w i t h a n o n s a t u r a t i n g c o n c e n t r a t i o n of ATP h a s a n e f f e c t o n ~ 0 . 5 f o r N a + a c t i v a t i o n Which i s s i m i l a r t o t h e e f f e c t of a n i n c r e a s e i n t h e ATP concent r a t i o n a t a g i v e n pH. A comparison between F i g . 5 and F i g . 2% shows t h a t t h e p a t t e r n of t h e e f f e c t o f a change i n t h e N a : K r a t i o on t h e pH t i t r a t i o n c u r v e i s t h e same i n t h e e q u i l i b r i u m and i n t h e h y d r o l y s i s e x periments. F i g u r e 5 shows t h a t a n i n c r e a s e i n t h e ATP concent r a t i o n from 0 . 1 t o 1 0 p M a t a N a : K r a t i o of 9 0 : 6 0 S h i f t s t h e pH t i t r a t i o n c u r v e t o w a r d a l o w e r pH v a l u e , d e c r e a s i n g PK. ATP t h u s f a c i l i t a t e s a d e p r o t o n a t i o n o f t h e s y s t e m and t h e r e b y f a c i l i t a t e s t h e t r a n s f o r m a t i o n from t h e K form t o t h e N a form a t a g i v e n N a : K r a t i o .

JENS CHR SKOU

0 150

50 100

100 50

150 NA O K

CONC, (MM)

F i g . 4 . A c t i v a t i o n of the Na,K-ATPase h y d r o l y s i s of ATP b y Na+ + K+ (Na+ + K+ = 150 mM) a t pH 5 . 9 , 7 . 4 , a n d 8 . 4 . A T P , 0.1 pM; Mg2+, 50 pM; b u f f e r s a s i n F i g . 2, 37OC ( S k o u , 1 9 7 9 ) . R e p r o d u c e d b y permission from B i o c h i m . B i o p h y s A c t a

.

111.

.

pH EFFECT ON RATE OF CONFORMATIONAL TRANSITION

ATP i n c r e a s e s t h e r a t e of t h e t r a n s f o r m a t i o n from t h e K form t o t h e Na form ( K a r l i s h e t a l . , 1978; K a r l i s h and Y a t e s , 1 9 7 8 ) . T h i s i s a l s o t h e case when pH i s i n c r e a s e d ( w i t h no ATP) (Table I ) . The change i n t h e f l u o r e s c e n c e of e o s i n i s used t o monitor t h e e f f e c t (Skou and Esmann, 1 9 8 1 ) . With t h e combination of c a t i o n s used i n t h e e x p e r i m e n t s , an i n c r e a s e i n pH from 7 . 4 t o 8 . 4 g i v e s a 5- t o 6-fold i n c r e a s e i n t h e r a t e b o t h a t 22OC and a t 4OC. The t)i f o r t h e t r a n s f o r m a t i o n from t h e K form t o t h e Na form (from K+ = 1 mM t o f i n a l K+ = 0 . 5 mM, N a + = 7 5 ITIM) a t pH 7 . 0 i s a b o u t 2 . 7 s e c a t 23OC i n e x p e r i m e n t s where t h e i n t r i n s i c f l u o r e s c e n c e of t r y p t o p h a n has been used t o monitor t h e c o n f o r m a t i o n a l changes ( K a r l i s h and Yates, 1 9 7 8 ) . The p r e s e n c e of 1 0 p~ ATP i n c r e a s e d t h e r a t e 4t o 5-fold ( r e a d from F i g . 5 i n K a r l i s h and Yates, 1 9 7 8 ) . T h i s i s t o be compared w i t h t h e 5- t o 6-fold i n c r e a s e i n r a t e by an i n c r e a s e i n pH from 7 . 4 t o 8.4 and which h a s an e f f e c t on K ~ f o. r Na+ ~ under s t e a d y - s t a t e c o n d i t i o n s

Na,K-ATPaseDISCRIMINATION BETWEEN Na+AND K+

329

Na:K mM

+Y

1 140:'0

+

z a

f

f

-

m m

t J 0

a n

o_ t-

o

I

t;

3

>

%t; a 2 + t 4

4 v

L

O

tW z

V

a

a W

5

6

7 PH

a

9

F i g . 5. The e f f e c t o f pH on the h y d r o l y s i s o f ATP a t d i f The a c t i v i t y i s g i v e n a s f e r e n t Na:K r a t i o s (Na+ + K+ = 1 5 0 mM). p e r c e n t o f the a c t i v i t y w i t h the o p t i m u m Na:K r a t i o f o r h y d r o l y s i s a t e a c h pH v a l u e . T h e ATP c o n c e n t r a t i o n was 0.1 p M , and f o r Na:K T h e v a l u e s w i t h 0.1 pM ATP a r e 90:60, 0.1 and 10 pM, pg2+ 5 0 pM. ( S k o u and r e p l o t t e d f r o m F i g . 4 . B u f f e r s a r e a s i n F i g . 2 , 37'C. E s m m n , 1 9 8 0 . ) Reproduced b y p e r m i s s i o n f r o m B i o c h i m . B i o p h y s . Acta.

comparable t o t h e e f f e c t of an i n c r e a s e i n t h e ATP conc e n t r a t i o n t o 4 p M a t pH 7 . 4 (compare F i g s . 4 and 6 ) . The r a t e of t h e r e v e r s e r e a c t i o n , t h e t r a n s f o r m a t i o n from t h e N a form t o t h e K form, i s d e c r e a s e d 5- t o 6 - f o l d A t 4'C t h e by an i n c r e a s e i n pH from 7 . 4 t o 8 . 4 a t 2 2 ' C . e f f e c t i s lower: t h e r a t e i s d e c r e a s e d 1 . 8 - f o l d , i . e . , t h e r a t e o f t h e t r a n s f o x m a t i o n i s less t e m p e r a t u r e - s e n s i t i v e a t pH 8.4 t h a n a t pH 7 . 4 . There a r e no measurements of t h e e f f e c t of ATP on t h e r a t e of t h e t r a n s f o r m a t i o n from t h e Na form t o t h e K form, and i t i s unknown whether ATP d e c r e a s e s t h i s r a t e , a s observed by an i n c r e a s e i n pH.

JENS CHR SKOU

330 TABLE I.

H a l f - T h e ( $1 f o r T r a n s f o r m a t i o n f r o m the K f o r m t o the Na Form o f the Enzyme and V i c e V e r s a a

T e m p e r a t u r e ('C)

t 4 (set)

PH

K form +Na form 22

4

7.4 8.4 7.4 8.4

0.53 f 0.01 0.100 k 0.002 4.18 k 0.07 0.69 f 0.03

b

Na f o r m -+ K f o r m (0.019 0.078 0.101 0.183

k k k k

0.001) 0.003 0.001 0.006

a

For the t r a n s f o r m a t i o n f r o m the K f o r m ( l o w f l u o r e s c e n c e ) t o the Na f o r m ( h i g h fluorescence), s y r i n g e 1 o f the s t o p - f l o w app a r a t u s c o n t a i n e d a t pH 7 . 4 , 3 0 mM h i s t i d i n e HCI, and a t pH 8 . 4 , 30 mM Tris-HC1, a n d a t e a c h pH, 2 mM 90 ug e n z y m e / m l , 0 . 5 p M eosin Y. S y r i n g e 2 c o n t a i n e d b u f f e r s a s i n s y r i n g e 1 , and 2 mM K+, 80 mM Na+, 0 . 5 pM eosin Y . For the t r a n d f o r m a t i o n from the Na form t o the K f o r m s y r i n g e 1 c o n t a i n e d b u f f e r s a s a b o v e , 20 mM Na*, 0.5 pM eosin Y , 90 p g e n z y m e / m l . S y r i n g e 2 c o n t a i n e d the same b u f f e r s a s i n s y r i n g e 1 , 20 mM Na+, 4 0 mM Kf, 0.5 p M eosin Y . T h e t i m e d e l a y was 7 and 0 msec, r e s p e c t i v e l y , a t 22'C, and 7 m s e c a t 4OC. E x c i t a t i o n was a t 530 nm, e m i s s i o n a t 560 nm, w i t h 10-nm s l i t f o r

+,

both.

bEach v a l u e i s b a s e d on a t l e a s t 3 d i f f e r e n t e x p e r i m e n t s and f o r e a c h e x p e r i m e n t , on 3-5 curves. The 0 . 0 1 9 - s e c v a l u e f o r t-4 f o r the t r a n s f o r m a t i o n f r o m the Na f o r m to the K f o r m a t pH 7 . 4 22'C, i s o f the o r d e r o f the t.4 f o r the r e l e a s e o f the eosin p r o b e f r o m the Na f o r m and may t h e r e f o r e be a maximum v a l u e f o r t.4 f o r the t r a n s f o r m a t i o n . Enzyme a s i n F i g . 6 ( S k o u , 1 9 8 2 ) .

IV.

pH VERSUS ATP EFFECT

There i s t h u s a s i m i l a r i t y between t h e e f f e c t of a n i n c r e a s e i n pH a t a n o n s a t u r a t i n g c o n c e n t r a t i o n of ATP and o f a n i n c r e a s e i n t h e ATP c o n c e n t r a t i o n a t a g i v e n pH on t h e N a + v s K+ t i t r a t i o n c u r v e s . There i s a l s o a s i m i l a r i t y between t h e e f f e c t of ATP ( K a r l i s h e t a l . , 1 9 7 8 ; K a r l i s h and Yates, 1 9 7 8 ) and of an i n c r e a s e i n pH on t h e r a t e of t h e t r a n s f e r from t h e K form t o t h e N a form. s u g g e s t i n g t h a t t h e b i n d i n g o f ATP t o t h e K form f a c i l i t a t e s release of p r o t o n s , d e c r e a s e s pK v a l u e s on t h e system, and t h e r e b y f a c i l i t a t e s t h e t r a n s f o r m a t i o n from t h e K form t o t h e N a form o f t h e system. There i s , however, a d i f f e r e n c e . A s s e e n from F i g s . 2 and 3 , K ~ f o. r ~N a + d e c r e a s e s c o n t i n u o u s l y when

331

Na.K-ATPase DISCRIMINATION BETWEEN Na+AND K+

150

B

A

E r 4

z a

CONTROL

0 m 4

0

I1

m

x

Y

100

s++ z 4

-

50

0

ATP

CONC.

(pM)

F i g . 6 . T h e concentration o f Na+ f o r half-maximum a c t i v a t i o n o f h y d r o l y s i s ( K o . 5 f o r Na+, Na+ + K+ = 1 5 0 mM) a t d i f f e r e n t ATP concentrations. ( A ) F o r control e n z y m e ( 0 ) , and f o r e n z y m e p r e i n c u b a t e d w i t h 0 . 4 mM p y r i d o x a l 5 - p h o s p h a t e + 1 5 0 mM Na+ f o r 30 m i n , pH 7 . 4 , 22OC ( A ) . T h e K O e 5 v a l u e s f o r Na+ a r e r e a d f r o m a series of curves l i k e t h e curves shown i n F i g s . 1 and 9 B , t e s t e d i n 3 0 mM h i s t i d i n e - H C 1 , pH 7 . 4 , 37'C. Enzyme i s a s i n F i g . 1 ; s p e c i f i c ATPase a c t i v i t y i s 1 4 6 2 p o l e s Pi/mg p r o t e i n / h r . ( B ) For control e n z y m e a t pH 7 . 4 ( 0 ) and pH 8 . 4 ( A ) . T h e v a l u e s a r e f r o m a n enzyme p r e p a r a t i o n f r o m ox b r a i n , a n d the v a l u e s a r e r e p l o t t e d f r o m A t pH 7 . 4 , 30 mM h i s t i d i n e - H C 1 , and a t pH F i g . 6 i n Skou (1979 ) 8 . 4 , 30 mM Tris-HC1 a s b u f f e r s ( S k o u . 1 9 8 2 ) . R e p r o d u c e d by p e r m i s s i o n from Biochim. Biophys. Acta

.

.

pH i s i n c r e a s e d , and a t t h e h i g h e r o f t h e two pK v a l u e s o f t h e system, a n i n c r e a s e i n pH h a s a pronounced e f f e c t on t h e decrease i n ~ 0 . 5 f o r N a + ; t h e ~ 0 . 5 f o r N a + i s 9 mM a t pH 9 . 4 . On t h e o t h e r hand, when ATP i s i n c r e a s e d , t h e e f f e c t on ~ 0 . 5 f o r N a + l e v e l s o f f o r i s r e p l a c e d by an i n c r e a s e i n ~ 0 . 5 a t t h e h i g h e r ATP c o n c e n t r a t i o n s ( F i g . 6). T h i s l a s t e f f e c t i s more pronounced a t pH 8 . 4 t h a n a t pH 7 . 4 . The minimum v a l u e f o r ~ 0 . 5 f o r N a + i s h i g h e r

332

JENS CHR SKOU

NABH,,

I

I

I

0

2

4

MINUTES F i g . 7 . ATP h y d r o l y s i s as a f u n c t i o n of t i m e b y e n z y m e w h i c h has been p r e i n c u b a t e d f o r 3 0 m i n i n a 3 0 mM N - e t h y l m o r p h o l i n e b u f f e r (pH 7 . 4 ) a t 22OC w i t h o u t ( A ) a n d w i t h 0 . 4 mM p y r i d o x a l 5 - p h o s p h a t e (PLP) + 1 5 0 mM Na+ ( 0 ) and w i t h 0 . 4 mM PLP + 150 mM Na+ b u t w i t h a d d i t i o n o f NaBH4 a t the end o f p r e i n c u b a t i o n ( 0 ) . T h e a c tivity was t e s t e d w i t h 3 mM ATP, 130 mM ??a+, 20 mM Kf, and 4 mM Mg2+ i n 30 mM h i s t i d i n e - H C 1 , pH 7 . 4 , 37'C. T h e e n z y m e was d i l u t e d 1:200 b y t r a n s f e r f r o m the preincubation t o the t e s t medium. Enzyme as i n F i g . 6 ( S k o u , 1 9 8 2 ) . R e p r o d u c e d b y permission f r o m B i o c h i m . Biophys. Acta

.

t h a n t h e v a l u e s o b t a i n e d by t h e pH i n c r e a s e ; a t pH 7 . 4 i t i s 4 8 mM w i t h r e c t a l g l a n d enzyme ( F i g . 6A) and 37 m M w i t h ox b r a i n enzyme ( F i g . 6B). Thus, ATP h a s two opposing e f f e c t s : o n e which d e c r e a s e s for N a + l i k e a

Na,K-ATPase DISCRIMINATION BETWEEN Na+AND K+

333

d e p r o t o n a t i o n of t h e system and w i t h a h i g h a f f i n i t y f o r ATP, and a n o t h e r which i n c r e a s e s ~ 0 . 5f o r N a + b u t w i t h a l o w a f f i n i t y f o r ATP ( s i n c e t h e l a t t e r e f f e c t i s more

pronounced when t h e pH i s i n c r e a s e d , i t seems r e l a t e d t o a d e p r o t o n a t i o n ) . Thus, i t seems a s i f i t i s t h e deprot o n a t i o n by ATP o f t h e group w i t h t h e lower o f t h e two pK v a l u e s which leads to a n i n c r e a s e i n ~ 0 . 5f o r N a + , whereas t h e d e p r o t o n a t i o n by ATP o f t h e group w i t h t h e higher PK, i n c o n t r a s t t o t h e effect of a deprotonation by a n i n c r e a s e i n pH, l e a d s t o a n i n c r e a s e i n ~ 0 . 5f o r Na+.

The d a t a s u g g e s t t h a t ATP n o t o n l y i n c r e a s e s t h e r a t e o f t r a n s f e r from t h e K form t o t h e N a form b u t , i n c o n t r a s t t o t h e e f f e c t o f an i n c r e a s e i n pH, a l s o i n creases t h e r a t e o f t h e t r a n s f e r from t h e N a form t o t h e K form ( o b s e r v e d i n p r e l i m i n a r y e x p e r i m e n t s ) , and t h a t t h e a f f i n i t y f o r t h e ATP e f f e c t on t h e r a t e o f t h e t r a n s f e r from t h e N a form t o t h e K f o r m i s lower t h a n t h e a f f i n i t y f o r t h e e f f e c t on t h e r a t e o f t h e t r a n s f e r from t h e K form t o t h e N a form. A low ATP c o n c e n t r a t i o n c o u l d g i v e a d e c r e a s e i n ~ 0 . 5f o r N a + which l e v e l s o f f o r i s r e v e r s e d a t a h i g h e r ATP c o n c e n t r a t i o n . The r e s u l t s seem t o r e q u i r e r e a c t i o n w i t h two ATP m o l e c u l e s e i t h e r simultaneously o r consecutively. What l i m i t s t h e r a t e of t u r n o v e r i s t h u s t h e req u i r e m e n t f o r ATP f o r t h e e f f e c t which i n c r e a s e s Ko.5 f o r N a + ( i . e . , t h e e f f e c t on t h e r a t e of t h e t r a n s f o r m a t i o n from t h e N a form t o t h e K f o r m ) , and n o t t h e r e q u i r e m e n t f o r ATP f o r t h e d e c r e a s e i n ~ 0 . 5f o r N T ( i . e . , t h e e f f e c t on t h e r a t e o f t h e t r a n s f o r m a t i o n from t h e K form t o t h e N a form; t h e ATP c o n c e n t r a t i o n which i s n e c e s s a r y f o r t h i s reaction is lower).

V.

EFFECT O F MODIFICATION W I T H PYRIDOXAL 5-PHOSPHATE

P y r i d o x a l 5-phosphate i n h i b i t s t h e enzyme r e v e r s i b l y . T h i s i s s e e n from F i g . 7 , where t h e enzyme h a s been i n c u b a t e d w i t h p y r i d o x a l 5-phosphate (PLP) i n t h e d a r k and t h e r e a f t e r t r a n s f e r r e d t o t h e t e s t medium w i t h no PLP ( d i l u t e d 1 : 2 0 0 ) . The i n i t i a l a c t i v i t y i s low b u t i n creases a s a f u n c t i o n of t i m e . The i n h i b i t i o n c a n be made i r r e v e r s i b l e by t e r m i n a t i n g t h e p r e i n c u b a t i o n by adding NaBH4 (Fig. 7 ) . With 0 . 4 mM PLP a t 2 2 O C , pH 7 . 4 , t h e i n h i b i t i o n o f t h e A T P a s e a c t i v i t y i n t h e p r e s e n c e of 150 mM N a + ? 3 mM ATP, of 150 mM K+ k 3 mlcr ATP, and of 3 mM ATP, r e s p e c t i v e l y , r e a c h e s e q u i l i b r i u m a f t e r 20-30 min o f p r e i n c u -

334

JENS CHR SKOU

ATP

ATP, K+ ATP,NA+

K+

0

10

20

30

MINUTES T h e ATPase a c t i v i t y a f t e r d i f f e r e n t times of p r e i n T h e enzyme was p r e i n c u b a t e d i n a 30 mM N - e t h y l m o r p h o l i n e HCl b u f f e r a t pH 7 . 4 , 22OC w i t h 10 mM CDTA, 0.4 mM PLP and w i t h 3 mM ATP ( a ) , 1 5 0 mM K' (O), 1 5 0 mM Na+ ( A ) , 1 5 0 mM + 3 mM ATP ( B ) , a n d w i t h 1 5 0 mM Na+ + 3 mM ATP ( 4 ) , r e s p e c t i v e l y , f o r the t i m e s shown on the f i g u r e . A f t e r p r e i n c u b a t i o n , 5 - f o l d excess o f NaBH was a d d e d ( 2 d r o p s o f octanol t o a v o i d 4 f o a m i n g ) a n d the e n z y m e was washed three t i m e s b y c e n t r i f u g a t i o n i n a 30 mM h i s t i d i n e - H C 1 b u f f e r , pH 6.8 a t ZoC, and f i n a l l y resusp e n d e d i n the same b u f f e r b u t w i t h 25% g l y c e r o l . T h e ATPase act i v i t y was m e a s u r e d w i t h 3 mM ATP, 20 mM Kf, 130 mM Na+, 4 mM My2+ i n 30 mM h i s t i d i n e - H C 1 , pH 7 . 4 , 37OC. Enzyme a s i n F i g . 6 ( S k o u , 1 9 8 2 ) . Reproduced b y p e r m i s s i o n f r o m B i o c h i m . B i o p h y s . A c t a Fig. 8 .

cubation w i t h PLP.

.

b a t i o n ( F i g . 8 ) . The i n a c t i v a t i o n i n t h e p r e s e n c e of 1 5 0 m K+ i s lower t h a n i n t h e p r e s e n c e of 150 m N a + . ATP p r o t e c t s p a r t l y a g a i n s t i n a c t i v a t i o n i n t h e p r e s e n c e of b o t h N a + and K+. I n t h e f o l l o w i n g e x p e r i m e n t s w i t h PLP, t h e enzyme h a s been p r e i n c u b a t e d w i t h 0 . 4 mM PLP a t 22OC, pH 7 . 4 , f o r 3 0 min i n t h e d a r k w i t h t h e l i g a n d s shown on t h e

Na,K-ATPaseDISCRIMINATION BETWEEN Na+AND K+

h

100

335

-

w

U

z

W

U

v) w

O

0

L

X

0

A

3

_I

.

L

L

W

v

w z

Y

z

1

V I w V w v t Y z o w 3 J V L

L

50

-

U I tY W

a : LL

L 4

-

0I

I

I

0

50

150

100

100 50

I

150 O

NA K

CONC, (MM)

I

0 150

I

I

1

50

100

150

100

50

O

NA K

CONC. (MM) Fig. 9. ( A ) T h e e f f e c t o f d i f f e r e n t N a : K r a t i o s (Na+ + Kf. = 1 5 0 mM) on the f l u o r e s c e n c e o f eosin i n the p r e s e n c e o f e n z y m e w h i c h has been p r e i n c u b a t e d w i t h 0 . 4 mM PLP f o r 30 m i n a t 22'C, pH 7 . 4 i n the p r e s e n c e o f 1 5 0 mM Na+ ( 0 ) and 1 5 0 mM K+ ( 0 ) and of control e n z y m e ( A ) . (30 mM h i s t i d i n e - H C l pH 7 . 4 , 22'C, 0 . 1 p M e o s i n ) . ( B ) T h e a c t i v a t i o n b y Na+ + K+ (Na' + @ = 150 mM) o f the h y d r o l y s i s o f ATP i n t h e same three e n z y m e p r e p a r a t i o n s a s i n F i g . 9A ( s y m b o l s a s i n F i g . 9 A ) . ATP c o n c e n t r a t i o n was 0.1 pM, Mg2+ 50 p M , h i s t i d i n e HCI 30 mM, pH 7 . 4 , n 0 C . Enzyme as i n F i g . 6 ( S k o u , 1 9 8 2 ) . R e produced b y permission from Biochim. Biophys. Acta.

336

JENS CHR SKOU

TABLE 11.

E f f e c t of M o d i f i c a t i o n b y PLPa

K

Control PLP, K+ PLP , ATP PLP, K+, ATP PLP, N a + PLP, N a + , ATP

0.5

for Na+

108 -+ 1 98 f 1 80 f 2 58 f 0 . 2 49 f 2 52 2 1

Activity control)

( % of

(mM)

(n=5) (n-3) (n=3) (n=3) (n=6) (n=3)

100 52 87 72 31 69

f 122 f 1.8 f 1.7 f 0.6 f 1.2

(-4) (n=3) (n=3) (n=9) (n=3)

a

T h e e f f e c t was m e a s u r e d i n the p r e s e n c e of 150 mM Kt f 3 mM ATP, o f 3 mM ATP, a n d of 150 mM Na+ f 3 mM ATP, r e s p e c t i v e l y , ( a ) on Ko.5 f o r Na' f o r the equilibrium d i s t r i b u t i o n between the K f o r m and the Na f o r m (Na+ + Kt = 150 mM) a n d ( b ) on the e n z y m e a c t i v i t y t e s t e d w i t h 3 mM ATP, 4 mM Mq2+, 130 mM Na+, 20 mM Kf, 30 mM h i s t i d i n e - H C 1 , pH 7 . 4 , 37OC. T h e Ko.5 v a l u e s for Na+ are taken f r o m curves l i k e those shown i n F i g . 9A. Enzyme i s a s i n F i g . 6 . (Skou, 1982).

f i g u r e s , f o l l o w e d by t h e a d d i t i o n of 5 mM NaBH4 and washing 3 t i m e s by c e n t r i f u g a t i o n . Enzyme m o d i f i e d w i t h PLP f o r 30 min under t h e cond i t i o n s s p e c i f i e d and w i t h t h e l i g a n d combinations shown i n F i g . 8 behaves a s a c o n t r o l enzyme b u t a t a h i g h e r pH. The ~ 0 . 5 f o r N a + i s d e c r e a s e d , an e f f e c t s e e n b o t h under e q u i l i b r i u m c o n d i t i o n s ( F i g , 9A) and under s t e a d y - s t a t e c o n d i t i o n s , where t h e h y d r o l y s i s of ATP i s used t o t e s t t h e e f f e c t ( F i g . 9B). The s t e a d y - s t a t e e f f e c t i n d i c a t e s t h a t t h e enzyme p r e p a r a t i o n a f t e r t h e m o d i f i c a t i o n i s n o t a m i x t u r e of m o d i f i e d i n a c t i v e and of normal a c t i v e enzyme; r a t h e r , i t i s t h e m o d i f i e d enzyme which h a s reduced a c t i v i t y . The d e c r e a s e i n ~ 0 . 5 f o r N a + when enzyme i s modif i e d by PLP d o e s n o t p a r a l l e l t h e decrease i n a c t i v i t y , b u t b o t h e f f e c t s are h i g h l y dependent on t h e combination of l i g a n d s i n t h e p r e i n c u b a t i o n m e d i u m ( F i g s . 8 and 9, and T a b l e 11). Enzyme which h a s been m o d i f i e d w i t h PLP i n t h e p r e s e n c e of N a + s t i l l responds t o an increase i n t h e ATP c o n c e n t r a t i o n i n t h e t e s t medium by an i n c r e a s e i n t h e ~ 0 . 5 f o r N a + ( F i g . 6A; n o t t e s t e d f o r enzyme m o d i f i e d i n t h e p r e s e n c e o f t h e o t h e r l i g a n d c o m b i n a t i o n s ) . A comp a r i s o n of g r a p h s A and B i n F i g . 6 shows t h a t t h e PLPm o d i f i e d enzyme behaves as a c o n t r o l enzyme b u t a t a h i g h e r pH. ATP p r o t e c t s a g a i n s t t h e d e c r e a s e i n enzyme a c t i v i t y which follows from t h e m o d i f i c a t i o n by PLP b o t h w i t h N a +

337

Na,K-ATPase DISCRIMINATION BETWEEN Na+AND K+

TABLE 111.

Half-Time ( t 5 ) f o r t h e Transformation from t h e K Form t o t h e Na Form and f o r t h e Reverse Reaction o f PLP Modified Enzymea

~~

Temperature ("C) 22

4

K form+Na form

PH 7.4

7.4

Control PLP, 'K PLP, ATP PLP,,'K ATP PLP, Na+ PLP, Na+, ATP Control PLP, 'K PLP , ATP PLP,,'K ATP PLP, Na' PLP, Na', ATP

0.45 0.46 0.37

N a form+K form

f 0.02 f 0.02 k 0.01

0.11

f 0.016

0.004

f 0.000

0.095 2 0.002 3.33 3.13 2.08 0.47 0.33 0.38

k 0.04 k 0.03

2 0.04 f 0.02 f 0.02 f 0.01

0.096 0.156 0.177 0.193 0.292 0.273

* 0.007 ? 0.007

f 0.004 f 0.006 f 0.012

f 0.007

aExperimental c o n d i t i o n s a s i n Table I, b u t with 300 ug of t h e modified enzyme/ml. The enzyme was p r e i n c u b a t e d f o r 30 min with t h e c o n c e n t r a t i o n s and combinations of PLP and l i g a n d s desc r i b e d i n F i g . 8. A t t h e end of p r e i n c u b a t i o n t h e enzyme was t r e a t e d a s d e s c r i b e d i n F i g . 8. Enzyme as i n F i g . 6 (Skou, 1 9 8 2 ) .

and w i t h K+ i n t h e p r e i n c u b a t i o n medium. But ATP i n t h e p r e i n c u b a t i o n medium h a s no e f f e c t on t h e "pH e f f e c t " of t h e m o d i f i c a t i o n by PLP i n t h e p r e s e n c e of N a + , and i n creases t h e "pH e f f e c t " when enzyme i s m o d i f i e d i n t h e p r e s e n c e o f K+ ( T a b l e 11). Thus, t h e r e must be a t l e a s t two d i f f e r e n t PLP-reactive g r o u p s on t h e enzyme. Howe v e r , enzyme m o d i f i e d i n t h e p r e s e n c e o f N a + b u t w i t h o u t ATP s t i l l r e s p o n d s t o a n i n c r e a s e i n t h e ATP c o n c e n t r a t i o n i n t h e t e s t m e d i u m by a decrease i n ~ 0 . 5 f o r Na+. I t means t h a t t h e r e a c t i o n w i t h PLP which l e a d s t o a decrease i n a c t i v i t y and which c a n be p r e v e n t e d by p r e i n c u b a t i o n i n t h e p r e s e n c e of ATP does n o t b l o c k t h e ATP s i t e . I t s u g g e s t s t h a t t h e A T P - s e n s i t i v e , PLP-reactive g r o u p s a r e n o t o n t h e ATP s i t e ; o r , t h a t t h e r e are two d i f f e r e n t ATP s i t e s , one which h a s PLP-reactive g r o u p s I t i n d i c a t e s t h a t t h e "pH e f and a n o t h e r which h a s n o t . f e c t " of PLP i s n o t due i n d i r e c t l y t o a n "ATP e f f e c t " o f PLP on an ATP s i t e b u t t o an e f f e c t o f a r e a c t i o n of PLP w i t h g r o u p s (amino g r o u p s ) t h a t are i n v o l v e d more d i r e c t l y i n t h e protonation-deprotonation r e a c t i o n .

JENS CHR SKOU

338

The "pH e f f e c t " of m o d i f i c a t i o n by PLP i s a l s o obs e r v e d i n t h e r a t e o f t r a n s f o r m a t i o n between t h e t w o forms ( T a b l e 111; c f . T a b l e I ) . Modificati'bn i n t h e p r e s e n c e o f K+, which h a s o n l y a s l i g h t e f f e c t o n ~ 0 f o, r N~a + ( i . e . , "pH e f f e c t " ) , h a s no e f f e c t on t h e r a t e o f t h e t r a n s f o r m a t i o n from t h e X form t o t h e N a form, b u t c a u s e s a s l i g h t d e c r e a s e i n t h e r a t e o f t h e r e v e r s e r e a c t i o n ( T a b l e 111). Wodificat i o n i n t h e p r e s e n c e of t h e o t h e r l i g a n d c o m b i n a t i o n s g i v e s a n i n c r e a s e i n t h e r a t e of t r a n s f o r m a t i o n from t h e K form t o t h e N a form and a d e c r e a s e i n t h e r a t e of t h e r e v e r s e r e a c t i o n , j u s t a s does a n i n c r e a s e i n pH ( T a b l e 111; c f . T a b l e 11). The e f f e c t s on t h e rates c o r r e l a t e w i t h t h e e f f e c t s o n ~ 0 . 5 f o r Na; ( T a b l e I I 1 ; c f . T a b l e I I ) . With K+ i n t h e p r e i n c u b a t i o h medium, i . e . , w i t h t h e enzyme i n t h e p r o t o n a t e d K form, t h e amino g r o u p s , which a r e i m p o r t a n t f o r t h e pH e f f e c t on t h e c o n f o r m a t i o n , a r e t h u s "hidden" f o r r e a c t i o n w i t h PLP. ATP, which i n t h e p r e s e n c e of K+ c o n v e r t s t h e enzyme 'to t h e N a form (Jgkgensen, 1 9 7 5 ) , e x p o s e s t h e amino g r o u p s , a s does p r e i n c u b a t i o n i n t h e p r e s e n c e o f N a + and o f N a + + ATP. However, from t h e p o i n t of view o f t h e l i g a n d e f f e c t o n t h e r e a c t i v i t y towards PLP, t h e c o n f o r m a t i o n i n t h e p r e s e n c e of K+ + ATP i s n o t i d e n t i c a l t o t h e N a form. N e i t h e r i s t h e c o n f o r m a t i o n i n t h e p r e s e n c e of ATP a l o n e i d e n t i c a l t o t h e N a form, s u g g e s t i n g t h a t t h e t r a n s f o r mation from t h e K form t o t h e N a form i s n o t a n e i t h e r / o r situation. PLP r e a c t s w i t h €-amino g r o u p s on l y s i n e (Colombo and Marcus, 1 9 7 4 ) , i . e . , t h e g r o u p s w i t h t h e h i g h e r PK v a l u e . However, t h e e f f e c t on ~ 0 . 5f o r N a + i s s e e n a t a pH which i s a t l e a s t 2 u n i t s l o w e r t h a n t h e PK v a l u e f o r I t means e i t h e r t h a t t h e PLP e f f e c t i s t h e s e groups. n o t on t h e €-amino g r o u p s , o r t h a t r e a c t i o n w i t h t h e s e ) g r o u p s f a c i l i t a t e s t h e d i s s o c i a t i o n (decreases p ~ of o t h e r groups which a r e i n v o l v e d i n t h e d e p r o t o n a t i o n p r o t o n a t i o n r e a c t i o n of importance f o r t h e t r a n s f o r m a t i o n between t h e two forms , i . e . , t h e g r o u p s w i t h t h e lower P K ,

VI.

INTERPWTATION

The p r o t o n e f f e c t on t h e c o n f o r m a t i o n , a l o n g w i t h t h e f a c t t h a t t h e i n v o l v e d amino g r o u p s are " h i d d e n " from r e a c t i o n w i t h PLP when t h e enzyme i s p r o t o n a t e d , s u g g e s t s t h a t t h e p r o t o n a t e d amino g r o u p s t a k e p a r t i n s a l t - b r i d g e f o r m a t i o n between and w i t h i n t h e p o l y p e p t i d e

339

Na,K-ATPaseDISCRIMINATION BETWEEN Na+AND K+

.

c h a i n s , a h e m o g l o b i n - l i k e s i t u a t i o n (Baldwin, 1 9 7 6 ) The d e p r o t o n a t e d , N a form ( E l ) i s t h e n t h e form w i t h t h e low d e g r e e of s a l t - b r i d g e f o r m a t i o n , i . e . , t h e r e l a x e d , R - s t r u c t u r e i n t h e n o t a t i o n by Monod e t a l . (19651, w i t h a h i g h a f f i n i t y f o r N a + ; a n d , a s s u g g e s t e d from t h e exp e r i m e n t s o n t h e e f f e c t of t h e c o n c e n t r a t i o n o f K+ on t h e r a t e o f t h e t r a n s f o r m a t i o n from t h e N a form t o t h e K form ( K a r l i s h e t a l . , 1 9 7 8 ) w i t h a low a f f i n i t y f o r K+ The p r o t o n a t e d K form ( E 2 ) i s t h e form w i t h t h e h i g h degree of salt-bridge formation, t h e tense T-structure, w i t h a h i g h a f f i n i t y f o r K + and low a f f i n i t y f o r N a + . The c a t i o n e f f e c t which l e a d s t o t h e c o n f o r m a t i o n a l change i s l o c a t e d on t h e i n t e r n a l s i t e s o f t h e s y s t e m (Skou and Esmann, 1980; K a r d i s h and P i c k , 1 9 8 1 ) . With e i t h e r 2 K+ o r 3 N a + bound (Glynn, 19681,

z

(m-n) H HmE2K2

+

( n - r ) H+ I

, HnE2KNa

II

EIKNa

L .

,

H E Na2

L

r 2

1 E1Na2-, E L

1N a 3

The s u c c e s s i v e b i n d i n g of N a + t o E 2 l e a d s t o weakeni n g and b r e a k i n g of t h e s a l t b r i d g e s , release of p r o t o n s , and a n i n c r e a s e d l i k e l i h o o d of c o n v e r s i o n from E 2 t o E l . E 2 N a 3 and E 1 K 2 a r e t o o u n s t a b l e t o e x i s t i n more t h a n m i n u t e amounts. ATP f a c i l i t a t e s t h e d e p r o t o n a t i o n by dec r e a s i n g P K v a l u e s , i . e . , f a c i l i t a t e s b r e a k i n g of t h e s a l t b r i d g e s and t h e r e b y t h e t r a n s f o r m a t i o n from t h e E 2 t o t h e E l form. ATP i n h i g h e r c o n c e n t r a t i o n s a p p a r e n t l y a l s o increases t h e r a t e o f t h e t r a n s f o r m a t i o n from t h e E l t o t h e E 2 form. I t i s t h e N a form ( E l ) w i t h Na+ bound which h a s cat a l y t i c a c t i v i t y , and i t would be o f i n t e r e s t t o know i f t h e H+ r e l e a s e d from ATP when i t i s h y d r o l y z e d by t h i s form t a k e s p a r t i n t h e r e p r o t o n a t i o n of t h e d e p r o t o n a t e d E l form and t h e r e b y i n c r e a s e s t h e r a t e o f t h e t r a n s f e r from t h e N a form ( E l ) t o t h e K form ( E 2 ) .

ACKNOWLEDGMENTS

I wish t o t h a n k t h e Danish Medical Research Council and t h e Ingeborg and Leo Dannins Foundation for S c i e n t i f i c Research f o r financial support.

340

JENS CHR SKOU

REFERENCES

Baldwin, J. M. ( 1 9 7 6 ) . A model o f c o - o p e r a t i v e oxygen b i n d i n g t o hemoglobin. B r . M e d . B u l l . 3 2 , 213-218. Colombo, G . , and Marcus, F. ( 1 9 7 4 ) . M o d i f i c a t i o n o f f r u c t o s e 1,6-diphosphatase w i t h p y r i d o x a l 5'-phosphate. Evidence f o r t h e p a r t i c i p a t i o n of l y s y l r e s i d u e s a t t h e a c t i v e s i t e . B i o c h e m i s t r y 1 3 , 3085-3091. Glynn, I. M. ( 1 9 6 8 ) . Membrane a d e n o s i n e t r i p h o s p h a t a s e and c a t i o n t r a n s p o r t . B r . M e d . B u l l . 2 4 , 156-169. Hegyvary, C . , and P o s t , R. L . ( 1 9 7 1 ) . B i n d i n g of a d e n o s i n e t r i p h o s p h a t e t o sodium and p o t a s s i u m i o n - s t i m u l a t e d a d e n o s i n e J. Biol C h e m . 2 4 6 , 5234-5240. triphosphatase. J + r g e n s e n , P. L. (1975) P u r i f i c a t i o n and c h a r a c t e r i z a t i o n o f (Na+,K+)-ATPase V. C o n f o r m a t i o n a l c h a n g e s i n t h e enzyme. T r a n s i t i o n s between t h e Na-form and t h e K-form s t u d i e d w i t h t r y p t i c d i g e s t i o n as a t o o l . B i o c h i m . B i o p h y s . A c t a 4 0 1 , . 399-4 15. K a r l i s h , S. J. D. ( 1 9 8 0 ) . C h a r a c t e r i z a t i o n o f c o n f o r m a t i o n a l changes i n (Na,K)-ATPase l a b e l e d w i t h f l u o r e s c e i n a t t h e a c t i v e s i t e . J . B i o e n e r g . B i o m e m b . 1 2 , 111-136. K a r l i s h , S. J. D. , and P i c k , U. ( 1 9 8 1 ) . S i d e d n e s s o f t h e e f f e c t s o f sodium and p o t a s s i u m i o n s on t h e c o n f o r m a t i o n a l s t a t e o f t h e sodium-potassium pump. J . P h y s i o l . (London) 3 1 2 , 505529. K a r l i s h , S. J . D . , and Yates, D. W. ( 1 9 7 8 ) . Tryptophan f l u o r e s c e n c e o f (Na+ + K+)-ATPase as a t o o l f o r s t u d y o f t h e enB i o c h i m . B i o p h y s . A c t a 5 2 7 , 115-130. zyme mechanism. K a r l i s h , S . J . D . , Yates, D. W . , and Glynn, I. M. ( 1 9 7 8 ) . Conf o r m a t i o n a l t r a n s i t i o n s between Na+-bound and K+-bound forms o f ( N a + + K+)-ATPase, s t u d i e d w i t h formycin n u c l e o B i o c h i m . B i o p h y s A c t a 5 2 5 , 252-264. tides. Monod, J . , Wyman, J . , and Changeux, J . P. ( 1 9 6 5 ) . On t h e n a t u r e J . Mol. B i o l . o f a l l o s t e r i c t r a n s i t i o n s : A p l a u s i b l e model. 1 2 , 88-228. Nbrby, J. G . , and J e n s e n , J. ( 1 9 7 1 ) . Binding of ATP t o b r a i n m i c r o s o m a l ATPase. B i o c h i m . B i o p h y s . A c t a 2 3 3 , 104-116. Skou, J . C. ( 1 9 5 7 ) . The i n f l u e n c e o f some c a t i o n s o n an a d e n o s i n e Biochim. Biophys. t r i p h o s p h a t a s e from p e r i p h e r a l n e r v e s . A c t a 2 3 , 394-401. Skou, J. C. ( 1 9 7 4 ) . E f f e c t o f ATP on t h e i n t e r m e d i a r y s t e p s o f t h e r e a c t i o n o f t h e ( N a + + K+)-dependent enzyme system. B i o c h i m . B i o p h y s . A c t a 3 3 9 , 246-257. Skou, J. C. ( 1 9 7 9 a ) . E f f e c t s o f ATP on t h e i n t e r m e d i a r y s t e p s o f t h e r e a c t i o n o f t h e ( N a + + K+)-ATPase. I V . E f f e c t s o f ATP on Xo.5 f o r N a + and on h y d r o l y s i s a t d i f f e r e n t p H and temperat u r e . B i o c h i m . B i o p h y s . A c t a 5 6 7 , 421-435. Skou, J. C. (197933). P r e p a r a t i o n o f membrane-bound and o f s o l u b i l i z e d ( N a + + K+)-ATPase from r e c t a l g l a n d s of Squalus a c a n t h i a s .

.

.

.

.

Na,K-ATPase DISCRIMINATION BETWEEN Na+AND K+

341

The e f f e c t o f p r e p a r a t i v e p r o c e d u r e s on p u r i t y , s p e c i f i c and B i o c h i m . B i o p h y s . A c t a 5 6 7 , 436-444. molar a c t i v i t y . Skou, J. C . , and Esnann, M. ( 1 9 8 0 ) . E f f e c t s o f ATP and p r o t o n s on t h e N a : K s e l e c t i v i t y of t h e ( N a + + K+)-ATPase s t u d i e d by l i g a n d e f f e c t s o n i n t r i n s i c and e x t r i n s i c f l u o r e s c e n c e . B i o c h i m . B i o p h y s . A c t a 6 0 1 , 386-402. Skou, J. C. , and Esmann, M. ( 1 9 8 1 ) . E o s i n , a f l u o r e s c e n t p r o b e o f Biochim. Biophys. ATP b i n d i n g t o t h e ( N a + + K+)-ATPase. A c t a 6 4 7 , 232-240. Skou, J. C. ( 1 9 8 2 ) . The e f f e c t of pH, o f ATP and of m o d i f i c a t i o n w i t h p y r i d o x a l 5-phosphate on t h e c o n f o r m a t i o n a l t r a n s i t i o n between t h e Na+-form and t h e K+-form o f t h e ( N a + + K+) ATPase. B i o c h i m . B i o p h y s . A c t a 6 8 8 , 369-380.

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CURRENT TOPICS IN MEMBRANES AND TRANSWRT. VOLUME 19

Characteristics of the Elecaic Eel Na,K-ATPase Phosphoprotein ATSUNORU YODA AND SHIZUKO YODA Depanmenr of Pharmacology University of WisconsinMedical School Madison, Wisconsin

I.

INTRODUCTION

The c l e a v a g e o f ATP by t h e N a , K - A T P a s e p r o c e e d s t h r o u g h p h o s p h o r y l a t e d i n t e r m e d i a t e s , t h e ADP- and K + - s e n s i t i v e phosphoenzymes (E1P and E 2 P ) . In a l l Na,K-ATPase p r e p a r a t i o n s known, E 2 P i s t h e m a j o r conpon e n t o f t h e p h o s p h o r y l a t e d form, whereas E 1 P i s t h e m a j o r component o n l y i n t h e p r e s e n c e of v e r y h i g h concent r a t i o n s of N a + o r i n a n enzyme p a r t i a l l y i n h i b i t e d by NEM. W e o b s e r v e d t h a t t h e e l e c t r i c e e l N a , K - A T P a s e p r e p a r e d w i t h o u t any d e t e r g e n t (Yoda and Yoda, 1 9 8 1 ) produced m o s t l y E 1 P i n t h e p r e s e n c e of 1 0 0 mM Na+, b u t t h e microsomes o f s h a r k r e c t a l g l a n d , a f a i r l y a c t i v e form of N a , K - A T P a s e when p r e p a r e d w i t h o u t d e t e r g e n t (Hokin e t a l . , 19731, produced m a i n l y E 2 P .

343

Copyright 0 1983 by Academic Press. Inc. All rights ofreproduction m any form reserved. ISBN 0-12-153319-0

344 11.

ATSUNOBU YODA AND SHIZUKO YODA

MATERIALS AND METHODS

I n t h i s s t u d y , t h e enzyme p h o s p h o r y l a t i o n w a s terminated a f t e r t h e p h o s p h o r y l a t i o n o f [ Y - ~ ~ATP P ] reached e q u i l i b r i u m i n t h e p r e s e n c e of Na+ and Mg2+ by t h e add i t i o n of 50 mM CDTA w i t h or w i t h o u t 5 0 vg/ml oligomyc i n , w i t h 1 mM ADP o r 2 mM K + added 0 . 5 sec l a t e r . The E1P and E 2 P p e r c e n t a g e s w e r e r o u g h l y c a l c u l a t e d from t h e r a t i o of t h e value of t h e dephosphorylated p r o t e i n 1 sec a f t e r t h e a d d i t i o n of ADP o r K+ t o t h e v a l u e of t h e p h o s p h o r y l a t e d p r o t e i n 1 sec a f t e r a d d i t i o n of water i n t h e absence o f ADP and K+. The p h o s p h o r y l a t i o n and dephosphorylation occurred i n t h e presence of a c o n s t a n t N a + c o n c e n t r a t i o n and a t a t e m p e r a t u r e of 4OC.

111.

RESULTS AND DISCUSSION

I n t h e p r e s e n c e of 1 0 0 mM N a + , a b o u t 7 0 % of t h e phosphoenzyme from t h e s h a r k enzyme w a s E2P ( i . e . , dep h o s p h o r y l a t e d by 2 mM K+ w i t h i n 1 sec) and a p p r o x i m a t e l y 3 0 % w a s ADP-sensitive ( i . e . , d e p h o s p h o r y l a t e d by 1 mM ADP w i t h i n 1 s e c ) , s i m i l a r t o t h e r e s u l t s r e p o r t e d f o r t h e kidney and b r a i n enzymes (Klodos and Ngkby, 1 9 7 9 ) . I n t h e e e l enzyme, however, a b o u t 8 0 % o f t h e In the phosphoenzyme was s e n s i t i v e t o b o t h ADP and.'K s h a r k and e e l enzymes, more t h a n 95% o f t h e phosphoenzymes were d e p h o s p h o r y l a t e d by t h e s i m u l t a n e o u s a d d i t i o n of ADP and K+. These d e p h o s p h o r y l a t i o n r e s u l t s f o r b o t h enzymes were n o t changed by t h e s u b s t i t u t i o n of unl a b e l e d ATP ( 2 mM) f o r 50 mM CDTA. The sum of t h e E 1 P and E2P p e r c e n t a g e s of t h e s h a r k enzyme was a b o u t l o o % , b u t t h a t of t h e eel enzyme w a s more t h a n 1 5 0 % , a s shown i n F i g . 1. Two d i f f e r e n t c o n c l u s i o n s c a n be drawn from t h e u n e x p e c t e d l y l a r g e sums of t h e e e l enzyme E1P and E2P p e r c e n t a g e s : (1) a s i g n i f i c a n t p e r c e n t a g e of t h e E 1 P may c o n v e r t t o E 2P o r v i c e v e r s a d u r i n g t h e dephosp h o r y l a t i o n p e r i o d (1 s e c ) ; o r ( 2 ) t h e e e l enzyme may produce a new t y p e of phosphoenzyme which i s b o t h K+and ADP-sensitive. To examine t h e s e p o s s i b i l i t i e s , t h e o l i g o m y c i n e f f e c t s on t h e d e p h o s p h o r y l a t i o n of t h e EP w e r e s t u d i e d , s i n c e s e v e r a l s t u d i e s have i n d i c a t e d t h a t oligomycin i s t h e i n h i b i t o r of t h e conversion of E1P t o E2P i n Na,KA T P a s e (Fahn e t a l . , 1 9 6 6 ) . I n t h e eel enzyme, 5 0 pg/ml oligomycin s u b s t a n t i a l l y reduced t h e K+ e f f e c t on t h e d e p h o s p h o r y l a t i o n r a t e , and reduced t h e sum of t h e E1P

ELECTRIC EEL Na,K-ATPase PHOSPHOPROTEIN

345

F i g . 1 . Percentages of E I P and E2P i n the phosphorylated Na,K-ATPases i n various Nat concentrations. The experimental cond i t i o n s and the calculations are shown i n the t e x t , and the open bar or the half-shadowed bar represents the r e s u l t obtained with or without oligornycin, respectively.

and E2P p e r c e n t a g e s from 1 6 0 t o 1 2 4 % , r e s p e c t i v e l y , b u t no such oligomycin e f f e c t s were o b s e r v e d i n t h e s h a r k enzyme. From t h e o l i g o m y c i n e f f e c t s , w e concluded t h a t t h e e e l enzyme formed mainly E I P from ATP i n t h e p r e s e n c e of 1 0 0 m N a + , and t h a t t h i s E 1 P c o n v e r t e d t o E2P and w a s t h e n d e p h o s p h o r y l a t e d by K+. A s shown i n t h e o t h e r Na,K-ATPase p r e a r a t i o n s ( P o s t e t a l . , 19751, an i n c r e a s e i n t h e N a p c o n c e n t r a t i o n l e d t o a h i g h e r E J P p e r c e n t a g e i n t h e s h a r k enzyme a l s o . t h e oligomyUnder s u c h c o n d i t i o n s ( 5 0 0 o r 7 0 0 mM Na'), c i n - i n h i b i t a b l e c o n v e r s i o n o f E 1 P t o E2P was a l s o obs e r v e d , s i m i l a r t o t h e e e l enzyme i n t h e p r e s e n c e o f 1 0 0 mM N a + . On t h e o t h e r hand, t h e e e l enzyme formed mainly E2P i n t h e p r e s e n c e of 1 0 mM N a + and d i d n o t show t h e oligomycin e f f e c t a s i n t h e s h a r k enzyme i n t h e p r e s e n c e o f 1 0 0 m Na+. W e t h e r e f o r e c o n c l u d e t h a t t h e d i f f e r e n c e s between e e l and s h a r k enzymes r e s u l t from t h e d i f f e r e n t a f f i n i t i e s of t h e two enzymes t o N a + . The c o n v e r s i o n of E1P t o E2P i n t h e e e l enzyme w a s s t i m u l a t e d by o u a b a i n . When 2 m~ o u a b a i n w a s added a f t e r t h e p h o s p h o r y l a t i o n w a s t e r m i n a t e d by 50 mM CDTA, t h e p h o s p h o r y l a t e d e e l enzyme bound w i t h t h e o u a b a i n . T h i s b i n d i n g w a s d e t e c t e d by t h e i n h i b i t i o n of A T P a s e ( 5 0 % a t

346

ATSUNOBU YODA AND SHIZUKO YODA

I n h i b i t i o n by Ouabain-EP I n t e r a c t i o n and t h e E f f e c t s of ADP, and Oligomycina

TABLE I .

K+,

C o n d i t i o n of p h o s p h o r y l a t i o n Na

Temp. ("C)

+

ATP

Mg

2+

--

I n h i b i t i o n o f Na,K-ATPase + 2 mM + 1 mM + 50 ug/ml Kf ADP oligomycin

(mM)

(mM)

(mM)

(%I

4 4 4

10 100 1000

0.02 0.02 0.10

2 2 10

49 64 12

0.3 0.1 2.0

45 42 lo

--

25 25 25

10 100 1000

0.02 0.02 0.10

2 2 10

54 71 66

1.0 7.5 54

45 31 14

54 50 5

a

(0)

--

--

T h e e x p e r i m e n t a l p r o c e d u r e i s s h o w n i n the t e x t .

4 O C , 7 1 % a t 25OC) a f t e r t h e removal of t h e unbound ouab a i n by a Sephadex G-50 column. A s shown i n T a b l e I , t h i s ouabain b i n d i n g was reduced w i t h K+, n o t w i t h ADP a t 4 O C , b u t a t 25OC, it was a l s o reduced w i t h ADP. Oligomycin a l s o p r e v e n t e d t h i s o u a b a i n b i n d i n g i n t h e These r e s u l t s p r e s e n c e of h i g h c o n c e n t r a t i o n s of N a + . a l s o s u g g e s t t h a t E2P c a n b i n d w i t h o u a b a i n a t 4OC, unl i k e E l P , and a t 25OC, b u t t h e r a p i d change o f E 1 P t o E 2 P o c c u r s a t 25OC w i t h o u a b a i n . From t h e s e r e s u l t s , w e concluded t h a t t h e e e l enzyme c h a r a c t e r i s t i c a l l y forms E1P-rich phosphoenzyme i n t h e p r e s e n c e of 1 0 0 mM N a + and p r o b a b l y h a s a s p e c i f i c a l l y h i g h e r a f f i n i t y f o r N a + t h a n o t h e r Na,K-ATPase p r e p a r a t i o n s . The c o n v e r s i o n of E 1 P t o E 2 P by K+ o r ouabain, r e s u l t i n g i n t h e dephosphorylation o r t h e bindof o u a b a i n , was a l s o o b s e r v e d i n t h e a b s e n c e of f r e e M9

ins+-

REFERENCES

Fahn, S., Kova-, G. J., and A bers, R. W. ( 3 6 ) . Sodium-potassiuma c t i v a t e d a d e n o s i n e t r i p h o s p h a t a s e o f E l ec trophorus e l e c t r i c organ. J . B i o l . Chem. 2 4 1 , 1882-1889. Hokin, L. E . , Dahl, J. L., Deupree, J. D . , Dixon, J. F., Hackney, J. F . , and Perdue, J. F. (1973). S t u d i e s on t h e c h a r a c t e r i z a t i o n o f t h e sodium-potassium t r a n s p o r t a d e n o s i n e t r i p h o s J . B i o l C h e m . 248 , 2593-2605. phatase

.

.

347

ELECTRIC EEL Na,K-ATPase PHOSPHOPROTEIN

+

Klodos, I . , and Ndrby, J . G. ( 1 9 7 9 ) . E f f e c t of K+ and L i on i n t e r m e d i a r y s t e p s i n t h e N a ,K-ATPase r e a c t i o n . In " N a , K ATPase: S t r u c t u r e and K i n e t i c s " (J. C. Skou a n d J. G. Ngkby, eds.) , p p . 331-342. Academic P r e s s , N e w York. P o s t , R. L . , Toda, G . , K u m e , S . , and T a n i g u c h i , K. ( 1 9 7 5 ) . Synthesis of a d e n o s i n e t r i p h o s p h a t e by N a , K-ATPase. J. Supraml. Struct. 3, 479-497. Yoda, A . , a n d Yoda, S. (1981). A new s i m p l e p r e p a r a t i o n method f o r Na,K-ATPase-rich membrane f r a g m e n t s . A n a l . Biochern. 110, 82-88.

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CURRENT TOPICS IN MEMBRANES AND TRANSPORT, VOLUME 19

Sulfhydryl Groups of Na,K-ATPase: Effects of N-Ethylmaleimide on Phosphorylation from ATP in the Presence of Na+ + Mg2' MIKAEL ESMA" AND I R E " KLODOS Institute of Biophysics University of Aarhus Aarhus. Denmark

Recent p u b l i c a t i o n s on t h e e f f e c t of N-ethylmaleimide (NEM) on t h e Na,K-ATPase a c t i v i t y have been i n conf l i c t w i t h r e s p e c t t o t h e molar a c t i v i t y of t h e m o d i f i e d enzyme (see Wallick e t a l . , 1 9 7 8 , and r e f e r e n c e s t h e r e i n ) . T h i s s t u d y , t o some e x t e n t , r e s o l v e s t h e r e p o r t e d discrepancies.

I.

EXPERIMENTAL DATA

Na,K-ATPase, p u r i f i e d from r e c t a l g l a n d s of S q u a l u s a c a n t h i a s , w a s t r e a t e d w i t h 0 . 1 mM NEM a t 37OC, p H 7 . 4 , i n t h e p r e s e n c e of 5 mM CDTA and 150 mM N a + o r K+, w i t h o r w i t h o u t 3 mM ATP. The r e a c t i o n was s t o p p e d

by t h e a d d i t i o n of m e r c a p t o e t h a n o l , and t h e a c t i v i t y of t h e enzyme w a s measured under o p t i m a l c o n d i t i o n s ( s e e Esmann, 1982, f o r d e t a i l s ) . NEM-modified enzyme was washed t h r e e t i m e s by c e n t r i f u g a t i o n t o remove l i g a n d s 349

Copyrighf 0 1983 by Academic Ress, Inc. AIL rights of reproduction in any form reserved. ISBN 0-12-153319-0

350

MIKAEL ESMANN AND IRENA KLODOS

TABLE I.

M o d i f i c a t i o n of Na,K-ATPase Time of Modification

ATPase

EP

L i g a n d s present

activitya

levela

d u r i n g m o d i f i c at i o n

(min)

(%I

Na', 0.1 xr& NEM N a + + ATP, 0 . 1 md ' NEM N a + + ATP, 0.8 mld NEM K+, 0.1 mM NEM K+ + ATP, 0.1 d NEM K+ + ATP, 0 . 1 mp1 NEM K+ + ATP, 0 . 1 mpI NEM K+ + ATP, 0 . 1 mPr NEM K+ + ATP, 0 . 1 mM NEM K+ + ATP, 0.1 mpI NEM

20 20 20 20 2 5 10 20 40 60

a

21 80 30

16 91 74 54 27 11 9

Molar activitya

83 90 83 77

25 89 47 20 92 85 77 65 61

100

57

16

89 72 42 20

Values given i n percent of control enzyme.

p r i o r t o t h e p h o s p h o r y l a t i o n experiments. C o n t r o l enzyme was t r e a t e d s i m i l a r l y , b u t w i t h o u t NEM. The s t e a d y - s t a t e l e v e l s of phosphoenzyme w e r e measured a s p r e v i o u s l y d e s c r i b e d (Klodos e t a l . , 1 9 8 1 ) . IIK+ s e n s i t i v i t y " means t h e s t e a d y - s t a t e l e v e l of EP i n t h e p r e s e n c e o f v a r y i n g c o n c e n t r a t i o n s of K', 0-2 mM ( a l l r e s u l t s a r e shown a f t e r t h e s u b t r a c t i o n of I I K + b l a n k , " c o n t a i n i n g 150 mM K+ i n s t e a d of Na+). The "ADP s e n s i t i v i t y " i s measured a f t e r a d d i t i o n of 2.5 m~ ADP t o t h e EP formed d u r i n g 1 0 s e c of p h o s p h o r y l a t i o n (see Klodos e t a l . I 1 9 8 1 ) .

11.

RESULTS

Table I shows t h e r e s u l t of modifying Na,K-ATPase w i t h NEM w i t h d i f f e r e n t l i g a n d s p r e s e n t d u r i n g t h e mod i f i c a t i o n . I n t h e p r e s e n c e of Na+ a l o n e , Na+ + ATP, o r K+ a l o n e , t h e d e c r e a s e i n Na,K-ATPase a c t i v i t y i s a l most p a r a l l e l t o t h e d e c r e a s e i n EP l e v e l , i . e . , t h e mol a r a c t i v i t y remains h i g h , 80-90% of t h e c o n t r o l enzyme. However, w i t h K+ + ATP p r e s e n t i n t h e m o d i f i c a t i o n medium, a l a r g e amount of EP i s r e t a i n e d i n s p i t e o f a l o s s of Na,K-ATPase a c t i v i t y . The molar a c t i v i t y i s t h u s d e c r e a s e d markedly, t h e e f f e c t b e i n g more pronounced a t l o n g e r i n c u b a t i o n t i m e s ( e . g . , 40-60 min)

.

SH-GROUPS OF THE Na,K-ATPase

351

K-SENSITIVITY

ADP-SENSITIVITY 100

50

K+ATP (20 M I N

N

-

z

10

a

K

w

1Na

Na+ATP C0NTR0L

5

A n

'5' i

B 'ii5'

i

K-CONCENTRAT I ON

*

(mM>

1

1

2

3

4

SECONDS

5

F i g . 1. ( A ) Steady-state l e v e l s o f EP i n t h e presence o f 150-148 mM Na+ and 0 - 2 mM K+ o f enzyme modified with NEM for 20 min i n t h e presence of Na+, Na+ + A T P , or K+ ( A ) , or i n t h e presence o f ?? + ATP ( 0 ) are shown together with t h e values f o r control enzyme ( 0 ) and f o r enzyme modified i n the presence of K+ + ATP f o r 60 min (*). S . E . are within t h e s i z e o f t h e symbols. ( B ) ADP s e n s i t i v i t y o f control enzyme and enzyme modified with NEM. 100% i s t h e EP l e v e l before the addition o f 2.5 mM ADP, and symbols are a s i n ( A ) .

The phosphoenzymes formed have been c h a r a c t e r i z e d w i t h r e s p e c t t o t h e i r K+ s e n s i t i v i t y and ADP s e n s i t i v i ty. F i g u r e 1 A shows t h a t t h e s t e a d y - s t a t e l e v e l o f EP i s much less s e n s i t i v e t o K+ when t h e enzyme h a s been m o d i f i e d i n t h e p r e s e n c e o f K+ + ATP t h a n w i t h any o f t h e o t h e r li and c o m b i n a t i o n s , and t h a t t h e e f f e c t o f NEM on t h e Kq s e n s i t i v i t y i s more marked a t l o n g e r i n c u b a t i o n times. I t i s a l s o s e e n t h a t enzyme m o d i f i e d i n t h e p r e s e n c e o f N a + , N a + + ATP, o r K+ a l o n e i s o n l y s l i g h t l y less K + - s e n s i t i v e t h a n t h e c o n t r o l enzyme ( t h e S.E. i s w i t h i n t h e s i z e o f t h e s y m b o l s ) . Conv e r s e l y , F i g . 1 B shows t h a t t h e ADP s e n s i t i v i t y o f enzyme m o d i f i e d w i t h NEM i n t h e p r e s e n c e o f K+ + ATP i s much h i g h e r t h a n f o r c o n t r o l enzyme o r enzyme m o d i f i e d i n t h e p r e s e n c e o f N a + , N a + + ATP, o r K+, i . e . , t h e

352

MIKAEL ESMANN AND IRENA KLODOS

amount of EP dephosphorylated r a p i d l y by ADP i s i n c r e a s e d . The e f f e c t i s more marked a t l o n g e r incubation t i m e s .

111.

CONCLUSIONS

1. M o d i f i c a t i o n o f Na,K-ATPase w i t h NEM i n t h e p r e s e n c e of K+ + ATP ( c o r r e s p o n d i n g t o an i n c o r p o r a t i o n of 3 moles of NEM p e r mole a-chain, see Esmann, 1 9 8 2 ) l e a d s t o an enzyme form w i t h low ATPase a c t i v i t y and a high EP l e v e l , i . e . , a l o w molar a c t i v i t y . 2. M o d i f i c a t i o n i n t h e p r e s e n c e of N a + , Na+ + ATP, o r K+ ( c o r r e s p o n d i n g t o an i n c o r p o r a t i o n of 4 moles of NEM p e r mole a-chain, see Esmann, 1 9 8 2 ) l e a d s t o an a l most p a r a l l e l d e c r e a s e i n t h e a c t i v i t y and EP l e v e l , i . e . , t h e molar a c t i v i t y remains h i g h . 3 . The experiments s u g g e s t t h a t t h e one SH-group n o t modified by NEM i n t h e p r e s e n c e of K+ + ATP i s ess e n t i a l t o phosphorylation, but t h a t the modification of t h e o t h e r 3 SH-groups i n a c t i v a t e s t h e o v e r a l l enzyme a c t i v i t y . The experiments s u g g e s t t h a t f o r t h e Na-ATPas t h i s i n a c t i v a t i o n i s probably a decreased a b i l i t y t o p e r form a E ~ P - E z P c o n f o r m a t i o n a l change, i n accordance w i t h t h e r e s u l t s of Fahn e t a l . ( 1 9 6 6 ) .

REFERENCES

Esmann, M. (1982). B i o c h i m . B i o p h y s . A c t a 688, 255-270. Fahn, S., Hurley, M. R , Koval, G . J . , and Albers, R. W. (1966). J . B i o l . C h e m . 241, 1890-1895. Klodos, I. , Nhrby, J. G., and P l e s n e r , I. W. (1981). B i o c h i m . B i o p h y s . A c t a 643, 463-482. W a l l i c k , E. T . , Anner, B. M . , Ray, M. V., and Schwartz, A. ( 1 9 7 8 ) . J . B i o l . C h e m . 253, 8778-8786.

C W N T TOPICS IN MEMBRANES AND TRANSPORT, VOLUME 19

Alternative Pathways of Phosphorylation of Na,K-ATPase Regulated by Na+ Ions on Both Sides of the Plasma Membrane HORST WALTER Blumenweg 10 Domtadt bei Ulm Federal Republic of Germany

I.

INTRODUCTION

S i d e d n e s s o f t h e phosphoenzyme o f Na,K-ATPase f o r N a + w a s s t u d i e d i n i n t a c t v e s i c l e s i s o l a t e d from k i d n e y medulla by a z o n a l g r a d i e n t c e n t r i f u g a t i o n p r o c e d u r e . I n t h i s v e s i c u l a r p r e p a r a t i o n t h e l e a k i n e s s of t h e v e s i c u l a r membrane w a s examined u s i n g t h e a c t i v a t i n g e f f e c t of l e a k - p r o d u c i n g a g e n t s o f N a , K - A T P a s e .

11.

METHODS AND DISCUSSION

I n t h e i n t a c t plasma membrane v e s i c l e s , t h e enzyme had t o be i n c u b a t e d f o r more t h a n 24 h r i n o r d e r t o act i v a t e t h e r a t e a t which t h e s t e a d y s t a t e o f phosphoenzyme formed from ATP was r e a c h e d . The t i m e r e q u i r e d t o a c t i v a t e t h e enzyme w a s c o n s i d e r a b l y s h o r t e n e d when t h e v e s i c l e s w e r e t r e a t e d with phospholipase A p r i o r to 353

Copyright 0 1983 by Academic Press, lnc. All righu of reproductionin m y form reserved. ISBN 0-12-153319-0

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HORST WALTER

p h o s p h o r y l a t i o n . The o b s e r v a t i o n s t h a t N a + a c t i v a t i o n of t h e f o r m a t i o n o f phosphoenzyme w a s slow, and t h a t t h e a c t i o n of [32P]ATP was completed w i t h i n 1 0 0 msec a t 0' were t a k e n as e v i d e n c e t h a t p h o s p h o r y l a t e d v e s i c l e s were i n s i d e - o u t w i t h r e s p e c t t o t h e o r i g i n a l o r i e n t a t i o n i n the cell. I n t h e e l e c t r o n microscope s m a l l v e s i c l e s (%0.5 p m ) a r e p r e s e n t which seemed t o have o r i g i n a t e d from i n v a g i n a t i o n s o f l a r g e r plasma membrane s h e e t s . These s p h e r i c a l v e s i c l e s were l a b e l e d by t h e SH-group-specific e l e c t r o n - d e n s e H g - p h e n y l a z o f e r r i t i n from t h e o u t s i d e s u r f a c e , p r e d o m i n a n t l y . I n i t i a l r a t e s of t h e phosphorylat i o n were measured u s i n g a rapid-mixing and quenching machine which a l l o w e d b o t h s i m u l t a n e o u s and s e r i a l a d d i t i o n of s u b s t r a t e s w i t h programmable t i m e i n t e r v a l s . The mixing chamber was t h e r m o s t a t i c a l l y c o n t r o l l e d a t O°C. When t h e v e s i c l e s , s a t u r a t e d w i t h 1 0 0 mM N a C 1 , were laced i n 5 0 0 mM N a C 1 , s h o r t l y b e f o r e t h e a d d i t i o n of [3gP] ATP t o i n i t i a t e p h o s p h o r y l a t i o n , a phosphoenzyme appeared which was c h a r a c t e r i z e d by a r a p i d r a t e of dep h o s p h o r y l a t i o n . However, i n t h e f i l t r a t e of t h e s o l u t i o n w i t h which p h o s p h o r y l a t i o n was s t o p p e d , t h e l e v e l of r e l e a s e d P i w a s n o t s i g n i f i c a n t l y e l e v a t e d compared t o the control. I n c o n t r a s t , t h e phosphoenzyme formed when N a + c o n c e n t r a t i o n w a s e q u a l on b o t h s i d e s of t h e membrane e x h i b i t e d a low r a t e of p h o s p h o r y l a t i o n ( t i m e constant of seconds) The r e a c t i o n o f Na,K-ATPase i n v o l v e s two conformat i o n s of E c P ( p r o b a b l y s u b s p e c i e s of E l Q P ) whose e q u i l i brium seems t o depend on t h e c o n c e n t r a t i o n o f N a + on t h e o u t s i d e s u r f a c e o f t h e membrane i n i n s i d e - o u t Furthermore, e i t h e r v e s i c l e s ( t h e cytoplasmic s i d e ) one of t h e phosphoenzyme c o n f o r m a t i o n s c o u l d a p p e a r as i n t e r m e d i a t e i n a branched mechanism of t h e N a , K - A T P a s e . When v e s i c l e s were p h o s p h o r y l a t e d i n 1 0 0 mM N a C l d u r i n g t h e i n i t i a l phase of p h o s p h o r y l a t i o n i n o r d e r t o q u i c k l y change t h e e x t r a v e s i c u l a r c o n c e n t r a t i o n of Na+, t h e r a p i d d e p h o s p h o r y l a t i o n c o u l d a l s o be o b s e r v e d . T h i s r e s u l t s u g g e s t e s t h a t s l o w and f a s t d e p h o s p h o r y l a t i n g phosphoenzymes w e r e i n t e r c o n v e r t a b l e , t h a t i s , t h e y app e a r e d i n series. These e x p e r i m e n t s i n d i c a t e a funct i o n a l f l e x i b i l i t y s i n c e t h e mechanism of t h e Na,K-ATPase shows b o t h s e r i a l and branched a s p e c t s .

.

.

ACKNOWLEDGMENT

Generous s u p p o r t of t h e Deutsche Forschungsgemeinschaft is acknowledged.

CURRENT TOPICS M MEMBRANES AND TRANSPORT, VOLUME 19

Structurally Different Nucleotide Binding Sites in Na,K-ATPase HERMANN KOEPSEU AND DORIS OUIG Max-Planck-lnstitut f r Biophysik Frankfun (Main) Federal Republic of Germany

I.

INTRODUCTION

I n f o r m a t i o n o n n u c l e o t i d e b i n d i n g s i t e s of p u r i f i e d membrane-bound Na,K-ATPase i s o l a t e d from t h e o u t e r m e d u l l a o f r a t k i d n e y s ( K o e p s e l l , 1 9 7 8 ) w a s o b t a i n e d by t h r e e i n d e p e n d e n t methods: (1) by m e a s u r i n g n u c l e o t i d e e f f e c t s on t h e i n h i b i t i o n of t h e enzyme w i t h t h e ATP a n a l o g 6-[(3-carboxy-4-nitrophenyl)thiol]-9B-U-ribof u r a n o s y l p u r i n e 5 I - t r i p h o s p h a t e ( H u l l a e t al., 1 9 7 8 ) (NbsGITP); ( 2 ) by m e a s u r i n g n u c l e o t i d e e f f e c t s on t h e enzyme i n h i b i t i o n w i t h t h e SH-group r e a g e n t 5 , 5 ' - d i t h i o b i s ( 2 - n i t r o b e n z o i c a c i d ) ( D T N B ) ; and ( 3 ) by a n a l y z i n g t h e e f f e c t s o f ADP and AMP-PNP o n t h e K+-dependent phosp h a t a s e a c t i v i t y of t h e N a , K - A T P a s e .

355

Copyright 0 1983 by Academic Press, Inc. All rights of reproductionin any form reserved.

ISBN 0-12-153319-0

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HERMANN KOEPSELL AND DORIS OLLIG

ATP ( m ) , ADP(0) or AMP-PNP(&) [mM] E f f e c t s o f d i f f e r e n t n u c l e o t i d e s on the irreversible a c t i v i t y by N b s 6 I T P ( a ) or DTNB ( b ) . Membrane-bound Na,K-ATPase (0.06 mg p r o t e i n / m l ) was i n c u b a t e d a t O°C w i t h 0.2 mM Nbs61TP or 24 P M DTNB i n the p r e s e n c e o f 5 0 mM T r i s - H C 1 , pH 8 . 5 , 2 mM EDTA, d i f f e r e n t n u c l e o t i d e s and choline c h l o r i d e a d d e d u p t o an ionic s t r e n g t h o f 1 9 0 mM. A f t e r a 2 0 - h r incubation the s a m p l e s were 50 t i m e s d i l u t e d and a s s a y e d f o r N a , K ATPase a c t i v i t y . V a l u e s are p r e s e n t e d a s p e r c e n t o f the control which was i n c u b a t e d u n d e r i d e n t i c a l l i g a n d c o n d i t i o n s . F i g . 1.

i n h i b i t i o n o f t h e Na,K-ATPase

11.

I N H I B I T I O N EFFECTS

A t pH 7 . 4 , Nbs61TP i n t e r a c t s s i m i l a r l y t o ATP w i t h a t l e a s t one n u c l e o t i d e - b i n d i n g s i t e of t h e Na,K-ATPase: i t i s hydrolyzed i n a K+- and Na+-dependent and ouabaini n h i b i t a b l e manner, shows n e g a t i v e c o o p e r a t i v i t y f o r t h e s u b s t r a t e dependence of t h e Nbs6ITP h y d r o l y s i s , and i s a c o m p e t i t i v e i n h i b i t o r of ATP ( H . Koepsell e t a l . , 1 9 8 2 ) . A t pH 8.5, Nbs6ITP r e a c t s i r r e v e r s i b l y w i t h t h e enzyme by forming a t h i o e t h e r l i n k a g e w i t h an SH-group o r a hydroxyl group of t y r o s i n e . When t h e e f f e c t of ATP on t h e i r r e v e r s i b l e i n h i b i t i o n by Nbs6ITP was measured i n t h e absence of Na+, K+ and Mg2+, i t was found t h a t t h e i n h i b i t i o n o f t h e enzyme w a s i n c r e a s e d a t low and d e c r e a s e d a t h i g h ATP c o n c e n t r a t i o n s . For t h e higha f f i n i t y e f f e c t a K 0 . 5 ATP) v a l u e of 3.2 p M w a s o b t a i n e d f o r t h e l o w - a f f i n i t y e f e c t , t h e K O . ~ ( A T P ) measured

1

NUCLEOTIDE BINDING SITES IN Na,K.ATPase

357

2.2 mM. When t h e n u c l e o t i d e s p e c i f i c i t y f o r b o t h eff e c t s was t e s t e d , it w a s found t h a t t h e l o w - a f f i n i t y p r o t e c t i v e e f f e c t was observed w i t h ATP, AMP-PNP, and ADP, whereas t h e h i g h - a f f i n i t y n u c l e o t i d e e f f e c t was observed w i t h ATP and ADP, b u t n o t w i t h AMP-PNP ( F i g . la). The e f f e c t s of v a r y i n g ATP c o n c e n t r a t i o n s on t h e i n h i b i t i o n of t h e Na,K-ATPase a c t i v i t y by DTNB w e r e measured under t h e same e x p e r i m e n t a l c o n d i t i o n s as i n t h e e x p e r i m e n t s w i t h Nbs6ITP. In contrast t o the Nbs6ITP e x p e r i m e n t s , it was found t h a t a d d i t i o n of 0.5-5 !AM ATP d i d n o t a f f e c t t h e i n h i b i t i o n of t h e Na,KATPase a c t i v i t y w i t h DTNB; however, h i g h e r ATP concent r a t i o n s (Ko.5 = 2 3 p M ) l e d t o an i n c r e a s e d enzyme i n h i b i t i o n . I n t h e p r e s e n c e of Na+, ATP d i d n o t i n c r e a s e b u t d e c r e a s e d the i n h i b i t i o n of t h e N a , K - A T P a s e by DTNB. For t h i s e f f e c t a K O , ATP) ~ v a l u e of 1 7 !AM w a s e s t i m a t e d . T e s t i n g f o r t h e s p e c i f f c i t y of t h e n u c l e o t i d e e f f e c t obs e r v e d i n t h e absence of Na+, it was found t h a t , a t v a r i a n c e w i t h t h e i n h i b i t i o n e x p e r i m e n t s w i t h Nbs61TP , o n l y ATP and AMP-PNP, b u t n o t ADP, were e f f e c t i v e ( F i g . lb) I t i s w e l l known t h a t n u c l e o t i d e s i n h i b i t t h e K+dependent phosphatase a c t i v i t y of t h e N a ,K-ATPase. S i n c e t h i s e f f e c t has been demonstrated f o r ADP a s w e l l as f o r AMP-PNP, we compared t h e k i n e t i c s of t h e i n h i b i t i o n w i t h b o t h n u c l e o t i d e s . Furthermore, it was t e s t e d whether o r n o t t h e e f f e c t s of ADP and AMP-PNP were add i t i v e . The K+-dependent phosphatase a c t i v i t y i n t h e p r e s e n c e of d i f f e r e n t n u c l e o t i d e s was measured i n t h e p r e s e n c e of 20 mM T r i s - H C 1 , pH 7 . 7 , 5 mM Mg2+, and 1 mM K+ w i t h p - n i t r o p h e n y l p h o s p h a t e a s s u b s t r a t e . The K+ c o n c e n t r a t i o n of 1 m M was employed s i n c e t h e n u c l e o t i d e e f f e c t s a r e s m a l l e r a t h i g h e r K+ c o n c e n t r a t i o n s . When t h e i n h i b i t i o n of t h e K+-dependent phosphatase a c t i v i t y by ADP and AMP-PNP was measured i n t h e p r e s e n c e of d i f f e r e n t s u b s t r a t e concentrations, competitive i n h i b i t i o n was observed i n b o t h cases, a s i t h a s been r e p o r t e d by Robinson ( 1 9 7 6 ) i n measurements w i t h 1 0 mM K + . The K i v a l u e s f o r ADP (0.12-0.13 mM) and f o r AMP-PNP (0.14-0.16 m M ) , which were e s t i m a t e d from c a l c u l a t e d r e g r e s s i o n l i n e s of t h e d a t a , were s i m i l a r . T e s t i n g whether o r n o t t h e ADP and AMP-PNP e f f e c t s a r e a d d i t i v e , it was found t h a t t h e r e l a t i v e i n h i b i t o r y e f f e c t of 5-80 !AM ADP on t h e K+-dependent p h o s p h a t a s e a c t i v i t y was t h e same whether o r n o t 1 0 0 o r 2 0 0 !AM AMP-PNP was p r e s e n t i n a d d i t i o n . Howe v e r , t h e r e l a t i v e i n h i b i t o r y e f f e c t of ADP was s i g n i f i c a n t l y reduced i f 1 0 0 o r 2 0 0 pM a d d i t i o n a l ADP was p r e s e n t ( F i g . 2 ) . When t h e i n h i b i t o r y e f f e c t of 5-80 pM AMP-PNP on t h e K+-dependent phosphatase a c t i v i t y was

.

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HERMANN KOEPSELL AND DORIS OLLIG

358

additional nucleotides present

t

lOOpM AMP-PNP 0 200pM AMP-PNP

100 pM ADP 200pM ADP

0

I

I

I

I

I

50

I

I

I

I

I *

100

ADP added [pM] Fig. 2.

Inhibition o f K+-dependent

phosphatase a c t i v i t y b y

ADP i n the p r e s e n c e and a b s e n c e o f a d d i t i o n a l n u c l e o t i d e s .

l@-

d e p e n d e n t p h o s p h a t a s e a c t i v i t y was m e a s u r e d a t 37OC i n the p r e s e n c e of 20 mM Tris-HC1 , pH 7 . 7 , 1 mM K C l , 5 mM M g C l ) , 5 mM p - n i t r o p h e n y l p h o s p h a t e and 50-80 pM ADP ( l i n e 2). In a d d i t i o n extra ADP ( l i n e s 4 and 5 ) or AMP-PNP was p r e s e n t ( l i n e s 1 and 3 ) . T h e a c t i v i t y m e a s u r e d i n the a b s e n c e o f the n u c l e o t i d e a m o u n t i n d i c a t e d on the a b s c i s s a ( l i n e 1 , 0 . 5 6 ; l i n e 2 , 1 . 0 ; l i n e 3, 0 . 3 7 ; l i n e 4 , 0.66; l i n e 5 , 0 . 5 4 ) was n o r m a l i z e d t o 1 , and the e f f e c t of 5-80 ~ . I M a d d i t i o n a l ADP was a n a l y z e d . R e g r e s s i o n l i n e s w h i c h w e r e c a l c u l a t e d from the d a t a a r e shown ( r e g r e s s i o n c o e f f i c i e n t s > 0.99, s t a n d a r d d e v i a t i o n s o f the s l o p e s
measured, t h e a d d i t i o n a l p r e s e n c e of 1 0 0 pM ADP d i d n o t a f f e c t t h e r e l a t i v e i n h i b i t i o n by AMP-PNP.

111.

DISCUSS I O N

The d a t a p r e s e n t e d p r o v i d e arguments f o r t h e exi s t e n c e of a t l e a s t two s i m u l t a n e o u s l y p r e s e n t nucleoF i r s t , it w a s t i d e - b i n d i n g s i t e s i n t h e Na,K-ATPase. found t h a t t h e i r r e v e r s i b l e i n t e r a c t i o n of t h e Na,KATPase s u b s t r a t e Nbs6ITP [which i n a l l p r o b a b i l i t y t a k e s p l a c e a t a n u c l e o t i d e b i n d i n g s i t e of t h e enzyme (H. Koepsell e t a l . , 1 9 8 2 1 1 i s a t nonhydrolyzing condit i o n s d i f f e r e n t i a l l y a f f e c t e d by low and h i g h nucleo-

NUCLEOTIDE BINDING SITES IN Na,K-ATPase

359

t i d e concentrations. For b o t h e f f e c t s d i f f e r e n t nucleot i d e s p e c i f i c i t i e s were d e m o n s t r a t e d . Second, a n u c l e o t i d e e f f e c t on t h e DTNB i n a c t i v a t i o n o f t h e Na,K-ATPase o b s e r v e d i n t h e a b s e n c e of N a + w a s s p e c i f i c f o r ATP and AMP-PNP b u t w a s n o t found w i t h ADP. This r e s u l t could be e x p l a i n e d by t h e a s s u m p t i o n t h a t b i n d i n g of ATP and AMP-PNP, b u t n o t of ADP, i n d u c e s a s t r u c t u r a l change of t h e enzyme, which may r e s u l t i n an a l t e r e d s e n s i t i v i t y t o DTNB. W e f e l l , however, t h a t a more p r o b a b l e e x p l a nation is t h a t t h i s nucleotide e f f e c t i s generated a t a n u c l e o t i d e - b i n d i n g s i t e where ADP d o e s n o t i n t e r a c t . T h i r d , t h e e f f e c t s o f ADP and AMP-PNP on t h e K+-dependent p h o s p h a t a s e a c t i v i t y were a d d i t i v e , which s u g g e s t s t h a t t h e y a r e due t o n u c l e o t i d e b i n d i n g t o d i f f e r e n t b i n d i n g s i t e s of t h e enzyme.

REFERENCES

H u l l a , F. W . , HEckel, M., Rack, M . , R i s i , S., and Dose, K. ( 1 9 7 8 ) . C h a r a c t e r i z a t i o n and a f f i n i t y l a b e l i n g of n u c l e o t i d e b i n d i n g s i t e s o f b a c t e r i a l plasma membrane a d e n o s i n e t r i p h o s p h a t a s e B i o c h e m i s t r y 1 7 , 823-828. (F1). Koepsell, H. ( 1 9 7 8 ) . C h a r a c t e r i s t i c s of a n t i b o d y i n h i b i t i o n of r a t k i d n e y (Na+-K+)-ATPase. J . Membr. B i o l . 4 4 , 85-102. Koepsell, H . , H u l l a , F. W . , and F r i t z s e h , G. ( 1 9 8 2 ) . D i f f e r e n t classes o f n u c l e o t i d e b i n d i n g s i t e s i n t h e ( N a + + K + ) ATPase s t u d i e d by a f f i n i t y l a b e l i n g and n u c l e o t i d e d e p e n d e n t SH-group m o d i f i c a t i o n s . J . Biol. C h e m . 2 5 7 , 10733-10741. Robinson, J. D. ( 1 9 7 6 ) . S u b s t r a t e s i t e s of t h e ( N a + + K+)-depenB i o c h i m . B i o p h y s . n - t a 4 2 9 , 1006-1019. d e n t ATPase.

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CURRENT TOPICS IN MEMBRANES AND TRANSPORT, VOLUME 19

Study of Na,K-ATPase with ATP Analogs WILHELM SCHONER, HARTMUT PAULS, ENGIN H. SERPERSU, ’ GEROLD REMPETERS, ROSEMARIE PAlZELT- WENCZLER, AND MARION HASSELBERG Institut pIr Biochemie und Endokrinologie Justus-Liebig-UniversitditGiessen Giessen, West Germany

I.

INTRODUCTION

+

The a c t i v e t r a n s p o r t of Na through c e l l u l a r memb r a n e s c a t a l y z e d by t h e sodium pump i s c o n s i d e r e d t o The K+ s t a r t i n t h e ATP-binding s i t e of Na,K-ATPase. t r a n s p o r t ends t h e r e (Grisham, 1 9 8 1 ) . Because of t h e c l o s e r e l a t i o n s h i p of ATP h y d r o l y s i s and a c t i v e lia+ t r a n s p o r t , s t u d i e s w i t h ATP a n a l o g s might be h e l p f u l f o r ( a ) t h e d e t e r m i n a t i o n of e s s e n t i a l amino a c i d s i n volved i n t h e r e c o g n i t i o n of ATP; ( b ) t h e t i t r a t i o n of t h e number of ATP-binding s i t e s of t h e membrane-bound enzyme; ( c ) t h e d e t e c t i o n of s t r u c t u r a l changes of t h e ATP-binding s i t e caused by t h e t r a n s p o r t s u b s t r a t e s and d i v a l e n t c a t i o n s ; and ( d ) t h e r e t a r d a t i o n of t h e t u r n over of t h e enzyme t o f a c i l i t a t e t h e s t u d y of t h e t r a n s -

lPresent a d d r e s s : Johns H o p k i n s M e d i c a l S c h o o l , D e p a r t m e n t of P h y s i o l o g i c a l C h e m i s t r y , Baltimore, M a r y l a n d . 361

Copyright 0 1983 by Academic Press,Inc. AU righu of reproductionin any form reserved. ISBN 0-12-1533190

WILHELM SCHONER eta/.

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p o r t o f i o n s . T h e r e f o r e , w e s t u d i e d , u s i n g Na,K-ATPase from p i g k i d n e y , t h e p r o p e r t i e s o f t h e f o l l o w i n g ATP a f f i n i t y l a b e l s : t h e disu1fi.de of t h i o i n o s i n e t r i p h o s p h a t e (s1noPPP)a ( P a t z e l t - W e n c z l e r and Schoner, 1980) and 3 '-0- [3- ( 4 - a z i d o - 2 - n i t r o p h e n y l ) p r o p i o n y l ] -ATP (N3ATP) (Rempeters and Schoner, 1980) I and t h e MgATP comp l e x a n a l o g s C r (111)-ATP and Co (NH3)4-ATP ( P a u l s e t a l . 1980a,b; S e r p e r s u et a l . , 1 9 8 0 ) . T h i s r e p o r t shows t h a t ATP a n a l o g s c a n be used f o r t h e s e p u r p o s e s .

11.

I

METHODS AND RESULTS

I n c u b a t i o n of Na,K-ATPase from p i g k i d n e y w i t h t h e MgATP-complex a n a l o g C r ( I I 1 ) - A T P r e s u l t s i n a slow i n a c t i v a t i o n ( P a u l s e t a l . I 1 9 8 0 a ) . A s t u d y of t h e depende n c e of t h i s p r o c e s s on t h e n a t u r e o f t h e MgATP-complex analog revealed t h a t a l l t e s t e d analogs i n a c t i v a t e t h e enzyme. The f o l l o w i n g d i s s o c i a t i o n c o n s t a n t s of t h e enzyme-metal-ATP complexes were o b t a i n e d from t h e k i n e t i c s of t h e i n a c t i v a t i o n : for the a,@,y-tridentate of C r A T P , 4 4 pM ( P a u l s e t a ] . , 1 9 8 0 a ) , and f o r t h e 8,y-bidentate of CrATP, 8 u M ; f o r t h e nonphosphorylating B,y-methylene C r A T P (CrAMP-PCP), 6 0 V M , and f o r Co(NH3)4ATP, 2 0 , 0 0 0 p ~ . These r e s u l t s s u g g e s t t h a t t h e enzyme f a v o r s a s t r a i h t p o l y p h o s p h a t e c h a i n i n t h e ATPb i n d i n g s i t e w i t h Mg2? bound between t h e y - and t h e 8-phosphate r e s i d u e s . S i n c e t h e chromium(II1) complex of t h e 8,y-methylene d e r i v a t i v e o f ATP i s a l s o a b l e t o i n a c t i v a t e t h e enzyme, i t seems l i k e l y t h a t t h e i n h i b i t i o n o f t h i s ATP a n a l o g i s due t o t h e f o r m a t i o n of a second n o n d i s s o c i a b l e CrAMP-PCP-enzyme complex ( r e a c t i o n (1)) The low a f f i n i t y of t h e bulky Co(NH3)4ATP f o r t h e enzyme may be i n d i c a t i v e o f a l i m i t e d s p a c e i n t h e t r i polyphosphate s u b s i t e of t h e ATP-binding s i t e . Na+ i o n s enhanced t h e i n a c t i v a t i o n p r o c e s s by C r A T P and i t s a n a l o g s half-maximally a t 2.6 mM. A s t i m u l a t i o n of t h e i n a c t i v a t i o n r e a c t i o n w a s a l s o s e e n by m i l l i m o l a r Mg2+ c o n c e n t r a t i o n s . These r e s u l t s are c o n s i s t e n t w i t h t h e A l b e r s - P o s t r e a c t i o n scheme of ( N a , K ) - A T P a s e . Since a c c o r d i n g t o t h i s scheme t h e p h o s p h o i n t e r m e d i a t e formed from ATP i s hydrolyzed r a p i d l y i n t h e p r e s e n c e of K + , w e s t u d i e d whether t h e s t a b l e p h o s p h o i n t e r m e d i a t e formed from [y-32P]CrATP ( P a u l s e t al., 1980a) i s hydrolyzed i n t h e p r e s e n c e o f K+ i o n s and whether K+ r e a c t i v a t e s t h e enzyme. While K+ i o n s d i d n o t r e a c t i v a t e t h e enzyme, h i g h c o n c e n t r a t i o n s o f N a + s l o w l y r e a c t i v a t e d i t by dep h o s p h o r y l a t i o n . T h i s Na+-enhanced r e a c t i v a t i o n c o u l d

.

TABLE I.

22 + + + + Comparison of t h e Na Uptake i n t o Na - o r (Na +K )-loaded Everted Human Red Blood C e l l s with ATP o r C r A TP a s Fuela

Intraves i c u l a r conditions 10 mM N a C l

100 100 100 100

mM NaCl mM NaCl m M NaCl mM NaCl

+

2 mM KC1

Energy source 20 !JM ATP 20 VM ATP + 0.2 mM vanadate 0.1 mM CrATP

+

0 . 2 mM ouabain

0.5 mM ATP + 0.2 mMADP 0.5 mM ATP + 0.2 mM ADP 0 . 1 mM CrATP

+

0 . 2 mM ouabain

0 . 1 mM CrATP

Incubation time (min)

Na

+

uptake

(nmo1e s/mg

8

20.6

8 8

8.7 8.7

60

2.3

60 60 60

0.8

2.6 1.1

aEverted red blood cells w e r e e q u i l i b r a t e d i n 2 5 mM Tris-HC1 a t p H 7 . 4 and 1 mM MgC12 with e i t h e r 1 0 mM NaCl + 2 mM KCl or 1 0 0 mM NaCl. A f t e r washing, 22Na uptake was measured a t 37OC b y i n c u b a t i n g t h e v e s i c l e s i n 25 mM T r i s - H C 1 p H 7 . 4 , 5 mM 22NaC1, 1 mM MgClz, a n d t h e a d d i t i o n s i n d i c a t e d . A l l d e t e r m i n a t i o n s w e r e done i n t r i p l i c a t e .

WILHELM SCHONER et el.

364

b e blocked by low K+ c o n c e n t r a t i o n s . These f i n d i n g s are summarized i n r e a c t i o n scheme (1), where E s t a n d s f o r N a , K - A T P a s e ( P a u l s e t al., 1 9 8 0 a ) :

luv Na+ E+CrATP

E*CrATP+

w2+

ADP

high Na+ E=CrATPAECr-E+Pi+Cr SP

3+

I@

(1)

The Na+-activated b u t K+-inhibited h y d r o l y s i s of CrATP ( r e a c t i o n ( 1 ) )shows p r o p e r t i e s s i m i l a r t o t h o s e of t h e Na+/Na+ exchange r e a c t i o n found i n r e d blood c e l l s . T h i s r e a c t i o n , which i s a p a r t i a l r e a c t i o n of t h e sodium pump, i s l i k e w i s e i n h i b i t e d by K+, and i s t h o u g h t t o req u i r e t h e f o r m a t i o n of a p h o s p h o i n t e r m e d i a t e t o d r i v e t h e Na+/Na+ exchange (Glynn and K a r l i s h , 1 9 7 6 ) . W e t h e r e f o r e i n v e s t i g a t e d whether CrATP might s u p p o r t t h e N a + u p t a k e i n t o e v e r t e d r e d blood c e l l s loaded w i t h e i t h e r 1 0 0 m M N a + o r 1 0 mM N a + + 2 mM K+. A s i s evident from Table I , CrATP s u p p o r t e d a o u a b a i n - i n h i b i t e d 2 2 N a + u p t a k e o n l y under c o n d i t i o n s i n which a N a + / N a + exchange i s o b s e r v e d . CrATP w a s n o t a b l e t o d r i v e t h e N a + u p t a k e i n t o K+-containing v e s i c l e s . Under t h e l a t t e r c o n d i t i o n a N a + / K + exchange s h o u l d o c c u r . I t t h e r e f o r e a p p e a r s t h a t i n t h e p r e s e n c e of CrATP o n l y a p a r t of t h e o v e r a l l c y c l e of t h e sodium pump i s working. The f i n d i n g t h a t Mg2+ enhanced t h e i n a c t i v a t i o n of N a , K - A T P a s e by a MgATP a n a l o g was r a t h e r unexpected. To a s c e r t a i n whether t h e ATP-binding s i t e might be a f f e c t e d by Mg2+ i n t h e m i l l i m o l a r range, w e s t u d i e d i t s a c t i o n on t h e i n a c t i v a t i o n of Na,K-ATPase by t h e p h o t o a f f i n i t y l a b e l 3'-0- [3- (4-azido-2-nitrophenyl)propionyl]-ATP (N3-ATP) and by t h e B,y-methylene d e r i v a t i v e of t h e d i s u l f i d e of t h i o i n o s i n e t r i p h o s p h a t e (sInoP-PCP)a. The former ATP a n a l o g l a b e l e d , as an ATP a f f i n i t y l a b e l , t h e a - s u b u n i t o f t h e enzyme (Rempeters and Schoner, 1 9 8 1 ) ; t h e l a t t e r r e a c t e d w i t h a s u l f h y d r y l group i n t h e ATP-binding s i t e of Na,K-ATPase ( P a t z e l t - W e n c z l e r and Schoner, 1 9 8 1 ) . Two d i f f e r e n t r e a c t i v e s u l f h y d r y l g r o u p s have been found w i t h ( s I ~ o P - P C P ) ~ i n t h e ATP-binding s i t e s . A r a p i d l y r e a c t i n g s u l f h y d r y l group i s found i n t h e h i g h - a f f i n i t y ATP-binding s i t e , whereas t h e lowa f f i n i t y ATP-binding s i t e c o n t a i n s a slowly r e a c t i n g s u l f h y d r y l group. Complete i n a c t i v a t i o n of t h e enzyme by (sInoPPP)2 i s reached when 4 SH-groups r e a c t w i t h t h e ATP a n a l o g (Patzelt-Wenczler and Schoner, 1 9 8 1 ) . The p r e s e n c e of Mg2+ enhanced t h e r e a c t i v i t y o f b o t h s u l f h y d r y l groups toward ( s I ~ o P - P C P ) ~ . A t 83 pM ATP a n a l o g , a half-maximal e f f e c t w a s s e e n a t 6 mM MgC12. N3-ATPI

STUDY OF Na,K-ATPase WITH ATP ANALOGS

365

which h o t o i n a c t i v a t e s t h e enzyme o n l y i n t h e p r e s e n c e o f Mg2'I showed i t s half-maximal e f f e c t a t 0 . 6 m M MgC12 a t a c o n c e n t r a t i o n of 5 0 U M ATP a n a l o g .

111.

CONCLUSIONS

From t h e r e s u l t s of s t u d i e s w i t h ATP a n a l o g s w e can r e a c h t h e f o l l o w i n g c o n c l u s i o n s : (1) t h e ATP-binding s i t e of Na,K-ATPase r e s i d e s on t h e a - s u b u n i t . ( 2 ) MgC12 a f f e c t s t h e c o n f o r m a t i o n o f t h e ATP-binding s i t e . ( 3 ) Experiments w i t h CrAMP-PCP i n d i c a t e t w o enzyme-MgATP ( 4 ) Since t h e Na,Kcomplexes d u r i n g ATP h y d r o l y s i s . A T P a s e i n a c t i v a t e d and phos h o r y l a t e d by C r A T P i s n o t r e a c t i v a t e d by K+ b u t by N a , i t a p p e a r s t h a t t h e t r a n s f e r r e d chromium f i x e s t h e p h o s p h o i n t e r m e d i a t e i n t h e E l c o n f o r m a t i o n a l s t a t e . The d e m o n s t r a t i o n o f a Na+/Na+exchange r e a c t i o n d r i v e n by CrATP i s c o n s i s t e n t w i t h t h i s conclusion.

Is

ACKNOWLEDGMENT T h i s work w a s s u p p o r t e d by t h e Deutsche Forschunqsqemeinshaf t , Bonn-Bad Godesberq. E.H.S. i s a F e l l o w o f t h e Alexander von H u m b o l d t Foundation.

REFERENCES

Glynn, J. M., a n d K a r l i s h , S. J. D. ( 1 9 7 6 ) . The sodium pump. Annu. Rev. P h y s i o l . 37, 13-55. Grisham, C. M. (1981). C h a r a c t e r i z a t i o n o f ATP b i n d i n g s i t e s o f sheep k i d n e y m e d u l l a ( N a + + K+)-ATPase u s i n g CrATP. J. Inorg. B i o c h e m . 1 4 , 45-57. P a t z e l t - W e n c z l e r , R . , and S c h o n e r , W. ( 1 9 8 1 ) . Evidence f o r t w o d i f f e r e n t reactive s u l f h y d r y l g r o u p s i n t h e ATP b i n d i n g s i t e s o f (Na' + K+)-ATPase. E u r . J. B i o c h e m . 1 1 4 , 79-87. P a u l s , H . , BredenbrGcker, B., and S c h o n e r , W. ( 1 9 8 0 a ) . I n a c t i v a t i o n o f ( N a + + K+)-ATPase by chromium(II1) complexes o f E u r . J. B i o c h e m . 1 0 9 , 523-533. nucleotide triphosphates.

366

WILHELM SCHONER e t d .

Pauls, H . , Serpersu, E . , and Schoner, W. (1980b). E f f e c t s of i o n s on t h e i n a c t i v a t i o n and r e a c t i v a t i o n of (Na' + K+)-ATPase by MgATP analogues. Hoppe-Seyler's 2. Physiol. Chem. 3 6 1 , 1 4 8 2 . Rempeters, G., and Schoner, W. (1980). P h o t o i n a c t i v a t i o n of (Na+ + K+) -ATPase by 3 ' -0-3- (4-azido-2-nitropheny1)propionyl ATP o r ADP, r e s p e c t i v e l y . Hoppe-Seyler's Z. Physiol. Chem. 3 6 1 , 1329-1330. Rempeters, G. , and Schoner, W. (1981). Evidence f o r a Mg2+-induced conformational change a t t h e ATP binding s i t e of (Na+ + K+)ATPase demonstrated w i t h a p h o t o r e a c t i v e ATP-analogue. Eur. J. Biochem. ( i n p r e s s ) . Serpersu, E . , P a u l s , H., and Schoner, W. (1980). I n a c t i v a t i o n of (Na+ + Kt)-ATPase by cobalt(II1)ATP and chromium(II1)ATP. Hoppe-Seyler's Z . Physiol. Chem. 3 6 1 , 1346.

CURRENT TOPICS IN MEMBRANES AND TRANSPORT, VOLUME 19

Affinity Labeling Studies of the ATP Binding Site of Canine Kidney Na,K-ATPase JAMES B. COOPER, CARL.JOHNSON, AND CHARLES G. WINTER Department of Biochemistry University of Arkansas College of Medicine Little Rock, Arkansm

I.

INTRODUCTION

P r e v i o u s work i n this l a b o r a t o r y h a s e s t a b l i s h e d 5'-p-fluorosulfonylbenzoyladenosine (FSEA) a s a n ATPbinding s i t e a f f i n i t y probe f o r t h e c a n i n e kidney N a , K A T P a s e (Cooper and W i n t e r , 1 9 8 0 ) . The enzyme w a s p u r i f i e d by a m o d i f i c a t i o n o f t h e method of J 6 r g e n s e n ( L i a n g and W i n t e r , 1 9 7 6 ) . FSBA and [3H]FSBA were s y n t h e s i z e d a s p r e v i o u s l y d e s c r i b e d (Cooper and W i n t e r , 1 9 8 0 ) . R e a c t i o n of FSBA w i t h N a , K - A T P a s e c a n b e c a r r i e d o u t a t 37OC by a d d i t i o n of t h e compound d i s s o l v e d i n s m a l l amounts of methanol o r d i m e t h y l s u l f o x i d e t o enzyme s u s p e n s i o n s b u f f e r e d a t pH 8 w i t h b a r b i t a l o r pH 8 . 5 w i t h bicine. R e a c t i o n of FSBA w i t h t h e enzyme and s o l v o l y s i s of t h e r e a g e n t o c c u r s i m u l t a n e o u s l y , so t h a t d e p l e t i o n of FSBA i n t h e r e a c t i o n m i x t u r e a f t e r a s i n g l e a d d i t i o n l i m i t s t h e e x t e n t o f r e a c t i o n w i t h t h e enzyme. Repeated a d d i t i o n s of FSBA c a n p r o d u c e e s s e n t i a l l y c o m p l e t e i n h i b i t i o n of enzyme a c t i v i t y . 367

Copyright 0 1983 by Academic Press, Inc. All rights of reproduction inany form reserved. ISBN 0-12-153319-0

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11.

JAMES 6. COOPER et el.

RESULTS AND DISCUSSION

R e a c t i o n o f t h e enzyme i n 0 . 1 M K C 1 w i t h FSBA i s s l i g h t l y more r a p i d t h a n i n 0 . 1 M N a C 1 . Inactivation of N a , K - A T P a s e a c t i v i t y by FSBA c a n be p r e v e n t e d by i n c l u s i o n of ATP i n t h e i n c u b a t i o n medium. When 0 . 1 M N a C l i s p r e s e n t , 2.5 pM ATP p r o t e c t s half-maximally a g a i n s t i n a c t i v a t i o n by FSBA. But c a r r y i n g o u t t h e i n c u b a t i o n i n 0 . 1 M K C 1 medium l e a d s t o much l a r g e r ATP r e q u i r e m e n t f o r half-maximal p r o t e c t i o n ( a b o u t 2 0 0 U M ) The a p p a r e n t d i s s o c i a t i o n c o n s t a n t s f o r ATP p r o t e c t i o n a g a i n s t FSBA i n h i b i t i o n t h e r e f o r e a r e t h o s e e x p e c t e d f o r t h e high- and l o w - a f f i n i t y forms of t h e ATP-binding s i t e . A t 1 0 0 pM c o n c e n t r a t i o n s i n 0 . 1 M N a C 1 , b o t h ATP and ADP are e f f e c t i v e p r o t e c t o r s , whereas AMP i s n o t , a f i n d i n g c o n s i s t e n t w i t h t h e known s p e c i f i c i t y of t h e Na,K-ATPase. I n h i b i t i o n o f t h e enzyme by FSBA i s i r r e v e r s i b l e . Addition of p r o t e c t i n g c o n c e n t r a t i o n s of n u c l e o t i d e a f t e r r e a c t i o n w i t h FSBA h a s a l r e a d y o c c u r r e d d o e s n o t r e s t o r e a c t i v i t y but simply prevents f u r t h e r i n a c t i v a t i o n . Study of t h e k i n e t i c s of r e a c t i o n i s c o m p l i c a t e d by s o l v o l y s i s of t h e r e a g e n t . However, measurement o f f l u o r i d e release from FSBA w i t h t h e a i f of a f l u o r i d e s p e c i f i c e l e c t r o d e p e r m i t s m o n i t o r i n g of t h e l o s s of r e a g e n t . Repeated small a d d i t i o n s of FSBA t o m a i n t a i n i t s c o n c e n t r a t i o n c o n s t a n t t o w i t h i n 215% a l l o w s d e t e r m i n a t i o n of t h e c o n c e n t r a t i o n dependence of FSBA i n h i b i t i o n . I n a c t i v a t i o n i s e s s e n t i a l l y l i n e a r w i t h FSBA c o n c e n t r a t i o n t h r o u g h 1 mM, t h e approximate s o l u b i l i t y l i m i t of t h e r e a g e n t . This finding i s c o n s i s t e n t with t h e f a c t t h a t FSBA f a i l s t o compete w i t h ATP f o r hydrol y s i s on t h e h i g h - a f f i n i t y ATP s i t e u s i n g Na-ATP ase a s s a y c o n d i t i o n s . T h i s r e s u l t i n d i c a t e s t h a t t h e appar e n t d i s s o c i a t i o n c o n s t a n t f o r FSBA must exceed 5 mM. To e s t a l i s h t h a t FSBA re a c ts c o v a l e n t l y a t t h e ATPb i n d i n g s i t e , t h e a b i l i t y o f [3H]ADP t o b i n d t o t h a t s i t e a f t e r t r e a t m e n t o f t h e enzyme w i t h FSBA w a s determined. A l o s s of ADP-binding c a p a c i t y i n d i r e c t proport i o n t o loss of N a , K - A T P a s e a c t i v i t y was o b t a i n e d . T h i s f i n d i n g i n d i c a t e s t h a t o c c u p a t i o n o f t h e ATP-binding s i t e by FSBA i r r e v e r s i b l y p r e v e n t s s u b s e q u e n t [ 3H] ADP b i n d i n g . I n a d d i t i o n , t h e r e l a t i v e s t o i c h i o m e t r y of s p e c i f i c (ATP-protectable) [ 3H] FSBA a t t a c h m e n t t o t h e enzyme w a s found t o b e 0.94 k 0 . 2 4 moles bound p e r mole of o u a b a i n bound. An a p p r o x i m a t e l y e q u i v a l e n t amount of n o n s p e c i f i c l a b e l i n g w a s a l s o found, b u t t h i s a t t a c h m e n t of FSBA t o t h e enzyme h a s no e f f e c t on Na,K-ATPase act i v i t y . Most of t h e n o n s p e c i f i c l a b e l i n g and a l l of t h e s p e c i f i c l a b e l i n g i s found i n t h e c a t a l y t i c ( a ) s u b u n i t .

.

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STUDIES OF THE ATP BINDING SITE OF CANINE KIDNEY

One c u r r e n t view o f N a , K - A T P a s e s t r u c t u r e r e l a t i n g t o i t s f u n c t i o n i s t h a t a s i n g l e a c t i v e s i t e on a + d i m e r i c a , B - u n i t c a n c a t a l y z e a l l a c t i v i t i e s of t h e Na pump w i t h o u t any r e q u i r e m e n t f o r a-a s u b u n i t i n t e r a c t i o n i n a n ~ 1 2 ~ 8t e2 t r a m e r i c u n i t (Smith e t a l . , 1980; Moczydlowski and F o r t e s , 1 9 8 1 ) . I f t h i s were t r u e , t h e n i n h i b i t i o n of N a , K - A T P a s e a c t i v i t y by FSBA s h o u l d b l o c k p - n i t r o p h e n y l p h o s p h a t a s e (pNPPase) a c t i v i t y i n e q u a l p r o p o r t i o n , s i n c e t h e l a t t e r a c t i v i t y i s c a t a l y z e d by t h e l o w - a f f i n i t y form o f t h e ATP-binding s i t e ( R o b i n s o n , 1 9 7 6 ) . An a , B - u n i t would need t o c y c l e between t h e s e two forms t o c a t a l y z e c o m p l e t e Na,K-ATPase a c t i v i t y . In a c t u a l i t y , l o s s of N a , K - A T P a s e a c t i v i t y (measured w i t h 5 mM ATP) a f t e r FSBA t r e a t m e n t i s r o u g h l y t w i c e a s ext e n s i v e a s l o s s of K+-phosphatase a c t i v i t y , w h e t h e r t h e t r e a t m e n t i s c a r r i e d o u t i n 0 . 1 M N a + or K+ medium. The same r e s u l t s are o b t a i n e d i f N a + - A T P a s e a c t i v i t y i s measured u s i n g 2 0 pM ATP. T h e r e i s no change i n appar e n t K~ for pNPP €or t h e s u r v i v i n g p h o s p h a t a s e a c t i v i t y . These r e s u l t s imply t h a t t h e f u n c t i o n a l a 2 8 2 pump u n i t s have two s i t e s f o r pNPP h y d r o l y s i s p e r e a c h f u n c t i o n a l ATP s i t e . B l o c k i n g t h e l a t t e r w i t h a n u c l e o t i d e a n a l o g s u c h a s FSBA i n h i b i t s Na-ATPase and Na,K-ATPase a c t i v i t y a l o n g w i t h one of t h e two pNPP s i t e s . The una f f e c t e d s i t e i s u n a b l e t o assume t h e c o n f o r m a t i o n req u i r e d t o p r o d u c e h i g h a f f i n i t y €or n u c l e o t i d e , e v e n i n These r e s u l t s a r e c o n s i s N a + medium c o n t a i n i n g no K + . t e n t w i t h a model o f Na,K-ATPase s t r u c t u r e h a v i n g e i t h e r (1) one pNPPase c a t a l y t i c s i t e on e a c h of t h e two d i f f e r e n t a-subunits i n t h e a282 tetramer, with r e s t r i c t i v e c o n f o r m a t i o n a l i n t e r a c t i o n s between them; or ( 2 ) two pNPPase s i t e s on a s i n g l e - s u b u n i t , w i t h t h e p o s s i b i l i t y t h a t these sites a l t e r n a t e as t h e ATPase c a t a l y t i c s i t e . I n t h e l a t t e r case, t h e o t h e r a - s u b u n i t of t h e ~ 1 2 B 2 t e t r a m e r would b e c a t a l y t i c a l l y i n a c t i v e .

ACKNOWLEDGMENT

S u p p o r t e d i n p a r t by N I H g r a n t 5S07RR5350.

REFERENCES

Cooper, J. B . , 165-174.

a n d W i n t e r , C. G.

(1980).

J . S u p r a n w l . Struct. 1 3 ,

370

JAMES 9. COOPER eta/.

Liang, S. M . , and W i n t e r , C. G ( 1 9 7 6 ) . B i o c h i m . B i o p h y s . A c t a 452, 552-565. Moczydlowski, E . G . , and F o r t e s , P. A . G. (1981). J. B i o l . Chem. 256, 2346-2356. Robinson, J. D. ( 1 9 7 6 ) . B i o c h i m . B i o p h y s . A c t a 429, 1006-1019. Smith, R. L . , Zinn, K . , and C a n t l e y , L. C . (1980). J. B i o l . Chem. 255, 9852-9859.

CURRENT TOPICS IN MEMBRANES AND TRANSPORT, VOLUME 19

31P[180]NMR Kinetic Analysis of l80Exchange Reaction between Pi and H,O Catalyzed by Na,K-ATPase A. STEPHEN DAHMS AND JOEUE E. MIARA Department of Chemistry and Molecular Biology Institute San Diego State Universiry San Diego. California

N a , K - A T P a s e from v a r i o u s s o u r c e s h a s been shown t o c a t a l y z e a r a p i d Mg2+- and K+-dependent exchange between t h e oxygens of medium i n o r g a n i c p h o s p h a t e ( P i ) and water, a PH i' OH exchange, which most p r o b a b l y a r i s e s t h r o u g h dynamic f o r m a t i o n and c o l l a p s e of t h e K+-complexed phosphoenzyme ( S h a f f e r e t a l . , 1 9 7 8 ; P e r e z e t a l . , 1 9 7 9 ) . The P i e H O H exchange r e a c t i o n is t h e most r a p i d r e a c t i o n known t o be c a t a l y z e d by t h e Na,K-ATPase, w i t h exchange c a p a c i t i e s a p p r o a c h i n g 20-50 t i m e s t h e maximal r a t e of ATP h y d r o l y s i s . The p r e s e n c e o f t h i s exchange h a s p r o v i d e d a p o t e n t means of a s s e s s i n g a d i s c r e t e port i o n o f t h e o v e r a l l c a t a l y t i c sequence o f t h e ATP hydrol y s i s r e a c t i o n and e l e m e n t s of t h e c o o p e r a t i v e i n t e r a c t i o n s o c c u r r i n g d u r i n g c a t a l y s i s : t h e s i d e d n e s s of K+ i n t e r a c t i o n s ; and t h e s t e p s a s s o c i a t e d w i t h K+ u p t a k e , K+/K+ exchange and P i d i s s o c i a t i o n . A s i m p l e scheme f o r t h e exchange i s g i v e n by

k-l

k-2

Copyright @ 1983 by Academic Press. Inc All rights of repduction in any form reserved. ISBN 0-12-i53319-11

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A. STEPHEN DAHMS AND JOELLE E.MlARA

According t o Boyer e t a l . (1977) and Hackney (1980) f o r g e n e r a l r e a c t i o n s of t h i s t y p e , t h e r a t i o R = k 2 / k - 1 d e t e r m i n e s t h e a v e r a g e number of oxygen atoms exchanged on a n enzyme-bound p h o s p h a t e b e f o r e i t i s r e l e a s e d t o t h e medium, assuming no r o t a t i o n a l res t r i c t i o n s on P i i n t h e E . P i complex e x i s t . When R > > 1, a l m o s t e v e r y P i b i n d i n g t o t h e enzyme w i l l ' l o s e i t s oxygen atoms b e f o r e d i s s o c i a t i o n o f P i from E - P i ; on t h e o t h e r hand, when R < < 1, o n l y o n e oxygen atom can be exchange p e r p r o d u c t i v e e n c o u n t e r o f P i w i t h t h e enzyme. The e x t e n t of water oxygen i n c o r p o r a t i o n i n t o medium i n o r g a n i c p h o s p h a t e h a s i n t h e p a s t been d e t e r mined by measurement of t h e t o t a l 1 8 0 p r e s e n t i n i s o l a t e d P i by c o n v e r s i o n o f t h e i n o r g a n i c p h o s p h a t e oxyg e n s t o C 0 2 and measurement o f t h e mass 4 6 / 4 4 r a t i o . T h i s e x p e r i m e n t a l approach h a s been v a l u a b l e b u t somewhat l i m i t e d i n t h a t i t c o u l d u n d e r e s t i m a t e t h e t r u e exchange r a t e due t o p o s s i b l e m u l t i p l e water oxygen i n s e r t i o n i f R > > 1; i n t h i s case i f l 8 0 - l a b e l e d P i b i n d s t o t h e enzyme, a l l o f i t s oxygens w i l l be r e l e a s e d t o t h e medium and f u r t h e r exchange of t h e enzyme-bound P i 1 6 0 4 w i l l n o t be d e t e c t e d . D e t e r m i n a t i o n of t h e r e l a t i v e g n i t u d e of R h a s been f a c i l i t a t e d by a n a l y s i s o f t h e ''0 s h i f t on t h e 31P NMR s i g n a l of P i (Cohn and Hu, 1 9 7 8 ) ; w i t h t h i s e x p e r i m e n t a l approach t h e enzyme i s i n c u b a t e d w i t h Pi1804, and t h e p a t t e r n o f f o r m a t i o n and d i s a p p e a r a n c e of t h e f i v e s p e c i e s Pi18O4, Pi1601803, pi16O218O2, ~ 1 6 0 3 ~ and ~ 0 ,P i 1 6 0 4 i s monitored as a f u n c t i o n o f r e a c t i o n t i m e ,. The a v a i l a b i l i t y of measuring t h e i n t e r m e d i a t e s p e c i e s has n o t only allowed t h e e v a l u a t i o n o f t h e part i t i o n i n g o f t h e M i c h a e l i s complex, E * P i , between f r e e enzyme and phosphoenzyme, b u t a l s o t h e e v a l u a t i o n of t h e r o l e of i o n i c e f f e c t o r s and membrane phase s t a t e i n det e r m i n i n g r e l a t i v e r a t e c o n s t a n t s f o r P i a s s o c i a t i o n and oxygen exchange. P i +HOH exchange r e a c t i o n c a t a l y z e d by t h e medium Na,K-ATPase i s o l a t e d from e e l e l e c t r o p l a x o r p o r c i n e and c a n i n e r e n a l o u t e r m e d u l l a h a s been assessed by u s i n g t h e 180 s h i f t o f t h e 31P NMR s i g n a l of P i . The 31P NMR spectrum of 98.9 atom % e x c e s s [180]phosphate was monit o r e d a t 23' as a f u n c t i o n of P i + H O H exchange reactioi time a t 2 9 . 2 9 4 2 MHz on a J M N PS 1 0 0 J E O L , equipped w i t h a d e u t e r i u m f i e l d l o c k o p e r a t i n g i n t h e FT mode; e a c h spectrum of 8 sweeps ( 2 min) was summed u s i n g a 500-Hz s p e c t r a l w i d t h ( K H 2 P l 8 0 4 , 4 0 mM; MgC12, 5 mM; EDTA, 1 m M ; microsomes, 3 mg; 0.05 M T r i s , f i n a l pH 7 . 2 . 15-25 min r e a c t i o n t i m e s ) . The d i s t r i b u t i o n of t h e l b O s p e c i e s (Pi1804 , ~ i 1 6 0 1 8 0 3 , Pi16021802 , Pi1603180 , and P i 1 6 0 4 a s a f u n c t i o n of r e a c t i o n t i m e c l e a r l y d e m o n s t r a t e s a

NMR KINETIC ANALYSIS OFOXYGEN-18 EXCHANGE REACTION

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c a s c a d e s e q u e n c e i n which a l l species s u b s e q u e n t t o P i 1 8 0 4 form and c o l l a p s e i n a p r o d u c t - p r e c u r s o r r e l a t i o n s h i p . T h e o r e t i c a l p l o t s o f a l l [18O]Pi s p e c i e s a s a f u n c t i o n of exchange r e a c t i o n t i m e were g e n e r a t e d , assuming a mechanism i n v o l v i n g f o u r s e q u e n t i a l exchange s t e p s , e a c h w i t h a n i d e n t i c a l exchange r a t e c o n s t a n t ; t h e e x p e r i m e n t a l d a t a a r e i n e x a c t agreement w i t h a mechanism c h a r a c t e r i z e d by R < < 1, i . e . , E . P i d i s s o c i a t e s more r a p i d l y t h a n i t forms t h e c o v a l e n t E-P complex, and a P i m o l e c u l e l o s e s o n l y one o f i t s o r i g i n a l 4 oxygens b e f o r e b e i n g r e l e a s e d t o t h e medium. Identical results have a l s o been o b t a i n e d u s i n g e r y t h r o c y t e g h o s t membrane N a ,K-ATPase and a n e l e c t r o n i m p a c t mass s p e c t r a l t e c h n i q u e employing trimethylsilylphosphate. An a l t e r n a t e i n t e r p r e t a t i o n of t h e d a t a i s t h a t E - P i i s l o c k e d i n a p a r t i c u l a r o r i e n t a t i o n a l l o w i n g o n l y o n e of t h e P i oxyg e n s t o p a r t i c i p a t e i n E-P f o r m a t i o n and s u b s e q u e n t oxygen exchange. T h i s seems u n l i k e l y c o n s i d e r i n g t h e mult i p l e p h o s p h o r u s oxygen i n s e r t i o n e v e n t s o b s e r v e d w i t h myosin s u b f r a g m e n t 1 ( S l e e p e t a l . , 1978; Webb e t a l . , 1978) and t h e a b i l i t y o f enzyme-bound P i of a l k a l i n e p h o s p h a t a s e ( t h e Zn enzyme c a t a l y z e s a slow P i e H O H c h a r a c t e r i z e d by ~ < < 1 Bock ; and Cohn, 1 9 7 8 ) t o undergo f r e e r o t a t i o n (Chlebowski e t a l . , 1 9 7 6 ) . S i n c e t h e nat i v e N a , K - A T P a s e e x c h a n g e s o n l y o n e o f t h e enzyme-bound P i oxygens p e r exchange s t e p , a n e x a c t a n a l y s i s o f R c a n n o t b e o b t a i n e d ; it i s l i k e l y t h a t k-1 i s 3 0 - 1 0 0 t i m e s g r e a t e r than k 2 . A s i n t h e c a s e o f a l k a l i n e p h o s p h a t a s e (Bock and Cohn, 1 9 7 8 ) , s i n c e t h e s t e a d y - s t a t e l e v e l of E2-P d u r i n g t h e medium PiH ' OH exchange r e a c t i o n i s low, i t i s l i k e l y t h a t k-2 > k 2 ; i f k l > k - 2 , k 2 i s t h e r a t e d e t e r m i n i n g s t e p i n t h e o v e r a l l medium oxygen exchange reaction. S i n c e t h e exchange c a p a c i t y i s 20-50 t i m e s t h e t u r n o v e r number o f t h e pump ( a b o u t 8000 min-1; Mardh and Z e t t e r q u i s t , 1 9 7 4 ) , k 2 i s a minimum of 1 6 0 , 0 0 0 m i n - l ( a 2 5 0 0 sec-1) u n d e r medium P i e H O H exchange c o n d i t i o n s ; s i n c e kVl i s 30-100 t i m e s g r e a t e r t h a n k 2 ( R < < 1 1 , a lower l i m i t of 80,000 sec-1 may b e a s s i g n e d t o k - 1 . It i s a p p a r e n t t h a t t h e Pi o f f r e a c t i o n may b e t h e f a s t e s t r e a c t i o n i n t h e e n t i r e c a t a l y t i c sequence. I t s h o u l d be emphasized t h a t t h e above e s t i m a t i o n of k - 1 a p p l i e s t o c o n d i t i o n s i n t h e a b s e n c e o f ATP h y d r o l y s i s , and t h a t t h e p r e v i o u s l y published rates f o r P i d i s s o c i a t i o n of 8000-18,000 min-1 (Mardh and L i n d a h l , 1 9 7 7 ; P l e s n e r and P l e s n e r , 1 9 8 1 ) a r e b a s e d upon d e p h o s p h o r y l a t i o n methods which d e t e r m i n e k-2 and n o t k - 1 . Replacement of K+ by T 1 + Rb+, o r NH4' d i d n o t a f f e c t t h e k i n e t i c p a t t e r n of 1 6 0 - l a b e l e d P i s p e c i e s i n d i c a t i n g no a p p a r e n t change i n R . Varying t h e t e m p e r a t u r e

374

A. STEPHEN DAHMS AND JOELLE E. MlARA

o v e r t h e 1 4 ' t o 40' r a n g e a l s o w a s w i t h o u t e f f e c t on R , i n d i c a t i n g a l a c k of e f f e c t of t h e membrane p h a s e s t a t e on t h e P i o f f r e a c t i o n ; t h e A r r h e n i u s p l o t w a s l i n e a r w i t h E a c t = 6 . 4 kcal/mole. P a r t i a l i n h i b i t i o n of t h e exchange w a s produced by 1 0 0 mM N a + ( v i a p o s i t i v e c o o p e r a t i v e i n h i b i t i o n a t a- and/or B-sites) b u t w i t h o u t apP u r i f i c a t i o n o f t h e r e n a l enzyme by p a r e n t a f f e c t on R . t h e SDS p r o c e d u r e of J # r g e n s e n ( 1 9 7 4 ) d i d n o t change t h e p a r t i t i o n c o e f f i c i e n t . A t pH 6 . 6 and below, however, t h e exchan e c a p a c i t y i s r e d u c e d by 7 0 % and t h e d i s t r i b u t i o n o f [ljO]s p e c i e s changes d r a m a t i c a l l y , i n d i c a t i n g 2 t o 3 oxygens e x c h a n g i n g p e r bound P i and s u g g e s t i n g t h a t f o r m a t i o n of E-P i s more r a p i d t h a n d i s s o c i a t i o n of P i from E-Pi. This i s i n d i r e c t analogy t o a l k a l i n e phosphatase (Shaffer e t a l . , 1978). S t u d i e s w i t h dival e n t c a t i o n s o t h e r than Mg2+ a r e i n p r o g r e s s . R v a l u e s have r e c e n t l y been d e t e r m i n e d f o r s e v e r a l o t h e r p h o s p h o h y d r o l a s e s c a p a b l e o f P i +HOH exchange. N a t i v e Zn a l k a l i n e p h o s p h a t a s e i s c h a r a c t e r i z e d by R < < 1 ( E a r g l e e t a l . , 1 9 7 7 ; Bock and Cohn, 1978) which increases t o 3 w i t h Co2+ r e p l a c e m e n t . Y e a s t i n o r g a n i c p y r o p h o s p h a t a s e and p r o s t a t i c a c i d p h o s p h a t a s e p o s s e s s R v a l u e s of 0 . 7 and <1, r e s p e c t i v e l y ( J a n s o n e t a l . , 1 9 7 9 ; Van E t t e n and R i s l e y , 1 9 7 8 ) . The s a r c o p l a s m i c r e t i c u l u m C a 2 + - A T P a s e and myosin s u b f r a g m e n t - 1 A T P a s e m a n i f e s t R v a l u e s o f 0.08-0.2 and 5 0 - 1 0 0 , r e s p e c t i v e l y (Boyer e t a l . , 1 9 7 7 ; S l e e p e t a l . , 1978; A r i k i and Boyer, 1 9 8 0 ; Webb e t a l . , 1 9 7 8 ) .

ACKNOWLEDGMENT

T h i s r e s e a r c h was s u p p o r t e d by U n i t e d S t a t e s P u b l i c H e a l t h R e s e a r c h G r a n t GM-22197, National S c i e n c e Foundation G r a n t GB-38671, 31P-spectra were and t h e C a l i f o r n i a Metabolic R e s e a r c h F o u n d a t i o n . o b t a i n e d on a n i n s t r u m e n t o b t a i n e d and m a i n t a i n e d u n d e r N I H RR-00708.

REFERENCES

A r i k i , M., and Boyer, P . D. ( 1 9 8 0 ) . B i o c h e m i s t r y 1 9 , 2001-2004. Bock, J. L., and Cohn, M. ( 1 9 7 8 ) . J. B i o l . Chem. 253, 4082-4085. Boyer, P. D . , d e M e i s , L., Carvalho, M., and Hackney, D. D. ( 1 9 7 7 ) . B i o c h e m i s t r y 16, 136-139. Chlebowski, J. F., A r m i t a g e , I. M . , Tusa, P. P., a n d Coleman, J. E. ( 1 9 7 6 ) . J. B i o l . Chem. 251, 1207-1216.

NMR KINETIC ANALYSIS OF OXYGEN-18 EXCHANGE REACTION

375

C o h , M . , and Hu, A . ( 1 9 7 8 ) . P r o c . N a t l . Acad. Sci. USA 7 5 , 200205. E a r g l e , D. H . , L i c k o , V . , a n d Kenyon, G . L. ( 1 9 7 7 ) . A n a l . B i o c h e m . 81, 186-195. Hackney, D . D . , S t e m p e l , K. E . , and Boyer, P. D. ( 1 9 8 0 ) . I n "Methods i n Enzymology" (Di L. P u r i c h , e d . ) , V o l . 6 4 , pp. 60-82. Academic P r e s s , N e w York. J a s o n , C. A . , Deqani, C., and Boyer, P. D. ( 1 9 7 9 ) . J . B i o l . Chem. 2 5 4 , 3743-3749. J d r q e n s e n , P . K. ( 1 9 7 4 ) . B i o c h i m . B i o p h y s . A c t a 3 5 6 , 36-54. M%rdh, S . , and L i n d a h l , S. ( 1 9 7 7 ) . J . B i o l . Chem. 2 5 2 , 8058-8061. M&dh, S . , and Z e t t e r q u i s t , 0. ( 1 9 7 4 ) . B i o c h i m . B i o p h y s . A c t a 3 5 0 , 473-483. P e r e z , B . , Miara, J . , and Dahms, A. S . ( 1 9 7 9 ) . In "Na,K-ATPase: S t r u c t u r e and K i n e t i c s " (J. Skou and J. Ndrby, e d s . ) , p p . 343-358. Academic P r e s s , N e w York. P l e s n e r , I . W . , and P l e s n e r , L. ( 1 9 8 1 ) . B i o c h i m . B i o p h y s . Acta 6 4 3 , 449-462. S h a f f e r , E . , A z a r i , J . , and D a h m s , A. S. ( 1 9 7 8 ) . J. B i o l . Chem. 2 5 3 , 5696-5704. S l e e p , J. A . , Hackney, D. D . , a n d Boyer, P . D. ( 1 9 7 8 ) . J. B i o l . Chem. 2 5 3 , 5235-5238. Van E t t e n , R . L., and R i s l e y , J. M. ( 1 9 7 8 ) . Proc. N a t . A c a d . S c i . USA 7 5 , 4784-4787. Webb, M. R., McDonald, G. G , , and Trentham, D. R. ( 1 9 7 8 ) . J. B i o l . Chern. 2 5 3 , 2908-2911.

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Part V

Conformational Changes, Structure/Function, and Active Site Probes

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CURRENT TOPICS IN MEMBRANES AND TRANSPORT, VOLUME 19

Principal Conformations of the a-Subunit and Ion Translocation PETER L. J0RGENSEN Institute of Physiology University of Aarhus Aarhus, Denmark

I.

INTRODUCTION

A conformational transition is a change in spatial arrangement of amino acid residues produced by rotation about covalent bonds, but not infolving breakage of chemical bonds except hydrogen bonds. The reason for our interest in these changes is not the detection of every motion in the proteins of the pure Na,K-ATPase, but the identification of structural changes which are coupled to energy conversion and ion translocation across the membrane. In absence of a detailed structural model of Na,K-ATPase, the transitions can be described in terms of differences between spatial arrangements of amino acid residues in states of the pump with different ligand affinities. Transitions of interest must correlate with the Na/K transport process with regard to ligand specificity and sensitivity to ouabain and vanadate. The time parameters must fit the rate constants of the transport reaction. With 377

Copyright 0 1983 by Academic Press. Inc. All rights of reproduction in any form reserved. ISBN 0-12-153319-0

PETER L. J0RGENSEN

378

these criteria, the study of conformational transitions may provide a shortcut to identification of protein components involved in the transport process. Two principal conformations of the a-subunit are defined by two patterns of tryptic cleavage and inactivation of enzymatic activity (J#rgensen, 1975) and by their different levels of tryptophan fluorescence intensity (Karlish and Yates, 1978). Differences in protein structure between the cation-induced dephospho forms (ElNa and E2K) are very similar to the differences between phospho forms (E1P and E2P) and the conformations are interconvertible through the action of ATP. Recently transitions between ElNa and E2K in the absence of other ligands have been related to cation binding (Jjdrgensen, 1982), occlusion of K+ or Rb+ (Beauge and Glynn, 19791, and presumably to dissipative cation fluxes (Anner, 1981; Karlish and Stein, 1981, 1982). The interesting aspect emerging from this is that the study of the cation-induced transitions in the absence of ligands other than Na+, K+, or Rb+ opens the possibility for dissecting the transport mechanism so that structural changes related to cation binding, occlusion, translocation, and release can be examined in the absence of energy-transducing reactions involving nucleotides, Pi, and Mg2+. In this topical review, the extent and mechanism of the cation-induced changes in protein structure are considered in relation to cation binding and the evidence for occlusion or translocation of cations in absence of other ligands. Next, these observations are related to the structure of the protein in ATP-bound forms, phospho forms, and inhibitorstabilized forms to see if there is evidence for structural changes in the a-subunit in addition to those described for the cation-induced conformations, ElNa and E2K.

11.

PROTEIN STRUCTURE

As a basis for the discussion of the structural changes it is necessary to summarize the data relevant to the subunit structure of Na,K-ATPase. Electron microscopy shows that the proteins of renal Na,K-ATPase are,organized in particles with a maximum diameter of 50 A , each containing one a-subunit and one 8-subunit. The particles are free to move in the plane of the bilayer, and the protein protrudes above the bilayer on

a-SUBUNITAND ION TRANSLOCATION

379

F i g . 1 . M o d e l s of the s t r u c t u r e o f Na,K-ATPase. Left: I n d e p e n d e n t f u n c t i o n o f a 8 - u n i t w i t h pathway for c a t i o n s p a s s i n g R i g h t : Redrawn f r o m t h r o u g h the s t r u c t u r e o f the a - s u b u n i t . K y t e ( 1 9 8 1 ) t o i l l u s t r a t e an o l i g o m e r i c ( a 8 ) ~s t r u c t u r e w i t h the c h a n n e l f o r c a t i o n s i n the s p a c e b e t w e e n the t w o a - s u b u n i t s .

both surfaces (Deguchi e t a l . , 1977). As illustrated in Fig. 1, the main question is whether a protomer a8-unit can form a pathway for cations across the membrane or whether active, coupled Na/K transport requires association between aB-units in the membrane. Independent catalytic function of a8-units is proposed from studies of ligand binding. The maximum capacities for binding of ATP or ouabain and phosphorylation fall in the range 2.8-5.1 nmoles/mg protein when the Lowry procedure is used for protein analysis (Jfdrgensen, 1974; Perrone e t a l . , 1975; Skou and Esman, 1979; Askari e t a ] . , 1980). With amino acid analysis the data fall in the range 5.2-5.7 nmoles/mg protein (Kyte, 1972; Moczydlowski and Fortes, 1981; Peters e t a l . , 1981) corresponding to masses of 175,000-192,000 gm protein/mole. More than one ligand may therefore be bound per (a8)2-unitI but binding of one ligand molecule per a@-unit is usually not realized, as the protein M~ are 106,000 for the a-subunit and 37,000-40,000 for the 8-subunit (Hastings and Reynolds, 1979; Esmann e t a l . , 1980; Sabatini e t a l . , 1981). A protomer a @ unit forms the minimum active soluble protein unit of Na,K-ATPase in C12E8 solution (Brotherus e t a l . , 19811, and the aB-unit forms the minimum asymmetric unit cell in the vanadate-induced two-dimensional crystal (Skiver e t a l . , 1981; Maunsbach e t a l . , this volume). In spite of these arguments for independent catalytic

380

PETER L. J0RGENSEN

functions of the a8-unit, it is not known if this unit can catalyze transport, and pathways for ions through aB-units have not been demonstrated. It is therefore important to evaluate the evidence for subunit interactions in the system. Associations between a@-units are observed both in detergent solution and in the membrane. In two-dimensional crystals, ( a 8 1 2-unit cells are seen in certain conditions (Hebert et a l . , 1982). In agreement with this, electron microscopy after freezefracture provides evidence for association between particles in the membrane (Deguchi et al., 1977; Maunsbach et a l . , 19791, but the functional significance of these interactions is not known. In detergent solution aggregates with molecular weight 380,000 (Hastings and Reynolds, 1979) or 265,000 (Esmann et a l . , 1980) are observed, but this may be due to unspecific aggregation of detergent micelles (Brotherus et a l . , 1981). Target size analysis by radiation inactivation consistently gives high molecular weights for Na,K-ATPase and lower masses for the partial reactions (Kepner and Macey, 1968; Ottolenghi et a l . , this volume; Bonting et al., this volume). Cross-linking data are also interpreted in favor of interaction between a-subunits in the membrane (Giotta, 1976; Askari et a l . , 1980). It is clear from this summary that the experimental problem is to distinguish functional protein-protein associations from unspecific aggregation in detergent solution or from random collisions between particles with free lateral mobility in the membrane. There are convincing arguments for independent catalytic functions of aB-units, but it is also quite likely that coupling between ATP hydrolysis and ion translocation requires the formation of an oligomeric (a8)2-complex.

111.

CATION-STABILIZED CONFORMATIONS OF THE a-SUBUNIT, ElNa AND E2K

Evaluation of the extent of the polypeptide chain rearrangements after adding cations alone shows that the transitions between El and E2 forms of the protein involve a significant number of residues in the asubunit. In addition to the trypsin-sensitive bonds and the tryptophan residues, there is evidence for structural changes involving sulfhydryl groups, ionizable groups, and intramembrane segments. The two forms have different affinities for nucleotides, and

a-SUBUNIT AND ION TRANSLOCATION

TRY “ 1 4

TRY

“4

381

TRY

(4 CHY

CHY

i

NH,-Gly -Arg -Asp

K -

L---78 NH, Ile -41 NH,Ala

I

Ser-Thr-Tyr-COOH Tyr COOH

-46 Kd L58 NH,Ala

K--$

COOH

F i g . 2 . L i n e a r map of a - s u b u n i t with p o s i t i o n o f s i t e s of p r i m a r y t r y p t i c c l e a v a g e (TRY) i n p r e s e n c e of KCI or N a C l ( J b r g e n s e n , 1 9 7 5 , 1 9 7 7 ) . C h y m o t r y p t i c c l e a v a g e (CHY) and l o c a t i o n of NH2- and COOH-terminal r e s i d u e s f r o m C a s t r o and F a r l e y ( 1 9 7 9 ) .

the structure of the ATP site may change from a hydrophobic pocket in ElNa to an open, hydrated structure in E2K.

A.

T R Y P T I C AND CHYMOTRYPTIC D I G E S T I O N

In a linear map of the a-subunit, the bonds exposed to primary tryptic cleavage are unique and welldefined points of reference (Jgkgensen, 1975, 1977; Giotta, 1975; Castro and Farley, 1979). At any one time only one or two bonds are exposed to cleavage. The tryptic splits have been located relative to the amino and carboxy terminal residues as shown in Fig. 2. Digestion in ghosts (Giotta, 1975) or in reconstituted vesicles (Karlish and Pick, 1981) shows that the trypsin-sensitive bonds are exposed at the cytoplasmic surface. Castro and Farley (1979) find that cleavage of bond 3 with chymotrypsin does not depend on Na+ or K’. We find that the chymotrypsin-sensitive bond is exposed in NaCl and completely protected from cleavage in KC1 medium. The monoexponential decay of Na,K-ATPase activity in E2K is related to cleavage of bond 1, while inacti-

382

PETER L. J0RGENSEN

vation of the K-phosphatase activity requires secondary cleavage of bond 2 within the same a-subunit. In E Na, the time course of tryptic inactivation is by two exponentials with equal fractions A and B lost in the two phases with rate constants a and B for cleavage of bond 2 and bond 3, respectively. The ratio of the rate constants a/@ is 15-25 at 37'C, but it is higher at 20'C because cleavage of bond 3 (rate constant 8 ) is more temperature sensitive than cleavage of bond 2 (Jdrgensen, 1975, 1977; Jdrgensen and Petersen, 1979; Karlish and Pick, 1981). As shown in Fig. 3, the rate constants a and B for cleavage of bond 2 and bond 3 are altered in parallel to saturation of sites for highaffinity binding of 86Rb ( K D = 7.5 p ~ )to Na,K-ATPase (Jdrgensen, 1982). In reconstituted vesicles the exchange of cations at the two membrane surfaces shows that it is Na+ and K+ or Rb+ at the cytoplasmic surface which stabilize the alternative patterns of tryptic digestion of the a-subunit (Karlish and Pick, 1981). Transition from ElNa to E2K involves protection of bond 3 and exposure of bond 1 near the middle of the a-subunit to trypsin while the position of bond 2 near the amino terminus is altered, so that cleavage of bond 2 becomes secondary to cleavage of bond 1 within the same a-subunit. The trypsin-sensitive bonds in the a-subunit are separated by intramembrane segments (Karlish et a l . , 1977; Jdrgensen et a l . , this volume). The bonds may therefore be located in loosely structured loops in domains which are protruding at the cytoplasmic surface. The changes in structure can be due to altered positions of side chains within the loops or to rearrangement of the cytoplasmic domains relative to each other.

describes

B.

INTRINSIC

PROTEIN

FLUORESCENCE

The intensity of intrinsic tryptophan fluorescence of the E K form is 2-3% higher than that of the EINa form. Ttis small but significant change in fluorescence can be due to changes in position of a single or two tryptophan residues from a relatively hydropholic microenvironment in EINa to a more hydrophobic state in E2K, since the change in intensity is accompanied by a shift of the spectrum of E2K to shorter wavelengths in comparison with the spectrum of ElNa (Karlish and Yates, 1978; Chetverin et al., 1980). This may be related to the degree of immersion of the protein into the membrane lipid. The tryptophan fluorescence levels are altered in parallel to saturation of two sites per a-subunit for

(Y-SUBUNITAND ION TRANSLOCATION

383

I .O 01

.-c .-cm

E

L

0.8

0.6

A c

..->

v

m 0)

m

CL

I-

0.5

0.4

?

Y

m-

z

0.3

0

c

.-0 ti P LL

0.2

0

10

20

40

60

80

Incubation time (min) F i g . 3. E f f e c t o f R b ' ions on t r y p t i c i n a c t i v a t i o n o f DiNa,K-ATPase a c t i v i t y i n p r e s e n c e o f 2 5 mM T r i s - C 1 , pH 7 . 5 . g e s t i o n a t 20°C was s t a r t e d by a d d i n g 0 . 2 1-19T P C K - t r y p s i n p e r 100 1 -19Na,K-ATPase i n a t o t a l v o l u m e o f 600 1-11. A t the i n d i c a t e d times a l i q u o t s o f 5 0 ~1 were m i x e d w i t h 2 0 p 1 b u f f e r c o n t a i n i n g 2 Ug t r y p s i n i n h i b i t o r . S a m p l e s c o n t a i n i n g 1 pg Na,K-ATPase Experimental p r o t e i n were t r a n s f e r r e d f o r a s s a y o f Na,K-ATPase. d a t a p o i n t s a r e p l o t t e d a n d curves a r e d r a w n a s c a l c u l a t e d by a T h e r a t i o o f the c o m p u t e r f o r the equation EA = A - e - a t + B.e-Bt. r a t e constants a/$ (@) i s p l o t t e d i n the i n s e t f o r c o m p a r i s o n w i t h t h e curve o f 86Rb b i n d i n g (----I t o n a t i v e Na,K-ATPase.

high affinity binding of 86Rb ( K D = 7.5 U M ) and t o the changes in tryptic cleavage in these conditions (Jpirgensen, 1982). C.

Sulfhydryl

Groups

Previously, only small changes in reactivity of sulfhydryl groups t o N-ethylmaleimide (NEM) were observed when K+ was exchanged €or Na+ in the absence of

PETER L. J0RGENSEN

384

ATP or Mg2+ (Skou, 1974; Schoot et a l . , 1980). Recently an "E2-sulfhydryl group" has been identified. The rate constant for inactivation of Na,K-ATPase by NEM is 2.5 times greater in the presence of K+ than with Na+, and labeling of a thiol group with radioactive NEM depends on the transition to the E2 conformation (Winslow, 1981). Additional evidence for involvement of a sulfhydryl group in the K-induced conformational transition is that thimerosal reversibly blocks the K-induced change in emission from fluorescein and tryptophan (Hegyvary and Jgirgensen, 1981) without affecting high affinity binding of 86Rb (Jfirgensen,1982). The thimerosal-sensitive group may thus be involved in a reaction step between the steps of the binding equilibrium and the conformational transition (see Section IV)

.

D.

pH D E P E N D E N C E

Titration of the pH dependence of the tryptophan fluorescence responses suggests the presence of an ionizable group with a PK in the range 6.5-7. Transition from ElNa to E2K causes uptake of a proton, a negative Bohr effect, most likely due to a change in PK of an unidentified ionizable group. Release of a proton from the protein, a positive Bohr effect, is elicited by transition from E2K to E1Na. The titrations show that protonation and deprotonation alter the equilibrium between the conformations. At pH above 9.5 the protein may be in the El form and at pH below 5.5 in the E2 form whether the cations are present or not (Skou and Esman, 1980). At pH 7-7.5, the transition from El to E2K may therefore involve both binding of K+ and uptake of a proton. E.

THE NUCLEOTIDE

BINDING REGION

I N EINa AND E2K

Conformations with the ATP binding region adapted for tight (E1Na) or weak (E2K) binding of nucleotide can be distinguished with radioactive ATP, ADP, and the fluorescent formycin analogs FTP or FDP (N6rby and Jensen, 1971; Hegyvary and Post, 1971; Kaniike et a l . , 1974; Karlish et a l . , 1978). Assuming that the steps for binding or release of nucleotides and cations are relatively fast, the change in fluorescence of formycin nucleotides is a convenient, but indirect tool for monitoring the rate of the conformational transitions in the protein.

(u-SUBUNITAND ION TRANSLOCATION

385

At high pH, fluorescein-isothiocyanate (FITC) attach covalently to the El form with high apparent affinity to an amino group in the 58K segment in vicinity of the ATP binding area (Karlish, 1980). With one fluorescein molecule per a-subunit, the transition from ElNa to E2K is accompanied by 20-25% quenching of fluorescence. The large size of this signal allows very detailed titrations of the interactions of Na+ and K+ with the enzyme. Titration with K+ alone gives hyperbolic curves and linear reciprocal plots suggesting interaction with a single type of cation site. In presence of Na+ the titration curves for K+ becomes sigmoid shaped, suggesting binding to multiple interacting sites (Hegyvary and Jdrgensen, 1981). In view of the observations discussed below it is likely that the fluorescein monitors the affinity or hydrophobicity in the ATP binding region rather than the state of the side chain to which it is covalently attached. Experiments with TNP-ATP (Moczydlowsky and Fortes, 1981) and eosine (tetrabromofluorescein) (Skou and Esmann, 1981) suggest that the conformational change induced by binding of Na+ creates a hydrophobic pocket for nucleotide binding, while binding of K+ either closes this pocket or transforms it into an open hydrated structure. Transfer of TNP-ATP or eosin from water to solvents of low polarity increases their fluorescence dramatically. Both compounds are potent inhibitors and bind with high affinity (KD = 0.2-0.7 p M ) to the ATP binding site in El. The emission maximum and fluorescence intensity of the complexes with Na,K-ATPase suggest that the ATP binding area in El has a polarity close to that in ethanol. F.

HYDROPHOBIC

LABELING

It is not known if the cation-induced transition is transmitted through the membrane from cytoplasmic to extracellular parts of the protein. Hydrophobic labels may monitor altered degrees of immersion of proteins into the lipid bilayer. In agreement with the transfer of tryptophan residues to a hydrophobic environment (Section III,B), labeling from within the lipid bilayer with iodonaphthylazide (INA) is 10-25% greater in E2K than in ElNa (Karlish et al., 1977), while transition to E2K reduces the incorporation of adamantane diazirine (AD) (Farley et a l . , 1080). As INA and AD label distinct segments of the a-subunit, this discrepancy may reflect that more of the 46K fragment and less of the 58K fragment lies embedded in the bilayer. It is not

386

PETER L. J0RGENSEN

known whether Na+ and K+ in absence of other ligands alter the structure of protein portions at the extracellular surface. G.

R E L A X E D OR N A T I V E C O N F O R M A T I O N

This is the conformation of lowest free energy which the a-subunit assumes in the absence of specific ligands. It is difficult to determine if El or E2 is the relaxed conformation because the equilibrium between the forms in the absence of specific cations (Na+, K+ or Rb+) depends on the pH (Skou and Esmann, 1980) and on unspecific ions like Tris-C1 or choline-Cl with sodiumlike or potassium antagonistic effects (Jdrgensen, 1975). At low ionic strength the protein assumes the E2 form in the absence of specific and unspecific cations suggesting that E2 is the relaxed conformation. However, in these conditions the affinity for Rb' and K+ is high with KD below 7 U M , and it is difficult to exclude that the shift to the E2 form is due to binding of contaminant K+.

IV.

CATION BINDING AND CONFORMATIONAL TRANSITION

As monitored by tryptic cleavage (Fig. 3) or by fluorescence from tryptophan and fluorescein, transition from El to E2Rb is parallel to occupancy of two high affinity sites for RbS per a-subunit (Jpkgensen, 1982). As calculated from data in Fig. 4, the capacity €or binding is 12.3 nmoles/mg protein with KD 7.5 U M . Binding of a single &+ ion is sufficient for transition to E2Rb since there is a close correlation between the binding of 86Rb and the parameters for the conformational state of the protein at low cation concentration. Monitoring of tryptophan fluorescence in a stopped flow fluorimeter shows that the transition from ElNa to E2K is fast (300 sec-1) while the transition from E2K to ElNa is very slow (0.3 sec-1). Titration of the effect of K+ on the rate of the transition to E2X suggested that binding is weak with a constant of 74 mM. The high apparent affinity of K+ binding has therefore been explained by coupling of weak binding of K+ to El with the conformational equilibrium which is poised in direction of E2K (Karlish and Yates, 1978; Karlish, 1980).

a-SUBUNIT AND ION TRANSLOCATION

387

-

.-c a

c

e

n

0

30

10

50

100

Concentration of 86RbCI (uM) F i g . 4 . E f f e c t o f m o d i f i c a t i o n w i t h t h i m e r o s a l on the b i n d i n g o f 86Rb a t e q u i l i b r i u m . P u r e r e n a l Na,K-ATPase ( 0 ) and t h i m e r o s a l enzyme (a) 50-70 ug p r o t e i n w e r e m i x e d w i t h 86Rb i n c o n c e n t r a t i o n s b e t w e e n 2 pM and 100 pM i n 25 mM T r i s - C 1 , pH 7 . 5 . Bound and f r e e 86Rb w e r e s e p a r a t e d b y c e n t r i f u g a t i o n . To e s t i m a t e unbound 86Rb t r a p p e d i n the p e l l e t s , 2 mM R b C l was added t o two t u b e s .

However, this scheme cannot explain the observation that thimerosal abolishes the conformational change (Hegyvary and Jfdrgensen, 1981) without affecting the affinity or the capacity for binding of F&J+ (Fig. 4 ) . Even more pertinent, recent binding studies show that Rb+ binds to Na,K-ATPase in the presence of 2 mM ATP with relatively high affinity ( K =~ 60 p ~ )(Jensen and Ottolenghi, this volume). To explain these observations one must assume that the El form exposes sites €or tight binding of Rb+ or K+ at the cytoplasmic surface and that an extra step is inserted between the binding equilibrium and the structural transition. One possibility for this extra step is protonation (Section 111,D). El

+

K+

f

EIK

+ H+

L7 HEIK f

HE2K

388

PETER L. J0RGENSEN

In this scheme high affinity binding of K+ at the cytoplasmic surface is followed by proton uptake and the transition from El to E2. The demonstration of high affinity sites for Rb+ in the absence of a conformational change suggests that tight binding may in part explain the slow release of Rb+ or K+ from E2K or E2Rb. A maximum rate of dissociation from the site can be estimated since the product of the equilibrium constant (kf-l-1 and the turnover rate (sec-1) cannot exceed the rate constant (108-109 M'lsec-1) in a diffusioncontrolled reaction (Lauger, 1980). With the affinity for Rb+ found in Fig. 4 to be 1.3 x lo5 M - 1 , the maximum rates calculated from this relationship are 3-5 orders of magnitude higher than the actual rate of release of R b from E2Rb. Retardation of the release of cation from E2Rb is therefore not only explained by tight binding, but may involve the transfer of cation sites from a secluded cavity to the membrane surface as proposed in the occlusion hypothesis (Post e t a l . , 1972; Beau96 and Glynn, 1979).

V.

OCCLUSION AND DISSIPATIVE FLUXES THROUGH THE PUMP PROTEIN

The term occlusion refers to the state of Rb+ or K+ after dephosphorylation of Na,K-ATPase (Post et a l . , 1972). As+monitored with trypsin, the conformation induced by K in absence of other ligands, E2K, has properties similar to those of the occluded complex after dephosphorylation (Jghgensen, 1975). With a rapid ion-exchange technique it can be shown that occluded E2(Rb) is formed after mixing Na,K-ATPase and Rb+ in absence of other ligands. The maximum capacity is 2.7 nmoles/mg protein at 500 !JM and the occluded Rb+ is released at a rate (0.2 sec-1) which is close to the rate of the transition from E2K to ElNa. The process is accelerated by ATP but not by Na+, and the occluded Rb+ does not exchange with Rb+ in solution until the conformational transition to ElRb is completed (Beau96 and Glynn, 1979; Glynn and Richards, this volume). These experiments suggest that the binding site for K+ moves from the cytoplasmic surface to a cavity within the protein where the bound cation is prevented from exchange with medium cation. As the capacity for occlusion is low (2-3 nmoles/mg protein) relative to the capacity for binding of Rb+ at equilibrium (10-13 nmoles/mg protein), the question can be raised if E2(K)

a-SUBUNIT AND ION TRANSLOCATION

389

represents a stable conformation or if it is a transitory form that can release K+ to the inside or to the outside. From experiments on red cells, the possibility has been discussed that part of the passive ion fluxes can take place through the molecular mechanism of the pump (Lew and BeaugB, 1979). Ouabain-insensitive, passive fluxes can be explained as unspecific leaks produced by the insertion of the protein in the bilayer (Mimms et al., 1981). In phospholipid vesicles, incorporation of Na,K-ATPase induces both ouabain-insensitive leaks and dissipative fluxes that are blocked by ouabain and vanadate (Karlish and Stein, 1981, 1982). At equal concentrations of Na+ and K+, the Na,K-ATPase transports more K+ than Na+ along the gradient, presumably reflecting that the affinity for binding of K+ is higher than that for Na+ (Anner, 1981). Vanadate-sensitive fluxes of Rb+ into vesicles loaded with Tris-C1 suggest that Rb+ is bound to cytoplasmic aspects prior to translocation. Addition of magnesium vanadate to the inside or the outside of the vesicles blocks about half of Rb/Rb exchange diffusion suggesting that the exchange is mediated by an equal number of pumps oriented in either direction (Karlish and Stein, 1981, 1982). The experiment in Fig. 5 demonstrates ouabainsensitive uptake of 22Na into vesicles with pumps operating slowly as carriers in a reverse mode, transporting Na+ and K+ along their gradients. The ouabain-sensitive flux is a saturable function of Na+ concentration with apparent K M close to 8 mM, while the unspecific flux with 10-4 M ouabain present in both phases is linearly related to the concentration of Na+. The rates of these fluxes are low, 0.1-0.4 sec-1, and it is important to distinguish passive fluxes from cation binding to Na,K-ATPase in the vesicles. The inhibition by vanadate and ouabain suggests that the passive fluxes are coupled to conformational transitions in the protein of Na,K-ATPase. In membrane fragments, transitions induced by K+ binding are coupled to occlusion in a cavity within the protein. In vesicles with gradients for K+ and Na+ across the membrane, cation binding and conformational transition are coupled to translocation and discharge of cation at the opposite phase in a dissipative flux along the cation gradient. The study of these slow, cation-induced transitions may prove to be important for the identification of structural elements involved in the translocation, but their relevance to the transport mechanism is not clear unless they can be related to energytransducing reactions.

390

PETER L. J0RGENSEN

1.5

1.0

0.5

0

0

5

10

15

20

Concentration of NaCl ( m M ) F i g . 5 . O u a b a i n - s e n s i t i v e , p a s s i v e f l u x o f 22Na i n t o vesicles r e c o n s t i t u t e d w i t h p u r e r e n a l Na,K-ATPase w i t h 4 5 mM K C l i n s i d e and N a C l v a r y i n g b e t w e e n 0 and 20 mM i n the o u t s i d e medium. C o n s t a n t i o n i c s t r e n g t h was m a i n t a i n e d w i t h T r i s - C 1 . R e c o n s t i t u t i o n and e x c h a n g e of m e d i a a s described b y K a r l i s h and P i c k ( 1 9 8 0 ) w i t h o u t o u a b a i n (a), w i t h 10-4 M o u a b a i n i n s i d e ( a ) , w i t h 10-4 M o u a b a i n o u t s i d e or i n both p h a s e s (A).

VI.

ATP- AND M ~ ~ + - B O U NCONFORMATIONS D OF THE

SUBUNIT

Data in the preceding sections establish relationships between cation-induced conformational transitions in the a-subunit and occlusion and ion translocation. This section examines whether the relationship can be carried further to the reaction steps involving ATP binding and phosphate transfer. The present treatise concentrates on protein structure and orientation and specificity of cation sites. Details concerning the effects of nucleotide binding and phosphorylation on cation exchange reactions are described in other articles (Glynn and Karlish, 1975; Simons, 1973; Cavieres and Glynn, 1979; Karlish and Stein, 1982). For

(u-SUBUNITAND ION TRANSLOCATION

391

F i g . 6 . M i n i m a l scheme i l l u s t r a t i n g the r e l a t i o n s h i p o f p r i n c i p a l c o n f o r m a t i o n s o f the p r o t e i n t o the r e a c t i o n s t a t e s o f Na,K-ATPase i d e n t i f i e d i n k i n e t i c s t u d i e s ( P o s t e t a l . , 1 9 7 2 ; f o r r e f e r e n c e s , see R o b i n s o n a n d F l a s h n e r , 1 9 7 9 ; C a n t l e y , 1 9 8 1 ) . The El f o r m exchanges for Na& i n the c y t o p l a s m i c p h a s e . E l i s p h o s p h o r y l a t e d f r o m MgATP, b u t not b y P i . T h e E2 f o r m i s p h o s p h o r y l a t e d b y P i , b u t not b y ATP. I t e x c h a n g e s Nazxt for i n the e x t r a c e l l u l a r m e d i u m . In E z K , K+ ions are o c c l u d e d , and b i n d i n g o f ATP a t a low a f f i n i t y s i t e a c c e l e r a t e s r e l e a s e o f Kcyt. Mg2+ i s r e q u i r e d for p h o s p h o r y l a t i o n , conversion between EIP and E z P , and f o r d e p h o s p h o r y l a t i o n .

eyt

guidance, Fig. 6 shows a minimal scheme with the positions of the principal conformations of the protein in a reaction scheme similar to that drawn by Post and co-workers (1972). The kinetic studies of these reactions have recently been reviewed (Robinson and Flashner, 1979; Cantley, 1981). A.

A T P B I N D I N G TO E 2 K

Binding of ATP with low affinity ( K + = 0.2 mM at 100 m M KC1) alters the monoexponential pattern of tryptic cleavage of E2K to a biphasic pattern similar to that observed for ElNa. ADP has a similar effect, and Mg2+ is not required (Jdrgensen, 1975). This transition is accompanied by a 2 - 3 % reduction in tryptophan fluorescence intensity (Karlish and Yates, 1978; Jbrgensen and Karlish, 1980). These requirements are similar to those previously found by Post e t a l . (1972)

PETER L. J0RGENSEN

392

f o r t h e a c t i v a t i n g e f f e c t of ATP o f t h e o c c l u d e d form of t h e s i t e f o r t r a n s l o c a t i o n of K+ o r Rb'. With f o r mycin n u c l e o t i d e s FTP and FDP t o monitor n u c l e o t i d e b i n d i n g ( K a r l i s h et a l . , 1 9 7 8 ) , o r w i t h t r y p t o p h a n f l u o r e s c e n c e ( K a r l i s h and Yates, 19781, it i s o b s e r v e d t h a t ATP accelerates t h e t r a n s i t i o n from E2K t o E l from a b o u t 0 . 2 sec-1 t o r a t e c o n s t a n t s o f a b o u t 6 0 sec-1 a t s a t u r a t i n g c o n c e n t r a t i o n s o f ATP. Release of o c c l u d e d Rb+ i o n s can b e determined i n t h e s e condit i o n s (Beaugk and Glynn, 1 9 7 9 ) . I t i s t h e r e f o r e proposed t h a t t h e e f f e c t of ATP is t o i n c r e a s e t h e r a t e of a t r a n s i t i o n which e x p o s e s t h e c a t i o n b i n d i n g s i t e a t t h e c y t o p l a s m i c s u r f a c e and releases K+ from t h e occluded state. B.

THE E I K - A T P

CONFORMATION

When N a + i s exchanged f o r K+ i n t h e p r e s e n c e of h i g h c o n c e n t r a t i o n s o f ATP, o n l y minor changes i n t h e b i p h a s i c p a t t e r n o f t r y p t i c d i g e s t i o n are o b s e r v e d (Jfdrgensen, 1 9 7 5 ) . Other p r o b e s o f p r o t e i n conformat i o n p r o v i d e e v i d e n c e f o r d i f f e r e n c e s i n s t r u c t u r e between E l N a - A T P and E l K - A T P . Sulfhydryl groups i n t h e a - s u b u n i t a r e p r o t e c t e d when K+ i s exchanged f o r N a + i n t h e p r e s e n c e of ATP ( H a r t and T i t u s , 1 9 7 3 ; Skou, 1 9 7 4 ; Winslow, 1 9 8 1 ) . The i n c r e a s e d a l k y l a t i o n of a n a c t i v i t y - a s s o c i a t e d s u l f h y d r y l group i s l i k e l y t o r e f l e c t f o r m a t i o n of a t e r n a r y complex o f N a , K - A T P a s e , ATP and K+ (Winslow, 1 9 8 1 ) . I n agreement w i t h t h i s , r e c e n t experiments provide evidence f o r simultaneous binding of Rb' and ATP on t h e enzyme ( J e n s e n and O t t o l e n g h i , The t h i s volume) w i t h KD = 6 0 p M f o r Rb+ a t 2 mM ATP. release of K+ or Rb+ from t h e b i n d i n g s i t e a t t h e c y t o p l a s m i c s u r f a c e may t h e r e f o r e r e q u i r e t h e combined p r e s e n c e o f ATP and Na+. C.

ATP BINDING

TO E l N a

High a f f i n i t y b i n d i n g of ATP t o E l N a c a u s e s o n l y minor changes i n s t r u c t u r e of t h e a - s u b u n i t . The p a t t e r n of b i p h a s i c t r y p t i c d i g e s t i o n of E l N a i s o n l y The r a t i o of t h e r a t e cons l i g h t l y a l t e r e d by ATP. s t a n t s a / B and t h e r e l a t i v e amount of t h e 78K fragment a r e i n c r e a s e d (Jfdrgensen, 1975; Jfdrgensen and P e t e r s e n , 1 9 7 9 ) . The t r y p t o p h a n f l u o r e s c e n c e l e v e l s of E l N a and ElATP a r e i d e n t i c a l ( K a r l i s h and Yates, 1 9 7 8 ) . ATP f a v o r s b i n d i n g of N a + o v e r K+ a t t h e c y t o p l a s m i c s i t e s i n t h e E l form presumably t h r o u g h a n e f f e c t on t h e pK

a-SUBUNIT AND ION TRANSLOCATION

393

of an ionizable group (Skou and Esmann, 1980). Deprotonation of this group shifts the equilibrium toward the ElNa form. High-affinity Na+ sites are accessible from the cytoplasmic surface in the ElATP form to catalyze phosphorylation from ATP, uncoupled Na+ efflux, and Na/Na exchange (Glynn and Karlish, 1975; Cavieres and Glynn, 1979). D.

Mg-BOUND FORMS OF THE P R O T E I N

Effects of Mg2+ on nucleotide binding and groupspecific reactions are reviewed by Bonting e t a l . (this volume). In the absence of other ligands Mg2+ reacts with low affinity to expose bond 3 in the asubunit to trypsin, but the Mg-bound form is classified as an El form since bonds 2 and 3 are exposed to cleavage (Jqjrgensen and Petersen, 1979) . Mg2+ reduces the affinity for binding of Rb+ and delays the rate of the transition from E2K or E2Rb to ElNa in a way suggesting that complex formation with Mg2+ may be part of the explanation for the occlusion (Hegyvary and Jplrgensen, 1981). Using fluorescein-Na,K-ATPase Steinberg e t a l . (this volume) found that Mn2+ has higher affinity than Mg2+ and suggest that Mn2+ produces a state of the enzyme which is closer to the E2 form than the Mg-bound form. Distinct effects of Mg2+ on fluorescence of sulfhydryl reagents have been observed (Gupte and Lane, 1979; Forgac, 1980). These groups have not been located within the protein and it is proposed that reaction with lipids may in part explain the fluorescence change.

VII.

PROTEIN CONFORMATIONS OF THE PHOSPHOENZYMES

Tryptic digestion and fluorescence experiments suggest that the major change in structure of the a-subunit occurs when E1P is transformed into E2P and not when phosphate is transferred from ATP to the protein (Jqjrgensen and Petersen, 1979; Jqjrgensen and Karlish, 1980). Taniguchi e t a l . (this volume) report transient changes in fluorescence from a maleimide derivative after addition of ATP. E1P produced at 2 M NaCl has a low fluorescence intensity and E2P a high intensity. With this probe, transitions between E1P and E2P are accompanied by the largest conformational changes of any elementary steps examined. In tryptic digestion

394

PETER L. J0RGENSEN

experiments the effects of individual ligands can be resolved in some detail. Binding of ATP in the presence of Mg2+ increases the rate constant for digestion in the second phase in Tris-C1 medium. Reaction of Na+ with El, MgEl, or ElATP seems not to alter the structure of the a-subunit. However binding of Na+ to MgEIATP causes removal of bonds 2 and 3 from the membrane surface while bond 1 becomes exposed in parallel to the formation of a phosphoenzyme. These titrations show that Na+ has a pronounced effect on the rate constant for cleavage of bond 2 (Jgirgensen and Petersen, 1979). This protection of bond 2 by Na+ is related to transition from E1P to E2P rather than to the Na-induced transfer of phosphate from ATP to the protein, since it can be shown that cleavage of bond 2 alters the poise of the equilibrium between El and E2 forms in the direction of the El forms for both the dephospho and phospho forms of the protein (Jgkgensen and Karlish, 1980). The increase in tryptophan fluorescence intensity accompanying phosphorylation from ATP in the presence of K+ reflects the formation of E2P which has the same fluorescence intensity as E2K. Conversely, the fluorescence intensity of E1P is similar to that of the El dephospho forms. The tryptophan fluorescence technique thus provides a simple and rapid assay of the steady-state proportions of E1P and E2P (Jph-gensenand Karlish, 1980). Other studies suggest that notable structure differences between E2P and E2K are detectable. Experiments with thimerosal show that formation of E2P involves chemical reactions that are different from those involved in the formation of E2K (Hegyvary and Jgkgensen, 1981). Phosphorylation is accompanied by a large change in fluorescence of reagents bound to sulfhydryl groups (Harris and Stahl, 19761, and the sulfhydryl groups available for alkylation in E2P are different from the number exposed in E2K (Winslow, 1981). In addition there is a large difference in ouabain binding affinity between the E2P and the E2K forms of the protein (Hansen, 1978). It is therefore likely that formation of E2P involves changes in structure of intramembrane and extracellular portions in addition to the structural differences between El and E2 forms that can be demonstrated in cytoplasmic portions of the a-subunit with trypsin. The interest in demonstrating structural changes in extracellular aspects of the protein is related to the hypothesis that only the phosphoenzyme in the E2 form can pick up or discharge cations at the extracellular surface.

a-SUBUNIT AND ION TRANSLOCATION

VIII.

395

INHIBITOR-STABILIZED CONFORMATIONS OF THE

-

a SUBUNIT

Analysis by tryptic digestion and tryptophan fluorescence shows that the protein must complete the transition to the structure in the Mg-bound form of E2K before vanadate can form the stable complex, Mg~E2Kavanadateand prevent further motion within the protein (Cantley et a l . , 1978; Jgkgensen and Karlish, 1980). A study of these reactions in reconstituted vesicles shows that both vanadate and potassium of this complex enters from the cytoplasmic phase (Karlish and Pick, 1981). High-affinity ouabain binding to E2P exposes a peptide bond within the a-subunit to cleavage by chymotrypsin (Castro and Farley, 1979). This split divides the 78K fragment into 35K and 40K fragments. The ouabain-bound conformation is also characterized by a very low level of fluorescence from fluorescein (Karlish, 1980; Hegyvary and J6rgensen, 1981; Steinberg et a l . , this volume). These observations suggest that ouabain stabilizes a unique conformation of the a-subunit that can be classified as a subconformation of E2P. The structural transitions in the a-subunit may be transmitted through intramembrane segments from the cytoplasmic portions of the protein to the ouabain binding area. The ligand dependence of fluorescence from anthroyl-ouabain bound to Na,K-ATPase is parallel to the results obtained by [3H]ouabain binding. Once bound to the enzyme the anthroyl-ouabain does not exhibit fluorescence changes dependent on protein conformation (Jesaitis and Fortes, 1980; Moczydlowsky, 1979).

IX.

CONCLUSIONS AND SPECULATIONS

The data discussed in this article show that transitions between El and E2 forms of the enzyme are accompanied by rearrangement of a significant number of residues in the a-subunit. These structural changes involve both the sites for cation binding (Section IV) and the nucleotide binding area (Section 111,E). The changes in tryptic digestion and the fluorescence responses are coupled to transitions between E l with cation sites for Na+ and K+ exposed at the cytoplasmic surface and E2 with K+ in the occluded state or with outside exposed cation sites. In absence of ligands

396

PETER L. JORGENSEN

other than Na+ and K+ or R b ' , equilibrium between these states depends on relative cation concentrations and on the cation gradients across the membrane (Section V). The tryptic digestion and fluorescence techniques monitor structural changes at the cytoplasmic surface. Probes for direct monitoring of the structure of transmembrane and extracellular protein portions are not available, but there is evidence that the transitions between E1P and E2P involve structural changes that are transmitted through the membrane (Section VII). From the viewpoint taken in this article the most pertinent gaps in our knowledge of the structure of the system concern the cation binding sites and the pathway for cations across the membrane. A system with binding sites for cations alternately exposed to the outside and to the inside, but not to both sides simultaneously, constitutes a carrier mechanism. The kinetics of a carrier may not be distinct from a channel with multiple conformational states, and both channel and carrier mechanisms are fast enough to account for the observed fluxes of Na+ and K+ through the pump (Lsuger, 1980). Two possible mechanisms for organization of the ionconducting pathway are considered. In the oligomeric model (Fig. 11, the channel is formed in the space between subunits. The alternate El and E2 conformations will then differ as result of small structural alterations in geometry of the channel presenting the cation sites to the inside or outside of the membrane. Alternatively, the channel may pass through a protomer a@-unit. The intramembrane portion of a single cr-subunit contains sufficient material to form a membrane pore. At present there is no compelling evidence favoring either of the two main possibilities. In view of recent progress in organizing Na,K-ATPase in membrane crystals (Skriver e t a l . , 1981) it is reasonable to assume that identification of cation pathways through the protein structure may soon come within reach.

EFERENCES

Anner, B. M. ( 1 9 8 1 ) . A K - s e l e c t i v e c a t i o n c h a n n e l formed by Na,K-ATPase i n liposomes. B i o c h e m . Int. 2, 365-371. A s k a r i , A . , Huang, W.-h., and A n t i e a u , J. M. (1980). Na+,K+ATPase: Ligand-induced c o n f o r m a t i o n a l t r a n s i t i o n s and alt e r a t i o n s i n s u b u n i t i n t e r a c t i o n s evidenced by c r o s s l i n k i n g s t u d i e s . B i o c h e m i s t r y 1 9 , 1132-1140. Beaug6, L. A . , and Glynn, I. M. (1979). Occlusion o f K i o n s i n t h e unphosphorylated sodium pump. Nature (London) 280, 510-512.

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Brotherus, J. B., Mhller, J. V., and Jdrgensen, P. L. (1981). Soluble and active Na,K-ATPase with maximum molecular mass 170,000+9000 daltons; formation of larger units by secondary aggregation. B i o c h e m . B i o p h y s . R e s . Commun. 100, 146-154. Cantley, L. C. (1981). Structure and mechanism of the Na,K-ATPase. C u r r . T o p . B i o e n e r g . 11, 201-237. Cantley, L. C., Cantley, L. G., and Josephson, L. (1978). A characterization of vanadate interactions with Na,K-ATPase. J. B i o l . Chem. 2 5 3 , 7361-7368. Castro, J., and Farley, R. A . (1979). Proteolytic fragmentation of the catalytic subunit of the sodium and potassium adenosine triphosphatase. J. B i o l . Chem. 2 5 4 , 2221-2228. Cavieres, J. D., and Glynn, I. M. (1979). Sodium-sodium exchange through the sodium pump: The roles of ATP and ADP. J. P h y s i o l . ( L o n d o n ) 2 9 7 , 637-645. Chetverin, A B., Venyaminov, S. Y., Emelyanenko, V. I., and Burstein, E. A. (1980). Lack of gross protein structure changes in the working cycle of (Na+,K+)-dependent adenosinetriphosphatase. E u r . J. B i o c h e m . 1 0 8 , 148-161. Deguchi, N., Jdrgensen, P. L., and Maunsbach, A. B. (1977). Ultrastructure of the sodium pump. Comparison of thin sectioning, negative staining, and freeze fracture of purified, membrane-bound Na,K-ATPase. J. C e l l B i o l . 7 5 , 619-634. Esmann, M., Christiansen, C., Karlsson, K.-A., Hansson, G. C., and Skou, J. C. (1980). Hydrodynamic properties of solubilized (Na' + K+)-ATPase from rectal glands of Squalus Acanthias. B i o c h i m . B i o p h y s . A c t a 6 0 3 , 1-12. Farley, R., Goldman, D. W , and Bayley, H. (1980). Identification of regions of the catalytic subunit of (Na-K)-ATPase embedded within the cell membrane. J. B i o l . Chem. 2 5 5 , 860864. Forgac, M. D. (1980). Characterization of a Mg2+-stabilized state of the (Na+ and K+) -stimulated adenosine triphosphatase using a fluorescent reporter group. J. B i o l . Chem. 2 5 5 , 1547-1553. Giotta, G. J. (1975). Native (Na+ + K+)-dependent adenosine triphosphatase has two trypsin-sensitive sites. J. B i o l . Chem. 2 5 0 , 5159-5164. Giotta, G. J. (1976). Quaternary structure of (Na+ + K+)-dependent adenosine triphosphatase. J. B i o l . Chem. 2 5 1 , 1247-1252. Glynn, I. M., and Karlish, S. J. D. (1975). The sodium pump. A n n u . R e v . P h y s i o l . 3 7 , 13-53. Gupte, S. S., and Lane, L. K. (1979). Reaction of purified (Na,K)ATPase with the fluorescent sulfhydryl probe 2-(4'-maleimidylani1ino)naphthalene 6-sulfonic acid. J. B i o l . Chem. 2 5 4 , 10362-10367. Hansen, 0 . (1978). The effect of sodium on inorganic phosphate and p-nitrophenyl-phosphate-facilitated ouabain binding to Na,K-ATPase. B i o c h i m . B i o p h y s . A c t a 5 1 1 , 10-22.

398

PETER L. JORGENSEN

Harris, W. E . , and S t a h l , W. L. ( 1 9 7 6 ) . I n t e r a c t i o n o f a new f l u o r e s c e n t r e a g e n t w i t h s u l f h y d r y l groups of t h e (Na+ + K+) - s t i m u l a t e d ATPase. Biochim. Biophys. A c t a 426, 325-334. H a r t , W. M., Jr., and T i t u s , E . 0. ( 1 9 7 3 ) . S u l f h y d r y l g r o u p s o f sodium-potassium t r a n s p o r t a d e n o s i n e t r i p h o s p h a t a s e . J. B i o l . Chem. 248, 4674-4681. H a s t i n g s , D. F . , and Reynolds, J . A. ( 1 9 7 9 ) . Molecular w e i g h t o f (Na+,K+)ATPase from s h a r k r e c t a l g l a n d . Biochemistry 1 8 , 817-821. H e b e r t , H . , J b r g e n s e n , P. L . , S k r i v e r , E . , and Maunsbach, A. B. ( 1 9 8 2 ) . C r y s t a l l i z a t i o n p a t t e r n s o f membrane-bound Na,K-ATPase. Biochim. Biophys. A c t a 689, 571-574. Hegyvary, C . , and J b r g e n s e n , P. L. (1981). Conformational changes of r e n a l sodium p l u s p o t a s s i u m i o n - t r a n s p o r t a d e n o s i n e t r i p h o s p h a t a s e l a b e l e d w i t h f l u o r e s c e i n . J. B i o l . Chem. 256, 6296-9303. Hegyvary, C., and P o s t , R . L. ( 1 9 7 1 ) . Binding o f ATP t o N a , K ATPase. J. B i o l . Chem. 246, 5234-5240. J e s a i t i s , A. J . , and F o r t e s , P. A. G. ( 1 9 8 0 ) . F l u o r e s c e n c e s t u d i e s of N a , K-ATPase labelled w i t h f l u o r e s c e i n m e r c u r i c acetate and a n t h r o y l o u a b a i n . J. B i o l . Chem. 255, 459-467. J b r g e n s e n , P. L. ( 1 9 7 4 ) . P u r i f i c a t i o n and c h a r a c t e r i z a t i o n of ( N a + + K+)-ATPase. I V . E s t i m a t i o n o f t h e p u r i t y and o f t h e m o l e c u l a r w e i g h t and p o l y p e p t i d e c o n t e n t p e r enzyme u n i t i n p r e p a r a t i o n s from t h e o u t e r medulla o f r a b b i t kidney. Biochim. Biophys. A c t a 356, 53-67. J b r g e n s e n , P. L. ( 1 9 7 5 ) . P u r i f i c a t i o n and c h a r a c t e r i z a t i o n o f (Na+,K+) -ATPase. V. Conformational changes i n t h e enzyme. T r a n s i t i o n s between t h e Na-form and t h e K-form s t u d i e d w i t h t r y p t i c d i g e s t i o n as a t o o l . Biochim. Biophys. A c t a 401, 399-415. J b r g e n s e n , P. L. ( 1 9 7 7 ) . P u r i f i c a t i o n and c h a r a c t e r i z a t i o n o f (Na' + K+) -ATPase. V I . D i f f e r e n t i a l t r y p t i c m o d i f i c a t i o n of c a t a l y t i c f u n c t i o n s o f t h e p u r i f i e d enzyme i n p r e s e n c e o f N a C l and KC1. Biochim. Biophys. A c t a 466, 97-108. J b r g e n s e n , P. L. ( 1 9 8 2 ) . High a f f i n i t y 86Rb b i n d i n g and s t r u c t u r a l changes i n t h e subunit o f Na,K-ATPase as d e t e c t e d w i t h t r y p t i c d i g e s t i o n and f l u o r e s c e n c e a n a l y s i s . Biochim. Biophys. A c t a 705, 38-47. J b r g e n s e n , P . L . , and K a r l i s h , S. J . D. ( 1 9 8 0 ) . D e f e c t i v e conformational response i n a s e l e c t i v e l y t r y p s i n i z e d (Na+ + K f ) ATPase s t u d i e d w i t h t r y p t o p h a n f l u o r e s c e n c e . Biochim. Biophys. A c t a 597, 305-317. J b r g e n s e n , P. L., and P e t e r s e n , J. ( 1 9 7 9 ) . P r o t e i n c o n f o r m a t i o n s of t h e p h o s p h o r y l a t e d i n t e r m e d i a t e s of p u r i f i e d Na,K-ATPase s t u d i e d w i t h t r y p t i c d i g e s t i o n and i n t r i n s i c f l u o r e s c e n c e as tools. I n "NaK-ATPase: S t r u c t u r e and K i n e t i c s " ( J . C . Skou Academic Press, New and J. G. Ndrby, e d s . ) , pp. 143-155. York.

399

a-SUBUNIT AND ION TRANSLOCATION

K a n i i k e , K . , Erdman, E . , and Schoner, W. ( 1 9 7 4 ) . D i f f e r e n t i a l m o d i f i c a t i o n of Na,K-ATPase by d i m e t h y l s u l f o x i d e . B i o c h i r n . B i o p h y s . A c t a 352, 275-286. K a r l i s h , S . J. D. ( 1 9 8 0 ) . C h a r a c t e r i z a t i o n of c o n f o r m a t i o n a l changes i n Na,K-ATPase labelled w i t h f l u o r e s c e i n a t t h e act i v e site. B i o e n e r g . B i o m e m b r . 1 2 , 111-136. K a r l i s h , S . J. D . , and P i c k , U. ( 1 9 8 1 ) . S i d e d n e s s of t h e e f f e c t s of sodium and potassium on t h e c o n f o r m a t i o n a l s t a t e o f t h e J. P h y s i o l . ( L o n d o n ) 312, 505-529. Na,K-pumps. K a r l i s h , S. J. D., and S t e i n , W. D. ( 1 9 8 1 ) . E f f e c t s o f ATP and phosphate on rubidium:rubidium exchange i n v e s i c l e s rec o n s t i t u t e d w i t h Na,K-ATPase. J. P h y s i o l . ( L o n d o n ) 3 1 9 , 55P. K a r l i s h , S. J . D. , and S t e i n , W. D. ( 1 9 8 2 ) . P a s s i v e rubidium f l u x e s mediated by N a , K - A T P a s e r e c o n s t i t u t e d i n t o p h o s p h o l i p i d v e s i c l e s when ATP- and p h o s p h a t e - f r e e J. P h y s i o l . 3 2 8 , 295-316. K a r l i s h , S. J. D . , and Yates, D. W. (1978). Tryptophan f l u o r e s c e n c e o f Na, K-ATPase as a t o o l f o r s t u d y of t h e enzyme mechanism. B i o c h i m . B i o p h y s . A c t a 5 2 7 , 115-130. K a r l i s h , S. J . D . , J d r g e n s e n , P. L . , and G i t l e r , C. (1977). Ident i f i c a t i o n o f a membrane-embedded segment of t h e l a r g e polyp e p t i d e c h a i n o f ( N a + , K+)ATPase. Nature ( L o n d o n ) 2 6 9 , 715-717. K a r l i s h , S . J . D . , Yates, D. W . , and Glynn, I. M. ( 1 9 7 8 ) . Conform a t i o n a l t r a n s i t i o n s between Na-bound and K-bound forms o f Na,K-ATPase, s t u d i e d w i t h formycin n u c l e o t i d e s . B i o c h i m . B i o p h y s . A c t a 525 , 252-264. Kepner, G. R . , and Macey, R. I. ( 1 9 6 8 ) . Membrane enzyme systems, m o l e c u l a r s i z e d e t e r m i n a t i o n s by r a d i a t i o n i n a c t i v a t i o n . B i o c h i r n . B i o p h y s . A c t a 1 6 3 , 188-203. Kyte, J. (1972). T i t r a t i o n o f t h e c a r d i a c g l y c o s i d e b i n d i n g s i t e of Na,K-ATPase. J. B i o l . Chern. 2 4 7 , 7634-7641. Kyte, J. ( 1 9 8 1 ) . Molecular c o n s i d e r a t i o n s r e l e v a n t t o t h e mechanism of a c t i v e t r a n s p o r t . N a t u r e ( L o n d o n ) 2 9 2 , 201204. Ltiuger, P. ( 1 9 8 0 ) . K i n e t i c p r o p e r t i e s o f i o n c a r r i e r s and chanJ. Membr. B i o l . 5 7 , 163-178. nels. Lew, V. L . , and Beaugd, L. ( 1 9 7 9 ) . P a s s i v e c a t i o n f l u x e s i n r e d c e l l membranes. In "Membrane T r a n s p o r t i n B i o l o g y , " ( D . D. G i e b i s c h , D. C. T o s t e s o n , and H. H . Ussing, e d s . ) , pp. 81-115. S p r i n g e r - V e r l a g , Berlin/New York. Maunsbach, A. B . , S k r i v e r , E . , and J d r g e n s e n , P. L. ( 1 9 7 9 ) . U l t r a s t r u c t u r e o f p u r i f i e d Na,K-ATPase membranes. I n "NaK-ATPase: S t r u c t u r e and K i n e t i c s " ( J . C . Skou and J. G. N$rby, e d s . ) , pp. 3-13. Academic P r e s s , New York. M i m m s , L. T . , Zampighi, G . , Nozaki, Y . , Tanford, C . , and Reynolds, J . A. ( 1 9 8 1 ) . P h o s p h o l i p i d v e s i c l e f o r m a t i o n and transmembrane p r o t e i n i n c o r p o r a t i o n u s i n g o c t y l g l u c o s i d e . B i o c h e m i s t r y 2 0 , 833-840.

J.

.

400

PETER L. JBRGENSEN

Moczydlowsky, E. (1979). The interaction of fluorescent ATP and ouabain derivations with the Na,K-ATPase, an approach to structure and mechanism. Dissertation, University of California, San Diego. Moczydlowski, E. G., and Fortes, P. A. G. (1981). Characterization of 2', 3' -0- (2,4,6-trinitrocyclohexadienylidine) adenosine 5I-triphosphate as a fluorescent probe of the ATP site of sodium and potassium transport adenosine triphosphatase. J. B i o l . Chem. 2 5 6 , 2346-2356. N4rby, J. G., and Jensen, J. (1971). Binding of ATP to Na,KATPase. B i o c h i m . B i o p h y s . A c t a 2 3 3 , 104-116. Perrone, J. R., Hackney, J. F., Dixon, J. F., and Hokin, L. E. (1975). Molecular properties of purified Na,K-ATPase and their subunits from the rectal gland of squalus acenthias and the electric organ of electrophorus electricus. J . B i o l . Chem. 2 5 0 , 4178-4184. Peters, W. H. M., Swarts, H. G. P., De Pont, J. J. H. H. M., Schuurmans Stekhoven, F. M. A. H., and Bonting, S. L. (1981). (Na+ + K+)ATPase has one functioning phosphorylation site per a-subunit. Nature ( L o n d o n ) 2 9 0 , 338-339. Post, R. L., Hegyvary, C., and Kume, S. (1972). Activation by adenosine triphosphate in the phosphorylation kinetics of sodium and potassium ion transport adenosine triphosphatase. J. B i o l . Chem. 2 4 7 , 6530-6540. Robinson, D., and Flashner, M. S. (1979). The Na,K-ATPase, enzymatic and transport properties. B i o c h i m . B i o p h y s . A c t a 5 4 9 , 145-176. Sabatini, D., Golman, D., Sabban, E., Sherman, J., Morimote, T., Kreibich, G., and Adesnik, M. (1982). Mechanisms for the incorporation of proteins into the plasma membrane. C o l d S p r i n g Harbor S y m p . Quant. B i o l . Vol. XIVI, 807-818. Schoot, B. M., Van Emst-De Vries, S. E., Van Haard, P. M. M., De Pont, J. J. H. H. M., and Bonting, S. L. (1980). Studies on NaK ATPase. Effect of cation-induced conformational changes on sulfhydryl group modification. B i o c h i m . B i o p h y s . A c t a 6 0 2 , 144-154. Simons, T. J. B. (1973). Potassium: Potassium exchange catalyses by the sodium pump in human red cells. J. P h y s i o l . ( L o n d o n ) 2 3 7 , 123-155. Skou, J. C. (1974). Effect of ATP on the intermediary steps of the reaction of the (Na+ + K+)-dependent enzyme system. I. Studied by the use of N-ethylmaleimide inhibition as a tool. B i o c h i m . B i o p h y s . A c t a 3 3 9 , 234-245. Skou, J. C . , and Esmann, M. (1979). Preparation of membrane-bound and of solubilized Na,K ATPase from rectal glands of Squalus Achanthius. The effect of preparative procedures on purity, specific and molar activity. B i o c h i m . B i o p h y s . A c t a 5 6 7 , 436-444.

(u-SUBUNITAND ION TRANSLOCATION

40 1

Skou, J. C., and Esmann, M. (19801. Effects of ATP and protons on the Na,K-selectivity of the Na,K-ATPase studied by ligand effects on intrinsic and extrinsic fluorescence. B i o c h i m . B i o p h y s . A c t a 6 0 1 , 386-402.

Skou, J. C., and Esmann, M. (1981). Eosin, a fluorescent probe of ATP binding to Na,K. ATPase. B i o c h i r n . B i o p h y s . A c t a 6 4 7 , 232-240.

Skriver, E., Maunsbach, A. B., and Jglrgensen, P. L. (1981). Formation of two-dimensional crystals in pure membrane-bound Na+,K+-ATPase. FEBS L e t t . 131, 219-222. Winslow, J. W. (1981). The reaction of sulfhydryl groups of sodium and potassium ion-activated adenosine triphosphatase with N-ethylmaleimide. J . B i o l . C h e m . 2 5 6 , 9522-9531.

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CURRENT TOPICS IN MEMBRANES AND TRANSPORT. VOLUME 19

Magnesium-InducedConformational Changes in Na,K-ATPase S. L. BONTING, H.G. P. SWARTS, W. H. M . PETERS, F. M. A. H. SCHUURMANSSTEKHOVEN, AND J. J. H. H. M. D E PONT Department of Biochemistry University of Nijmegen Nijmegen , The Netherlands

I.

INTRODUCTION

It is a well-known fact that Na,K-ATPase requires free Mg2+ in millimolar concentration for optimal overall ATPase activity. It is generally agreed that Mg2+ in micromolar concentrations is involved in phosphorylation and its reversal, the ADP-ATP exchange (Klodos an Skou, 1977; Swann and Albers, 1978). Tightly bound Ilg is known to be required for the K+-stimulated dephosphorylation of the phosphoenzyme (Fukushima and Post, 1978). Yet, the requirement of millimolar Mg2+ for optimal activity of the enzyme is not well understood. Originally, it was believed that millimolar Mg2+ shifts the equilibrium from an ADP-sensitive, K+-insensitive phospho intermediate, El plr P I to an FBP-insensitive, K+-sensitive phospho intermediate, E2-P (Post et a l . , 1969). However, it was shown that Mg2+, either at millimolar or micromolar Mg2' levels, does not affect the reactivity of the phospho intermediate to K+ or ADP (Klodos and Skou, 1975, 1977). This would suggest that

?I+

403

Copyright 0 1983 by Academic Press. Inc. All rights of reproduction in any form reserved. ISBN 0-12-l53319-0

S. L. BONTING eta/.

404

high Mg2+ exerts its effect on the overall reaction beyond the ElQP +E2-P transition. Evid nce for a conformational effect in the enzyme of Mg" and M g 2 + + ATP in millimolar concentrations has been presented (Jgkgensen and Petersen, 1979). It has also been shown that millimolar Mg2+ increases cooperative interactions between the Na+ sites in the overall ATPase reaction (Robinson, 1972), which could mean that high Mg2+ affects subunit interactions in the enzyme. We shall here present evidence for effects of millimolar M g 2 + on nucleotide binding and group-specific modifications, but first new findings about subunit structure and the stoichiometry of nucleotide binding and phosphorylation will be discussed in view of their relevance to our understanding of the Mg2+ effects.

11.

SUBUNIT CHARACTERIZATION

In all experiments here presented we have used a highly purified Na,K-ATPase preparation obtained from rabbit kidney outer medulla according to the method of JBrgensen (1974). The subunits have been separated by subjecting the enzyhe solubilized in 6% SDS to gel filtration on a Sephadex G - 2 0 0 column. A clear separation of the a- and 8-subunits is obtained (Fig. 1). The bulk of the phospholipids trail behind, but 2-5% of them are left in the subunit fractions and cannot be removed by repeated gel filtration of the protein. Quantitative two-dimensional thin layer chromatography of the residual phospholipids demonstrates that their composition in the a- and 8-subunit fractions is virtually the same as in the original enzyme preparation (Table I). This means that there is no specific binding of phospholipids to the subunits. The amino acid compositions of the a- and 8-subunits do not differ much from each other and from that of the intact enzyme, except for alanine, tyrosine, histidine, and lysine (Table 11). Both subunits are glycoproteins, but the total amount of sugar residues in the a-subunit is only 2 0 % of that in the 8-subunit (Table 111). From the weight percentages of protein, carbohydrate, and phospholipid after removal of SDS by dialysis or ion exchange chromatography their partial specific volumes have been calculated. Use of these data permitted the determination of the molecular weights of the detergentfree subunits by means of sedimentation equilibrium

405

I

I

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a,

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,

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I

1

0.3-

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I

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Q

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.

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i

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-2

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0

300

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F i g . 1 . P r o t e i n ( x ) and p h o s p h o l i p i d (0) p a t t e r n s o f the s o l u b i l i z e d Na ,K-ATPase a f t e r g e l f i l t r a t i o n on S e p h a d e x G-200 s u p e r f i n e ( f r o m Peters e t a l . , 1 9 8 1 a ) .

TABLE I. Phospholipid Composition of Isolated a- and @-Subunits and Intact Enzymea Phospholipid composition ( % of total)

Phospholipid Sphingomyelin Phosphatidylcholine Phosphatidylserine Phosphatidylinositol Phosphatidylethanolamine pg P/mg protein

b

a-Subunit

B-Subunit

Total enzyme

(n=3)

(n=3)

(n=15)

23 31 11 1 33

f 6 f 5 2 2 1 f 7

*

d.74

17 37 11 1 33

f 4

18 36 13 6 28

f 2

f 2.5

f 1 +_

4.5

f 0.6 f 0.7

2 0.9 0.3

*

f 1.0

33.1

1.66

a V a l u e s a r e g i v e n w i t h s t a n d a r d error of the mean; n i s the number o f p r e p a r a t i o n s a n a l y z e d ( f r o m Peters e t a 1 , 1 9 8 1 a ) . bData d e r i v e d f r o m D e P o n t e t a l . ( 1 9 7 8 ) .

.

S. L. BONTING et el.

406

TABLE 11.

Amino Acid Composition of a- and 8-Subunits and I n t a c t

Enzymea Amino a c i d composition (moles/100 moles amino a c i d s ) Amino a c i d Aspartic acid Threonine Serine Glutamic a c i d Pro1ine Glycine Alanine Cysteine Valine M et h i on i n e Isoleucine bucine Tyrosine phenylalanine Tryptophan Histidine Lysine Arginine

a-Subunit 10.5 6.0 7.2 10.4 4.6 8.0 8.2 0.9 6.7 2.3 6.5 9.3 2.3 4.0 1.6 1.9 5.6 4.7

&Subunit 9.9 4.1 6.6 11.8 5.8 9.9 4.3 0.7 5.7 2.1 5.0 7.1 4.8 5.3 2.4 0.5 9.9 4.7

I n t a c t enzyme 9.95 5.6 6.95 10.7 4.6 8.65 7.2 0.8 6.2 2.6 5.8 9.35 2.7 4.5

- b 1.6 6.7 4.8

a Averages for a n a l y s e s on two p r e p a r a t i o n s a f t e r 4 - , 6-, 24-, 48- and 72-hr h y d r o l y s i s . The v a l u e s f o r s e r i n e a r e e x t r a p o l a t e d t o z e r o t i m e o f h y d r o l y s i s , t h o s e for v a l i n e and i s o l e u c i n e t o i n f i n i t e t i m e o f h y d r o l y s i s . Average s t a n d a r d e r r o r i n t h e v a l u e s i s 0.13 mole/100 mole amino a c i d s (from P e t e r s et a l . , 1981a). bNot determined.

(Table IV). Protein molecular weights of 120,600 for the a-subunit and of 4 2 , 8 0 0 for the 8-subunit are thus obtained.

111.

SUBUNIT STOICHIOMETRY

Widely varying molar subunit ratios have been proposed for the Na,K-ATPase complex: ~ 2 8 1 ,a 2 8 2 r a ~ f . 3 3 ~ a 2 8 4 and a8PX (for references, see Peters e t a l . , 1981a). The wide variation in ct/8 molar ratios is due to technical shortcomings: the unreliability of molecular weight determinations of glycoproteins by SDS-poly-

MAGNESIUM-INDUCEDCONFORMATIONAL CHANGES

TABLE 111.

407

Carbohydrate Composition of a- and 8-Subunits

a

Carbohydrate composition (moles/100 mole amino a c i d s ) Carbohydrate

a- Subuni t

B-Subunit

Mannos e Galactose Glucose Glucosamine Galactosamine Sialic acid

0.3 0.9 0.9 2.0

f 0.1 f 0.1 f 0.2 2 0.2 - b 0.35 f 0.05

2.5 2 0 . 1 5.5 2 0.4 2.0 2 0.5 1 0 . 1 f 0.5 0 . 3 f 0.1 3.2 f 0.2

Total

4.4

23.6 f 0.8

2 0.3

a Averages for mu1 tiple determinations on four preparations a r e given with standard error of the mean (from Peters et al., 1981a). bNot detectable.

TABLE I V .

Molecular Weights of Detergent-Free ~~

a-Subuni t Total P r o t e i n + CHO P r o te in only

f 4.5 133 131 f 4.5 120.6 f 4.6

(n=7)

______

a- and ,&Subunits a ~

~~

$-Subunit 63.5 f 1 . 5 61.8 2 1 42.8 f 1

(n=5)

a

Determined by sedimentation equilibrium method (kilograms) (from Peters e t a l . , 1981a).

acrylamide gel electrophoresis and gel filtration, and the error of protein weight ratio determination by Coomassie brilliant blue staining due to the fact that the a- and 6-subunits show different degrees of staining. We have, therefore, applied three independent methods for determining the a / B molar ratio after separation of the a- and B-subunits: (1) determination of the a/B protein weight ratio through amino acid analysis (Table V), (2) determination of the a / $ 280-nm light absorbance ratio (Table VI) , ( 3 ) for those amino acids which showed the largest difference in the a- and

S. L. BONTING eta/.

408

TABLE V.

P r o t e i n Weight Ratio of a- and B-Subunita

Subunit r a t i o assumed

Weight r a t i o calculated

Weight r a t i o determined

1.41 ? 0.06b 1.88 f 0.08b 2.82 f 0.12 4.23 f O.lgb 5.64 ? 0.25b

"161 a1.5B1

a24

3.04 2 0.06

a Weight ratios are calculated from the subunit molecular weights shown in Table IV. The weight ratio is determined by amino acid analysis of the separated a- and @-subunits with standard error of the mean given for five determinations in five different preparations (from Peters et al., 1981a). bSignificantly different from the determined weight ratio (P 0.001).

TABLE V I .

a

Absorbance Ratio of a- and @-Subunits

280-run absorbance r a t i o

Subunit r a t i o Assumed

Calculated

Determined

0.92 f 0.07b 1.23 f O.Ogb

"1 B2 O181.5 "181

1.85 ?: 0.14b

"1.581

2.02 f 0.06

(n=8)

2.77 f 0.21b 3.69 ? 0.27b

a2 81

a Absorbance ratios are calculated from the molar absorption coefficients, determined from the absorbance at 280 nm of the separated subunit fractions through division by the molar concentration. The subunit molar concentration is determined via amino acid analysis and division of the protein weight per 1 by the subunit protein molecular weight, given in Table IV. The = 143,000 6000 liter-mole-l-cm-l(n=4), the €6 = 78,000 f 5000 liter.mole'l-cm-l (n=4). The 280-nm absorbance ratio is determined on the separately pooled a- and B-subunit fractions. Averages with standard error of the mean for n different preparations are given (from Peters et a1 1981a). bsignificantly different from the determined absorbance ratio (P 0.01).

*

MAGNESIUM-INDUCEDCONFORMATIONAL CHANGES

409

TABLE VII. Comparison of C a l c u l a t e d and Determined Contents of Four Amino Acids i n t h e N a t i v e Enzymea

Enzyme

Alanine (7.2) Calculated

a “182 a181.5 a181 “1.581

6.6 6.85 7.2 7.45 7.6

0,281

0.6 0.35 0.0 0.25 0.4

Tyrosine (2.7) Calculated A

Histidine (1.6) Calculated A

Lysine (6.7) Calculated A

3.3 3.15 2.95 2.75 2.6

1.35 1.4 1.55 1.65 1.7

7.35 7.05 6.7 6.4 6.2

0.6 0.45 0.25 0.05 0.1

0.25 0.2 0.05 0.05 0.1

0.65 0.35 0.0 0.3 0.5

Aa”

0.53k0.08b 0.34-+0.05b 0.08f.O.06 0.16+0.065 0.28f0.10b

a

Based on a v e r a g e amino a c i d molecular weights, c a l c u l a t e d from t h e amino a c i d composition (Table 11) and t h e p r o t e i n molecul a r weights (Table I V ) , t h e a - s u b u n i t would c o n t a i n 1090 and t h e @-subunit 374 amino a c i d s . Using t h e amino a c i d compositions, given i n Table 11, t h e mole % f o r i n t a c t enzyme can be c a l c u l a t e d A i s the absolute difference a t v a r i o u s assumed a/B molar r a t i o s . between t h e c a l c u l a t e d and determined (value i n p a r e n t h e s e s ) amino a c i d c o n t e n t i n moles/100 moles amino a c i d s (from P e t e r s e t a l . , 1981a). 0.05). b S i g n i f i c a n t l y d i f f e r e n t from zero (P

8-subunits, the measured amino composition in the intact enzyme was compared with that of an enzyme with various theoretical subunit compositions (Table VII). In all cases an a/B molar ratio of 1 is the only one which fits the data. This ratio yields a protein molecular weight for the a $ complex of 163,400. By radiation inactivation of the intact Na,K-ATPase complex, we find a molecular weight of 332,000 k 1,500. Hence, the total Na,K-ATPase complex must consist of an “282 tetramer with a calcuThis figure lated protein molecular weight of 3 2 7 , 0 0 0 . falls in the range of molecular weights (280,000380,000) reported by other investigators for the shark rectal gland enzyme, analyzed in detergent-solubilized form by sedimentation equilibrium centrifugation (Esmann e t a l . , 1979; Hastings and Reynolds, 1979) and for the erythrocyte enzyme analyzed by means of radiation inactivation (Ellory e t a l . , 1979).

S. L. BONTING et 81.

410

IV.

STOICHIOMETRY OF NUCLEOTIDE BINDING AND PHOSPHORYLATION

The stoichiometry of nucleotide binding and phosphorylation is of fundamental importance for our understanding of the enzyme mechanism. The commonly found ATP binding and phosphorylation capacities of 3.6-4.3 nmoles/mg protein yield a stoichiometry of 0.9-1.08 moles/mole enzyme on the basis of the previously accepted molecular weight of 250,000. Since the a-subunit binds ATP and is phosphorylated by it, and there are two a-subunits in the total enzyme complex, a stoichiometry of 1 mole/mole enzyme suggests a "half-of-the-sites" mechanism, as originally proposed by Repke and Schon (1973) and Stein e t a l . (1973). However, the phosphorylation and binding capacities are based on the Lowry protein determination with bovine serum albumin as a reference. For Na,K-ATPase from rabbit kidney outer medulla this leads to protein values 35% higher than those obtained by means of amino acid analysis (Peters e t a l . , 1981b). Moreover, since the protein molecular weight for the a262 tetramer is 327,000 rather than 250,000, the ATP binding and phosphorylation capacities based on Lowry protein determinations should be multiplied by a factor 0,327 x 1.35 = 0.44 instead of 0.25 for conversion to a molar basis. This factor has been applied in the following studies on nucleotide binding and phosphorylation. Nucleotide binding has been studied with [3H]ATP and its nonphosphorylating analogue AMP-PNP. A filtration procedure (Yamaguchi and Tonomura, 1979) has been used which permits the use of high nucleotide levels (up to 0.3 mM) Binding of ATP in the absence of Mg2+ gives a homogeneous Scatchard plot (Fig. 2) with a KD value of 0.13 W M . A similar lot is obtained for AMP-PNP (Fig. 3) in the absence of Mgs+; here the KD value is 1.9 P M . These KD values agree with those previously reported by Hegyvary and Post (1971) and N#rby and Jensen (1971) €or ATP binding (0.1-0.2 L I M ) and Robinson (1980) for AMP-PNP binding (4.2 p ~ ) . We find a binding capacity for ATP of 3.9 nmoles/mg protein and for AMP-PNP of 4.0 nmoles/mg protein, which is equivalent to 1.7-1.8 moles/mole Na,K-ATPase complex. Maximal ATP phosphorylation levels have been determined for the same type of Na,K-ATPase preparations, from which ATP, added for protection during purification had not been removed by washing. This prevents a partial l o s s (21%) of phosphorylation capacity (Peters e t a l . , 1981b), although the binding capacity does not show

.

MAGNESIUM-INDUCEDCONFORMATIONAL CHANGES

41 1

( Na+-K+)-ATPase

ATP-bi nding

a 2o

d 5

I

b:O.l3pM

0

I

G

5 E

c- o

2

I

I

0

05

3 4 5 nmol ATP, mg-' p r o t e l n I

10 1.5 20 mol ATP. mol-' enzyme

Fig. 2. B i n d i n g of ATP t o Na,K-ATPase i n the absence of Mg2+.EDTA ( 2 mM) i s p r e s e n t i n the i n c u b a t i o n m e d i u m (22'C) cont a i n i n g 0.25 mg p r o t e i n - m l - l a t 0.4-2 ~ . I M ATP or 1 mg p r o t e i n - m l - I a t 10-300 pM ATP, a n d 40 mM i r n i d a z o l e - H C l (pH 7 . 0 ) . T h e KD v a l u e d e r i v e d from the s l o p e i s 0.13 V M , a n d the b i n d i n g c a p a c i t y d e r i v e d from the i n t e r c e p t on the a b s c i s s a i s 1.7 m o l e s / m o l e enz y m e (from S c h u u r m a n s S t e k h o v e n e t a l . , 1981).

much of this effect. The problem of the residual nonradioactive ATP has been circumvented by addition of excess (1-2.5 mM) [y-32P]ATP. A phosphorylation capacity of 4.2 nmoles/mg protein is thus obtained, equivalent to 1.85 moles per mole of the a262 tetramer. S o we conclude that the enzyme contains two highaffinity sites €or nucleotide binding as well as for phosphorylation, implying that the "half-of-the-sites" theory is no longer tenable. This does not exclude that under physiological conditions the two a6-units may influence each other, possibly leading to their operation out of phase.

412

S. L. BONTING eta/.

( Na' -K' 1- ATPase A M PPN P - binding

a Z a n2

1.00

6

EDTA

7

2 30.75

.-C

P)

c

g 0.50

E"

d

0.25

a 2

6 #

!

0

4 5 6 7 0 nmol AMPPNP.mg-' protein

3

2

1

C

I

1

I

0

0.5

10

1

I

2.5

3.0 35 mol AMPPMP. mol-' enzyme

1.5

2.0

F i g . 3 . B i n d i n g of AMP-PNP t o Na,K-ATPase i n the absence of Mg2+-EDTA (2 RIM) i s p r e s e n t i n the incubation medium (22OC) cont a i n i n g 1 mg p r o t e i n - m l - l , 1-300 l~iV AMP-PNP, and 40 mM i m i d a z o l e HCl (pH 7 . 0 ) . T h e KD v a l u e i s 1.9 ?JMand the b i n d i n g c a p a c i t y 1.8 moles/mole e n z y m e (from Schuurmans S t e k h o v e n e t a l . , 1 9 8 1 ) .

V.

Mg2+ EFFECTS ON NUCLEOTIDE BINDING

We then proceeded to study the effects of millimolar Mg2+ on nucleotide binding. Since in the presence of Mg2+, ATP elicits phosphorylation, we have used the nonphosphorylating ATP analog AMP-PNP to study binding per se. In the presence of 5 mM Mg2+ the Scatchard plot for AMP-PNP binding becomes nonlinear and can be resolved in two linear plots, representing the simultaneous occurrence of two sets of different binding sites, one with a low KD and one with a high KD (Fig. 4). By means of an iterative mutual correction pro-

413

MAGNESIUM-INDUCEDCONFORMATIONALCHANGES

a 2 -9 2

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;,3 0 7 5 -

"\ I

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1

1

1

-

-

5mM Mg2+

-

c

0

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_ _ _ _ _ _, -

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1'0

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'

,

0

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1'5 2'0 2'5 3'0 3'5 mol AMPPNP. mol" enzyme

F i g . 4 . B i n d i n g o f AMP-PNP t o Na,K-ATPase i n the p r e s e n c e of 5 mM Mg2+. E x p e r i m e n t a l c o n d i t i o n s a s i n F i g . 3 , e x c e p t t h a t 5 mM MgC12 r e p l a c e s the EDTA. T h e c u r v e d p l o t h a s been r e s o l v e d i n t o t w o l i n e a r p l o t s ( d a s h e d l i n e s ) b y t h r e e f o l d m u t u a l correct i o n . T h e h i g h - a f f i n i t y p l o t y i e l d s a KD v a l u e o f 3 . 4 pM and a b i n d i n g c a p a c i t y o f 1.81 m o l e s / m o l e e n z y m e , the l o w - a f f i n i t y p l o t a KD o f 0.15 mM and a b i n d i n g c a p a c i t y o f 1.85 moles/mole e n z y m e ( f r o m Schuurmans S t e k h o v e n e t a l . , 1 9 8 1 ) .

cedure K~ values of 3 . 4 and 150 ?JM and binding capacities of 1.81 and 1.85 mole/mole enzyme are derived for the high-affinity and low-affinity nucleotide binding sites, respectively. This means that in the presence of millimolar Mg2+ the enzyme acquires two low-affinity nucleotide binding sites in addition to the two highaffinity sites already operating in the absence of Mg2+. We have also determined the affinity of the sites for Mg2+ in the induction of the extra nucleotide binding sites. A linear double reciprocal plot of M 2+induced AMP-PNP binding capacity versus total Mgq+ concentration (Fig. 5) is obtained. The intercept with the ordinate gives a maximal induced extra nucleotide binding capacity (E-Amax) of 1.7 moles/mole enzyme, in

S. L. BONTING eta/,

414 ( Na+-K+) -ATPase A M P P N P -binding

-1

0

F i g . 5. E f f e c t o f Mg2+ on the l o w - a f f i n i t y AMP-PNP b i n d i n g c a p a c i t y . A series o f b i n d i n g p l o t s l i k e the one shown i n F i g . 4 h a s been d e t e r m i n e d a t d i f f e r e n t Mg2+ c o n c e n t r a t i o n s . T h e s e h a v e been a n a l y z e d f o r h i g h - a f f i n i t y and l o w - a f f i n i t y b i n d i n g a s exp l a i n e d i n the l e g e n d o f F i g . 4 . The d a t a a r e p r e s e n t e d i n a d o u b l e r e c i p r o c a l p l o t . T h e maximal b i n d i n g c a p a c i t y o f the lowa f f i n i t y s i t e s (E-Amax), d e r i v e d f r o m the i n t e r c e p t on the o r d i n a t e , is 1.7 m o l e s / m o l e e n z y m e . T h e KD v a l u e f o r Mg2+, d e r i v e d from the i n t e r c e p t on the a b s c i s s a i s 0.8 RIM. T h e a b s c i s s a shows the r e c i p r o c a l t o t a l My c o n c e n t r a t i o n ( f r e e p l u s AMP-PNP comp l e x e d ) ( f r o m Schuurmans Stekhoven e t a 1 , 1 9 8 1 ) .

.

good agreement with the earlier value. The intercept with the abscissa gives a KD value for Mg2+ of 0.8 mM, which agrees with the Km values for the overall Na,K-ATPase activity (0.8 mM) and for the p-nitrophenylphosphatase reaction (0.9 mM) and with the KD value of 0.8 mM obtained from Be2+ inactivation in the absence of substrate (Robinson, 1974). The binding capacity at the high-affinity substrate sites is independent of the Mg2+ concentration and remains at a constant level of 1.8 moles/mole enzyme for this preparation. In addition to increasing the nucleotide binding capacity, Mg2+ also decreases the affinity for nucleotides at both types of sites, maximally at 2 mM Mg2+ (Fig. 6). For the preparation used in Fig. 5 the KD for the high-affinity sites increases from 1.5 to 4 . 2 V M (an

MAGNESIUM-INDUCEDCONFORMATIONAL CHANGES

415

( Nat-Kt) - A T P a s e A M P P N P - binding

2+ Fig. 6 . Effect of Mg on the KD value of high-affinity and low-affinity AMP-PNP binding to Na,K-ATPase. The series of binding plots obtained in the experiment of Fig. 5 has been analyzed for high-affinity and low-affinity binding as explained in the legend of Fig. 4 . On the abscissa the total Mg concentration (free p l u s AMP-PNP complexed is shown, on the left ordinate the KD for low-affinity AMP-PNP binding, and on the right ordinate the KD for high-affinity AMP-PNP binding. (From Schuurmans Stekhoven et al., 1981.)

effect also observed by Robinson and Flashner, 1979 and the KD for the low-affinity sites from 60 to 260 !AM. This effect on the affinity may be exerted via "affinity regulating" Mg2+ sites, which are different from the "capacity regulating" Mg2+ sites saturated only above 5 mM Mg2+ (Figs. 4 and 5). The high-affinity and low-affinity sites further distinguish themselves from each other in their sensitivity to ouabain. After ouabain binding in the presence of 5 mM Mg2+ and 5-25 P M ouabain ( K D = 1.1 P M ) , the capacity for high-affinity nucleotide binding is reduced by 50-78% without effect on the KD for AMP-PNP, whereas the capacity for low-affinity nucleotide binding remains unaffected, except for a rise in KD for the nucleotide.

416

S. L. BONTING eta/.

100 P,

90-

0 .-

'0

80-

0

c

7

0"

.6

c

70-

60-

9 L .C 50-

-

E" 'g

LO-

€ c C

E

a

302010 -

O

L' + &

+&

+;

+i

+cDTA 1 +; I -d, -log [ATP]

++

+6I

+51

+LI

+3 1 -log [ADP]

Fig. 7. Protective effect of ATP (A) and ADP ( B ) against inactivation by butanedione. Na,K-ATPase (30 pg protein-ml-l)is incubated during 30 min at 25OC in 50 mM sodium borate buffer (pH 7.5) containing either 5 mM Mg2+ or 5 mM CDTA, 4 mM butanedione and ATP or ADP in the indicated concentrations expressed in moles/liter-Na.K-ATPase is determined on 20-pl aliquots, following 16-fold dilution in the assay medium at pH 7.5 and 37OC. Results are expressed as percent of inhibition obtained in the absence of added nucleotide. Results represent means and standard errors of three experiments. (From De Pont et al., 1977.)

VI

.

Mg2+ EFFECTS ON GROUP-SPECIFIC MODIFICATIONS

The effects of millimolar Mg2+ on nucleotide binding clearly indicate the occurrence of Mg2+-induced conformational changes. This conclusion is further supported by effects of Mg2+ on group-specif ic modif ications of the enzyme by butanedione, N-ethylmaleimide (NEM), 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), and trinitrobenzene sulfonate (TNBS). Butanedione modifies

MAGNESIUM-INDUCED CONFORMATIONAL CHANGES

~

41 7

NEM inhibition vs. time of preincubation 100

..I c 80 0 m m

60

a

k

a

40

Y I

m

z m .-

time of preincubation (min) F i g . 8 . Inhibition o f Na,K-ATPase b y N - e t h y l m a l e i m i d e . T h e reaction m i x t u r e f o r p r e i n c u b a t i o n w i t h NEM c o n t a i n e d 7.5 p g protein-ml-', 2 mM CDTA (0) or 5 mM MgCl2 (0), 0.7 mM NEM, 2 5 mM i m i d a z o l e - H C 1 (pH 7 . 5 ) . A f t e r v a r y i n g t i m e s of p r e i n c u b a t i o n a t 37OC a l i q u o t s a r e a s s a y e d f o r Na,K-ATPase a c t i v i t y at pH 7 . 4 and 37'C after a d d i t i o n o f a f i v e f o l d m o l a r excess d i t h i o e r y t h r i t o l . (From S c h o o t e t a l . , 1 9 7 7 . )

the guanidino group of arginine, cysteinyl-sulfhydryl groups of NEM and DTNB, and TNBS amino groups. In the absence of Mg2+, protection by ATP in the high-affinity range of 6-35 U M (De Pont e t al., 1977; Schoot e t a l . , 1977, 1978) is observed, indicating that these groups are located in or near the high-affinity ATP binding center. Mg*+ enhances inactivation by butanedione by decreasing the affinity for protecting nucleotides as shown for ATP and ADP in Fig. 7. It may do so via the "affinity regulating" sites demonstrated in Fig. 6. The accelerating effect on inactivation by NEM and DTNB is shown in Figs. 8 and 9. Reactive SHgroups are not only located in the high-affinity nucleotide binding center, but also in the low-affinity binding center, which is protected by ATP in 30-fold higher concentrations (Patzelt-Wenczler and Schoner, 1981). The same appears to be true for the amino groups involved

418

S. L. BONTING etel.

2+ Fig. 9. Effect of Mg and CDTA on the inhibition of Na,K-ATPase and the number of sulfhydryl groups modified by 5,5'dithiobis(2-nitrobenzoic acid). The reaction mixture consists of 25 mM imidazole-HC1 (pH 7.5), 2 mM CDTA ( 0 ) or 2 mM CDTA + 10 mM MgC12 (e) , 0.2 mg proteinvml-I and DTNB (indicated here as Nbsz) in the concentration range given at the abscissa. After incubation at 37'C for 1 hr, a 5O-pl aliquot is diluted threefold in imidazole buffer and assayed for Na,K-ATPase activity at 37OC and pH 7 . 4 . The remaining incubation medium is centrifuged for 15 min at 16,000 x g, and the number of SH groups is determined from the absorbance at 412 nm. Curves represent averages from two experiments. (From Schoot et a l . , 1980).

MAGNESIUM-INDUCEDCONFORMATIONAL CHANGES

( N& + K+)

0

20

419

- A T P ~ S(TN ~ Bs

40

60 Time( min)

F i g . 1 0 . E f f e c t of Mg2+ on t i m e d e p e n d e n c e of Na,K-ATPase i n a c t i v a t i o n b y TNBS. T h e e n z y m e ( 0 . 4 mq p r o t e i n - m l - l ) i s i n c u b a t e d a t 20°C w i t h 100 pM TNBS i n 0 . 2 M t r i e t h a n o l a r n i n e (pH 8 . 5 ) w i t h (m) or w i t h o u t (0) 3 mM Mg2+. The r e a c t i o n i s s t o p p e d b y g e l f i l t r a t i o n , a n d e l u t i n g p r o t e i n a s s a y e d f o r Na,K-ATPase a c t i v i t y a t 37OC a n d pH 7 . 4 . R e p r e s e n t a t i v e f o r 3 e x p e r i m e n t s . ( J . J . H . H . M . D e P o n t , S . E . V a n Emst-De V r i e s , a n d S . L . B o n t i n g , hitherto u n p u b l i s h e d r e s u l t s . )

420

S. L. BONTING eta/.

in enzyme activity and modified by TNBS in the presence or absence of Mg2+ (Fig. 10). It is striking that half-maximal protection against TNBS inactivation is afforded by ATP at 6 M in the absence of Mg2+, indicating binding to the high-affinity sites, but only at 1.2 mM in the presence of 5 mM Mg2+, indicating binding to the low-affinity sites. The halfmaximal Mg2+ effect is exerted at 1 mM Mg2+ (J. J. H. H. M. De Pont, S. E. Van Emst-De Vries and S. L. Bonting, unpublished observations), which suggests that binding to the "capacity regulating" sites (KD for Mg2+, 0.8 mM; Fig. 5) is involved.

VI I. COMCLUS IONS The following conclusions can be drawn from the evidence presented: 1. The Na,K-ATPase complex ( a 2 8 2 tetramer, protein MW 327,000) from rabbit kidney outer medulla contains two ouabain-sensitive, high-affinity nucleotide binding sites, at which phosphorylation by ATP takes place. 2. Mg2+, when binding to "capacity regulating" sites (KD = 0.8 mM), induces a conformational change, which gives rise to two additional binding sites. These are ouabain-insensitive, low-affinity, nonphosphorylating sites. 3. Mg2+ lowers the affinity of both high-affinity and low-affinity substrate binding, apparently via "affinity regulating" Mg2+ sites. Mg2+ , when binding to "affinity regulating" 4. and/or "capacity regulating" sites, has accelerating effects on group-specific modification of arginine, sulfhydryl, and amino groups, located in or near the high-affinity as well as the low-affinity nucleotide binding centers. 5. These findings indicate that Mg2+ exerts profound conformational changes in the enzyme. The primary step in the reaction mechanism at which millimolar Mg2+ acts, particularly in inducing the low-affinity nonphosphorylating nucleotide binding sites, has not yet been resolved. Grosse et al. (1979) place the additional lowaffinity binding of ATP at the start of the reaction cycle, where it would enhance K+-dependent dephosphorylation and transformation of the liberated site into a high-affinity ATP phosphorylating site. The companion

MAGNESIUM-INDUCED CONFORMATIONAL CHANGES

421

site would simultaneously decrease its affinity and thus release its product ADP in an anticooperative "flip-flop" mechanism (Grosse et a l . , 1978). Kinetic studies have indeed suggested stimulation by high ATP of the hydrolysis of a presumed K+-dependent, acid-labile phospho intermediate (Froehlich et a l . , 1976). In our opinion, high Mg2+ might act at the E2K -+ E1K transition, which follows K+-stimulated hydrolysis of E2-P. This assumption is based on the following argument. High ATP would drive the E2K E1K transition in nonphosphorylating fashion (Post et a l . , 19721, whereas p-nitrophenyl phosphate hydrolysis, competing with ATP hydrolysis at low-affinity sites (Robinson, 19801, would occur by reversal of the E2K E1K transition (Blostein et a l . , 1979). It has a Km for Mg2+ of 0.9 mM (Robinson, 1974), which matches the KD (0.8 mM) at the "capacity regulating" sites. Obviously, this point requires further investigation. -+

-+

mEERENCES Blostein, R., Pershadsingh, H. A., Drapeau, P., and Chu, L. ( 1 9 7 9 ) . Side-specificity of alkali cation interactions with Na,KATPase. Studies with inside-out red cell membrane vesicles. In "Na,K-ATPase: Structure and Kinetics" (J. C. Skou and Academic Press, New York. J. C. Ndrby, eds.), pp. 233-245. De Pont, J. J. H. H. M., Schoot, B. M., Van Prooyen-Van Eeden, A., and Bonting, S. L. ( 1 9 7 7 ) . An essential arginine residue in the ATP-binding centre of (Na++K+)-ATPase. Biochim. Biophys. Acta 4 8 2 , 213-227. De Pont, J. J. H. H. M., Van Prooyen-Van Eeden, A., and Bonting, S. L. ( 1 9 7 8 ) . Studies on (Na++K+)-activated ATPase. XXXIX. Role of negatively charged phospholipids in highly purified (Na++K+)-ATPase from rabbit kidney outer medulla. Biochim. Biophys. Acta 508, 464-477. E l l o r y , J. C., Green, J. R., Jarvis, S. M., and Young, J. D. (1979). Measurement of the apparent molecular volume of membrane-bound transport systems by radiation inactivation. J. Physiol. (London) 295, 1OP-11P. Esmann, M., Skou, J. C., and Christiansen, C. ( 1 9 7 9 ) . Solubilization and molecular weight determination of the (Na++K+)ATPase from rectal glands of S q u a l u s acanthias. Biochim. Biophys. Acta 567 , 410-420. Froehlich, J. P., Albers, R. W., Koval, G. J., Goebel, R., and Berman, M. ( 1 9 7 6 ) . Evidence for a new intermediate state in the mechanism of (Na++K+)-adenosine triphosphatase.

422

s. L. BONTING eta/.

J . B i o l . Chem. 251, 2186-2188. Fukushima, Y., and Post, R. L. (1978). Binding of divalent cation to phosphoenzyme of sodium- and potassium-transport adenosine triphosphatase. J. B i o l . Chem. 2 5 3 , 6853-6862. Grosse, R., Eckert, K., Malur, J., and Repke, K. R. H. (1978). Analysis of function-related interactions of ATP, sodium and potassium ions with Na+- and K+-transporting ATPase studied with a thiol reagent as tool. A c t a B i o l . Med. G e r . 3 7 , 83-96. Grosse, R., Rapoport, T., Malur, J., Fischer, J., and Repke, K. R. H. (1979). Mathematical modelling of ATP, K+, and Na+ interactions with (Na++K+)-ATPase occurring under equilibrium conditions. B i o c h i m . B i o p h y s . A c t a 550, 500-514. Hastings, D. F., and Reynolds, A. (1979). Molecular weight of (Na+,K+)-ATPase from shark rectal gland. B i o c h e m i s t r y 1 8 , 817-821. Hegyvary, C., and Post, R. L. (1971). Binding of adenosine triphosphate to sodium and potassium ion-stimulated adenosine triphosphatase. J. B i o l . Chem. 246, 5234-5240. Jphgensen, P. L. (1974). Purification and characterization of (Na++K+)-ATPase. 111. Purification from the outer medulla of mammalian kidney after selective removal of membrane components by sodium dodecylsulphate. B i o c h i m . B i o p h y s . A c t a 3 5 6 , 36-52. Jgkgensen, P. L. , and Petersen, J. (1979). Protein conformations of the phosphorylated intermediates of purified Na,K-ATPase studied with tryptic digestion and intrinsic fluorescence as tools. In "Na,K-ATPase: Structure and Kinetics" (J. C. Skou and J. G. N$rby, eds.), pp. 143-155. Academic Press, New York. Klodos, I. , and Skou, J. C. (1975). The effects of Mg2+ and chelating agents on intermediary steps of the reaction of Na+,K+-activated ATPase. B i o c h i m . B i o p h y s . A c t a 3 9 1 , 474-485. Klodos, I . , and Skou, J. C. (1977). The effect of chelators on Mg2+,Na+-dependent phosphorylation of (Na++K+)-activated ATPase. B i o c h i m . B i o p h y s . A c t a 481 , 667-679. NZrby, J. G., and Jensen, J. (1971). Binding of ATP to brain microsomal ATPase. Determination of the ATP-binding capacity and the dissociation constant of the enzyme-ATP complex as a function of K+ concentration. B i o c h i m . B i o p h y s . A c t a 2 3 3 , 104-116. Patzelt-Wenczler, R., and Schoner, W. (1981). Evidence for two different reactive sulfhydryl groups in the ATP-binding sites of (Na++K+)-ATPase. Eur. J. B i o c h e m . 114, 79-87. Peters, W. H. M., De Pont, J. J. H. H. M., Koppers, A., and Bonting, S. L. (1981a). Studies on (Na++Kf)-activated ATPase. XLVII. Chemical composition, molecular weight and molar ratio of the subunits of the enzyme from rabbit kidney outer medulla. B i o c h i m . B i o p h y s . A c t a 641, 55-70.

MAGNESIUM-INDUCEDCONFORMATIONALCHANGES

423

P e t e r s , W. H. M . , SwartS, H. G. P . , D e P o n t , J. J. H. H. M . , Schuurmans Stekhoven,-h. M. A. H . , and B o n t i n g , S. L. (1981b) (Na++K+) -ATPase h a s one f u n c t i o n i n g phosphorylat i o n s i t e per a - s u b u n i t . N a t u r e ( L o n d o n ) 2 9 0 , 338-339. Post, R. L., K u m e , S . , Tobin, T . , O r c u t t , B . , and Sen, A. K. ( 1 9 6 9 ) . F l e x i b i l i t y o f a n a c t i v e c e n t e r i n sodium-plusp o t a s s i u m a d e n o s i n e t r i p h o s p h a t a s e . J. G e n . P h y s i o l . 5 4 , 306s-326s. P o s t , R. L . , Hegyvary, C., and Kume, S. ( 1 9 7 2 ) . A c t i v a t i o n by a d e n o s i n e t r i p h o s p h a t e i n t h e p h o s p h o r y l a t i o n k i n e t i c s of sodium and p o t a s s i u m i o n t r a n s p o r t a d e n o s i n e t r i p h o s p h a t a s e . J. B i o l . C h e m . 247, 6530-6540. Repke, K. R. H . , and Schb;n, R. (1973). F l i p - f l o p model of ( N a , K ) - A T P a s e f u n c t i o n . Acta B i o l . M e d . G e r . 31, K19-K30. Robinson, J. D. ( 1 9 7 2 ) . D i v a l e n t c a t i o n s as a l l o s t e r i c m o d i f i e r s B i o c h i m . B i o p h y s . Acta o f t h e (Na++K+) -dependent ATPase. 2 6 6 , 97-102. Robinson, J. D. (1974). N u c l e o t i d e and d i v a l e n t c a t i o n i n t e r a c t i o n s w i t h the (Na++K+)-dependent ATPase. Biochim. Biophys. A c t a 3 4 1 , 232-247. Robinson, J. D. ( 1 9 7 6 ) . S u b s t r a t e s i t e s of t h e (Na++K+)-dependent B i o c h i m . B i o p h y s . A c t a 4 2 9 , 1006-1019. ATPase. Robinson, J. D. ( 1 9 8 0 ) . Binding t o t h e h i g h - a f f i n i t y substrate s i t e of the (Na++K+)-dependent ATPase. J . B i o e n e r g . B i o m e m b r . 1 2 , 165-174. Robinson, J . D . , and F l a s h n e r , M. S. ( 1 9 7 9 ) . C a t i o n and nucleot i d e i n t e r a c t i o n s w i t h the Na,K-ATPase. I n "Na,K-ATPase: S t r u c t u r e and K i n e t i c s " (J. C. Skou and J. G. Ndrby, e d s . ) , pp. 275-285. Academic Press, New York. Schoot, B. M . , S c h o o t s , A. F. M., D e P o n t , J. J . H. H. M . , Schuurmans Stekhoven, F. M. A. H . , and B o n t i n g , S. L. ( 1 9 7 7 ) . XLI E f f e c t s of S t u d i e s on (Na++K+) - a c t i v a t e d ATPase. N-ethylmaleimide on o v e r a l l and p a r t i a l r e a c t i o n s . Biochim. B i o p h y s . A c t a 483, 181-192. Schoot, B. M . , D e P o n t , J. J. H. H. M . , and Bonting, S. L. ( 1 9 7 8 ) . S t u d i e s on (Na++K+)-activated ATPase. X L I I . Evidence f o r Biochim. Biotwo classes o f e s s e n t i a l s u l f h y d r y l groups. p h y s . A c t a 522, 602-613. Schoot, B. M . , Van Emst-De V r i e s , S . E . , Van Haard, P. M. M . , D e Pont, J. J. H. H. M . , and Bonting, S. L. ( 1 9 8 0 ) . S t u d i e s on (Na++K+)- a c t i v a t e d ATPase. XLVI. E f f e c t o f cation-induced c o n f o r m a t i o n a l changes on s u l f h y d r y l group m o d i f i c a t i o n . B i o c h i m . B i o p h y s . A c t a 6 0 2 , 144-154. Schuurmans Stekhoven, F. M. A. H . , S w a r t s , H. G. P . , D e P o n t , J. J. H. H. M . , and Bonting, S. L. ( 1 9 8 1 ) . S t u d i e s on Magnesium i n d u c e s two low(Na++K+)-activated ATPase. XLV. a f f i n i t y non-phosphorylating n u c l e o t i d e b i n d i n g s i t e s p e r molecule. B i o c h i m . B i o p h y s . A c t a 6 4 9 , 533-549. S t e i n , W. D., L i e b , W. R . , K a r l i s h , S. J. D . , and E i l a m , Y . ( 1 9 7 3 ) . A model f o r a c t i v e t r a n s p o r t of sodium and p o t a s s i u m i o n s as

.

.

424

S. L. BONTING eta/.

mediated by a t e t r a m e r i c enzyme. P r o c . N a t l . Acad. Sci. U.S A . 7 0 , 275-278. Swann, A. C . , and Albers, R. W. (1978). Sodium and potassium iondependent adenosine t r i p h o s p h a t a s e of mammalian b r a i n . I n t e r a c t i o n s o f magnesium i o n s w i t h t h e phosphatase s i t e . Biochim. Biophys. A c t a 5 2 3 , 215-227. Yamaguchi, M . , and Tonomura, Y. (1979). Simultaneous b i n d i n g of t h r e e N a + and two K+ i o n s t o Na+,K+-dependent ATPase and changes i n i t s a f f i n i t i e s f o r t h e i o n s induced by t h e format i o n of a phosphorylated i n t e r m e d i a t e . J. Biochem. ( T o k y o ) 86, 509-523.

CURRENT TOPICS IN MEMBRANES AND TRANSPORT, VOLUME 19

Rubidium Movements in Vesicles Reconstituted with Na,K-ATPase, Measured in the Absence of ATP and Pi, in the Presence of Either Ligand, and in the Presence of Both Ligands: Role of the iiOccluded State’’ in Allowing for the Control of the Direction of Ion Movements S. J . D. W I S H Departnzent of Biochemistry W e i m n n Institute of Science Rehovot. Israel

W. D. STEIN Depanment of Biochemistry Hebrew University Jerusalem, Israel

In this chapter we will discuss the role of the major nonphosphorylated conformations of the Na/K-pump in the movement of potassium ions, and the implications of this role €or sodium/potassium pumping. The experiments involve the measurements of ion fluxes into and out of phospholipid vesicles containing the reconstituted Na,K-ATPase from pig kidney. In an earlier paper (Karlish and Pick, 19811, it was shown that such vesicles sustain the conventional ATP-dependent Na/K exchanges. In the present chapter we use a study of the rubidium movements in the presence of ATP or Pi, of both ATP and Pit and in the absence of ATP and Pi, to throw light on the mechanism of action of the pump and the physiological control of the direction of ion pumping and the associated ATP breakdown. The experi‘ D e p a r t m e n t of B i o c h e m i s t r y , Weizrnann I n s t i t u t e of Science , Rehovot , I s r a e l . 2Departrnent of B i o c h e m i s t r y , Hebrew U n i v e r s i t y , J e r u s a l ern, Israel. 425

Copyright 0 1983 by Academic Press, Inc. All rights of reproduction in nny form reserved. ISBN 0-12-153319-0

426

S. J. D. KARLISH AND W. D. STEIN

mental method we have used is summarized in Table I. [An extended account of the studies discussed here will be found in Karlish and Stein (1982a,b) and Karlish e t a l . (1982)l.

I.

CONFORMATIONAL TRANSITIONS OF THE Na,K-ATPase

Physical techniques, in particular fluorescence spectroscopy (Karlish et al., 1978; Karlish and Yates, 1978) , but including as well the use of controlled proteolytic digestion (Jpkgensen, 1975), have identified two clearly different conformations of the Na/K pump enzyme, El and E2. The salient features of the interconversions between these two forms are summarized in the top scheme of Fig. 1. The El form binds Na or K competitively, from the cytoplasmic side. Sodium binds with high affinity (KNa = 1 mM), K with a low intrinsic affinity (KK % 75 mM). If K binds, a spontaneous conformational transition takes the potassium into the species E2-(K), in which the potassium ions are occluded. Kinetic experiments show that the conformational transition in the direction E1.K + E2-(K) is fast (with a rate constant of about 300 sec-l at room temperature), while that in the opposite direction The equilibrium is is very slow (about 0.3 sec'l) thus set very far in the direction of E2.(K) (KC % 1,000). A very important piece of information is that ATP binds to the El forms with a high affinity

.

TABLE I. A.

B. C.

a Methods for Flux Measurements

Vesicles are prepared by 1. Mixing cholate-solubilized pig kidney Na,K-ATPase with soybean phospholipid vesicles 2. Freezing rapidly in liquid nitrogen and thawing at room temperature 3 . Sonicating briefly The exterior medium is replaced by one of choice by brief centrifugation on small columns of Sephadex G-50 preequilibrated with the appropriate medium. Transport assays involve either measurement of 22Na or 86Rb uptake into the vesicles, or efflux of isotope from preloaded vesicles. Separation of vesicles from the medium is performed on small columns of Dowex 50X-8.

a

See Karlish and Pick (1981).

Rb FLUXES THROUGH RECONSTITUTED Na, K PUMPS

427

SCHEME FOR CONFORMATIONAL TRANSITIONS KNa - I m M

KK-75mM

KC

-

1000

F i g . 1 . Schemes f o r c o n f o r m a t i o n a l t r a n s i t i o n s ( u p p e r h a l f ) and f o r K/K e x c h a n g e ( l o w e r h a l f ) , m e d i a t e d b y the Na/K pump. K N a , K K : and KC a r e the v a l u e s of the e q u i l i b r i u m c o n s t a n t s f o r the e q u i l i b r i a w r i t t e n b e l o w e a c h s y m b o l . S u b s c r i p t s c y t and e x t r e f e r t o ions b i n d i n g a t the c y t o p l a s m i c and e x t e r n a l s u r f a c e s , r e s p e c t i v e 1y .

(less than 1 P M ) , but to the occluded forms with a low affinity (half millimolar). Thus ATP shifts the conformational equilibrium away from the occluded species, and stabilizes El. This is reflected in the very large stimulation of the rake of the transition E2.(K) -+ El-K by ATP. The lower scheme of Fig. 1 shows how these conformational transitions have been thought, conventionally, to participate in active K transport and in K/K exchange (Glynn and Karlish, 1975). This scheme makes a number of predictions which we have tested with the reconstituted ATPase vesicles preparation. First, potassium ions or the competing sodium ions should combine with sites on El which are oriented toward the cytoplasm. Binding of Na from the cytoplasmic surface should leave the enzyme in the El form, but binding of potassium from the cytoplasm should produce the occluded conformation, E2. (K). This prediction has been verified by studying the results of controlled tryptic digestion of such vesicles (Karlish and Pick, 1981). Second, on this scheme, movement of potassium out of the occluded form toward the cell exterior should require phosphorylation by inorganic phosphate (Pi) (conversely, potassium at the extracellular sites induced a rapid dephosphorylation), while we have seen that the conformational transition E2.(K) to El-K is accelerated by ATP. These proposed roles of ATP and Pi explain the conventional finding that active K+ transport in the forward direction is stimulated by ATP, while K/K exchange has been thought

428

S.J. D. KARLISH AND W. D. STEIN

to require, mandatorily, both ATP and phosphate (Simons, 1974; Sachs, 1981). The second major prediction, therefore, is that we should observe no K/K exchange in the complete absence of either Pi or ATP. The third prediction was made at the previous conference on the Na/K pump (Stein, 19791, and concerns the behavior of the system at high concentrations of either ATP or phosphate. Now according to the scheme of Fig. 1, ATP stabilizes the El forms, while Pi stabilizes E2 forms. In such a situation variation of the concentration of either ATP or Pi, at a fixed concentration of the other ligand, should show stimulation of K/K exchange at low levels of ligand, but eventually inhibition of exchange at high enough concentrations of ligand. The pump is then trapped totally in one or other of the two alternative conformations. On this scheme, ATP and Pi should show symmetric behavior, and most importantly, should compete with one another for binding to the occluded form E2-K. [Preliminary verifications of this prediction have been reported by Karlish and Stein (1981) for vesicles and by Eisner and Richards (1981) for red cell ghosts.]

11.

RUBIDIUM FLUXES IN THE ABSENCE OF ATP OR OF Pi

We have seen that the conventional scheme for Rb/Rb exchange (we treat here potassium and rubidium indifferently, rubidium being a very good marker for potassium fluxes), Fig. 1, would predict no flux of ion in the absence of ATP and Pi. We found, to our surprise, that the reconstituted sodium pump showed, at low levels of rubidium, a very noticeable Rb/Rb exchange (Fig. 2 ) , in the absence of added ligand. This flux was entirely absent in vesicles made with Na,K-ATPase pretreated with ouabain. The ouabain-sensitive component (inset to Fig. 2 ) represents a saturable flux, and is superimposed on a linear flux which appears to be a simple ion leak. Now such a flux of rubidi’um could be carried on pumps oriented either inside-out or right-side-out, with respect to their orientation in the intact cell (Fig. 3 ) . When the pump inhibitor, vanadate, is added to such reconstituted vesicles, externally added vanadate (vanadateo) inhibits exactly one-half the saturable rubidium flux (Fig. 4), while all of the saturable flux is inhibited only if vanadate is added to both sides of the vesicles (vanadatei+o! (Fig. 4). Thus a vesicle preparation seems to contain an equal number of pumps oriented

Rb FLUXES THROUGH RECONSTITUTED Na, K PUMPS

- - - 13 . 0

429

/

Rb, .rnM F i g . 2. O u a b a i n - s e n s i t i v e 86Rb u p t a k e i n t o Rb-loaded vesicles a t d i f f e r e n t Rbo c o n c e n t r a t i o n s . Two s e t s of reconstit u t e d vesicles w e r e p r e p a r e d , both c o n t a i n i n g 100 mM R b C l , and 2.5 mM MgCl2, b u t one set was p r e p a r e d f r o m Na,K-ATPase p r e i n c u b a t e d f o r 1 hr a t 2OoC w i t h 2 mM o u a b a i n , 2 mM M q C 1 2 , and 1 mM p h o s p h a t e ( T r i s ) . T h e 86Rb u p t a k e a s s a y was i n i t i a t e d b y a d d i n g 40 ~1 o f the vesicles s u s p e n s i o n t o 40 p 1 of a s o l u t i o n c o n t a i n i n g R b C l + T r i s - H C 1 ( t o t a l c o n c e n t r a t i o n 128 mM) m i x e d i n p r o p o r t i o n s t o p r o d u c e the d e s i r e d f i n a l c o n c e n t r a t i o n o f R b C l , a f i x e d amount of 8 6 R b , and MgCl2, 2 mM ( f i n a l c o n c e n t r a t i o n o f 1 mM). For a s s a y of the vesicles p r e p a r e d w i t h e n z y m e p r e i n c u b a t e d w i t h o u a b a i n , the r e a c t i o n m i x t u r e a l s o c o n t a i n e d o u a b a i n , 1 mM. A f t e r 4 m i n , the s u s p e n s i o n s w e r e removed t o the Dowex columns.

S. J. D. KARLISHAND W. D. STEIN

430

RIGHT- SIDE OUT

Vo no d a t e

IN-SIDE OUT

n No ext. Rb ext. Oua bain

W Rb ext. Na ext.

A

PiT P Vanadate Rb cyt. Na cyt.

Fig. 3. Schematic diagram of the orientation of sodium pumps in vesicles and the sites of binding of the ligands, substrates, and inhibitors. The sidedness of ligand binding to Na/K pumps reconstituted into phospholipid vesicles is shown. The lollipop is a sodium pump, the large circle a vesicle into which the pump has been reconstituted.

in either direction. These vesicles demonstrate a net flux of rubidium ions (Fig. 5 ) . This net flux is, of course, carried by both right-side-out and inside-out oriented pumps. Figure 5 shows that the inside-out pumps (inhibited by externally added vanadate) carry the major part of this net flux. As the right half of Fig. 5 shows, the net flux component which is sensitive to externally added vanadate shows a sigmoidal dependence on the concentration of rubidium. That these measurements of rubidium uptake by reconstituted pumps represent transport and not binding of rubidium was shown by studies of the time course of Rb movements (Fig. 6) into Rb-free or Rb-loaded vesicles. Rubidium net uptake (lower curve of Fig. 6) took an extended time and reached a level consistent with the water content of such a vesicle preparation. In contrast, uptake into Rb-loaded vesicles reached quite high levels from which labeled rubidium gradually left the cell (Fig. 6, upper curve) to reach the level found in vesicles that had not been preloaded, a typical picture for a countertransport, carrier-mediated system. The mechanism of these passive fluxes of rubidium has been clarified by investigation of the vanadate-sensitive Rb uptake into vesicles loaded with different monovalent cations (Table 11). Rubidium uptake into Tris-loaded, choline-loaded, or lysine-loaded vesicles is about the same and repre-

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86Rb/Rb e x c h a n g e t h r o u g h i n s i d e - o u t and r i g h t - s i d e pumps: Fig. 4 . Vanadate,-sensitive d e p e n d e n c e on Rbo c o n c e n t r a t i o n . Two sets o f r e c o n s t i t u t e d vesicles w e r e p r e p a r e d , one c o n t a i n i n g 150 mM RbCl the other c o n t a i n i n g 150 mM R b C l ; 5 0 p M v a n a d a t e ( T r i s ) , and 1.5 mM MgC12. A f t e r cent r i f u g a t i o n t w i c e on c o l u m n s o f S e p h a d e x G - 5 0 the f i r s t set was d i v i d e d a n d 200 W M MgCl2 + 6 0 0 p M v a n a d a t e ( T r i s ) was added t o one h a l f - s e t ( i . e . , the + v a n a d a t e , s a m p l e ) and also t o the s e t o f vesicles c o n t a i n i n g v a n a d a t e i ( i - e . , the + v a n a d a t e i + o s a m p l e ) . T h e a s s a y was i n i t i a t e d by a d d i n g 40 1.11 of vesicles (control, or + vanadate,, or + vanadatei+,) t o 4 0 p 1 o f the a p p r o p r i a t e RbCl + Tris-HC1 m i x t u r e ( t o t a l c o n c e n t r a t i o n 150 mM), c o n t a i n i n g also a f i x e d amount o f 86Rb. A f t e r 2-min i n c u b a t i o n , the s u s p e n s i o n was a p p l i e d t o the Dowex c o l u m n s for a n a l y s i s .

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R b, -mM F i g . 5. V a n a d a t e , - s e n s i t i v e net R b f l u x i n t o Rb-free vesicles: d e p e n d e n c e on Rbo. R e c o n s t i t u t e d vesicles w e r e p r e p a r e d c o n t a i n i n g 1 5 0 mM T r i s - H C 1 , pH 7.0. A f t e r c e n t r i f u g a t i o n on S e p h a d e x G - 5 0 , the s u s p e n s i o n was d i v i d e d , and 2 mM M q C l , + 1 mM v a n a d a t e ( T r i s ) was added t o one half-set. T h e assay was p e r f o r m e d a s i n F i g . 4 , e x c e p t t h a t the f l u x t i m e was r e d u c e d t o 1 m i n i n order t o e n s u r e t h a t B6Rb u p t a k e was l i n e a r w i t h t i m e ( c f . F i g . 3 ) .

Rb FLUXESTHROUGH RECONSTITUTED Na, K PUMPS

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Fig. 6. Equilibration or countertransport of 86Rb into Rbfree or Rb-loaded vesicles. Two sets of reconstituted vesicles were prepared, containing either 150 mM Tris-HCL; 3 mM MgCl2 or 130 mM Tris-HC1; 3 mM MgC12 and 20 mM RbCl. 200 1-11 of each vesicle suspension was mixed with 400 p 1 of 150 mM Tris-HC1 containing RbCl (+ 86Rb), 150 pM (final concentration of 100 PM). A t the times indicated, 50 ~1 samples were removed to Dowex columns for analysis of the vesicle radioactivity content.

sents the net uptake of Rb. Uptake into vesicles containing Rb, K, C s , Na, or Li is increasingly stimulated along that series and reaches fivefold. Interestingly, the exchange of Rb and Na here measured is in the opposite direction to the direction of ATP-driven pumped movements in the intact cell. Were such movements to be as rapid as those of the pumped fluxes, they would constitute an unacceptable leak for an effectively working pump. In Table 11, we record the turnover numbers of these pump-mediated leaks and parallel measurements of the ATP-driven Na/K pump rates, and the ATP, Pi-requiring Rb/Rb exchanges. One can see that the ligand-free fluxes are very slow, of the order of 1% of the ATP-linked fluxes. The rates of ligand-free fluxes, 0.2-0.3 sec-1, are comparable to the rate of the E2*(K) -+ El-K transition measured on the isolated Na,K-ATPase, spectroscopically. How can we explain these passive fluxes in terms of the familiar conformational transitions of Fig. 1? We must assume that the occluded E2-(Rb) will occasionally undergo a conformational transition bringing the ion-binding (and releasing) sites to the extracellular surface. Thus, the ATP- and Pa-independent Rb/Rb exchange would then involve osciflations along this path ,

S.J. D. KARLISH AND W. D. STEIN

434

E f f e c t s of D i f f e r e n t I n t e r n a l C a t i o n s on Vanadate,s e n s i t i v e and - i n s e n s i t i v e Uptakea

TABLE 11.

R b a 6 uptake/min

Internal cation Tris

Choline Lysine

Rb K

cs Na Li

Vanadate,sensitive 255 f 18 317 f 1 0 297 f 1 8 429 798 867 1406 1611

i:

4 t 22 5 22 k 25 i: 55

Vanadate,insensitive 312 ? 4 380 f 2 208 f 1 3

f 3 f 3 f 11 f 7 600 f 1 8

638 692 1352 430

a

E i g h t sets o f r e c o n s t i t u t e d v e s i c l e s were p r e p a r e d , cont a i n i n g 150 mM of T r i s - H C 1 (pH 7.01, choline-C1, lysine-HC1 (pH 7.01, R b C l , K C 1 , C s C 1 , NaC1, o r L i C 1 , r e s p e c t i v e l y . The vanadate,-sensitive and - i n s e n s i t i v e f l u x e s were measured a s i n Fig. 4 , o v e r 1 min, a t a f i n a l e x t e r n a l Rbo (+ 86F&) concentrat i o n of 2 mM.

while the net flux would be possible if the unloaded species El and E2 were able to interconvert while not bound to rubidium. This is depicted in the top scheme of Fig. 7 while the case of Rb/Na exchange is depicted in the lower half of that figure. All of these schemes are, kinetically, simple carriers (Lieb and Stein, 1974). We have seen that there is independent physical evidence that the conformational transitions symbolized by the rate constant a in Fig. 7 is much faster than that for the rate constant b. There is evidence also that step d is fast (Karlish and Stein, 1982a). NOW, the step c is common to all the three modes of Rb movement: net flux in the 1 -+ 2 direction, exchange for external Rb, and exchange for Na. This being so, it cannot be the rate-limiting step for all of these three modes, having as they do different rates. In particular, c cannot be the rate-limiting step in the slower two of these modes, namely net Rb flux and Rb/Rb exchange. One can thus argue that the net flux in the 1 -+ 2 direction is rate-limited by the step f, Rb/Rb exchanges by step b , and the Rb/Na exchange possibly by c. That this latter identification can, indeed, be made

TABLE 111.

ATP-dependent 22Na/F& exchange

Derived Turnover Numbersa tb

(ATP + Pi)-stimulated Vanadate,-sensitive 86Rb/Rb exchange 86E&/Rb exchange ~~

43

a

7

0.2s

Vanadate,-sensitive net 86Rb uptake (ATP- and Pi-free conditions) ~~

Vanadate,-sensitive 86Rb/Na exchange ~~

0.15

0.63

The values have been calculated from the experimental data in Table I11 of Karlish and Stein (1982a), and corrected slightly as follows to take into account lack of full saturation or inhibition by ions: ATP-dependent Ha uptake is activated only 91% by 30 mM NaCl (see Karlish and Pick, 1981). (ATP f phosphate)-stimulated Rb/Rb exchange is activated 90% at 25 mM R b C l and is inhibited by about 15% at 1 mM free Mgo ions (see Karlish et a l . , 1982). The slow vanadate,-sensitive Rb fluxes are activated only by about 80% at 2 mM external R b C l (see Karlish and Stein, 1982a). holes R b or Na/sec/mole phosphoenzyme.

S. J. D. KARLISH AND W. D. STEIN

436

EXTRACELLULAR FACE

CYTOPLASMIC FACE

f Net Rb flux

El Rbcytj

Rb- Rb exchange

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El .Rb

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Rbext.

a_ E,*(Rb) c _ E,*Rb

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C' A

7 E2.Na

F i g . 7 . Schemes f o r c a t i o n movements and a s s o c i a t e d conform a t i o n a l t r a n s i t i o n s f o r n e t R b f l u x e s and R b / R b e x c h a n g e s ( u p p e r h a l f ) and f o r Rb/Na e x c h a n g e s ( l o w e r h a l f ) . The s y m b o l s a , b , c , e t c . , are the r a t e c o n s t a n t s f o r t h e s t e p s i n d i c a t e d . T h e conf o r m a t i o n s w i t h c a t i o n b r a c k e t e d a r e the o c c l u d e d f o r m s .

is supported by a consideration of early experiments by Post e t a l . (1972) who found that the rate of the E2.(X) + El-X transition depended on the nature of the occluded ion, X I and was very fast for X being sodium. 111.

RUBIDIUM FLUXES IN THE PRESENCE OF EITHER ATP OR OF Pi

If the interpretation offered above of the ligandfree fluxes is a correct one, we might expect to see effects of ATP or Pi acting separately, since (according to Fig. 1) these ligands greatly accelerate the rates of the conformational transitions that take Rb from the occluded state to the cytoplasmic (for ATP) or extracellular (for P - ) surfaces. Figure 8 shows, indeed, the effects of aaded ATP on the exchange of Rb with internal Rb, while Fig. 9 shows the effect of ATP on the net flux. In all cases we see the expected stimulation by ATP, but we also see (especially clearly at the lower Rb levels) inhibition of Rb fluxes by higher ATP levels. The

Rb FLUXESTHROUGH RECONSTITUTED Na, K PUMPS

437

-

Rb, =20mM

-

'

'

-

'

I

'

I

0.02 r

'

I::

'

:

-

+

I

/

+

t

4

c

t

1

-

-c

-

Rb, = 2mM

0

'

1

1

1

1

1

'

1

1

'

/

+AM+

0.006 -

-

a.

0.002

-

0 0

+

-0-

t

+ -

-

Rb, =0.2mM

"

"

"

'

"

'

I

0.02 0.04 0.06

0.08

A*L&

0.10 0.3

I

3

9

ATP- mM

Fig. 8. Effects of ATP on vanadate,-sensitive Rb/Rb exchange at different Rbo concentrations. Reconstituted vesicles containing 150 mM RbCl were prepared and centrifuged twice on columns of Sephadex G- 5 0 . 1 mM vanadate (Tris) + 5 mM MgC12 was added to one portion of the suspension. The 86Rb uptake was measured by mixing 40 l.11 of vesicles with 40 p 1 of reaction medium containing the appropriate mixture of Tris-HC1 + RbCl at a total concentration of 150 mM, a fixed amount of 86Rb, and ATP (Tris) at twice the indicated final concentration. g6Rb flux was measured for 2 min, at Rbo 0.2, 2.0, or 20 mM, respectively, with the different ATP concentrations. The vanadate-insensitive 86Rb flux was measured in the absence of ATP at all three Rbo concentrations, and the value was subtracted from the values of the total 86Rb uptake at all the ATP concentrations.

stimulations are expected if ATP speeds up a ratelimiting step ( b in Rb/Rb exchange, f in net flux). Inhibition occurs [in the way predicted by Stein (1979) at the high concentrations of ATP, because ATP stabilizes the El conformation, decreasing the steady-state

S.J. D. KARLISH AND W. D. STEIN

430

I

100

I

200 A T P - pLM

1

loo0 4000

Fig. 9. Effects of ATP on vanadateo-sensitive net Rb uptake at different Rb, concentrations. Reconstituted vesicles were prepared, containing 150 mM Tris-HC1, pH 7.0. 86Rb uptake was measured for 1 min, in the way described in Fig. 8.

level of the occluded state E2-(Rb), and lowering its ability to contribute to the flux. The four- to fivefold maximal stimulation by ATP seen in Fig. 7 suggests that in such circumstances it is step c which now becomes rate-limiting, so that if b is about 0.2 sec-1, step c has a rate constant of about 1 sec-l. The figures depict a clear antagonism between Rb and ATP, in that as the Rb concentration is lowered, the achievable stimulation of flux by ATP is lowered, inhibition is much increased, and all effects of ATP are found at lower ATP levels. These effects are readily understandable if ATP drives the pump enzyme out of the occluded state and into El forms, while Rb, conversely, drives the system into the occluded form. At any ATP concentration, net movements of Rb are affected as if

Rb FLUXES THROUGH RECONSTITUTED Na, K PUMPS

50

I

I

0

I

I

I

I

2

439

I

I

I

I

I

I

I

I

4 6 Phos p h a t e,- m M

I

I

I

I

0

I

I

10

F i g . 1 0 . E f f e c t s of p h o s p h a t e on v a n a d a t e o - s e n s i t i v e net R b u p t a k e . R e c o n s t i t u t e d vesicles w e r e p r e p a r e d c o n t a i n i n g 150 mM T r i s - H C 1 pH 7 . 0 . 86Rb u p t a k e was m e a s u r e d over 1 m i n a s i n F i g . 4 , i n a r e a c t i o n s o l u t i o n c o n s i s t i n g of T r i s - H C 1 , 1 5 0 mM; R b C l ( + 8 6 R b ) , 2 mM; MgCl2, 50 VM or 5 mM; and p h o s p h a t e ( T r i s ) a s i n d i c a t e d .

they were occurring at far lower Rb concentrations in comparison with the exchange flux. For net movements, entry into the occluded state is driven by Rb from one face of the membrane only, and Rb is thus a far less effective antagonist of ATP. As far as effects of phosphate are concerned, these, too, are consistent with the hypothesis that Pi stimulates the transition out of the occluded state. The data (Fig. 10) show a small stimulation of Rb movements by Pi, at low concentrations of magnesium. The stimulation would be expected to be small if, indeed, net Rb movement here was only partly rate-limited by the rate constant e. If b and c are comparable in magnitude, in the absence of P., then increasing the magnitude of c by adding phosphate should approximately double the net flux, which is almost the result found. High magnesium ions allow an inhibitory effect of Pi to be revealed, an effect indicating perhaps the formation of the "K-insensitive" form of the phosphorylated pump described by Post

S.J. D. KARLISHAND W. D. STEIN

440

(1975). Inhibition of the net flux by very high levels of Pi (at low Mg levels) would not be expected in this net mode since it is not the amount of the occluded state that now limits the flux. We see that the effects of Pi (and of ATP) in stimulating the Rb fluxes are in each case limited by the flux reaching a maximum as some other step becomes rate-limiting. What happens if both ATP and Pi are present to stimulate movement out of the occluded state in both directions? et al.

IV.

RUBIDIUM FLUXES IN THE PRESENCE OF BOTH ATP AND Pi

The data on the combined effects of ATP and Pi are easily understandable on the basis of their effects on the system when each is present alone. At different fixed levels of ATP, increasing concentrations of Pi stimulate Rb/Rb exchange (Fig. ll), while at the lower levels of ATP, inhibitions are also seen. The effect of increasing ATP concentrations at fixed Pi levels (Fig. 12) show a perfectly symmetrical behavior. The stimulations followed by inhibitions are just what we would expect and have understood from the behavior of the separate ligands. The stimulations are always larger than in the "one ligand only" systems, since the rate-limiting step here is larger (being itself stimulated by the presence of the fixed ligand). The absence of inhibition at high levels of both ligands needs special comment, however. These results can only mean that ATP and Pi are able to bind simultaneously to the enzyme, and that they do not compete for the occluded form, but can bind to it both at once. The models of Figs. 1 and 7 have to be extended to take account of this possibility. What we have done is to generalize the scheme of Fig. 1 to include also the possibility that ATP can bind, additionally, to all E2 forms, while Pi can, additionally, bind to all El forms, and that such forms can bind our two ligands independently and (hence) simultaneously. The model we arrive at is given in Fig. 13. It looks formidable, but is in fact very simple. Along any long edge of the figure are equilibria such as those depicted in Fig. 1, in which the state of ligand binding is invariant, with either none, one ATP only, one Pi only, or both ATP and Pi bound. Movement from one long edge to another represents addition or release of ligand. To analyze the kinetics of this model we have used the methods of King and Altman

Phosphate

-

mM

Fig. 11. D ependence of Rb/Rb e x c h a n g e on p h o s p h a t e c o n c e n t r a t i o n a t d i f f e r e n t f i x e d ATP concent r a t i o n s . R e c o n s t i t u t e d vesicles c o n t a i n i n g 140 mM T r i s - H C 1 pH 7 . 0 , 10 mM RbCl w e r e p r e p a r e d and w e r e c e n t r i f u g e d t w i c e on c o l u m n s o f S e p h a d e x G-50 e q u i l i b r a t e d w i t h 1 5 0 mM Tris -HC1, 5 mM R b C l , and 2 mM MgCl2. 86Rb u p t a k e was m easured over 2 m i n a f t e r m i x i n g 40 p 1 o f vesicles w i t h 40 p 1 of r e a c t i o n m i x t u r e . The f i n a l r e a c t i o n s o l u t i o n c o n s i s t e d o f Tris-H C1, 150 mM; RbCl (+ 8 6 R b ) , 5 mM; MgCl2, 2 mM, d i f f e r e n t f i x e d c o n c e n t r a t i o n s o f e q u i m o l a r m i x t u r e s o f ATP p l u s MgC12 and v a r i a b l e p h o s p h a t e ( T r i s ) Where n e c e s s a r y e x t r a Tris-HC1 was added t o make the o s m o l a r i t y 400 mosmoles i n a l l c o n d i t i o n s . E x p e r i m e n t s A and B w e r e p e r f o r m e d i n the same c o n d i t i o n s b u t on d i f f e r e n t d a y s .

.

r

i

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Pi = 0.5 mu

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1

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1

1500

I

2000

F i g . 1 2 . Dependence of Rb/Rb e x c h a n g e on ATP c o n c e n t r a t i o n a t d i f f e r e n t f i x e d p h o s p h a t e c o n c e n t r a tions. T h i s experiment was d o n e i n p r e c i s e l y the same way a s i n F i g . 11 e x c e p t t h a t t h e p h o s p h a t e ( T r i s ) c o n c e n t r a t i o n was k e p t c o n s t a n t w h i l e the ATP-Mg was v a r i e d a s shown.

Rb FLUXES THROUGH RECONSTITUTED Na, K PUMPS

443

(1956) and of Cha (19681, while assuming that the ratelimiting steps are everywhere the conformational transitions (signified with double arrows in the figures), addition and release of ions and ligands being assumed very fast. We also assumed that the effect of addition of ATP is, in every situation, only to increase the rate of that transition which takes cation out of the occluded state towards the cytoplasmic surface, while Pi always increases the rate of transition out of the occluded state towards the extracellular surface. The equation used and rate constants are recorded in Table IV. Computer predictions of this model gave curves such as seen in Fig. 11 (lower curve €or the effect of ATP at different fixed Pi levels, and upper curve for the effect of Pi at different fixed ATP levels). While we have not attempted to fit the data of Figs. 11 and 12 exactly to the model, by appropriate variation of the rate constants, we do feel that the overall shape of the computed curves is a pretty good description of the shape of the experimental data, justifying our hypothesis that ATP and Pi behave in an independent fashion, exerting their kinetic effects separately and independently. Note especially that the values of the kinetic constants that we have used in this computer prediction are all derived independently of the data of Figs. 11 and 12. The values €or the rate constants b and c come from experiments in the absence of added ligands, a comes from conformational transition experiments, while d is given by the rate of dephosphorylation of the isolated enzyme. The rate constant bA, representing the rate of exit from the occluded state in the presence of ATP, is given by the independent physical measurements of conformational transitions, while c P I the transition stimulated by added P , is consistent with studies on the isolated enzyme. IThe full development of the model and the justification of the choice of rate constants used is given in the extended papers of Karlish and Stein (1982a,b) and Karlish et al. (1982) ] Clearly the effects of ATP and Pi, at the level of analysis suitable for the data recorded here, can be quite simply described: ATP speeds up some 300-fold the rate of egress o f Rb from the occluded state toward the cytoplasmic surface, regardless of whether Pi is or is not bound to the enzyme. Inorganic phosphate speeds up by some 100-fold the rate of egress of Rb from the occluded state toward the cell exterior, regardless of whether ATP is or is not bound to the pump enzyme. ATP does this and hence stabilizes El forms of the enzyme. Inorganic phosphate similarly stabilizes phosphorylated forms of the enzyme. Enzyme can simultaneously bind ATP and Pi.

.

Rb binds to unloaded El or E2 forms

E, (Rb) forms contain occluded Rb ions ATP binds tightly to El forms and weakly to E,

forms

Phosphate is bound tightly in phosphorylated forms and weakly in ionically bound forms. Conformational Transitions

E l -Rb forms

a ,E2-(Rb) forms

Rate 0

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b = bP
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E2-Rb forms

d ,E2-(Rb) forms

d dA 5 dP = d

AP

mechanism for K / K e x c h a n g e t h r o u g h the Na/K pump i n the p r e s e n c e or a b s e n c e o f a , b , c , and d a r e r a t e c o n s t a n t s . R a t e c o n s t a n t s w i t h s u p e r s c r i p t s A , P , or AP r e f e r t o interconversions b e t w e e n f o r m s w i t h e i t h e r A T P , or p h o s p h a t e , or w i t h both l i g a n d s bound, r e s p e c t i v e l y . Fig. 13.

A

ATP a n d / o r p h o s p h a t e .

TABLE IV.

V -

S t e a d y - S t a t e S o l u t i o n f o r U n i d i r e c t i o n a l F l u x f r o m S i d e 1 to S i d e 2 o f F i g . 1 3 a ' b ' c

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-

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.

<+

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+

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<+

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K + P L

a A s i n the scheme f o u n d b y the m e t h o d s o f C h a ( 1 9 6 8 ) a n d K i n g a n d A l t m a n ( 1 9 5 6 ) . a f o r E l - R b + E2' ( R b ) , 300 sec-l; b f o r E 2 - ( R b ) -+ E l - R b , 0.3 sec-l; b R a t e constants u s e d : bA for E 2 - ( R b ) + E l - R b . A T P , 100 sec-l; c for E 2 - ( R b ) -+ E z ' R b , 1 sec-l; c p for E z - ( R b ) - P -+ E 2 . P - R b , 100 sec-1; d f o r E z - R b -+ E 2 - ( R b ) , 2 4 0 sec-1.

";,

C P

4 are intrinsic

8

P

K a r e b i n d i n g conR b b i n d i n g constants, a s s u m e d t o equal 50 mM. L' L . s t a n t s o f ATP a n d p h o s p h a t e f o r E 2 ' ( R b ) , a s s u m e d t o equal 300 pM or 3 mM, r e s p e c t i v e l y . F o r t h e c a l c u l a t i o n o f F i g . 1 4 , R b /# a n d RbZ/K: a r e a s s u m e d e q u a l t o 0.1; ATP/K: a n d P/K: a r e v a r i e d .

1

1

446

S. J. D. KARLISHAND W. D. STEIN I

I

1

C

4

11

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2

-

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0

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Fig. 14. Computer simulations of the effect on Rb/Rb exchange of variable ATP or phosphate concentrations at fixed concentrations of the complementary ligand. Explanation and details of the calculations are given in the text. Note that if we assume $ = 200-400 pM, then the fixed ATP concentrations in Fig. 14 (upper) are 2-4 p M , 20-40 pM, and 2-4 mM, respectively, and the range of variable ATP concentrations in Fig. 14 (lower) is 0-(0.60-1.2)rnM. Similarly for Kf = 1 - 3 mM, the fixed phosphate concentrations in Fig. 14 (lower) are 0, 0.1-0.3 mM, and 10-30 mM, respectively, and the range of variable phosphate in Fig. 14 (upper) is 0-(3-9)mM.

Rb FLUXESTHROUGH RECONSTITUTED Na, K PUMPS

V.

447

PHYSIOLOGICAL IMPLICATIONS OF THE LIGAND EFFECTS AND THE OCCLUDED STATE

Can one conceive of a physiological role for the occluded state and for the effects of ATP and of Pi on the rates of transition out of the occluded state? We believe that one can answer this question in the affirmative, in both its parts. The well-known effects of high levels of ATP on the rate of ATP splitting by the pump enzyme and on the rate of cation pumping (Glynn and Karlish, 1975) show that the transition out of the occluded state is normally rate-limiting in the absence of added ATP. The role of the occluded state is thus to slow down the rate of ion movements by the pump enzyme in situations where ligands are in short supply. The occluded state is an energy well into which the ions fall. Egress from this energy well is held low in the absence of added ligands, so that leak of ions by the pump enzyme is held to a minimum in the absence of regulating ligands. The addition of regulating ligands, however, is not merely to speed up the rate of pumping by speeding up the rate of egress from the occluded state. Rather, it is the direction of egress from the occluded state and hence the direction of ion movements that is controlled by the nature of the regulatory ligand. If ATP is present in excess, egress of potassium from the occluded state occurs toward the cell interior, i.e., in the direction which will lead to most effective pumping (Honig and Stein, 1979). In the presence of excess P., egress of potassium is in the direction of reversai of the pump, leading to incorporation of the regulatory Pi into newly synthesized ATP. The vectorial nature of the pump, whether it be most effective in pumping or in using ion gradients for ATP synthesis, is controlled by the nature of the ligand present, ATP or Pi. We feel that this is the first case in which the direction of action of a pumping system has been shown to be a target for physiological control.

ACKNOWLEDGMENT

W e are i n d e b t e d to Mrs. R. Goldshlegger f o r e x c e l l e n t t e c h nical assistance.

448

S.J. D. KARLISH AND W. D. STEIN

REFERENCES

Char S. (1968). A simple method f o r d e r i v a t i o n of r a t e equations f o r enzyme-catalyzed r e a c t i o n s under t h e r a p i d equilibrium assumption o r combined assumptions of equilibrium and J. B i o l . C h e m . 243, 820-825. steady-state. Eisner, D. A , , and Richards, D. E. (1981). E f f e c t s of ATP and phosphate on potassium-potassium exchange i n r e d c e l l g h o s t s . J . P h y s i o l . Commun. ( i n press). Glynn, I . M . , and K a r l i s h , S . J. D. (1975). The sodium pump. Annu. Rev. P h y s i o l . 37, 13-55. Honig, B . , and S t e i n , W. D. (1979). Design p r i n c i p l e s f o r a c t i v e t r a n s p o r t systems. J. T h e o r . B i o l . 75, 299-306. Jgkgensen, P. L. (1975). P u r i f i c a t i o n and c h a r a c t e r i z a t i o n of (Na+ + K+)ATPase. V I . D i f f e r e n t i a l t r y p t i c modification of c a t a l y t i c f u n c t i o n s of t h e p u r i f i e d enzyme i n t h e presence B i o c h i m . B i o p h y s . A c t a 466, 97-108. o f NaCl and KC1. K a r l i s h , S. J. D . , and Pick, U . (1981). Sidedness of t h e e f f e c t s o f sodium and potassium i o n s on t h e conformational s t a t e of t h e sodium-potassium pump. J . P h y s i o l (London) 312, 505-529. K a r l i s h , S . J. D . , and S t e i n , W. D . (1981). E f f e c t s of ATP and phosphate on Rb-Rb exchange i n v e s i c l e s r e c o n s t i t u t e d with (Na,K)ATPase. J. P h y s i o l . Commun. ( i n p r e s s ) . K a r l i s h , S. J. D., and S t e i n , W. D. (1982a). Passive Rb f l u x e s mediated by t h e (Na,K)ATPase r e c o n s t i t u t e d i n t o phospholipid v e s i c l e s : ATP- and phosphate-free c o n d i t i o n s . J. P h y s i o l . (London) (submitted f o r p u b l i c a t i o n ) K a r l i s h , S. J. D , and S t e i n , W. D. (198233). E f f e c t s of e i t h e r ATP o r of phosphate on p a s s i v e Rb f l u x e s mediated by (Na,K)ATPase r e c o n s t i t u t e d i n t o phospholipid v e s i c l e s . J. P h y s i o l . (London) (submitted f o r p u b l i c a t i o n ) K a r l i s h , S. J. D . , and Yates, D. W. (1978). Tryptophan f l u o r e s cence of (Na+ + K+)ATPase a s a t o o l f o r study of t h e enzyme mechanism. B i o c h i m . B i o p h y s . A c t a 527, 111-130. K a r l i s h , S . J. D . , Yates, D. W . , and Glynn, I. M. (1978). Conformational t r a n s i t i o n s between Na+-bound and K+-bound forms of (Na+ + K+)ATPase s t u d i e d w i t h formycin n u c l e o t i d e s . B i o c h i m . B i o p h y s . A c t a 525, 252-264. K a r l i s h , S. J. D . , Lieb, W. R . , and S t e i n , W . D. (1982). E f f e c t s of ATP and phosphate, i n combination, on Rb-Rb exchange mediated by (Na,K)ATPase r e c o n s t i t u t e d i n t o phospholipid v e s i c l e s . J . P h y s i o l . (London) (submitted f o r publication). King, E. L . , and Altman, C. (1956). A schematic method of d e r i v i n g t h e r a t e laws f o r enzyme catalyzed r e a c t i o n s . J. P h y s . Chern. 6 0 , 1375-1379. Lieb, W. R . , and S t e i n , W. D. (1974). Testing and c h a r a c t e r i s i n g t h e simple c a r r i e r . B i o c h i m . B i o p h y s . A c t a 373, 178-196.

.

.

.

Rb FLUXESTHROUGH RECONSTITUTED Na, K PUMPS

449

Post, R. L., Hegevary, C., and Kume, S. (1972). Activation by adenosine triphosphate in the phosphorylation kinetics of sodium and potassium ion transport adenosine triphosphatase. J . Biol. Chem. 247, 6530-6540. Post, R. L., Toda, G., and Rogers, F. (1975). Phosphorylation by inorganic phosphate of sodium plus potassium ion transport adenosine triphosphatase. Four reactive states. J. Biol. Chem. 250, 691-701. Sachs, J. R. (1981). Mechanistic implications of the potassiumpotassium exchange carried out by the sodium-potassium pump. J. Physiol. (London) (in press). Simons, T. J. B. (1974). Potassium-potassium exchange catalyzed by the sodium pump in human red cells. J. Physiol. (London) 237, 123-155. Stein, W. D. (1979). Half-of-the-site reactivity and the Na, K-ATPase. In "Na, K-ATPase: Structure and Kinetics (J. C. Skou and J. G. N&rby, eds.), pp. 475-486. Academic Press, New York.

This Page Intentionally Left Blank

CURRENT TOPICS IN MEMBRANESAND TRANSPORT, VOLUME 19

Eosin: A Fluorescent Probe of ATP Binding to Na,K-ATPase J. C. SKOUA~JD M.ESMA" Institute of Biophysics University of Aarhus Aarhus, Denmark

I.

RESULTS AND DISCUSSION

E o s i n b i n d s n o n c o v a l e n t l y t o t h e Na,K-ATPase (Skou and Esmann, 1 9 8 1 ) . I n t h e p r e s e n c e of K+ i t s a f f i n i t y i s low, and t h e b i n d i n g of e o s i n h a s p r a c t i c a l l y no e f f e c t on i t s f l u o r e s c e n c e ( F i g . 1 ) . Sodium opens up a h i g h - a f f i n i t y binding s i t e f o r e o s i n , w i t h a consequent i n c r e a s e i n f l u o r e s c e n c e , a s h i f t i n t h e e x c i t a t i o n maximum from 5 1 8 t o 5 2 4 run, i n t h e e m i s s i o n maximum from 538 t o 5 4 2 nm, and w i t h a s h o u l d e r a p p e a r i n g a t a b o u t 4 9 0 nm o n t h e e x c i t a t i o n c u r v e , F i g . 1. T h i s e f f e c t on t h e f l u o r e s c e n c e i s s i m i l a r t o t h e effect of d i s s o l v i n g e o s i n i n a l c o h o l i n s t e a d of water. With 150 m M N a + 1 e o s i n molecule i s bound w i t h a h i g h a f f i n i t y ( K D 0 . 4 5 V M ) p e r 32P b i n d i n g s i t e ( F i g . 2A). The d i f f e r e n c e i n f l u o r e s c e n c e w i t h enzyme + Na+ v e r s u s enzyme + K+ i s o b s e r v e d u s i n g e o s i n Y ( F i g . 11, B, and S , b u t n o t u s i n g t h e c h e m i c a l l y c l o s e l y r e l a t e d compounds e r y t h r o s i n B , e r y t h r o s i n Y , p h l o x i n e , and p h l o x i n e B (Skou and Esmann, 1 9 8 1 ) . 451

Copyright 0 1983 by Academic Press, Inc. All rights of reproductionin any form m ~ e d . ISBN 0-12-153319-0

452

J. C. SKOU AND M. ESMANN

Fig. 1. Fluorescence excitation and emission spectra of 30 nM eosin in a 30 mM histidine buffer at pH 7.2 without and with 0.1 m g Na,K-ATPase/ml with 12 mM Kt and 20 mM Na+, respectively, The specific activity of the Na,K-ATPase was 1270 pmoles at 22'C. Pi/mg protein/hr and with 2.1 nmoles 32P labeling sites p e r milligram protein. A 10-nm slit was used for both excitation and emission. Reproduced by permission from Skou and Esmann, 1981).

ATP (and ADP) p r e v e n t t h e h i g h - a f f i n i t y b i n d i n g of e o s i n , and e o s i n i s c o m p e t i t i v e w i t h ATP f o r t h e ATP hyd r o l y s i s i n t h e p r e s e n c e of N a + (+ Mg2+) (Skou and Esmann, 1 9 8 1 ) . E o s i n , l i k e ATP, i n c r e a s e s t h e N a " r e l a t i v e t o K+ a f f i n i t y on t h e i n t e r n a l s i t e s of the system (Skou and Esmann, 1 9 8 1 ) . T h i s e f f e c t of e o s i n i s o b s e r v e d b o t h under c o n d i t i o n s where t h e system is n o t t u r n i n g o v e r and under c o n d i t i o n s where ATP is h y d r o l y z e d .

W t

(NI310tld 6"J/10su)

N 0

NIS03 ONflOE

rY)

t

/ r

/'

N

454

J. C. SKOU AND M. ESMANN

The experiments suggest that the high-affinity eosin binding site is an ATP binding site and that it is located in an environment with a low polarity, i.e., the conformational change induced by Na+ opens up a high-affinity site for ATP. The experiments su gest furthermore that the ATP which increases the Na' relative to K+ affinity on the internal sites is not the ATP which is hydrolyzed, i.e., in a turnover cycle in the presence of Na+ + K+, the system reacts with two different ATP molecules. Mg2+ has the same effect as Na+ on the fluorescence of eosin in the presence of enzyme. However, binding experiments show that with 5 mM Mg2+, the high-affinity binding, which is G-strophanthin sensitive, gives an upward curved line in a Scatchard plot with an intercept at 4.2 nmoles of binding sites per 2.1 nmoles 32P labelin sites, i.e., 2 high-affinity eosin binding sites per 33P labeling site. The data can be resolved according to a model with two equal populations of sites and with a KD of 0.25 and 1.7 p ~ , respectively (Fig. 2B). The Mg2+-dependent , high-affinity binding sites are vanadate sensitive (Fig. 2C) and ATP sensitive. Na+ gives a slight, while KS gives a pronounced, decrease in affinity of eosin in the presence of 5 mM Mg2+ (Fig. 2D) , but in the presence of K+ there are still two vanadate-sensitive binding sites per 32P labeling site KD is 17 pM.

F i g . 2. ( A ) Scatchard p l o t o f eosin binding i n t h e presence o f 150 mM Na+ and 150 mM K+, respectively, t o Na,K-ATPase, with 2.1 nmoles 3 2 p labeling s i t e s per m i l l i g r a m protein. 0.8 mg protein i n 30 mM h i s t i d i n e b u f f e r (pH 7 . 2 , 23OC) was incubated f o r 5 min with 1 5 0 mM N a f or 150 mM K+ i n the d a r k and with d i f f e r e n t concentrations of eosin. A f t e r preincubationtheenzyme was separated from the supernatant b y centrifugation a t 60,000 rpm f o r 1 hr. The f r e e eosin concentration and the amount o f eosin bound t o the enzyme was calculated from the fluorescence o f the supernatant and o f a control without enzyme. The r e s u l t s given i n F i g . 2A are representative f o r the r e s u l t s obtained. ( B , C , and D) Scatchard p l o t s o f the d i f f e r e n c e i n binding: ( B ) with Mg2+ 5 mM, P i 0 . 2 5 mM without and with 1 mM ouabain; (C) with Mg2+ 5 mM Na' 150 mM and with Mg2+ 5 mM Kc 150 vanadate 10-2 mM; (D) w i t h Mg2' 5 mM I?150 mM without and w i t h vanadate mM. The preincubation time i n ( B ) , ( C ) , and ( D ) was 30 min. The values shown are d i f f e r e n c e s between curves based on means o f 3 determinations. Figure 2A reproduced b y permission from Skou and Esmann, 1 9 8 1 .

EOSIN: PROBE OF ATP BINDING TO Na,K-ATPase

455

These results indicate that the conformation of the system in the presence of Mg2+ is different not only from that in the presence of K+ but also from the conformation in the presence of Na+; furthermore, that the conformation seen in the presence of Mg2+ + Na+ is different from the conformation seen in the presence of Mg2+ + K+, and that the conformation in the presence of Mg2+ + K+ is different from that induced by ouabain which from the point of view of eosin binding is identical to the conformation induced by vanadate. It is tempting to suggest from the effects of Na+, K+, ATP, ouabain, and vanadate on the two high-affinity eosin binding sites, which are seen in the presence of Mg2+, that they are ATP binding sites.

REFERENCES

Skou, J. C., and E s m a n n , M . (1981). Eosin, a fluorescent probe of ATP b i n d i n g to the (Na+ + K+)-ATPase. Biochirn. B i o p h y s . A c t a 6 4 7 , 232-240.

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CURRENT TOPICSIN MEMBRANES AND TRANSPORT. VOLUME 19

Interactionof Divalent Cations with Fluorescein-LabeledNa,K-ATPase MARCIA STEINBERG' AND JAMES G. KAPAKOS Department of Pharmacology State University of New York Upstate Medical Center Syracuse, New York

P A R M A C. SEN The Home1 Institute University of Minnesota Austin, Minnesota

I.

INTRODUCTION

Mg2+, in addition to its presumed role with ATP as the Mg-ATP substrate, is also an important regulator of the Na,K-ATPase. Acting through low affinity sites (X0.5 = 0.5-1 mM) , Mg2+ (1) inhibits nucleotide binding (Robinson and Flashner, 1979a); (2) raises the ~ 0 . 5for Na+ for enzyme phosphorylation and the Na-ATPase reaction (Flashner and Robinson, 1979); and ( 3 ) inhibits ADP/ATP exchange (Fahn et a l . , 1966). Mn2+ can partially substitute for Mg2+ in the Na,K-ATPase and K-phosphatase reactions and can itself support phosphatase activity in the absence of K+ (Robinson, 1981). ' P u b l i s h i n g p r i o r t o 1981 a s Marcia S. F l a s h n e r .

457

Copyright b 1983 by Academic Ress, Inc. All rights of repduction in any form reserved. ISBN 0-12-153319-0

MARCIA STEINBERG eta/.

458

We have studied the interaction of Mg2+ and Mn 2+ with Na,K-ATPase labeled with fluorescein isothiocyanate (FITC), a probe that has been used to distinguish the Ei(Na) and E2 (K) forms of the enzyme (Karlish, 1980). From the observed effects of Mn2+ and Mg2+ on (1) fluorescence changes due to K+ and Na+, (2) fluorescence in the absence of other ligands, and (3) fluorescence quenching produced by ouabain, we conclude that Mn2+ is a more potent selector than Mg2+ of the E2 conformational state of the Na,K-ATPase. A preliminary account of these studies is presented here.

11.

METHODS

The enzyme preparation was obtained from the outer medulla of dog kidney following Jbrgensen's procedure (1974). The specific activity was in the range of 15-25 umoles Pi/min mg protein. Protein was measured and labeling with 10 pM FITC was done at pH 9.0 for 30 min in the presence of 100 mM NaCl as previously described (Sen et al., 1981), except that the reaction was stopped by passage over Sephadex G-25. Fluorescence measurements were made with 20 pg/ml enzyme in 50 mM Tris, pH 7.5 at 25' on a Aminco-Bowman Ratio I1 spectrofluorimeter equipped with a magnetic stirring assembly. Excitation was at 490 nm and emission at 515 nm. 111.

RESULTS AND DISCUSSION

In contrast to Mg2+, which has almost no effect on fluorescence intensity, Mn2+ quenches the fluorescence of FITC-labeled enzyme in a saturable manner. A double reciprocal plot of the data is linear, with a calculated Kd(Mn) = 89 p M (Fig. 1). The maximal quenching in this experiment is 5%, about 30% of that produced maximally by K+. When the fluorescence quenching due to K+ and the subsequent reversal by Na+ were examined in the presence of divalent cations, the following results were obtained (Fig. 2) : (1) Both Mg2+ and Mn2+ reduce the K+-induced quenching (~0,5(Mg)= 0.32 mM, Mn2+ is a better inhibitor of the ~0.5 (Mn) = 99 pM) K+ quenching than Mg2+ (maximal inhibition for Mn2+ = 86%; for Mg2+ = 33%). (2) Both Mg2+ and Mn2+ in-

.

1.6

F i g . 1 . D o u b l e r e c i p r o c a l p l o t of the Mn2+-induced f l u o r e s c e n c e q u e n c h i n g . V a r i o u s concent r a t i o n s of MnClz w e r e added t o F I T C - l a b e l e d e n z y m e and the f l u o r e s c e n c e r e c o r d e d and corrected f o r d i l u t i o n . Inset, s a t u r a t i o n c u r v e for Mn2+ e f f e c t .

2+ 2+ + + F i g . 2 . S a t u r a t i o n c u r v e s f o r the e f f e c t s o f Mg and M n on the K and Na i n d u c e d f l u o r e s c e n c e c h a n g e s . V a r i o u s c o n c e n t r a t i o n s o f Mg2+ (A) or Mn2+ ( B ) were added t o F I T C l a b e l e d enzyme, f o l l o w e d b y a d d i t i o n of 20 mM K C I , then 75 mM NaCl, and the f l u o r e s c e n c e was recorded. Note t h a t Na+ p r o d u c e s an i n c r e a s e i n f l u o r e s c e n c e . T h e open circles r e g r e s e n t the r e d u c t i o n of t h a t i n c r e a s e by t h e d i v a l e n t c a t i o n s .

EFFECTS OF DIVALENTCATIONS ON (Na+ K)-ATPase

46 1

3.0

v)

m

-0 Y

'.-I / 0

2

4 [OUABAIN]

6

8

10

x

2+ 2+ Fig. 3. Effect of Mg and Mn on the pseudo-first-order rate constant (min-1) for ouabain-induced quenching of fl uorescence. Various concentrations of ouabain were added to FITClabeled enzyme in the presence of either 3.3 mM Mg2+ (0)or 1 mM Mn2+ (e), and the rate of fluorescence change recorded.

hibit the reversal of the K+ quenching by Na+, with ~ 0 . 5 values similar to those given above. In this case, however, the inhibition is similar with both divalent cations, i.e., about 9 0 -95 % of the reversal is abolished. Addition of ouabain to FITC-labeled enzyme has no effect on fluorescence 'n the absence of divalent cations. If, however, Mg3+ or Mn2+ is present, ouabain quenches the fluorescence to the same level as that produced by K+ (about -15-20%). These changes are dependent on ouabain concentration, with Kd values in the range of 0.5-3.0 x 10-6 M (results not shown). In contrast to the rapid fluorescence changes seen with Na+, K+, and Mn2+, the ouabain quenching is relatively slow, with half-times on the order of minutes, enabling us to follow the process continuously on a conventional fluorimeter. When the time course is examined

462

MARCIA STEINBERG et el.

in this way the following is observed. The rate of quenching is significantly greater with Nn2+ present than with Mg2+. The pseudo-first-order rate constant, k' is 2- to 3-fold higher with Mn2+ over a range of ouabain concentrations (Fig. 3). These results indicate that (1) the fluorescence changes seen are due to ouabain binding to the enzyme, ( 2 ) oua ain binds in the absence of phosphorylation, and ( 3 ) Mnb+ increases the rate of ouabain binding relative to Mg2+. Ca2+ also supports the fluorescence quenching by ouabain with about the same rate constants as seen with Mg2+ (results not shown). Taken together, these observations suggest that Mn2+ produces a state of the enzyme that is closer to EZ than does Mg2+. Both divalent cations anta onize sodium binding, an effect seen previously for Mg9+ in kinetic studies (Flashner and Robinson, 1979). Ouabain is believed to bind to an E2 or E2-P conformation (Robinson and Flashner, 197933); Mn2+ is shown here to facilitate this process, in accord with its effect of increasing the sensitivity of enzymatic activity to ouabain inhibition (Robinson, 1981). Hegyvary and Jdrgensen (1981) could distinguish an enzyme conforrnation (E2-P)sMg.ouabain with a very low fluorescence; FITC enzyme should thus be suitable for studying binding of ouabain to different conformations of the Na,K-ATPase.

ACKNOWLEDGMENT This work was supported by USPHS Grant GM-25033 and New York State Health Research Council Grant 9-058.

REFERENCES Fahn, S., Koval, G. J., and Albers, R. W. (1966). J . B i o l . Chem. 241, 1882-1889. Flashner, M. S . , and Robinson, J. D. (1979). A r c h . B i o c h e m . B i o p h y s . 192, 584-591. Hegyvary, C . , and Jbrgensen, P. L. (1981). J. B i o l . C h e m . 2 5 6 , 6296-6303. Jbrgensen, P. L. (1974). B i o c h i m . B i o p h y s . A c t a 356, 36-52. Karlish, S. J. D. (1980). J . B i o e n e r g . B i o m e m b r . 1 2 , 111-136. Robinson, J. D. (1981). B i o c h i m . B i o p h y s . A c t a 642, 405-417. Robinson, J. D., and Flashner, M. S. (1979a). In "Na,K-ATPase:

EFFECTS OF DIVALENT CATIONS ON (Na+ K)-ATPase

463

S t r u c t u r e and K i n e t i c s " (J. C. Skou and J. G . ~ + r b y ,e d s . ) , pp. 275-285. Academic P r e s s , New York. Robinson, J. D . , and F l a s h n e r , M. S . (197913). B i o c h i m . Biophys. Acta 549, 145-176. Sen, P. C., Kapakos, J. G . , and S t e i n b e r q , M. (1981). Arch. Biochem. Biophys. 211, 652-661.

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CURRENT TOPICS IN MEMBRANES AND TRANSPORT, VOLUME 19

Cation Activation of Na,K-ATPase after Treatment with Thimerosal MANISHA D. MONE AND JACK H.KAPLAN Department of Physiology University of Pennsylvania Philadelphia, Pennsylvania

I.

INTRODUCTION

The importance of sulfhydryl groups in maintaining the functional integrity of the Na,K-ATPase has been well established since the work of Fahn et a l . (1966). These workers showed that modification of one set of -SH groups on the enzyme by N-ethylmaleimide leads to specific changes in the functional catalytic properties of the Na,K-ATPase. It was shown that the treatment with N-ethylmaleimide blocks the usual sequential transition from phosphoenzyme which is ADP sensitive and K insensitive (ElP) to one which is K sensitive and ADP insensitive (E2P). The major consequence of this modification is, along with inhibition of Na,K-ATPase activity, an increase in the partial reaction which depends upon the level of ElP, i.e., ATP/ADP exchange. More recently the effects of another class of -SH group reagents, organomercurials, have been studied and apparently a different subset of -SH groups on the enzyme is involved. Henderson and Askari (1976, 1977) have 465

Copyright 0 1983 by Academic Press, Inc All rights of reproduction In any form reserved ISBN 0.12- I533 I94

MANISHA D.MONE AND JACK H. KAPIAN

466

shown that treatment of Na,K-ATPase enzyme with thimerosal (ethylmercurithiosalicylate) results in a significant inhibition of the overall Na,K-ATPase activity, without significantly inhibiting Na-ATPase, ATP:ADP exchange, or K-stimulated p-nitrophenylphosphatase (PNPPase) activities. In fact the last of these showed some stimulation. Furthermore, these workers claimed that the normal-complex Na-activation kinetics seen in the Na-ATPase activity, became monotonic after mercurial modification. Their interpretation was that mercurial modification disrupted the normal interchain protein interactions and that the dissociated chains show simple noninteractive cation-activation effects. We have reinvestigated the properties of the thimerosalmodified enzyme, and focused our attention on a cationindependent, ouabain-sensitive activity that is produced (or enhanced?) by mercurial treatment.

11.

RESULTS AND DISCUSSION

Treatment of a partially purified prepar tion of Na,K-ATPase from canine renal outer medulla with 10 mM thimerosal for 10 min (OOC) results in about 85-95% inhibition of the Na,K-ATPase activity, with about 10% inhibition of the Na-ATPase activity (measured with 100 mM Na), 5% inhibition of ATP:ADP exchange activity (also with 100 mM Na) and a stimulation (about 40%) of K-stimulated PNPPase activity (measured with 5 0 mM K). Treatment with dithiothreitol reverses the inhibition of Na,K-ATPase activity, and all other activities return to the same levels as untreated enzyme. When the cation concentration-dependence of these activities is examined and compared to that of untreated enzyme, a somewhat unexpected effect is seen. In Fig. 1 the Na dependence of the ATPase activity of the untreated enzyme is compared with that of the enzyme after modification with thimerosal. Apparently, although the activity in the range 20-140 m~ Na is not greatly altered, a new activity is introduced which does not depend upon the presence of Na ions. A similar effect is seen when the Na-dependence of the ATP:ADP exchange is examined. The unmodified enzyme shows a triphasic dependence on Na ions, as previously described (Beaugtj and Glynn, 1979; Kaplan et al., 1981; Kaplan and Hollis, 1980), while the thimerosal-treated enzyme shows an enhanced activity in the absence of Na, which is inhibited on the addition of Na ions up to about 10 mM Na; at

CATION ACTIVATION OF Na,K-ATPaseWITH THIMEROSAL

467

MOLES R/MG/MIN

mM Na 0.

F i g . 1 . Na-ATPase a c t i v i t y of t r e a t e d 0 a n d control e n z y m e A s s a y e d a t 37OC w i t h 2 mM A T P , 1 mM Mg.

higher Na concentrations, the treated and untreated enzymes show similar activities. Recent work which has established the sidedness of the effects of Na in this partial reaction would suggest that the extracellular Na sites are unaltered as a result of thimerosal modification (Kaplan and Hollis, 1980). The affinity of the unmodified enzyme for ATP in the Na-ATPase reaction is high, ~ 9 . 5for ATP is 2-3 P M in the presence of 100 mM Na. This high affinity is also observed with the modified enzyme under the same conditions. However, in the absence of added Na ions (where the total Na in the system is less than 0.1 mM), the modified enzyme has a ~ 0 . 5 for ATP of about 90 U M . The most parsimonious interpretation of these observations is that rather than introducing a completely new site for ATP hydrolysis, thimerosal treatment modifies a preexisting site for ATP. One possibility is that the nonhydrolytic low-affinity binding site for ATP on E2, usually associated with the release of occluded K, in the modified enzyme hydrolyzes ATP with a relatively low affinity for ATP in the absence of Na ions. The pattern of K-activation of the PNPPase activity also

468

MANISHA D. MONE AND JACK H.KAPLAN

suggests that modifications to the E 2 forms of the enzyme have occurred. The overall shape of the K-activation curve of the PNPPase activity is similar in control and thimerosal-modified enzyme. The stimulation of the PNPPase activity referred to above can entirely be accounted for by the appearance of a ouabainsensitive, K-independent PNPPase activity following treatment with thimerosal. The question of the extent to which the properties of the thimerosal-modified enzyme shed light on the reaction mechanism of the Na-pump protein must await further work. However, it is clear that these profound changes in cation-activation kinetics are brought about by modification of a small number of -SH groups. Preliminary studies using DTNB to measure the -SH group content of the enzyme have shown that the SDSsolubilized enzyme contains 32-35 -SH groups per 250,000 daltons. After treatment with thimerosal, 4-5 of these have been modified.

ACKNOWLEDGMENTS

T h i s work w a s supported by Grant i n Aid 79787 from t h e American Heart A s s o c i a t i o n , and N I H HL 28457.

REFERENCES Beaug6, L. A . , and Glynn, I. M. (1979). Sodium i o n s , a c t i n g a t h i g h a f f i n i t y e x t r a c e l l u l a r s i t e s , i n h i b i t sodium-ATPase a c t i v i t y of t h e sodium pump by slowing dephosphorylation. J. P h y s i o l . (London) 2 8 9 , 17-31. Fahn, S . G . , Koval, G. , and A l b e r s , R. W. (1966). Sodium-potassiuma c t i v a tea adenos i n e t r i p ho s p h a t a se o f e l e c t ropho ru s e lec t r i c organ. I. An a s s o c i a t e d sodium-activated t r a n s p h o s p h o r y l a t i o n J. B i o l . Chem. 2 4 1 , 1882-1889. Henderson, G. R . , and A s k a r i , A. (1976). T r a n s p o r t ATPase: Thimerosal i n h i b i t s t h e Na+K-dependent ATPase a c t i v i t y w i t h o u t d i m i n i s h i n g t h e Na-dependent A T P a s e a c t i v i t y . B i o c h e m . B i o p h y s . R e s . Commun. 6 9 , 499-505. Henderson, G. R., and A s k a r i , A. (1977). T r a n s p o r t ATPase: F u r t h e r s t u d i e s on t h e p r o p e r t i e s of t h e t h i m e r o s a l t r e a t e d enzyme. A r c h . B i o c h e m . B i o p h y s . 1 8 2 , 221-226.

.

CATION ACTIVATION OF Na,K-ATPasr WITH THIMEROSAL

469

Kaplan, J. H., and Hollis, R. J. (1980). External Na dependence of ouabain-sensitive ATP:ADP exchange initiated by photolysis of intracellular caged-ATP in human red cell ghosts. Nature (London) 288, 587-589. Kaplan, J. H., Hollis, R. J., and Mone, M. D. (1981). The regulation of Na pump-mediated ATP:ADP exchange by extracellular Na ions. Adv. Physiol. Sci., Proc. Int. Conqr., 2 8 t h , 1 9 8 0 , Vol. 6, pp. 293-298.

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CURRENT TOPICS IN MEMBRANES AND TRANSPORT. VOLUME 19

Alteration of Conformational Equilibria in Na,K-ATPase by Glutaraldehyde Treatment DAVID M . CHIPMAiV, E. ELHAiVAiVY, R. BERGER, AND A . LEV Department of Biology Ben Gurion University Beer S h e w . Israel

I.

INTRODUCTION

The Na,K-ATPase has multiple, interacting ligand sites whose kinetic expression is complicated by conformational transitions. The experiments reported here were designed to clarify the behavior of some of the monovalent cation sites, by shifting the protein conformational equilibrium strongly to one side or the other. Since the enzyme conformations El and E2 presumably differ in the configurations of peripheral groups (J6rgensen, 1 9 7 5 ) as well as of the active site, cross-linking of such groups with a bifunctional reagent should change the relative stabilities of the two conformations (Nucci et a l . , 1978; Enns and Chan, 1978). The properties of microsomal electroplax preparations after treatment with glutaraldehyde in media containing Na+ or K+ support this hypothesis.

47 1

Copynght Q 1983 by Academic Prerr. Inc. All riphraof repruducrioninrny form rewrvd. ISBN 0-12-153319-0

DAVID M. CHIPMAN eta/.

472

+

t

TABLE I. Effect of Glutaraldehyde Treatment in Na -and K -rich Media Activity after glutaraldehyde treatmenta Reaction

in 30 mM KC1 t10 mM P;

in 100 mM NaCl +3 mM ATP

Na,K-ATPase c p-Nitrophenylphosphatase d Dinitrophenylphosphatase e ADP-ATP exchange f Phosphorylation from [Y-~~P]ATP

10% 56% 57% 40% 40%

17% 27% 21% 60% 70%

b

a Microsomal enzyme from T o r p e d o ocellata electroplax (5 mg/ml) treated 20 min at 25OC with 0.035% glutaraldehyde in 100 mM imidazole buffer (pH 7.0) + 1 mM EDTA and indicated additional ligands. The reaction was stopped with borohydride, and enzyme was spun down and resuspended twice in sucrose-EDTA. Controls were treated identically except that glutaraldehyde was added after borohydride. Aliquots were taken for assays as indicated, at 25'C, pH 7.0. b3 mM ATP, 100 mM NaC1, 20 mM KC1, 3 mM MgC12, 60 mM imidazole. c5 mM PNPP, 30 mM KC1, 10 mM MgC12, 100 mM imidazole. d0.6 mM DNPP, 40 mM RbC1, 10 mM MgC12, 200 mM imidazole. e1.25 mM [3H]ADP, 5 mM ATP, 100 mM NaC1, 0.15 mM MgC12, 200 mM imidazole. =20 pM [Y-~~PIATP, 100 mM NaC12, 5 mM MgC12, 60 mM imidazole, PH 7.5, O ' C for 20 sec.

11.

RESULTS AND DISCUSSION

The kinetics of the disappearance of Na,K-ATPase and K+-dependent p-nitrophenyl phosphatase (K-PNPPase) activity on exposure to glutaraldehyde are complex, and depend on the ligands present in the medium. Under all conditions Na,K-ATPase is lost more rapidly. ATP or inorganic phosphate afford only partial protection against glutaraldehyde inactivation, even at rather high concentrations. Table I compares the catalytic properties of enzyme treated with glutaraldehyde in K+-rich and Na+-rich media. The persistence of ADP:ATP exchange activity and phosphorylation from ATP suggests that the nucleotide sites are not destroyed by glutaraldehyde treatment, and

ALTERATION OF CONFORMATIONALEQUILIBRIA

473

4-

F i g . 1 . K d e p e n d e n c e o f PNPPase a c t i v i t y of control enz y m e ( o p e n s y m b o l s ) a n d e n z y m e t r e a t e d ( a t 9 mg p r o t e i n / m l ) w i t h 0.03% g l u t a r a l d e h y d e ( c l o s e d s y m b o l s ) i n v a r i o u s m e d i a : V, A , 30 mM KCl; , 0 , 100 mM N a C l + 3 mM A T P . N a t i v e e n z y m e ( 0 ) a n d t w o controls a r e f i t t o the s a m e c u r v e .

requires an alternative explanation for the preferential loss of Na,K-ATPase activity. The ligand dependence of the reactions catalyzed by the enzyme sheds light on the nature of the modification. Figure 1 shows the [K+] dependence of the PNPPase activity before and after glutaraldehyde treatment. The apparent affinity for K+ and the cooperativity of the activation are dramatically altered after glutaraldehyde treatment in KC1. Similar results (not shown) are obtained for Rb+ activation of the dinitrophenylphosphatase (DNPPase) reaction. The [K+] dependence of the residual Na,K-ATPase activity (not shown) is unchanged, however. Some kinetic parameters for the enzyme treated in the presence of K+ are given in Table 11. Although the glutaraldehyde reaction is complex, it appears that the same process which leads to the change in the [K+] dependence of the phosphatase activities is responsible for the loss of Na,K-ATPase activity. Figure 2 compares the time courses of the processes in question. The behavior of the enzyme treated with glutaraldehyde in the presence of KC1 (Table21 can be rationalized if the major effect of the reagent is to shift the conformational equilibrium toward E2. The [ K + ] or [a+]

474

DAVID M. CHIPMAN eta/.

TABLE 11. Some Kinetic Properties of Enzyme Cross-linked in the Presence of K+ a b

Reaction

Control

Glutaraldehyde-treated in KC1

p-Nitrophenylphosphatase Relative activity ~ 0 . 5 (K+) nH (K+)

100% 4.5 mM 1.9

60-90% 0.5-0.7 mM

1.0

Dinitrophenylphosphatase

Relative activity ~ 0 . 5 (m+) "H (Rb+)

100% 4.5 mM 2.0

60% 0.33 mM 1.0

ADP-ATP exchange Relative activity

100%

40%

Na,K-ATPase Relative activity K0.5 (K+) "H (K+) Calculated turnover number per phosphorylation site

100% 0.55 mM 1.0

10% 0.5 f . 2 mM 1 f .3

35 sec-l

a

Enzyme treated in 30 mM KC1, 10 inM phosphate as described + in Table I. Residual K in resuspended enzyme samples measured by flame emission spectrometry results in 5 4 X M extraneous K+ in the assays. bSee Table I for assay conditions. CThe PNPPase activity was a l s o examined after treatment under a wide range of glutaraldehyde/protein ratios, and the range of results indicates this.

dependence of the reactions of the treated and native enzymes can be understood on the basis of a scheme involving a single type of K+ site for activation of all the reactions. ~ 0 . 5for the phosphatase activity in the altered enzyme probably reflects the intrinsic K+affinity of the E form. In the native enzyme the conformational equilibrium favors El (Karlish et a l . , 1978), and leads to a high apparent ~ 0 . 5and to cooperativity for K+ activation of the phosphatase reactions.

ALTERATION OF CONFORMATIONAL EQUILIBRIA

1.0

>-

t L

6 U

5 W

.

475

-1.0

; I I

m

57

'm

m

0.5

W

a

0.75mMx+

No.K-ATmw

0

-0.59

2

1

D

0

3

3

F i g . 2 . Comparison of the t i m e c o u r s e f o r l o s s o f ATPase a c t i v i t y and c h a n g e s i n the & d e p e n d e n c e of PNPPase a c t i v i t y on g l u t a r a l d e h y d e t r e a t m e n t . Enzyme ( 2 . 4 m g / m l ) was t r e a t e d w i t h 0.24% g l u t a r a l d e h y d e i n 30 mM KCI and b u f f e r a t pH 7 . 0 . Aliquots quenched a t v a r i o u s times were examined for Na,K-ATPase a c t i v i t y ( 0 ) and PNPPase a c t i v i t y a t 30 mM K+ ( m ) and 0.75 mM K+ (0). I n the r i g h t - h a n d p a n e l ATPase a c t i v i t y i s r e p l o t t e d a s a f u n c t i o n o f the r a t i o of PNPPase a c t i v i t i e s a t 0.75 and 30 mM K+.

Under the conditions for Na,K-ATPasz activity, on the other hand, the interconversion El E2(K) is not at equilibrium (Cantley, 1981). Potassium is bound via E2P ( resumably a rapidly reversible process) and ~ 0 . 5 for 'K activation of ATPase activity in both native and altered enzymes reflects the intrinsic affinity of this state of the enzyme.

ACKNOWLEDGMENT This work was supported by the Commission for Basic Research, Israel Academy of Sciences.

REFERENCES Cantley, L. C. (1981). Structure and mechanism of the (Na,K)ATPase. C u r r . Top. B i o e n e r g . 2 , 201-237. Enns, C. A., and Chan, W. W. C. (1978). Stabilization of the relaxed state of aspartate transcarbamylase by modification

476

DAVID M.CHIPMAN eta/.

with a bifunctional reagent. J. B i o l . Chern. 2 5 3 , 2511-2513. Jplrgensen, P. L. (1975). Purification and characterization of (Na+,K+)-ATPase. V. Conformational changes in the enzyme. Transitions between Na-form and K-form studied with tryptic digestion as a tool. B i o c h i r n . B i o p h y s . Acta 4 0 1 , 399-415. Karlish, S. J. D., Yates, D. W., and Glynn, I. M. (1978). Conformational transitions between Na+-bound and K+-bound forms of (Na+ + K+)-ATPase, studied with formycin nucleotides. B i o chirn. B i o p h y s . Acta 5 2 5 , 252-264. Nucci, R., Raia, C. A., Vaccaro, C., Sepe, S., Scarano, E., and Rossi, M. (1978). Freezing of dCMP aminohydrolase in the activated conformation by glutaraldehyde. J . Mol. B i o l . 1 2 4 , 133-145.

CURRENT TOPICS IN MEMBRANES AND TRANSWRT. VOLUME 19

Conformational Transition between ADP-Sensitive Phosphoenzyme and Potassium-Sensitive Phosphoenzyme KAZUYA TANIGUCHI, KUNIAKI SUZUKI, AND SHOICHI IIDA Department of Pharmacology Hokkuido University School of Dentistry Supporo, J a p n

I.

INTRODUCTION

Numerous experimental evidence has been accumulated to show that Na+,K-ATPase can exist in at least two conformations, E l and E2 (Glynn and Karlish, 1975; Jgkgensen and Karlish, 1980; Glynn et a l . , 1979; Karlish, 1979; Stahl and Harris, 1979; Post, 1979; Askari et a l . , 1980). To understand better the mechanism of energy coupling between hydrolysis of ATP and Na/K transport, it is very important to ascertain the conformational differences of various reaction intermediates. The difference between ADP-sensitive phosphoenzyme (ElP) and K-sensitive phosphoenzyme (E2P) is especially interesting, because synthesis of ATP (Taniguchi and Post, 1975) has been reported with the phosphoenzyme formed from Pi (via E1P). We (Taniguchi et a l . , 1980) reported that ~-[p-(2-benzimidazolyl)phenyllmaleimide (B1PM)-treated Na,K-ATPase preparations were sufficiently suitable for following conformational states of Na,K-ATPase accompanying ATP hydrolysis. In 477

Copynght 0 1983 by Academic Pres. Inc All rightsof reproduction in any form reserved ISBN 0-12-I53319-0

KAZUGA TANIGUCHI eta/.

470

this paper we measured the change in the fluorescence intensity induced by various ligands of Na,K-ATPase. ATP-induced transient changes in fluorescence intensity (Taniguchi et a l . , 1980) were related to measurements of the amount of phosphoenzyme at varying concentrations of Na+ and to the sensitivity of the phosphoenzyme to ADP and K+. It is known that high concentrations of Na+ shift the equilibrium between E1P and E2P to the former (Taniguchi and Post, 1975; Post, 1977).

11.

RESULTS AND DISCUSSION

ATP induced a reversible increase or decrease in the fluorescence of treated enzyme in the resence of Mg2+ with low or high concentrations of Na , respectively (Table I). Addition of one or two of these ligands simply reduced the fluorescence. Addition of the third ligand induced remarkable changes which could not be predicted from the sum of the negative fluorescence changes induced by each ligand. ADP could not replace ATP in inducing these changes. When ATP was added to the treated Preparation in the presence of 0.4 mM Mg with 16 mM Na , E2P was produced, and the fluorescence transiently increased to the highest level. After exhaustion of ATP, the fluorescence decreased to the original level. This cycle could be repeated by the readdition of ATP. The time-dependent decrease in the fluorescence intensity was shown to be due to the decrease in the amount of E2P. Addition of ouabain to E2P completely stabilized the fluorescence at the highest level which was equal to that of E2P. On the other hand, when ATP was added to the preparation in the presence of 0.4 mM Mg with 2 M Na+, E1P was produced and the fluorescence decreased to the lowest level. After exhaustion of ATP the fluorescence increased to the original level. This cycle also could be repeated by the readdition of ATP. The time-dependent increase in the fluorescence was shown not only to be due to the decrease in the amount of EiP, but also to the increase in the amount of a nonphosphorylated intermediate which seemed to be produced significantly in the simultaneous presence of Mg2+, ATP, ADP, and high concentrations of Na+. Addition of ouabain to E1P increased the fluorescence from the lowest to the highest level which was equal to that of E2P. These findings and others indicate that the transition of E1P to E2P is accompanied by the largest conformational changes of any

P

ADP- AND POTASSIUM-SENSITIVEPHOSPHOENNME

TABLE I.

F l u o r e s c e n c e Change Induced by L i g a n d ( s ) of Na,K-ATPase

Mg:

0.43

0

0

0

Na:

Control +ATP +ADP +ADP+K +ATP +ouabain

479

0.0 -0.520.2 '-0.4

0.43

0

16 -0.2kO.1 -0.8 -0.6

-2.120.4 -3.2 -3.2

-2.920.4 -0.620.5 -3.2f0.5 -1* 220.2 -0.6f0.5

a

0.45 2000

0.0"

-0.5* -0.3*

o.o* -2.320.3* -0.7*

+4.720.1*

The p r o t e i n (70 pg) w a s suspended i n 7 m l of a s o l u t i o n cont a i n i n g 20 mM imidazole-HC1 (pH 7.4) , 0 . 1 mM EDTA-Tris and 25 mM The sample and r e f e r e n c e c e l l s s u c r o s e w i t h o r w i t h o u t 2000 mM N a + . c o n t a i n e d 3.2 m l o f t h e s u s p e n s i o n . The l i g a n d s f u r t h e r added were 1 2 . 8 )11 o f 2 M Na', 3 p 1 o f 30.1 mM ATP-Tris, 3 l.11o f 30.1 mM ADPT r i s , 2 l.11o f 686.4 mM MgC12, 4 p 1 o f 1 . 6 M KC1, and 100 p l o f 1 5 m ouabain f o r t h e sample c e l l . The same volume o f w a t e r w a s added t o t h e r e f e r e n c e c e l l t o m a i n t a i n c o n s t a n t sample volumes i n b o t h c e l l s . The d a t a shown are % v a l u e s ; 100% v a l u e s o f t h e f l u o r e s c e n c e i n t e n s i t y were t a k e n from t h e d i f f e r e n c e between t h e f l u o r e s c e n c e i n t e n s i t y a t 365 nm and t h a t a t 500 nm o f t h e r e f e r e n c e sample i n t h e absence o f b o t h Mg and N a + o r i n t h e p r e s e n c e o f 2000 mM Na+(*) I n t h e p r e s e n c e o f Mg, N a and ATP, t h e f l u o r e s c e n c e i n t e n s i t y changed w i t h time as d e s c r i b e d i n t h e t e x t . The d a t a shown are t h e v a l u e s o b s e r v e d immediately a f t e r t h e a d d i t i o n o f t h e t h i r d l i g a n d .

.

elementary steps examined. We also compared the fluorescence change of BIPM-treated enzyme with tryptophan fluorescence of native enzyme (Karlish, 1979). The results suggest the microenvironments of tryptophan and sulfhydryl residues are similar in E2P but different in E1P.

ACKNOWLEDGMENT

Supported by G r a n t from t h e M i n i s t r y o f E d u c a t i o n S c i e n c e and C u l t u r e of J a p a n .

480

KAZUGA TANlGUCHl eta/.

REFERENCES

+ +

Askari, A,, Huang, W., and Antieau, J. M. (1980). Na ,K -ATPase; ligand-induced conformational transitions and alterations in subunit interactions evidenced by cross-linking studies. Biochemistry 19, 1132-1140. Glynn, I. M., and Karlish, S. J. D. (1975). The sodium pump. Annu. Rev. Physiol. 37, 13-55. Glynn, I. M., Karlish, S. J. D., and Yates, D. W. (1979). The use of formycin nucleotides to investigate the mechanism of N a ' , K+-ATPase. In "Na,K-ATPase: Structure and Kinetics" (J. C. Skou and J. G. Nbrby, eds.), pp. 101-113. Academic Press, New York. J$rgensen, P. L., and Karlish, S. J. D. (1980). Defective conformational response in a selectively trypsinized (Na++K+)ATPase studied with tryptophan fluorescence. Biochim. Biophys. Acta 597, 305-317. Karlish, S. J. D. (1979). Cation induced conformational states of Na+, K+-ATPase studied with fluorescent probes. In "Na,KATPase: Structure and Kinetics" (J. C. Skou and J. G. Nbrby, eds.), pp. 115-128. Academic Press, New York. Post, R. L. (1977). Titration of sodium against potassium by their action on (Na+, K+) transport ATPase. FEBS-Symp. 42, 352-362. Post, R. L. (1979). A perspective on sodium and potassium ion transport adenosine triphosphatase. In "Cation Flux Across Membranes" (Y. Mukohata and L. Packer, eds.), pp. 3-11. Academic Press, New York. Stahl, W. L., and Harris, W. E. (1979). A fluorescent sulfhydryl probe for studying conformational changes of the Na+, K+ATPase. In "Na,K-ATPase: Structure and Kinetics" (J. C. Skou and J. G. Ndrby, eds.), pp. 158-167. Academic Press, New York. Taniguchi, K., and Post, R. L. (1975). Synthesis of adenosine triphosphate and exchange between inorganic phosphate and adenosine triphosphate in sodium and potassium ion transport adenosine triphosphatase. J. Biol. Chem. 250, 3010-3018. Taniguchi, K., Suzuki, K., and Iida, S. (1980). ATP dependent reversible conformational change of Na+, K+-ATPase modified with N-(p-(2-Benzimidazolyl)phenyl)maleimide. J. Biochem. (Tokyo) 88, 609-612.

CURRENT TOPICS IN MEMBRANES AND TRANSPORT, VOLUME 19

Relation between Red Cell Membrane Na,K-ATPase and Band 3' ERIC T. FOSSEL AND A. K. SOLOMON Biophysical Laborarory Harvard Medical School Boston, Massachusetts

I.

INTRODUCTION

Evidence has previously been presented (Fossel and Solomon, 1977, 1979) which shows a functional linkage between the Na,K-ATPase which spans the human red cell membrane and two intracellular glycolytic enzymes, glyceraldehyde-3-phosphate dehydrogenase (G3PDH) and monophosphoglycerate mutase (MPGM). We have further suggested that the three components [Na,K-ATPase/band 3/G3PDH] are linked together so closely that conformational information can be transferred through this complex from the outside of the cell to the inside. The present experiments are designed to establish the role of band 3 as the protein responsible for information transfer between the extracellular ouabain binding site of the Na,K-ATPase and the cytoplasmic glycolytic enzymes. ' T h i s c o m m u n i c a t i o n i s a p r e c i s of r e s u l t s g i v e n i n a p a p e r by E . T . Ebbssel and A . K . Solomon ( 1 9 8 1 ) . B i o c h i m . B i o p h y s . A c t a 6 4 9 , 557-571. 48 1

Copyright 0 1983 by Academic Press. Inc. All right5 of reproduction in any form reserved. ISBN 0-12-153319-0

482

11.

ERIC T. FOSSEL AND A. K. SOLOMON

RESULTS AND DISCUSSION

In view of the presence of binding sites for G3PDH and aldolase on band 3 (Strapazon and Steck, 1976; McDaniel et al., 1974; Kliman and Steck, 1980), we first carried out experiments to determine whether conformational information could be transferred between band 3 and the glycolytic enzymes, using the 31P resonance shift of 2,3-diphosphoglycerate (2,3-DPG) to report on the conformation of MPGM for which 2,3-DPG is an obligatory substrate. The inhibitor, 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid (DIDS) is a specific inhibitor of anion transport in band 3. Treatment of red cells with DIDS affects the resonance of 2,3-DPG as does treatment with the cardiac glycoside cation transport inhibitor, ouabain, which is specific to the Na,K-ATPase. The DIDS experiments show that band 3 is coupled to the glycolytic enzyme system. Resonance shifts induced by the cardiac glycoside alone are modulated by addition of the anion transport inhibitor, which indicates that there is also coupling in the red cell between the Na,K-ATPase and band 3, as indicated in Fig. 1. In a further study of the linkage between the Na,K-ATPase and band 3, band 3 was separated from the membrane and partially purified following the technique of Yu and Steck (1975). This partially purified preparation was shown to exhibit a small amount of Na,K-ATPase activity by measurements of ouabain-inhibitable dephosphorylation of ATP. When G3PDH was added to this partially purified band 3 preparation, addition of cardiac glycosides caused shifts in the 31P resonance of glyceraldehyde 3-phosphate, which reports on the G3PDH conformation. These experiments indicate that there is coupling between the Na,K-ATPase and band 3 in the separated preparation and suggest that the anion and cation transport systems may be closely related spatially and functionally in the intact red cell. The existence of enzyme complexes linking ion transport to the metabolic enzymes that form ATP is not limited to red cells and may be of general occurrence in nature. In cardiac muscle, Grosse et al. (1980) have found an interaction between the Na,K-ATPase and creatine phosphokinase that exhibits many of the properties that characterize the enzyme complex in the human red cell. As these authors point out, there are significant advantages in efficiency from the apposition of the energy production and transport steps in cardiac muscle which, of course, apply equally to the red cell. In the case of the red cell, Parker and Hoffman (1967) and Mercer and

RED CELL MEMBRANE Na,K-ATPaseAND BAND 3

Fig. 1 .

483

schematic arrangement of some red cell components.

Dunham (1981) have shown that the pump ATP is in a pool segregated from the rest of the ATP in the cytoplasm. The juxtaposition of the glycolytic enzyme complex and the Na,K-ATPase could place the phosphoglycerate kinase which is thought to produce the pump ATP close to the phosphorylating site on the ATPase. The anion transport properties of band 3 could deliver the Pi to the site where it is needed for the phosphorylation reaction.

404

ERIC T. FOSSEL AND A. K. SOLOMON

ACKNOWLEDGMENTS This research has been supported in part by Grant 5 RO1 GM 15692 from the National Institutes of Health, in part by a grantin-aid from the American Heart Association, 76-900, and in part by Contract C-670 from the National Science Foundation. This work was done during the tenure of one of us (E.T.F.) of an Established Investigatorship of the American Heart Association.

REFERENCES

.

Biochirn. B i o p h y s . A c t a Fossel, E. T., and Solomon, A. K. 1977) 4 6 4 , 82-92. Fossel, E. T., and Solomon, A. K. 1979). B i o c h i r n . B i o p h y s . A c t a 5 5 3 , 142-153. Grosse, R., Spitzer, E., Kupriyanov, V. V., Saks, V. A., and Repke, K. R. H. (1980). B i o c h i r n . B i o p h y s . A c t a 6 0 3 , 142-156. Kliman, H. J., and Steck, T. L. (1980). J. B i o l . C h e m . 2 5 5 , 6314-6321. McDaniel, C. F., Kirtley, M. E., and Tanner, M. J. A. (1974). J. B i o l . C h e m . 2 4 9 , 6478-6485. Mercer, R. W., and Dunham, P. €3. (1981). F e d . P r o c . , F e d . Am. SOC. Exp. B i o l . 4 0 , 613. Parker, J. C., and Hoffman, J. F. (1967). J. G e n . P h y s i o l . 5 0 , 893-916. Strapazon, E., and Steck, T. L. (1976). B i o c h e m i s t r y 1 5 , 14211424. Yu, J., and Steck, T. L. (1975). J. B i o l . C h e m . 2 5 0 , 9170-9175.

Part VI

Reaction Mechanism and Kinetic Analysis

This Page Intentionally Left Blank

CURRENT TOPICS IN MBMBRANES AND TRANSPORT. VOLUME 19

Kinetic Analyses and the Reaction Mechanism of the Na,K-ATPase JOSEPH D. ROBINSON Department of Pharmacology State University of New York Upstate Medical Center Syracuse, New York

I.

INTRODUCTION

With t h e Na,K-ATPase, a s i n t h e s t u d y o f most enzymes, k i n e t i c a n a l y s e s have s u g g e s t e d p l a u s i b l e react i o n schemes d e r i v e d from e x a m i n a t i o n o f s u b s t r a t e , p r o d u c t , a c t i v a t o r , and i n h i b i t o r i n t e r a c t i o n s . Such e a r l y s t u d i e s , c o u p l e d w i t h more r e c e n t measurements o f l i g a n d b i n d i n g t o t h e enzyme, have l e d t o a formul a t i o n ( F i g . 1) m o d i f i e d i n o n l y one s i g n i f i c a n t f e a t u r e from t h e o r i g i n a l A l b e r s - P o s t scheme proposed a decade and a h a l f ago. T h i s f o r m u l a t i o n i s p r e s e n t e d h e r e b o t h a s a n exemplary e f f o r t t o accommodate t h e v a r i o u s phenomena d e s c r i b e d o v e r t h e y e a r s , and as a s p e c i f i c i n t e r p r e t a t i o n a g a i n s t which c o n f l i c t i n g obs e r v a t i o n s and c o n s t r u c t s c a n be p i t t e d . For t h i s purpose a b r i e f d e s c r i p t i o n o f t h e scheme i s p r e s e n t e d i n t h e n e x t s e c t i o n , which i s t h e n f o l l o w e d by a c o l l e c t i o n of c o n t r a d i c t o r y i n t e r p r e t a t i o n s , p o s s i b l e anomalies, and o b s e r v a t i o n s c o n s i d e r e d d i f f i c u l t t o i n c o r p o r a t e w i t h i n t h e framework o f t h e proposed model. D e t a i l e d 485

Copynght 0 1983 by Academic Press, Inc. All rights of reproduction in any form resewed. ISBN 0-12-153319-0

JOSEPH D. ROBINSON

486

presentations of the experimental support for this scheme are available in recent reviews by Glynn and , Cantley Karlish (1975), Robinson and Flashner (1979~) (1981), and Schuurmans Stekhoven and Bonting (1981). Before such an analysis, however, two cautionary notes should be emphasized. First, it is commonly acknowledged that kinetic analysis can exclude certain reaction mechanisms, rather than prove an alternative. Even this interpretation may be too optimistic. With a complex reaction process the presumed constraints of one model may be relieved merely by an ancillary adjustment. This is well illustrated by an earlier debate over whether the transport kinetics ruled out a ping-pong (or, in transport terminology, "consecutive") scheme for Na+-then-K+ transport; Sachs (1980) showed that a plausible modification of the kinetic scheme, providing a minor parallel pathway for uncoupled Na+ efflux, allowed the major pathway to enjoy the pingpong mode while fitting the observed data. Second, the concern about anomalies--about observations that seem inconsistent with the current formulation--should be tempered by the realization that anomalies are a common companion of scientific practice (Kuhn, 1970). Although such inconsistencies may indicate the need for a major revision, apparent anomalies may also reflect merely a circumscribed defect readily accommodated by minor readjustments.

11.

A MODIFIED ALBERS-POST REACTION SCHEME

The reaction schemes originally formulated by Albers (1967) and by Post (Post et a l . , 1969) proposed two forms of the phosphorylated enzyme, El-P and E2-P. El-P was readily dephosphorylated by ADP but not water, as exemplified in Na+-activated ADP/ATP exchange, and thus exhibited high available free energy. Ez-P, on the other hand, was readily dephosphorylated by water in the presence of K+, but not by ADP; this lower state of available free energy was subsequently emphasized by demonstration of rapid equilibration between Pi and

E2-P .

Addition of two dephosphorylated forms for symmetry, El and E2, led to the cyclic scheme:

KINETIC ANALYSES AND REACTION MECHANISM

E2

487

+E2-P

This is the essence not only of the Albers-Post proposal, but also, as Jencks (1980; this volume) has pointed out, of the general scheme for coupled vectorial processes. For a rapid overall reaction rate it is essential that the various intermediate forms be present, under physiological conditions, at more or less comparable concentrations and energy levels, otherwise the very stable states would predominate. With this formulation of the Na,K-ATPase reaction scheme the two horizontal steps represent rapid equilibrium forms but with different chemical specificities for dephosphorylation, ADP versus water. These two horizontal steps are linked by the vertical transport steps in which conversion of cation specificity and reorientation of cation binding/discharge sites occur. It is El forms, with sites accessible to the c toplasm, to which Na+ binds tightly and from which KT is discharged, whereas it is E2 forms, with sites accessible extracellularly, to which K+ binds tightly and from which Na+ is discharged. The requirements for coupling underlie the stricture that Na+ be discharged from E2-P rather than El-P, ensuring the efficient linkage of transport to catalysis: this stricture is also embodied in the formulations for Na+/Na+ exchange and uncoupled Na+ efflux in which Na+ discharge must also be from E2-P (see below). A current formulation (Fig. 1) begins with ATP bound to El, in a ternary complex with Mg2+ (since the details of Mg2+ binding and dissociation are uncertain, the presence of divalent cation is usually ignored in such schemes). Addition of Na+, to high-affinity sites accessible from the cytoplasm, triggers the readily reversible phosphorylation of the enzyme and the dissociation of ADP, leaving Na+ bound to El-P. The transport step of the formulation coincides with isomerization of El-P to E2-P and the transformation of the Na+ sites to low-affinity ones accessible from the exterior. Na+ is released, and K + , by a ping-ping mechanism, is only then accepted at high-affinity sites accessible

488

JOSEPH D. ROBINSON

F i g . 1 . Modified Albers-Post reaction sequence. In t h i s scheme (Albers, 1967; Post e t a l . , 1969, 1972; Karlish e t a l . , 1978; Robinson and Flashner, 1979c; Smith e t a l . , 1980; Moczydlowski and Fortes, 1981) the Na,K-ATPase reaction with ATP concentrations s u f f i c i e n t t o f i l l only the h i g h - a f f i n i t y subs t r a t e s i t e s follows forms 1-8p l u s 11 and 12, whereas with ATP concentrations s u f f i c i e n t t o f i l l a s well the low-a€finity subs t r a t e s i t e s the reaction follows forms l-g. The Na-ATPase reaction follows forms I-6- p l u s 13 and 12. The El enzyme conformations are considered t o have t h e i r monovalent cation s i t e s accessible t o the cytoplasm, whereas the E 2 conformations have t h e i r s i t e s accessible t o the extracellular medium.

from the exterior. K+ binding to these sites triggers dephosphorylation by water. The translocation of K+ is accomplished by the isomerization of E2 to El and the transformation of the K+ sites to low-affinity ones accessible from the cytoplasm. The major modification to the original Albers-Post proposal is the inclusion of a quasi-stable form of the enzyme with "occluded K+," E2 (K), which releases K+ readily only after ATP binds to a low-affinity site on the E2 form. This binding then facilitates conversion of E2 to El and release of K+: E2(K)

+

ATP

+

E2(K)ATP

+

EIATP

+ K-I-

KINETIC ANALYSES AND REACTION MECHANISM

489

Justification for this proposed role of ATP arose from studies indicating a mutual antagonism between K+ and ATP binding to the enzyme, the ability of ATP to liberate trapped K+ from an occluded form, E2(K), and biphasic substrate-velocity plots for the Na,K-ATPase reaction interpretable as high-affinity sites (comparable to those at which ATP phosphorylates the enzyme) and low-affinity sites (comparable to those at which ATP antagonizes certain actions of K+) If rapid equilibrium existed between El and E2 forms, then binding ATP solely to El forms would be sufficient to pull the reaction away from E2(K). The lack of rapid interconversion between E2 and El (necessary to prevent a leakage pathway for K+ backward through the transport cycle) is alleviated by the binding of ATP to E2(K), which then drives the reaction to ElATP with the liberation of K+. The reaction scheme, in its ordered sequential mechanism (with respect to K+, Pi, and ATP), also displays a second check against a backward leakage of K+: extracellular K+ equilibrates with E2-P but not E2. The dephosphorylation triggered by K+ binding drives K+ to its occluded form, E2(K), from which it can either progress to EJ + K+ (cytoplasmic) in the step accelerated by ATP binding to E2, or to E2-P + K+ (extracellular), in the step dependent on Pi. This reaction sequence may be more readily visualized in the form of a Cleland diagram of the reaction progress (Fig. 2), in which the E2 forms are indicated by a thickening of the horizontal arrow. Three alternative reaction pathways, and transport modes, are also incorporated into the formulation. (1) Na-ATPase activity and uncoupled Na+ efflux are rep-

.

Na

ADP

I

Na

E-P forms

-I

Fig. 2. A Cleland diagram of the Na,K-ATPase reaction sequence of Fig. 1. The shaded portion of the arrow represents the E 2 enzyme conformations; the numbers refer to the specific forms of Fig. 1.

490

JOSEPH D. ROBINSON

r e s e n t e d by forms 1-6, 1 3 , and 1 2 ( F i g . 1), a pathway o m i t t i n g i n t e r a c t i o n s o ~ A T Pw i t h E 2 forms and of K+ with e i t h e r E l o r E2. ( 2 ) ADP/ATP exchange is r e p r e s e n t e d by forms 1 2 and 1 - 4 , whereas Na+/Na+ exchange r e q u i r e s , i n a d d Z i o n , forms 5 and 6 . The r e q u i r e m e n t f o r N a + d i s c h a r g e t o t h e e x t e r i o r fFom E 2 forms, i n b o t h uncoupled N a + e f f l u x and N a + / N a + exchange, r e p r e s e n t s t h e n e c e s s a r y c o n s t r a i n t t o e n s u r e c o u p l i n g and p r e v e n t l e a k a g e t h r o u g h t h e pump. ( 3 ) P i / w a t e r exchange i s r e p r e s e n t e d by forms _ 6-8 - , whereas K+/K+ exchange req u i r e s , i n a d d i t i o n , forms 9 and 1 0 , r e p r e s e n t i n g a g a i n t h e r e q u i r e m e n t f o r E1/E2 i s o m e r i z a t i o n t o a c h i e v e t r a n s p o r t . T h i s f o r m u l a t i o n a l s o d e m o n s t r a t e s why ATP s t i m u l a t e s K+/K+ exchange b u t n o t P i / w a t e r exchange. A f u r t h e r r e a c t i o n c a t a l y z e d by t h e enzyme, Kp h o s p h a t a s e a c t i v i t y , c a n be superimposed on forms 1 and 8 i n which t h e a c y l p h o s p h a t e of t h e p h o s p h a t a s e subs t r a t e r e p l a c e s t h e a c y l phosphate of E2-P. B e f o r e c o n s i d e r i n g s p e c i f i c a s p e c t s o f t h i s formul a t i o n , one f u r t h e r i s s u e s h o u l d be i n t r o d u c e d h e r e , t h a t o f half-sites-reactivity/flip-flop mechanisms/rec i p r o c a t i n g - d i m e r mechanisms. E s t i m a t e s of m o l e c u l a r w e i g h t s u g g e s t t h a t t h e f u n c t i o n a l enzyme i n s i t u i s a t l e a s t e q u a l t o a n ( a f 3 )2 o l i g o m e r (see S e c t i o n I I 1 , D ) . On t h e o t h e r hand, i n i t i a l measurements of ATP b i n d i n g , o u a b a i n b i n d i n g , v a n a d a t e b i n d i n g , and p h o s p h o r y l a t i o n i n d i c a t e d t h a t t h e stoichiometry of such i n t e r a c t i o n s , based on t h e c a l c u l a t e d w e i g h t of t h e a - p e p t i d e , was o n e p e r t w o a - p e p t i d e s of t h e enzyme complex ( a c o n c l u s i o n now d i s p u t e d : see S e c t i o n 1 1 1 , A ) . These d a t a , and t h e b i p h a s i c s u b s t r a t e - v e l o c i t y p l o t s c o n s i s t e n t with negative cooperativity, suggested t h a t only half of t h e a - p e p t i d e s f u n c t i o n s i m u l t a n e o u s l y , and t h a t t h e r e a c t i o n mechanism might e i t h e r be c a t a l y z e d i n p a r t by one and i n p a r t by t h e o t h e r a - p e p t i d e , o r be c a t a l y z e d i n t o t o by e a c h a - p e p t i d e b u t w i t h t h e s e a c t i n g o u t o f p h a s e . A p o s s i b l e v e r s i o n of t h e l a t t e r c o n c e p t , i n which e a c h a - p e p t i d e c a t a l y z e s t h e e n t i r e r e a c t i o n sequence of F i g . 2 , i s d e p i c t e d i n F i g . 3 . Arguments f o r and a g a i n s t s u c h d i m e r i c mechanisms w i l l be c o n s i d e r e d below.

KINETIC ANALYSESAND REACTION MECHANISM

No

ADP

I

K

NO

E - P forms

P i ATP

K

491

NO

4

NO

ADP

I

K

P . ATP

I

E-P forms

E - P forms

7

K

Pi ATP

K

No

ADP

No

K

Pi ATP

K

No

ADP

F i g . 3 . A d i m e r i c reaction p r o c e s s . T h e u p p e r a n d lower arrows each r e p r e s e n t the reaction sequence of one monomer of a d i r n e r i c e n z y m e , w i t h the reaction sequence of F i g s . 1 a n d 2 proc e e d i n g s e p a r a t e l y on e a c h monomer, b u t o u t of p h a s e .

111.

UNCERTAINTIES, ALTERNATIVES, AND ANOMALIES

A.

LIGAND B I N D I N G S I T E S

1.

S i t e s for ATP

N$rby and J e n s e n ( 1 9 7 1 ) and Hegyvary and P o s t (1971) d e m o n s t r a t e d h i g h - a f f i n i t y s i t e s f o r ATP w i t h a Kd i n t h e a b s e n c e o f o t h e r l i g a n d s n e a r 0 .1 - 0 .2 PM. N a + i n c r e a s e d b i n d i n g somewhat, whereas K + i n c r e a s e d t h e Kd a p p r e c i a b l y . With t h e n o n h y d r o l y z a b l e a n a l o g AMP-PNP,I Robinson (1980b) showed t h a t micromolar quant i t i e s o f MgC12 d e c r e a s e d t h e Kd o n l y s l i g h t l y , a n d , a s i n t h e a b s e n c e o f MgC12, K + i n c r e a s e d t h e Kd a p p r e c i a bly. On t h e o t h e r hand, w i t h t h e n o n h y d r o l y z a b l e a n a l o g TNP-ATP, Moczydlowski and F o r t e s ( 1 9 8 1 a ) found a s m a l l e r i n c r e a s e i n Kd i n t h e p r e s e n c e of K + , and t h i s i n c r e a s e w a s f u r t h e r r e d u c e d by a d d i n g MgC12. O t h e r r e a g e n t s considered t o bind t o the high-affinity s u b s t r a t e sites i n c l u d e f l u o r e s c e i n i s o t h i o c y a n a t e ( K a r l i s h , 1 9 8 0 ) and e o s i n (Skou and Esmann, 1 9 8 1 ) . ' A b b r e v i a t i o n s : AMP-FCP, the 6-y m e t h y l e n e a n a l o g of ATP; AMP-PNP, the 6-y i m i d o analog of ATP; NBD-Cl, 7 - c h l o r o - 4 - n i t r o b e n zo-2-oxa-1,3-diazole; NPP, p - n i t r o p h e n y l p h o s p h a t e ; TNP-ATP, 2',3 '-0- (2,4,6-trinitrocyclohexadienylidine) d e r i v a t i v e of ATP.

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Based on c u r r e n t e s t i m a t e s o f p r o t e i n c o n t e n t and molecular w e i g h t , Moczydlowski and F o r t e s (1981a) c a l c u l a t e d t h a t one h i g h - a f f i n i t y s i t e w a s p r e s e n t f o r each a - p e p t i d e of t h e enzyme. T h i s a g r e e s w i t h t h e ree v a l u a t i o n of one p h o s p h o r y l a t i o n s i t e (Peters et al., 1 9 8 1 ) and one vanadate-binding s i t e (Smith e t a l . , 1980) p e r a - p e p t i d e . Although t h e Kd v a l u e f o r t h e h i g h - a f f i n i t y s i t e s corresponded w i t h t h e K~ f o r t h e Na-ATPase r e a c t i o n (Neufeld and Levy, 1 9 6 9 1 , i t w a s t h r e e o r d e r s of magnit u d e lower t h a n t h e K~ commonly observed f.or t h e Na,K-ATPase r e a c t i o n i n e a r l y s t u d i e s ( e . g . , Robinson, 1 9 6 7 ) , even c o n s i d e r i n g t h e e f f e c t s of K+ on t h a t Kd (Hegyvary and P o s t , 1 9 7 1 ) . Kanazawa et a l . ( 1 9 7 0 ) subs e q u e n t l y showed b i p h a s i c Lineweaver-Burk p l o t s of Mg-ATP c o n c e n t r a t i o n v e r s u s v e l o c i t y , i n t e r p r e t a b l e a s high- and l o w - a f f i n i t y s u b s t r a t e s i t e s , o r a s c a t a l y t i c and r e g u l a t o r y s i t e s ( s i n c e t h e former mediated enzyme p h o s p h o r y l a t i o n whereas t h e l a t t e r a major i n c r e a s e i n vmax) The l o w - a f f i n i t y s i t e s , r e p r e s e n t i n g a K m on t h e o r d e r of 0 . 1 mM, a r e t e c h n i c a l l y d i f f i c u l t t o demons t r a t e . N e v e r t h e l e s s , s t u d i e s showing ATP b i n d i n g i n t h i s range were d e s c r i b e d by Yamaguchi and Tonomura ( 1 9 8 0 b ) . Bonting et al. ( t h i s volume) r e p o r t e d t h a t i n t h e p r e s e n c e of 5 mM MgCl a second b i n d i n g s i t e f o r AMP-PNP was demonstrable w i t 8 a Kd n e a r 0.15 mM; t h e s t o i c h i o m e t r y w a s one p e r a - p e p t i d e , t h u s i n d i c a t i n g one high- and one l o w - a f f i n i t y s i t e on each a - p e p t i d e . Whether t h e high- and l o w - a f f i n i t y s u b s t r a t e s i t e s c o e x i s t o r a r e s e q u e n t i a l l y a v a i l a b l e , whether they a r e s t r u c t u r a l l y d i s t i n c t o r a r e interconverted, and whether ( i n l i g h t of t h e t e c h n i c a l d i f f i c u l t i e s i n t h e b i n d i n g e x p e r i m e n t s ) t h e s t o i c h i o m e t r y should be one or more s i t e s p e r a - p e p t i d e , are a l l a r g u a b l e i s s u e s . K i n e t i c models w i t h i n t e r c o n v e r s i o n of highand l o w - a f f i n i t y s i t e s c a n produce t h e b i p h a s i c s u b s t r a t e - v e l o c i t y p l o t s (Moczydlowski and F o r t e s , 1 9 8 1 ; Smith e t a l . , 1 9 8 0 ) . Skou and Esmann ( 1 9 8 1 ) d e s c r i b e d d i s t i n c t e f f e c t s of two classes of n u c l e o t i d e s i t e s , which might be a v a i l a b l e s i m u l t a n e o u s l y o r seq u e n t i a l l y . Garrahan e t a l . ( t h i s volume) showed t h a t t h e nonhydrolyzable a n a l o g AMP-PCP c o u l d , a t c o n c e n t r a t i o n s s u f f i c i e n t t o occupy t h e l o w - a f f i n i t y s i t e s , s t i m u l a t e A T P a s e a c t i v i t y , presumably c a t a l y z e d a t t h e h i g h - a f f i n i t y s i t e s . Although it i s n o t e a s y t o v i s u a l i z e AMP-PCP f i r s t occupying t h e l o w - a f f i n i t y r e g u l a t i n g s i t e s t o s t i m u l a t e , and t h e n v a c a t i n g t h e s i t e s t o ATP as t h e y a r e c o n v e r t e d t o t h e h i g h - a f f i n i t y

-

KINETIC ANALYSES AND REACTION MECHANISM

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c a t a l y t i c s i t e s , t h e two c l a s s e s o f s i t e s do d i s p l a y d i f f e r e n t s e l e c t i v i t i e s (Robinson, 1 9 7 6 a ) . Other arguments f o r d i s t i n c t c l a sse s o f nucleot i d e s i t e s a r i s e from n u c l e o t i d e e f f e c t s on t h e Kp h o s p h a t a s e r e a c t i o n (Robinson, 1 9 7 6 a ; K o e p s e l l and O l l i g , t h i s volume; W i n t e r , t h i s volume), and from n u c l e o t i d e m o d i f i c a t i o n of c e r t a i n chemical i n a c t i v a t i o n s of t h e enzyme a t c o n c e n t r a t i o n s e q u i v a l e n t t o occupancy o f t h e h i g h - a f f i n i t y s i t e s ( e . g . , p r o t e c t i o n a g a i n s t a c e t i c a n h y d r i d e (Robinson and F l a s h n e r , 1 9 7 9 a ) ) o r of t h e l o w - a f f i n i t y sites (e.g., p r o t e c t i o n a g a i n s t NBD-C1 (Grosse et a l . , 1979) and methylene b l u e (Robinson, 1 9 7 6 a ) ) . Moreover, Kaplan and Mone ( t h i s volume) showed t h a t a f t e r t r e a t m e n t w i t h t h i m e r o s a l t h e enzyme c a t a l y z e d A T P a s e a c t i v i t i e s n o t o n l y a t h i g h a f f i n i t y sites ( < 5 WM) but a l s o a t lower-affinity sites (>70 pM). Another a s p e c t o f n u c l e o t i d e b i n d i n g , p o s s i b l y d i s t i n c t from t h e above i s s u e s , i s t h e v e r y t i g h t bindi n g o f ATP t o t h e enzyme i n f e r r e d from t r a n s i e n t k i n e t i c s t u d i e s ( F r o e h l i c h et al., t h i s volume), and p e r h a p s a l s o a p p a r e n t i n measurements o f maximal phosphorylat i o n by [32P]ATP ( P e t e r s e t al., 1 9 8 1 ) . 2.

Mq

2+

Sites

I n t h e a b s e n c e o f n u c l e o t i d e s , Grisham and Mildvan ( 1 9 7 4 ) found one h i g h - a f f i n i t y b i n d i n g s i t e f o r Mn2+

(presumably, now, p e r a - p e p t i d e ) , f o r which Mg2+ comThis value i s i n accord with p e t e d w i t h a K i o f 1 mM. t h e Kd f o r Mg2+ found i n B e 2 + i n a c t i v a t i o n e x p e r i m e n t s (Robinson, 1973) and t h e K, f o r t h e K-phosphatase react i o n (Robinson, 1 9 7 4 ) . On t h e o t h e r hand, micromolar Mg2+ i n c r e a s e d t h e b i n d i n g o f micromolar AMP-PNP, c l e a r l y i n d i c a t i n g a n i n t e r a c t i o n i n t h i s c o n c e n t r a t i o n r a n g e (Robinson, 1 9 8 0 b ) . H i g h - a f f i n i t y Mg2+ s i t e s c a n n o t be a t t r i b u t e d s o l e l y t o b i n d i n g through t h e n u c l e o t i d e , €or Fukushima and P o s t (1978) showed t h a t d i v a l e n t c a t i o n s remain t i g h t l y bound t o E2-P a f t e r d i s s o c i a t i o n of ADP from El-P. Hobbs e t a l . ( t h i s volume) s u g g e s t e d t h a t bindi n g o f d i v a l e n t c a t i o n t o E1.ATP is i n v o l v e d i n t h e t i g h t b i n d i n g o f ATP (see S e c t i o n I I I , A , l ) , a s p a r t o f t h e r e a c t i o n sequence. The p o s s i b i l i t y of a second d i v a l e n t c a t i o n b i n d i n g t o t h e enzyme-nucleotided i v a l e n t c a t i o n complex (Grisham, t h i s volume) may b e a r on t h i s i s s u e . The f a t e o f t h i s Mg2+ bound t o E2-P i s u n c e r t a i n . Smith et ai. (1980) showed t h a t t h e d i v a l e n t c a t i o n bound t i g h t l y i n t h e p r e s e n c e o f v a n a d a t e d i s s o c i a t e d

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in parallel with the vanadate, but how closely vanadate release resembles phosphate release (see Section III,A, 3 ) and how the Kd at that step compares to Mg2+ levels i n v i v o are uncertain. Since Mg2+ favors the E2 conformational family, dissociation or complexing with bound ATP may be necessary for conversion to El forms (Robinson, this volume) Despite the evidence for high-affinity binding of Mg2+ in the presence of nucleotides, there remains evidence also for the simultaneous existence of the lowaffinity sites demonstrable in the absence of nucleotides. !$?us, in contrast to micromolar Mg2+, millimolar Mg decreased AMP-PNP binding (Robinson, 1980b) and inhibited both enzyme phosphorylation and Na-ATPase activities measured with micromolar ATP (Flashner and Robinson, 1979). Although the ~ 0 . 5values correspond well with those for binding in the absence of nucleotides and for activating the K-phosphatase reaction (Robinson and Flashner, 1979b), an alternative explanation for these latter effects is action through occupancy of some other ligand site. For example, Mg2+ appears to compete with Na+ for its sites in the Na,K-ATPase reaction and with K+ for its sites in the K-phosphatase reaction (Robinson, 1969, 1972; Parodi e t a l . , this volume), although the Ki appears to be far higher than 1 mM (Robinson, 1975a).

.

3.

Phosphate Sites

Inorganic phosphate interacts with an apparent Kd near 2 mM, as demonstrated in such processes as inhibition of K-phosphatase activity (Robinson, 1970; this volume) and of vanadate binding (Cantley e t a l . , 1978) and stimulation of K+/K+ exchange (Simons, 1975). Presumably the binding site corresponds to that from which Pi is released and through which Pi phosphorylates the enzyme (Taniguchi and Post, 1975). Because of the resemblance of vanadate to phosphate as a leaving group, Cantley e t a l . (1978) proposed that vanadate bound to and inhibited the enzyme as a transition state analog. The binding, although not covalent, is several orders of magnitude tighter than for Pi, requires divalent cations, and is stimulated by K+. For both inhibition by Pi and.binding of vanadate Mn2+ is superior to Mg2+ (Robinson, 1981; this volume). On the other hand, Hansen (this volume) showed that the coordinate release of vanadate-ouabain from the enzyme was not stimulated by ATP, whereas the release of ouabain after binding in the presence of Pi was

KINETIC ANALYSES AND REACTION MECHANISM

495

s t i m u l a t e d by ATP: t h u s a s t r i c t p a r a l l e l i s m between E*phosphate and E - v a n a d a t e does n o t h o l d . The number of h i g h - a f f i n i t y vanadate-binding s i t e s i s now e q u a t e d t o one p e r a - p e p t i d e , and occupancy b l o c k s ATP b i n d i n g t o t h e h i g h - a f f i n i t y s u b s t r a t e s i t e s (Smith e t a l . , 1 9 8 0 ) . T h i s argument t h u s f a v o r s a s i n g l e s u b s t r a t e s i t e w i t h a l t e r n a t i n g h i g h and low a f f i n i t y f o r ATP. F r o e h l i c h e t a l . ( t h i s volume), howe v e r , found c o o p e r a t i v i t y i n i n h i b i t i o n o f Na,K-ATPase a c t i v i t y by submicromolar v a n a d a t e , i n d i c a t i n g more t h a n one s i t e f o r vanadate. 4.

~ a + Sites

Garay and Garrahan (1973) d e s c r i b e d a c t i v a t i o n of t h e sodium pump through t h r e e e q u i v a l e n t s i t e s f o r Na+, a c c e s s i b l e from t h e cytoplasm, w i t h a Kd of 0 . 2 mM. A s i m i l a r a f f i n i t y was a p p a r e n t f o r Na+ s i t e s a c t i v a t i n g Na,K-ATPase, Na-ATPase, and enzyme p h o s p h o r y l a t i o n (Robinson, 1 9 7 7 a ; F l a s h n e r and Robinson, 1 9 7 9 ) . Nevert h e l e s s , F o s t e r and Ahmed ( 1 9 7 6 ) and F o s t e r et a l . ( t h i s volume) r e p o r t e d f u r t h e r a c t i v a t i o n of enzyme p h o s p h o r y l a t i o n by one o r two a d d i t i o n a l classes o f s i t e s w i t h f a r lower a f f i n i t y (Kd = 2 4 - 4 0 m M ) , whose l o c a l i z a t i o n i s unknown. Low-affinity s i t e s t h r o u g h which Na+ s t i m u l a t e s p h o s p h o r y l a t i o n must be compared w i t h l o w - a f f i n i t y e x t e r n a l s i t e s f o r N a + through which Na-ATPase, uncoupled Na+ e f f l u x , ADP/ATP exchange, and Na+/Na+ exchange a r e a c t i v a t e d (Glynn and K a r l i s h , 1 9 7 6 ; B l o s t e i n , t h i s volume: Kaplan, t h i s volume). I t i s plausible t h a t these external sites a r e r e l a t e d t o t h o s e a t which Na+ i s d i s c h a r g e d and ATP s y n t h e s i s from E2-P i s d r i v e n (Taniguchi and P o s t , 1 9 7 5 ) . N e v e r t h e l e s s , a fundamental d i s c r e p a n c y i s a l s o app a r e n t : h i g h c o n c e n t r a t i o n s of Na+ can s t i m u l a t e d ADP-dependent d e p h o s p h o r y l a t i o n , i n t e r p r e t a b l e a s N a + d r i v i n g E2-P back t o E l - P I whereas h i g h c o n c e n t r a t i o n s of Na+ c a n a l s o s t i m u l a t e ALP-independent dephosphorylat i o n , i n t e r p r e t a b l e a s E2- decomposing t o E 2 and P i (Hara and Nakao, 1 9 8 1 ) . What i s t h e r e l a t i o n s h i p between t h e N a + s i t e s m e d i a t i n g t h e s e t w o d i v e r s e e f f e c t s and t h e o t h e r c a t i o n s i t e s ? I n a d d i t i o n , a s i n g l e , higha f f i n i t y , e x t e r n a l s i t e f o r Na+ was demonstrated by C a v i e r e s and E l l o r y (19751, who s u g g e s t e d t h a t it might r e p r e s e n t t h e "missing" s i t e i n a t r a n s p o r t mechanism c o n v e r t i n g t h r e e Na+ s i t e s i n t o two K+ s i t e s . Three h i g h - a f f i n i t y b i n d i n g s i t e s f o r Na+ have a l s o been d e s c r i b e d (Kaniike e t a l . , 1 9 7 6 ; Yamaguchi and Tonomura, 1980a; Tonomura e t a l . , t h i s volume: Matsui e t a l . , t h i s volume).

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5.

K+ S i t e s

The sodium pump i s a c t i v a t e d through s i t e s f o r K + a c c e s s i b l e from t h e e x t e r i o r , probably t w o f o r each f u n c t i o n a l u n i t a l t h o u g h occupancy o f both i s n o t req u i r e d (Sachs, 1 9 7 7 ) . These correspond t o higha f f i n i t y s i t e s (Ko.5 % 0 . 1 m ) a c t i v a t i n g t h e Na,KATPase, termed 8-sites (Robinson, 1 9 7 5 a ) . Three o t h e r c l a s s e s o f s i t e s have been named. The K-phosphatase r e a c t i o n i s a c t i v a t e d by m o d e r a t e - a f f i n i t y s i t e s ( ~ 0 . 5% 2 m ) , r e c e n t l y shown t o be a c c e s s i b l e from t h e cytoplasm (Drapeau and B l o s t e i n , 1 9 8 0 ) , and termed a - s i t e s (Robinson, 1 9 7 5 a ) . These s i t e s seem c l o s e l y r e l a t e d t o t h e "occluded K+ s i t e s , " f i r s t desc r i b e d by P o s t e t a l . ( 1 9 7 2 ) and c o n s i d e r e d t o be s p a t i a l l y between t h e e x t e r n a l e n t r y s i t e f o r K+ t r a n s p o r t and t h e c y t o p l a s m i c d i s c h a r g e s i t e , w i t h a c c e s s gained a f t e r enzyme p h o s p h o r y l a t i o n . E x i t from t h e occluded s i t e s , d e s i g n a t e d E ( K ) , was p o s t u l a t e d as slow e x c e p t i n t h e p r e s e n c e of ATP, which, through occupying t h e l o w - a f f i n i t y s u b s t r a t e s i t e s , markedly i n c r e a s e d t h e r a t e of d i s s o c i a t i o n . T h i s f o r m u l a t i o n , t h e major m o d i f i c a t i o n t o t h e o r i g i n a l Albers-Post scheme ( S e c t i o n 11), i s s u p p o r t e d by t h e e x p l i c i t experiments of Beauge and Glynn (197933) showing such changes i n r a t e . The only s i g n i f i c a n t r e v i s i o n of t h e P o s t e t a l . ( 1 9 7 2 ) f o r m u l a t i o n a r i s e s from r e a l i z a t i o n t h a t K+ can r e a c h t h e occluded s i t e s n o t o n l y from t h e e x t e r i o r a f t e r enzyme p h o s p h o r y l a t i o n b u t a l s o from t h e c y t o plasm i n t h e absence of p h o s p h o r y l a t i o n (Beaug6 and Glynn, 1 9 7 9 b ) . With t h i s m o d i f i c a t i o n , t h e l i n k between a-sites and occluded s i t e s i s obvious i n such phenomena a s mutual antagonism between K+ and ATP a t t h e lowa f f i n i t y s u b s t r a t e s i t e s (Robinson, 1 9 6 7 , 197533); a c t i v a t i o n of t h e K-phosphatase r e a c t i o n by c y t o p l a s m i c K+ i n t h e absence of enzyme p h o s p h o r y l a t i o n by ATP b u t by e x t e r n a l K+ i n t h e p r e s e n c e of such p h o s p h o r y l a t i o n (Drapeau and B l o s t e i n , 1 9 8 0 ) ; and p o t e n t i a t i o n of vanad a t e i n h i b i t i o n by c y t o p l a s m i c K+ i n t h e absence of p h o s p h o r y l a t i o n b u t by e x t e r n a l K+ i n i t s p r e s e n c e (Robinson and Mercer, 1 9 8 1 ) . Because of t h e slow e q u i l i b r a t i o n between c y t o plasmic K+ and t h e occluded s i t e s ( K a r l i s h , 1 9 8 0 ) , est i m a t e s from k i n e t i c s t u d i e s of t h e Kd f o r K+ a t t h e a - s i t e s o r occluded s i t e s a r e i m p r e c i s e . Thus, t h e "0.5 f o r a c t i v a t i o n of t h e K-phosphatase r e a c t i o n (Robinson, 1 9 6 9 ) i s 2 mM (no ATP p r e s e n t ) , whereas f o r a c t i v a t i o n of K+/K+ exchange (Simons, 1 9 7 4 ) i t i s 1 0 m M . By t h e above a n a l y s i s b o t h v a l u e s should be f o r e n t r y from and t o t h e same c l a s s e s of s i t e s . Both v a l u e s ,

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moreover, are f a r lower t h a n t h e K0.5 f o r r e v e r s a l of t h e pump w i t h ATP s y n t h e s i s , 0 . 3 M (Robinson e t a l . , 19771, which s h o u l d r e f l e c t K+ b i n d i n g t o i t s c y t o p l a s m i c d i s c h a r g e s i t e s , termed y - s i t e s (Robinson, 1975a). The A l b e r s - P o s t r e a c t i o n scheme ( F i g . 1) p e r m i t s t h e h i g h - a f f i n i t y e x t e r n a l 8 - s i t e s t o e x i s t o n l y on E2-P. I n t h e a b s e n c e o f p h o s p h o r y l a t i o n , t h e n , K+ b i n d i n g s h o u l d b e t h r o u g h a - s i t e s / o c c l u d e d s i t e s , which number 2 p e r a - p e p t i d e ( M a t s u i e t a l . , 1977; C a n t l e y e t a l . , 1978; Matsui e t a l . , t h i s volume; J @ r g e n s e n , t h i s volume) o r 2-3 ( J e n s e n and O t t o l e n g h i , t h i s v o l u m e ) . I n t h e p r e s e n c e o f enzyme p h o s p h o r y l a t i o n t h e a f f i n i t y i n c r e a s e d (Tonomura e t a l . , t h i s v o l u m e ) , c o n s i s t e n t w i t h t h e a v a i l a b i l i t y o f B-sites on E z - P , as d e m o n s t r a t e d i n e a r l i e r measurements of Kd f o r K+ S t i l l unresolved i s whether t h e cytoplasmic N a + s i t e s are i d e n t i c a l t o t h e c y t o p l a s m i c K+ s i t e s , a l though t h e d i s c r e p a n c y i n numbers of b i n d i n g s i t e s , 3 v e r s u s 2 , a r g u e s a g a i n s t s u c h i d e n t i t y . Tonomura e t a l . ( t h i s volume) found c o e x i s t i n g N a + and K+ s i t e s , i n r a t i o of 3:2, a l t h o u g h o t h e r a p p r o a c h e s s u g g e s t compet i t i o n between N a + and K+ ( P a r o d i e t a l . , t h i s volume; Robinson, 1 9 7 7 a ) . 6.

S i t e s for

the P h o s p h a t a s e S u b s t r a t e

K-phosphatase a c t i v i t y r e f l e c t s t h e K + - a c t i v a t e d dephosphorylation s t e p s i n t h e Na,K-ATPase r e a c t i o n : t h e s u b s t r a t e s , phosphoric a c i d anhydrides such as a c e t y l phosphate o r p-nitrophenyl phosphate, s u b s t i t u t e f o r t h e enzyme a c y l p h o s p h a t e . The a p p r o a c h t o t h e hyd r o l y t i c s i t e a p p e a r s t o be t h r o u g h t h e u s u a l p h o s p h a t e d i s c h a r g e s t e p . Thus, P i i s a c o m p e t i t o r toward NPP (Robinson, 1 9 7 0 ) and NPP a n t a g o n i z e s v a n a d a t e b i n d i n g (Robinson and Mercer, 1 9 8 1 ) . A p p a r e n t c o m p e t i t i o n by ATP a t t h e l o w - a f f i n i t y s u b s t r a t e s i t e s ( R o b i n s o n , 1976a) may, however, b e due m e r e l y t o s h i f t i n g t h e E2/E1 equilibrium. P a r o d i e t a l . ( t h i s volume) r e p o r t e d a n o r d e r e d a d d i t i o n o f Mg2+ t h e n NPP t o t h e enzyme. B.

C O N F O R M A T I O N A L FORMS

Each s t e p i n t h e r e a c t i o n s e q u e n c e ( F i g . 1) c a n be c o r r e c t l y c o n s i d e r e d a d i s t i n c t c o n f o r m a t i o n . Grouping t h e s e i n t o two f a m i l i e s , E l and E 2 ( o r i n t o f o u r f a m i l i e s by s e g r e g a t i n g t h e p h o s p h o r y l a t e d s t a t e s ) , a r o s e from c o n s i d e r a t i o n of t h e d i f f e r e n c e i n a p p a r e n t e n e r g y

490

JOSEPH D. ROBINSON

level of E l - P and Ez-P, with symmetrical dephosphorylated forms, E2 and El, added for balance. These distinctions have been correlated with a variety of properties, recently in terms of the pattern of tryptic digestion (Jgkgensen, 1975) and the intensity of intrinsic tryptophan fluorescence (Karlish and Yates, 1978): in these examples K+ favors one form, often equated to E2, whereas no ligands or Na+ favors another, often equated to E l . Various other ligands similarly influence the apparent equilibria between the presumed E 2 and El families, including nonionic detergents and divalent cations (Robinson, 1980a, 1981; this volume), pH and ionic strength (Jensen and Ottolenghi, this volume: Skou, this volume). Nevertheless, different representations of the conformational families are distinguishable by their various ligand sensitivities: for example, the K+ pattern of tryptic digestion follows the hierarchy K+ > Mg2+ (Robinson, this volume) , whereas vanadate binding follows the hierarchy Mn2+ > Mg2+ > K+ (Robinson and Mercer, 1981). The division into conformational families is also justified, as discussed in Section 11, on considerations of coupling transport to catalysis (Jencks, 1980; this volume) and of the heuristic value of portraying one family, El, with cation sites facing the cytoplasm and the other, E2, with sites facing the exterior (Post e t a l . , 1969). This latter rationale is not always followed even by its proponents (e.g., Post e t a l . , 1972, 1975), and is clearly not applicable to the occluded K+ form when designated E2(K) since this conformation has its sites accessible, albeit slowly, to the cytoplasm and not to the exterior.2 If grouping the conformations ought to include reference to sidedness of their sites, then it seems necessary to label the occluded form El(K), or, recognizing its slow communication to the interior, reclassify it distinctly from E l and E2 as E O ( K ) . On the other hand, the form with occluded K+ is that giving the tryptic digestion and tryptophan fluorescent response often defined as "E2." Occluded forms may also exist in the Na+-transport steps (Karlish e t a l . , 1978; Glynn and Richards, this volume). 2 W i t h the E l enzyme f o r m e n t r y o f xi? t o the o c c l u d e d s i t e s f r o m t h e c y t o p l a s m is r a p i d , w h e r e a s r e l e a s e t o t h a t s i d e i s slow. In the a b s e n c e of p h o s p h o r y l a t e d enzyme f o r m s , both r e l e a s e t o the e x t r a c e l l u l a r medium and a c c e s s f r o m t h a t s i d e i s e x t r e m e l y s l o w ; c o n s e q u e n t l y l e a k a g e t h r o u g h the pump i s l e s s t h a n 1% o f the o p t i mal r a t e o f a c t i v e t r a n s p o r t ( K a r l i s h and S t e i n , t h i s v o l u m e ) .

KINETIC ANALYSES AND REACTION MECHANISM

C.

REACTION SEQUENCE

1.

A l t e r n a t i v e Pathways

499

Two a l t e r n a t i v e pathways are d e p i c t e d i n F i g . 1, f o r Na,K-ATPase i n t h e absence of ATP c o n c e n t r a t i o n s s u f f i c i e n t t o fill t h e low-affinity s u b s t r a t e sites, and f o r Na-ATPase a c t i v i t y . Beyond such c o n s i d e r a t i o n s , r e a d i l y accommodated by t h i s f o r m u l a t i o n , a r e two o t h e r c l a s s e s of problems. F i r s t , t h e decomposition of t h e p h o s p h o r y l a t e d enzyme c a n b e b i p h a s i c ( F r o e h l i c h e t a l . , t h i s volume; Klodos e t a l . , t h i s volume; Hobbs e t a l . , t h i s volume), s u g g e s t i n g d i s t i n c t , a l t e r n a t i v e pathways f o r dephosp h o r y l a t i o n ( F r o e h l i c h e t a l . , t h i s volume; Klodos e t a l . , t h i s volume; Lowe and Reeve, t h i s volume). Such p a r a l l e l pathways c o u l d be f u n c t i o n a l l y u n r e l a t e d , a s i n t h e case of isoenzymes o r p a r t i a l d e n a t u r a t i o n . Since t h e r e l a t i v e t u r n o v e r s are v a s t l y d i f f e r e n t , i f t h e a l t e r n a t i v e pathways r e p r e s e n t some s o r t of coord i n a t e d system t h e n t h e c o u p l i n g must be v e r y weak ( a t least after i s o l a t i o n ) . A r e l a t e d consideration i s whether t h e r e a r e more t h a n t h e u s u a l l y d e p i c t e d two forms of E-P (Hobbs e t a l . , t h i s volume; Klodos e t a l . , t h i s volume). An e a r l i e r i n d i c a t i o n t h a t ADP s e n s i t i v i t y developed o n l y s l o w l y d u r i n g t h e c o u r s e of enzyme p h o s p h o r y l a t i o n h a s now been r e c o n c i l e d w i t h t h e s e q u e n t i a l o c c u r r e n c e of E l - P f o l l o w e d by E2-P (Fukushima and Nakao, t h i s volume) Second, d o u b t s have p e r s i s t e d a b o u t whether t h e p h o s p h o r y l a t e d enzyme i s a r e q u i r e d i n t e r m e d i a t e f o r t h e Na,K-ATPase r e a c t i o n . Recently, t h e s e concerns have c e n t e r e d on u n c e r t a i n t i e s a b o u t whether t h e obs e r v e d r a t e s of p h o s p h o r y l a t i o n and of dephosphorylat i o n a r e c o n s i s t e n t w i t h t h e s t e a d y - s t a t e r a t e of ATP h y d r o l y s i s and w i t h t h e e a r l y b u r s t of dephosphorylat i o n i n t r a n s i e n t state k i n e t i c experiments ( F r o e h l i c h e t a 1 , t h i s volume; Lowe and Reeve, t h i s volume; P l e s n e r , t h i s volume). P l e s n e r e t a l . (1981) have c r i t i c i z e d t h e e a r l i e r c a l c u l a t i o n s o f Mardh and E l - P + E2-P * E 2 Lindahl (1977) supporting t h e E l sequence. The r e s o l u t i o n of t h e s e i s s u e s i s n o t c u r r e n t l y c l e a r . A s i d e from t h e d i f f i c u l t i e s of e x a c t l y d u p l i c a t i n g s t e a d y - s t a t e c o n d i t i o n s d u r i n g measurements of p h o s p h o r y l a t i o n and d e p h o s p h o r y l a t i o n , t h e r e i s t h e p o s s i b i l i t y t h a t t i g h t l y bound ATP c o n t i n u e s t o phosp h o r y l a t e t h e enzyme even a f t e r t h e d i l u t i o n w i t h unl a b e l e d ATP t h a t i s added t o p e r m i t measurement o f dep h o s p h o r y l a t i o n ( F r o e h l i c h e t a l . , t h i s volume; Lowe

.

.

-f

JOSEPH D. ROBINSON

500

and Reeve, t h i s volume) , t h e r e b y d i s t o r t i n g estimates o f E-P c o n c e n t r a t i o n and decay. A f u r t h e r p o s s i b i l i t y i s t h e p a r t i c i p a t i o n of a n a c i d - l a b i l e enzyme-phosphate complex (Lowe and Reeve, t h i s volume; P l e s n e r , t h i s volume; F r o e h l i c h e t al., 1976). O v e r a l l , t h e v i r t u e s of t h e m o d i f i e d A l b e r s - P o s t scheme ( F i g . 1) recommend t h e accommodation o f t h e s e a b e r r e n t d a t a w i t h as l i t t l e r e v i s i o n as p o s s i b l e . 2.

Ping-Pong

Sequences

A ping-pong mechanism (which i n t r a n s p o r t t e r m i nology i s o f t e n r e f e r r e d t o a s " c o n s e c u t i v e " ) d e s c r i b e s t h e sequence i n which r e a c t a n t A b i n d s and t h e f i r s t p r o d u c t i s r e l e a s e d b e f o r e r e a c t a n t B b i n d s . T h i s sequence i s r e p r e s e n t e d by a f a m i l y o f p a r a l l e l l i n e s i n Lineweaver-Burk p l o t s as one r e a c t a n t i s v a r i e d a t s e v e r a l f i x e d l e v e l s of t h e o t h e r ; such p a r a l l e l p l o t s occur whenever a n i r r e v e r s i b l e s t e p (such as, i n i n i t i a l v e l o c i t y s t u d i e s , p r o d u c t release) i n t e r v e n e s between t h e a d d i t i o n of two r e a c t a n t s . The m o d i f i e d A l b e r s - P o s t r e a c t i o n sequence ( F i g s . 1 and 2 ) i n d i c a t e s t h a t p a r a l l e l p l o t s c o n s i s t e n t w i t h ping-pong s e q u e n c e s s h o u l d o c c u r w i t h t h e r e a c t a n t p a i r s Na+/K+ ( N a + release i n t e r v e n e s ) , K+/ATP ( P i rel e a s e i n t e r v e n e s ) , and ATP/Na+ (K+ release i n t e r v e n e s ) . Sachs (1980) argued t h a t t h e N a + / N a + and K+/K+ exchange p r o p e r t i e s o f t h e t r a n s p o r t system s t r o n g l y s u g g e s t a ping-pong r e a c t i o n sequence, and d e m o n s t r a t e d t h a t t h e f a i l u r e t o f i n d t h e expected p a r a l l e l p l o t s c o u l d b e a t t r i b u t e d t o t h e c o e x i s t e n c e of an a l t e r n a t i v e r e a c t i o n pathway r e p r e s e n t i n g uncoupled N a + e f f l u x . Robinson (1967) showed p a r a l l e l p l o t s o f t h e app a r e n t Km f o r ATP ( i n t h e c o n c e n t r a t i o n r a n g e t o f i l l t h e l o w - a f f i n i t y s u b s t r a t e s i t e ) as a f u n c t i o n o f K+, c o n s i s t e n t w i t h t h e i n t e r v e n t i o n o f an i r r e v e r s i b l e s t e p . T h i s i s most l i k e l y due t o t h e release o f Pi ( a s shown i n F i g s . 2 and 3 ) r a t h e r t h a n t o t h e release o f K+ s i n c e i n t h e s e e x p e r i m e n t s " s i d e d n e s s " w a s l o s t , and t h u s K+ w a s p r e s e n t b o t h a s " s u b s t r a t e " and " p r o d u c t . " R e c e n t l y , t h e s e e x p e r i m e n t s have been r e p e a t e d i n prepar a t i o n s where K+ w a s a v a i l a b l e o n l y a t t h e e x t e r i o r , a s " s u b s t r a t e , " i n r e d b l o o d c e l l g h o s t s ( E i s n e r and R i c h a r d s , t h i s volume) and i n d i a l y z e d s q u i d axons (Beaugd and DiPolo, t h i s volume): i n b o t h cases t h e d a t a are c o n s i s t e n t w i t h t h e ping-pong sequence. Moreo v e r , E i s n e r and R i c h a r d s showed t h a t a d d i n g P i , thereby making t h e release o f P i r e v e r s i b l e i n i n i t i a l v e l o c i t y s t u d i e s , changed t h e p l o t s from a p a r a l l e l t o a n

KINETIC ANALYSES AND REACTION MECHANISM

501

i n t e r s e c t i n g p a t t e r n . G a r r a h a n e t a l . ( t h i s volume) found p a r a l l e l p l o t s i n s t u d i e s w i t h ATP f i l l i n g o n l y t h e h i g h - a f f i n i t y s u b s t r a t e s i t e s ; t h e s e d a t a a r e cons i s t e n t w i t h t h e a l t e r n a t i v e pathway of F i g . 1. I t s h o u l d b e n o t e d t h a t f o r schemes i n which t h e low- a n d high-affinity s u b s t r a t e sites are not interconverted, i . e . , ATP b i n d i n g t o t h e l o w - a f f i n i t y s i t e i s n o t d i r e c t l y t r a n s f o r m e d i n t o ATP b i n d i n g t o t h e h i g h - a f f i n i t y s i t e , t h a t a ping-pong r e l a t i o n s h i p between K + and ATP w i l l o c c u r as w e l l . With r e g a r d t o t h e ATP/Na+ p a i r , G a r r a h a n e t a 1 ( t h i s volume) d i d n o t f i n d t h e p r e d i c t e d p a r a l l e l f a m i l y o f p l o t s , and found i n s t e a d no e f f e c t of ATP on t h e ~ 0 . 5 f o r N a + o r v i c e v e r s a ; however, " s i d e d n e s s " w a s n o t p r e s e r v e d i n t h e s e s t u d i e s and t h u s N a + w a s p r e s e n t b o t h as " s u b s t r a t e " and " p r o d u c t . I' Robinson ( 1 9 7 7 a ) d i d f i n d a c h a n g e i n ~ 0 . 5 f o r N a + as ATP w a s v a r i e d , b u t , a g a i n , " s i d e d n e s s , " s e p a r a t i n g c a t i o n s as " s u b s t r a t e s " from c a t i o n s as " p r o d u c t s , I 1 w a s l o s t . In any case, G a r r a h a n e t a l . ( t h i s volume) p r o p o s e d t h a t N a c o u l d b i n d t o E ( K ) t o s p e e d d i s s o c i a t i o n of K + . P l e s n e r and P l e s n e r ( t h i s volume) a l s o a r g u e d f o r t h e s i m u l t a n e o u s p r e s e n c e o f N a + and K + , i n a c c o r d w i t h t h e b i n d i n g measurements o f Tonomura e t a l . ( t h i s volume). The d i s c r e p a n c y w i t h t h e a p p a r e n t ping-pong s e q u e n c e f o r N a + / K + ( a b o v e ) and w i t h o t h e r b i n d i n g s t u d i e s showing c o m p e t i t i o n between t h e s e c a t i o n s ( e . g . , J e n s e n and O t t o l e n g h i , t h i s volume) i s u n r e s o l v e d .

.

3.

A D P / A T P and

N a + / N a + Exchanges

Reexamination of e a r l i e r e x p e r i m e n t s c o n f i r m e d t h e s e q u e n c e E l - P t o E2-P (Fukushima and Nakao, t h i s volume), i n accord w i t h o r i g i n a l formulations of an A D P - s e n s i t i v e phosphoenzyme t h a t p a r t i c i p a t e s i n ADP/ATP exchange p r e c e d i n g t h e A D P - i n s e n s i t i v e E2-P (Fahn e t a l . , 1 9 6 6 a , b ) . The p o s s i b i l i t y o f a d d i t i o n a l p h o s p h o r y l a t e d forms h a s a l s o been p r o p o s e d (Hobbs e t a l . , t h i s volume; Klodos e t al., t h i s v o l u m e ) . I n h i b i t i o n o f ADP/ATP exchange on a d d i n g MgCl2 i s d i f f i c u l t t o analyze because o f t h e i n t e r r e l a t e d changes i n Mg2+, Mg-ADP , and Mg-ATP c o n c e n t r a t i o n s . F r e e ADP, r a t h e r t h a n Mg-ADP, i s r e q u i r e d (Beauge and Glynn, 1979a; F r o e h l i c h e t a l . , t h i s v o l u m e ) , b u t b o t h Mg-ATP and f r e e Mg2+ p r o b a b l y a l s o i n h i b i t (Robinson , 1 9 7 6 b ) . Moreover, a l t h o u g h Mg-ATP might i n h i b i t m e r e l y by competing w i t h ADP, it i s t h e n d i f f i c u l t t o a c c o u n t f o r t h e a b i l i t y o f s u l f h y d r y l r e a g e n t s and o l i g o m y c i n t o r e l i e v e s u c h i n h i b i t i o n (Fahn e t a ] . , 1 9 6 6 a , b & + A l t e r n a t i v e l y , Robinson (1976b) p r o p o s e d t h a t Mg and

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Mg-ATP i n h i b i t e d by b i n d i n g t o t h e l o w - a f f i n i t y subs t r a t e s i t e s , f a v o r i n g conformations removed from t h e s t e p s c a t a l y z i n g exchange. R e l a t e d t o t h e s e i s s u e s i s t h e a b i l i t y of K+ t o s t i m u l a t e ADP/ATP exchange, i n t e r p r e t e d a s K+ r e l i e v i n g i n h i b i t i o n due t o Mg-ATP ( a t t h e l o w - a f f i n i t y s u b s t r a t e s i t e s ) by t h e antagonism between K+ and ATP b i n d i n g a t t h o s e s i t e s (Robinson, 1 9 7 7 b ) . Whether K+ might r e l i e v e i n s t e a d by b i n d i n g a t s t e p s 1 0 t o 8 of t h e Albers-Post f o r m u l a t i o n ( F i g . 1 1 , t h e r e by a s s r s t i n g t h e n e c e s s a r y d i s s o c i a t i o n of ATP from t h e h i g h - a f f i n i t y s i t e s , r e q u i r e s q u a n t i t a t i v e examination. A f u r t h e r u n c e r t a i n t y a r i s e s from t h e a b i l i t y of e x t e r n a l N a + t o s t i m u l a t e ADP/ATP exchange (Kaplan, t h i s volume), s i n c e ADP/ATP exchange, a c c o r d i n g t o F i g . 1, i s c a t a l y z e d by E l forms whereas e x t e r n a l Na+ s i t e s a r e p r e s e n t o n l y on E 2 forms. Na+/Na+ exchange i s r e l a t e d t o ADP/ATP exchange, b u t r e q u i r e s , i n a d d i t i o n , p a r t i c i p a t i o n of t h e E 2 forms t o a l l o w r e l e a s e and a c c e p t a n c e of e x t e r n a l Na+. C a v i e r e s and Glynn ( 1 9 7 9 ) showed t h a t t h e nonhydrolyzable a n a l o g AMP-PNP could n o t s u b s t i t u t e f o r ATP, s u p p o r t i n g t h e f o r m u l a t i o n t h a t p h o s p h o r y l a t i o n by ATP i s a s s o c i a t e d w i t h Na+ movement t o t h e e x t e r i o r , and d e p h o s p h o r y l a t i o n by ADP i s a s s o c i a t e d w i t h Na+ movement t o t h e cytoplasm. On t h e o t h e r hand, DeWeer e t a l . ( t h i s volume) demons t r a t e d t h a t Na+/Na+ exchange i s f a s t e r t h a n ADP/ATP exchange, s u g g e s t i n g t h a t ADP i s n o t r e l e a s e d w i t h each c y c l e of Na+ t r a n s p o r t t o t h e e x t e r i o r , i n c o n t r a s t + t o t h e mechanism o f F i g . 1, where ADP l e a v e s b e f o r e Na K a r l i s h e t a l . ( 1 9 7 8 ) had s u g g e s t e d p r e v i o u s l y t h a t ADP might bind t o E2-P, t h e r e b y a c c e l e r a t i n g i t s c o n v e r s i o n t o E l - P w i t h o u t t r a n s p h o r y l a t i o n o c c u r r i n g . The p r e s e n c e of ADP-binding s i t e s on E2-P i s u n c e r t a i n , and it i s p o s s i b l e t h a t l o c a l i z e d t r a p p i n g of n u c l e o t i d e s could account f o r t h e a p p a r e n t low exchange r a t e w i t h o u t e a s i n g t h e s t r i c t u r e t h a t ADP i s r e l e a s e d from E l - P .

.

4.

Pi/Water

and K+/K+

Exchanges

l 8 0 exchange between P i and water has t h e c h a r a c t e r i s t i c s e x p e c t e d from F i g . 1: a requirement f o r Mg2+ ( ~ 0 . 5 = 0 . 7 m M ) and s t i m u l a t i o n by K+ ( K 0 . 5 = 0 . 9 m M ) , w i t h no requirement f o r n u c l e o t i d e s ( P e r e z e t a l . , 1 9 7 9 ) . The c h a r a c t e r i s t i c s of K+/K+ exchange, on t h e o t h e r hand, f i t less w e l l , a s f i r s t p o i n t e d o u t by S t e i n ( 1 9 7 9 ) . K+/K+ exchange r e q u i r e s , i n a d d i t i o n t o K+ a t each membrane f a c e , P i and ATP ( o r even one of i t s nonhydrolyzable a n a l o g s ) ; t h e s e r e a c t a n t s a r e i n accord w i t h a pathway o v e r s t e p s 6-10 p l u s of F i g . 1, i n which P i can p h o s p h o r y l a t e tE enzyme t o p r o v i d e t h e

L

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Fig. 4 . Alternative sequences in the hydrolytic steps of the Na,K-ATPase reaction. This diagram differs from Fig. 1 in (a) indicating the presence of a noncovalent enzyme phosphate complex, E . P , analogous to the enzyme-vanadatecomplex, (b) including the possibility, suggested by the e / K i exchange experiments (Sachs, 1 9 8 1 ; Karlish and Stein, this volume), that ATP can bind before Pi is discharged (dashed arrows), and (c) indicating the enzyme forms bearing occluded K as E o ( K ) . The latter distinction emphasizes that, unlike E z - P , access of monovalent cations is from the cytoplasm; on the other hand, some indicators of conformational state group the EO forms with E2-P (Jdrgensen, 1 9 7 5 ; Karlish et al., 1 9 7 8 ) .

e x t e r n a l a c c e p t a n c e / d i s c h a r g e s i t e s of Ez-P, w h e r e a s ATP, b i n d i n g t o t h e l o w - a f f i n i t y substrate s i t e s , f o r c e s t h e enzyme toward El w i t h i t s c y t o p l a s m i c acceptance/discharge sites (Fig. 4 ) . The problem a r i s e s i n t h e q u a n t i t a t i v e r e l a t i o n s h i p between ATPand P i - m e d i a t e d s t i m u l a t i o n o f exchange: ATP and P i a t high c o n c e n t r a t i o n s should i n h i b i t , b u t t h e y do n o t (Simons, 1 9 7 4 , 1 9 7 5 ) . S t e i n ( 1 9 7 9 1 , S a c h s (19811, and K a r l i s h and S t e i n ( t h i s volume) d e m o n s t r a t e d t h a t s u c h l a c k of i n h i b i t i o n r e q u i r e s a f o r m u l a t i o n w i t h i n t e r m e d i a t e s b i n d i n g P i and ATP s i m u l t a n e o u s l y . Sachs (1981) p r o p o s e d a model i n which ATP, P i , and c y t o p l a s m i c K+ can b i n d i n random o r d e r , w i t h exchange o c c u r r i n g o n l y a f t e r a l l t h r e e l i g a n d s h a v e bound. On t h e o t h e r hand, K a r l i s h and S t e i n ( t h i s volume) f i t t e d t h e i r d a t a t o a formulation t h a t includes four p a r a l l e l a l t e r n a t i v e s , one w i t h n e i t h e r P i n o r ATP, o n e s w i t h e a c h a l o n e , and o n e w i t h b o t h t o g e t h e r , p l u s t h e p o s s i b i l i t y of c o m p o s i t e pathways. A c o m p o s i t e i s shown i n F i g . 4 , i n which t h e s o l i d a r r o w s d e p i c t t h e s t a n d a r d s e q u e n c e of F i g . 1, m o d i f i e d t o show a n o n c o v a l e n t l y bound enzyme-phosphate ( E 0 . P ) on t h e o c c l u d e d enzyme form ( i . e . , w i t h c a t i o n s i t e s a v a i l a b l e , a l b e i t p o o r l y , t o t h e c y t o p l a s m ) , and w i t h ATP b i n d i n g t o s u c h a d e p h o s p h o r y l a t e d form. The

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dashed arrows p r o v i d e a p a r a l l e l a l t e r n a t i v e w i t h ATP and P i bound s i m u l t a n e o u s l y . Two problems w i t h t h i s scheme are a p p a r e n t . ( a ) The a p p a r e n t ping-pong sequence o f K+/ATP a d d i t i o n s h o u l d n o t o c c u r i f t h e dashed pathway i s q u a n t i t a t i v e l y s i g n i f i c a n t (see S e c t i o n I I I , C , 2 ) ; q u i t e p o s s i b l y s u c h a pathway i s minor i n t h e Na,K-ATPase r e a c t i o n p r o c e s s . ( b ) How b o t h E - P and EeATP are t o f i t i n t o t h e same l o w - a f f i n i t y s u b s t r a t e s i t e is u n c l e a r ; one s o l u t i o n t o t h i s problem i s t o invoke b i n d i n g o f p h o s p h a t e and ATP a t s e p a r a t e sites, c o n s i s t e n t with a d d i t i o n a l nucleotide binding s i t e s (see S e c t i o n I I I , A , l ) on t h e same o r d i f f e r e n t a - p e p t i d e s (see S e c t i o n I V ) . Vanadate i n h i b i t s ATP b i n d i n g t o h i g h - a f f i n i t y s u b s t r a t e s i t e s (Smith, 1981a) , b u t even if v a n a d a t e b i n d i n g c o r r e s p o n d s t o E - P forms ( C a n t l e y e t a l . , 19781, t h i s d o e s n o t e x c l u d e ATP bindi n g t o t h e low-affinity s u b s t r a t e s i t e s i n t h e presence of E S P . 5.

A c t i v a t i o n o f t h e K-Phosphatase P1 u s Na+

R e a c t i o n b y ATP

I n t h e a b s e n c e of N a + , ATP i n h i b i t s t h e K-phosphat ase r e a c t i o n e i t h e r by d i r e c t c o m p e t i t i o n w i t h t h e subs t r a t e NPP f o r t h e same s i t e , presumably t h e low a f f i n i t y s i t e of E o ( K ) , o r by ATP s h i f t i n g t h e e q u i l i b r i u m away from t h e forms b i n d i n g NPP. I n t h e presence of N a + however, low c o n c e n t r a t i o n s of ATP c a n s t i m u l a t e t h e K-phosphatase r e a c t i o n t h r o u g h a r e d u c t i o n i n t h e ~ 0 . 5 f o r K+ (Robinson, 1 9 6 9 ) . S i n c e ATP p h o s p h o r y l a t e s t h e enzyme under t h e s e c o n d i t i o n s , i t i s p l a u s i b l e t h a t s t i m u l a t i o n r e s u l t s from ATP a t t h e h i g h - a f f i n i t y subs t r a t e s i t e s p r o d u c i n g t h e E2-P form w i t h i t s higha f f i n i t y s i t e s f o r K+ (Robinson, 1 9 7 5 a ) ; t h i s i n t e r p r e t a t i o n i s s u p p o r t e d by t h e d e m o n s t r a t i o n t h a t i n t h e p r e s e n c e o f ATP p l u s N a + t h e K-phosphatase i s a c t i v a t e d t h r o u g h e x t e r n a l r a t h e r t h a n c y t o p l a s m i c s i t e s (Drapeau and B l o s t e i n , 1 9 8 0 ) . The u n c e r t a i n t y a b o u t t h e mechanism a r i s e s from t h e r e c o g n i t i o n t h a t s e v e r a l NPP molecules a r e hydrot h u s s e q u e n t i a l p h o s p h o r y l a t i o n by l y z e d f o r each ATP: one ATP f o l l o w e d by h y d r o l y s i s of one NPP i s i n s u f f i c i e n t . Two f o r m u l a t i o n s can e x p l a i n t h e l o n g - l a s t i n g ( a ) There c a n be d i s e f f e c t of p h o s p h o r y l a t i o n by ATP. t i n c t s i t e s f o r p h o s p h o r y l a t i o n and NPP h y d r o l y s i s , on t h e same o r d i f f e r e n t p e p t i d e s , w i t h NPP h y d r o l y s i s n o t r e q u i r i n g d e p h o s p h o r y l a t i o n o f E2-P (Robinson, 1 9 7 6 a ) . T h i s i n t e r p r e t a t i o n i s s u p p o r t e d by s t u d i e s i n d i c a t i n g m u l t i p l e b i n d i n g s i t e s (see S e c t i o n s I I I , A , l and I I I , A , 6 ) , b u t r e q u i r e s l o o s e c o u p l i n g between s u c h s i t e s i n

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t h a t t h e t u r n o v e r r a t e s must b e d i f f e r e n t . (b) A l t e r n a t i v e l y , t h e a c t i v a t i n g e f f e c t of phosphorylation c o u l d p e r s i s t even a f t e r d e p h o s p h o r y l a t i o n . C a n t l e y (1981) p r o p o s e d t h a t K' t h r o u g h b i n d i n g t o t h e h i g h a f f i n i t y e x t e r n a l K+ s i t e s o f E2-P t h e r e b y g a i n s access t o t h e o c c l u d e d s i t e s o f E o ( K ) a t which NPP h y d r o l y s i s is activated: consequently, because o f t h e slow d i s s o c i a t i o n from E o ( K ) , t h e enzyme r e m a i n s a c t i v a t e d f o r s e v e r a l c y c l e s o f NPP h y d r o l y s i s . T h i s i s an a p p e a l i n g model, and t h e c o n s t r a i n t on t h e K+-phosphatase react i o n , t h a t K+ need n o t d i s s o c i a t e a f t e r e a c h NPP hyd r o l y s i s , is reasonable. D.

DIMERIC

ENZYME MODELS

Whether t h e f u n c t i o n a l enzyme i s a s i n g l e a 6 comp l e x o r a n (a812 dimer i s u n r e s o l v e d . The s t r o n g e s t e v i d e n c e f o r t h e dimer i s from r a d i a t i o n i n a c t i v a t i o n e x p e r i m e n t s , i n d i c a t i n g a minimal molecular w e i g h t f o r t h e N a , K - A T P a s e n e a r 3 0 0 , 0 0 0 ( E l l o r y e t al., 1979; O t t o l e n g h i e t a l . , t h i s v o l u m e ) . Such a v a l u e i s cons i s t e n t w i t h r e s u l t s from s e d i m e n t a t i o n e q u i l i b r i u m ( H a s t i n g s and Reynolds, 1 9 7 9 ; Esmann e t al., 19801, f r e e z e - f r a c t u r e e l e c t r o n microscopy (Maunsbach e t a l . , 19791, and c r o s s - l i n k i n g ( K y t e , 1975; A s k a r i e t al., 1980) e x p e r i m e n t s . Moreover, c e r t a i n c h e m i c a l m o d i f i c a t i o n e x p e r i m e n t s r e v e a l l o s s e s of i n t e g r a l f r a c t i o n s of enzyme a c t i v i t y ( e . g . , G r o s s e et al., 1 9 7 9 ; Sen e t a l . , 1 9 8 1 ) , and c e r t a i n b i n d i n g s t u d i e s i n d i c a t e f r a c t i o n a l r e a c t i v i t y ( e . g . , A s k a r i and Huang, 1 9 8 1 ) . I f i n d e e d t h e f u n c t i o n a l enzyme c o n t a i n s two catal y t i c a - p e p t i d e s t h e q u e s t i o n arises: what f u n c t i o n a l a d v a n t a g e i s c o n f e r r e d by s u c h a d i m e r i c s t a t e ? A t t e m p t s t o d e p i c t t h e two a - p e p t i d e s o p e r a t i n g o u t o f p h a s e (see S e c t i o n 11; F i g . 3 ) r e v e a l no o b v i o u s catal y t i c advantages. F o r example, a d d i t i o n o f ATP d o e s n o t s t i m u l a t e dephosphorylation as experiments h a l t i n g enzyme p h o s p h o r y l a t i o n w i t h e i t h e r EDTA o r u n l a b e l e d ATP r e v e a l (see S e c t i o n I I I , C , l ) , n o r d o e s a d d i n g ATP a c c e l e r a t e d i s s o c i a t i o n of v a n a d a t e (Smith et a l . , 1 9 8 0 ) . Moreover, a l l t h e a - p e p t i d e s a r e c a p a b l e o f b e i n g p h o s p h o r y l a t e d a t t h e same t i m e (Peters et a l . , 1 9 8 1 ) and t h e enzyme maximally p h o s p h o r y l a t e d by ATP c a n n o t b e p h o s p h o r y l a t e d f u r t h e r by P i (Schuurmans Stekhoven e t a l . , 1 9 8 0 ) ; t o what e x t e n t s u c h e x p e r i m e n t s must r e f l e c t c o n d i t i o n s of "normal" N a , K - A T P a s e a c t i v i t y may, however, b e a r g u e d . Other r a t i o n a l e s i n c l u d e a f u n c t i o n f o r a dimeric complex i n forming t h e c a t i o n t r a n s p o r t c h a n n e l between

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t h e p e p t i d e s o r i n f a c i l i t a t i n g i n s e r t i o n of t h e newly s y n t h e s i z e d complex i n t o t h e membrane. It is d i f f i c u l t t o v i s u a l i z e a dimeric complex c a t a l y z i n g ATP h y d r o l y s i s on e a c h s u b u n i t i n a random f a s h i o n w i t h no i n t e r a c t i o n . F o r o t h e r s y s t e m s , p e r h a p s anal o g o u s , Macara and C a n t l e y (1981) argued f o r independe n c e of each s u b u n i t i n t h e d i m e r i c a n i o n exchange t r a n s p o r t e r of r e d blood c e l l membrane band 3 , whereas Ikemoto e t a l . ( 1 9 8 1 ) found w i t h t h e s a r c o p l a s m i c r e t i culum C a - A T P a s e t h a t t r a n s p o r t w a s c o n s i s t e n t w i t h two s u b u n i t s o p e r a t i n g o u t o f phase.

IV.

CONCLUSIONS

The m o d i f i e d r e a c t i o n sequence of F i g . 1 ( o r t h e f u r t h e r m o d i f i c a t i o n of F i g . 4 ) have b o t h t h e o r e t i c a l a p p e a l and e x p e r i m e n t a l s u p p o r t . Remaining problems and u n c e r t a i n t i e s i n c l u d e t h e p o s s i b i l i t y of d i m e r i c c a t a l y t i c and t r a n s p o r t s y s t e m s , a s w e l l a s o f m u l t i p l e d i s t i n c t s i t e s f o r t h e n u c l e o t i d e s u b s t r a t e and t h e i r r e l a t i o n s h i p t o s u c h p r o c e s s e s a s ADP/ATP exchange, Mg2+-modif i e d r e a c t i o n s , ATP s t i m u l a t i o n o f t h e Kp h o s p h a t a s e a c t i v i t y , and t h e a p p a r e n t r e q u i r e m e n t f o r s i m u l t a n e o u s b i n d i n g of ATP and P i i n K+/K+ exchange. A l t e r n a t i v e i n t e r p r e t a t i o n s , o r t h e i n c l u s i o n of quant i t a t i v e l y minor p a r a l l e l pathways, may be s u f f i c i e n t t o accommodate t h e s e a p p a r e n t a n o m a l i e s w i t h i n t h e c o n v e n t i o n a l scheme.

ACKNOWLEDGMENT The author's experiments were supported by USPHS research grant NS-05430.

REFERENCES Albers, R. W. (1967). Biochemical aspects of active transport. A n n u . Rev. Biochem. 36, 727-756. Askari, A. , and Huang, W. H. (1981). Na+,K+-ATPase: (Ca2+ + ouabainl-dependent phosphorylation by Pi. FEBS L e t t . 126, 215-218.

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+ +-

A s k a r i , A . , Huang, W. H . , and A n t i e a u , J. M. ( 1 9 8 0 ) . N a , K ATPase: Ligand i n d u c e d c o n f o r m a t i o n a l t r a n s i t i o n s and a l t e r n a t i o n s i n s u b u n i t i n t e r a c t i o n s e v i d e n c e d by crossl i n k i n g s t u d i e s . B i o c h e m i s t r y 1 9 , 1132-1140. Beaug6, L. A . , and Glynn, I. M. ( 1 9 7 9 a ) . Sodium i o n s , a c t i n g a t h i g h - a f f i n i t y e x t r a c e l l u l a r s i t e s , i n h i b i t sodium-ATPase a c t i v i t y of t h e sodium pump by s l o w i n g d e p h o s p h o r y l a t i o n . J . P h y s i o l . ( L o n d o n ) 2 8 9 , 17-31. Beaug6, L. A . , and Glynn, I. M. ( 1 9 7 9 b ) . O c c l u s i o n o f K i o n s i n the u n p h o s p h o r y l a t e d sodium pump. N a t u r e ( L o n d o n ) 2 8 0 , 510-512. C a n t l e y , L. C . (1981). S t r u c t u r e and mechanism o f the ( N a , K ) ATPase. C u r r . T o p . B i o e n e r g . 1 1 , 201-237. C a n t l e y , L. C . , J r . , C a n t l e y , L. G . , and J o s e p h s o n , L. ( 1 9 7 8 ) . A c h a r a c t e r i z a t i o n o f vanadate i n t e r a c t i o n s with t h e J . B i o l . Chem. 2 5 3 , 7361-7368. (Na,K)-ATPase. C a v i e r e s , J. D . , and E l l o r y , J . C. ( 1 9 7 5 ) . A l l o s t e r i c i n h i b i t i o n o f t h e sodium pump by e x t e r n a l sodium. N a t u r e ( L o n d o n ) 2 5 5 , 338-340. C a v i e r e s , J. D . , and Glynn, I . M. ( 1 9 7 9 ) . Sodium-sodium exchange t h r o u g h t h e sodium pump. J . P h y s i o l . ( L o n d o n ) 2 9 7 , 637-645. Drapeau, P . , and B l o s t e i n , R. ( 1 9 8 0 ) . I n t e r a c t i o n s o f K+ w i t h J. B i o l . Chem. 2 5 5 , 7827-2834. (Na,K)-ATPase. E l l o r y , J. C . , Green, J . R . , Jarvis, S . M . , and Young, J. D. ( 1 9 7 9 ) . Measurement o f t h e a p p a r e n t m o l e c u l a r volume of membrane-bound t r a n s p o r t s y s t e m s by r a d i a t i o n i n a c t i v a t i o n . J . P h y s i o l . ( L o n d o n ) 2 9 5 , 1OP-UP. Esmann, M . , C h r i s t i a n s e n , C . , K a r l s s o n , K. A . , Hansson, G. C . , and Skou, J. C. ( 1 9 8 0 ) . Hydrodynamic p r o p e r t i e s of a s o l u b i l i z e d (Na+ + K+)-ATPase from r e c t a l g l a n d s of s q u a l u s a c a n t h i a s . B i o c h i m . B i o p h y s . A c t a 6 0 3 , 1-12. Fahn, S . , Koval, G. J . , and A l b e r s , R W. ( 1 9 6 6 a ) . Sodiump o t a s s i u m - a c t i v a t e d a d e n o s i n e t r i p h o s p h a t a s e o f electrop h o r u s e l e c t r i c organ. J . Biol. Chem. 2 4 1 , 1882-1889. Fahn, S . , H u r l e y , M. R . , Koval, G . J . , and Albers, R. W. ( 1 9 6 6 b ) . Sodium-potassium-activated a d e n o s i n e t r i p h o s p h a t a s e o f e l e c t r o p h o r u s e l e c t r i c o r g a n : E f f e c t s o f N-ethyl ma1eimi.de and o t h e r s u l f h y d r y l r e a g e n t s . J . B i o l . Chem. 2 4 1 , 1890-1895. 2+ F l a s h n e r , M. S . , and Robinson, J. D. ( 1 9 7 9 ) . E f f e c t s o f Mg on a c t i v a t i o n of t h e ( N a + + K+)-dependent ATPase by N a + . A r c h . B i o c h e m . B i o p h y s . 1 9 2 , 584-591. F o s t e r , D . , and Ahmed, K. ( 1 9 7 6 ) . Na+-dependent p h o s p h o r y l a t i o n Biochim. Biophys. o f t h e r a t b r a i n ( N a + + K+)-ATPase. A c t a 429, 258-273. F r o e h l i c h , J. P . , A l b e r s , R. W . , Koval, G. W . , Goebel, R . , and Berman, M. ( 1 9 7 6 ) . Evidence f o r a new i n t e r m e d i a t e s t a t e i n t h e mechanism o f ( N a + + K+)-adenosine t r i p h o s p h a t a s e . J . B i o l Chem. 2 5 1 , 2186-2188.

.

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Fukushima, Y., and Post, R. L. (1978). Binding of divalent cation to phosphoenzyme of sodium- and potassium-transport adenosine triphosphatase. J. B i o l . Chem. 2 5 3 , 6853-6862. Garay, R. P., and Garrahan, P. J. (1973). The interaction of sodium and potassium with the sodium pump in red cells. J. P h y s i o l . ( L o n d o n ) 2 3 1 , 297-325. Glynn, I. M. , and Karlish, S. J. D. (1975). The sodium pump. A n n u . R e v . P h y s i o l . 3 7 , 13-55. Glynn, I. M., and Karlish, S . J. D. (1976). ATP hydrolysis associated with an uncoupled sodium flux through the sodium pump. J. P h y s i o l . ( L o n d o n ) 2 5 6 , 465-496. Grisham, C. M., and Mildvan, A. S . (1974). Magnetic resonance and kinetic studies of the mechanism of sodium and potassium ion-activated adenosine triphosphatase. J . B i o l . Chem. 2 4 9 , 3187-3197. Grosse, R., Rapoport, T., Malur, J., Fischer, J., and Repke, K. R. H. (1979). Mathematical modeling of ATP, K+, and Na+ interactions with (Na+ + K+) -ATPase occurring under equilibrium conditions. B i o c h i m . B i o p h y s . A c t a 5 5 0 , 500-514. Hara, Y., and Nakao, M. (1981). Sodium ion discharge from pig kidney Na+,K+-ATPase. J . B i o c h e m . ( T o k y o ) 9 0 , 923-931. Hastings, D. F., and Reynolds, J. A. (1979). Molecular weight of (Na+,K+)ATPase from shark rectal gland. B i o c h e m i s t r y 18, 817-821. Hegyvary, C., and P o s t , R. L. (1971). Binding of adenosine triphosphate to sodium and potassium ion-stimulated adenosine triphosphatase. J . B i o l . Chem. 2 4 6 , 5234-5240. Ikemoto, N., Miyao, A., and Kurobe, Y. (1981). Further evidence for an oligomeric calcium pump by sarcoplasmic reticulum. J. B i o l . Chem. 2 5 6 , 10809-10814. Jencks, W. P. (1980). The utilization of binding energy in coupled vectorial processes. A d v . E n z y m o l . 51, 75-106. Jbrgensen, P. L. (1975). Purification and characterization of (Na+,K+)-ATPase. V. Transitions between the Na-form and the K-form studied with tryptic digestion as a tool. B i o c h i m . B i o p h y s . A c t a 4 0 1 , 399-415. Kanazawa, T., Saito, M., and Tonomura, Y. (1970). Formation and decomposition of a phosphorylated intermediate in the reaction of Na+-K+-dependent ATPase. J . B i o c h e m . ( T o k y o ) 6 7 , 693-711. Kaniike, C., Lindenmayer, G. E., Wallick, E . T., Lane, L. K., and Schwartz, A. (1976). Specific sodium-22 binding to a purified adenosine triphosphatase. J. B i o l . Chem. 2 5 1 , 47944196. Karlish, S. J. D. (1980). Characterization of conformational changes in (Na,K)-ATPase labeled with fluorescein at the active site. J. B i o e n e r g . B i o m e m b r . 1 2 , 111-136. Karlish, S . J. D., and Yates, D. W. (1978). Tryptophan fluorescence of (Na+ + K+)-ATPase as a tool for study of the enzyme mechanism. B i o c h i m . B i o p h y s . A c t a 5 2 7 , 115-130.

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Karlish, S. J. D. , Yates, D. W. , and Glynn, I. M. (1978). Conformational transitions between Na+-bound and K+-bound forms of (Na+ + K+)-ATPase studied with formycin nucleotides. B i o c h i m . B i o p h y s . A c t a 5 2 5 , 252-264. Kuhn, T. S. (1970) "The Structure of Scientific Revolutions." Univ. of Chicago Press, Chicago. Kyte, J. (1975). Structural studies of sodium and potassium ionactivated adenosine triphosphatase. J. B i o l . Chem. 2 5 0 , 7443-7449. Macara, I. G., and Cantley, L. C. (1981). Mechanism of anion exchange across the red cell membrane by band 3. B i o c h e m i s t r y 2 0 , 5695-5701. Mardh, S., and Lindahl, S. (1977). On the mechanism of sodiumand potassium-activated adenosine triphosphatase. J . B i o l . Chem. 2 5 2 , 8058-8061. Maunsbach, A. B., Skriver, E., and Jdrgensen, P. L. (1979). In "Na,K-ATPase: Structure and Kinetics" (J. C. Skou and J. G. Ndrby, eds.), pp. 3-13. Academic Press, New York. Moczydlowski, E. G., and Fortes, P. A. G. (1981a). Characterization of 2',3'-0-(2,4,6-trinitrocyclohexadienylidine) adenosine 5I-triphosphate as a fluorescent probe of the ATP site of sodium and potassium adenosine triphosphatase. J . B i o l . Chem. 2 5 6 , 2346-2356. Moczydlowski, E. G , and Fortes, P. A. G. (1981b). Inhibition of sodium and potassium adenosine triphosphatase by 2',3'-0(2,4,6-trinitrocyclohexadienylidene) adenine nucleotides. J . B i o l . Chern. 2 5 6 , 2357-2366. Neufeld, A. H., and L e v y , H. M. (1969). A second ouabainsensitive sodium-dependent adenosine triphosphatase in brain microsomes. J . B i o l . Chem. 2 4 4 , 6493-6497. N$rby, J. G., and Jensen, J. (1971). Binding of ATP to brain microsomal ATPase. B i o c h i m . B i o p h y s . A c t a 2 3 3 , 104-116. Perez, B., Miara, J., and D a b s , A. S. (1979). Probes at the medium and intermediate water oxygen exchange reactions of the Na,K-ATPase. In "Na,K-ATPase: Structure and Kinetics" (J. C. Skou and J. G. Ndrby, eds.), pp. 343-358. Academic Press, New York. Peters, W. H. M., Swarts, H. G. P., dePont, J. J. H. H. M., Schuurmans Stekhoven, F. M. A. H., and Bonting, S. L. (1981) (Na+ + K+)-ATPase has one functioning phosphorylation site per a subunit. N a t u r e (London) 2 9 0 , 338-339. Plesner, I. W., Plesner, L., N$rby, J. G., and Klodos, I. (1981). The steady-state kinetic mechanism of ATP hydrolysis catalyzed by membrane-bound (Na+ + K+)-ATPase from ox brain. B i o c h i r n . B i o p h y s . A c t a 6 4 3 , 483-494. Post, R. L., Kume, T., Tobin, T., Orcutt, B., and Sen, A. K. (1969). Flexibility of an active center in sodium-pluspotassium adenosine triphosphatase. J . Gen. P h y s i o l . 5 4 , 306-326s.

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P o s t , R. L., Hegyvary, C . , and Kume, S . (1972). A c t i v a t i o n by adenosine t r i p h o s p h a t e i n t h e p h o s p h o r y l a t i o n k i n e t i c s of sodium and potassium i o n t r a n s p o r t adenosine t r i p h o s p h a t a s e . J. B i o l . Chem. 2 4 7 , 6530-6540. P o s t , R. L . , Toda, G . , Kume, S . , and Taniguchi, K . ( 1 9 7 5 ) . Synt h e s i s o f adenosine t r i p h o s p h a t e by way of potassiums e n s i t i v e phosphoenzyme of sodium, potassium adenosine t r i p h o s p h a t a s e . J. S u p r a m o l . S t r u c t . 3 , 479-497. Robinson, J. D. ( 1 9 6 7 ) . K i n e t i c s t u d i e s on a b r a i n microsomal adenosine t r i p h o s p h a t a s e : Evidence s u g g e s t i n g conformational changes. B i o c h e m i s t r y 6 , 3250-3258. Robinson, J. D. ( 1 9 6 9 ) . K i n e t i c s t u d i e s on a b r a i n microsomal adenosine t r i p h o s p h a t a s e . 11. Potassium-dependent phosp h a t a s e a c t i v i t y . B i o c h e m i s t r y 8 , 3348-3355. Robinson, J. D. (1970). Reaction sequence of t h e K+-dependent phosphatase. B i o c h i m . B i o p h y s . A c t a 2 1 2 , 509-511. Robinson, J. D. ( 1 9 7 2 ) . D i v a l e n t c a t i o n s as a l l o s t e r i c m o d i f i e r s of t h e (Na+ + K+)-dependent ATPase. B i o c h i m . B i o p h y s . A c t a 2 6 6 , 97-102. Robinson, J. D. (1973). V a r i a b l e a f f i n i t y o f t h e ( N a + + K + ) dependent ATPase f o r potassium. A r c h . B i o c h e m . B i o p h y s . 1 5 6 , 232-243. Robinson, J . D. (1974). Nucleotide and d i v a l e n t c a t i o n i n t e r a c t i o n s w i t h t h e (Na+ + K+)-dependent ATPase. B i o c h i m . B i o p h y s . A c t a 3 4 1 , 232-247. + Robinson, J. D. (1975a). F u n c t i o n a l l y d i s t i n c t c l a s s e s o f K s i t e s on t h e (Na+ + K+) -dependent ATPase. B i o c h i m . B i o p h y s . A c t a 384, 250-264. Robinson, J. D. (1975b). I n t e r a c t i o n s between K+ and ATP b i n d i n g t o t h e (Na+ + K+) -dependent ATPase. B i o c h i m . B i o p h y s . A c t a 3 9 7 , 194-206. Robinson, J. D. (1976a). S u b s t r a t e s i t e s o f t h e (Na+ + K + ) dependent ATPase. B i o c h i r n . B i o p h y s . A c t a 4 2 9 , 1006-1019. Robinson, J. D. (197633). The ( N a + + K+)-dependent ATPase: Mode o f i n h i b i t i o n of ADP/ATP exchange a c t i v i t y by MgC12. B i o c h i m . B i o p h y s . A c t a 4 4 0 , 711-722. Robinson, J. D. ( 1 9 7 7 a ) . Na+ s i t e s o f t h e ( N a + + K+)-dependent ATPase. B i o c h i m . B i o p h y s . A c t a 4 8 2 , 427-437. Robinson, J. D. (1977b). K+ s t i m u l a t i o n o f ADP/ATP exchange c a t a l y z e d by t h e (Na' + K+) -dependent ATPase. B i o c h i m . B i o p h y s . A c t a 4 8 4 , 161-168. Robinson, J . D. (1980a). S e n s i t i v i t y of t h e (Na+ + K+)-ATPase t o state-dependent i n h i b i t o r s . B i o c h i m . B i o p h y s . A c t a 598, 543-553. Robinson, J . D. (1980b). Binding t o t h e h i g h - a f f i n i t y s u b s t r a t e s i t e of t h e (Na' + K+)-dependent ATPase. J . B i o e n e r g . B i o m e m b r . 1 2 , 165-174. Robinson, J. D. (1981). S u b s t i t u t i n g manganese f o r magnesium a l t e r s c e r t a i n r e a c t i o n p r o p e r t i e s of t h e ( N a + + K+)-ATPase. B i o c h i m . B i o p h y s . A c t a 6 4 2 , 405-417.

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51 1

Robinson, J. D., and Flashner, M. S. (1979a). Modification of the (Na+ + Kt)-dependent ATPase by acetic anhydride and trinitrobenzene sulfonate. A r c h . B i o c h e m . B i o p h y s . 1 9 6 , 350-362. Robinson, J. D., and Flashner, M. S. (1979b). Cation and nucleotide interactions with the Na,K-ATPase. In "Na,KATPase: Structure and Kinetics" (J. C. Skou and J. G. Nbrby, eds.), pp. 275-285. Academic Press, New York. Robinson, J. D., and Flashner, M. S. (1979~). The (Na+ + K+)activated ATPase: Enzymatic and transport properties. B i o c h i m . B i o p h y s . A c t a 5 4 9 , 145-176. Robinson, J. D., and Mercer, R. W. (1981). Vanadate binding to the (Na+K)-ATPase. J. B i o e n e r g . B i o m e m b r . 1 3 / 205-218. Robinson, J. D., Hall, E. S., and Dunham, P. B. (1977). Reversal of the Na-K pump and apparent affinity for intracellular potassium. N a t u r e ( L o n d o n ) 2 6 9 , 165-167. Sachs, J. R. (1977). Kinetics of the inhibition of the Na-K pump by external sodium. J. P h y s i o l . ( L o n d o n ) 2 6 4 , 449-470. Sachs, J. R. (1980). The order of release of sodium and addition of potassium in the sodium-potassium pump reaction mechanism. J. P h y s i o l . (London) 3 0 2 , 219-240. Sachs, J. R. (1981). Mechanistic implications of the potassiunpotassium exchange carried out by the sodium-potassium pump. J. P h y s i o l ( L o n d o n ) 31 6 , 263-277. Schuurmans Stekhoven, F., and Bonting, S. L. (1981). Transport adenosine triphosphatases. P h y s i o l . R e v . 61, 1-76. Schuurmans Stekhoven, F. M. A. H., Swarts, H. G. P., dePont, J. J. H. H. M., and Bonting, S. L. (1980). Studies on the (Na' + K+)-activated ATPase. B i o c h i m . B i o p h y s . A c t a 5 9 7 ,

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Sen, P. C., Kapakos, J. G., and Steinberg, M. (1981). Modification of (Na+ + K+) -dependent ATPase by fluorescein isothiocyanate. A r c h . B i o c h e m . B i o p h y s . 2 1 1 , 652-661. Simons, T. J. B. (1974). Potassium-potassium exchange catalyzed by the sodium pump in human red cells. J. P h y s i o l . ( L o n d o n ) 2 3 7 , 123-155. Simons, T. J. B. (1975). The interaction of ATP-analogues possessing a blocked y-phosphate group with the sodium pump in human red cells. J . P h y s i o l . ( L o n d o n ) 2 4 4 , 731-739. S o u , J. C., and Esmann, M. (1981). Eosin, a fluorescent probe of ATP binding to the ( N a t + Kt)-ATPase. B i o c h i m . B i o p h y s . A c t a 6 4 7 , 232-240. Smith, R. L., Zinn, K., and Cantley, L. C. (1980). A study of the vanadate-trapped state of the (Na,K)-ATPase. J. B i o l . Chem. 2 5 5 , 9852-9859. Stein, W. D. (1979). Half-of-the-sites reactivity and the Na,K-ATPase. I n "Na,K-ATPase: Structure and Kinetics" (J. C. Skou and J. G. Ngkby, eds.), pp. 475-486. Academic Press, New York.

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Taniguchi, K . , and P o s t , R. L. (1975). S y n t h e s i s o f a d e n o s i n e t r i p h o s p h a t e and exchange between i n o r g a n i c phosphate and adenosine t r i p h o s p h a t e i n sodium and potassium i o n t r a n s J. B i o l . C h e m . 2 5 0 , 3010-3018. p o r t adenosine t r i p h o s p h a t e . Yamaguchi, M., and Tonomura, Y . (1980a). Binding of monovalent c a t i o n s t o N a + , K+-dependent ATPase p u r i f i e d from p o r c i n e kidney. I. Simultaneous b i n d i n g of t h r e e sodium and two potassium i o n s . J . B i o c h e m . ( T o k y o ) 8 8 , 1365-1375. Yamaguchi, M . , and Tonomura, Y . (1980b). Binding o f monovalent c a t i o n s t o N a + , K+-dependent A T P a s e p u r i f i e d from p o r c i n e kidney. 11. A c c e l e r a t i o n of t r a n s i t i o n from a K+-bound form t o a Na+-bound form. J. B i o c h e m . ( T o k y o ) 8 8 , 13771385.

CURRENT TOPICS IN MEMBRANES AND TRANSPORT, VOLUME 19

Evidence for Parallel Pathways of Phosphoenzyme Formation in the Mechanism of ATP Hydrolysis by Electrophorus Na,K-ATPase JEFFREY P. FROEHLICH National lnstitute on Aging National Institutes of Health Gerontology Research Center Baltimore City Hospituls Baltimore, Maryland

A" S. HOBBSAND R. WAMVEALBERS National Institute of Neurological and Communicative Disorders and Stroke National Institures of Heulth Berhesda, Maryland

I.

INTRODUCTION

I n a p a p e r p r e s e n t e d a t t h e Second I n t e r n a t i o n a l C o n f e r e n c e on t h e P r o p e r t i e s and F u n c t i o n o f t h e N a , K A T P a s e , Klodos and Ndrby ( 1 9 7 9 ) showed t h a t t h e measured r a t e of ATP h y d r o l y s i s i n N a , K - A T P a s e from ox b r a i n was f a s t e r t h a n t h e e x p e c t e d r a t e c a l c u l a t e d from t h e amount o f K + - s e n s i t i v e phosphoenzyme (E2-P) and i t s t u r n o v e r r a t e . A s i m i l a r f i n d i n g was r e p o r t e d a t t h e p r e v i o u s c o n f e r e n c e by Tonomura and Fukushima ( 1 9 7 4 ) who s u g g e s t e d t h a t a n u n d e r e s t i m a t i o n of t h e r a t e of dep h o s p h o r y l a t i o n and h e n c e ATP h y d r o l y s i s m i g h t r e s u l t from t h e p r e s e n c e of t i g h t l y bound ATP t h a t c o n t i n u e s t o p r o d u c e phosphoenzyme a f t e r r e p h o s p h o r y l a t i o n of t h e enzyme i s p r e v e n t e d by d i l u t i o n o f t h e l a b e l e d f r e e s u b s t r a t e . T h i s would n o t a c c o u n t f o r t h e d i s c r e p a n c y r e p o r t e d by Klodos and Ndrby s i n c e t h e y measured t h e decay o f t h e phosphoenzyme i n t h e p r e s e n c e o f ADP which s h o u l d h a v e p r e v e n t e d t h e f o r m a t i o n of E2-P from E l - P by A s an i n c r e a s i n g t h e c o n v e r s i o n o f El-P t o EIATP. 513

JEFFERY P. FROEHLICH eta/.

514

a l t e r n a t i v e e x p l a n a t i o n , t h e y proposed t h a t i n o r g a n i c p h o s p h a t e ( P i ) m i g h t a l s o be produced by t h e d i r e c t breakdown of E l - P t o E l + P i o r by a p a r a l l e l pathway o f ATP h y d r o l y s i s i n v o l v i n g a n a c i d - l a b i l e i n t e r m e d i a t e . W e have p r e v i o u s l y shown ( F r o e h l i c h e t a l . , 1 9 7 9 ; Hobbs e t a l . , 1 9 8 0 a ) t h a t t h e e e l enzyme m a n i f e s t s a s i m i l a r i n e q u a l i t y between t h e r a t e of P i p r o d u c t i o n d u r i n g t h e b u r s t p h a s e and p r e d i c t e d r a t e c a l c u l a t e d from t h e decay o f E2-P. I n t h e p r e s e n t a r t i c l e , w e have extended o u r e a r l i e r s t u d i e s t o i n c l u d e measurements o f P i r e l e a s e u n d e r c o n d i t i o n s p r e v e n t i n g r e p h o s p h o r y l a t i o n of t h e e n zyme. The r e s u l t s show t h a t when EDTA o r e x c e s s u n l a b e l e d ATP i s added t o t h e phosphoenzyme, P i r e l e a s e e x c e e d s t h e amount of E-P decay s u p p o r t i n g t h e e x i s t e n c e of a p o o l of bound n u c l e o t i d e t h a t d o e s n o t r a p i d l y exchange w i t h ATP i n t h e medium. I n a d d i t i o n , a comparison of t h e obs e r v e d p a t t e r n of E-P decay v e r s u s t i m e w i t h t h e p r e d i c t e d p a t t e r n s f o r enzyme mechanisms c o n t a i n i n g more t h a n one phosphoenzyme i n t e r m e d i a t e r e v e a l e d t h a t t h e mechanism of ATP h y d r o l y s i s i n e e l Na,K-ATPase v e r y l i k e l y i n v o l v e s more t h a n o n e pathway f o r phosphoenzyme formation.

11.

METHODS

E e l e l e c t r i c o r g a n N a , K - A T P a s e was p r e p a r e d a s p r e v i o u s l y d e s c r i b e d (Goldman and A l b e r s , 1 9 7 3 ) . Samples o f t h e enzyme were f r o z e n i n l i q u i d n i t r o g e n and s t o r e d a t -80'. Rapid mixing e x p e r i m e n t s were c a r r i e d o u t u s i n g t h e c h e m i c a l quench-flow a p p a r a t u s d e s c r i b e d by F r o e h l i c h et a l . ( 1 9 7 6 b ) . The t i m e c o u r s e of d e p h o s p h o r y l a t i o n of Na,K-ATPase was measured by p h o s p h o r y l a t i n g t h e enzyme f o r 1 1 6 msec i n a medium c o n t a i n i n g 1 0 0 mM N a C 1 , 20 mM K C 1 , 3 mM MgC12, 0 . 1 mM EDTA, 50 mM T r i s - H C 1 (pH 7 . 5 ) , and [y-32P]ATP a t d i f f e r e n t c o n c e n t r a t i o n s ( 1 0 - 2 0 0 V M ) , and t h e n a d d i n g EDTA ( 3 . 3 3 o r 1 0 m M ) , u n l a b e l e d ATP ( 3 . 3 3 o r 10 m M ) , o r EDTA p l u s ATP t o p r e v e n t t h e enzyme from becoming r e p h o s p h o r y l a t e d . The enzyme was e q u i l i b r a t e d w i t h K+ b e f o r e h a n d t o p r e v e n t p a s s i v e d i f f u s i o n from c o n t r o l l i n g t h e r a t e of d e p h o s p h o r y l a t i o n of Na,K-ATPase w i t h s e q u e s t e r e d K+ s i t e s . F o r d e t e r m i n a t i o n of t h e r a t e o f ATP d i s s o c i a t i o n , t h e enzyme (1 mg/ml) was i n c u b a t e d w i t h 1 0 0 ~ I M [y-32P]ATPI 1 0 0 mM N a C 1 , 2 mM EDTA, and 6 0 mM T r i s - H C 1 (pH 7 . 5 ) f o r 1 m i n t and t h e n l o a d e d i n t o t h e machine and r a p i d l y mixed w i t h 1 0 mM ATP t o p r e v e n t r e b i n d i n g o f t h e l a b e l e d s u b s t r a t e a f t e r it had d i s s o c i a t e d . The amount of El[y-32P]ATP complex r e m a i n i n g

515

PARALLEL PATHWAYS OF PHOSPHOENZYMEFORMATION

a f t e r a v a r i a b l e t i m e of e x p o s u r e t o u n l a b e l e d ATP w a s m o n i t o r e d by s u b s e q u e n t l y a d d i n g 3 mM MgC12 t o t h e r e a c t i o n m i x t u r e and m e a s u r i n g t h e amount o f phosphoenzyme a c c u m u l a t e d d u r i n g a 25 msec i n t e r v a l . A b l a n k w a s p r e p a r e d by mixing t h e enzyme w i t h [y-32P]ATP a f t e r it had been d e n a t u r e d by a c i d . Acid-quenched s a m p l e s o f t h e r e a c t i o n mix u r e w e r e a s s a y e d f o r [32P]- l a b e l e d phosphoenzyme and L3$P] P i acc o r d i n g t o p r e v i o u s l y d e s c r i b e d method ( F r o e h l i c h e t a l . , 1976a) e x c e p t t h a t t h e c h a r c o a l c o n c e n t r a t i o n w a s 2 % and t h e e x t r a c t i o n p r o c e d u r e r e p e a t e d twice. I n some experiments c e n t r i f u g a t i o n i n s t e a d o f f i l t r a t i o n w a s used t o remove t h e c h a r c o a l w i t h s i m i l a r r e s u l t s . N a , K - A T P a s e a c t i v i t y w a s measured u s i n g t h e c o u p l e d enzyme a s s a y s y s t e m i n which t h e c o n v e r s i o n of NADH t o NAD was u s e d t o m o n i t o r t h e f o r m a t i o n of ADP ( A l b e r s e t a l . , 1 9 6 8 ) . When v a n a d a t e w a s p r e s e n t , t h e enzyme w a s a l l o w e d t o r e a c t w i t h t h e i n h i b i t o r f o r 15 min p r i o r t o measuring t h e a c t i v i t y . S i m u l a t i o n s o f t h e t i m e c o u r s e o f phosphoenzyme f o r m a t i o n and P i release were c a r r i e d o u t u s i n g MLAB ( K n o t t and Reese, 1 9 7 2 ) , which employs a n i t e r a t i v e l e a s t s q u a r e s method f o r g e n e r a t i n g t h e b e s t f i t t o a set o f data points. The r o u t i n e p r o v i d e s a mean v a l u e f o r t h e b e s t f i t and a s t a n d a r d e r r o r which w a s u s e d t o j u d g e w h e t h e r two sets of d a t a a r e s i g n i f i c a n t l y d i f f e r e n t .

111. A.

RESULTS DEPHOSPHORYLATION OF THE PHOSPHOENZYME BY AND A T P

EDTA

I n a n e a r l i e r s t u d y (Hobbs e t a l . , 1980a) w e showed t h a t when e l e c t r i c o r g a n Na,K-ATPase i s p h o s p h o r y l a t e d by ATP i n t h e a b s e n c e of K+ and d e p h o s p h o r y l a t e d by t h e a d d i t i o n of EDTA o r e x c e s s u n l a b e l e d A T P , t h e t i m e c o u r s e of E-P decay i s monophasic. When K+ was p r e s e n t d u r i n g p h o s p h o r y l a t i o n , t h e d e p h o s p h o r y l a t i o n t i m e c o u r s e showed b i p h a s i c k i n e t i c s w i t h a s m a l l residuum ( < l o % ) t h a t dec a y e d v e r y s l o w l y . An a d d i t i o n a l f i n d i n g w a s t h a t t h e p r e d i c t e d r a t e of E-P decay o b t a i n e d by s i m u l a t i o n o f t h e e a r l y P i b u r s t w a s t w i c e as f a s t as t h e observed i n i t i a l r a t e o f E-P d e c a y . T h i s s u g g e s t e d t h a t n o t a l l o f t h e P i measured d u r i n g t h e b u r s t p h a s e a r o s e from t u r n o v e r of t h e phosphoenzyme. A s an a l t e r n a t i v e e x p l a n a t i o n w e p r o posed t h a t f o l l o w i n g t h e a d d i t i o n o f EDTA o r A T P , phos-

JEFFERY P. FROEHLICH eta/.

516 I

I

13

i

.o*!

1.2 1.1

1.o

.9

0

250

0

500

.a

TIME lmsecl F i g . 1 . Phosphoenzyme d e c a y and i n o r g a n i c p h o s p h a t e r e l e a s e f o l l o w i n g the a d d i t i o n o f EDTA or excess u n l a b e l e d ATP. E l e c t r o p h o r u s Na,K-ATPase ( 0 . 5 m g / m l ) was p h o s p h o r y l a t e d i n a m e d i u m cont a i n i n g 100 mM N a C l , 2 0 mM K C l , 3 mM MgC12, 6 0 mM T r i s - H C 1 (pH 7.5), a n d 2 0 pM [ Y - ~ ~ P ] A T P A . f t e r 1 1 6 m s e c o f p h o s p h o r y l a t i o n , either 1 0 mM ( f i n a l concentration) EDTA ( 0 ) or 1 mM ( f i n a l c o n c e n t r a t i o n ATP ( 0 ) w a s a d d e d a n d d e p h o s p h o r y l a t i o n w a s a l l o w e d t o p r o c e e d for the a d d i t i o n a l t i m e s i n d i c a t e d i n the f i g u r e b e f o r e q u e n c h i n g w i t h a c i d ( 2 . 2 5 % p e r c h l o r i c a c i d a n d 2 mM P i ) . P h o s p h o e n z y m e d e c a y i s s h o w n on the l e f t a n d Pi r e l e a s e on the r i g h t .

p h o r y l a t i o n i s n o t immediately b l o c k e d b u t c o n t i n u e s f o r a s h o r t t i m e a f t e r w a r d due t o a p o o l of bound n u c l e o t i d e t h a t d o e s n o t r a p i d l y exchange w i t h ATP i n t h e medium. I f bound n u c l e o t i d e i s r e s p o n s i b l e f o r t h e a p p a r e n t s l o w i n g of t h e i n i t i a l r a t e o f d e p h o s p h o r y l a t i o n , t h e n P i p r o d u c t i o n f o l l o w i n g t h e a d d i t i o n o f EDTA s h o u l d exceed t h e t o t a l amount of E-P decay. The r e s u l t s of t h i s exp e r i m e n t , d e p i c t e d i n F i g . 1, show t h a t P i r e l e a se d u r i n g d e p h o s p h o r y l a t i o n d o e s i n f a c t exceed E-P decay d u r i n g b o t h t h e i n i t i a l and f i n a l p h a s e s o f t h e r e a c t i o n . Moreover, t h e i n i t i a l and f i n a l r a t e s of P i release a r e s i m i l a r t o t h e r a t e s f o r t h e c o r r e s p o n d i n g p h a s e s o f E-P decay ( T a b l e I ) . EDTA o r ATP added s e p a r a t e l y produced e s s e n t i a l l y i d e n t i c a l p a t t e r n s of d e p h o s p h o r y l a t i o n . When EDTA and ATP were b o t h p r e s e n t d u r i n g dephosphorylat i o n , t h e t o t a l amount of P i r e l e a s e w a s a b o u t twice t h e amount o f E-P d e c a y ( n o t shown). S i n c e t h i s r e p r e s e n t s a b o u t one-half t h e t o t a l P i p r o d u c t i o n measured w i t h EDTA o r ATP a l o n e , it a p p e a r s t h a t b o t h a g e n t s are more e f f e c t i v e i n preventing rephosphorylation than e i t h e r one alone.

517

PARALLEL PATHWAYS OF PHOSPHOENZYME FORMATION

TABLE I.

1

E f f e c t o f S u b s t r a t e Concentration on P h o s p h o e n z y m e D e c o m p o s i t i o n P r o d u c e d by the A d d i t i o n o f EM'A or ATPa

50

0.041 0.071 0.242

200

0.366

10

0.018

0.013

0.010

160 162

0.161 0.275

0.032 0.071 0.064

0.011

101 157

0.038

0.010 0.029

7.8 7.9 8.6 6.7

0.133 0.39 1.04

--

a E l e c t r o p l a x Na,K-ATPase ( 0 . 5 m g / m l ) w a s p h o s p h o r y l a t e d f o r 116 m s e c i n a m e d i u m c o n t a i n i n g 100 mM NaCl, 20 mM K C l , 3 mM M g C l z , 0.1 mM EDTA, 6 0 mM T r i s - H C 1 (pH 7 . 5 ) , a n d [ y - 3 2 P ] A T P a s i n d i c a t e d i n c o l u m n 1 . D e p h o s p h o r y l a t i o n w a s i n i t i a t e d b y the a d d i t i o n o f 10 mM EDTA. I n the e x p e r i m e n t a t 50 U M [ y - 3 2 P ] A T P , p h o s p h o r y l a t i o n w a s c a r r i e d o u t a t 1 mM M9Cl2 a n d d e p h o s p h o r y l a t i o n i n i t i a t e d b y the a d d i t i o n of 3 . 3 3 mM EDTA a n d 3 . 3 3 mM ATP. T h e s u b s c r i p t s f , s , and r r e f e r t o the f a s t , s l o w , a n d r e s i d u a l c o m p o n e n t s of the d e p h o s p h o r y l a t i o n reaction, r e s p e c t i v e l y . APi r e f e r s t o the a m o u n t of p h o s p h a t e r e l e a s e d d u r i n g the 5 4 2 msec t i m e i n t e r v a l f o l l o w i n g the a d d i t i o n of EDTA or ATP.

W e examined t h e e f f e c t of v a r y i n g t h e c o n c e n t r a t i o n of ATP used t o p h o s p h o r y l a t e t h e enzyme on t h e p a t t e r n of d e p h o s p h o r y l a t i o n produced by EDTA and ATP i n t h e p r e s e n c e of K+. The r e s u l t s of t h e s e e x p e r i m e n t s , summ a r i z e d i n T a b l e I , show t h a t a s t h e ATP c o n c e n t r a t i o n w a s r a i s e d , t h e p r o p o r t i o n of phosphoenzyme t u r n i n g o v e r r a p i d l y i n c r e a s e d t o g e t h e r w i t h t h e t o t a l amount of E-P f o r m a t i o n . Between 1 and 2 0 0 U M ATP, t h e r a t i o o f r a p i d l y t o s l o w l y d e c a y i n g phosphoenzyme i n c r e a s e d from a l : l t o 4.5:1 w i t h o u t a s i g n i f i c a n t change i n t h e r a t e of e i t h e r component. I n o r g a n i c p h o s p h a t e f o r m a t i o n a l s o i n c r e a s e d w i t h [ATP] ( T a b l e I , c o l . 8 ) s u c h t h a t 5 0 V M A T P , t h e t o t a l amount of Pi r e l e a s e w a s a p p r o x i m a t e l y e q u a l t o t h e a c t i v e s i t e c o n c e n t r a t i o n estimated from t h e maximum l e v e l of p h o s p h o r y l a t i o n measured i n t h e p r e s e n c e of oligomycin ( ~ nmole/mg). 1 Thus, t h e c a p a c i t y o f c a t a l y t i c s i t e s would a p p e a r t o be s u f f i c i e n t t o a c c o u n t f o r t h e amount of P i r e l e a s e d u r i n g dephosphorylation.

JEFFERY P.FROEHLICH eta/.

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7 1

TIME (rnsecl F i g . 2 . P h o s p h o e n z y m e d e c a y and Pi r e l e a s e f o l l o w i n g the a d d i t i o n of ADP. E l e c t r o p h o r u s Na,K-ATPase was p h o s p h o r y l a t e d a s d e s c r i b e d i n the l e g e n d t o F i g . 1 . A f t e r 116 m s e c , d e p h o s p h o r y l a t i o n w a s i n i t i a t e d b y the a d d i t i o n of 1 mM ADP ( f i n a l concentration) and the r e a c t i o n w a s a l l o w e d to p r o g r e s s f o r the a d d i t i o n a l t i m e s shown i n the f i g u r e b e f o r e q u e n c h i n g w i t h a c i d . T h e f i g u r e on the l e f t s h o w s p h o s p h o e n z y m e d e c a y a n d t h e f i g u r e on the r i g h t s h o w s concomitant P i r e l e a s e . I n s e t : E f f e c t of M q 2 + c o n c e n t r a t i o n on d e p h o s p h o r y l a t i o n b y ADP. R e a c t i o n c o n d i t i o n s were the s a m e a s d e s c r i b e d a b o v e e x c e p t t h a t the Mg2+ d u r i n g d e p h o s p h o r y l a t i o n w a s 20 mM { @ ) ; the Mg2+ w a s 3 mM t h r o u g h o u t ( A ) i t h e e n z y m e w a s d e p h o s p h o r y l a t e d b y t h e a d d i t i o n o f 1 0 mM EDTA p l u s 1 mM ADP ( 0 ) . T h e s e m i l o g p l o t s of p h o s p h o e n z y m e d e c a y versus t i m e shown i n t h e i n s e t h a v e been n o r m a l i z e d t o f a c i l i t a t e c o m p a r i s o n .

B.

D E P H O S P H O R Y L A T I ON O F T H E PHOSPHOENZYME BY A D P

E x p e r i m e n t s s i m i l a r t o t h o s e w i t h EDTA and ATP were c a r r i e d o u t u s i n g m i l l i m o l a r ADP t o p r e v e n t r e p h o s p h o r y l a t i o n o f t h e enzyme by [ Y - ~ ~ P I A T P . I n t h e e x p e r i m e n t shown i n F i g . 2 a d d i t i o n of 1 mM ADP t o t h e p h o s p h o r y l a t e d enzyme produced a t r i p h a s i c p a t t e r n of E-P d e c a y , i n which o v e r o n e - h a l f of t h e phosphoenzyme d i s a p p e a r e d The i n i t i a l p h a s e of d e c a y was much f a s t e r rapidly. t h a n o b s e r v e d i n t h e p r e s e n c e of EDTA o r ATP and P i p r o d u c t i o n o c c u r r e d o n l y d u r i n g t h e i n t e r m e d i a t e and slow p h a s e s of d e c a y . The a b s e n c e of P i r e l e a s e d u r i n g t h e i n i t i a l phase i m p l i e s t h a t during t h a t i n t e r v a l phosphate i s b e i n g t r a n s f e r r e d f r o m t h e enzyme t o ADP t o form ATP.

PARALLEL PATHWAYS OF PHOSPHOENZYME FORMATION

519

When t h e Mg2+ c o n c e n t r a t i o n w a s d e c r e a s e d d u r i n g dephosp h o r y l a t i o n by t h e a d d i t i o n o f EDTA ( i n s e t , F i g . 2 ) , t h e p r o p o r t i o n o f r a p i d l y decaying phosphoenzyme i n c r e a s e d . I n c r e a s i n g t h e Mg2+ c o n c e n t r a t i o n t o 2 0 m M d u r i n g dep h o s p h o r y l a t i o n produced t h e o p p o s i t e e f f e c t . S u b t r a c t i o n of t h e slow p h a s e o f decay, which had a r a t e s i m i l a r t o t h e slow component i n F i g . 1, gave a n a p p r o x i m a t e l y s t r a i g h t l i n e w i t h a s l o p e o f 1 8 sec-1. T h i s i n t e r m e d i a t e p h a s e o f EP decay c o u l d be due t o t h e c o n v e r s i o n o f ElPADP t o ElATP i f t h e r e v e r s a l o f phosp h o r y l a t i o n during t h e r a p i d i n i t i a l phase i s incomplete. I n t h i s case, t h e r a t e o f d i s s o c i a t i o n of ATP from t h e c a t a l y t i c s i t e would c o n t r o l t h e k i n e t i c s of r e v e r s a l o c c u r r i n g s u b s e q u e n t t o t h e r a p i d p h a s e o f dephosphoryla t i o n . T h i s seems u n l i k e l y , however, s i n c e t h e r a t e c o r r e s p o n d i n g t o t h e i n t e r m e d i a t e p h a s e of dephosphoryla t i o n i s 2 t o 3 times smaller t h a n t h e measured r a t e of ATP d i s s o c i a t i o n (see b e l o w ) . The f a c t t h a t t h e combined i n t e r m e d i a t e and slow p h a s e s a c c o u n t f o r a b o u t t h e same f r a c t i o n o f t h e t o t a l phosphoenzyme a s t h e slow component i n F i g . 1 s u g g e s t s t h a t t h e y may a l l be i n t h e same p a t h way. A s i n t h e EDTA d e p h o s p h o r y l a t i o n e x p e r i m e n t , P i rel e a s e f o l l o w i n g t h e a d d i t i o n o f ADP exceeded t h e amount o f E-P decay ( F i g . 2 ) . I n c r e a s i n g t h e ADP c o n c e n t r a t i o n from 1 t o 5 mM d i d n o t s i g n i f i c a n t l y a f f e c t t h e amount o f P i r e l e a s e c o n t r a r y t o o u r e x p e c t a t i o n t h a t some reduction i n P i should occur a s a r e s u l t of an i n c r e a s e i n When t h e s e ext h e r a t e o f t r a n s i t i o n of E2-P t o E l - P . p e r i m e n t s were r e p e a t e d w i t h EDTA added t o t h e ADPcontaining s o l u t i o n t o g i v e a f i n a l ( a f t e r mixing) r a t i o o f EDTA:Mg2+ of 5:1, t o t a l P i r e l e a s e w a s less t h a n ( a t 5 mM ADP) o r e q u a l t o ( a t 1 mM ATP) t h e amount of phosphoenzyme d e c a y ( T a b l e 11). These r e s u l t s s u g g e s t t h a t ADP (predominant s p e c i e s when EDTA i s p r e s e n t ) b i n d s more t i g h t l y t o E l - P t h a n MgADP (predominant s p e c i e s when EDTA i s a b s e n t ) , t h e r e b y d e c r e a s i n g t h e c o n v e r s i o n o f E l - P t o E2-P and d e c r e a s i n g t h e amount of P i produced by bound ATP d u r i n g d e p h o s p h o r y l a t i o n . Adenosine d i p h o s p h a t e , i n a d d i t i o n t o p r e v e n t i n g P i r e l e a s e by i n c r e a s i n g t h e f o r m a t i o n of E l A T P and El-PADP, can a l s o i n h i b i t ATP h y d r o l y s i s by c o m p e t i t i o n f o r t h e c a t a l y t i c s i t e . To t e s t how e f f e c t i v e ADP i s a t p r e v e n t i n g ATP b i n d i n g , p h o s p h o r y l a t i o n and P i release were measured a f t e r mixing t h e enzyme w i t h a s o l u t i o n cont a i n i n g [y-32P]ATP and 1 5 0 - f o l d e x c e s s o f ADP. A s s e e n i n T a b l e 11, t h e amount o f P i produced u n d e r t h e s e cond i t i o n s i s a b o u t one-half t h e amount produced when ADP i s added t o t h e enzyme a f t e r i t h a s been p h o s p h o r y l a t e d . These r e s u l t s i n d i c a t e t h a t a b o u t one-half of t h e P i

JEFFERY P. FROEHLICH eta/.

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TABLE 11.

E f f e c t of ADP and EDTA on De Novo and Continued Phosphoenzyme Formation and Pi Releasea Reaction c o n d i t i o n

Na,K-ATPase + 1.5 mM MgC12 + 6.5 ~.IMATP + 1 mM ADP Phosphorylated N a I K-ATPase + 2 mM MgC12 + 1 mM ADP Phosphorylated Na, K-ATPase + 1 mM MgC12 + 1 mM ADP + 3.33 mM EDTA Phosphorylated Na, K-ATPase + 1 m M MgC12 + 5 mM ADP + 3.33 mM EDTA

AE-P (nmole/mg)

APi (nmole/mg)

0.004

0.188

0.087

0.375

0.105

0.099

0.102

0.013

a

E l e c t r o p l a x Na,K-ATPase (0.9-1.2 m g / m l ) suspended i n a medium c o n t a i n i n g 3 mM MgC12 was mixed with an equal volume o f s u b s t r a t e medium c o n t a i n i n g 13 pM [y-32P]ATP and 2 mM ADP to s i m u l a t e t h e c o n d i t i o n s o f dephosphorylation Both s y r i n g e s cont a i n e d 100 mM NaCl 20 mM K C l and 60 mM Tris-HCl (pH 7 . 4 ) . Phosphoenzyme formation and P i r e l e a s e w e r e t e r m i n a t e d a f t e r 540 msec by t h e a d d i t i o n of a c i d . I n a n o t h e r t y p e o f experiment t h e enzyme was phosphorylated a s d e s c r i b e d i n t h e l e g e n d t o F i g . 1 and then mixed with ADP or ADP p l u s EDTA to i n i t i a t e dephosphorylation. The r e a c t i o n was t e r m i n a t e d with a c i d a f t e r 5 4 0 msec. The c o n c e n t r a t i o n s of r e a g e n t s g i v e n above a r e t h e f i n a l c o n c e n t r a t i o n s a f t e r mixing.

.

r e l e a s e d d u r i n g d e p h o s p h o r y l a t i o n comes from n u c l e o t i d e t h a t is bound t o t h e enzyme a t t h e t i m e of a d d i t i o n of ADP C.

.

S I M U L A T I O N OF PHOSPHOENZYME FORMATION A N D

DECOMPOSITION

The p r e - s t e a d y - s t a t e t i m e c o u r s e of ATP h y d r o l y s i s i n e l e c t r i c o r g a n Na,K-ATPase e x h i b i t s an e a r l y b u r s t phase t h a t c o i n c i d e s w i t h a t r a n s i e n t decay i n t h e phosphoenzyme l e v e l (Hobbs e t a l . , 1 9 8 0 a , b ) . I n s i m u l a t i n g t h i s b e h a v i o r w e assumed t h a t t h e t r a n s i t i o n of El-P t o E2-P w a s much f a s t e r t h a n h y d r o l y s i s of E2-P so t h a t t h e model c o n t a i n e d o n l y t h e K + - s e n s i t i v e form of t h e phosphoenzyme. However, t h e e x p e r i m e n t i n F i g . 2 i n d i c a t e s t h a t t h e predominant form of t h e phosphoenzyme when b o t h N a + and K+ are p r e s e n t i s t h e ADP-sensitive phosphoenzyme, s u g g e s t i n g t h a t c l e a v a g e of E2-P i s f a s t e r t h a n

PARALLEL PATHWAYS OF PHOSPHOENNME FORMATION

521

TIME hnsecl F i g . 3. S i m u l a t e d t i m e c o u r s e s o f phosphoenzyme f o r m tion and P i r e l e a s e . T h e t i m e c o u r s e s o f p h o s p h o e n z y m e f o r m a t i o n ( E l - P + E2-P) and P i r e l e a s e ( E z - P i + P i ) w e r e s i m u l a t e d u s i n g the m e c h a n i s m g i v e n i n the t e x t and the f o l l o w i n g e n z y m e s i t e concent r a t i o n and set of r a t e c o n s t a n t s : (-), [Eo] = 1 X 10-6 M ; k l / k - l = 1 x lo7 M - l s e c - l / 3 5 sec-l; k2/k-2 = 1 5 0 sec-l/O; k3/k-3 = 350 sec-l/O; k4/k-4 = 1000 sec-l/O; k~Jk-5 = 1000 sec-l/O; kg/k-6 = 1 2 sec-l/O. (---), T h e p a r a m e t e r s had the same v a l u e s a s i n the p r e v i o u s c a s e e x c e p t t h a t k5 = 1 0 sec’l. The s u b s t r a t e conc e n t r a t i o n was t h e same i n e v e r y c a s e (= 10 pM). The actual data ( E - P c l o s e d c i r c l e s , P i open c i r c l e s ) were o b t a i n e d i n a n e x p e r i m e n t i n w h i c h electric o r g a n Na,K-ATPase ( 0 . 5 m g / m l ) i n a s o l u t i o n c o n t a i n i n g 100 mM NaCl, 20 mM K C I , 3 mM MgClz, and 60 mM T r i s HCI, pH 7 . 5 , was m i x e d w i t h a n e q u a l v o l u m e o f a n i d e n t i c a l s o l u t i o n b u t w i t h 20 V M [ Y - ~ ~ P ] A Tand P w i t h o u t e n z y m e . The r e a c t i o n was a l l o w e d t o p r o g r e s s f o r the i n d i c a t e d t i m e s before q u e n c h i n g w i t h a c i d .

t h e phosphoenzyme c o n v e r s i o n r e a c t i o n .

When t h e model and K+-

was expanded t o i n c l u d e b o t h t h e ADP-sensitive

s e n s i t i v e phosphoenzymes, v i z . ADP E-,,

+ ATP + E l A T P

’i

& E 1 P + E 2 P + E2.Pi

E2

-

(1)

(2)

(3)

(4)

(5)

+E l (6)

s i m u l a t i o n of t h e P i b u r s t r e u i r e d t h a t k3 = 350 sec-’ when k l / k - 1 = 1 x 107 k-lsec-q/35 sec’l, k 2 = 150 sec-1,

522

JEFFERY P. FROEHLICH eta/.

k 4 = 1000 sec-l, k5 = 1000 sec-’, k6 = 1 2 sec-’, and a l l o f t h e r e m a i n i n g r e v e r s e r a t e c o n s t a n t s were s e t e q u a l t o z e r o ( F i g . 3 ) . T h i s c h o i c e of r a t e c o n s t a n t s gave a s t e a d y - s t a t e r a t i o o f t h e A D P - s e n s i t i v e t o K+s e n s i t i v e phosphoenzyme o f a b o u t 3 t o 1; however, t h e p r e d i c t e d r a t e o f E2-P h y d r o l y s i s w a s r o u g h l y 3 t i m e s l a r g e r t h a n t h e measured v a l u e (kapp ’L 3 0 0 sec-1; Hobbs e t a l . , 1 9 8 0 a ) . Although r e v e r s i n g t h e v a l u e s f o r k 3 and k 4 g a v e an e q u a l l y good f i t t o t h e t r a n s i e n t b u r s t , t h e p r e d i c t e d r a t i o of E l - P t o E2-P s t r o n g l y f a v o r e d t h e K + - s e n s i t i v e i n t e r m e d i a t e which c o n t r a s t s with t h e observed behavior (Fig. 2 ) . Thus n e i t h e r a s s i g n m e n t gave an e n t i r e l y s a t i s f a c t o r y f i t t o a l l of t h e d a t a and a n i m p o r t a n t c o n c l u s i o n i s t h a t t h e r a t e of phosphoenzyme i n t e r c o n v e r s i o n must be f a i r l y l a r g e t o a c c o u n t f o r t h e r a p i d i n i t i a l p r o d u c t i o n of P i . The b i p h a s i c p a t t e r n of EP decay i n F i g . 1 s u g g e s t s t h a t p h o s p h o r y l a t i o n of e l e c t r i c o r g a n Na,K-ATPase by ATP p r o d u c e s m u l t i p l e forms o f t h e K + - s e n s i t i v e phosphoenzyme. I n o r d e r t o d e t e r m i n e what m e c h a n i s m ( s ) m i g h t give rise t o t h i s behavior we t e s t e d various consecutive and p a r a l l e l pathway models f o r c o n f o r m i t y w i t h t h e obs e r v e d p a t t e r n of EP d e c a y . I n t h e s e s i m u l a t i o n s phosphoenzyme f o r m a t i o n w a s modeled a c c o r d i n g t o t h e mechani s m and r a t e c o n s t a n t s g i v e n i n T a b l e I11 ( t h e mechanism w a s i d e n t i c a l t o t h e above scheme b u t d i d n o t i n c l u d e a p r o d u c t complex s t a t e ) . A f t e r t h e phosphoenzyme had acc u m u l a t e d f o r 1 1 6 msec, d e p h o s p h o r y l a t i o n w a s s i m u l a t e d by s e t t i n g k l = 0 and a l l o w i n g t h e l e v e l s of t h e i n t e r mediates t o decay t o zero. The r e s u l t s of t h i s a n a l y s i s , p r e s e n t e d i n T a b l e 111, show t h a t f o r schemes c o n t a i n i n g one p h o s p h o r y l a t e d intermediate (without r e v e r s i b l e hydrolytic cleavage , mechanism 1) o r c o n s e c u t i v e p h o s p h o r y l a t e d i n t e r m e d i a t e s (mechanism 2) y i e l d s t r a i g h t l i n e p l o t s of E-P decay v e r s u s t i m e o r p l o t s which c u r v e downward from t h e o r i g i n . S t r a i g h t s e m i l o g p l o t s are a l s o o b t a i n e d when t h e r e a r e p a r a l l e l pathways of E-P f o r m a t i o n and t h e r a t e c o n t r o l l i n g s t e p s have s i m i l a r r a t e s (mechanism 3 ) . If t h e s e r g t e s are s u f f i c i e n t l y d i f f e r e n t , however , t h e p l o t bends upward from t h e o r i g i n (mechanism 6 ) . Another case i n which t h e s e m i l o g p l o t s l o w s upward c u r v a t u r e i s one i n which t h e a c i d - s t a b l e phosphpenzyme i s r e v e r s i b l y c l e a v e d t o a n o n c o v a l e n t p r o d u c t complex ( E 2 ’ P i ) which r e l e a s e s P i a t a s l o w r a t e (mechanism 4 ; u p p e r dashed and u p p e r d o t t e d l i n e s i n F i g . 4 ) . The s i m u l a t i o n s i n F i g . 4 were o b t a i n e d by s e t t i n g t h e r a t e o f r e v e r s a l of E2-P h y d r o l y s i s t o 1 5 sec’l and t h e r a t e of P i release t o 1 0 sec-l. Although t h e s e v a l u e s gave a r e a s o n a b l y good f i t of t h e P o s t - A l b e r s mechanism t o t h e o b s e r v e d

PARALLEL PATHWAYS OF PHOSPHOENZYME FORMATION

TABLE 111.

523

P a t t e r n of P h o s p h o e n z y m e Decay f o r Mechanisms I n c l u d i n g One o r More P h o s p h o e n z y m e Intermediatea Pho sphoen zyme decay p a t t e r n

Mechanism

(1) E2-P

E2

+

( 2 ) El-P

E2-P

(3) El-P

4 E2-P

Pi

NC, CD E2

+

E2 k' E i + Pi El-P Ei-P ' k k2 ( 4 ) E2-P -&E 2 * P i E2 + P i k- 1 ( 5 ) E -P E2-P 4 E2 + P

+ +

i

El

+

+ P

NC, CD

P.

k k' = k k

1

>> k

2

,b

NC, CD

cu

k-l

cu

i

i

cu

aEnzyme m e c h a n i s m s w e r e c h a r a c t e r i z e d a c c w r d i n g t o w h e t h e r the

n p l o t o f l o g C E . - P v e r s u s t i m e ( w h e r e n i s the number o f p h o s p h o r y l i 1 a t e d i n t e r m e d i a t e s ) c u r v e d upward f r o m t h e o r i g i n (CU), c u r v e d Simudownward f r o m t h e o r i g i n ( C D ) , or showed no c u r v a t u r e ( N C ) l a t i o n s w e r e c a r r i e d o u t u s i n g the Post-Albers m e c h a n i s m :

.

ADP El

+

ATP

P.

+ E I A T P & El-P (1)

(2)

E2-P

(3)

E (4)

2

+El (5)

and the f o l l o w i n g r a t e c o n s t a n t s : k l / k - l = l o 7 M - l s e c - l / 3 5 sec-l; k 2 / k - 2 = 1 5 0 sec-'/O; k j / k - ? = 350 sec-l/O; k4/k-4 = 1000 sec-l/O; k s / k - s = 1 2 sec-l/O. The t i m e c o u r s e o f phosphoenzyme decomposit i o n was s i m u l a t e d b y a l l o w i n g p h o s p h o e n z y m e t o a c c u m u l a t e a c c o r d i n g t o the a b o v e r a t e c o n s t a n t s f o r 100 msec and then s e t t i n g kl = 0 and a l l o w i n g the i n t e r m e d i a t e s t o d e c a y t o z e r o . In testi n g the s i m p l e c o n s e c u t i v e mechanism (mechanism 2 ) , k3 and k g w e r e a l l o w e d t o h a v e the f o l l o w i n g v a l u e s : 5 0 , 100, 2 0 0 , 300, 400, 500, a n d 1 0 0 0 sec-l. T h i s y i e l d s a 7 x 7 m a t r i x c o n t a i n i n g 49 d i f f e r e n t c o m b i n a t i o n s o f v a l u e s f o r k3 and k 4 .

JEFFERY P. FROEHLICH eta/.

524

s I

Ly

0.151

41.75

O.lO/

11.70 I b-

T

TIME lmsecl F i g . 4 . S i m u l a t e d t i m e c o u r s e of d e p h o s p h o r y l a t i o n f o r r e a c t i o n m e c h a n i s m s w i t h reversible and irreversible h y d r o l y t i c c l e a v a g e s t e p s . The t i m e c o u r s e of d e p h o s p h o r y l a t i o n p r o d u c e d b y EDTA was s t i m u l a t e d u s i n g the mechanism g i v e n i n the t e x t and the f o l l o w i n g set of r a t e c o n s t a n t s . Upper d o t t e d and d a s h e d l i n e s : k l / k - l = 1 x lo7 M - I s e c - l / 3 5 sec-l; k 2 / k - 2 = 1 5 0 sec-l/O; k3/k-3 = 1000 sec-l/O; k 4 / k - 4 = 350 s e c - 1 / 1 5 sec-1; k 5 / k - 5 = 1 0 sec-l/O; k 6 / k - = 1 2 sec-l/O. T h e e n z y m e and s u b s t r a t e c o n c e n t r a M. Lower d o t t e d tions w e r e [EoY = 1 X 10-6 M ; [ATP] = 10 X and d a s h e d l i n e s : the r a t e c o n s t a n t s , e n z y m e and s u b s t r a t e concent r a t i o n s w e r e i d e n t i c a l t o the p r e v i o u s c a s e w i t h the f o l l o w i n g exc e p t i o n s : k 4 / k - 4 = 350 sec-l/O; k 5 / k - 5 = 1000 sec-l/O. T h e s o l i d l i n e i s a c u r v e drawn b y e y e t h r o u g h d a t a o b t a i n e d i n a n e x p e r i m e n t s i m i l a r t o t h a t d e s c r i b e d i n the l e g e n d t o F i g . 1 .

t i m e c o u r s e s o f E-P f o r m a t i o n and i n i t i a l P i , p r o d u c t i o n , t h e f i n a l r a t e of P i release (dashed l i n e , F i g . 3 ) w a s t o o slow because o f t h e p r e s e n c e o f two slow s t e p s i n s u c c e s s i o n , namely E 2 ' P i -+ E 2 + P i ( 1 0 sec'l) and E 2 -f E l ( 1 2 sec-1). The f i t c a n be improved by combining t h e two slow s t e p s i n t o o n e so t h a t P i release i s depenSince t h e d e n t on t h e r a t e o f c o n v e r s i o n of E 2 t o El. r a t e of d i s a p p e a r a n c e of t h e slow component i s d e p e n d e n t on t h e r a t e o f d i s s o c i a t i o n of P i from E 2 - P j r accelerat i o n o f E 2 t o El by ATP s h o u l d i n c r e a s e t h e r a t e of d i s -

PARALLEL PATHWAYS OF PHOSPHOENZYME FORMATION

525

a p p e a r a n c e o f t h e slow phosphoenzyme. In contrast to t h i s , w e o b s e r v e d t h a t d e p h o s p h o r y l a t i o n by EDTA o r excess u n l a b e l e d ATP (1 m M ) produced similar r e s u l t s ( F i g . 1). These c o n s i d e r a t i o n s s u g g e s t t h a t r e v e r s i b l e h y d r o l y t i c c l e a v a g e of E2-P i s n o t t h e p r i n c i p a l c a u s e of t h e b i p h a s i c decay p a t t e r n i n F i 1. I n s u p p o r t of t h i s c o n c l u s i o n , measurements o f 01 exchange between H 2 0 and m e d i u m P i u n d e r c o n d i t i o n s s i m i l a r t o o u r s f a i l e d t o produce d e t e c t a b l e l e v e l s of i n t e r m e d i a t e exchange ( S . Dahms, p e r s o n a l communication). The lower dashed l i n e i n F i g . 4 , which obeys monop h a s i c k i n e t i c s , r e p r e s e n t s t h e t i m e c o u r s e of phosphoenzyme decay produced by t h e P o s t - A l b e r s mechanism f o r t h e c a s e i n which t h e c l e a v a g e o f E2-P i s i r r e v e r s i b l e . T o t a l P i , p r o d u c t i o n d u r i n g d e p h o s p h o r y l a t i o n (lower dashed l i n e , F i g . 4 ) was between 2 and 3 times t h e amount of phosphoenzyme decay, i n agreement w i t h t h e o b s e r v e d b e h a v i o r . Because ATP a c c e l e r a t e s t h e c o n v e r s i o n o f E 2 t o E l ( P o s t e t a l . , 1 9 7 2 ) a g r e a t e r p r o p o r t i o n of t h e enzyme s h o u l d be i n t h e E l s t a t e a t h i g h s u b s t r a t e concent r a t i o n s . T h i s would l e a d t o an i n c r e a s e i n t h e s t e a d y s t a t e l e v e l of E l A T P and c o u l d e x p l a i n why t h e amount o f P i release p r e s e n t d u r i n g d e p h o s p h o r y l a t i o n i n c r e a s e s w i t h ATP c o n c e n t r a t i o n ( T a b l e I ) . Another mechanism which y i e l d s a semilog p l o t w i t h upward c u r v a t u r e i s o n e i n which E l - P c a n s i m u l t a n e o u s l y h y d r o l y z e d i r e c t l y t o E l + P i o r undergo c o n v e r s i o n t o E2-P. T h i s e x p l a n a t i o n seems u n l i k e l y , however, s i n c e t r e a t m e n t o f t h e enzyme w i t h o l i g o m y c i n , which b l o c k s t h e c o n v e r s i o n of E l - P t o E2-PI s t r o n g l y i n h i b i t s P i p r o d u c t i o n (Hobbs et a l . , 1 9 8 3 ) .

3.

D.

KINETICS

OF D I S S O C I A T I O N O F BOUND A T P

The o b s e r v a t i o n t h a t P i release e x c e e d s E-P decay i n t h e dephosphorylation experiments suggests t h a t t h e r e i s a p o o l o f bound n u c l e o t i d e t h a t d o e s n o t r a p i d l y exchange w i t h u n l a b e l e d ATP when t h e l a t t e r i s added i n e x c e s s t o p r e v e n t r e p h o s p h o r y l a t i o n of t h e enzyme by l a b e l e d ATP.To t e s t t h i s p o s s i b i l i t y , w e examined t h e k i n e t i c s of d i s s o c i a t i o n of l a b e l e d ATP from t h e enzyme i n a p u l s e c h a s e exp e r i m e n t . The complex between t h e enzyme and ATP w a s formed by manually mixing e l e c t r o p l a x microsomes w i t h n t h e p r e s e n c e of 1 0 0 mM NaC1, 6 0 mM 5 0 y M [ Y - ~ ~ P ] A Ti P T r i s - H C 1 (pH 7 . 5 ) , and 2 mM EDTA, t h e l a t t e r b e i n g added t o p r e v e n t endogenous Mg2+ from a c t i v a t i n g phosphorylat i o n . The e n z y m e - s u b s t r a t e complex w a s a l l o w e d t o r e a c t w i t h e x c e s s u n l a b e l e d ATP f o r v a r i o u s f i x e d times, a f t e r which Mg2+ w a s added and p h o s p h o r y l a t i o n was a l l o w e d t o

JEFFERY P. FROEHLICH eta/.

526

n 0

n I w Y

\+ n

0-

I

Lu

Y

I

I

I

I

I

0

200

400

600

800

TIME (msecl F i g . 5 . T i m e c o u r s e of [Y-32P]ATP d i s s o c i a t i o n m o n i t o r e d b y the d i s a p p e a r a n c e o f 3 2 P - 1 a b e l e d p h o s p h o e n z y m e f o l l o w i n g a n u n l a b e l e d ATP c h a s e . T h e e n z y m e - s u b s t r a t e c o m p l e x w a s f o r m e d b y i n c u b a t i n g eel Na,K-ATPase (1 m g / m l ) w i t h 100 I.IM [ y - 3 2 P ] A T P , {A) 0 or ( 0 ) 100 mM N a C l , 2 mM EDTA, a n d 6 0 mM T r i s - H C l ( p H 7 . 5 ) f o r 1 m i n a t 21'. T h e e n z y m e w a s l o a d e d i n t o the m a c h i n e a n d r a p i d l y m i x e d w i t h a s o l u t i o n c o n t a i n i n g 20 mM ATP ( f i n a l c o n c e n t r a t i o n 10 m M ) , ( A ) 0 o r ( 0 ) 100 mM N a C l , 2 mM EDTA, a n d 6 0 mM T r i s - H C 1 ( pH 7 . 5 ) . A f t e r the i n d i c a t e d t i m e s , ( 0 ) 10 mM MgC12 or {A) 10 mM MgC12 a n d 100 mM NaCl w a s a d d e d a n d p h o s p h o r y l a t i o n w a s a l l o w e d t o p r o c e e d f o r 25 m s e c before q u e n c h i n g w i t h a c i d . A s i m i l a r r e s u l t w a s obtained when 2 0 0 ~ . I M [ Y - ~ ~ P ] A TwPa s p r e s e n t . T h e d a t a a r e normalized a s an a i d to comparison.

p r o c e e d f o r 25 msec. A b l a n k w a s p r e p a r e d by d e n a t u r i n g t h e enzyme w i t h a c i d p r i o r t o a d d i n g t h e remaining comp o n e n t s of t h e r e a c t i o n m i x t u r e . A s shown i n F i g . 5 , t h e l e v e l of 32P-labeled phosphoenzyme, r e f l e c t i n g t h e amount of El[y-32P]ATP p r e s e n t a t t h e t i m e of a d d i t i o n o f M g 2 + , decreased b i p h a s i c a l l y w i t h i n c r e a s i n g t i m e of exposure t o u n l a b e l e d ATP. The r a p i d p h a s e of ATP d i s s o c i a t i o n , which a c c o u n t e d f o r 85% o f t h e t o t a l phosphoenzyme, had

PARALLEL PATHWAYS OF PHOSPHOENZYME FORMATION

527

a r a t e o f 42 sec'l s i m i l a r t o t h e v a l u e s o b t a i n e d by computer s i m u l a t i o n ( F r o e h l i c h e t a l . , 1 9 7 6 a ) o r by d i rect measurement u s i n g f l u o r e s c e n t s u b s t r a t e a n a l o g s ( K a r l i s h e t a l . , 1 9 7 8 ) . A s l o w component w a s o b s e r v e d f o l l o w i n g t h e r a p i d p h a s e which decayed a t a r a t e 8 0 t i m e s s l o w e r . When t h e e n z y m e - s u b s t r a t e complex w a s formed i n t h e a b s e n c e o f N a and l a t e r mixed w i t h N a + and Mg2+ t o i n i t i a t e p h o s p h o r y l a t i o n , t h e r a t e o f d i s a p p e a r a n c e o f t h e f a s t component remained t h e same b u t t h e amount of t h e slow component d e c r e a s e d . Because p h o s p h o r y l a t i o n i s d e p e n d e n t on N a + , t h i s r a i s e d t h e p o s s i b i l i t y t h a t t h e s l o w component m i g h t a c t u a l l y r e p r e s e n t p h o s p h o p r o t e i n r a t h e r t h a n t i g h t l y bound ATP. When a b l a n k w a s p r e p a r e d by m i x i n g a s o l u t i o n c o n t a i n i n g t h e enzyme, [ Y - ~ ~ P I A T P and , N a C l w i t h a c i d f o l l o w e d by Mg2+ p l u s c o l d ATP, 3 2 P - l a b e l e d p h o s p h o p r o t e i n e q u a l t o t h e amount of s l o w l y d i s a p p e a r i n g E-P w a s found i n t h e acid precipitate. Shamoo a n d Brodsky ( 1 9 7 1 ) and R. L. P o s t ( p e r s o n a l communication) have o b t a i n e d e v i d e n c e f o r t h e p r e s e n c e o f u n h y d r o l y z e d ATP i n a c i d p r e c i p i t a t e s o f Na,K-ATPase p r e v i o u s l y exposed t o l a b e l e d ATP. To det e r m i n e i f t h e r a d i o a c t i v i t y i n t h e a c i d p r e c i p i t a t e of t h e p u l s e c h a s e e x p e r i m e n t i s due t o t r a p p e d ATP, t h e e x p e r i m e n t w a s r e p e a t e d u s i n g [14C]ATP i n s t e a d o f [y-32P]ATP a s s u b s t r a t e . Although s i g n i f i c a n t l y more r a d i o a c t i v i t y w a s found i n t h e samples t h a n i n t h e b l a n k , N a + had no e f f e c t on t h e amount of l a b e l i n g , i n d i c a t i n g t h a t t h e s l o w component i n t h e p u l s e c h a s e e x p e r i m e n t i s p r i m a r i l y d u e t o phosphoenzyme r a t h e r t h a n t o bound ATP. An i m p o r t a n t q u e s t i o n c o n c e r n i n g t h e r a t e c o n s t a n t f o r ATP d i s s o c i a t i o n i s w h e t h e r i t i s s u f f i c i e n t l y s l o w t o a c c o u n t f o r t h e i n c r e m e n t of P i p r o d u c t i o n measured d u r i n g d e p h o s p h o r y l a t i o n . To answer t h i s q u e s t i o n , w e s i m u l a t e d P i release d u r i n g d e p h o s p h o r y l a t i o n a f t e r a l lowing t h e phosphoenzyme t o a c c u m u l a t e u n d e r c o n d i t i o n s where bound ATP c o u l d d i s s o c i a t e a t r a t e s v a r y i n g between 1 0 and 1 0 0 0 sec-l. The r e s u l t s of t h e s e s i m u l a t i o n s ( T a b l e I V ) show t h a t f o r r a t e s o f ATP d i s s o c i a t i o n o f 200 sec-1 o r l e s s , t h e amount o f P i release w a s twice t h e E-P l e v e l and t h a t P i release became s t o i c h i o m e t r i c w i t h EP d e c a y o n l y f o r v e r y l a r g e v a l u e s o f t h e d i s s o c i a t i o n rate c o n s t a n t ( > l o 0 0 sec-1). For a d i s s o c i a t i o n r a t e of 4 2 sec-1 t h e r a t i o of P i release t o E-P d e c a y l i e s between 2.5 and 3 , which i s w i t h i n t h e e x p e r i m e n t a l W e c o n c l u d e from t h i s t h a t t h e measured r a t e of range. ATP d i s s o c i a t i o n i s s u f f i c i e n t l y s l o w t o p r e v e n t r a p i d d e p l e t i o n of t h e bound n u c l e o t i d e p o o l .

JEFFERY P. FROEHLICH eta/.

528

TABLE I V .

0 10 35 75 100 200 3 00 500 1000

E f f e c t of Varying t h e ATP D i s s o c i a t i o n Rate C o n s t a n t on S i m u l a t e d P i Release d u r i n g Dephosphorylation

0.064 0.064 0.064 0.062 0.061 0.058 0.056 0.051 0.042

0.030 0.030 0.029 0.029 0.028 0.026 0.026 0.024 0 020

0.094 0.094 0.093 0.091 0.089 0.084 0.082 0.075 0.062

0.094 0.091 0.081 0.070 0.065 0.051 0.044 0.035 0.025

3.21 3.03 2.79 2.41 2.32 1.96 1.69 1.46 1.25

aPhosphoenzyme f o r m a t i o n and Pi release w e r e s i m u l a t e d u s i n g the Post-Albers m e c h a n i s m (see t e x t ) and the f o l l o w i n g set of r a t e c o n s t a n t s (Hobbs e t a l . , 1 9 8 0 a ) : kl = lo7 M - l sec-l; kz/k-2 = 150 sec-l/O; k3/k-3 = 350 sec-l/O; k4/k-4 = 1000 sec-l/O; kg/k-5 = 1000 sec-l/O; k6/k-6 = 12 sec-l/O. k2 was a l l o w e d to h a v e the v a l u e s g i v e n i n column 1 . [ E o ] = 10-6 M; [ATP] = 10 X M. A f t e r 100 m s e c o f p h o s p h o r y l a t i o n kl was s e t t o 0 and the interm e d i a t e s w e r e a l l o w e d t o d e c a y f o r 500 msec. E.S and E-PT refer t o the e n z y m e - s u b s t r a t e c o n c e n t r a t i o n a n d phosphoenzyme (El-P + Ez-P) level a t the s t a r t of d e p h o s p h o r y l a t i o n and APi i s the amount of P i r e l e a s e d i n the 500-msec d e p h o s p h o r y l a t i o n i n t e r v a l .

E.

I N H I B I T I O N BY

VANADATE O F E l e c t r o p h o r u s N a , K - A T P A S E

Vanadate w i l l r e a c t w i t h Na,K-ATPase i n t h e presencc of Mg2+ and K+ t o form a slowly r e v e r s i b l e i n h i b i t i o n complex ( C a n t l e y e t a l . , 1 9 7 8 ) . Our e a r l i e r s t u d i e s (Hobbs e t a l . , 1980b) showed t h a t vanadate a t v e r y low c o n c e n t r a t i o n s i n h i b i t s a l l b u t a s m a l l r e s i d u a l phosphoenzyme which i s o n l y blocked a t h i g h e r c o n c e n t r a t i o n s F i g u r e 6 shows t h e e f f e c t of v a n a d a t e c o n c e n t r a t i o n on ATPase a c t i v i t y measured i n t h e p r e s e n c e of 75 mM NaC1, 25 mM K C 1 , 3 mM MgC12, 6 0 mM T r i s - H C 1 (pH 7.51, and 500 LIM ATP. Because t h e enzyme h a s a v e r y h i g h a f f i n i t y f o r v a n a d a t e , it i s n e c e s s a r y t o work a t a low s i t e concent r a t i o n ( a b o u t 2 nM) t o a v o i d having s i m i l a r i n h i b i t o r and s i t e c o n c e n t r a t i o n s . The d a t a ( c l o s e d c i r c l e s ) were f i t t o a two-component H i l l e q u a t i o n (open c i r c l e s ) , a l lowing a l l of t h e p a r a m e t e r s t o v a r y w i t h o u t c o n s t r a i n t . From t h e H i l l c o e f f i c i e n t s , t h e i n t e r a c t i o n of t h e i n h i b i t o r w i t h Na,K-ATPase a t low (nanomolar) c o n c e n t r a t i o n s a p p e a r t o be c o o p e r a t i v e ( n = 1 . 6 9 ) whereas i n h i b i t i o n a t

PARALLEL PATHWAYS OF PHOSPHOENZYME FORMATION

100'

i1z

5

1

2 5 z -

a?

I

I

A

Q

80 A

8

529

I

70.1 % (Na,KJATPase KO,= .0134 n = 1.69

-

60-

9)

29.9%

0

40-

'

0

KO5= .284 n = 1.14

0 g a

201

0

-

-

l U

.01 [VANADATE] micromolar F i g . 6 . I n h i b i t i o n of Na,K-ATPase b y v a n a d a t e . E l e c t r o p h o r u s Na,K-ATPase (0.005 m g / m l ) was s u s p e n d e d i n a medium c o n t a i n i n g 75 mM NaCl , 2 5 mM K C l , 3 mM MgC12, 60 mM T r i s - H C l (pH 7 . 5 1 , and ammonium v a n a d a t e a t the c o n c e n t r a t i o n s i n d i c a t e d i n the f i g u r e . A f t e r a 15-min i n c u b a t i o n , 500 pM A T P , 2 . 5 mM PEP, NADH, and 5 pg e a c h o f l a c t i c a c i d d e h y d r o g e n a s e and p y r u v a t e k i n a s e were a d d e d t o the r e a c t i o n m i x t u r e and ADP f o r m a t i o n was m o n i t o r e d b y measu r i n g the conversion o f NADH t o NAD a t 340 m. A b l a n k ( m i n u s Na+) was s u b t r a c t e d f r o m the v e l o c i t y determined i n the p r e s e n c e o f Na+ and K+ a t e a c h v a n a d a t e c o n c e n t r a t i o n . C l o s e d circles r e p r e s e n t the a c t u a l d a t a p o i n t s and o p e n circles r e p r e s e n t the best c o m p u t e r f i t t o a two-component H i l l e q u a t i o n :

%

ATPase ( v a n a d a t e )

= (100%

-

A) KY

+

+ (A/ (I)n

w h e r e ( 1 0 0 - A ) and ( A ) r e p r e s e n t the f r a c t i o n a l amount o f ATPase a c t i v i t y a t e a c h v a n a d a t e b i n d i n g s i t e , ( I ) is the inhibitor conc e n t r a t i o n , K1,2 i s the v a n a d a t e c o n c e n t r a t i o n a t h a l f - m a x i m a l i n h i b i t i o n , and n and m a r e the H i l l c o e f f i c i e n t s . The o p e n t r i a n g l e s r e f e r t o a c t u a l d a t a points corrected f o r the maximum amount o f vana d a t e t h a t c a n b i n d t o the e n z y m e a s s u m i n g a n e n z y m e s i t e d e n s i t y of 1 n m l e / m g .

530

JEFFERY P. FROEHLICHeta/.

h i g h e r c o n c e n t r a t i o n s may i n v o l v e o n l y a s i n g l e s i t e (n = 1 . 1 4 ) . Care must be t a k e n i n a n a l y z i n g t h e s e d a t a because a t v a n a d a t e c o n c e n t r a t i o n s c l o s e t o t h e e s t i mated s i t e c o n c e n t r a t i o n t h e amount o f f r e e i n h i b i t o r may b e s i g n i f i c a n t l y less t h a n t h e added amount which is plotted i n the figure. I f a c o r r e c t i o n i s made f o r t h e maximum amount of v a n a d a t e t h a t c o u l d be bound (assumed e q u a l t o t h e e s t i m a t e d s i t e c o n c e n t r a t i o n ) , t h e n t h e c u r v e a t low c o n c e n t r a t i o n s i s s h i f t e d t o t h e l e f t by a n amount i n d i c a t e d by t h e open t r i a n g l e s . The H i l l c o e f f i c i e n t f o r t h e c o r r e c t e d c u r v e , 1.25, may b e an underestimate of t h e t r u e value. The low c o n c e n t r a t i o n o f enzyme r e q u i r e d i n t h e s e s t u d i e s p r e c l u d e d examining t h e e f f e c t o f v a n a d a t e on E-P f o r m a t i o n a t low i n h i b i t o r c o n c e n t r a t i o n s . However, a t 20 P M v a n a d a t e o n l y a b o u t 8 0 % of the phosphoenzyme formation w a s i n h i b i t e d , i n d i c a t i n g t h e presence of a l o w - a f f i n i t y vanadate binding site.

IV.

DISCUSSION

I n t h e s e e x p e r i m e n t s P i release o c c u r r i n g d u r i n g dep h o s p h o r y l a t i o n of e l e c t r o p h o r u s Na,K-ATPase was measured i n c o n j u n c t i o n w i t h E-P decay a l l o w i n g a d i r e c t q u a n t i t a t i v e comparison o f t h e two r e a c t i o n s . The r e s u l t s show t h a t P i release, l i k e E-P decay, i s b i p h a s i c and t h a t t h e rates o f t h e c o r r e s p o n d i n g p h a s e s are s i m i l a r . Our p r e v i o u s a t t e m p t s (Hobbs e t al., 1980a) t o s i m u l a t e Pi p r o d u c t i o n d u r i n g t h e b u r s t phase o f ATP h y d r o l y s i s i n d i c a t e d t h a t d e p h o s p h o r y l a t i o n o f E2-P w a s t a k i n g p l a c e more r a p i d l y t h a n t h e a c t u a l r a t e measured u n d e r condit i o n s where r e p h o s p h o r y l a t i o n w a s p r e v e n t e d by c h e l a t i o n of f r e e Mg2+ o r by i s o t o p e d i l u t i o n . W e s u g g e s t e d t h a t t h i s d i s c r e p a n c y might be due t o t h e c o n t i n u e d f o r m a t i o n of E l - P from E l A T P , o c c u r r i n g a f t e r t h e a d d i t i o n o f EDTA o r u n l a b e l e d ATP. T h i s p r e d i c t i o n was confirmed i n t h e p r e s e n t s t u d y by showing t h a t P i p r o d u c t i o n l i n k e d t o the r a p i d p h a s e of d e p h o s p h o r y l a t i o n 1 s g r e a t e r t h a n t h e amount of phosphoenzyme p r e s e n t i n t h a t p h a s e . In order f o r t h e P i o r i g i n a t i n g from bound ATP t o b e r e l e a s e d d u r i n g d e p h o s p h o r y l a t i o n , t h e r a t e c o n s t a n t f o r t h e rate AT1 d i s s o c i a t i o n must b e small compared t o t h a t of t h e compef i n g r e a c t i o n , i . e . , p h o s p h o r y l a t i o n . When w e s i m u l a t e d P i release d u r i n g d e p h o s p h o r y l a t i o n u s i n g a n ATP d i s s o c i a t i o n rate c o n s t a n t s i m i l a r t o t h e observed rate ( 4 2 sec'l), w e o b t a i n e d a t o t a l p r e d i c t e d amount of P i release t h a t a g r e e d w i t h t h e measured v a l u e ( T a b l e I V ) .

PARALLEL PATHWAYS OF PHOSPHOENZYME FORMATION

531

W e t h e r e f o r e c o n c l u d e t h a t t h e measured r a t e i s s l o w enough t o p r e v e n t r a p i d d e p l e t i o n o f t h e E l A T P complex

a n d it i s n o t n e c e s s a r y t o p o s t u l a t e t h e e x i s t e n c e of an a d d i t i o n a l t i g h t l y bound complex t o a c c o u n t f o r t h e greater-than-stoichiometric P i p r o d u c t i o n o b s e r v e d d u r i n g dephosphorylation. W e o b t a i n e d r e s u l t s w i t h ADP i n t h e d e p h o s p h o r y l a t i o n e x p e r i m e n t s which were d i f f i c u l t t o e x p l a i n u s i n g t h e c o n v e n t i o n a l Na,K-ATPase mechanism. Following t h e d i s a p p e a r a n c e o f t h e r a p i d component (presumably r e f l e c t i n g r e v e r s a l of p h o s p h o r y l a t i o n ) a s l o w component w a s obs e r v e d which a c c o u n t e d f o r 3 0 - 5 0 % of t h e t o t a l E-P depending on t h e l e v e l of Mg2+ ( F i g . 2 ) . S i n c e t h i s ADPi n s e n s i t i v e component t u r n s o v e r v e r y s l o w l y (%18 sec’l) i n t h e p r e s e n c e o f K + , i t i s p r o b a b l y n o t i n t h e main h y d r o l y t i c pathway b u t i n a s e p a r a t e pathway t h a t hydrol y z e s ATP a t a much s l o w e r r a t e . The s i m i l a r i t y i n t h e r a t e s of t h e slow components o b t a i n e d w i t h EDTA and ADP s u g g e s t s t h a t t h e two phosphoenzymes a r e i n t h e same pathway. The s l i g h t l y f a s t e r r a t e o f d i s a p p e a r a n c e o f t h e s l o w component f o l l o w i n g t h e a d d i t i o n o f ADP can be e x p l a i n e d by assuming t h a t ADP, i n a d d i t i o n t o r e v e r s i n g p h o s p h o r y l a t i o n , a l s o a c t i v a t e s t h e r e v e r s a l of El-P + E2-P by combining w i t h E l - P , t h u s a l l o w i n g E2-P t o dec a y i n b o t h t h e f o r w a r d and r e v e r s e d i r e c t i o n s . The f a c t t h a t d e p h o s p h o r y l a t i o n by ADP d o e s n o t p r o d u c e a compon e n t w i t h a r a t e o f 100-150 sec-1 f o l l o w i n g t h e r a p i d i n i t i a l p h a s e o f d e c o m p o s i t i o n s u g g e s t s t h a t most o f t h e phosphoenzyme i n t h e main c a t a l y t i c pathway i s ADPsensitive (El-PI. If this interpretation is correct, t h e n m o s t o f what remains a f t e r t h e d i s a p p e a r a n c e o f t h e ADP-sensitive phosphoenzyme i s t h e A D P - i n s e n s i t i v e phosphoenzyme i n t h e s e c o n d pathway. Because t h i s phosphoenzyme t u r n s o v e r v e r y s l o w l y , ATP h y d r o l y s i s c a l c u l a t e d from i t s c o n c e n t r a t i o n and t u r n o v e r r a t e w i l l be less t h a n t h e measured o v e r a l l r a t e o f ATP h y d r o l y s i s . This c o u l d a c c o u n t f o r t h e d i s c r e p a n c y between t h e measured and c a l c u l a t e d r a t e s o f ATP h y d r o l y s i s r e p o r t e d by Klodos and Nbrby ( 1 9 7 9 ) . Although t h e c o n t i n u e d f o r m a t i o n o f phosphoenzyme by bound n u c l e o t i d e c a n e x p l a i n t h e r e s u l t s w i t h EDTA, i t i s u n c l e a r how t h i s would a c c o u n t f o r t h e g r e a t e r t h a n - s t o i c h i o m e t r i c P i release o b s e r v e d a f t e r t h e a d d i t i o n o f ADP. According t o t h e c l a s s i c a l scheme, t h e f o r m a t i o n o f E2-P from E l - P s h o u l d b e r e d u c e d i n t h e p r e s e n c e of ADP due t o t h e i n c r e a s e i n t h e c o n v e r s i o n of E l - P I n t h e e x p e r i m e n t shown i n F i g . 2 , Mg2+ w a s t o EIATP. present during dephosphorylation s o t h a t a s i g n i f i c a n t f r a c t i o n of t h e n u c l e o t i d e - d i p h o s p h a t e w a s p r e s e n t a s MgADP. When EDTA w a s added t o g e t h e r w i t h ADP s o t h a t

532

JEFFERY P. FROEHLICH eta/.

t h e amount of Mg2+ a v a i l a b l e t o complex w i t h ADP was reduced, t h e e x t e n t o f t h e i n i t i a l r e a c t i o n w i t h t h e phosphoenzyme was i n c r e a s e d and P i p r o d u c t i o n w a s s t o i c h i o metric w i t h t h e slow p h a s e of E-P d e c a y . These r e s u l t s s u g g e s t t h a t ADP b i n d s more t i g h t l y t o El-P and i s t h e r e f o r e a b e t t e r s u b s t r a t e f o r t h e r e v e r s a l of p h o s p h o r y l a t i o n t h a n MgADP. Beauge and Glynn ( 1 9 7 9 ) a r r i v e d a t a s i m i l a r c o n c l u s i o n b a s e d on t h e r e s u l t s of s t u d i e s of t h e e f f e c t s of Mg2+ on ATP-ADP exchange. The f i n d i n g t h a t EDTA and ATP t o g e t h e r a r e more e f f e c t i v e i n p r e v e n t i n g r e p h o s p h o r y l a t i o n t h a n EDTA a l o n e s u g g e s t s t h a t eel microsomal Na,K-ATPase c o n t a i n s v e r y t i g h t l y bound o r o c c l u d e d Mg2+. C o n s i s t e n t w i t h t h i s p o s s i b i l i t y , w e o b s e r v e d t h a t when Na,K-ATPase w a s i n c u n t h e p r e s e n c e of N a + and 2 mM b a t e d w i t h [ Y - ~ ~ P I A Ti P EDTA, [32P]P l a b e l i n g o f t h e enzyme c o u l d s t i l l b e d e t e c t e d ( F i g . 5 ) . W e have a l s o r e c e n t l y shown t h a t i n h i b i t i o n o f t h e s l o w l y d e c a y i n g hosphoenzyme by vanad a t e o c c u r s i n t h e a b s e n c e of Mgs+, s u g g e s t i n g t h a t t h e Mg2+ which i s r e q u i r e d f o r i n h i b i t i o n i s t i g h t l y bound t o t h e enzyme ( u n p u b l i s h e d o b s e r v a t i o n ) . A f r e q u e n t c r i t i c i s m of t r a n s p o r t models d e r i v e d from quenched f l o w d a t a i s t h a t t h e ATP c o n c e n t r a t i o n s u s e d e x p e r i m e n t a l l y a r e w e l l below t h e p h y s i o l o g i c r a n g e , r e q u i r i n g e x t r a p o l a t i o n of t h e r e s u l t s t o much higher s u b s t r a t e l e v e l s . I n the present study, the bip h a s i c i t y found i n t h e EDTA d e p h o s p h o r y l a t i o n e x p e r i m e n t a t 1 L I M ATP w a s a l s o p r e s e n t a t s u b s t r a t e c o n c e n t r a t i o n s 5 0 and 2 0 0 times h i g h e r . Although h i g h e r s u b s t r a t e conc e n t r a t i o n s were n o t t e s t e d , t h e t r e n d o f t h e d a t a i n Table I c l e a r l y i n d i c a t e s t h a t i n t h e m i l l i m o l a r range, a l a r g e p r o p o r t i o n of t h e enzyme w i l l be p h o s p h o r y l a t e d and t u r n i n g o v e r v e r y r a p i d l y . T h i s i s i n c o n t r a s t t o t h e view h e l d by P l e s n e r e t al. (1981) which assumes t h a t ATP h y d r o l y s i s a t p h y s i o l o g i c ATP c o n c e n t r a t i o n s p r o c e e d s w i t h o u t t h e f o r m a t i o n of a n a c i d - s t a b l e phosphorylated intermediate. An i m p o r t a n t q u e s t i o n r a i s e d by t h e p r e s e n c e o f r a p i d l y and s l o w l y d e c a y i n g phosphoenzymes i n t h e dephosp h o r y l a t i o n r e a c t i o n produced by t h e a d d i t i o n of EDTA o r u n l a b e l e d ATP c o n c e r n s t h e f u n c t i o n a l r e l a t i o n s h i p of t h e s e components. I n an e a r l i e r communication (Hobbs e t a l . , 1 9 8 0 a ) we e l i m i n a t e d enzyme c o n t a m i n a t i o n and s e q u e s t r a t i o n o f I@-activated dephosphorylation sites a s p o s s i b l e e x p l a n a t i o n s f o r t h e b i p h a s i c decay p a t t e r n . A t h i r d p o s s i b i l i t y i s t h a t t h e two components are due t o isozymes i n t h e p r e p a r a t i o n . Isozymes have been found i n h i g h l y p u r i f i e d f r a c t i o n s of N a , K - A T P a s e p r e p a r e d from mammalian b r a i n (Sweadner, 1 9 7 9 ) ; however, t h e r e i s no e v i d e n c e f o r isozymes i n microsomes c o n t a i n i n g e l e c t r i c

533

PARALLEL PATHWAYS OF PHOSPHOENZYME FORMATION

o r g a n Na,K-ATPase. A f o u r t h a l t e r n a t i v e is phosphorylat i o n i n a pathway t h a t r u n s p a r a l l e l t o t h e main c a t a l y t i c pathway. The p r e s e n c e o f a s i d e r e a c t i o n h a s been p r e v i o u s l y proposed by P o s t e t a l . (1975) t o a c c o u n t f o r s i m i l a r b e h a v i o r found i n t h e g u i n e a p i g k i d n e y N a , K ATPase. They showed t h a t by r a i s i n g t h e Mg2+ c o n c e n t r a t i o n d u r i n g p h o s p h o r y l a t i o n t h e amount of s l o w l y d e c a y i n g phosphoenzyme i n c r e a s e d w h i l e r a i s i n g t h e N a + c o n c e n t r a t i o n had t h e o p p o s i t e e f f e c t . We d i d n o t examine t h e N a + - dependence o f t h e s l o w l y d e c a y i n g phosphoenzyme; however, t h e o b s e r v a t i o n t h a t i t i s p r e s e n t i n t h e r e l a t i v e l y s m a l l amounts and t h a t i t s r a t e o f t u r n o v e r i s slow i n t h e p r e s e n c e of ADP o r u n l a b e l e d ATP s u g g e s t s t h a t i t may b e i d e n t i c a l t o t h e K + - i n s e n s i t i v e , ADPi n s e n s i t i v e phosphoenzyme r e p o r t e d by P o s t e t a1 (1975). Although t h e mechanism o f f o r m a t i o n of t h i s s p e c i e s i s u n c l e a r , t h e f a c t t h a t i t decomposes w i t h a r a t e s i m i l a r t o t h a t found i n t h e phosphoenzyme formed i n t h e a b s e n c e of K+ (4-5 sec-1; Hobbs e t a l . , 1980a) s u g g e s t s t h a t i t s d e p h o s p h o r y l a t i o n s i t e s a r e u n a v a i l a b l e t o b i n d K+, poss i b l y b e c a u s e of c o m p e t i t i o n w i t h a n o t h e r i o n . P a r a l l e l p h o s p h o r y l a t i o n pathways may a l s o o c c u r i n a dimer mechanism i n which a d j a c e n t s u b u n i t s become s i m u l t a n e o u s l y p h o s p h o r y l a t e d . I n t h i s t y p e of mechanism, c a t a l y t i c c o u p l i n g between t h e s u b u n i t s w i l l p r e v e n t them from c o - e x i s t i n g i n i d e n t i c a l c h e m i c a l o r c o n f o r m a t i o n a l s t a t e s so t h a t o n e s u b u n i t c o m p l e t e s t h e r e a c t i o n c y c l e ahead of t h e o t h e r . Because e a c h s u b u n i t u n d e r g o e s one c y c l e of ATP h y d r o l y s i s f o r each c y c l e o f t h e d i m e r , t h e p r o d u c t f l u x e s i n t h e two s u b u n i t s w i l l be e q u a l . F o r each s t e p A - B i n t h e c a t a l y t i c cycle, t h e product f l u x i s d e f i n e d a s ki[A] - k,i[B] where k i and k - i are t h e r a t e c o n s t a n t s f o r t h e f o r w a r d and r e v e r s e r e a c t i o n s , res p e c t i v e l y . Assuming t h a t d e p h o s p h o r y l a t i o n i s e s s e n t i a l l y i r r e v e r s i b l e , t h e n t h e p r o d u c t f l u x f o r t h e react i o n pathway l e a d i n g t o t h e r a p i d l y d e c a y i n g component i s kf[E-Pf] = (162 s-1) (0.38 nmole/mg) o r 5.83 nmole/mg/sec (Table I ) w h i l e t h e corresponding product f l u x f o r t h e s l o w l y d e c a y i n g component i s ( 7 . 9 s-1 x .032 nmole/mg/sec) The f a c t t h a t t h e s e p r o d u c t f l u x e s o r . 2 4 nmole/mg/sec. a r e unequal s t r o n g l y s u g g e s t s t h a t t h e o b s e r v e d b i p h a s i c i t y of d e p h o s p h o r y l a t i o n i s n o t due t o a dimer. Although it a p p e a r s u n l i k e l y t h a t t h e r a p i d and slow components o f d e p h o s p h o r y l a t i o n measured i n t h e p r e s e n c e o f EDTA a r e due t o c o u p l e d i n t e r a c t i o n s i n a dimeric enzyme, t h e f a c t t h a t v a n a d a t e i n h i b i t i o n a p p e a r s t o be weakly c o o p e r a t i v e a t low c o n c e n t r a t i o n s s u g g e s t s t h a t t h e r e may b e some t y p e of s u b u n i t - s u b u n i t i n t e r a c t i o n i n v o l v e d i n t h e r a p i d l y t u r n o v e r o v e r f r a c t i o n . T h i s app a r e n t c o o p e r a t i v i t y c a n n o t be t a k e n a s p r i m a f a c i e e v i dence f o r t h e involvement of two s u b u n i t s , b u t i t i s

.

JEFFERY P.FROEHLICH eta/.

534

s t r o n g l y s u g g e s t i v e s i n c e v a n a d a t e a p p e a r s t o a c t by b i n d i n g t o t h e p r o d u c t ( P i ) s i t e . Although C a n t l e y e t a l . (1978) o b t a i n e d no e v i d e n c e f o r t h e involvement o f more t h a n o n e h i g h - a f f i n i t y s i t e i n t h e SDS-purified kidney Na,K-ATPase, t h e s o l u b i l i z a t i o n p r o c e d u r e may have a b o l i s h e d t h e s u b u n i t i n t e r a c t i o n . Examination o f t h e v a n a d a t e b i n d i n g c h a r a c t e r i s t i c o f n a t i v e microsomes may be h e l p f u l i n d e c i d i n g whether t h e mechanism of i n h i b i t i o n involves a s i n g l e vanadate binding s i t e o r m u l t i p l e i n t e r a c t i n g sites. RGFERENCES Albers, R. W . , Koval, G. J., and S i e g e l , G. J. (1968). S t u d i e s on t h e i n t e r a c t i o n of ouabain and o t h e r c a r d i o a c t i v e s t e r o i d s with sodium-potassium-activated adenosine t r i p h o s p h a t e Mol. Pharmacol. 4 , 324-336. Beaug6, L. A. , and Glynn, I. M. (1979). Sodium i o n s , a c t i n g a t high a f f i n i t y e x t r a c e l l u l a r s i t e s , i n h i b i t sodium ATPase act i v i t y o f t h e sodium pump by slowing dephosphorylation. J . Physiol. (London) 289, 17-31. Cantley, L. C . , J r . , Cantley, L. G., and Josephson, L . (1978). A c h a r a c t e r i z a t i o n of vanadate i n t e r a c t i o n s with t h e (Na,K) ATPase. J. B i o l . Chem. 253, 7361-7368. Froehlich, J. P . , Albers, R. W . , Koval, G . J . , Goebel, R . , and Bennan, M. (1976a). Evidence f o r a new i n t e r m e d i a t e s t a t e i n t h e mechanism of t h e (Na++K+)-adenosine t r i p h o s p h a t a s e . J . Biol. Chem. 251, 2186-2188. F r o e h l i c h , J. P . , S u l l i v a n , J. V., and Berger, R. L. (197613). A chemical quenching apparatus f o r studying r a p i d r e a c t i o n s . A n a l . Biochem. 73, 331-341. Froehlich, J. P . , Albers, R. W . , and Hobbs, A S. (1979). K+induced t r a n s i t i o n between t h e r a p i d l y and slowly r e a c t i n g In "Na,K-ATPase: Strucconformations of t h e Na,K-ATPase. t u r e and K i n e t i c s " (J. C . Skou and J. G. Nbrby, e d s . ) , pp. 129-142. Academic Press, N e w York. Goldman, S. S. , and Albers, R. W. (1973). Sodium-potassium a c t i vated adenosine t r i p h o s p h a t a s e . I X . The r o l e o f phospholipids. J. B i o l . Chem. 2 4 8 , 867-874. Hobbs, A. S . , Albers, R. W . , and Froehlich, J. P. (1980a). Potassium-induced changes i n phosphorylated and dephosphorylat e d of (Na,K)-ATPase observed i n t h e t r a n s i e n t s t a t e . J . Biol. Chem. 255, 3395-3402. Hobbs, A. S., F r o e h l i c h , J. P., and Albers, R. W. (1980b). I n h i b i t i o n by vanadate of t h e r e a c t i o n s c a t a l y z e d by t h e (Na+ + K+)stimulated ATPase. J . Biol. Chem. 255, 3724-3727. Hobbs, A. S. , A l b e r s , R. W. , and F r o e h l i c h , J. P. (1983). J. Biol Chem., i n p r e s s .

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PARALLEL PATHWAYS OF PHOSPHOENZYME FORMATION

535

K a r l i s h , S. J. D . , Yates, D. W. , and Glynn, I. M. ( 1 9 7 8 ) . Element a r y s t e p s o f the ( N a + + K+)-ATPase m e c h a n i s m s t u d i e d w i t h f o n y c i n nucleotides. B i o c h i m . B i o p h y s . A c t a 5 2 5 , 230-251. Klodos, I . , and Ngirby, J. G. ( 1 9 7 9 ) . E f e c t of L i + and K+ on t h e I n "Na ,Ki n t e r m e d i a r y steps of t h e N a ,K-ATPase r e a c t i o n . ATPase: S t r u c t u r e and K i n e t i c s " (J. C. Skou and J. G. Ngirby, e d s . ) , pp. 331-342. Academic P r e s s , N e w York. K n o t t , G . D. ( 1 9 7 9 ) . ---a mathematical modelling t o o l . C o m p u t . Programs B i o m e d . 1 0 , 271-280. P l e s n e r , I . W . , P l e s n e r , L., Nbrby, J. G . , and Klodos, I. ( 1 9 8 1 ) . The s t e a d y - s t a t e k i n e t i c mechanism o f ATP h y d r o l y s i s catal y z e d by membrane-bound ( N a + + K+)-ATPase from ox b r a i n . 111. Minimal model. B i o c h i r n . B i o p h y s . A c t a 6 4 3 , 483-494. P o s t , R. L., Hegevary, C. , and K u m e , S. ( 1 9 7 2 ) . A c t i v a t i o n by a d e n o s i n e t r i p h o s p h a t e i n t h e p h o s p h o r y l a t i o n k i n e t i c s of sodium and potassium i o n t r a n s p o r t a d e n o s i n e t r i p h o s p h a t a s e J. B i o l . Chem. 2 4 7 , 6530-6540. P o s t , R. L . , Toda, G . , and Rogers, F. N . ( 1 9 7 5 ) . P h o s p h o r y l a t i o n by i n o r g a n i c p h o s p h a t e o f ( N a + + K+) - a d e n o s i n e t r i p h o s p h a tase. Four r e a c t i v e s t a t e s . J . Biol. Chem. 2 5 0 , 691-701. Shamoo, A. E . , and Brodsky, W. A. ( 1 9 7 1 ) . I d e n t i f i c a t i o n o f i n t a c t ATP bound t o ( N a + + K+)ATPase. B i o c h i m . B i o p h y s . A c t a 2 4 1 , 846-856. Sweadner, K. J. ( 1 9 7 9 ) . Two m o l e c u l a r forms of ( N a + + K + ) s t i m u l a t e d ATPase i n b r a i n . S e p a r a t i o n and d i f f e r e n c e i n J. B i o l . Chem. 2 5 4 , 6060a f f i n i t y f o r strophanthidin. 6067. Tonomura, Y., and Fukushima, Y. ( 1 9 7 4 ) . K i n e t i c p r o p e r t i e s of p h o s p h o r y l a t e d i n t e r m e d i a t e s i n t h e r e a c t i o n o f Na+,K+ATPase. Ann. N . Y . A c a d . S c i . 2 4 2 , 92-105.

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CURRENT TOPICS IN MEMBRANES AND TRANSPORT, VOLUME 19

Evaluation of the Reaction Mechanism of the Sodium Pump by Steady-State Kinetics JOHN R. SACHS Department of Medicine State University of New Yorkat Stony Brook Stony Brook, New York

I.

INTRODUCTION

Extensive investigation of the partial biochemical reactions and of the ion exchanges carried out by the Na pump has supported the reaction mechanism for the overall transport cycle originally proposed by Post and Albers (Robinson and Flashner, 1979). Although the partial reactions support the model, it is still possible that they are not, in fact, part of the main reaction pathway but are side reactions. For a partial reaction to be considered part of the overall reaction mechanism it must in the first place be shown to be kinetically competent; discussion of this point occurs elsewhere in this volume. In addition it must be shown that the partial reaction occurs while the overall reaction is proceeding. The utility of the steadystate kinetic approach to the evaluation of a reaction mechanism lies in its ability to- distinguish between plausible mechanisms which are consistent with the known 537

Copyright 0 1983 by Academic Press. Inc.

AU rightsof reproduction in any form nsnvd. ISBN 0-12-153319-0

JOHN R. SACHS

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partial reactions. In particular, by a steady-state approach it is frequently possible to determine the order in which substrates add and products are released while the overall reaction is proceeding. In the Post-Albers mechanism, the order of addition and release of Na and K during the transport cycle is NaC

NaO

KO

KC

in which Na first adds to the enzyme at the cytoplasmic surface (Na,) and is then released at the extracellular surface (Nag). This is followed by the addition of K at the outside surface (KO) and its subsequent release intracellularly (Kc). The sequence is then repeated. This mechanism is called ping-pong by Cleland (1963). It turned out, however, that the kinetic behavior which resulted when the concentration of intracellular Na and extracellular K were varied independently was not that predicted from the ping-pong model (Hoffman and Tosteson, 1971; Garay and Garrahan, 1973; Chipperfield and Whittam, 1976). When the velocity of the Na-K exchange was measured as a function of external K at a number of fixed concentrations of intracellular Na in the absence of internal K and external Na the variation in the kinetic parameters was consistent with a sequential mechanism (Sachs, 1977) :

KO

NaC

KC

NaO

Internal Na and external K must bind simul aneously before either is transported. This sequential mechanism is difficult to reconcile with the post-Albers scheme. However, the existence of an uncoupled Na outflux in red cells when external Na and K are both absent makes the result ambiguous. The kinetic data are equally well fit by a modified ping-pong mechanism:

REACTION OF Na PUMP BY STEADY STATE KINETICS

NaC

NaO

KO

539

KC

k

in which the reverse arrow labeled k represents the uncoupled Na outflux (Sachs, 1979). This model is consistent with the Post-Albers scheme. The fit is equally good for either model and therefore this type of steadystate kinetic experiment cannot distinguish between the ping-pong and the sequential mechanism.

11.

THE Na-Na AND K-K EXCHANGES

Demonstration of a Na-Na exchange in the absence of external K and of a K-K exchange in the absence of internal Na offers strong support for the ping-pong mechanism provided that the exchanges are part of the overall reaction pathway and provided that they are true exchanges--i.e., that they occur by the mechanism Ei \

- = '

EiS -EoS

7Eo

in which Ei and Si are enzyme and substrate at the inner cell surface and Eo and So are enzyme and substrate at the outer cell surface. Garrahan and Garay (1976) have suggested that the exchanges are not, in fact, true exchanges but rather occur by a mechanism in which internal and external sites must be filled simultaneously before transport in either direction occurs:

JOHN R. SACHS

540

E

K2

+

K1

Si +SE

+

+

1I.

1

ES

+

I.

So+SES+

aK2

E

+

SOut 1

+

Skn

in which E represents the pump, SyUt is originally intracellular Na or K which moves to the outside, S&n is originally extracellular Na or K which moves into the cell, K1 and K 2 are dissociation constants for the combination of inside and outside ions with the pump, and a is a factor by which combination of the pump with one ion (Na or K) changes the dissociation constant for the second. This mechanism will reproduce the major characteristics of the Na-Na and K-K exchanges: movement of the ion in one direction will depend on the presence of the same ion on the opposite side of the membrane, the exchange rate will be a saturable function of the inside and outside ion concentration, and the magnitude of the ion movement in one direction will equal the magnitude of its movement in the opposite direction. Clearly, the second mechanism does not support the ping-pong mechanism for the overall transport reaction. It is possible to devise a kinetic test to distinguish between the two mechanisms. The test involves measurement of the exchange at varying concentrations of the ion at one side of the membrane and several fixed concentrations at the other side. The kinetic parameters apparent Vm and apparent K+ for the ion at the side at which its concentration varies are then estimated. The first mechanism predicts that both apparent vm and apparent K+ increase as the fixed concentration of the ion increases, but that the ratio apparent vm/apparent K+ remains constant. For the second mechanism apparent Vm and apparent K+ also increase, but the ratio will not, in general, remain constant. In the special case in which c1 = 1 (i.e., combination of the pump with an ion at one side does not alter the affinity of the site at the other side), the apparent K+ will not

REACTIONOF Na PUMP BY STEADY STATE KINETICS

541

TABLE I. Kinetic Parameters for K-K Exchange at Two Constant Intracellular K Concentrationsa m' (mole/literRBC) (mole/literRBC, hr) KC

1.4 27.2

0.058 0.291

K

4

for outside K

R

(mM)

( Vm/K+ 1

0.0196 0.0887

3.3

3.0

a

Ouabain-sensitive K influx was measured in cells free of Na and with the indicated intracellular concentrations of Na at varying external K concentrations. The parameters were estimated by a least squares fit of a Michaelis-Menten equation to the data.

vary with the fixed concentration at the other side. When the experiment was performed (Sachs, 1981) the results in Table I were obtained. Variation of intracellular K changed both apparent Vm and apparent K+ for outside K in the same proportion; the result is consistent with a true exchange mechanism. For technical reasons it is not possible to perform the same experiment with the Na-Na exchange. Although it is clear from these results that the K-K exchange has the kinetic characteristics of a true exchange, it is not so clear that the exchange is in the overall reaction pathway. Stein (1979) pointed out that, since the K-K exchange is dependent on both intracellular phosphate and ATP, failure of either of these substrates to inhibit the exchange as its concentration is increased at a fixed high concentration of the other must mean that there is an intermediate in the exchange which binds ATP and phosphate at the same time. The observation that the substrates do not inhibit when both are present at high concentration now seems secure (Simons, 1974; Sachs, 1981; Kharlish and Stein, this volume). Circumstances which result in stimulation of the K-K exchange by inorganic phosphate have been shown to result in phosphorylation of the active site (Post et a l . , 1975), and the site at which ATP stimulates the K-K exchange has many of the characteristics of the lowaffinity site which appears during hydrolysis of ATP in the presence of Na and K. Since there is evidence that, during a transport cycle, phosphate is released before ) , it is probable ATP adds (Eisner and Richards, that the form of the p m p which is an intermediate in the K-K exchange and which simultaneously binds phosphate and ATP may not appear during the overall reaction

JOHN R. SACHS

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cycle and that the K-K exchange may, therefore, represent a side reaction. If this turns out to be so, the presence of the exchange cannot be used as support for the ping-pong reaction mechanism.

111.

ORDER OF ADDITION AND RELEASE OF SUBSTRATES AND PRODUCTS

Some evidence about the order of release of Na and addition of K at the external pump surface can be obtained from a steady-state kinetic study of the characteristics of pump inhibition by oligomycin (Sachs, 1980). The argument depends on whether an inhibitor is a noncompetitive inhibitor or an uncompetitive inhibitor with respect to a particular substrate. If an inhibitor combines equally well with enzyme forms which occur before and after the addition of a particular substrate, inhibition is noncompetitive with respect to that substrate; the apparent vm is reduced by the inhibitor but the apparent K+ for the substrate is not changed. On the other hand, if the inhibitor binds preferentially with enzyme forms which occur in the reaction pathway after the addition of the substrate, then inhibition is uncompetitive; both the apparent Vm and the apparent K+ are reduced and, if inhibition is completely uncompetitive, they are reduced by the same proportion. When the effect of oligomycin on the Na,K-ATPase activity of broken membrane preparations was investigated, it was found that inhibition is uncompetitive with ATP. Similarly, oligomycin inhibition of the Na-K exchange in intact cells is uncompetitive with internal Na. Inhibition by oligomycin of the Na-Na exchange carried out by intact cells in K-free solutions is uncompetitive with external Na. These results can be interpreted with the help of the following diagram: ATP

ADP

Since inhibition is uncompetitive with both intracellular Na and ATP, oligomycin must combine preferentially with enzyme forms which occur after the addition of these substrates. Similarly, since inhibition is uncompetitive with respect to external Na, the inhibitor

REACTION OF Na PUMP BY STEADY STATE KINETICS

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must combine w i t h enzyme forms which o c c u r b e f o r e t h e release o f N a t o t h e o u t s i d e . S i n c e o l i g o m y c i n does n o t i n h i b i t t h e ADP-ATP exchange, it must combine e q u a l l y w e l l w i t h t h e enzyme forms E - A T P - N a , E - P - N a , and any forms which o c c u r between. The o b s e r v a t i o n t h a t o l i g o m y c i n d o e s n o t i n h i b i t t h e K-K exchange when t h e measurement i s made i n Na-free c e l l s , b u t does i n h i b i t i f t h e measurement i s made i n c e l l s w i t h low conc e n t r a t i o n s of N a means t h a t t h e i n h i b i t o r d o e s n o t combine w i t h any E2 form of t h e pump. Oligomycin i n h i b i t i o n o f t h e N a - K exchange i s unc o m p e t i t i v e w i t h r e s p e c t t o e x t e r n a l K i f t h e measurement i s made i n Na-free s o l u t i o n s b u t u n c o m p e t i t i v e i f t h e measurement i s made i n high-Na s o l u t i o n s . The res u l t s can be i n t e r p r e t e d w i t h t h e h e l p of t h e following diagram:

I n Na-free s o l u t i o n s breakdown of E - P - N a t o E * P i s i r r e v e r s i b l e and E - P - N a ( t h e form which combines w i t h o l i g o m y c i n ) forms o n l y a f t e r a d d i t i o n of e x t e r n a l K and c o m p l e t i o n of a n o v e r a l l t r a n s p o r t c y c l e . Since t h e form which combines w i t h oligomycin o c c u r s i n t h e r e a c t i o n sequence o n l y a f t e r a d d i t i o n o f e x t e r n a l K , i n h i b i t i o n i s uncompetitive with r e s p e c t t o e x t e r n a l K . On t h e o t h e r hand, i f t h e s o l u t i o n c o n t a i n s N a , E - P - N a can be formed e i t h e r by way of t h e complete c y c l e a f t e r a d d i t i o n of K o r by r e v e r s a l o f t h e s t e p a t which N a i s released t o t h e o u t s i d e ; i n h i b i t i o n i s t h e r e f o r e nonc o m p e t i t i v e w i t h r e s p e c t t o e x t e r n a l K . The c o n c l u s i o n t h a t e x t e r n a l K c a n add t o t h e enzyme a f t e r N a i s released f o l l o w s from t h i s . Beauge and DiPolo (19811, u s i n g p e r f u s e d s q u i d axons, and E i s n e r and R i c h a r d s (19811, u s i n g r e s e a l e d r e d c e l l g h o s t s , have shown t h a t a s i n t e r n a l ATP i s i n c r e a s e d , t h e a p p a r e n t K+ f o r e x t e r n a l Rb o r K i n c r e a s e s ; i n red c e l l g h o s t s it was a l s o shown t h a t i n c r e a s i n g These ree x t e r n a l K i n c r e a s e s t h e a p p a r e n t K+ f o r ATP. s u l t s suggest t h a t i n t h e t r a n s p o r t cycle t h e addition of K a t t h e o u t s i d e and t h e a d d i t i o n of ATP a r e s e p a r a t e d by an i r r e v e r s i b l e s t e p . E i s n e r and R i c h a r d s ( 1 found t h a t t h e s e e f f e c t s were s e e n o n l y i n phosphatef r e e g h o s t s ; i f t h e g h o s t s w e r e h i g h i n p h o s p h a t e , there w a s l i t t l e e f f e c t o f i n c r e a s i n g ATP on t h e a p p a r e n t K+ f o r e x t e r n a l K . A d d i t i o n o f p h o s p h a t e , t h e n , makes t h e s t e p between t h e a d d i t i o n of K and t h e a d d i t i o n of ATP

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i r r e v e r s i b l e , which i n d i c a t e s t h a t phosphate i s r e l e a s e d between t h e a d d i t i o n of t h e two s u b s t r a t e s . Taking t h e s e r e s u l t s t o g e t h e r w i t h t h e r e s u l t s from t h e o l i g o mycin i n h i b i t i o n experiments p r o v i d e d i r e c t s t e a d y - s t a t e k i n e t i c e v i d e n c e f o r t h e f o l l o w i n g sequence a s p a r t of the overall transport cycle

The ping-pong mechanism p r e d i c t s t h a t K i s r e l e a s e d a t t h e i n s i d e b e f o r e Na adds. There i s no s t e a d y - s t a t e k i n e t i c e v i d e n c e t h a t t h i s sequence does i n f a c t o c c u r . However, t h e r e are s e v e r a l o b s e r v a t i o n s which would most e a s i l y be e x p l a i n e d i f t h e o r d e r of release of K and a d d i t i o n of Na where t h e r e v e r s e : i . e . , a d d i t i o n of Yamaguchi and Tonomura Na b e f o r e t h e r e l e a s e of K. ( 1 9 8 0 ) found t h a t t h e dephosphorylated enzyme simult a n e o u s l y bound 3 Na and 2 Rb i o n s . I f t h i s form of t h e enzyme i s i n t h e main r e a c t i o n sequence, it most l i k e l y c o r r e s p o n d s t o enzyme w i t h i o n s bound a t t h e i n t e r n a l s u r f a c e . Garrahan e t a l . ( t h i s volume) have p r e s e n t e d s t e a d y - s t a t e k i n e t i c e v a l u a t i o n of t h e N a , K dependent ATPase r e a c t i o n which s u g g e s t s t h a t Na adds b e f o r e K i s r e l e a s e d . I t should be p o i n t e d o u t , howe v e r , t h a t i n t e r n a l K does n o t d i r e c t l y a f f e c t t h e maximal v e l o c i t y of t h e Na-K exchange a t s a t u r a t i n g concent r a t i o n s of i n t e r n a l N a : p r e v i o u s r e p o r t s (Garay and Garrahan, 1975; Sachs e t a l . , 1 9 7 4 ) t h a t i n t e r n a l K i n c r e a s e s t h e Na-K exchange r a t e probably can be e x p l a i n e d by an i n c r e a s e d i n t r a c e l l u l a r ATP c o n c e n t r a t i o n produced by K r e s u l t i n g from i t s s t i m u l a t i o n of t h e a c t i v i t y of g l y c o l y t i c enzymes (Sachs, 1 9 8 2 ) . F u r t h e r i n v e s t i g a t i o n of t h e complicated i n t e r a c t i o n between i n t e r n a l Na, K , and ATP w i l l be n e c e s s a r y b e f o r e a f i r m c o n c l u s i o n a b o u t t h e o r d e r of a d d i t i o n of Na and r e l e a s e of K can be drawn.

ACKNOWLEDGMENT This work w a s supported by a g r a n t AM-19185 from t h e United S t a t e s Public Health Service.

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REFERENCES Beaug6, L . , and DiPolo, R. (1981). The e f f e c t s of ATP on t h e i n t e r a c t i o n s between monovalent c a t i o n s and t h e sodium pump J . P h y s i o l . (London) 314, 457-480. i n d i a l y z e d s q u i d axons. C h i p p e r f i e l d , A. R . , and Whittam, R. (1976). The c o n n e c t i o n between ion-binding s i t e s of t h e sodium pump. J . P h y s i o l . (London) 260 , 371-385. C l e l a n d , W. W. (1963). The k i n e t i c s of enzyme c a t a l y z e d r e a c t i o n s w i t h t w o or more s u b s t r a t e s o r p r o d u c t s . I. Nomenclature and rate e q u a t i o n s . B i o c h i m . B i o p h y s . Acta 67, 104-137. E i s n e r , D. A . , and Richards, D. E. ( 1. K i n e t i c e v i d e n c e i n f a v o r of a c o n s e c u t i v e model of t h e sodium pump. E i s n e r , D. A., and Richards, D. E. (1981). The i n t e r a c t i o n of potassium i o n s and ATP on t h e sodium pump of r e s e a l e d r e d c e l l g h o s t s . J. P h y s i o l . (London) 319, 403-418. Garay, R. P., and Garrahan, P . J . (1973). The i n t e r a c t i o n of sodium and potassium w i t h t h e sodium pump i n r e d c e l l s . J. Physiol (London) 231, 297-325. Garay, R. P . , and Garrahan, P. J. (1975). The i n t e r a c t i o n of a d e n o s i n e t r i p h o s p h a t e and i n o r g a n i c phosphate w i t h t h e sodium pump i n r e d c e l l s . J . P h y s i o l . (London) 249, 51-67. Garrahan, P. J . , and Garay, R . P. (1976). The d i s t i n c t i o n between s e q u e n t i a l and simultaneous models f o r sodium and potassium Curr. T o p . Membr. Transp. 8 , 19-97. transport. Hoffman, P. G . , and Tosteson, D. C. (1971). A c t i v e sodium and potassium t r a n s p o r t i n h i g h potassium and low potassium sheep r e d c e l l s . J . G e n . P h y s i o l . 58, 438-466. ). Conformational K h a r l i s h , S. D., P i c k , U . , and S t e i n , W. D. ( changes and c a t i o n t r a n s p o r t i n p h o s p h o l i p i d v e s i c l e s recons t i t u t e d w i t h (Na,K)ATPase. P o s t , R. L . , Toda, G . , and Rogers, F. N. (1975). P h o s p h o r y l a t i o n by i n o r g a n i c phosphate of sodium p l u s potassium i o n t r a n s p o r t adenosinetriphosphatase. Four r e a c t i v e s t a t e s . J. B i o l . Chem. 250, 691-701. + + Robinson, J. D., and F l a s h n e r , M. S . (1979). The (Na + K 1Enzymatic and t r a n s p o r t p r o p e r t i e s . a c t i v a t e d ATPase. B i o c h i r n . B i o p h y s . Acta 549, 145-176. Sachs, J . R . (1977). K i n e t i c e v a l u a t i o n of t h e Na-K pump r e a c t i o n J . P h y s i o l . (London) 273, 489-514. mechanism. Sachs, J. R. (1979). A modified c o n s e c u t i v e model f o r t h e Na-K In "Na,K-ATPase: S t r u c t u r e and K i n e t i c s " (J. C . pump. Skou and J. G. N#rby, e d s . ) , pp. 463-473. Academic P r e s s , New York. Sachs, J . R. (1980). The order of r e l e a s e of sodium and a d d i t i o n o f potassium i n t h e sodium-potassium pump r e a c t i o n mechanism. J . P h y s i o l . (London) 302, 219-240. Sachs, J. R. (1981). Mechanistic i m p l i c a t i o n s of t h e potassiumpotassium exchange c a r r i e d o u t by t h e sodium-potassium pump. J. P h y s i o l . (London) 316, 263-277.

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Sachs, J . R. ( 1 9 8 2 ) . I n t e r n a l p o t a s s i u m s t i m u l a t e s t h e sodiumpotassium pump by i n c r e a s i n g c e l l ATP c o n c e n t r a t i o n . J . P h y s i o l . (London) 319, 515-528. r e a c t i v i t y and t h e N a , K S t e i n , W. D. (1979). H a l f - o f - t h e - s i t e s ATPase. In Na,K-ATPase: S t r u c t u r e and K i n e t i c s " ( J . C . Skou and J. G. Ngkby, e d s . ) , pp. 475-486. Academic P r e s s , New York. Yamaguchi, M., and Tonomura, Y. ( 1 9 8 0 ) . B i n d i n g o f monovalent c a t i o n s t o N a + , K+-dependent ATPase p u r i f i e d from p o r c i n e kidney. I. Simultaneous b i n d i n g of t h r e e sodium and two potassium o r rubidium i o n s t o t h e enzyme. J. B i o c h e m . ( T o k y o ) 88, 1365-1375.

CURRENTTOPICS IN MEMBRANES AND TRANSPORT. VOLUME 19

Kinetic Evidence in Favor of a Consecutive Model of the Sodium Pump D. A. EISNER AND D. E. RICHARDS Physiological Laboratory University of Cambridge Cambridge. England

I.

INTRODUCTION

C u r r e n t models of t h e sodium pump s u g g e s t t h a t t h e e f f l u x of sodium i o n s from t h e c e l l i s accompanied by t h e f o r m a t i o n of a phosphoenzyme, which i s t h e n hydrol y z e d i n a r e a c t i o n a c c e l e r a t e d by e x t e r n a l p o t a s s i u m ions. The p o t a s s i u m i o n s t h e n become o c c l u d e d w i t h i n t h e enzyme, and t h e i r release a t t h e e x t r a c e l l u l a r s u r f a c e i s a c c e l e r a t e d by ATP a c t i n g a t a l o w - a f f i n i t y s i t e ( P o s t e t a l . , 1 9 7 2 ; K a r l i s h et a l . , 1978; BeaugB and Glynn, 1 9 7 9 ) . I f t h i s scheme i s c o r r e c t , t h e app a r e n t a f f i n i t y of t h e o v e r a l l r e a c t i o n s h o u l d depend on t h e ATP c o n c e n t r a t i o n . E x p e r i m e n t s on n o n s i d e d p r e p a r a t i o n s showed t h e e x p e c t e d r e s u l t (Robinson, 1 9 6 7 ; Skou, 1 9 7 4 1 , a s d i d recent e x p e r i m e n t s i n t h e s q u i d axon (BeaugB and D i P o l o , 1 9 7 9 , 1981) however, work i n r e d c e l l s h a s been n e g a t i v e (Glynn, 1956; Beau# and D e l C a m p i l l o , 1976; Garay and G a r r a h a n , 1 9 7 5 ) . Another p r e d i c t i o n of t h e model i s t h a t phosp h a t e , by a c t i n g a s a p r o d u c t i n h i b i t o r , s h o u l d r e d u c e 547

Copynghr 0 1983 by Academic Press, Inc All nghts of reproduction in any form r e ~ e ~ e d ISBN @12-1533194

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t h e a p p a r e n t a f f i n i t y f o r e x t e r n a l K i o n s . P i I howe v e r , h a s been r e p o r t e d t o have no e f f e c t on t h e app a r e n t a f f i n i t y f o r e x t e r n a l K i o n s (Garay and Garrahan, 1975; Robinson e t al., 1 9 7 8 ) . I n t h e l i g h t of t h i s d i s a g r e e m e n t , t h e e f f e c t s of ATP and P i o n t h e ext e r n a l K a f f i n i t y are worth i n v e s t i g a t i n g . W e decided t o s t u d y t h e e f f e c t s of t h e s e l i g a n d s on t h e i n f l u x of K i n t o r e s e a l e d g h o s t s p r e p a r e d from human red blood c e l l s . A p r e l i m i n a r y a c c o u n t of p a r t of t h e s e e x p e r i ments h a s a l r e a d y been p u b l i s h e d , and a f u l l a c c o u n t w i l l a p p e a r s h o r t l y ( E i s n e r and R i c h a r d s , 1980, 1981, 1982).

11.

METHODS

The p r o c e d u r e t o p r e p a r e r e s e a l e d g h o s t s was a s f o l l o w s : human r e d b l o o d c e l l s were ( a ) d e p l e t e d of ATP by i n c u b a t i o n a t 37OC f o r 24-36 h r i n t h e absence o f n u t r i e n t s ; (b) f u r t h e r d e p l e t e d of ATP by incubat i o n a t 37OC w i t h i n o s i n e iodoacetamide; (c) i n c u b a t e d w i t h n y s t a t i n i n a N a medium, t o r e d u c e t h e K c o n t e n t t o a b o u t 30 p ~ . A f t e r t h i s t h e c e l l s were l y s e d i n a medium c o n t a i n i n g 1 5 mf N a , c h o l i n e , b u f f e r , Mg, ATP, and a n ATP-regenerating system (Glynn and K a r l i s h , 1 9 7 6 ) . The g h o s t s were t h e n r e s e a l e d , washed, and suspended i n i c e - c o l d media c o n t a i n i n g 5 mM N a , c h o l i n e , b u f f e r , Mg, and d i f f e r e n t c o n c e n t r a t i o n s of r a d i o a c t i v e R b , w i t h o r w i t h o u t o u a b a i n . The i n f l u x w a s measured by warming t h e g h o s t s t o 37OC and incubating f o r 1 hr.

111.

RESULTS AND D I S C U S S I O N

I n a f i r s t s e t of e x p e r i m e n t s w e measured Rb i n f l u x i n t o ghosts containing d i f f e r e n t concentrations of ATP, and suspended i n media c o n t a i n i n g d i f f e r e n t c o n c e n t r a t i o n s of Rb. The r e s u l t i n g c u r v e s were s i g moid and w e l l d e s c r i b e d by e u a t i o n s of t h e form When (Rb i n f l u x ) 1 / 2 = [Rb] (Vmax)l Y 2 / (Kap + [Rb] ) analyzed i n a double r e c i p r o c a l p l o f , t h e s t r a i g h t l i n e s o b t a i n e d a t d i f f e r e n t ATP c o n c e n t r a t i o n s were p a r a l l e l . The v a l u e s of vmax and .xapp o b t a i n e d a r e t a b u l a t e d below.

.

CONSECUTIVE MODEL OFTHE SODIUM PUMP

'ma, 1 10 100 3000

549

(mmol/liter/hr) 0.084 0.34 1.53 2.62

14 33 68 96

0.021 0.018 0.018 0.017

The d a t a show t h a t ( i ) t h e i n c r e a s e i n ATP c o n c e n t r a t i o n p r o d u c e s n o t o n l y t h e e x p e c t e d change i n vrnax, b u t a l s o i n c r e a s e s t h e Kapp f o r e x t e r n a l Rb; ( i i ) a l though t h e Vmax changes o v e r a r a n g e of more t h a n ( t h e g r a d i e n t of 3 0 - f o l d , t h e v a l u e s of (Vmax)'/2/xa t h e Lineweaver-Burk p l o t ) remain r e g g r k a b l y c o n s t a n t . A n a l y s i s of t h e o r i g i n a l d a t a shows, f u r t h e r m o r e , t h a t t h e f r a c t i o n a l i n c r e a s e i n R b e f f l u x produced by i n c r e a s i n g ATP depends on t h e Rb c o n c e n t r a t i o n ; as Rb d e c r e a s e s toward z e r o , t h e Rb i n f l u x becomes more and more i n d e p e n d e n t of t h e ATP c o n c e n t r a t i o n . A s i m i l a r e x p e r i m e n t , b u t i n t h e p r e s e n c e of 5 mM P i , showed t h a t ( a ) P i i n h i b i t e d R b i n f l u x ; ( b ) P i i n creased t h e K a f o r R b ; ( c ) t h e p a t t e r n of p a r a l l e l l i n e s i n t h e a%sence of P i was changed t o a n i n t e r s e c t i n g p a t t e r n i n t h e presence of P i ; (d) t h e effects of P i w e r e more o b v i o u s a t t h e lower ATP c o n c e n t r a t i o n s . Taken t o g e t h e r , t h e s e r e s u l t s a r e c o n s i s t e n t w i t h a c o n s e c u t i v e model o f t h e sodium pump, i n which a dep h o s p h o r y l a t i o n r e a c t i o n c a t a l y z e d by e x t e r n a l K (Rb) i s f o l l o w e d by b i n d i n g o f ATP t o a l o w - a f f i n i t y s i t e t o r e l e a s e K (Rb) t o t h e i n t r a c e l l u l a r medium, and where P i i s r e l e a s e d a t a s t e p p r e v i o u s t o t h e b i n d i n g o f ATP a t t h e low-affinity site. The f a i l u r e o f p r e v i o u s s t u d i e s t o o b s e r v e t h e s e e f f e c t s i n i n t a c t c e l l s might be a t t r i b u t e d t o s e v e r a l ( i ) i n s u f f i c i e n t r e d u c t i o n o f ATP; r e a s o n s , namely: ( i i ) a s t h e e f f e c t s o f ATP on t h e a f f i n i t y f o r e x t e r n a l Rb v a r y w i t h t h e s q u a r e r o o t of t h e e f f e c t s o f Vmax, t h e s i z e o f t h e e x p e c t e d change i n K~~~ would be s m a l l f o r a Vmax change of a b o u t 2 - f o l d , a s o b s e r v e d i n t h o s e s t u d i e s ; ( i i i ) t h e u s e of phosphate- and potassiumc o n t a i n i n g c e l l s must have c o m p l i c a t e d p r e v i o u s e x p e r i ments and o b s c u r e d t h e e f f e c t s o f ATP on t h e K a f f i n i t y .

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D. A. EISNERAND D. E. RICHARDS

ReFERENCES

Beaugd, L. A . , and D e l Campillo, E. (1976). The ATP dependence of a ouabain s e n s i t i v e sodium e f f l u x a c t i v a t e d by e x t e r n a l sodium, potassium and l i t h i u m i n human r e d c e l l s . Biochim. Biophys. Acta 433, 547-554. Beaug6, L. A . , and DiPolo, R . ( 1 9 7 9 ) . Sidedness of t h e ATP-Na+-K+ i n t e r a c t i o n s w i t h t h e N a f pump i n s q u i d axons. Biochim. Biophys. Acta 553, 495-500. Beaug6, L. A . , and DiPolo, R. (1981). The e f f e c t s o f ATP on t h e i n t e r a c t i o n s between monovalent c a t i o n s and t h e sodium pump i n d i a l y z e d s q u i d axons. J. P h y s i o l . (London) 314, 457-480. Beauge, L. A . , and Glynn, I . M. (1979). Occlusion of K i o n s i n t h e unphosphorylated sodium pump. N a t u r e (London) 280, 510-512. E i s n e r , D. A . , and R i c h a r d s , D. E. (1980). Decreasing t h e conc e n t r a t i o n of ATP i n c r e a s e s t h e a p p a r e n t a f f i n i t y o f t h e J . Physiol sodium pump f o r e x t r a c e l l u l a r potassium. (London) 317, 56P. E i s n e r , D. A , and R i c h a r d s , D. E. (1981). The i n t e r a c t i o n of potassium i o n s and ATP on t h e sodium pump of r e s e a l e d r e d c e l l g h o s t s . J . P h y s i o l . (London) 319, 403-418. E i s n e r , D. A . , and R i c h a r d s , E. E . (1982). I n h i b i t i o n of t h e N a pump by i n o r g a n i c phosphate i n r e s e a l e d r e d c e l l g h o s t s . J . P h y s i o l . (London) 326, 1-10. Garay, R. P., and Garrahan, P . J . (1975). The i n t e r a c t i o n o f a d e n o s i n e t r i p h o s p h a t e and i n o r g a n i c phosphate w i t h t h e sodium pump i n r e d c e l l s . J. P h y s i o l . (London) 249, 51-67. Glynn, I. M. (1956). S dium and potassium movements i n human r e d cells. J. P h y s i o l . (London) 134, 278-310. Glynn, I . M . , and K a r l i s h , S . J. D. (1976). ATP h y d r o l y s i s a s s o c i a t e d w i t h an uncoupled sodium f l u x through t h e sodium pump: Evidence f o r a l l o s t e r i c e f f e c t s o f i n t r a c e l l u l a r ATP and e x t r a c e l l u l a r sodium. J. P h y s i o l . (London) 256, 465-496. K a r l i s h , S . J. D . , Yates, D. W . , and Glynn, I. M. (1978). Conf o r m a t i o n a l t r a n s i t i o n s between Na+-bound and K+-bound forms o f (Na+ + Kf)-ATPasel s t u d i e d w i t h formycin n u c l e o t i d e s . Biochim. Biophys. Acta 525, 252-264. P o s t , R. L., Hegyvary, C . , and K u m e , S. (1972). A c t i v a t i o n by adenosine t r i p h o s p h a t e i n t h e p h o s p h o r y l a t i o n k i n e t i c s o f sodium and potassium i o n t r a n s p o r t adenosine t r i p h o s p h a t a s e . 3 . B i o l . Chem. 247, 6530-6540. Robinson, J. D. ( 1 9 6 7 ) . K i n e t i c s t u d i e s on a b r a i n microsomal adenosine t r i p h o s p h a t a s e . Evidence s u g g e s t i n g conformational changes. Biochemistry 6 , 3250-3258. Robinson, J. D . , F l a s h n e r , M. S . , and Marin, G. K. ( 1 9 7 8 ) . I n h i b i t i o n of t h e (Na+ + K+)-dependent ATPase by i n o r g a n i c phosphate. Biochim. Biophys. Acta 509, 419-428.

.

CONSECUTIVE MODEL OF THE SODIUM PUMP

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Skou, J. C. (1974). Effect of ATP on the intermediary steps of the reaction of the (Na+ + K+)-dependent enzyme system. 11. Effect of a variation in the ATP/Mq2+ ratio. B i o c h i m . B i o p h y s . A c t a 339, 246-257.

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CURRENT TOPICS IN MEMBRANES AND TRANSPORT, VOLUME 19

Kinetic Models of Na-Dependent Phosphorylation of Na,K-ATPase from Rat Brain DONALD M. FOSTER O h E. Teague Veterans* Center and Texas A 8M College of Medicine Temple, Texas

STANLEY J. RUSSELL Depariment of Mechanical and Aerospace Engineering Arizona State University Temple, Arizona

KHALIL AHMED Veterans Administration Medical Center and University of Minnesota School of Medicine Minneapolis, Minnesota

I.

INTRODUCTION

I n agreement w i t h t r a n s p o r t s t u d i e s ( P o s t and J o l l y , 1957; P o s t e t al., 1 9 6 0 ) Na+-dependent k i n e t i c s of t h e enzyme have i n d i c a t e d t h a t m u l t i p l e Na+ i o n s i n t e r a c t w i t h i t . K i n e t i c models f o r Na+ i n t e r a c t i o n on 2 o r 3 e q u i v a l e n t s i t e s (Ahmed e t a l . , 1 9 6 6 ; Lindenmayer e t al., 1 9 7 4 ) and 2 o r 3 n o n e q u i v a l e n t s i t e s ( F o s t e r and Ahmed, 1 9 7 6 ) have been proposed. The l a t t e r was b a s e d upon t h e correspondence between s t e a d y - s t a t e Na+dependent phosphoenzyme f o r m a t i o n as a f u n c t i o n of Na+ c o n c e n t r a t i o n s . I n t h i s a r t i c l e a f u r t h e r a n a l y s i s of t h e s e models i s p r e s e n t e d . The approach p r e s e n t e d may p r o v i d e a u s e f u l t o o l t o k i n e t i c a l l y c h a r a c t e r i z e Na+ i n t e r a c t i o n s w i t h t h e enzyme i n r e l a t i o n t o a c t i v e s i t e E-P i n t e r m e d i a t e s .

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DONALD M. FOSTER eta/.

METHODS

A l l of t h e methods e s s e n t i a l l y were t h e same a s d e s c r i b e d by u s p r e v i o u s l y (Foster and Ahmed, 1 9 7 6 ; Ahmed and Judah, 1 9 6 4 ; Quarfoth e t al., 1 9 7 6 ) . The E-P v e r s u s NaS d a t a were a n a l y z e d by S c a t c h a r d p l o t , H i l l p l o t , double r e c i p r o c a l p l o t , and a m u l t i p l e e q u i l i b r i u m t i t r a t i o n c u r v e based on t h e f o l l o w i n g e q u a t i o n :

where [E-P] i s t h e s t e a d y - s t a t e l e v e l , [ E l i s t h e est i m a t e d t o t a l amount of enzyme p r e s e n t , and ~i i s t h e c l a s s i c a l a s s o c i a t i o n c o n s t a n t ( F o s t e r and Ahmed, 1 9 7 6 ; Klotz and Hunston, 1 9 7 1 ) .

111.

RESULTS AND DISCUSSION

A S c a t c h a r d p l o t of t h e d a t a w a s markedly curved inward toward t h e o r i g i n , i n d i c a t i n g m u l t i p l e N a + i n t e r a c t i o n s ( d a t a now shown). Double r e c i p r o c a l and H i l l p l o t s were n o n l i n e a r which w e r e d i v i d e d i n t o t h r e e reg i o n s by i n s p e c t i o n and s t r a i g h t l i n e - f i t t e d by l i n e a r r e g r e s s i o n i n each r e g i o n . The n u l l h y p o t h e s i s t h a t any two sample s l o p e s of a d j a c e n t segments came from a popul a t i o n of e q u a l s l o p e s was r e j e c t e d a t approximately 90% c o n f i d e n c e l e v e l f o r t h e H i l l p l o t and a t a 9 9 % c o n f i dence l e v e l f o r t h e double i n v e r s e p l o t . The m u l t i s i t e p l o t , based on e q u a t i o n s d e r i v e d from t h e law of mass a c t i o n f o r m u l t i p l e b i n d i n g of s m a l l molecules t o p r o t e i n s ( K l o t z , 1 9 4 6 ) , w a s used t o t e s t models t o f i t t h e e x p e r i m e n t a l d a t a . C e r t a i n assumptions employed i n t h i s approach have been d i s c u s s e d ( F o s t e r and Ahmed, 1 9 7 6 ) . The r e s u l t s of a t h r e e - s i t e ( o r t h r e e - t e r m ) p l o t a r e shown i n F i g . 1, which i n c l u d e s c u r v e s f o r e q u i v a l e n t (dashed) and n o n e q u i v a l e n t (cont i n u o u s ) s i t e s . S i m i l a r models were t e s t e d f o r two s i t e s . I t was e v i d e n t t h a t t h e t w o - o r three-nonequival e n t - s i t e models gave a b e t t e r f i t t h a n t h e e q u i v a l e n t s i t e models. However, d i s t i n c t i o n between two- o r three-nonequivalent-site models, even w i t h a d d i t i o n a l Na+ l e v e l s a n a l y z e d i n t h i s s t u d y , i s d i f f i c u l t on a s t a t i s t i c a l b a s i s alone.

KINETIC MODELS OF Na+Sites ON Na,K-ATPase

2l 2.0

555

,a*/+ ,-

Fig. 1 . Multiple-site models for steady-state phosphoenzyme as a function of Na+. The experimental data are represented by discrete points, whereas theoretical models to fit these data are represented by lines. Theoretical models were derived from multiple equilibrium equations described in Section 11: (1 three-nonequivalent-si te model; (----- ) three-equivalent-site model.

Robinson ( 1 9 7 7 )

,

u s i n g d i f f e r e n t methods, found

s i m i l a r i t i e s among v a l u e s of k i n e t i c c o n s t a n t s o b t a i n e d by him and by u s ( F o s t e r and A b e d , 1 9 7 6 ) . Based on e f f e c t s o f Na+ o n t r a n s f o r m a t i o n s of E-P i n t e r m e d i a t e s , t h r e e n o n e q u i v a l e n t N a + i n t e r a c t i o n s have been p r o p o s e d (Hara and Nakao, t h i s volume; Klodos et a l . , t h i s v o l u m e ) . A l s o , 22Na+ b i n d i n g t o p u r i f i e d N a , K - A T P a s e h a s r e v e a l e d t h r e e n o n e q u i v a l e n t ( M a t s u i e t al., t h i s volume) o r t h r e e b i n d i n g s i t e s f o r Na+ (Tonomura e t al., t h i s v o l u m e ) . Thus, i t a p p e a r s t h a t N a + i n t e r a c t s w i t h t h i s enzyme i n a m u l t i p l i s t i c f a s h i o n commensurate w i t h i t s t r a n s p o r t d u r i n g one pump c y c l e . However, t h e p r e c i s e l o c a t i o n o r f u n c t i o n o f e a c h of t h e s e s i t e s i s unc l e a r and would r e q u i r e f u r t h e r a n a l y s i s . Nonetheless, u n d e r t h e g i v e n e x p e r i m e n t a l c o n d i t i o n s , d i s t i n c t i o n between h i g h - and l o w - a f f i n i t y Na+ s i t e s o n t h e enzyme can be made.

DONALD M.FOSTER eta/.

556

REFERENCES Ahmed, K . , and Judah, J. D. (1964). P r e p a r a t i o n of l i p o p r o t e i n s c o n t a i n i n g cation-dependent ATPase. Biochim. Biophys. Acta 93, 603-613. Ahmed, K . , Judah, J . D . , and S c h o l e f i e l d , P. G. (1966). I n t e r a c t i o n o f sodium and potassium w i t h a cation-dependent adenos i n e t r i p h o s p h a t a s e system from r a t b r a i n . Biochim. Biophys. Acta 120, 351-360. F o s t e r , D., and Ahmed, K. (1976). Na+-dependent p h o s p h o r y l a t i o n o f t h e r a t b r a i n ( N a + + K+)-ATPase. P o s s i b l e nonequivalent a c t i v a t i o n s i t e s f o r Na'. Biochim. Biophys. Acta 429, 258273. Lindenrnayer, G. E . , Schwartz, A., and Thompson, H. K . , Jr. (1974). A k i n e t i c d e s c r i p t i o n f o r sodium and potassium e f f e c t s on ( N a + + K+)-adenosine t r i p h o s p h a t a s e : A model f o r a twononequivalent s i t e potassium a c t i v a t i o n and an a n a l y s i s of m u l t i e q u i v a l e n t site models f o r sodium a c t i v a t i o n . J. P h y s i o l (London) 236, 1-28. K l o t z , I. M. (1946). The a p p l i c a t i o n o f t h e law o f m a s s a c t i o n t o b i n d i n g by p r o t e i n s . I n t e r a c t i o n s w i t h calcium. Arch. Biochem. Biophys. 9, 109-117. K l o t z , I. M . , and Hunston, D. L. (1971). P r o p e r t i e s o f g r a p h i c a l r e p r e s e n t a t i o n s of m u l t i p l e classes o f b i n d i n g sites. Biochemistry 10, 3065-3069. P o s t , R. L . , and J o l l y , P. C. (1957). The l i n k a g e o f sodium, and ammonium a c t i v e t r a n s p o r t a c r o s s t h e human e r y t h r o c y t e memb r a n e . Biochim. Biophys. Acta 25, 118-128. P o s t , R. L., Merritt, C. R . , Kinsolving, C. R . , and A l b r i g h t , C. D. (1960). Membrane adenosine t r i p h o s p h a t a s e as a p a r t i c i p a n t i n t h e a c t i v e t r a n s p o r t of sodium and potassium i n t h e human e r y t h r o c y t e . J. B i o l . C h e m . 235, 1796-1802. Quarfoth, G . , Ahmed, K . , F o s t e r , D., and Zieve, L. (1976). A c t i o n of m e t h a n e t h i o l on membrane (Na+,K+)-ATPase of r a t b r a i n . Biochem. Pharmacol. 25, 1039-1044. D. (1977). N a + sites o f t h e ( N a + + K+)-dependent Robinson, ATPase. Biochim. Biophys. Acta 4 8 2 , 427-437.

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J.

CURRENT TOPICS IN MEMBRANES AND TRANSPORT, VOLUME 19

Reinvestigation of the Sequence of Sensitivity of Phosphoenzyme of Na,K-ATPase to ADP and K+ during Presteady State of the Phosphorylation by ATP Y. FUKUSHIMA AND M. NAKAO Laboratory ofActive Transpon National Institute for Physiology Sciences Okaraki, Japan

I.

INTRODUCTION

In the Post cycle of the reaction of Na,K-ATPase, it has been proposed that the two major conformational states of the phosphoenzyme, El-P and E2-P, appear sequentially (Glynn and Karlish, 1975). According to this mechanism, El-P is formed first, if the enzyme is phosphorylated from ATP. El-P splitting is accelerated by ADP. El-P then becomes Ez-P, whose hydrolysis is accelerated by .'K Experiments which utilized a rapid quenching method have yielded results which do not conform to the Post cycle described above (Fukushima and Tonomura, 1973). In particular, it was found that ADP accelerated the splitting of phosphoenzyme only after a long period of phosphorylation, rather than at the early stage of phosphorylation. This experiment, along with others, indicated that the initial ADP insensitivity corresponds to the stabilization of the ADP-bound phosphoenzyme. 557

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In the present experiments, we reinvestigated the time-dependent change of the sensitivity of pig kidney Na,K-ATPase to either ADP or K+ in the presteady state of the phosphor lation by [32P]ATP (10 PM). In order to add ADP or Ky to the phosphoenzyme before the steady state was reached, the rate of phos horylation was slowed by replacement of Mg2+ by Ca3+ at O°C and pH 7.4. This method precluded the need for a rapid quenching method. Furthermore, ADP-sensitive phosphoenzyme could be accumulated to a measurable level without modification of the enzyme by an SH reagent such as N-ethylmaleimide. Our observations agreed with the Post sequence (Fukushima and Nakao, 1981).

11.

RESULTS AND DISCUSSION

In order to test for sensitivity to ADP or K+ during the initial stage of the phosphorylation, we added an excess amount of unlabeled ATP to monitor the splitting of the phosphoenzyme. Unlabeled ATP did not inhibit the labeling of the ATPase instantaneously in the initial stage. The labeling of the enzyme continued to increase for several seconds after a chase with unlabeled ATP, and thereafter showed a decrease. The rate of exchange between free and bound ATP is probably slow enough to allow bound [32P]ATP to phosphorylate the enzyme. Prolonged phosphorylation was noticeable only at the initial stage, since the amount of enzyme.ATP complex was greater at the beginning of the reaction than in the steady state. This delayed inhibition does not necessarily justify the reported heterogeneity of loosely and tighly bound enzyme-ATP complexes (Kanazawa et a l . , 1970; Fukushima and Tonomura, 1973). There is no difficulty in the explanation of the present observation by a homogeneous precursor of the phosphoenzyme. Sensitivity to ADP or to K+ was tested by simultaneous addition of 2.5 mM ADP or 1.5 mM KC1 with the unlabeled ATP. In the presence of 1 mM Ca2+ and 270 mM Na+, all of the phosphoenzyme formed during a 1-sec reaction period was split by ADP. With an increase of the labeling time, the portion of the ADP-sensitive phosphoenzyme decreased, while the K+-sensitive portion increased. In order to obtain a more quantitative estimation of El-P and E2-P by the chase experiment, we reduced the Na+ concentration to 126 mM. The degree of shift of the equilibrium between El-P and E2-P by the removal

SEQUENCE OF SENSITIVITY OF PHOSPHOENZYME TO ADP AND K +

559

of one component a p p a r e n t l y l e s s e n e d i n t h e p r e s e n c e of a lower c o n c e n t r a t i o n of Na+. Under t h e s e c o n d i t i o n s , it took approximately 1 0 sec t o r e a c h t h e s t e a d y s t a t e of p h o s p h o r y l a t i o n . Delayed i n h i b i t i o n , f o l l o w i n g t h e a d d i t i o n of u n l a b e l e d ATP, was s e e n i n t h e c h a s e o n l y a t 1 sec. A f t e r a 2-sec l a b e l i n g of t h e enzyme, a s i m p l e d e c r e a s e of t h e 32P-labeled phosphoenzyme w a s observed f o l l o w i n g t h e f i r s t - o r d e r s p l i t t i n g . W e t h e r e f o r e made a s e m i l o g a r i t h m i c p l o t of t h e amount of t h e 32P-labeled phosphoenzyme ( o r d i n a t e a x i s ) a g a i n s t t i m e f o r each c h a s e experiment performed d u r i n g t h e p r o g r e s s of t h e p h o s p h o r y l a t i o n . The v a l u e of t h e o r d i n a t e a x i s i n t e r c e p t of t h e r e g r e s s i o n l i n e i n c r e a s e d w i t h t h e l a b e l i n g t i m e i n t h e c h a s i n g w i t h ADP, whereas it dec r e a s e d i n t h e c h a s i n g w i t h K+. About 40% and 20% of t h e t o t a l phosphoenzyme w e r e r a p i d l y s p l i t by ADP (E1-P), whereas 6 0 % and 8 0 % were s e n s i t i v e t o K+ (E2-P) a t 2and 2 0 - s e c p h o s p h o r y l a t i o n r e a c t i o n p e r i o d s , r e s p e c t i v e ly. According t o a r e c e n t s t u d y of ADP b i n d i n g (Yamag u s h i and Tonomura, 1 9 7 8 1 , t h e r e t e n t i o n of p r o d u c t ADP on t h e phosphoenzyme depended upon t h e c o n c e n t r a t i o n of K+. C l e a r l y , f u r t h e r s t u d i e s a r e needed f o r a b e t t e r u n d e r s t a n d i n g of t h e r e a c t i o n of Na,K-ATPase i n i t i a t e d i n t h e p r e s e n c e of b o t h Na+ and K+. I n t h e p r e s e n t experiments, we did not chase t h e l a b e l i n g by a c h e l a t i n g r e a g e n t , s i n c e t h e Ca-phosphoenzyme becomes i n e r t when i t r e l e a s e s Ca2+ and s t a b l e w i t h r e s p e c t t o t h e phosphate bond i n t h e p r e s e n c e of a c h e l a t o r (Fukushima and P o s t , 1 9 7 8 ) . However, we c o u l d u s e t h e s e p r o p e r t i e s t o compare t h e a f f i n i t y of E l - P and E2-P t o Ca2+. When e x c e s s amount of CDTA w a s added t o t h e phosphoenzyme a t t h e beginning of t h e p h o s p h o r y l a t i o n , t h e phosphoenzyme became e x t r e m e l y s t a b l e . I n c o n t r a s t , o n l y a s m a l l f r a c t i o n of t h e phosphoenzyme was s t a b i l i z e d by CDTA a t t h e s t e a d y s t a t e . The a f f i n i t y of E l - P t o Ca2+ appeared t o be lower t h a n t h a t of E2-P (Fukushima and Nakao, 1 9 8 0 ) .

REFERENCES Fukushima, Y . , and Nakao, M. (1980). Changes i n a f f i n i t y of Na+- and K+-transport ATPase f o r d i v a l e n t c a t i o n s d u r i n g i t s r e a c t i o n sequence. J. Biol. Chem. 255, 7813-7819. Fukushima, Y., and Nakao, M. ( 1 9 8 1 ) . T r a n s i e n t s t a t e i n t h e p h o s p h o r y l a t i o n o f sodium- and p o t a s s i u m - t r a n s p o r t adenosine t r i p h o s p h a t a s e by adenosine t r i p h o s p h a t e . J. Biol Chem. 256, 9136-9143.

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Fukushima, Y., and Post, R. L. (1978). Binding of divalent cation to phosphoenzyme of sodium- and potassium-transport adenosine triphosphatase. J. B i o l . Chem. 253, 6853-6862. Fukushima, Y., and Tonomura, Y. (1973). Two kinds of high energy phosphorylated intermediate, with and without bound ADP, in the reaction of Na+-K+-dependent ATPase. J. B i o c h e m . ( T o k y o ) 7 4 , 135-142. Glynn, I. M., and Karlish, S. J. D. (1975). The sodium pump. Annu. R e v . P h y s i o l . 37, 13-55. Kanazawa, T., Saito, M., and Tonomura, Y. (1970). Formation and decomposition of a phosphorylated intermediate in the reaction of Na+-K+ dependent ATPase. J. B i o c h e m . (Tokyo) 6 7 , 693-711. Yamagushi, M., and Tonomura, Y. (1978). Binding of a adenosine diphosphate to reaction intermediates in the Na+,K+-dependent ATPase from porcine kidney. J. B i o c h e m . ( T o k y o ) 8 3 , 977-987.

CURRENT TOPICS IN MEMBRANES A N D TRANSPORT, VOLUME 19

Interaction of Na+,K+ and ATP with Na,K-ATPase P. J. GARRAHAN, R. ROSSI, AND A. F. REGA Departamento de Quimica Bioldgica Facultad de Farmacia y Bioquimica Universidad & Buenos Aires Buenos Aires, Argentina

I.

INTRODUCTION

Although a great deal of experimental information on the elementary steps of the hydrolysis of ATP catalyzed by the Na,K-ATPase is available (see, for instance, Karlish et a l . , 19781, few attempts have been made to see to what extent reaction schemes based on results from experiments on partial reactions are able to predict the steady-state kinetic behavior of the enzyme.

11.

MATERIALS AND METHODS

In the experiments reported here we studied the interactions of ATP, a nonhydrolyzable analog of ATP adenylylmethylene diphosphonate (AMPPCP), Na+, and K4 with the Na,K-ATPase and confronted the results with 561

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the predictions of the current schemes for the hydrolysis of ATP by the Na,K-ATPase (see, for instance, Karlish et a l . , 1978). Na,K-ATPase was prepared from dog kidney red outer medulla by the simpler of the two procedures described by Jgkgensen (1974). ATPase activity was measured by the release of [32P]Pi from [y-32P]ATP at 37OC and at pH 7.4 and 150 mM total salt concentration. Confirming observations by other authors, the substrate curve of the ATPase can be described by the sum of two Michaelis equations--one with low Km (0.25 VM) and Vmax (0.5 pmole/mg/min) and the other with high Km (100-200 pM) and Vmax (7-9 pmoles/mg/min)

.

111.

RESULTS AND DISCUSSION

AMPPCP acts as a competitive inhibitor of ATP at the high-affinity component of the substrate curve of the ATPase. At high (100 VM) concentration of ATP, AEIPPCP enhances ATPase activity. In the presence of 3 mM AMPPCP the substrate curve of the ATPase loses its biphasic shape and becomes a single hyperbola. These findings support the idea that the high-affinity component of the substrate curve expresses the combination of ATP at a catalytic site and that the low-affinity component expresses the combination of ATP at a site from which the nucleotide activates the ATPase without undergoing hydrolysis. At nonlimiting concentrations of Na+ both the Km and the Vmax for ATP at the low-affinity site increase with the concentration of K+ along rectangular hyperbolae which are half-maximal at about 1.5 m~ K+. Hence, the ratio Km/Vmax for ATP is independent of the concentration of K+. A similar effect is exerted by ATP on the ~ 0 . 5and the maximum effect of K+. These findings are consistent with the idea that the interactions between K+ and ATP at the noncatalytic site follow pingpong kinetics. In the presence of saturating concentrations of AMPPCP it is likely that the noncatalytic site remains fully occupied and that ATPase activity is governed by the occupation of the catalytic site by ATP. This would allow one to study the interactions between K+ and ATP at this site. Experiments performed in media with 3 mM AMPPCP suggest that the interactions between K+ and ATP at the catalytic site also follow ping-pong kinetics.

INTERACTION OF Na+,K+,AND ATP WITH Na,K-ATPase

563

It is generally accepted that in the presence of Na+, ATP at the catalytic site phosphorylates the El conformer of the ATPase and that K+ acts by accelerating the hydrolysis of the phosphoenzyme. There is evidence that after dephosphorylation K+ remains bound in an "occluded" state to the E2 conformer of the enzyme and that rapid release of K+ requires binding of ATP or of ATP analogs at a low-affinity site (see Beauge and Glynn, 1980). In agreement with the experimental results, steady-state rate equations derived from a kinetic scheme based on this hypothesis predict the biphasic response to AT , the effects of AMPPCP, and the ping-pong kinetics between K+ and ATP at both the catalytic and the regulatory sites. The kinetic analysis does not allow one to decide whether the two sites for ATP are physically distinct or represent different states of the same site. In sharp contrast with the observed interactions between K+ and ATP, at nonlimiting K+ concentrations, changes in Na+ concentration only alter the vmax and have no detectable effect on the Km for ATP at the lowaffinity site. Likewise, changes in the concentration of ATP only affect the maximum effect of Na+. This kind of interaction is not predicted by the reaction scheme mentioned before since if Na+ only acted to promote phosphorylation, the interaction between Na+ and ATP at the regulatory site should follow ping-pong kinetics. It would seem therefore that the kinetic scheme has to be modified to account for the interactions between Na+ and ATP. One way to do this is to assume that not only phosphorylation but also the (E2K)oc,luded + E2 + Kf transition requires Na+ and that this transition is rate-limiting. More experimental evidence is needed to test the validity of this hypothesis. However, even if this proposal proved to be false, it is useful since it demonstrates that in a system that interacts with many ligands the existence of ping-pong kinetics between two ligands is not sufficient evidence for proposing fully consecutive reaction schemes.

ACKNOWLEDGMENT

Supported by g r a n t s from CONICET, SUBCYT, and Fundaci6n R o e m m e r s ( A r g e n t i n a ) and PNUD/UNESCO RLA 78/024.

P.J. GARRAHAN eta/.

564

REFERENCES Beaug6, L. A., and Glynn, I. M. (1980). The equilibrium between different conformations of the unphosphorylated sodium pump: Effects of ATP and of potassium ions and their relevance to potassium transport. J. P h y s i o l . (London) 2 9 9 , 367-383. J$rgensen, P. K. (1974). Purification and characterization of (Na+ + K+)ATPase. 111. Purification from the outer medulla of mamaluan kidney after selective removal of membrane components by sodium dodecylsulphake. B i o c h i m . B i o p h y s . A c t a 356, 36-52. Karlish, S. J. D., Yates, D. W., and Glynn, I. M. (1978). Conformational transitions between Na+-bound and K+-bound forms of the (Na+-K+) ATPase studied with formycin nucleotides. B i o c h i r n . B i o p h y s . A c t a 5 2 5 , 252-264.

CURRENT TOPICS IN MEMBRANES AND TRANSPORT, VOLUME 19

Sodium Ion Discharge From Pig Kidney Na,K-ATPase YUKICHI HARA AND MAKOTO NAKAO Tokyo Medical and Dental University School of Medicine Yushimu, Bunkyo-ku, Tokyo,Japan

I.

INTRODUCTION

It is suggested that sodium ions bound to the cytoplasmic surface of Na,K-ATPase and translocated to the extracellular surface are discharged from the enzyme during the transphosphorylation of the enzyme (El-P -+ E 2 - P ) (Albers, 1967; Glynn and Karlish, 1975; Post et a l . , 1975). But points which remain unclear include how many sodium ions are released and what is the mode of release of sodium ions. Recently, the model, accounting for the effect of Na+ on the steadystate ratio between El-P and E2-P was proposed (Klodos et a l . , 1981). In this article we will describe the effects of various concentrations of Na+ on the El-P T- E2-P equilibrium.

-

565

Copyright 0 1983 by Academichess, Inc. MI rights of reproduction in any form R S ~ N ISBN 0-12-153319-0

~ .

YUKlCHl HARAAND MAKOTO NAKAO

566

METHODS

11.

A s a workin h y p o t h e s i s w e us d Scheme 1, where k 2 , and k3 w e r e a p p a r e n t f i r s t - o r d e r r a t e c o n s t a n t s . The l e v e l s of E l - P and E2-P were measured from t h e sens i t i v i t y t o ADP and K+, r e s p e c t i v e l y . The r a t e c o n s t a n t kl,

kl

k3

E~P-Ez

+

Pi

was c a l c u l a t e d from an a p p a r e n t f i r s t - o r d e r r a t e cons g a n t f o r t h e i n i t i a l phase of E-P d e p h o s p h o r y l a t i o n ( k d e ) and t h e l e v e l of E2-P f r a c t i o n . The r a t e c o n s t a n t k 2 was c a l c u l a t e d from k3 and t h e r a t e c o n s t a n t of E2-P d e p h o s p h o r y l a t i o n which w a s observed i n t h e p r e s e n c e of ADP. The r a t e c o n s t a n t k l was c a l c u l a t e d on t h e b a s e s of s t e a d y - s t a t e e q u a t i o n k l = [Ez-P] ( k 2 + k 3 ) / [ E l - P ] . A l l experiments were c a r r i e d o u t a t O°C. Details are g i v e n by Hara and Nakao ( 1 9 8 1 ) . k

111.

A.

RESULTS AND DISCUSSION

E f f e c t of N a

+

on E l - P a n d E - P L e v e l s 2

The E l - P f r a c t i o n i n c r e a s e d a s Na+ c o n c e n t r a t i o n i n c r e a s e d i n t h e r a n g e of 2 0 mM t o 1 . 4 M . A s t h e sum of t h e s e two f r a c t i o n s was n e a r l y 1 o v e r a wide range of Na+ c o n c e n t r a t i o n s , it i s l i k e l y t h a t E-P produced i n t h e p r e s e n c e of Na+, Mg2+, and ATP c o n s i s t s of ess e n t i a l l y o n l y two components, i . e . , E l - P and E2-P. B.

E f f e c t of N a

+

on k 1 l k 2 '

kylf and kde

The r a t e c o n s t a n t kde was found t o i n c r e a s e w i t h i n c r e a s i n g Na+ c o n c e n t r a t i o n , r e a c h a maximum a t 0 . 4 M NaC1, and t h e n d e c r e a s e a t h i g h e r Na+ c o n c e n t r a t i o n . The r a t e c o n s t a n t k3 was found t o i n c r e a s e w i t h i n c r e a s i n g Na+ c o n c e n t r a t i o n and s a t u r a t e a t 1 M NaC1. The r a t e c o n s t a n t k 2 was found t o i n c r e a s e w h i l e kl decreased with i n c r e a s i n g N a + concentration. I f t h e Na+ i o n s a r e r e l e a s e d one by one i n a s t e p w i s e manner, Scheme 2 c a n be proposed. I n t h e scheme, E-PI and E-PI' a r e p h o s p h o r y l a t e d i n t e r m e d i a t e s b i n d i n g

SODIUM ION DISCHARGE FROM PIG KIDNEY

Na* step 1

567

Na* step 2

Na* step 3

two and one Na ions, respectively, K1 and ~2 are the dissociation constants of the quasi-equilibrium reactions, step 1 and step 3 , respectively, ki is the fifstorder rate constant of the E-P' + E-P" conversion, k2 I is the second-order rate constant of E-P" + Naf -+ E-P conversion, and K1 is much smaller and K2 is much larger than "a+]. Apparent first-order rate constants of the El-P 7 E2-P transition (kl) and that of the E2-P + El-P transition (k2) fof: this scheme may be,expressed as follows: kl = Klk2/[Na+] I k2 = "a] 2k2/K2. Since kl was directly proportional to the reciprocal of Na+ concentration, k2 to the square, and K ~ the ~ equilib~ , rium constant between El-P and Ez-P, to he third power, the Na+ dependence of kl, k2, and kapp can be explained by Scheme 2.

IV.

CONCLUSIONS

The conclusions to be drawn from our results are that the levels of E-Ps other than El-P or E2-P were negligible and that the Na+ dependence of the El-P and E2-P levels and of the E2-P El-P transition rate constant could be explained by the reaction scheme in which three Na+ ions are released in a highly cooperative manner. -+

REFERENCES Albers, R. W. (1967). Biochemical aspects of active transport. Annu. R e v . B i o c h e m . 3 6 , 727-756. Glynn, I. M., and Karlish, S. J. D. (1975). The sodium pump. Annu. R e v . P h y s i o l . 37, 13-55. Hara, Y., and Nakao, M. (1981). Sodium ion discharge from pig kidney Na+, K+-ATPase; Na+-dpeendency of the EIP , "E2P equilibrium in the absence of KC1. J . B i o c h e m . (Tokyo) 90, 923-9 31.

568

YUKlCHl HARA AND MAKOTO NAKAO

Klodos, I . , Nplrby, J. G . , and P l e s n e r , I. W. ( 1 9 8 1 ) . The s t e a d y s t a t e k i n e t i c mechanism of ATP h y d r o l y s i s c a t a l y z e d by membrane-bound (Na+ + K+)-ATPase from ox b r a i n . 11. K i n e t i c c h a r a c t e r i z a t i o n of p h o s p h o i n t e r m e d i a t e s . Biochim. Biophys Acta 643, 463-482. P o s t , R. L . , Toda, G . , Kume, S . , and T a n i g u c h i , K. (1975). Synt h e s i s o f a d e n o s i n e t r i p h o s p h a t e by way o f potassiums e n s i t i v e phosphoenzyme of sodium, p o t a s s i u m a d e n o s i n e t r i phosphatase. J. S u p r a m o l . Struct. 3, 479-497.

CURRENT TOPICS IN MEMBRANES AND TRANSPORT, VOLUME 19

ADP Sensitivity of the Natiie and Oligomycin-Treated Na,K-ATPase A" S. HOBBS AND R. WAYNEALBERS National Institute of Neurological and Communicative Disorders and Stroke National Institutes of Health Bethesda, Maryland

JEFFREY P. FROEHLICH National Institute on Aging National Institutes of Health Gerontology Research Center Baltimore City Hospitals Baltimore, Maryland

I.

INTRODUCTION

A large body of experimental evidence supports the participation of two phosphorylated forms in the main catalytic pathway of the Na,K-ATPase--ElaP, an ADPsensitive form, and E2-P, a K+-sensitive one. In this study, we used rapid-quenching techniques (Froehlich e t a l . , 1976) to measure formation and breakdown of E1%P in microsomal enzyme obtained from E l e c t r o p h o r u s e l e c t r i c u s both while the enzyme was fully activated by Na+, K+, Mg2+, and ATP, and in the presence of oligomycin, an inhibitor believed to act mainly by blocking the E1QP +. E2-P transition.

569

Copyright 0 1983 by Academic Press, IIIF. All rights of reproduction in any form reserved. ISBN 0-12-1533190

ANN S.HOBBS

570

1 .o

0.5

0

0

0.275

0.55

SECONDS AFTER ADP ADDITION F i g . 1. D e p h o s p h o r y l a t i o n of o l i g o m y c i n - t r e a t e d Na,K-ATPase b y 5 mM ADP. Enzyme (1 m g / m l ) s u s p e n d e d i n 100 mM NaCl, 3 mM MgC12, o l i g o m y c i n (0.1 m g / m l , added i n e t h a n o l , f i n a l c o n c e n t r a t i o n 0 . 5 % ) , 60 mM T r i s - H C 1 (pH 7.5), and 0.1 mM EDTA was mixed f o r either 6 or 1 1 6 m s e c w i t h 10 pM [y-32P]ATP, then w i t h 5 mM ADP for the t i m e shown on the a b s c i s s a , then w i t h 2.25% perchloric a c i d and 1.5 mM i n o r g a n i c p h o s p h a t e t o s t o p the r e a c t i o n . T o t a l E-P p r e s e n t a f t e r 6 m s e c ( 0 ) o f p h o s p h o r y l a t i o n t i m e was 37% of t h a t p r e s e n t a f t e r 116 m s e c ( 0 ) ( d a t a a r e n o r m a l i z e d ) . Inset shows i n i t i a l t i m e p o i n t s on a n expanded s c a l e .

11.

METHODS

When 5 mM ADP is added to oligomycin-treated enzyme which has been phosphorylated in the presence of 10 p~ ATP, approximately two-thirds of the phosphoenzyme disappears rapidly and the remainder disappears at a much slower rate (Fig. 1). Since oligomycin blocks the E1%P + E2-P transition, the fast rate presumably reflects the establishment of a new equilibrium between E1*ATP and El%P, as ADP reverses the phosphorylation reaction. Although the disappearance of the rapid fraction occurs too quickly for accurate measurement of its rate, estimation of the forward reaction (E-ATP -t E-P) at 150 sec'l (Froehlich e t a l . , 1976), coupled with the extent and rate of disappearance of E-P in Fig. 1, sug-

ADP SENSITIVITY OF Na,K-ATPase

571

.

gests that the rate constant for E,p ADP + E-ATP is >300 sec-l. Both computer simulations (Froehlich et a l . , 1976) and direct measurements (Froehlich et al., this volume) of the rate of ATP dissociation in the presence and absence of oligom cin indicate that it occurs at a rate of 35-50 secThus, this reaction is too fast to account for the slowly disappearing E-P.

1.

111.

RESULTS AND DISCUSSION

The proportion of rapidly and slowly decaying intermediates is independent of the time of incubation with ATP. In two experiments, one in which phosphorylation was allowed to proceed for 6 msec and the other for 116 msec prior to the addition of ADP, the relative proportions were identical. This shows (1) that ADP is released rapidly from the enzyme once E-P has been formed, and (2) that the two species, "sensitive" and "insensitive" to ADP, equilibrate rapidly. In the oligomycin-treated enzyme, very little Pi (<0.001 nmole/mg) is released during 542 msec of reaction with ADP. Thus, the slow component probably arises as a consequence of the perturbation of the equilibrium between El and El-ATP following ADP binding and reversal of the phosphorylation reaction. Because measured ATP release is too fast to account for the rate of disappearance of the slow component, there must be an additional slow step in the mechanism that is rate-limiting:

El

+

ATP

2

E1*ATP

Ei-ATP

E;Zp

EISP

+

ADP

In this scheme, Ei-ATP represents a second enzymesubstrate complex which is formed rapidly from El-ATP, but has a very slow reversal. Its formation probably requires Mg2+, which was not present when the ATP dissociation measurements were made. The uninhibited enzyme shows a similar pattern of dephosphorylation when 5 mM ADP is added during steadystate turnover. In contrast to experiments in which excess unlabeled ATP, EDTA, or both are used to stop the reaction and 50-70% of the E-P decays with a rate of 100-150 sec-1, when 5 mM ADP is added, 82% of the E-P disappears at a rate of 300 sec-1 or greater. This suggests that a substantial fraction of the E-P measured under steady-state turnover conditions is in the E1%P form, and that the rate at which E1%P is converted to E2-P is slower than the rate at which E2-P breakdown occurs.

572

ANN S. HOBBS

I n t h e case of t h e n a t i v e enzyme, t h e s l o w (ADP i n s e n s i t i v e ) component might be t h o u g h t t o r e p r e s e n t Ez-P i n a s e q u e n t i a l pathway of ATP h y d r o l y s i s . We view t h i s a s u n l i k e l y because it h a s a slow r a t e of hyI n previous d r o l y s i s , d e s p i t e t h e p r e s e n c e of K+. s t u d i e s , K+-activated h y d r o l y s i s of phosphoenzyme formed i n t h e p r e s e n c e of Na+ was shown t o be v e r y r a p i d (>300 sec-1, Hobbs et al., 1 9 8 0 ) . One p o s s i b i l i t y i s t h a t t h i s slow component a r i s e s i n a p a r a l l e l pathway which may be coupled t o o r independent of t h e main c a t a l y t i c pathway. The f a c t t h a t t h e p r o d u c t f l u x through t h i s pathway ( d e f i n e d a s t h e r a t e of t h e slow component t i m e s i t s c o n c e n t r a t i o n ) i s s m a l l (%0.6 nmole/mg/sec) compared t o t h e t o t a l ATPase a c t i v i t y (6-8 nmoles/mg/sec) supp o r t s t h e view t h a t t h e t w o pathways a r e i n d e p e n d e n t .

REFERENCES Froehlich, J. P., Albers, R. W., Koval, G. J., Goebel, R., and Berman, M. (1976). Evidence for a new intermediate state in the mechanism of the (Na+ + K+)-adenosine triphosphatase. J. Biol. Chem. 251, 2186-2188. Hobbs, A. S., Albers, R. W., and Froehlich, J. P. (1980). Potassium-induced changes in phosphorylation and dephosphorylation of (Na+ + K+)-ATPase observed in the transient state. J. Biol. Chem. 255, 3395-3402.

CURRENT TOPICS IN MEMBRANESAND TRANSPORT. VOLUME 19

Three (at Least) Consecutive Phosphointermediates of Na-ATPase I. KLODOS, J. G. N0RBY, AND N. 0.CHRISTIANSEN Institute ofBiophysics University of Aarhus Aarhus, Denmark

I.

INTRODUCTION

The present work is an elaboration of the recently published kinetic characterization of the phosphoenzymes of Na,K-ATPase (Klodos et a l . , 1981). In that article we showed results of phosphorylation-dephosphorylation experiments at constant Na+ concentration and evaluated them according to the simplest model containing two phosphointermediates--an ADP-sensitive El-P and a K+-sensitive E2-P.

11.

RESULTS

Here we have further xami ed the interco version of the phosphointermediates as well as their hydrolysis at varying “a+]. Nearly 100% of the Na K-ATPase from ox brain was phosphorylated with 25 U M [5zP]ATP, 1 mM 573

Copyright 0 I983 by Afadcmic Press, Inc. All rights of reproductionin any form reserved. ISBN 0-12-1533194

I. KLODOS eta/.

574

2+ Mg , and "a+] from 20 t o 300 mM a t O°C. The dephosphorylation of t h e s e r a d i o a c t i v e phosphointermediates w a s followed under f o u r d i f f e r e n t c o n d i t i o n s ( c f . F i g . 1), namely, a f t e r a d d i t i o n of (1) 1 m ATP, ( 2 ) 2.5 m ADP, ( 3 ) 2.5 m ADP + N a + up t o 6 0 0 m , o r ( 4 ) 1 mM ATP + 20 m K+. An increase i n "a+] caused ( a ) an i n c r e a s e i n t h e d e p h o s p h o r y l a t i o n r a t e of E-P ( e x p e r i ments 1, 2 , and 3 ) , ( b ) a n i n c r e a s e i n t h e amount of E-P r a p i d l y d e p h o s p h o r y l a t e d by ADP--"ADP-sensitive" E-P ( e x p e r i m e n t s 2 and 3 ) , and ( c ) a d e c r e a s e i n t h e amount of E-P r a p i d l y d e p h o s p h o r y l a t e d by h i g h K+--"K+s e n s i t i v e " E-P (experiment 4 ) . S i n c e , a t a g i v e n "a+], t h e sum of t h e d e t e r m i n e d c o n c e n t r a t i o n s of t h e c l a s s i c a l " K + - s e n s i t i v e E2-P" and "ADP-sensitive E l % P " i s always g r e a t e r t h a n 1 0 0 % (see F i g . 11, w e concluded t h a t t h e r e must be a t h i r d E-P s ecies, which i s d e p h o s p h o r y l a t e d b o t h by ADP and h i g h KP Moreover, t h e amount of "ADP-sensitive" E-P i n creases n o t o n l y w i t h "a+] d u r i n g p h o s p h o r y l a t i o n b u t a l s o w i t h "a+] d u r i n g d e p h o s p h o r y l a t i o n ( e x p e r i m e n t 3 ) . A l i k e l y e x p l a n a t i o n i s t h a t t h e t h i r d E-P s p e c i e s i s a " N a + - s e n s i t i v e E l - P , " which i n high-Na+ media i s conv e r t e d r a p i d l y t o t h e "ADP-sensitive E1%P." W e have used t h e three-compartment model, shown i n Table I , t o s i m u l a t e t h e e x p e r i m e n t a l r e s u l t s under t h e f o l l o w i n g premises: ( I ) b o t h E l - P ( " N a + - s e n s i t i v e " E-P) and E2-P are d e p h o s p h o r y l a t e d r a p i d l y w i t h K + , s o t h a t E l % P i s t h e o r d i n a t e i n t e r c e p t of t h e "slow component" i n exp e r i m e n t 4 ; (11) w i t h 6 0 0 mM N a + b o t h E1%P and E l - P a r e dephosphorylated r a p i d l y i n ADP e x p e r i m e n t s , so t h a t E2-P c a n be o b t a i n e d from t h e i n t e r c e p t of t h e "slow component" i n experiment 3 ; (111) t h e s l o p ? o f t h e "ATP curve" (experiment 1) g i v e s estimates of k-A, k g , and kc (see Klodos e t a l . , 1981) ; ( I V ) from t h e same r e f e r e n c e , t h e s l o p e o f t h e "slow" p a r t of t h e "ADP curve" ( e x p e r i ment 2 ) w i l l be between kc and k c + k-C; and (V) h a v i n g E1$P and an estimate of E 2 - P , E l - P i s g i v e n . From t h e s t e a d y - s t a t e c o n d i t i o n s t h e remaining c o n s t a n t s can now be estimated. Some r e s u l t s of t h e c u r v e - f i t t i n g proc e d u r e ( u s i n g t h e d i f f e r e n t i a l and s t e a d y - s t a t e equat i o n s c o r r e s p o n d i n g t o t h e model) are shown i n Table I.

.

111.

CONCLUSIONS

1. An i n t e r m e d i a t e between t h e c l a s s i c a l ADPs e n s i t i v e E1QP and t h e K + - s e n s i t i v e E z - P , a N a + - s e n s i t i v e E l - P , h a s been i d e n t i f i e d . By r e a c t i o n w i t h N a +

-

THREE CONSECUTIVE PHOSPHOINTERMEDIATESOF Na-ATPase

575

[ PHOSPHORYLATION I [ I DEPHOSPHORYLATION 1

7 CEPI, %;/At

Enzyme

(110 n M )

ATP32

(

Mg2+

(

25 p M ) [E,PI? 1 mM)

1

t=O addition

of

40

Na+ (tex. 150 mM 1

:Na'

ooc

I steady

state

I

K + (20mM) All curves are simulated

Fig. 1.

Experimental plan and results for [Na']

= 150 mM.

(apparent K O , ~= 450 mM) Na+-sensitive E -P is converted to El%P. This reaction may be responsibie for the known effect of Na+ on the Ei-P/E2-P ratio and may play a key role in Na+-Na+ exchange. 2. Both El-P and E2-P are rapidly dephosphorylated by.'K The ratelconstant for the disappyarance of El%P should then be kqA + k A (kA is about 10k-A) in the presence of K+ + ATP. We find a rate constant of about k-A, indicating that K+ reacts also with E1%P to form a slowly decaying phosphoenzyme which is not converted to El-P (or E2-P).

-IE;P ADP

I

1°C

Model 1

I. KLODOS eta/.

576

TABLE I. Steady-State Concentrations of the Three Phosphoenzymes and Values for the Two Rate Constants Showing Prominent Dependence on "a+] (cf. Model above).

20 50 150 300 450 600

12 13 19 32 41 51

45 45 43 40 38 34

43 42 38 28 21 15

0.1 0.13 0.41 0.94 1.3 1.95

0.025 0.04 0.07 0.12 0.165 0.23

REFERENCES Klodos, I., Nbrby, J. G . , and Plesner, I. W. (1981). The steadystate kinetic mechanism of ATP hydrolysis catalysed by membrane-bound (Na+ + K+)-ATPase from ox brain. 11. Kinetic characterization of phosphointermediates. Biochim. Biophys. A c t a 6 4 3 , 463-482.

CURRENT TOPICS IN MEMBRANES AND TRANSPORT, VOLUME 19

Aspects of the Presteady State Hydrolysis of ATP by Na,K-ATPase A . G.LO WEAND L. A. REEVE Deparimenr of Biochemistry University of Manchester Manchester. England

I.

INTRODUCTION

An e a r l y b u r s t i n t h e h y d r o l y s i s of low (micromolar) c o n c e n t r a t i o n s of ATP by t h e Na,K-ATPase has been r e p o r t e d w i t h enzymes p r e p a r e d from b o t h e l e c t r i c e e l ( F r o e h l i c h e t a l . , 1 9 7 6 ) and p i g b r a i n (Lowe and Smart, 1 9 7 8 ) . T h i s phenomenon i s most simply e x p l a i n e d by p o s t u l a t i n g t h a t d u r i n g a s i n g l e c y c l e of enzyme act i v i t y t h e release of i n o r g a n i c phosphate ( P i ) from ATP i s a r e l a t i v e l y f a s t p r o c e s s and t h a t t h i s i s followed by a s u b s t a n t i a l l y slower s t e p which i s r a t e - l i m i t i n g I f the cycle f o r t h e s t e a d y - s t a t e h y d r o l y s i s of ATP. of ATPase a c t i v i t y i s r e p r e s e n t e d a s f o l l o w s :

577

Copyright 0 1983 by Academic Press, Inc. All rights of reproductionin any form nscrvcd. ISBN 0-12-153319-0

A. G.LOWE AND L. A. REEVE

578

-* ATP

ADP

EIATP

I

E,

+ E2-Pi

“77-

El-P

Jt -

.. E2-P

this hypothesis demands that all steps from El up to and including formation of E2-Pi are fast, whereas one of the steps E20Pi + E and E2 -t E is much slower under conditions in whic2 the early h r s t of Pi release is observed. In keeping with this, the fluorescence studies of Karlish et al. (1976) have shown that the transition from E2 (which is stabilized by K+) to El (stabilized by Na+) is a slow process likely to be rate-limiting for ATPase activity at low trinucleotide concentrations. Post et al. (1972) also suggested that the rate-limiting step in hydrolysis involves the ATPpromoted dissociation of K+ from an occluded E2.K+ complex.

11.

DISCUSSION

Further investi ations have revealed antagonistic effects of Na+ and Kq consistent with the above interpretation. Pre-exposure of the pig brain Na,K-ATPase to 20 mM KC1 before mixing with [y-32P]ATP has been found to suppress the early burst of Pi release and this effect of K+ is decreased b simultaneous preexposure of the enzyme to both Ky and Na+. Reversal of K+ suppression by Na+ is concentration-dependent, 80 mM Na+ being sufficient to restore half of the early burst of Pi in the presence of 20 mM K+. These effects of Na+ and K+ on the early burst of Pi release are mirrored by their effects on the p-nitrophenylphosphatase (pNPPase) activity of the same enzyme since Na+ inhibits K+-dependent pNPPase activity with a ~ 0 . 5 of about 40 mM in the presence of 10 mM KC1. This result is satisfactorily accounted for if the E2 form of the enzyme is active as a pNPPase, whereas the El form has no pNPPase activity.

ASPECTS OF PRESTEADY STATE HYDROLYSIS OF ATP

579

While the rate of the transition E2 to El can satisfactorily account for the early burst of Pi release, the question of the enzymic precursor of Pi is less easily resolved by the results of pre-steady-state experiments. Using a simple model for ATP hydrolysis,

El

+

ATP

- %1 kl

k-l

EIATP

k- 2

ADP

:s pi

E2-P

E2

k- 3

k4

7

k- 4

Lowe and Smart (1978) derived integrated rate equations and showed that the early burst of Pi must be accompanied by a complementary overshoot in its precursor, and that the magnitude of this predicted overshoot was greater than that actually observed for the phosphoenzyme. This finding raises the possibility that ATP hydrolysis can occur by a branched or parallel pathway in which the immediate precursor of Pi can be either E2P or another unidentified intermediate. We have investigated this possibility by comparing the early time courses of ( i ) formation of phosphoenzyme, ( i i ) the release of [ 32P] Pi from [ y-32P]ATP after quenching with perchloric acid, and ( i i i ) the release of [32P]Pi from [y-32P]ATP after quenching with excess unlabeled ATP and allowing hydrolysis of any enzymic intermediates (including phosphoenzyme) before addition of perchloric acid. The results of these experiments have shown that the amount of [32P]Pi released after quenching with cold ATP exceeds the sum of the amounts of phosphoenzyme found and [32P]Pi released on quenching with perchloric acid 10-30 msec after mixing a NaI- and deoxycholateextracted pig brain Na,K-ATPase with [y-32P]ATP. This result is consistent with the existence of an enzymic precursor of [ 32P]Pi other than the phosphoenzyme, although the errors in experiments of this type make it uncertain whether the sum of the phosphoenzyme and the new precursor can account quantitatively for the size of the early burst of Pi release. The nature of this new putative intermediate is not known, but a relatively tightly bound enzyme-ATP complex (E'ATP) (cf. tightly bound ATP in the hydrolysis of ATP by myosin) and an acid-labile form of phosphoenzyme are two possibilities. An intermediate of this type does not necessarily demand a substantial adjustment in the interpretation of Na+ and K+ transport in terms of conformational changes in the Na,K-ATPase. E'ATP could be incorporated into conventional schemes as follows:

580

A. G. LOWE AND L. A. REEVE

Jl E2

ADP

rj

F -

E2*Pi

+E2-P

E2'Pi

I n such a scheme there w o u l d be t w o precursors of P i and N a + t r a n s p o r t m i g h t occur d u r i n g e i t h e r of t h e t r a n s i t i o n s , E l - P -t E2-P and E i A T P E;.Pi. -f

REFERENCES

F r o e h l i c h , J. P . , A l b e r s , R. W . , Koval, G. J . , Goebel, R . , and Berman, M. (1976). Evidence f o r a new i n t e r m e d i a t e s t a t e i n t h e mechanism of (Na' + K+) -adenosine t r i p h o s p h a t a s e . J. B i o l . C h e m . 2 5 1 , 2186-2188. K a r l i s h , S . J. D . , Glynn, I . M., and Yates, D. W. (1976). T r a n s i e n t k i n e t i c s of ( N a + + K+)-ATPase s t u d i e d w i t h a f l u o r e s c e n t probe. N a t u r e (London) 263, 251-253. Lowe , A. G . , and Smart, J. W. (1978). The pre-steady s t a t e hyd r o l y s i s of ATP by p o r c i n e b r a i n ( N a + + Kf)-dependent ATPase. B i o c h i r n . B i o p h y s . Acta 4 8 1 , 695-705. P o s t , R. L . , Hegyvary, C . , and K u m e , S. (1972). A c t i v a t i o n by adenosine t r i p h o s p h a t e i n t h e p h o s p h o r y l a t i o n k i n e t i c s of sodium and potassium i o n t r a n s p o r t ade nos i n e t r ipho spha t a s e . J. Biol. Chern. 2 4 7 , 6530-6540.

-

CURRENT TOPICS IN MEMBRANES AND TRANSPORT, VOLUME 19

Identity of the Na Activation Sites in ATPase with the K Activation Sites in p-Nitrophenylphosphatase L. A. PARODI, J. F. PNCUS, L. JOSEPHSON, D. J. SORCE, AND S. R. SIMON Department of Biochemistry State University of New York at Stony Brook Stony Brook, New York

I.

INTRODUCTION

Na,K-ATPase has t h e c a p a c i t y t o c a t a l y z e t h e hyd r o l y s i s of a number of s u b s t r a t e s i n a d d i t i o n t o ATP, i n c l u d i n g a series of s u b s t i t u t e d a r o m a t i c p h o s p h a t e s , such a s p - n i t r o p h e n y l phosphate. The h y d r o l y s i s of t h e s e o r g a n i c p h o s p h a t e s i s s t r o n g l y a c t i v a t e d by K i o n s , l e a d i n g many workers t o compare t h e s o - c a l l e d n i t r o p h e n y l p h o s p h a t a s e r e a c t i o n (NPPase) t o t h e Kdependent h y d r o l y s i s of t h e phosphoenzyme i n t e r m e d i a t e formed from r e a c t i o n w i t h ATP (Skou, 1 9 7 4 ; Glynn and K a r l i s h , 1975; Gache et a l . , 1 9 7 6 ) . However, t h e r e are some s t r i k i n g d i f f e r e n c e s i n t h e c h a r a c t e r i s t i c s of t h e s e two r e a c t i o n s (Gache e t a l . , 1 9 7 6 ; Swann and A l b e r s , 1978; Robinson, 1 9 6 9 ; Hobbs and D e Weer, 1 9 7 6 ; Koyal et a l . , 1 9 7 1 ; S h a f f e r e t a l . , 1 9 7 8 ) . W e have und e r t a k e n a d e t a i l e d c h a r a c t e r i z a t i o n of t h e a c t i v a t i o n of ATPase a c t i v i t y by Na and K i o n s and t h e NPPase ac-

581

Copyrigbt 0 1983 by Academic Rem,Inc. All rights of repmduction in any form nscrvcd. ISBN 0-12-1533198

582

L. A. PARODI eta/.

t i v i t y by K i o n s t o r e s o l v e some of t h e c o n f l i c t s remaining i n t h e i n t e r p r e t a t i o n of t h e s e two r e a c t i o n s .

11.

MATERIALS AND METHODS

O u r p r e p a r a t i o n of Na,K-ATPase i s d e r i v e d from e l e c t r i c u s , prepared e s s e n t i a l l y according t o t h e method of C a n t l e y e t al. ( 1 9 7 8 ) . Enzyme a s s a y s were E.

c a r r i e d o u t w i t h automated i n s t r u m e n t a t i o n a c c o r d i n g t o t h e methods of Josephson e t a l . ( 1 9 7 4 ) and P a r o d i et a l . ( 1 9 7 9 ) . Data were analyzed a c c o r d i n g t o t h e nonlinear l e a s t squares f i t t o the H i l l equation d e s c r i b e d by A t k i n s ( 1 9 7 3 ) . A c t i v a t i o n of ATPase a c t i v i t y by Na and K i o n s i s b e s t d e s c r i b e d by a model of c o o p e r a t i v e a c t i v a t i o n a t m u l t i p l e sites. Models proposing o b l i g a t e b i n d i n g t o multiple noninteracting sites p r i o r t o c a t a l y s i s g e n e r a t e n o n l i n e a r H i l l p l o t s and a r e i n c o n s i s t e n t w i t h o u r r e s u l t s (Lindenmayer e t a l . , 1 9 7 4 ) . A s judged by t h e l i n e a r i n c r e a s e s i n t h e a p p a r e n t a c t i v a t i o n cons t a n t f o r each c a t i o n w i t h i n c r e a s i n g c o n c e n t r a t i o n s of t h e o t h e r , N a i o n s can be d e s c r i b e d a s c o m p e t i t i v e i n h i b i t o r s of K a c t i v a t i o n , and K i o n s a s c o m p e t i t i v e i n h i b i t o r s of N a a c t i v a t i o n . Ca i o n s a l s o s t r o n g l y i n h i b i t t h e ATPase r e a c t i o n , combining w i t h ATP t o form an a p p a r e n t l y nonhydrolyzable complex, and s e l e c t i v e l y competing w i t h Na i o n s f o r t h e s i t e s i n v o l v e d i n a c t i v a t i o n of t h e enzyme p h o s p h o r y l a t i o n reaction. T h i s c o m p e t i t i v e i n h i b i t i o n of Na a c t i v a t i o n i s s t r o n g l y pH-dependent, i n c r e a s i n g w i t h i n c r e a s i n g pH. Ca i o n s appear t o have l i t t l e d i r e c t e f f e c t on K i o n a c t i v a t i o n of ATPase. Na a c t i v a t i o n of t h e ATPase r e a c t i o n i s a l so i n h i b i t e d by Mg i o n s , b u t u n l i k e t h e i n h i b i t i o n by K and Ca, t h e p a t t e r n o f i n h i b i t i o n c a n n o t be d e s c r i b e d a s simple c o m p e t i t i v e . The s l o p e of t h e H i l l p l o t s i s i n c r e a s e d , and t h e dependence of t h e a p p a r e n t a c t i v a t i o n c o n s t a n t f o r Na i o n s on Mg c o n c e n t r a t i o n i s hyperb o l i c . I n h i b i t i o n by Mg i o n s i n c r e a s e s w i t h i n c r e a s i n g pH. A c t i v a t i o n of ATPase a c t i v i t y by K i o n s i s comp l e t e l y i n s e n s i t i v e t o Mg i o n c o n c e n t r a t i o n . By d e t e r m i n i n g t h e c o n c e n t r a t i o n s of f r e e NPP and Mg i o n s w i t h a n eriochrome b l u e t i t r a t i o n method, w e have been a b l e t o d e s c r i b e a c c u r a t e l y t h e dependence of t h e NPPase r e a c t i o n on s u b s t r a t e c o n c e n t r a t i o n . The t r u e s u b s t r a t e s a r e f r e e Mg i o n s and f r e e NPP, which b i n d i n an o b l i g a t e o r d e r w i t h Michaelis-Menten k i n e t ics, t h e d i v a l e n t c a t i o n binding f i r s t . Determination

IDENTITY OF THE Na ACTIVATION SITES IN ATPase

583

of t h e M i c h a e l i s p a r a m e t e r s a t d i f f e r e n t pH v a l u e s rev e a l e d t h e p r e s e n c e of secondary Mg s i t e s t o which t h e metal i s bound more s t r o n g l y a t h i g h e r pH v a l u e s and which a r e i n h i b i t o r y t o t h e NPPase r e a c t i o n . E x t e n s i v e c h a r a c t e r i z a t i o n of t h e e f f e c t s of d i f f e r e n t K i o n conc e n t r a t i o n s on t h e b i n d i n g of Mg i o n s and NPP p e r m i t t e d s e p a r a t i o n of two e f f e c t s of K i o n s on t h e NPPase react i o n . The a f f i n i t y of t h e enzyme f o r NPP i s reduced by a f a c t o r of approximately 4 , w h i l e t h e c a t a l y t i c cons t a n t i s i n c r e a s e d o v e r 15-fold when t h e K i o n concenBoth t h e s e t r a t i o n i s i n c r e a s e d from 2 mM t o 1 0 0 mM. e f f e c t s d i s p l a y a marked s i g m o i d a l c o n c e n t r a t i o n dependence, b u t a c t i v a t i o n of c a t a l y s i s o c c u r s a t s i g n i f i c a n t l y lower c o n c e n t r a t i o n s t h a n does i n h i b i t i o n of subs t r a t e b i n d i n g , s u g g e s t i n g t h a t t h e t w o e f f e c t s may i n v o l v e d i s t i n c t s i t e s . A c t i v a t i o n of c a t a l y s i s by K ions i s b e s t f i t t o t h e H i l l equation. Ca ions i n h i b i t t h i s a c t i v a t i o n i n t h e same way a s t h e y i n h i b i t N a a c t i v a t i o n of A T P a s e , d i s p l a y i n g s i m p l e c o m p e t i t i v e i n h i b i t i o n k i n e t i c s w i t h i n c r e a s i n g a f f i n i t y a t i n c r e a s i n g pH. The i n h i b i t o r y p r o p e r t i e s of Mg i o n s r e f e r r e d t o above may be a s c r i b e d t o i n h i b i t i o n of K i o n a c t i v a t i o n of c a t a l y s i s . The dependence of t h i s i n h i b i t i o n on Mg i o n c o n c e n t r a t i o n was found t o be h y p e r b o l i c , w i t h i n c r e a s i n g a f f i n i t y a t i n c r e a s i n g pH, a s w a s found f o r Mg i n h i b i t i o n of Na a c t i v a t i o n i n t h e ATPase r e a c t i o n . Also, Mg i o n s i n c r e a s e d t h e s l o p e of t h e H i l l p l o t s f o r K i o n a c t i v a t i o n of NPPase t o v a l u e s a s g r e a t a s 2.3. These v a l u e s s u g g e s t t h a t no fewer t h a n t h r e e K i o n s are i n volved i n t h e NPPase r e a c t i o n . F u r t h e r e v i d e n c e t h a t a c t i v a t i o n of NPPase by K i o n s may i n v o l v e s i t e s w i t h p r o p e r t i e s v e r y l i k e t h o s e i n v o l v e d i n N a a c t i v a t i o n of ATPase comes from t h e e f f e c t s of t h e p e s t i c i d e Kepone on t h e two r e a c t i o n s . T h i s compound i n h i b i t s b i n d i n g o f ATP and NPP, and i n h i b i t s a c t i v a t i o n of t h e ATPase r e a c t i o n by b o t h Na and K i o n s a s w e l l a s K a c t i v a t i o n of t h e NPPase r e a c t i o n . By s o r t i n g o u t t h e s e m u l t i p l e i n h i b i t o r y e f f e c t s w e have been a b l e t o show t h a t Kepone i n h i b i t i o n of N a a c t i v a t i o n i n t h e A T P a s e r e a c t i o n i s f i t by a n o n l i n e a r s e t of p a r a m e t e r s which a l s o d e s c r i b e t h e i n h i b i t i o n of K a c t i v a t i o n of t h e NPPase r e a c t i o n . The i n h i b i t i o n of K i o n a c t i v a t i o n of t h e ATPase r e a c t i o n i s l i n e a r w i t h Kepone c o n c e n t r a t i o n and cannot be f i t t o t h e s e p a r a m e t e r s . By measuring K a c t i v a t i o n of NPPase a c t i v i t y i n t h e p r e s e n c e of N a + ATP, w e have been a b l e t o d e m o n s t r a t e a d r a m a t i c a l t e r a t i o n i n t h e c h a r a c t e r i s t i c s of t h i s a c t i v a t i o n p r o c e s s . The s l o p e of t h e H i l l p l o t f o r K a c t i v a t i o n d r o p s t o 1, w i t h a marked i n c r e a s e i n a p p a r e n t a f f i n i t y f o r K i o n s . T h i s a f f i n i t y i s now i n s e n s i t i v e

L. A. PARODI eta/.

584

t o Ca o r Mg i o n c o n c e n t r a t i o n s a t a l l pH v a l u e s , a s was observed f o r K i o n a c t i v a t i o n of ATPase a c t i v i t y . These r e s u l t s are i n e x c e l l e n t agreement w i t h t h e f i n d i n g s of B l o s t e i n ' s l a b o r a t o r y , which r e p o r t e d t h a t act i v a t i o n of N P P a s e by K i o n s o c c u r r e d on c y t o p l a s m i c f a c i n g s i t e s i n r e s e a l e d r e d c e l l g h o s t s when N a + ATP were a b s e n t , b u t on e x t r a c e l l u l a r f a c i n g sites when Na + ATP were p r e s e n t ( B l o s t e i n and Chu, 1 9 7 7 ; Drapeau and B l o s t e i n , 1 9 7 9 ) . Our d a t a s u g g e s t t h a t t h e inward f a c i n g s i t e s f o r K a c t i v a t i o n of NPPase have t h e same p r o p e r t i e s a s t h e inward f a c i n g s i t e s f o r N a a c t i v a t i o n of ATPase, and may w e l l b e t h e v e r y same sites.

111.

CONCLUSIONS

W e propose t h a t t h e evidence h e r e i s a l l c o n s i s t e n t w i t h a model of c o o p e r a t i v e c a t i o n a c t i v a t i o n l i k e t h a t of Monod et al. ( 1 9 6 5 ) . I n t h i s model, c a t i o n s a c t i v a t e by s t a b i l i z i n g a c a t a l y t i c a l l y a c t i v e conformation of t h e enzyme, b u t a r e n o t r e q u i r e d f o r t h a t conformat i o n . The model r e q u i r e s two c o n s t a n t s , one of which d e s c r i b e s t h e e q u i l i b r i u m between c a t a l y t i c a l l y a c t i v e and i n a c t i v e conformations i n t h e absence of c a t i o n s and t h e o t h e r of which d e s c r i b e s t h e r e l a t i v e a f f i n i t i e s of t h e two conformations f o r each of t h e c a t i o n s . I n t h e absence of c a t i o n s , t h e c a t a l y t i c a l l y i n a c t i v e conformat i o n predominates. An a c t i v a t i n g c a t i o n i s bound p r e f e r e n t i a l l y t o t h e c a t a l y t i c a l l y a c t i v e conformation. A c a t i o n which a c t s a s a s i m p l e c o m p e t i t i v e i n h i b i t o r i s bound a t t h e same s i t e s , b u t n e i t h e r p r e f e r e n t i a l l y t o t h e a c t i v e nor t o t h e i n a c t i v e conformation. Mg i o n s , which produce n o n l i n e a r i n h i b i t i o n w i t h i n c r e a s i n g coo p e r a t i v i t y of t h e r e s i d u a l a c t i v a t i o n , a r e bound p r e f e r e n t i a l l y t o t h e i n a c t i v e conformation. I n t h e p r e s e n c e of ATP, t h e enzyme a c q u i r e s t h e c a t a l y t i c a c t i v i t y of p h o s p h o r y l a t i o n , and t h e t h r e e inward f a c i n g s i t e s b i n d Na i o n s p r e f e r e n t i a l l y i n t h e c a t a l y t i c a l l y a c t i v e conf o r m a t i o n . I n t h e absence of ATP, t h e enzyme a c q u i r e s t h e c a t a l y t i c a c t i v i t y of a phosphatase, and t h e same t h r e e sites bind K ions p r e f e r e n t i a l l y i n t h e c a t a l y t i c a l l y a c t i v e conformation. W e have been a b l e t o f i t o u r ATPase and N P P a s e d a t a t o t h i s model, u s i n g a r a t i o of i n a c t i v e t o a c t i v e conformations i n t h e absence of cat i o n s of 5000. An e s t i m a t e d s e l e c t i v i t y of t h e i n a c t i v e conformation o v e r t h e a c t i v e conformation of 1 0 - f o l d f o r Mg i o n s reproduces t h e h y p e r b o l i c p a t t e r n s of i n h i b i t i o n of N a a c t i v a t i o n of ATPase and K a c t i v a t i o n of NPPase.

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IDENTITY OF THE Na ACTIVATION SITES IN ATPase

The application of these parameters to the model also generates activation curves of increasing cooperativity at increasing Mg ion concentrations. Further studies are needed to provide direct experimental evidence to correlate enzyme conformational equilibria with cooperativity of cation activation of the ATPase and NPPase reactions.

REFERENCES

A t k i n s , G. L. ( 1 9 7 3 ) . E u r . J. B i o c h e m . 3 3 , 175. B l o s t e i n , R . , and Chu, L. ( 1 9 7 7 ) . J. B i o l . Chem. 2 5 2 , 3035. C a n t l e y , L. C . , Gelles, J . , and J o s e p h s o n , L. (1978). B i o c h e m i s t r y 1 7 , 418. Drapeau, P . , and B l o s t e i n , R. ( 1 9 7 9 ) . F e d . P r o c . , Fed. Am. SOC. E x p . B i o l 3 8 , 1041. Gache, C . , R o s s i , B., and Lazdunski, M. ( 1 9 7 6 ) . E u r . J. B i o c h e m . 6 5 , 293. Glynn, I. M., and K a r l i s h , S. J. D. ( 1 9 7 5 ) . A n n u . Rev. P h y s i o l . 37, 13. Hobbs, A. S . , and D e Weer, P. ( 1 9 7 6 ) . A r c h . B i o c h e m . B i o p h y s . 1 7 3 , 386. Josephson, L., Mangold, J. H . , and Simon, S. R. ( 1 9 7 4 ) . A n a l . B i o c h e m . 6 0 , 312. Koyal, D., Rao, S. N . , and A s k a r i , A. (1971) Biochim. Biophys. A c t a 2 2 5 , 11. Lindenmayer, G. E . , Schwartz, A . , and Thompson, H. K. ( 1 9 7 4 ) . J . P h y s i o l . (London) 2 3 6 , 1. Monod, J . , Wyman, J . , and Changeux, J. P. ( 1 9 6 5 ) . J. Mol. B i o l . 1 2 , 88. P a r o d i , L. A . , P i n c u s , J. F . , Josephson, L . , and Simon, S. R. ( 1 9 7 9 ) . Fed. P r o c . , Fed. Am. SOC. E x p . B i o l . 3 8 , 242. Robinson, J. D. ( 1 9 6 9 ) . B i o c h e m i s t r y 8 , 3348. S h a f f e r , E . , A z a r i , J . , and D a h m s , S . ( 1 9 7 8 ) . J. B i o l . Chem. 2 5 3 , 5696. Skou, J. C. (1974). B i o c h i m . B i o p h y s . A c t a 339, 234. Swann, A . C . , and A l b e r s , R. W. ( 1 9 7 8 ) . B i o c h i m . B i o p h y s . A c t a 5 2 3 , 215.

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CURRENT TOPICS IN MEMBRANES AND TRANSPORT, VOLUME 19

On the Existence of Two Distinct Hydrolysis Cycles for Na,K-ATPase with Only One Active Substrate Site IGOR W. PLESNER Depanmeni of Physical Chemistry Aarhus Univer~siiy Kenisk Institute Aarhus, Denmark

I.

INTRODUCTION

R e c e n t l y a new minimal model f o r t h e h y d r o l y t i c a c t i o n of N a , K - A T P a s e h a s been proposed ( P l e s n e r et a l . , 1 9 8 1 ) . The r i g h t - h a n d c y c l e i n t h e model ( F i g . 1) r e p r e s e n t s t h e a c t i o n o f t h e Na-enzyme ( i . e . ? a t micromolar s u b s t r a t e and no K + ) , whereas t h e l e f t - h a n d c y c l e r e p r e s e n t s t h e e s s e n t i a l f e a t u r e of t h e Na,K-enzyme (i.e.? a t millimolar substrate plus K+). The p r e s e n t c o n t r i b u t i o n p r e s e n t s a s h o r t a c c o u n t of a p r o c e d u r e f o r e x t r a c t i n g v a l u e s of a l l t h e r a t e c o n s t a n t s on t h e b a s i s of t h e k i n e t i c p a r a m e t e r s o b t a i n e d from s t e a d y s t a t e e x p e r i m e n t s . These a r e n e c e s s a r y f o r a s t u d y o f t h e e n e r g e t i c s of t h e system, and a s t u d y of t h e i r f u n c t i o n a l dependence on N a + and K+ may y i e l d informat i o n on t h e p r e c i s e mode of i n t e r a c t i o n of t h e s e l i g a n d s w i t h enzyme.

587

Copynght 0 1983 by Academic F'ress, Inc. All rights of reproduction in any form reserved. ISBN 0-12-1533190

+

El MA

F i g . 1 . A m i n i m a l m o d e l f o r the a c t i o n of Na,K-ATPase, c o n t a i n i n g t w o d i s t i n c t h y d s : E l + ElMA + EIP + E2P -t E l ( t h e Na-enzyme) and E2K + E2KMA + E x -t E2K ( t h e Na,K-

E2KMAM

TWO HYDROLYSIS CYCLES FOR Na,K-ATPase

11.

589

RESULTS AND DISCUSSION

I t may b e shown t h a t if it i s assumed t h a t ( a ) t h e enzyme-substrate complex EMA (E = E l o r E2K) i s the same w h e t h e r o b t a i n e d d i r e c t l y ( E + Em) or by t h e r o u t e E + EA + EMA, and ( b ) t h a t t h e r a t e c o n s t a n t f o r a d d i t i o n of Mg2+ t o ATP i s i n d e p e n d e n t of w h e t h e r o r n o t ATP i s bound t o a n enzyme u n i t , t h e n a l l t h e r a t e cons t a n t s i n d i c a t e d on F i g . 1 may be c a l c u l a t e d (when a l s o e x p e r i m e n t s on K+ i n h i b i t i o n o f t h e Na-enzyme a r e t a k e n i n t o account). The v a l u e o f t h e r a t e c o n s t a n t k 2 f o r t h e Na-enzyme (which i s found w i t h o u t r e f e r e n c e t o o t h e r p o s s i b l e c y c l e s ) shows t h a t i t i s t o o s m a l l t o y i e l d a h y d r o l y s i s r a t e l a r g e enough by i t s e l f t o a c c o u n t f o r t h e r a t e of t h e Na,K-enzyme a t s a t u r a t i n g s u b s t r a t e c o n c e n t r a t i o n s . S i n c e t h e s t e p E 1 P -+ E 2P a l s o h a s been shown ( P l e s n e r e t a l . , 1981) t o b e i n c a p a b l e o f p r o v i d i n g s u f f i c i e n t n e t f l u x , t h i s c o r r o b o r a t e s t h a t t h e h y d r o l y s i s w i t h K+ and m i l l i m o l a r substrate c o n d i t i o n s must f o l l o w a n ent i r e l y d i f f e r e n t k i n e t i c cycle. I n a d d i t i o n , it i s found t h a t t h e i n t e r m e d i a t e E2KP must be p r e s e n t a t s t e a d y state a t nonzero c o n c e n t r a t i o n b u t i s a c i d - l a b i l e (and t h e r e f o r e n o t d e t e c t e d by t h e u s u a l p r o c e d u r e f o r d e t e r m i n i n g p h o s p h o r y l a t e d enzyme). The model shown i n F i g . 1 i s c a p a b l e of e x h i b i t i n g t h e e x p e r i m e n t a l l y found n o n l i n e a r d o u b l e - r e c i p r o c a l p l o t (when a s u f f i c i e n t l y l a r g e r a n g e of s u b s t r a t e conc e n t r a t i o n s i s c o n s i d e r e d ) even though a n enzyme u n i t a t any t i m e h a s o n l y o n e a c t i v e s u b s t r a t e s i t e . Such a p l o t , c a l c u l a t e d from t h e model i n F i g . 1 w i t h t h e r a t e c o n s t a n t s found as d e s c r i b e d , i s shown i n F i g . 2 . It may be shown t h a t t h e n e c e s s a r y c o n d i t i o n f o r t h i s f e a t u r e i s t h a t t h e two h y d r o l y s i s c y c l e s ( E l + ElMA + E1P + E2P + E l and E2K + E2KMA + Ex E 2 K ) a r e doubly c o n n e c t e d . The c u r v e i n F i g . 2 r e f l e c t s , g o i n g from r i g h t t o l e f t , a gradual t r a n s i t i o n , as t h e s u b s t r a t e c o n c e n t r a t i o n i s i n c r e a s e d , from " a l l f l u x i n t h e N a c y c l e " t o " a l l f l u x i n t h e Na,K-cycle." A more d e t a i l e d a c c o u n t o f t h i s work, w i t h t a b l e s o f r a t e c o n s t a n t s , h a s a p p e a r e d e l s e w h e r e ( P l e s n e r and Plesner, 1981). -f

590

IGOR W. PLESNER

'I-

- enzyme

(Na

15

54-

3-

CK'I

= 20 mM

2-

= 15 rnM 'hMgATP1 I

20

I

I

40

I

I

60

I

I

80

I

(mM-1)

-i '

100 200 300 400 500

-1 -1 Fig. 2 . Double-reciprocal p l o t (v v e r s u s [MgATP] ) comp u t e d u s i n g the m o d e l i n F i g . 1 a n d the r a t e c o n s t a n t s d e r i v e d b y the p r o c e d u r e d e s c r i b e d (see Plesner and P l e s n e r ( 1 9 8 1 ) for d e tails).

REFERENCES

P l e s n e r , 'I. W . , a n d P l e s n e r , L. (1981). B i o c h i m . B i o p h y s . A c t a 648, 231-246. P l e s n e r , I . W. , Plesner, L., N g k b y , J. G . , a n d Klodos , I. (1981) B i o c h i m . B i o p h y s . A c t a 643, 483-494.

.

CURRENT TOPICSm MEMBRANES AND TRANSPORT, VOLUME 19

Kinetic Analysis of the Effects of Na+ and K on Na,K-ATPase LISELOnE PLESNER Institute Biophysical Aarhus University Aarhus. Denmark

IGOR W. PLESNER Department of Physical Chemistry Aarhus University Kenisk Insritute Aarhus, Denmark

I.

INTRODUCTION

R e c e n t l y a k i n e t i c mechanism of Na,K-ATPase, exh i b i t i n g two d i s t i n c t h y d r o l y t i c c y c l e s , was proposed ( P l e s n e r et al., 1 9 8 1 ) . The model, shown i n F i q . 1, d i d n o t t a k e a c c o u n t of t h e a c t i o n of N a + and K , and t h e purpose o f o u r p r e s e n t work i s an e x t e n s i o n of t h e model t o i n c l u d e a l s o t h e e f f e c t s of t h e s e i o n s . The p r e s e n t c o n t r i b u t i o n p r e s e n t s r e s u l t s from a s t u d y of t h e e f f e c t of N a + and K+ on t h e s l o p e s and o r d i n a t e i n t e r c e p t s of Lineweaver-Burk p l o t s of s t e a d y s t a t e k i n e t i c d a t a f o r t h e Na-enzyme ( i . e . , a t micromolar s u b s t r a t e c o n c e n t r a t i o n ) and f o r t h e Na,K-enzyme ( i . e . , a t m i l l i m o l a r s u b s t r a t e ) . The c o n d i t i o n s of t h e experiments and t h e a s s a y p r o c e d u r e s were as d e s c r i b e d e a r l i e r ( P l e s n e r and P l e s n e r , 1 9 8 1 ) . When it i s r e c a l l e d t h a t a v a r i a t i o n w i t h l i g a n d c o n c e n t r a t i o n of t h e s l o p e and of t h e o r d i n a t e i n t e r c e p t of t h e primary ( d o u b l e - r e c i p r o c a l ) p l o t i n d i c a t e s 59 1

Copyright 0 1983 by Academic &ss. Inc. All rights of reproduction in any f o n reserved. ISBN 0-12-153319-0

592

LISELOTTE PLESNER AND IGOR W. PLESNER

F i g . 1 . The p r o p o s e d m i n i m a l k i n e t i c model f o r t h e h y d r o l y t i c a c t i o n o f Na ,K-ATPase.

1

slope

min

.

i03

12.5 -

10 -

Na - enzyme

7.5 -

5-

-

25 -

I

100

200

300

rnM Na'

100

200

300

mM Na'

F i g . 2 . Na-enzyme: O r d i n a t e i n t e r c e p t s ( a ) and s l o p e s ( b ) f r o m Lineweaver-Burk p l o t s o f s t e a d y - s t a t e d a t a ( s u b s t r a t e i s Mg-ATP) a s f u n c t i o n s o f the Na' concentration a t d i f f e r e n t con[Mgfree ] = 7 mM, p n = 7 . 4 , 37OC. centrations o f K'.

ANALYSIS OF EFFECTS OF Na+and K+on Na,K-ATPase

593

Na,K - enzyme

I

100

200

300

mM Na'

300 mM Na'

50

100

mM K'

Fig. 3. Na,K-enzyme: Ordinate intercepts from LineweaverBurk plots (a) as a function of ' a N at different K+ concentrations and (b) as functions of K+ at different N ' a concentrations.

l i g a n d i n f l u e n c e on enzyme i n t e r m e d i a t e s p r i o r t o and subsequent t o s u b s t r a t e b i n d i n g , r e s p e c t i v e l y , a q u a l i t a t i v e i n t e r p r e t a t i o n of t h e r e s u l t s i s as f o l l o w s .

11.

THE Na-ENZYME

A d d i t i o n of Na+ i n c r e a s e s t h e a p p a r e n t Vmax wherea s K + d e c r e a s e s i t ( F i g . 2 a ) . The l a t t e r e f f e c t i s c o u n t e r a c t e d by N a + a t h i g h c o n c e n t r a t i o n . Na+ t h e n a c c e l e r a t e s dephosphor l a t i o n ( c f . Klodos e t al., t h i s volume) , whereas t h e K' e f f e c t i s due t o t h e accumulat i o n of E2K which is n o t c a t a l y t i c a l l y a c t i v e a t micromolar s u b s t r a t e c o n c e n t r a t i o n . Hence Na+ and K + comp e t e f o r t h e E 2P form (Fig. l ) - - K + increases the slope ( F i g . 2 b ) , b u t t h i s e f f e c t i s c o u n t e r a c t e d by Na+. T h i s i n d i c a t e s t h a t Na+ and K+ compete € o r t h e same s i t e on t h e s u b s t r a t e - f r e e enzyme. Thus, t h e i n t e r a c -

LISELOTTE PLESNER AND IGOR W. PLESNER

594

t i o n of Na+ and K+ i n t h e two d i f f e r e n t p a r t s of t h e mechanism i s s t r i c t l y c o m p e t i t i v e .

111.

THE Na,K-ENZYME

I t i s s e e n ( F i g . 3 a , b ) t h a t when one of t h e c a t i o n s i s p r e s e n t a t l o w c o n c e n t r a t i o n , vap$ i s d e c r e a s e d a t i n c r e a s i n g c o n c e n t r a t i o n s of t h e o a e r i o n . Conversely, when one c a t i o n i s p r e s e n t a t h i g h c o n c e n t r a t i o n , addit i o n of t h e o t h e r i o n c a u s e s an i n c r e a s e i n Both i o n s , t h e n , a r e i n v o l v e d i n t h e sequence E2MA + Ex -t E2K. A s i m p l e e q u i l i b r i u m argument shows t h a t t h e observed q u a l i t a t i v e f e a t u r e s are o b t a i n e d onl y i f Na+ and K+ are s i m u l t a n e o u s l y p r e s e n t on a t l e a s t one of t h e a c t i v e s p e c i e s .

v$gg.

REFERENCES

P l e s n e r , I. W . , P l e s n e r , L., Ndrby, J. G., and Klodos, I. (1981). B i o c h i m . Biophys. Acta 643, 483-494. P l e s n e r , L., and P l e s n e r , I. W. (1981). B i o c h i m . B i o p h y s . A c t a 643, 449-462.

CURRENT TOPICS IN MEMBRANES AND TRANSFORT, VOLUME 19

Divalent Cations and Conformational States of Na,K-ATPase JOSEPH D. ROBINSON Deparrment of Pharmacology State Universiry of New York Upstate Medical Center Syracuse, New York

I.

INTRODUCTION

The r e a c t i o n s e q u e n c e o f t h e Na,K-ATPase i n v o l v e s s e q u e n t i a l changes i n conformational states: E l -+ El-P -f E2-P -+ E2 + E l ( f o r r e v i e w , see Robinson a n d F l a s h n e r , 1 9 7 9 ) . D i v a l e n t c a t i o n s bound t o ATP form t h e s u b s t r a t e complex, y e t f r e e Mg2+ can a l s o mod i f y e n z y m a t i c p r o c e s s e s d i r e c t l y by f a v o r i n g t h e E2/E2-P c o n f o r m a t i o n a l s t a t e s (Robinson, 1 9 8 1 ) . Such e f f e c t s , m e d i a t e d t h r o u g h s i t e s w i t h a ~ 0 . 5 of 0 . 1 - 1 mM (Robinson a n d F l a s h n e r , 1 9 7 9 ) , a r e e x e m p l i f i e d by ( i ) t h e Mg r e q u i r e m e n t f o r K-phosphatase act i v i t y ( P i t t s and A s k a r i , 1971) and v a n a d a t e b i n d i n g ( C a n t l e y e t al. , 1978) , and t h e Mg-induced " K - p a t t e r n " of t r y p t i c d i g e s t i o n ( T a b l e I ) , a l l m a n i f e s t a t i o n s of t h e E2 c o n f o r m a t i o n a l f a m i l y ; and ( i i ) t h e Mg2+ i n h i b i t i o n o f ADP/ATP exchange a c t i v i t y (Fahn et al., 19661, c a t a l y z e d by t h e E l f a m i l y .

595

Copyright 0 1983 by Academic Press. Inc. All rightsof reproduction inany form reserved. ISBN 0-12-153319-0

JOSEPH D. ROBINSON

596

TABLE I.

E f f e c t s o f Cations on T r y p t i c Digestiona

Additions

Residual K-phosphatase a c t i v i t y (percentage o f c o n t r o l a c t i v i t y without t r y p s i n ) ~

10 mM NaCl 10 mM NaCl 10 mM NaCl 10 mM KC1

+ +

3 mM MgC12 3 mM MnCl2

26 42 51 54

a

The enzyme preparation from dog kidney medulla was incubated with trypsin for 10 min at 37OC in 30 mM histidine-HCl/Tris (pH 7.8), 100 mM choline chloride, and the concentrations of cations indicated. Digestion was halted by adding a molar excess of trypsin inhibitor, and residual K-phosphatase activity then measured 3 mM nitroin media containing 30 mM histidine.HCl/Tris (pH 7.8), phenyl phosphate, 3 mM MgCl2, and 10 mM KCI.

Mn2+ i s an even b e t t e r s e l e c t o r of E 2 s t a t e s t h a n i s Mg2+, a s e x e m p l i f i e d by ( i ) t h e lower Kd f o r vanad a t e b i n d i n g (Robinson, 1 9 8 1 ) and b e t t e r s e l e c t i o n of t h e "K-form" of t r y p t i c d i g e s t i o n (Table I ) ; and ( i i ) t h e g r e a t e r i n h i b i t i o n of ADP/ATP exchange ( J . D. Robinson, un u b l i s h e d o b s e r v a t i o n s ) . These d a t a i n d i c a t e t h a t Mg5+ s h i f t s t h e e q u i l i b r i u m between E l and E 2 s t a t e s by b i n d i n g more t i g h t l y t o E 2 , and t h a t Mn2+ s h i f t s t h e e q u i l i b r i u m s t i l l f u r t h e r by a more e x c l u s i v e [ I n t h e s e experiments m i l l i m o l a r MnC12 binding t o E2. corresponds t o micromolar f r e e Mn2+ due t o c h e l a t i o n by h i s t i d i n e (Robinson, 1981) . ]

11.

DISCUSSION

For t h e Na,K-ATPase r e a c t i o n d i v a l e n t c a t i o n s t h u s can i n h i b i t by h i n d e r i n g E 2 t o E l c o n v e r s i o n s r e q u i r e d i n t h e c a t a l y t i c c y c l e . S i n c e Mn2+ i s a more p o t e n t s e l e c t o r of E 2 , it t h e n s h o u l d be a p o o r e r c o f a c t o r i n t h e r e a c t i o n , as it i s ( F i g . 1 ) . On t h e o t h e r hand, t h e K-phosphatase r e a c t i o n app a r e n t l y i s c a t a l y z e d by E 2 states w i t h o u t any r e q u i r e ment f o r E 2 t o E l c o n v e r s i o n s . Mn2+, as t h e b e t t e r s e l e c t o r of E 2 s t a t e s , t h e n should be t h e b e t t e r cof a c t o r . I n s t e a d , Mn2+ is less good t h a n Mg2+ ( F i g . 1 ) .

597

DIVALENT CATIONS AND CONFORMATIONAL STATES

1.2r

1

K-Phosphatase

/@

‘,2r

Na,K-ATPase

1.0’

0.8.

/

0.61

0 L, 0.I

I

I

I .o

I

,

10

[Mg CI,]

0 LI

I

0.1

or [MnC12]

I

I .o

I

1

10

(mM)

F i g . 1 . E f f e c t s of d i v a l e n t c a t i o n s on e n z y m a t i c a c t i v i t y . T h e e n z y m e from d o g k i d n e y m e d u l l a was i n c u b a t e d a t 37OC w i t h , ( i ) f o r the K - p h o s p h a t a s e a s s a y , 30 mM h i s t i d i n e . H C l / T r i s (pH 7 . 8 ) , 3 mM n i t r o p h e n y l p h o s p h a t e , 10 mM K C I , and the c o n c e n t r a t i o n s of M g C l 2 ( 0 ) or MnCl2 ( @ ) i n d i c a t e d ; a n d ( i i ) f o r the Na,K-ATPase a s s a y , the same r e a c t a n t s e x c e p t t h a t ATP r e p l a c e d n i t r o p h e n y l p h o s p h a t e and 90 mM NaCl w a s a d d e d .

Two o b s e r v a t i o n s b e a r on why Mn2+ i s t h e p o o r e r cofactor: ( i ) w i t h Mn2+ t h e K d f o r v a n a d a t e b i n d i n g i s 8 - f o l d l o w e r t h a n w i t h Mg2+ ( R o b i n s o n , 1 9 8 1 ) ; and ( i i ) t h e ~i €or p h o s p h a t e a s a c o m p e t i t i v e i n h i b i t o r of t h e K-phosphatase r e a c t i o n i s 2 - f o l d l o w e r , 0 . 7 m~ w i t h MnC12 v e r s u s 1 . 3 mM w i t h MgC12. S i n c e v a n a d a t e i s a h i g h l y p o t e n t a l t h o u g h n o n c o v a l e n t i n h i b i t o r of b o t h A T P a s e and p h o s p h a t a s e r e a c t i o n s , p r o b a b l y a s a t r a n s i t i o n - s t a t e a n a l o g of P i a s a l e a v i n g group ( C a n t l e y e t a l . , 1 9 7 8 ) , t h e s e o b s e r v a t i o n s t h u s sugg e s t t h a t d i v a l e n t ca i o n s , p a r t i c u l a r l y Mn2+, i n h i b i t by s t a b i l i z i n g an Ez:$ complex. C o r r e s p o n d i n g l y , i n t h e Na,K-ATPase r e a c t i o n , where d i v a l e n t c a t i o n i s bound a f t e r P i d i s s o c i a t e s (Fukushima and P o s t , 1978) , a r o l e of ATP a t t h e “ l o w -

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a f f i n i t y s u b s t r a t e s i t e " may be t o f a c i l i t a t e conver.M .M sion to El through t h e r e a c t i o n : E2 + ATP -+ E1.iTp.

ACKNOWLEDGMENT This work was supported by U.S.P.H.S. NS-05430.

research grant

REFERENCES Cantley, L. C., Jr., Cantley, L. G . , and Josephson, L. (1978). A characterization of vanadate interactions with the (Na,K)-ATPase. J. B i o l . Chem. 2 5 3 , 7361-7368. Fahn, S., Koval, G. J., and Albers, R. W. (1966). Sodiumpotassium-activated adenosine triphosphatase of electrophorus electric organ. J . B i o l . C h e m . 2 4 1 , 1882-1889. Fukushima, Y., and Post, R. L. (1978). Binding of divalent cation to phosphoenzyme of sodium- and potassium-transport adenosine triphosphatase. J. B i o l . C h e m . 253, 6853-6862. Pitts, B. J. R., and Askari, A. (1971). A fluorimetric assay method for the K+-phosphatase associated with the (Na+ + K ) activated ATPase. B i o c h i m . B i o p h y s . A c t a 227, 453-459. Robinson, J. D. (1981). Substituting manganese for magnesium alters certain reaction properties of the (Na+ + K+)-ATPase. B i o c h i m . B i o p h y s . A c t a 6 4 2 , 405-417. Robinson, J. D., and Flashner, M. S. (1979). The (Na+ + K+)activated ATPase: Enzymatic and transport properties. B i o c h i m . B i o p h y s . A c t a 5 4 9 , 145-176. +

Part VII

Ion Translocation and Reaction Mechanism

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CURRENT TOPICS IN MEMBRANES A N D TRANSPORT, VOLUME 19

Na,K-ATPase: Reaction Mechanisms and Ion Translocating Steps PAUL DE WEER Depanment of Physiology and Biophysics Washingion University School of Medicine St. Louis, Missouri

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INTRODUCTION

The purpose of t h i s c h a p t e r i s t o summarize b r i e f l y from t h e l i t e r a t u r e some selected f a c t s t h a t may have b e a r i n g on t h e q u e s t i o n : To what e x t e n t , and i n how much d e t a i l , can t h e " b i o p h y s i c a l " o b s e r v a t i o n s on t h e o p e r a t i o n of t h e sodium pump ( i . e . , i o n t r a n s p o r t and c u r r e n t measurements) be r e c o n c i l e d w i t h what i s known about t h e "biochemical" r e a c t i o n mechani s m of t h e Na,K-ATPase? T h i s s e l e c t i o n i s by n e c e s s i t y t o p i c a l s i n c e it i s mainly i n s p i r e d by t h e a r t i c l e s p r e s e n t e d d u r i n g t h i s meeting a t t h e s e s s i o n devoted t o t h e r e l a t i o n s h i p between enzyme mechanisms and i o n t r a n s l o c a t i o n . F l u x e s i n r e c o n s t i t u t e d liposomes w i l l n o t be reviewed a t l e n g t h h e r e s i n c e o t h e r s w i l l d i s c u s s them e l s e w h e r e i n t h i s volume.

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DO CARDIOTONIC STEROIDS I N H I B I T Na,K-ATPase AND PUMP-MEDIATED FLUXES TO THE SAME EXTENT?

T h i s q u e s t i o n may appear s u r p r i s i n g , s i n c e t h e consensus i s t h a t c a r d i o t o n i c s t e r o i d s block pump f l u x e s by a r r e s t i n g t h e ATPase machinery. Y e t a c u r i ous o b s e r v a t i o n made on s q u i d g i a n t axon ( B r i n l e y and M u l l i n s , 1 9 6 8 ; Beaug6 and M u l l i n s , 1 9 7 6 ) had s u g g e s t e d t h a t s t r o p h a n t h i d i n , i n a d d i t i o n t o a r r e s t i n g t h e "norm a l " o p e r a t i o n o f t h e A T P a s e and i t s concomitant f l u x e s , could induce a n o v e l mode of i o n exchange through t h e pump. Consequently, k i n e t i c and s t o i c h i o metric c o n c l u s i o n s based on f l u x d i f f e r e n c e measurements might be i n e r r o r . R e c e n t e x p e r i m e n t s (Beaug6 and DiPolo, 1981a) show, however, t h a t t h e r e e x i s t s a strophanthidin-resistant, ATP-dependent sodium e f f l u x i n s q u i d axon which p r o b a b l y a c c o u n t s f o r t h e e a r l i e r observations.

111.

OVERALL PUMP STOICHIOMETRY

K i n e t i c o b s e r v a t i o n s a l o n e are n o t s u f f i c i e n t t o e s t a b l i s h s t o i c h i o m e t r i e s . I t may be g r a t i f y i n g t o find H i l l coefficients t h a t f i t one's expectations but, i f t h e y do n o t , c a u t i o n i s a d v i s e d when drawing conclus i o n s concerning t h e pump's s t o i c h i o m e t r y . A c t u a l f l u x and/or e l e c t r i c a l measurements m u s t be c a r r i e d o u t simultaneously w i t h ATP h y d r o l y s i s measurements. The commonly a c c e p t e d Na:K:ATP s t o i c h i o m e t r y of 3:2:1 f o r t h e "normal" mode of o p e r a t i o n of t h e sodium pump ( G a r rahan and Glynn, 1967d) remains unchallenged, and has r e c e i v e d s u p p o r t from experiments c a r r i e d o u t on p u r i f i e d Na,K-ATPase r e c o n s t i t u t e d i n t o liposomes (Goldin, 1977). The 3:2 s t o i c h i o m e t r y f o r Na:K exchange h a s rec e i v e d some s u p p o r t from i s o t o p e f l u x measurements i n o t h e r c e l l s , b u t it must be r e a l i z e d t h a t t h e operat i o n a l d e f i n i t i o n of "pump-mediated K+ f l u x " i s o f t e n a d i f f i c u l t m a t t e r f o r t h e f o l l o w i n g reason. I n every i n s t a n c e t e s t e d , i n c l u d i n g e r y t h r o c y t e s (Hoffman et al., 1 9 7 9 ) , t h e N a : K exchange pump has proved e l e c t r o g e n i c , and a r r e s t i n g t h e pump w i l l l e a d t o c e l l d e p o l a r i z a t i o n ; t h i s , i n t u r n , may modify p a s s i v e K+ i n f l u x i n a way determined by t h e r e c t i f y i n g p r o p e r t i e s of t h e c e l l memb r a n e ' s potassium conductance. A p e r h a p s more promising approach t o e s t a b l i s h i n g t h e Na:K s t o i c h i o m e t r y i s t h e

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s i m u l t a n e o u s measurement of e l e c t r o g e n i c pump c u r r e n t sodium e f f l u x (Cooke et a l . , 1 9 7 4 ) . Pump c u r r e n t can be c a l c u l a t e d i n d i r e c t l y from membrane pot e n t i a l changes and membrane conductance, o r measured d i r e c t l y a s t h e e x t r a c u r r e n t r e q u i r e d t o clamp memb r a n e p o t e n t i a l a t a g i v e n ( u s u a l l y r e s t i n g ) v a l u e when t h e pump i s e i t h e r s t i m u l a t e d o r i n h i b i t e d . Pump s t i m u l a t i o n h a s u s u a l l y been achieved i n t h e p a s t by p r e s s u r e - o r i o n t o p h o r e t i c i n j e c t i o n of sodium s a l t s b u t , u n l e s s t h e anion i s very c a r e f u l l y chosen, t h e r e i s a danger of a r t i f a c t s a r i s i n g from d i f f u s i o n potent i a l s c r e a t e d by t h e i n j e c t e d a n i o n ( f o r a c r i t i q u e , see D e Weer, 1 9 7 5 ) . N o such problem e x i s t s i f t h e pump c u r r e n t i s determined a s t h e clamp c u r r e n t r e q u i r e d t o compensate f o r sudden blockage of t h e pump ( D e Weer, 1 9 7 4 1 , provided t h e b l o c k i n g a g e n t ( e . g . , o u a b a i n ) h a s no e f f e c t on membrane conductance. The work of Nelson and Lederer ( t h i s volume) on t h e sodium pump of i n t e r n a l l y p e r f u s e d g i a n t b a r n a c l e muscle f i b e r s i l l u s t r a t e s t h i s procedure. A f e w y e a r s ago t h e n o t i o n a r o s e t h a t t h e pump's N a : K s t o i c h i o m e t r y might be v a r i a b l e depending on memb r a n e p o t e n t i a l and i o n i c c o n d i t i o n s . C o n s i d e r i n g t h e t e c h n i c a l d i f f i c u l t y of measuring s t o i c h i o m e t r i e s acc u r a t e l y , t h e i s s u e s h o u l d be c o n s i d e r e d u n r e s o l v e d . Even w i t h v e r y a c c u r a t e measurements of t r a c e r N a f l u x and pump c u r r e n t , it may b e v e r y d i f f i c u l t t o d i s t i n g u i s h between genuine " v a r i a b l e s t o i c h i o m e t r y " and t h e s i m u l t a n e o u s o c c u r r e n c e of v a r i a b l e p r o p o r t i o n s o f , s a y , e l e c t r o g e n i c N a : K exchange and (presumably e l e c t r o n e u t r a l ) Na:Na exchange. Another l i n e of r e s e a r c h w i t h b e a r i n g on t h e probl e m of i o n t r a n s l o c a t i o n s t o i c h i o m e t r y i s d i r e c t d e t e r m i n a t i o n of t h e number of i o n s bound t o t h e enzyme p e r c a t a l y t i c u n i t . Most s t u d i e s so f a r have examined e q u i l i b r i u m b i n d i n g s t o i c h i o m e t r i e s ( s e e H a s t i n g s and Skou, 1 9 8 0 , f o r a r e c e n t e x a m p l e ) , b u t bound i o n s w i l l n o t n e c e s s a r i l y be t r a n s l o c a t e d d u r i n g pump t u r n o v e r . The a t t e m p t s of Glynn and co-workers (Beauge and Glynn, 1979a; Glynn and R i c h a r d s , t h i s volume) t o d e t e r m i n e t h e number of "occluded" i o n s bound p e r t r a n s p o r t c y c l e , on t h e o t h e r hand, may h e l p e s t a b l i s h t h e a c t u a l t r a n s l o c a t i o n s t o i c h i o m e t r y s i n c e occluded ( i. e . , s l o w l y r e l e a s a b l e ) i o n s presumably a r e "on t h e i r way" from one s i d e of t h e membrane t o t h e o t h e r .

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IV.

OVERALL PUMP K I N E T I C S

I t i s f a i r t o say t h a t the Albers-Post (Albers, 1 9 6 7 ; P o s t e t a l . , 1 9 6 9 ) k i n e t i c model o f t h e N a , K A T P a s e , w i t h minor v a r i a t i o n s , i s w i d e l y a c c e p t e d (see Glynn et a l . , 1979, f o r a n updated v e r s i o n ) . T h a t i s , t h e A T P a s e enzyme a p p e a r s t o be c o n s e c u t i v e l y phosp h o r y l a t e d by ATP i n a s o d i u m - r e q u i r i n g s t e p and dephosp h o r y l a t e d i n a p o t a s s i u m - c a t a l y z e d s t e p . I t would t h e n seem l o g i c a l t o e x p e c t a c o n s e c u t i v e mechanism f o r t h e i o n t r a n s l o c a t i o n c y c l e a s w e l l , w i t h sodium e x p o r t a l -

t e r n a t i n g w i t h p o t a s s i u m u p t a k e . I n f a c t , T r e v o r Shawls (1954) c o n s e c u t i v e model f o r t h e sodium pump l o n g a n t e d a t e s t h e b i o c h e m i c a l model f o r t h e N a , K - A T P a s e , and one may wonder t o what e x t e n t Shawls model h a s guided t h e b i o c h e m i s t s ' e x p e r i m e n t s . Much i n t e r e s t w a s a r o u s e d , ion transport t h e r e f o r e , when c a r e f u l e v a l u a t i o n of t h e k i n e t i c s i n r e d b l o o d c e l l s (Hoffman and T o s t e s o n , 1 9 7 1 ; Garay and Garrahan, 1973; C h i p p e r f i e l d and W h i t t a m , 1976; Sachs, 1 9 7 7 ) suggested a simultaneous t r a n s p o r t model, i n which t h e pump enzyme t u r n s o v e r w i t h b o t h sodium and p o t a s s i u m t r a n s l o c a t i o n s i t e s o c c u p i e d , r a t h e r t h a n a c o n s e c u t i v e model. I n g e n i o u s models c a p a b l e of s a t i s f y i n g b o t h t h e a p p a r e n t c o n s e c u t i v e k i n e t i c s of phosphorylation/dephosphorylation and t h e a p p a r e n t s i multaneous f l u x k i n e t i c s were proposed by S t e i n e t a l . (1973) and by Repke and Schbn (1973; see a l s o S t e i n , 1 9 7 9 ) . Such models r e l i e d on t h e t h e n p r e v a i l i n g not i o n t h a t t h e pump enzyme w a s a dimer of c a t a l y t i c subu n i t s ; t h e i n d i v i d u a l s u b u n i t s were assumed t o undergo phosphorylation/dephosphorylation s e q u e n c e s , b u t o u t o f phase s o t h a t p h o s p h o r y l a t i o n of one s u b u n i t i s concom i t a n t w i t h d e p h o s p h o r y l a t i o n of t h e o t h e r . J u s t when i t appeared t h a t b i o c h e m i c a l k i n e t i c s and f l u x k i n e t i c s c o u l d b e t h u s r e c o n c i l e d , Sachs (1979) reexamined t h e same d a t a a n a l y z e d p r e v i o u s l y ( S a c h s , 1977) and conc l u d e d t h a t , i f allowance i s made f o r some "uncoupled" sodium e f f l u x , i . e . , e f f l u x o f sodium w i t h o u t accompanyi n g K e n t r y (Garrahan and Glynn, 1967b; Lew et a l . , 1973; Glynn and K a r l i s h , 1 9 7 6 1 , t h e f l u x k i n e t i c s can be i n t e r p r e t e d , a f t e r a l l , on t h e b a s i s of a s t r i c t l y c o n s e c u t i v e model. O t h e r s (Smith e t a l . , 1980; Moczydlowski and F o r t e s , 1981) have shown t h a t i t i s p e r f e c t l y p o s s i b l e t o a c c o u n t f o r t h e h i g h and low a f f i n i t y of t h e N a / K pump f o r ATP w i t h o u t r e s o r t i n g t o a d i m e r i c model. The p r o s p e c t s f o r a s i n g l e u n i t a r y model t h a t a c c o u n t s f o r t h e b i o c h e m i c a l s t e p s as w e l l a s t h e known f l u x modes ( N a : K exchange; ADP-dependent N a : N a exchange; Pi-dependent K:K exchange, e t c . ) , look b r i g h t e r again.

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A s i f on c u e , c e r t a i n s t r u c t u r a l f i n d i n g s (see elsewhere i n t h i s volume) a r e b e g i n n i n g t o c a s t d o u b t on t h e n o t i o n t h a t t h e o p e r a t i o n a l u n i t o f t h e sodium pump i s an A T P a s e dimer.

V.

TRANSPORT CORRELATES O F Na-ATPase

O u a b a i n - s e n s i t i v e Na,K-ATPase w i l l h y d r o l y z e ATP w i t h o u t t h e b e n e f i t o f p o t a s s i u m i o n s (Czerwinski et a l . , 1967; Neufeld and Levy, 1969; B l o s t e i n , 1 9 7 0 ) . Unlike t h e Na,K-stimulated a c t i v i t y , which h a s b o t h a low and a h i g h a f f i n i t y f o r ATP, t h e N a - s t i m u l a t e d act i v i t y d i s p l a y s a s i n g l e , h i g h - a f f i n i t y (<1 P M ) K O , ~ f o r ATP. The sodium dependence of ATP h y d r o l y s i s i s q u i t e complex: The a c t i v i t y c u r v e rises r a p i d l y a t v e r y l o w N a c o n c e n t r a t i o n s , ~ 0 . 5< 1 m~ ( P o s t et a l . , 1972; M&rdh and P o s t , 1977; Beaug6 and Glynn, 197933; F l a s h n e r and Robinson, 19791, r e a c h e s a p l a t e a u around 5-10 mM N a ( P o s t et a l . , 1972; M&dh and P o s t , 1 9 7 7 ; Beauge and Glynn, 1979b1, t h e n c o n t i n u e s t o r i s e w i t h i n c r e a s i n g sodium c o n c e n t r a t i o n s . I t h a s been p o i n t e d o u t by Beauge and Glynn (1979b) t h a t a c u r v e w i t h a p l a t e a u o r i n f l e c t i o n p o i n t r e f l e c t s a t least t h r e e k i n d s of sites: h i g h - a f f i n i t y s t i m u l a t i o n , medium-affinity i n h i b i t i o n , and l o w - a f f i n i t y s e c o n d a r y s t i m u l a t i o n . A t low ATP l e v e l s , p o t a s s i u m i n h i b i t s ATP h y d r o l y s i s (Kanazawa et al., 1967; Czerwinski e t a l . , 1967) and t h i s i s m o s t s a t i s f a c t o r i l y a c c o u n t e d f o r by t h e model of P o s t e t a l . (1972) i n which t h e enzyme becomes t r a p p e d i n a n " o c c l u d e d p o t a s s i u m " form. I n t r a c e l l u l a r sodium i s r e q u i r e d f o r enzyme phosp h o r y l a t i o n , and t h e sodium a f f i n i t y f o r t h i s a c t i v i t y i s v e r y h i g h ( B l o s t e i n , 1 9 7 9 ) . T h i s , and o t h e r e v i d e n c e s u c h as K a p l a n ' s f i n d i n g , r e p o r t e d i n t h i s volume, t h a t ADP:ATP exchange i n e r y t h r o c y t e s s a t u r a t e s a t 2 mM N a i l makes i t v e r y l i k e l y t h a t t h e i n i t i a l , h i g h - a f f i n i t y s t i m u l a t i o n o f N a - A T P a s e a c t i v i t y r e p r e s e n t s an a c t i o n a t i n t r a c e l l u l a r sodium s i t e s . Granted t h a t i n t r a c e l l u l a r sodium s i t e s are s a t u r a t e d a t v e r y low "a], it i s c o n v e n i e n t t o examine t h e p o s s i b l e f l u x c o r r e l a t e s o f Na-ATPase under t h r e e e x p e r i m e n t a l c o n d i t i o n s : i n t h e a b s e n c e of e x t e r n a l sodium, and i n t h e p r e s e n c e o f low and h i g h e x t e r n a l sodium c o n c e n t r a t i o n s . Pump-mediated ATP h y d r o l y s i s i n t h e t o t a l a b s e n c e of e x t e r n a l p o t a s s i u m o r sodium h a s been i d e n t i f i e d (Glynn and K a r l i s h , 1976) w i t h ';uncoupled" sodium e f f l u x (Garrahan and Glynn, 196733). Glynn and K a r l i s h

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showed t h a t t h i s e f f l u x h a s a v e r low K0.5 f o r ATP ( < 1P M ) , i s i n h i b i t e d a t low [ATPY by e x t e r n a l p o t a s sium, and t r a n s p o r t s two t o t h r e e N a i o n s p e r ATP s p l i t . I f Nao- and KO- independent sodium e f f l u x i s t r u l y "unc o u p l e d , " it s h o u l d be e l e c t r o g e n i c and have d e t e c t a b l e e f f e c t s on membrane p o t e n t i a l . T h i s q u e s t i o n i s examined i n t h e p r e s e n t volume by D i s s i n g and Hoffman. These a u t h o r s show t h a t , c o n t r a r y t o e x p e c t a t i o n , "uncoupled" sodium e f f l u x i s e l e c t r o n e u t r a l , presumably because a n i o n s accompany t h e sodium i o n s on t h e i r p a t h through t h e pump. A o u a b a i n - s e n s i t i v e t r a n s p o r t of s u l f a t e , which o n l y p a r t l y a c c o u n t s f o r t h e pump's elect r o n e u t r a l i t y , i s found i n t h e absence of c h l o r i d e . I t i s n o t immediately a p p a r e n t how t h i s unexpected new act i v i t y can be f i t i n t o t h e c u r r e n t model f o r t h e sodium pump, b u t t h e f i n d i n g i s r e m i n i s c e n t of t h a t by Baker ( 1 9 6 4 ) of an Nai-dependent, o u a b a i n - s e n s i t i v e l o s s of g l u t a m a t e and a s p a r t a t e from c r a b n e r v e b a t h e d i n Nafree, K-free s o l u t i o n . KO-independent sodium e f f l u x i s i n h i b i t e d by low e x t e r n a l sodium c o n c e n t r a t i o n s (Garrahan and Glynn, T h i s phenomenon h a s been c o n v i n c i n g l y 1967a) ( F ' m - . i d e n t i f i e d by Glynn and K a r l i s h (1976) a s r e f l e c t i n g t h e i n h i b i t o r y a c t i o n of l o w "a] ( ~ m 5 ) on Na-ATPase a c t i v i t y r e s p o n s i b l e f o r t h e p l a t e a u i n t h e sodium a c t i v a t i o n c u r v e mentioned e a r l i e r . When Na-ATPase a c t i v i t y was p l o t t e d a g a i n s t e x t e r n a l "a] ( F i g . 1), o n l y i n h i b i t i o n ( 0 - 5 m ) followed by s t i m u l a t i o n ( > 5 mM) w a s s e e n (Glynn and K a r l i s h , 1 9 7 6 ; L e e and B l o s t e i n , 1 9 8 0 1 , confirming the notion t h a t t h e high-affinity stimulation by N a of Na-ATPase a c t i v i t y i n unsided p r e p a r a t i o n s ref l e c t s b i n d i n g of Na+ t o i n t e r n a l s i t e s . How Nao might i n h i b i t Na-ATPase (and concomitant sodium e f f l u x ) i s n o t immediately a p p a r e n t from t h e c u r r e n t r e a c t i o n schemes f o r Na,K-ATPase. Beau96 and Glynn (1979b) found t h a t 5 m~ Na s t r o n g l y i n h i b i t s t h e r a t e of dephosphoryl a t i o n of p h o s p h o r y l a t e d p i g kidney A T P a s e i n K-free media ( F i g . 1 1 , and have proposed t h i s as t h e mechanism whereby low [Na] i n h i b i t s Na-ATPase. High e x t e r n a l sodium l e v e l s , i n t h e nominal absence of potassium a n d P , promote pump-mediated Na:Na exchange --a s w e l l a s ATP h y d r o l y s i s ( F i g . 1) ( L e e and B l o s t e i n , 1 9 8 0 ; see a l s o B l o s t e i n i n t h i s volume). I t i s very l i k e l y t h a t t h i s l a t t e r a c t i v i t y r e p r e s e n t s t h e ( > 5 m ) arm of t h e Na-ATPase a c t i v a t i o n c u r v e . high-"a] What i s less c l e a r i s t h e p r e c i s e mechanism of t h i s ATP-hydrolyzing Na:Na exchanging mode of o p e r a t i o n of t h e sodium pump. Sodium could be pumped inward a s a congener of K + , f o l l o w i n g t h e u s u a l E2-P -+ E 2 + P i p a t h way; one would t h e n e x p e c t a 3:2 0utward:inward s t o l -

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NA-ATPASE (1) ADP:ATP EXCHANGE (2) NA EFFLUX; ADP PRESENT ( 3 )

E-P

HYDROLYSIS

RATE (UNSIDED)

(6)

/

/ / / /

EXTRACELLULAR SODIUM F i g . 1 . S c h e m a t i c summary o f the e f f e c t o f external s o d i u m ions on v a r i o u s a c t i v i t i e s c a t a l y z e d b y the Na,K-ATPase. T h e numbers i n p a r e n t h e s e s r e f e r t o t h e f o l l o w i n g l i t e r a t u r e s o u r c e s : ( 1 ) G l y n n and K a r l i s h , 1 9 7 6 ; ( 2 ) K a p l a n and H o l l i s , 1 9 8 0 ; ( 3 ) Garr a h a n and G l y n n , 1 9 6 7 a ; (4) L e e and B l o s t e i n , 1 9 8 0 ; (5) S a c h s , 1 9 7 2 a ; ( 6 ) Beau96 and G l y n n , 1 9 7 9 b . T h e sole p u r p o s e of the g r a p h i s t o c o m p a r e the s h a p e s o f the c u r v e s , not the a b s o l u t e m a g n i tudes. T h e f i r s t four a c t i v i t i e s a r e i n i t i a l l y i n h i b i t e d w i t h h i g h a f f i n i t y , and then s t i m u l a t e d w i t h l o w a f f i n i t y and w i t h o u t evidence o f saturation. T h e next t w o a c t i v i t i e s a r e not i n h i b i t e d , o n l y s t i m u l a t e d b y Na,. T h e l a s t a c t i v i t y (E-P b r e a k d o w n ) i s i n i t i a l l y s t r o n g l y i n h i b i t e d , then m o d e r a t e l y s t i m u l a t e d .

c h i o m e t r y . A l t e r n a t i v e l y , N a : N a exchange i n t h e abs e n c e of ADP c o u l d p r o c e e d v i a t h e E l - P pathway f o r ADP-ATP exchange, b u t w i t h s u b s t i t u t i n g € o r ADP;

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such mechanism would c a l l f o r an e l e c t r o n e u t r a l N a : N a exchange. S t i l l o t h e r r e t u r n pathways can be e n v i s i o n e d . W e w i l l r e t u r n t o t h i s q u e s t i o n when examining t h e ADP:ATP exchange phenomenon. A contribution t o t h e problem of t h e o u t : i n s t o i c h i o m e t r y of N a : N a exchange i s provided by Forgac and Chin ( t h i s volume). I t a p p e a r s t h a t t h e pump enzyme, r e c o n s t i t u t e d i n t o l i p o s o m e s , can g e n e r a t e a membrane p o t e n t i a l when pumping sodium i n t h e absence of K + , s u g g e s t i n g an o u t : i n N a : N a s t o i c h i o m e t r y g r e a t e r t h a n u n i t y . Moreover, t h e ( n e t t r a n s p o r t ) : (ATP h y d r o l y s i s ) s t o i c h i o m e t r y d e c l i n e d with i n c r e a s i n g N a c o n c e n t r a t i o n , s u g g e s t i n g t h a t , as "a] i s i n c r e a s e d , t h e sodium t r a n s p o r t mode s h i f t s from an e l e c t r o g e n i c mechanism ("uncoupled" and/or " N a : K-like"?) toward an e l e c t r o n e u t r a l one ( " c o n v e n t i o n a l " N a : N a exchange?) Whereas i n h i b i t i o n of Na-ATPase i n e r y t h r o c y t e s c o u l d be r a t i o n a l i z e d as r e s u l t i n g from a Na-induced r e d u c t i o n of E-P breakdown r a t e , a c o n v e r s e mechanism c a n n o t e a s i l y be invoked i n t h e case of h i g h [ N a ] , s i n c e E-P breakdown i s enhanced no more t h a n 2 - f o l d between 5 and 150 m N a ( P o s t et a l . , 1972; Beau96 and Glynn, 1 9 7 9 b ) , whereas N a e f f l u x and Na-ATPase are s t i m u l a t e d many-fold ( F i g . 1 ) . I t must b e remembered, however, t h a t E-P breakdown r a t e s were determined on u n s i d e d p r e p a r a t i o n s from p i g o r g u i n e a p i g k i d n e y , a t a much lower temperat u r e , and t h a t t h e p r e c i s e dependence o f s t e a d y - s t a t e E-P l e v e l s on [Na] w a s unknown. In sided erythrocyte t h a t high p r e p a r a t i o n s , L e e and B l o s t e i n (1980)nd [Nal0, i f a n y t h i n g , d e c r e a s e d s t e a d y - s t a t e E-P l e v e l s . I t i s s a f e t o conclude t h a t , i f t h e s i d e d n e s s o f t h e three-pron e d e f f e c t o f N a on ATP h y d r o l y s i s i n t h e abs e n c e of Kq i s u n d e r s t o o d , t h e p r e c i s e mechanisms i n volved are n o t .

.

VI. A.

Na:Na EXCHANGE AND ADP:ATP EXCHANGE C H A R A C T E R I S T I C S OF N a : N a E X C H A N G E

The sodium pump c a n engage i n N a : N a exchange (Caldw e l l et a l . , 1960; Garrahan and Glynn, 1967a; Baker et a l . , 1 9 6 9 ; D e Weer, 1 9 7 0 ; Kennedy and D e Weer, 1 9 7 6 ) . T h i s exchange a p p e a r s t o be one-for-one (Garrahan and Glynn , 1967a) and e l e c t r o n e u t r a l (Abercrombie and D e Weer, 1978)--not n e c e s s a r i l y a redundant s t a t e m e n t , c o n s i d e r i n g t h e f i n d i n g s of D i s s i n g and Hoffman ( t h i s volume). I t i s i n h i b i t e d by e x t e r n a l K+, half-maximal i n h i b i t i o n o c c u r r i n g a t t h e ~ 0 . 5f o r a c t i v a t i o n of po-

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tassium i n f l u x ( G a r r a h a n and Glynn, 1 9 6 7 ~ ) . I t req u i r e s ADP ( D e Weer, 1970; Glynn a n d Hoffman, 1971) a n d a l s o ATP ( C a v i e r e s a n d Glynn, 1979) , b u t t h e l a t t e r i s n o t h y d r o l y z e d ( G a r r a h a n a n d Glynn, 1 9 6 7 d ) . Kennedy et a l . (see e l s e w h e r e i n t h i s volume) f o u n d a ~ 0 . 5f o r ADP o f a b o u t 350 U M , i n d e p e n d e n t o f [ATP] i n t h e r a n g e The exchange i s s t i m u l a t e d by N a o w i t h l o w 0.4-1 mM. a f f i n i t y a n d by N a i w i t h h i g h a f f i n i t y ( K ~ 3 m ~ ) ,a n d i s n o t i n h i b i t e d by h i g h [ N a ] i (Garay a n d G a r r a h a n , 1973) a l t h o u g h e a r l i e r o b s e r v a t i o n s had s u g g e s t e d t h e contrary. I n t r a c e l l u l a r K s t i m u l a t e s Na:Na e x c h a n g e w i t h l o w a f f i n i t y (Garay and G a r r a h a n , 1973; S a c h s , 1 9 8 1 b ) . I n t r a c e l l u l a r Mg s t i m u l a t e s N a : N a e x c h a n g e w i t h h i g h a f f i n i t y ( ~ 0 . 5 9 U M ) and h a s a n i n h i b i t o r y e f f e c t a t c o n c e n t r a t i o n s above m i l l i m o l a r ( F l a t m a n and Lew, 1981; and see e l s e w h e r e i n t h i s volume). Oligomyc i n a l s o i n h i b i t s N a : N a e x c h a n g e ( G a r r a h a n and Glynn, 1967d) Q,

Q,

.

B.

CHARACTERISTICS

OF ADP : A T P EXCHANGE

I n view o f i t s r e q u i r e m e n t f o r ADP and o t h e r c h a r a c t e r i s t i c s , i t h a s l o n g a p p e a r e d a t t r a c t i v e t o assume ( D e Weer, 1970; Glynn a n d Hoffman, 1971) t h a t Na:Na e x c h a n g e i s t h e f l u x correlate o f sodium-dependent ADP:ATP e x c h a n g e , a well-documented r e a c t i o n c a t a l y z e d by t h e Na,K-ATPase (Skou, 1 9 6 0 ; Fahn et a l . , 1 9 6 6 a ; S t a h l , 1 9 6 8 ) , and which presumably r e p r e s e n t s t h e rev e r s a l of t h e f i r s t p a r t o f t h e A l b e r s - P o s t r e a c t i o n scheme ( A l b e r s , 1967; P o s t e t a l . , 1 9 6 9 ) : ATP N a i

ADP Nao +

J . J .

El

, -

L

E1-ATP-Na + E

I

.

1=P-ADP*Na

The ATP r e q u i r e m e n t of t h i s r e a c t i o n u n d o u b t e d l y ref l e c t s t h e need f o r p h o s p h o r y l a t e d enzyme i n t h e backward d i r e c t i o n . T r e a t m e n t o f t h e enzyme w i t h N-ethylmaleimide or o l i g o m y c i n , b o t h o f which i n h i b i t t h e ATPase a c t i v i t y , leaves t h e ADP:ATP e x c h a n g e a c t i v i t y unharmed o r e n h a n c e d (Fahn e t a l . , 1 9 6 6 a , b ; B l o s t e i n , 1970). Of p a r t i c u l a r i n t e r e s t f o r o u r p r e s e n t p u r p o s e are t h e e f f e c t s o f N a + , K + , a n d Mg2+ on t h e e x c h a n g e react i o n . Magnesium i o n s s t i m u l a t e a t l o w (micromolar f r e e ) and i n h i b i t t h e r e a c t i o n a t h i g h ( m i l l i m o l a r ) co n cen t rat i o n s (Fahn et al., 1966a; Robinson, 1976; Yamaguchi

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and Tonomura, 1 9 7 7 ; Beauge and Glynn, 197933). The act i v a t i n g e f f e c t undoubtedly r e f l e c t s t h e h i g h a f f i n i t y f o r Mg i n t h e enzyme p h o s p h o r y l a t i o n s t e p ( P o s t e t al., 1 9 6 5 ) , b u t t h e i n h i b i t o r y s t e p i s somewhat more c o n t r o v e r s i a l . O r i g i n a l l y , i t was t h o u g h t ( A l b e r s , 1 9 6 7 ; P o s t et al., 1 9 6 9 ) t h a t m i l l i m o l a r c o n c e n t r a t i o n s of Mg2+ drove t h e E l - P + E2-P e q u i l i b r i u m t o t h e r i g h t , b u t experiments of Klodos and Skou (1975) have r e n d e r e d t h a t t h e s i s u n t e n a b l e . Robinson ( 1 9 7 6 ) has proposed a l t e r n a t i v e schemes based on a d i m e r i c enzyme model. Within t h e framework of t h e Albers-Post scheme, howe v e r , it a p p e a r s (see D e Weer e t al. elsewhere i n t h i s volume) t h a t Mg2+ could s t i m u l a t e and i n h i b i t by a c t i n g a t a s i n g l e s i t e , provided ATP and Mg add t o t h e enzyme in t h a t o r d e r , a r e q u i r e m e n t f o r which t h e r e i s some e v i d e n c e m d h and P o s t , 1 9 7 7 ) :

El

ATP

Mg

J.

J.

, -

L

E1*ATP

E1*MgATP

* *

A t l o w c o n c e n t r a t i o n s , Mg w i l l a l l o w t h e f o r m a t i o n of phosphoenzyme. A t h i g h c o n c e n t r a t i o n s , Mg w i l l r e t a r d t h e r e l e a s e of l a b e l e d ATP formed from l a b e l e d ADP. A second i o n of i n t e r e s t i s potassium. Stimulat i o n o f ADP:ATP exchange by K has been d e s c r i b e d (Banerjee and Wong, 1 9 7 2 ; Robinson, 1 9 7 7 ) ; such stimu-

l a t i o n only o c c u r s i n t h e p r e s e n c e of sodium and under c o n d i t i o n s where Mg i s i n h i b i t o r y . A p o s s i b l e mechanism f o r t h i s a c t i o n i s t h e d i s p l a c e m e n t , by K , of t h e E1OATP + E z - A T P e q u i l i b r i u m t o t h e r i g h t ( K a r l i s h e t al., 1 9 7 8 ; Beauge and Glynn, 1980; Jldrgens e n and K a r l i s h , 1 9 8 0 ) , where ATP i s weakly bound and l a b e l , presumably, more r e a d i l y r e l e a s e d . A s f o r t h e e f f e c t s of sodium on ADP:ATP exchange, t h e s i t u a t i o n seems a s complex as t h a t f o r Na-ATPase. There i s a h i g h - a f f i n i t y ( ~ 0 . 5= 1 - 2 mM) s t i m u l a t i o n (Fahn e t al., 1966a; Wildes e t al., 1973; Beauge and Glynn, 1 9 7 9 b ) , followed by a d i p a t 5-10 mM Na, f o l l o w e d a g a i n by l o w - a f f i n i t y s t i m u l a t i o n (BeaugB and Glynn, 197933; Kaplan and H o l l i s , 1 9 8 0 ) . A curve t h a t rises, d i p s , and rises a g a i n c l e a r l y r e f l e c t s a t l e a s t t h r e e e f f e c t s : h i g h - a f f i n i t y s t i m u l a t i o n , medium-affinity i n h i b i t i o n , and l o w - a f f i n i t y s t i m u l a t i o n . The s i m i l a r i t y between t h e sodium a c t i v a t i o n c u r v e s f o r Na-ATPase and ADP:ATP exchange i s s t r i k i n g . Here a l s o , a t t e m p t s have been made t o a s s e s s t h e s i d e d n e s s of t h e t h r e e sodium e f f e c t s on ADP:ATP exchange j u s t mentioned.

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Attempts t o measure pump-mediated ADP:ATP exchange i n a " s i d e d " p r e p a r a t i o n such a s r e s e a l e d e r y t h r o c y t e g h o s t s have long been f r u s t r a t e d by t h e p r e s e n c e of o t h e r enzymes t h a t c a t a l y z e t h e same r e a c t i o n , and by h y d r o l y s i s o f ATP d u r i n g p r e p a r a t i o n of t h e c e l l s . These problems have been overcome w i t h more r e f i n e d methods f o r p r e p a r i n g " c l e a n " r e s e a l e d g h o s t s and t h e u s e of i n h i b i t o r s ( C a v i e r e s and Glynn, 1 9 7 9 ; Kaplan and H o l l i s , 1 9 8 0 ) ; r e a c t i o n s were i n g e n i o u s l y i n i t i a t e d e i t h e r by suddenly a l l o w i n g Mg2+ i n t o t h e p r e v i o u s l y magnesium-free g h o s t s ( C a v i e r e s , 1 9 8 0 ; and e l s e w h e r e i n t h i s volume) or by p h o t o l y s i s of "caged" ATP (Kaplan and H o l l i s , 1 9 8 0 ; a l s o s e e Kaplan e l s e w h e r e i n t h i s volume). The f i n d i n g s from K a p l a n ' s l a b o r a t o r y can be summarized a s f o l l o w s : Nai s t i m u l a t e s w i t h v e r y h i g h a f f i n i t y ( ~ 0 . 5 < 2 m M ) and does n o t i n h i b i t ; Nao i n h i b i t s between 0 and 5 m M and t h e n s t i m u l a t e s i n app r o x i m a t e l y l i n e a r f a s h i o n ( F i g . 1) and KO i n h i b i t s w i t h an a f f i n i t y s i m i l a r t o t h a t of i t s i n h i b i t o r y act i o n on Na:Na exchange. The h i g h - a f f i n i t y s t i m u l a t i o n by Nai undoubtedly r e f l e c t s Nai r e q u i r e m e n t f o r enzyme p h o s p h o r y l a t i o n , and KO presumably i n h i b i t s ADP:ATP exchange by s h i f t i n g t h e E l - P * E2-P e q u i l i b r i u m t o t h e r i g h t . The mode of a c t i o n of e x t e r n a l sodium i s , a s i t w a s i n t h e case of Na-ATPase, l e s s c l e a r . S t i m u l a t i o n of ADP:ATP exchange by h i g h can be r a t i o n a l i z e d a s r e s u l t i n g from a sodium-induced s h i f t of t h e El-P =+ E2-P e q u i l i b r i u m t o t h e l e f t (Taniguchi and P o s t , 1975; K u r i k i and Racker, 1 9 7 6 ; Hara and Nakao, 1 9 8 1 ) , b u t it i s d i f f i c u l t t o f i t i n h i b i t i o n of ADP:ATP exchange by l o w i n t o the current schemes w i t h o u t a d d i t i o n a l a d h o c assumptions. C.

R E L A T I O N S H I P BETWEEN N a r N a EXCHANGE AND A D P : A T P EXCHANGE

The p a r a l l e l s between N a : N a exchange and ADP:ATP exchange a r e s t r i k i n g . Both r e q u i r e ADP as w e l l a s ATP; a r e i n h i b i t e d by low e x t e r n a l [K] and s t i m u l a t e d by h i g h (presumably i n t e r n a l ) [ K ] ; are s t i m u l a t e d w i t h h i g h a f f i n i t y by Nai which is n o t i n h i b i t o r y a t h i g h concent r a t i o n s ; and are s t i m u l a t e d by high Nao c o n c e n t r a t i o n s . These s i m i l a r i t i e s a r e s t r o n g s u p p o r t f o r t h e view t h a t t h e two a c t i v i t i e s s h a r e a common p a t h . Some of t h e d i s s i m i l a r i t i e s can a l s o be accommodated w i t h o u t t o o much d i f f i c u l t y . For example, oligomycin i n h i b i t s Na:Na exchange i n e r y t h r o c y t e s , b u t n o t ADP:ATP exchange; t h i s would be e x p e c t e d i f ADP and Nao were r e l e a s e d i n t h a t o r d e r , and oligomycin a c t e d between t h e two s t e p s (Glynn and Hoffman, 1 9 7 1 ) :

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( A c o r o l l a r y of t h i s o r d e r i n g i s t h a t s i f f i c i e n t l y h i g h l e v e l s of ADP s h o u l d i n h i b i t N a : N a exchange w i t h o u t i n h i b i t i n g ADP:ATP exchange; t h i s has n o t y e t been observed e x p e r i m e n t a l l y . ) S i m i l a r l y , f r e e Mg2+ concent r a t i o n s o v e r a f e w t e n s micromolar i n h i b i t ADP:ATP exchange b u t n o t Na:Na exchange: b u t a g a i n , p r o p e r o r d e r i n g of A T P , Mg, and N a i a d d i t i o n w i l l a c c o u n t f o r these o b s e r v a t i o n s :

( A c o r o l l a r y of t h i s o r d e r i n g i s t h a t s u f f i c i e n t l y h i g h l e v e l s o f N a i s h o u l d i n h i b i t ADP:ATP exchange w i t h o u t i n h i b i t i n g Na:Na exchange.) I f t h e o r d e r i n g s d i s c u s s e d above are c o r r e c t , i t i s o b v i o u s t h a t t h e r e i s no r e a s o n t o e x p e c t a f i x e d s t o i c h i o m e t r i c r e l a t i o n s h i p between Na:Na exchange and ADP:ATP exchange as measured w i t h i s o t o p e s . I n t h i s c o n t e x t , it i s worth p o i n t i n g o u t t h a t i n s q u i d axon no o u a b a i n - s e n s i t i v e ADP:ATP exchange w a s d e t e c t e d w h i l e Na:Na exchange w a s t a k i n g p l a c e ( D e Weer e t a l . , t h i s volume), whereas i n e r y t h r o c y t e g h o s t s c o n s i d e r a b l e ADP:ATP exchange a c t i v i t y w a s measured i n t h e a b s e n c e of e x t e r n a l Na, where obvio u s l y no m e a s u r a b l e N a : N a exchange can t a k e place (Kaplan and H o l l i s , 1 9 8 0 ; Kaplan, t h i s volume).

VII.

RELATIONSHIP BETWEEN Na-ATPase AND ADP:ATP EXCHANGE

Although t h e r e a r e s e v e r a l d i f f e r e n c e s between t h e e f f e c t s of v a r i o u s a g e n t s ( e . g . , i n t e r n a l K ; i n t e r n a l Mg; o l i g o m y c i n ) on Na-ATPase and ADP:ATP exchange, t h e r e i s a s t r i k i n g s i m i l a r i t y between t h e sodium a c t i v a t i o n c u r v e f o r Na-ATPase a c t i v i t y and t h a t f o r ADP:ATP exchange a c t i v i t y (BeaugB and Glynn, 197933). Both c u r v e s d i s p l a y a h i g h - a f f i n i t y s t i m u l a t i n g a c t i o n of N a i , and h i g h - a f f i n i t y i n h i b i t o r y and l o w - a f f i n i t y s t i m u l a t i n g a c t i o n s of Nao. There a p p e a r s t o be no problem i n

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a s s i g n i n g t h e h i g h - a f f i n i t y N a i e f f e c t on b o t h ATPase and exchange t o t h e enzyme p h o s p h o r y l a t i o n s t e p r e q u i r e d by b o t h a c t i v i t i e s . The s i m i l a r i t y of t h e l o w - a f f i n i t y s t i m u l a t i o n of b o t h Na-ATPase and ADP:ATP exchange by N a o c o u l d b e a c o i n c i d e n c e s i n c e t h e s e e x p e r i m e n t s have n e v e r been done under e x a c t l y i d e n t i c a l c o n d i t i o n s ( e . g . , ADP i s p r e s e n t i n t h e exchange e x p e r i m e n t s ) . I f it i s n o t a c o i n c i d e n c e , and t h e s i m i l a r i t y r e f l e c t s a s i n g l e k i n e t i c e v e n t , and i f one a t t e m p t s t o f i t t h i s o b s e r v a t i o n w i t h i n t h e A l b e r s - P o s t framework i n c l u d i n g t h e documented e f f e c t s of N a o on t h e E l - P / E 2 - P e q u i l i b r i u m , t h e concluvia s i o n seems i n e s c a p a b l e t h a t Na-ATPase m u s t p r o c e e d d i r e c t E l - P breakdown r a t h e r t h a n v i a E2-P w i t h N a o act i v a t i n g i n a Ko-like manner. ADP:ATP exchange and NaA T P a s e would t h e n r e p r e s e n t a l t e r n a t e r e t u r n pathways f o r E l - P + E l , t h e p h o s p h a t e a c c e p t o r b e i n g ADP i n one c a s e and H 2 0 i n t h e o t h e r . I t w i l l be r e c a l l e d t h a t ADP:ATP exchange can t a k e p l a c e w i t h and w i t h o u t sodium i n t h e e x t e r n a l medium and t h a t Na-ATPase a c t i v i t y can o c c u r w i t h o r w i t h o u t e x t e r n a l sodium p r e s e n t . Taken t o g e t h e r , t h e s e o b s e r v a t i o n s s u g g e s t t h a t E l - P c a n rev e r t t o El w i t h or w i t h o u t ADP, and w i t h o r w i t h o u t Nao, (1) "conl e a d i n g t o f o u r p o s s i b l e modes o f o p e r a t i o n : v e n t i o n a l " (ADP-requiring) Na:Na exchange w i t h o u t hyd r o l y s i s : ( 2 ) " u n c o u p l e d " e f f l u x w i t h o u t ATP h y d r o l y s i s : ( 3 ) Na:Na exchange w i t h ATP h y d r o l y s i s : and ( 4 ) " u n c o u p l e d " e f f l u x w i t h ATP h y d r o l y s i s . Only t h e s e c o n d mode h a s n o t been e x p l i c i t l y d e s c r i b e d , b u t it c o u l d have been p r e s e n t i n " u n c o u p l e d " f l u x e x p e r i m e n t s where ADP w a s p r e s e n t , s u c h as t h o s e o f G a r r a h a n and Glynn ( 1 9 6 7 a ) . A r e q u i r e m e n t of t h i s model i s t h a t oligomyc i n , i n a d d i t i o n t o p r e v e n t i n g N a r e l e a s e on t h e o u t s i d e , b e assumed t o b l o c k d e p h o s p h o r y l a t i o n o f E l - P by H 2 0 , b u t n o t by ADP. A s f o r t h e h i g h - a f f i n i t y i n h i b i t i o n by Nao of NaATPase, ADP:ATP exchange, and sodium e f f l u x i n t o K-free media, t h e r e i s no o b v i o u s p r o v i s i o n f o r s u c h a mechani s m i n t h e c u r r e n t schemes. I n a d d i t i o n t o t h e e v i d e n c e a l r e a d y q u o t e d , C a v i e r e s and E l l o r y (1975) a l s o have d e s c r i b e d e x p e r i m e n t s t h a t l e d them t o p o s t u l a t e an ext e r n a l h i g h - a f f i n i t y i n h i b i t o r y s i t e f o r sodium. P o s t e t a ] . ( 1 9 7 2 ) have d e s c r i b e d , i n u n s i d e d k i d n e y ATPase, a s t i m u l a t i n g e f f e c t ( i n a d d i t i o n t o t h e more f a m i l i a r i n h i b i t o r y o n e ) o f K+ on N a , K - A T P a s e a t low ATP c o n c e n t r a t i o n s , and Beauge et a l . ( 1 9 7 9 ) h a v e i d e n t i f i e d t h a t e f f e c t a s due t o e x t e r n a l K d i s p l a c i n g SOdium from an i n h i b i t o r y s i t e . T h e r e i s a c l e a r n e e d , t h e n , f o r a h i g h - a f f i n i t y a c t i o n of sodium i n any r e a l i s t i c model of t h e sodium pump. E i t h e r an a d d i -

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t i o n a l s i t e f o r sodium must be p o s t u l a t e d , o r t h e c u r r e n t schemes must be adapted t o accommodate t h e o b s e r v a t i o n s . The f o l l o w i n g i s a t e n t a t i v e p r o p o s a l f o r a mechanism by which t h e c u r r e n t Albers-Post scheme could e x h i b i t h i g h - a f f i n i t y i n h i b i t i o n by e x t e r n a l sodium. L e t t h e E l - P * E2-P e q u i l i b r i u m , i n t h e absence of e x t e r n a l sodium o r potassium, be v e r y much t o t h e r i g h t . Nao s t a b i l i z e s t h e E l - P form by b i n d i n g t o it w i t h low a f f i n i t y . Now assume t h a t t h e r a t e c o e f f i c i e n t of d e p h o s p h o r y l a t i o n (by H 2 0 o r ADP) of i o n f r e e E l - P i s much h i g h e r t h a n t h a t of E l - P f u l l y s a t u r a t e d w i t h sodium: t h e s m a l l amount of i o n - f r e e E l - P t h a t e x i s t s i n t h e absence of Nao and KO w i l l s u s t a i n an a p p r e c i a b l e d e p h o s p h o r y l a t i o n r a t e ( i . e . , ATPase a c t i v i t y o r ADP :ATP exchange , and concomitant Na e f f l u x ) . E l e v a t i o n of Nao w i l l p r o g r e s s i v e l y remove t h e l a b i l e i o n - f r e e E l - P from t h e p o o l and e v e n t u a l l y r e p l a c e it w i t h a much l a r g e r p o p u l a t i o n of f u l l y ( t r i p l y ? ) s o d i u m - s a t u r a t e d , more s l o w l y dephosphorylati n g El-P-Na3, l e a d i n g a g a i n t o an a p p r e c i a b l e dephosphorylation r a t e . (For t h i s mechanism t o d i s p l a y higha f f i n i t y i n h i b i t i o n followed by l o w - a f f i n i t y s t i m u l a t i o n , it i s obvious t h a t v a n i s h i n g d e p h o s p h o r y l a t i o n r a t e c o e f f i c i e n t s must b e a s s i g n e d t o E l - P - N a and E l - P -Na2. )

-

VIII.

RELATIONSHIP BETWEEN Na:Na EXCHANGE AND

Na:K EXCHANGE E x t e r n a l potassium i n h i b i t s N a : N a exchange (Garrahan and Glynn, 1967c) and ADP:ATP exchange (Kaplan, t h i s volume) a s i t s t i m u l a t e s Na:K exchange. These e f f e c t s are e a s i l y r a t i o n a l i z e d w i t h i n t h e Albers-Post framework a s r e f l e c t i n g t h e e f f e c t s of ext e r n a l sodium and potassium on t h e E l - P -+ E2-P e q u i l i b r i u m . I n t r a c e l l u l a r ADP, on t h e o t h e r hand, w i l l induce Na:Na exchange ( p r o v i d e d KO i s n o t s a t u r a t i n g ) w i t h o u t much e f f e c t on ongoing Na:K exchange, a t l e a s t when ATP i s i n t h e m i l l i m o l a r range (Kennedy and D e Weer, 1 9 7 7 ; D e Weer e t al., 1 9 7 9 ) . A t lower [ATP], t h e e f f e c t of ADP i s t o induce Na:Na exchange and t o i n h i b i t Na:K exchange (Kennedy et al., t h i s volume). The r e l e v a n t p o r t i o n of t h e pump c y c l e i s a s f o l l o w s :

613

BIOPHYSICAUBIOCHEMICALCORRELATIONS

E1*ATP

Nai

ADP

J.

4

E1-ATP*Na+E

1=P .ADP.Na

4

Ki + I ATP

I I

- + I

I I

pi +

I

I

E1-P-Na

11 +

NaO

E2-P

I1

c

KO

E2-P*K

At high ATP levels, the hydrolysis of E2-P (lower line) is probably rate-limiting. For ADP to have little effect on the population of enzyme molecules in the El-P and E2-P forms, it appears that, under those conditions, the steady-state distribution of enzyme forms in the upper line must remain biased toward the right, so that a relatively large increase in the traffic from E1-P.Na to E1.ATP (i.e., Na influx) has little effect on the absoltte amounts.of enzyme present in the El-P .Na , E2-P, and E2-P*K forms.

IX.

"OCCLUDED" FORMS AND I O N TRANSPORT

Experiments published in 1972 by Post and collaborators showed that the rate of rephosphorylation of freshly dephosphorylated Na,K-ATPase depended on the nature of the cation (K+,Rb+, Li+) that had catalyzed the dephosphorylation. These authors concluded that K+ and its congeners remain "occluded" in the enzyme for some time after dephosphorylation, the rate of deocclusion depending on the nature of the cation. They also concluded that ATP accelerates (with low affinity) the release of the occluded cation. This model has several verifiable "biophysical" consequences. One of them is that physical "trapping" of K congeners should be measurable if sufficiently long-lived; Glynn and Richards review this question elsewhere in this volume. Other "flux" consequences are kinetic. At high [ATP], deocclusion is not rate-limiting, and the overall transport velocity will be rate-limited by the saturation of E2-P with K+ or its congeners. At low [ATP], overall transport velocity could be rate-limited by the

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d e o c c l u s i o n v e l o c i t y . There i s no a p r i o r i r e a s o n t o e x p e c t any c o r r e l a t i o n between t h e a f f i n i t y of K+ and congeners f o r t h e e x t e r n a l E2-P s i t e , and t h e tendency of t h e s e i o n s t o remain occluded. Beauge and DiPolo ( 1 9 7 8 ; 1981b; and e l s e w h e r e i n t h i s volume) have shown t h a t i n s q u i d axon t h e o r d e r of e f f e c t i v e n e s s of K+ and congeners a s a c t i v a t o r s o f t h e sodium pump indeed d i f f e r s depending on whether [ATP] i s 3 m~ o r 30-50 U M . Also, a t low [ATP], s i n c e t h e r a t e - l i m i t i n g s t e p i s deo c c l u s i o n , t h e a p p a r e n t a f f i n i t y of K+ and congeners a t t h e e x t e r n a l s i t e w i l l i n c r e a s e . T h i s w a s found t o be t h e c a s e f o r K+ i n s q u i d axons (Beaug6 and DiPolo, 1979, 1981b; and t h i s volume) and f o r Rb+ i n e r y t h r o c y t e s ( E i s n e r and R i c h a r d s , 1 9 8 0 , 1 9 8 1 b ) . Conversely, low [KIo, by making d e p h o s p h o r y l a t i o n r a t e - l i m i t i n g , w i l l i n c r e a s e t h e a p p a r e n t a f f i n i t y f o r ATP, a p r e d i c t i o n made and v e r i f i e d by E i s n e r and R i c h a r d s (1981b). F i n a l l y , i n view of t h e r e c i p r o c a l e f f e c t s of K+ and ATP on t h e e q u i l i b r i u m between t h e dephosphoenzyme forms E l + E 2 (see Glynn et al., 1 9 7 9 ; K a r l i s h , 1 9 7 9 ; Beauge and Glynn, 1 9 8 0 ) , one might e x p e c t h i g h K t l e v e l s t o reduce t h e pump t u r n o v e r r a t e a t l o w [ATP? b u t n o t a t h i g h [ATP]. T h i s was v e r i f i e d i n s q u i d axon by Beaug6 and DiPolo ( 1 9 8 1 b ) .

X.

BIOCHEMICAL CORRELATES O F K:K EXCHANGE

The a b i l i t y of t h e sodium pump t o engage i n Na:Na exchange i n t h e absence of potassium, and i n K:K exchange i n t h e absence of sodium, h a s been a s t r o n g a r gument i n s u p p o r t of " c o n s e c u t i v e " models of t h e pump. Y e t , j u s t a s a s t r a i g h t f o r w a r d a p p l i c a t i o n of t h e ext a n t v e r s i o n (Glynn et a l . , 1 9 7 9 ) of t h e Albers-Post model does n o t y e t accommodate a l l c h a r a c t e r i s t i c s of Na:Na and ADP:ATP exchange, it i s f a i r t o s a y t h a t n o t a l l p r e d i c t i o n s of t h e c o n s e c u t i v e model w i t h r e s p e c t t o K:K exchange have been unambiguously v e r i f i e d e i t h e r . O u a b a i n - s e n s i t i v e K:K exchange h a s been w e l l s t u d i e d i n human e r y t h r o c y t e s (Glynn, 1957; P o s t and Sen, 1965; Glynn et al., 1 9 7 0 ; Sachs, 1972b, 1980, 1981a; Simons, 1 9 7 4 , 1975; E i s n e r and R i c h a r d s , 1 9 8 1 a ) . The exchange depends on t h e p r e s e n c e of i n t r a c e l l u l a r orthophosphate ( P o s t and Sen, 1965; Glynn et al., 1 9 7 0 ) w i t h a ~ 0 . 5= 1-2 mM (Simons, 1 9 7 4 ; Sachs, 1981a1, and a l s o on t h e p r e s e n c e of ATP, which i s n o t hydrolyzed (Simons, 1 9 7 4 ) , w i t h a ~ 0 . 55 1 0 0 P M (Simons, 1 9 7 4 ; Sachs, 1 9 8 1 a ) . ATP can be r e p l a c e d w i t h nonhydrolyzable

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a n a l o g s (Simons, 19751, s u g g e s t i n g t h a t enzyme phosp h o r y l a t i o n by ATP i s n o t r e q u i r e d f o r t h e r e a c t i o n . E x t e r n a l K+ a c t s w i t h h i g h a f f i n i t y : ~ 0 . 5< 0 . 1 mM i n t h e absence of e x t e r n a l sodium ( S a c h s , 1 9 8 1 a ) ; and i n t e r n a l K+ w i t h low a f f i n i t y : ~ 0 . 5% 1 0 mM (Simons, 1 9 7 4 ) . I n t e r n a l sodium i s a s t r o n g i n h i b i t o r (Simons, 1974). The q u e s t i o n a t hand i s whether t h e s e o b s e r v a t i o n s f i t t h e Albers-Post model, and what o t h e r v e r i f i a b l e p r e d i c t i o n s t h e model makes f o r K:K exchange. The requirement f o r P i i s r e g a r d e d a s an e x p r e s s i o n of t h e r e v e r s a l of K-catalyzed enzyme d e p h o s p h o r y l a t i o n (Glynn et al., 1 9 7 0 ) . The requirement f o r ATP was s u r p r i s i n g a t f i r s t , b u t i n t e r p r e t e d by Simons ( 1 9 7 4 ) a s p o s s i b l y r e f l e c t i n g t h e ATP-induced a c c e l e r a t i o n of K+ release from t h e lloccludedl' potassium-E2 form d e s c r i b e d by P o s t e t al. ( 1 9 7 2 ) . These f e a t u r e s w e r e e x p l i c i t l y i n c l u d e d by K a r l i s h e t al. (1978) i n t h e i r modified v e r s i o n of t h e Albers-Post c y c l e . The r e l e v a n t p a r t of t h e c y c l e ( i n t h e p r e s e n c e of [ATP] over a few micromolar) f o l l o w s : Ki

-

J.

E1.ATP

EIK-ATP $E2 (K) -ATP

m

+

+ E2 (K)

'i

KO

J.

1.

E2=P.K

E2=P

-

*

S t e i n ( 1 9 7 9 ) h a s p o i n t e d o u t t h a t such a model p r e d i c t s n o t o n l y t h e observed dependencies of K:K exchange on ATP and P i , b u t a l s o secondary i n h i b i t o r y e f f e c t s a s i n c r e a s i n g c o n c e n t r a t i o n s of ATP o r Pi f o r c e t h e enzyme p o p u l a t i o n i n t o one o r t h e o t h e r " c o r n e r . " A t t h e t i m e of S t e i n ' s a n a l y s i s , no such i n h i b i t i o n s had been d e s c r i b e d . I n s p e c t i o n of t h e model shows (see S t e i n , 1 9 7 9 , f o r a d e t a i l e d a n a l y s i s ) t h a t any i n h i b i t o r y e f f e c t o f , s a y , ATP would depend on t h e p r e v a i l i n g l e v e l s of P i , K i , and KO. ATP and P i s h o u l d a l s o a f f e c t t h e a p p a r e n t a f f i n i t i e s f o r K i and K O , i n o p p o s i t e d i r e c t i o n s (ATP i n c r e a s i n g Km f o r KO and d e c r e a s i n g t h a t f o r K i , and v i c e v e r s a i n t h e case o f P i ) . Some of t h e s e q u e s t i o n s have r e c e n t l y been s p e c i f i c a l l y i n v e s t i g a t e d . Sachs ( 1 9 8 1 a ) , working w i t h r e d blood c e l l s , found no i n h i b i t o r y e f f e c t of P i on K : K exchange up t o [ P i ] = 6 0 mN and no i n h i b i t o r y e f f e c t of ATP a t c o n c e n t r a t i o n s a s high a s 6 . 2 m ~ and , h a s concluded t h a t s i m u l t a n e o u s b i n d i n g of ATP and orthophosphate i s required t o account f o r t h i s observation. Since " t h e r e a p p e a r s t o be no e v i d e n c e e i t h e r f o r o r a g a i n s t t h e e x i s t e n c e " of c a t a l y t i c s u b u n i t s t h a t can b i n d ATP and o r t h o p h o s p h a t e s i m u l t a n e o u s l y , Sachs (1981a) prop o s e s a model i n which K:K t r a n s l o c a t i o n i s c a r r i e d o u t

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by an ATPase dimer, one s u b u n i t of which i s phosphoryla t e d , and t h e o t h e r b i n d s ATP. A s h o r t n o t e of E i s n e r and Richards ( 1 9 8 1 a ) , on t h e o t h e r hand, c l a i m s t h a t i n h i b i t i o n by ATP can be s e e n provided [Xi] i s s u f f i c i e n t l y low, and i n h i b i t i o n by P i provided [ATP] i s s u f f i c i e n t l y low. Recent work by K a r l i s h et a l . (1982) on ATP- and Pi-stimulated Rb:Rb exchange by Na,K-ATPase r e c o n s t i t u t e d i n t o liposomes may reconcile t h e s e f i n d i n g s . K a r l i s h et a l . ( 1 9 8 2 ) f i n d t h a t , a t low f i x e d [ATP] o r [ P i ] , Rb:Rb i s f i r s t s t i m u l a t e d and t h e n i n h i b i t e d by i n c r e a s i n g c o n c e n t r a t i o n s of P i o r ATP, r e s p e c t i v e l y . A t s u f f i c i e n t l y high f i x e d l e v e l s of one, i n c r e a s i n g t h e o t h e r s u b s t r a t e o n l y s t i m u l a t e s exchange. These k i n e t i c s a r e i n t e r p r e t e d on t h e b a s i s of a model t h a t a l l o w s s e v e r a l a l t e r n a t e pathways €or K:K exchange, one of v h i c h r e q u i r e s t h a t b o t h ATP and P i be bound t o t h e enzyme. Unlike Sachs ( m a ) , who p o s t u l a t e s a dimer, K a r l i s h e t a l . (1982) p o s t u l a t e independent and s i m u l t a n e o u s b i n d i n g of ATP and P i t o (presumably) a s i n g l e c a t a l y t i c u n i t . T h e i r model i s q u i t e g e n e r a l and comp l e x , b u t i t s e s s e n t i a l f e a t u r e w i t h r e g a r d t o ATP- and P i - s t i m u l a t e d K : K exchange i n i n t a c t c e l l s can be reduced t o t h e f o l l o w i n g :

I

L

The lower p a t h r e p r e s e n t s t h e " c o n v e n t i o n a l " Albers-Post pathway , which p r e d i c t s i n h i b i t i o n of K:K exchange by h i g h c o n c e n t r a t i o n s of e i t h e r ATP o r P i ( S t e i n , 1 9 7 9 ) . The upper pathway a l l o w s f o r t h e s i m u l t a n e o u s b i n d i n g of ATP and o r t h o p h o s p h a t e , and p r e d i c t s no i n h i b i t i o n of K:K exchange when b o t h [ATP] and [ P i ] are h i g h . Obvio u s l y , more work w i l l be r e q u i r e d t o apply t h e v a r i o u s tests t h a t w i l l v a l i d a t e o r i n v a l i d a t e competing models.

XI.

CONCLUSION

I t i s obvious t h a t t h e Albers-Post model, expanded t o i n c l u d e t h e l o w - a f f i n i t y s t i m u l a t i o n of potassium deo c c l u s i o n by ATP, has been extremely u s e f u l both i n t h e

BIOPHYSICAUBIOCHEMICALCORRELATIONS

617

i n t e r p r e t a t i o n of e x p e r i m e n t a l " f l u x " f i n d i n g s and i n t h e d e s i g n of c r i t i c a l t e s t s . The a p p a r e n t c o n t r a d i c t i o n between o v e r a l l ATPase k i n e t i c s and Na/K t r a n s p o r t k i n e t i c s a p p e a r s t o be r e s o l v a b l e w i t h o u t t h e need t o invoke h a l f - o f - t h e - s i t e s b e h a v i o r . Both h i g h and low a f f i n i t i e s f o r ATP can be accommodated by a s i n g l e - u n i t model. Na:Na exchange, ADP:ATP exchange, and K : K exchange a r e p r e d i c t e d by t h e model. Many of t h e v a r i o u s i o n and n u c l e o t i d e i n t e r a c t i o n s a r e q u a l i t a tively interpretable. Problems remain, however. There i s no unambiguous scheme f o r Na-ATPase a c t i v i t y ( E l - P breakdown or E2-P breakdown, o r b o t h ? ) . The model l a c k s an obvious l o c u s €or t h e h i g h - a f f i n i t y i n h i b i t o r y e f f e c t of e x t e r n a l sodium on ATPase, sodium e f f l u x , and ADP:ATP exchange. The s t r i k i n g s i m i l a r i t i e s between t h e k i n e t i c s of NaATPase and t h o s e of ADP:ATP exchange remain p u z z l i n g . A s f o r t h e p r e c i s e mechanism of K:K exchange and i t s requirement f o r ATP and o r t h o p h o s p h a t e , t h e f i n a l v e r d i c t i s n o t i n . None of t h e s e unanswered q u e s t i o n s , however, a p p e a r t o c a l l f o r a complete o v e r h a u l of t h e c u r r e n t way of t h i n k i n g about t h e sodium pump. A more d i s c o n c e r t i n g prospect i s t h e p o s s i b i l i t y t h a t anion movements might t a k e p l a c e v i a t h e pump machinery. a posteriori

ACKNOWLEDGMENT

The a u t h o r ' s r e s e a r c h i s s u p p o r t e d by N I H g r a n t N S 11223.

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622

PAUL DE WEER

Neufeld, A. H . , and Levy, H. M. (1969). A second o u a b a i n - s e n s i t i v e sodium-dependent adenosine t r i p h o s p h a t a s e i n b r a i n microsomes. J. B i o l . C h e m . 244, 6493-6497. P o s t , R. L . , and Sen, A. K. (1965). A n enzymatic mechanism of act i v e sodium and potassium t r a n s p o r t . J . Histochem. C y t o c h e m . 13, 105-112. P o s t , R. L . , Sen, A. K . , and R o s e n t h a l , A. S. (1965). A phosp h o r y l a t e d i n t e r m e d i a t e i n adenosine triphosphate-dependent sodium and potassium t r a n s p o r t a c r o s s kidney membranes. J. B i 0 1 . C h m . 24, 1437-1445. P o s t , R. L . , Kume, S . , Tobin, T . , O r c u t t , B . , and Sen, A. K. (1969) F l e x i b i l i t y of an a c t i v e c e n t e r i n sodium-plus-potassium J . G e n . P h y s i o l . 54, 306s-326s. adenosine t r i p h o s p h a t a s e . P o s t , R. L. Hegyvary, C . , and K u m e , S . (1972). A c t i v a t i o n by adenosine t r i p h o s p h a t e i n t h e p h o s p h o r y l a t i o n k i n e t i c s of sodium and potassium i o n t r a n s p o r t adenosine t r i p h o s p h a t a s e . J. B i o l . C h e m . 247, 6530-6540. Repke, K. R. H. , and Schiin, R. (1973). F l i p - f l o p model of ( N a K ) ATPase f u n c t i o n . Acta B i o l . Med. G e r . 3 1 , K19-K30. Robinson, J. D. (1976). The ( N a + + K+)-dependent ATPase. Mode of i n h i b i t i o n of ADP/ATP exchange a c t i v i t y by MgC12. B i o c h i m . B i o p h y s . A c t a 440, 711-722. Robinson, J . D. (1977). K+ s t i m u l a t i o n of ADP/ATP exchange c a t a l y z e d by t h e ( N a + + K+)-dependent ATPase. B i o c h i m . B i o p h y s . A c t a 484, 161-168. Sachs, J. R . (1972a). Sodium movements i n t h e human r e d blood c e l l . J. G e n . P h y s i o l . 56, 322-341. Sachs, J. R. (197233). Recoupling t h e Na-K pump. J . C l i n . Invest. 51, 3244-3247. Sachs, J. R. (1977). K i n e t i c e v a l u a t i o n o f t h e Na-K pump r e a c t i o n mechanism. J. P h y s i o l . (London) 273, 489-514. Sachs, J. R. (1979). A modified c o n s e c u t i v e model f o r t h e Na-K pump. I n "Na,K-ATPase: S t r u c t u r e and K i n e t i c s " (J. C. Skou Academic P r e s s , and J. G. Ndrby, e d s . ) , pp. 463-473. New York. Sachs, J. R. (1980). The o r d e r o f release o f sodium and a d d i t i o n of potassium i n t h e sodium-potassium pump r e a c t i o n mechanism. J. P h y s i o l . (London) 302, 219-240. Sachs, J. R. (1981a). Mechanistic i m p l i c a t i o n s of t h e potassiumpotassium exchange c a r r i e d o u t by t h e sodium-potassium pump. J. P h y s i o l . (London) 3 1 6 , 263-277. Sachs, J. R . (1981b). I n t e r n a l potassium s t i m u l a t e s t h e sodiumpotassium pump by i n c r e a s i n g c e l l ATP c o n c e n t r a t i o n . J. P h y s i o l . (London) 319 , 515-528. Sh.aw, T. I. (1954). Sodium and potassium movements i n r e d c e l l s . Ph.D. T h e s i s , Cambridge U n i v e r s i t y . Simons, T. J. B. (1974). Potassium:potassium exchange c a t a l y s e d by t h e sodium pump i n human r e d c e l l s . J. P h y s i o l . (London) 237 , 123-155.

BIOPHYSICAUBIOCHEMICAL CORRELATIONS

J.

623

Simons, T. B. (1975). The i n t e r a c t i o n o f ATP-analogues p o s s e s s i n g a blocked y-phosphate group w i t h t h e sodium pump i n human r e d cells. J. P h y s i o l . (London) 2 4 4 , 731-739. Skou, J . C. ( 1 9 6 0 ) . F u r t h e r i n v e s t i g a t i o n s on a Mg++ + Na+-activ a t e d adenosine t r i p h o s p h a t a s e , p o s s i b l y r e l a t e d t o t h e act i v e , l i n k e d t r a n s p o r t of Na+ and K+ a c r o s s t h e n e r v e membrane. B i o c h i m . B i o p h y s . A c t a 4 2 , 6-23. Smith, R. L . , Zinn, K . , and C a n t l e y , L. C. ( 1 9 8 0 ) . A s t u d y of t h e vanadate-trapped s t a t e of t h e (Na,K)-ATPase. Evidence a g a i n s t J. B i o l . C h e m . 2 5 5 , i n t e r a c t i n g n u c l e o t i d e s i t e models. 9852-9859. S t a h l , W. L. (1968) Sodium s t i m u l a t e d [14C] adenosine d i p h o s p h a t e a d e n o s i n e t r i p h o s p h a t e exchange a c t i v i t y i n b r a i n microsomes. J. N e u r o c h e m . 1 5 , 511-518. S t e i n , W. D. ( 1 9 7 9 ) . H a l f - o f - t h e - s i t e s r e a c t i v i t y and t h e Na,K-ATPase. In "Na,K-ATPase: S t r u c t u r e and K i n e t i c s " (J. C. Skou and J. G. Nqkby, e d s . ) , pp. 475-406. Academic Press, N e w York. S t e i n , W . D., L i e b , W. R . , K a r l i s h , S. J . D . , and E i l a m , Y. ( 1 9 7 3 ) . A model f o r a c t i v e t r a n s p o r t of sodium and potassium i o n s a s P r o c . N a t l . Acad. S c i . mediated by a tetrameric enzyme. U.S.A. 70, 275-278. T a n i g u c h i , K . , and P o s t , R. L. ( 1 9 7 5 ) . S y n t h e s i s o f a d e n o s i n e t r i p h o s p h a t e and exchange between i n o r g a n i c phosphate and a d e n o s i n e t r i p h o s p h a t e i n sodium and potassium i o n t r a n s p o r t adenosine t r i p h o s p h a t a s e . J. B i o l . C h e m . 2 5 0 , 30103018. Wildes, R. A . , Evans, H. J . , and Chiu, J. ( 1 9 7 3 ) . E f f e c t s o f cat i o n s on t h e adenosine diphosphate-adenosine t r i p h o s p h a t e exchange r e a c t i o n c a t a l y z e d by r a t b r a i n microsomes. B i o c h i m . B i o p h y s . A c t a 3 0 7 , 162-168. Yamaguchi, M . , and Tonomura, Y . ( 1 9 7 7 ) . K i n e t i c s t u d i e s on t h e ADP-ATP exchange r e a c t i o n c a t a l y z e d by N a + , K+-dependent ATPase. J. B i o c h e m . (Tokyo) 8 1 , 249-260.

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CURRENT TOPICS IN MEMBRANES AND TRANSPORT. VOLUME 19

Existence and Role of Occluded-Ion Forms of Na,K-ATPase I. M. G L ~ N A ND. D E. RICHARDS Physiological Laboratory University of Cambridge Cambridge, England

I.

INTRODUCTION

It has always seemed likely that in the course of its normal working cycle the sodium pump passes through states in which Na+ ions or K+ ions are trapped within the pump molecule so that they are unable to exchange with ions in either the extracellular or the intracellular medium. This article is concerned with two sets of relevant experiments. The first set shows that K+ ions, or more precisely Rb+ ions, can be occluded within the unphosphorylated form of the pump, and can enter and leave the occluded state by two different routes. The experiments of the second set are much more preliminary; they seem to show that Na+ ions can be occluded within the E l - P form of the phosphoenzyme, that is, the form that is attacked by ADP but is unaffected by K+ ions. The existence and, so far as they are known, the properties of these occluded-ion forms support the hypothesis that the Albers-Post chemical cycle is able to drive ion transport because of alterations 625

Copyright 0 1983 by Academic Press. Inc. All rights of reproduction in any form reserved. ISBN 0-12-1533190

I. M. GLYNN AND D. E. RICHARDS

626

in the accessibility of the ion binding sites--alterations that are brought about by the transfer of phospho groups and by changes in conformation between the El and E2 forms of the enzyme in both its phosphorylated and unphosphorylated states (see Karlish et al., 1978; Glynn et al., 1979).

11.

OCCLUSION OF K+ (OR F&+)

IONS

The first clear suggestion that Kf ions could be occluded within the dephosphoenzyme came from the experiments of Post et a l . (1972). They studied the rate of rephosphorylation of enzyme that had just been dephosphorylated and found that the rate of rephosphorylation differed depending on which congener of potassium had been used to catalyze the hydrolysis. Furthermore, this was true even if the experiments were done in such a way that during rephosphorylation the conditions were identical. In other words, the enzyme appeared to remember which cation had catalyzed the hydrolysis. To explain this "memory," they suggested that the catalyzing ions remained trapped within the enzyme molecule and were released only later after a slow conformational change. Since they also found that the enzyme became capable of rephosphorylation sooner if high concentrations of ATP were present, they suggested that the slow conformational change was accelerated by the binding of ATP at low-affinity sites. This hypothesis was extremely attractive, though, of course, the experimental evidence proved only that the hydrolysis products were different depending on the nature of the catalyzing ions, not that the difference lay in the nature of ions occluded within the enzyme molecule. The hypothesis of Post et a l . received strong, although indirect, support from experiments with the fluorescent ATP and ADP analogs formycin triphosphate and formycin diphosphate (Karlish et a l . , 1978) and from studies of the intrinsic fluorescence of the enzyme (Karlish and Yates, 1978). These fluorescence studies, which were done on Na,K-ATPase prepared from pig kidney outer medulla, showed that the change in the form of the enzyme when it was transferred from a predominantly potassium to a predominantly sodium medium was remarkably slow (k = 0.2 sec-1 at 2OoC) and was accelerated by nucleotides in high concentration. Both the slowness of the change and the acceleration by nucleotides suggested that in potassium media the enzyme

ROLE OF OCCLUDED-ION FORMS OF Na,K-ATPase

627

e x i s t s i n a form t h a t i s i d e n t i c a l w i t h t h e h y p o t h e t i c a l o c c l u d e d K form. The s l o w n e s s o f t h e f l u o r e s c e n c e changes i n t h e abs e n c e of n u c l e o t i d e s s u g g e s t e d t h a t i t might be p o s s i b l e t o t e s t f o r o c c l u s i o n d i r e c t l y by s u s p e n d i n g enzyme i n a s u i t a b l e medium c o n t a i n i n g 42K o r , more c o n v e n i e n t l y 86Rb, f o r c i n g i t r a p i d l y down a cation-exchange column, and measuring t h e amount of r a d i o a c t i v i t y t h a t emerged. Whether t h e t e c h n i q u e worked would depend on whether i t proved p o s s i b l e t o f i n d a f l o w r a t e t h a t w a s f a s t enough f o r most o f t h e enzyme t o e s c a p e from t h e column b e f o r e i t s c o n f o r m a t i o n had changed, y e t w a s slow enough f o r t h e r e s i n t o be a b l e t o remove n e a r l y a l l o f t h e free 86Rb+ i o n s . I n t h e e v e n t , t h e t e c h n i q u e d i d work (see T a b l e I ) . Enzyme suspended i n a Na+-free medium cont a i n i n g 1 0 0 ~.IM 86RbC1 c a r r i e d more r a d i o a c t i v i t y t h r o u g h a c a r b o x y l i c r e s i n column t h a t enzyme suspended i n a s i m i l a r medium c o n t a i n i n g ATP o r ADP a t c o n c e n t r a t i o n s known t o be s u f f i c i e n t t o c o n v e r t t h e enzyme i n t o t h e E l form (Beau96 and Glynn, 1979b, 1 9 8 0 ) . Subsequent e x p e r i m e n t s showed t h a t 1 5 mM N a + , which would a l s o be exp e c t e d t o c o n v e r t t h e enzyme t o t h e E l form, c a u s e d a s i m i l a r r e d u c t i o n i n t h e amount of r a d i o a c t i v i t y c a r r i e d t h r o u g h t h e column. The e a r l y e x p e r i m e n t s w e r e v e r y e x t r a v a g a n t of enzyme, a b o u t 1 mg b e i n g r e q u i r e d p e r t r i a l . F o r t u n a t e l y , w e d i s c o v e r e d t h a t w e c o u l d o b t a i n e q u a l l y good r e s u l t s w i t h o n l y 1 / 2 0 of t h e amount of enzyme by u s i n g a s u l f o n i c acid r e s i n (Dowex 50W, 100-200 or 200-400 mesh, 8% c r o s s - l i n k e d ) i n s t e a d of t h e c a r b o x y l i c a c i d r e s i n . W e a l s o m o d i f i e d t h e a p p a r a t u s by i n c o r p o r a t i n g a g e a r b o x between t h e s t e p p i n g motor and t h e f r i c t i o n c l u t c h i n t h e s y r i n g e d r i v e , so t h a t w e c o u l d v a r y t h e c o n t a c t t i m e w i t h t h e r e s i n between 2 0 0 msec and 2 0 sec. F i g u r e 1 shows t h e r e s u l t s of a n e x p e r i m e n t i n which w e v a r i e d t h e t i m e s p e n t by t h e enzyme i n p a s s i n g down t h e r e s i n column and r e l a t e d t h i s t o t h e amount o f o c c l u d e d Rb+ i n t h e enzyme emerging from t h e column (Glynn and R i c h a r d s , 1 9 8 0 ) . ( I n t h i s and o t h e r e x p e r i ments t h e amount o f o c c l u d e d Rb+ w a s e s t i m a t e d from t h e r e d u c t i o n i n t h e amount o f r a d i o a c t i v i t y emerging from t h e column t h a t w a s c a u s e d by i n c l u d i n g s u f f i c i e n t N a + o r n u c l e o t i d e i n t h e o r i g i n a l enzyme s u s p e n s i o n t o conv e r t v i r t u a l l y a l l of t h e enzyme i n t o t h e E l form.) The l o s s o f Rb+ f o l l o w e d a s i n g l e e x p o n e n t i a l w i t h a r a t e c o n s t a n t ( a t 20') of a b o u t 0 . 2 sec-1, which i s i n good agreement w i t h estimates o f t h e r a t e c o n s t a n t o f t h e c o n f o r m a t i o n a l change from t h e E2 t o t h e E l form obtained f r o m fluorescence experiments.

TABLE I.

Experiment

+

Nucleotide in suspension medium

1 2 2

+

Retention of Rb by Na,K-ATPase a f t e r the Removal of Free Rb through a Cation-Exchange Resinab Protein content of effluent (mg)

None ATP

0.628 0.657

None

0.568 0.572 0.582

mpJ

2 mM ATP 2 mM ADP

* _+

0.025 0.027

? 0.019

f 0.018 0.010

_+

Rb content of effluent (nmole) 0.992 0.293

_+

_+

0.051 0.003

f 0.042 0.351 f 0.025 0.318 0.012 1.160

*

by Passage o f t h e Enzyme

Extra

Rb i n absence o f nucleotide (nmole)

E x t r a Rb (nmole p e r mg p r o t e i n )

f 0.051

1.12 f 0.09

0. 825c? 0.044

1.45 f 0.09

0.699

-

-

-

-

-

a F r o m Beau96 and G l y n n , 1 9 7 9 b . b R e p r i n t e d b y p e r m i s s i o n from N a t u r e , V o l . 2 8 0 , p p . 510-512, C o p y r i g h t ( c ) 1 9 7 9 M a m i l l a n Journals Limited. %lculated b y s u b t r a c t i n g the a v e r a g e o f the Rb contents o f the ATP and ADP s a m p l e s f r o m the a v e r a g e Rb content o f the N u c l e o t i d e - f r e e s a m p l e s .

ROLE OF OCCLUDED-ION FORMS OF Na,K-ATPase

+

zE=

0.1

J ,

-

0

a

Fig. 1.

I

629

I

5 10 Time s p e n t on column (seconds)

R e l e a s e o f Rb'

f r o m Na,K-ATPase.

I

15

The graph shows

the amount o f Rb+ c a r r i e d t h r o u g h the resin c o l u m n b y the e n z y m e , a s a f u n c t i o n o f the t i m e s p e n t b y the e n z y m e i n p a s s i n g down the

c o l u m n . Na,K-ATPase ( s p e c i f i c a c t i v i t y 1 2 . 4 p m o l e s / m g / m i n ) , p r e p a r e d f r o m p i g k i d n e y o u t e r m e d u l l a b y the m e t h o d of J d r g e n s e n ( 1 9 7 4 ) , was s u s p e n d e d a t a c o n c e n t r a t i o n o f 8 0 pg/rnl i n a medium c o n t a i n i n g 100 pM 86RbC1, 100 mM T r i s / T r i s - C l (pH 7 . 4 a t 20QC) , 0.5 mM EDTA ( T r i s s a l t ) , w i t h or w i t h o u t 1 5 mM NaCl. Portions o f e n z y m e s u s p e n s i o n , 0.5 m l i n v o l u m e , w e r e p a s s e d a t d i f f e r e n t s p e e d s t h r o u g h c o l u m n s o f Dowex SOW x 8 s u l f o n i c resin (100-200 mesh) i n the K f f o r m . T h e t e m p e r a t u r e was 19OC. T h e d i m e n s i o n s and p r e p a r a t i o n o f the c o l u m n s , and the p r o c e d u r e f o r p a s s i n g the e n z y m e t h r o u g h t h e m w e r e a s d e s c r i b e d b y Beau96 and G l y n n ( 1 9 7 9 b ) . T h e e f f l u e n t s w e r e a n a l y z e d f o r 86Rb and t o t a l p r o t e i n . Each p o i n t r e p r e s e n t s t h e d i f f e r e n c e (+SE) b e t w e e n the mean o f three d e t e r m i n a t i o n s w i t h no NaCl i n the s u s p e n d i n g medium a n d three d e t e r m i n a t i o n s w i t h 15 mM NaCl i n the s u s p e n d i n g medium. From t h e s l o p e of the r e g r e s s i o n l i n e , the r a t e c o n s t a n t f o r r e l e a s e o f o c c l u d e d Rb+ = 0 . 2 0 see-1.

630

I. M. GLYNN AND D. E. RICHARDS

+

By measuring t h e amount o f Rb c a r r i e d t h r o u g h t h e column under d i f f e r e n t c o n d i t i o n s , w e were a l s o a b l e t o examine t h e e f f e c t s o f v a r i o u s l i g a n d s and i n h i b i t o r s on t h e r a t e of t h e c o n f o r m a t i o n a l change l e a d i n g t o Rb+ r e l e a s e (Glynn and R i c h a r d s , 1 9 8 0 ) . An i m p o r t a n t negat i v e r e s u l t w a s t h a t t h e r a t e of release was t h e same whether t h e enzyme was p a s s e d down a K+-loaded o r a Na+l o a d e d column. T h i s i m p l i e s t h a t t h e r a t e of release w a s t h e same whether t h e enzyme w a s i n a Na+-containing o r a K+-containing medium. S i n c e w e know t h a t , a t t h e N a + c o n c e n t r a t i o n s t h a t t h e enzyme would have e x p e r i e n c e d i n p a s s i n g down t h e Na+-loaded column, t h e e q u i l i b r i u m form of t h e enzyme i s t h e E l form, w e c a n c o n c l u d e t h a t t h e e f f e c t of N a + i o n s on t h e e q u i l i b r i u m between E l and E z a r i s e s s o l e l y from a s l o w i n g of t h e r a t e o f convers i o n o f E l t o E 2 , and n o t from an a c c e l e r a t i o n of t h e r a t e o f c o n v e r s i o n of E 2 t o E l . O u r n e x t t a s k was t o d i s c o v e r whether ATP o r ADP a c c e l e r a t e d t h e c o n f o r m a t i o n a l change l e a d i n g t o Rb+ rel e a s e . T h i s was t e c h n i c a l l y a more d i f f i c u l t problem. W e knew t h a t t h e n u c l e o t i d e s d i s p l a c e t h e e q u i l i b r i u m between E l and E2 t o t h e El f o r m , b u t t h a t c o u l d r e f l e c t a n e f f e c t on e i t h e r t h e f o r w a r d o r t h e backward r a t e c o n s t a n t s . To d e m o n s t r a t e t h a t t h e n u c l e o t i d e s a c c e l e r a t e t h e c o n v e r s i o n o f E 2 t o E l , it w a s n e c e s s a r y t o add n u c l e o t i d e o n l y j u s t b e f o r e t h e enzyme e n t e r e d t h e res i n , s o t h a t t h e t o t a l p e r i o d between e x p o s u r e t o ATP and emergence from t h e r e s i n w a s s m a l l compared t o t h e t i m e c o n s t a n t f o r t h e c o n f o r m a t i o n a l change ( a b o u t 5 s e c ) . W e d i d t h i s by p u t t i n g a b o u t 0 . 2 m l of Sephadex G-25, loaded w i t h n u c l e o t i d e , j u s t above t h e r e s i n . When t h e enzyme s u s p e n s i o n w a s f o r c e d t h r o u g h t h e column, t h e n u c l e o t i d e i n i t i a l l y p r e s e n t i n t h e v o i d volume of t h e Sephadex w a s swept away, b u t f u r t h e r n u c l e o t i d e d i f f u s e d from t h e Sephadex p a r t i c l e s . P r e l i m i n a r y e x p e r i ments showed t h a t , a t t h e flow r a t e s w e were u s i n g , t h e enzyme s u s p e n s i o n emerging from t h e column c o n t a i n e d n u c l e o t i d e a t a p p r o x i m a t e l y o n e - q u a r t e r of t h e i n i t i a l c o n c e n t r a t i o n i n t h e Sephadex. By v a r y i n g t h e nucleot i d e c o n c e n t r a t i o n , and s e e i n g how much o c c l u d e d Rb+ emerged a t t h e bottom of t h e r e s i n , w e were a b l e t o show t h a t b o t h ADP and ATP g r e a t l y a c c e l e r a t e d t h e r e l e a s e of Rb+. Both a c t e d w i t h a low a f f i n i t y and, s i n c e Mg w a s a b s e n t and EDTA w a s p r e s e n t , b o t h presumably acted w i t h o u t p h o s p h o r y l a t i n g t h e enzyme (Glynn and R i c h a r d s , 1980).

ROLE OF OCCLUDED-ION FORMS OF Na,K-ATPase

111.

631

A SECOND ROUTE TO THE OCCLUDED Rb+ FORM

The e x p e r i m e n t s d e s c r i b e d above show t h a t ( i ) N a , K A T P a s e i n i t s u n p h o s p h o r y l a t e d form i n a Na+-free m e d i u m can o c c l u d e Rb+ i o n s , ( i i ) t h e c o n f o r m a t i o n a l change t h a t a l l o w s Rb+ i o n s t o be r e l e a s e d i s v e r y s l o w ( k = 0 . 2 sec-1 a t 20'1, and ( i i i ) t h e r a t e o f r e l e a s e i s a c c e l e r a t e d by n u c l e o t i d e s , a c t i n g w i t h o u t phosphor y l a t i o n and w i t h a low a f f i n i t y . The enzyme i n a Na+-free Rb+-containing medium t h e r e f o r e h a s a l l t h e p r o p e r t i e s o f t h e h y p o t h e t i c a l o c c l u d e d K + form p o s t u l a t e d by P o s t et a l . I t i s i m p o r t a n t t o n o t e , however, t h a t w e r e a c h e d o u r o c c l u d e d i o n form by q u i t e a d i f f e r e n t r o u t e . W e s i m p l y p u t u n p h o s p h o r y l a t e d enzyme i n a Na+-free, Rb+-containing medium; P o s t e t a l . g e n e r a t e d t h e i r o c c l u d e d K + form by p h o s p h o r y l a t i n g t h e enzyme i n a high-Na+ medium, and t h e n a l l o w i n g t h e phosphoenzyme If the t o b e h y d r o l y z e d i n t h e p r e s e n c e o f K+ i o n s . form i n which t h e enzyme e x i s t s i n a Rb+-containing, Na+-free m e d i u m i s i d e n t i c a l w i t h t h e h y p o t h e t i c a l occ l u d e d i o n form o f P o s t e t a l . , it s h o u l d be p o s s i b l e t o u s e o u r r a p i d ion-exchange t e c h n i q u e t o d e m o n s t r a t e t h e e x i s t e n c e of o c c l u d e d Rb+ i o n s when t h e o c c l u d e d Rb+ form i s g e n e r a t e d by, what I a m a f r a i d w e have t a k e n t o c a l l i n g somewhat i r r e v e r e n t l y , t h e " P o s t a l " r o u t e . The d i f f i c u l t y w i t h a t t e m p t i n g t o d e m o n s t r a t e occ l u d e d Rb+ i o n s i n newly formed dephosphoenzyme i s t h a t some ATP must be p r e s e n t i n o r d e r t o p h o s p h o r y l a t e t h e enzyme i n t h e f i r s t p l a c e , b u t i f t h e r e i s t o o much t h e c o n f o r m a t i o n a l change w i l l be a c c e l e r a t e d , and any occ l u d e d Rb+ form t h a t i s g e n e r a t e d w i l l l o s e i t s Rb+ bef o r e t h e enzyme emerges from t h e column. I f , however, one u s e s v e r y low c o n c e n t r a t i o n s of ATP i n t h e enzyme s u s p e n s i o n , a11 o f t h e ATP w i l l be h y d r o l y z e d b e f o r e t h e e x p e r i m e n t s t a r t s , because t h e enzyme c o n c e n t r a t i o n i s h i g h and N a + , Mg2+, and Rb+ a r e a l l p r e s e n t . To a v o i d t h e s e d i f f i c u l t i e s , w e suspended t h e enzyme i n a s o l u t i o n c o n t a i n i n g 1 5 mM N a + , 1 0 0 mM T r i s b u f f e r (pH 7.41, 1 mM Mg2+, and 1 0 0 p~ 86Rb+, and added ATP t o it by p a s s i n g t h e s u s p e n s i o n t h r o u g h a l a y e r o f Sephadex j u s t above t h e r e s i n (Glynn and R i c h a r d s , 1 9 8 1 ) . The Sephadex w a s l o a d e d w i t h a s o l u t i o n s i m i l a r i n c o m p o s i t i o n t o t h a t u s e d f o r s u s p e n d i n g t h e enzyme, b u t c o n t a i n i n g i n a d d i t i o n 4 0 pM ATP o r , i n t h e c o n t r o l columns, 40 p M ADP. The r e s u l t s of one e x p e r i m e n t a r e summarized i n T a b l e 11. They show t h a t i n t h e p r e s e n c e of a low conc e n t r a t i o n o f ATP, e x t r a r a d i o a c t i v i t y w a s carried t h r o u g h t h e column. ADP w a s i n e f f e c t i v e . F u r t h e r exp e r i m e n t s (Glynn and R i c h a r d s , 1981) showed t h a t t h e

632

I. M. GLYNNAND D. E. RICHARDS

TABLE 11.

+

Experiment Showing Occlusion of Rb i n a High-Na+, Mg-Containing Medium When ATP Is P r e s e n t a

Nucleoti.de i n Sephadex ~~~~

~

None 40 ~ . I MATPb 40 pM ADP

Rb i n e f f l u e n t from r e s i n ( m o l e per mg p r o t e i n ) ~~

0.30 ? 0.05 1.15 t 0.01 0.31 ?c 0.02

a Enzyme, suspended i n a medium containing 100 mM T r i s b u f f e r (pH 7.4), 15 mM N a C l , 1 . 5 mM MgCl2, 0.5 mM EDTA, and 100 pM 86RbC1 a t room temperature, was forced f i r s t through Sephadex loaded with ATP or ADP a t the concentrations shown, and then immediately through Dowex 50W resin i n the Na form. The e f f l u e n t s were analyzed f o r radioactivity and protein content. Each f i g u r e i n the table represents the mean ( 2 S E ) o f four determinations. bThe concentration o f nucleotide experienced b y the enzyme i s probably only about one-quarter o f the concentration i n the Sephadex.

e f f e c t of ATP was n o t seen i n t h e absence of Mg, and t h a t ATP could n o t be r e p l a c e d by t h e nonphosphorylating Furthermore, i f t h e ATP c o n c e n t r a t i o n a n a l o g AMP-PNP. was i n c r e a s e d , less e x t r a Rb+ emerged from t h e column-presumably because of t h e a c c e l e r a t i n g e f f e c t of ATP on t h e c o n f o r m a t i o n a l change t h a t r e l e a s e s occluded Rb+.

IV.

STOICHIOMETRY

So f a r , n o t h i n g has been s a i d about t h e s t o i c h i o metry of Rb+ o c c l u s i o n . T o d e t e r m i n e how many Rb+ i o n s a r e occluded p e r p h o s p h o r y l a t i o n s i t e (or ouabain bindi n g s i t e ) , it i s n e c e s s a r y t o s o l v e t h r e e problems. F i r s t , t h e amount of occluded Rb+ emerging a t t h e b o t tom of t h e r e s i n column must be c o r r e c t e d f o r t h e Rb+ l o s t w h i l e t h e enzyme i s on t h e column. S i n c e w e know b o t h t h e r a t e c o n s t a n t f o r t h e l o s s of Rb+ from t h e enzyme and t h e f l o w r a t e down t h e column, t h i s c o r r e c t i o n p r e s e n t s l i t t l e d i f f i c u l t y and i s i n any c a s e r a t h e r small ( a b o u t 2 0 % ) . A second problem i s t o a s s e s s , from t h e amount of Rb+ occluded a t any g i v e n Rb+ c o n c e n t r a t i o n , how much would be occluded a t s a t u r a t i n g Rb+ conc e n t r a t i o n s . T o do t h i s , w e have e s t i m a t e d t h e amount of Rb+ occluded a t RbS c o n c e n t r a t i o n s between 1 2 ~ . I M and 5 0 0 p ~ . The r e l a t i o n between t h e amount of Rb+ occluded

ROLE OF OCCLUDED-ION FORMS OF Na,K-ATPase

633

+

and t h e R b c o n c e n t r a t i o n i s s l i g h t l y S-shaped a t v e r y low Rb+ c o n c e n t r a t i o n s and g i v e s a c u r v e d S c a t c h a r d p1ot.l The f i g u r e s can b e f i t t e d r a t h e r w e l l by assumi n g t h a t , f o r o c c l u s i o n t o o c c u r , e i t h e r two o r t h r e e i d e n t i c a l b i n d i n g s i t e s must b e o c c u p i e d by Rb+, and t h e e s t i m a t e d amount of Rb+ o c c l u d e d a t s a t u r a t i n g conc e n t r a t i o n s i s n o t very d i f f e r e n t whichever assumption i s made. The t h i r d problem i n d e t e r m i n i n g t h e s t o i c h i o m e t r y i s t o estimate t h e number of p h o s p h o r y l a t i o n o r o u a b a i n b i n d i n g s i t e s p e r m i l l i g r a m of enzyme, and t h i s h a s been done ( i ) by m e a s u r i n 32P i n c o r p o r a t i o n from [32P]ATP, ( i i ) by m e a s u r i n g 3 3P i n c o r p o r a t i o n from [ 3 2 P ] P i i n t h e p r e s e n c e of Mg and o u a b a i n , ( i i i ) by m e a s u r i n g o u a b a i n b i n d i n g i n t h e p r e s e n c e o f ATP, Mg, and N a + , and ( i v ) by m e a s u r i n g o u a b a i n b i n d i n g i n t h e p r e s e n c e o f Mg and i n o r g a n i c p h o s p h a t e . A l l f o u r methods g a v e s i m i l a r r e s u l t s and t h e s t o i c h i o m e t r y worked o u t , s u r p r i s i n g l y , as 3.0 2 0 . 1 Rb+ i o n s bound p e r phosphorylation o r ouabain binding s i t e . W e have a l s o done one e x p e r i m e n t i n which w e comp a r e d t h e amount o f Rb+ o c c l u d e d by t h e d i r e c t r o u t e w i t h t h e amount o c c l u d e d by t h e r o u t e i n v o l v i n g phosp h o r y l a t i o n and d e p h o s p h o r y l a t i o n . The estimate of t h e amount o c c l u d e d by t h e r o u t e i n v o l v i n g p h o s p h o r y l a t i o n and d e p h o s p h o r y l a t i o n was 2 . 6 f 0 . 1 , b u t t h i s estimate s h o u l d b e r e g a r d e d a s a l o w e r l i m i t , s i n c e w e c a n n o t be s u r e t h a t a l l of t h e enzyme would have been phosphoryl a t e d and d e p h o s p h o r y l a t e d .

V.

THE ROLE OF THE OCCLUDED K + FORM

The e x p e r i m e n t s d e s c r i b e d above show t h a t t h e r e are two r o u t e s t o t h e o c c l u d e d Rb+ form--and t h e r e f o r e p r e sumably t o t h e o c c l u d e d K+ form--of t h e enzyme. If we i g n o r e m u l t i p l e i o n b i n d i n g , t h e s e two r o u t e s c o u p l e d back-to-back may b e w r i t t e n :

+ + El

K

L 7

El-K

L

E2-K

7

E2-P.K -E

P

+ K+

2-

'imrg

' I n one experiment, i n which we took great care t o exclude a l l traces of M q by using 2 mM CDTA, the saturation curve was not i n f l e c t e d and the i n f l e c t i o n was restored b y the addition o f Mg. Mg d i d not much a l t e r the saturation l e v e l , however.

634

I. M.GLYNN AND D. E. RICHARDS

W e know from a good d e a l of i n d i r e c t e v i d e n c e , and from some d i r e c t e v i d e n c e ( B l o s t e i n and Chu, 1 9 7 7 ) , t h a t t h e s i t e s a t which K+ i o n s c a t a l y z e t h e h y d r o l y s i s of phosIf phoenzyme are e x t r a c e l l u l a r and of h i g h a f f i n i t y . t h e s i t e s a t which K+ i o n s b i n d t o E l were i n t r a c e l l u l a r and of l o w a f f i n i t y , t h e two r o u t e s t o t h e occluded K+ form of t h e enzyme would p r o v i d e a mechanism w i t h many of t h e p r o p e r t i e s r e q u i r e d t o a c c o u n t f o r K + f l u x e s t h r o u g h t h e pump a c t i n g i n i t s v a r i o u s modes--Na+-K+ exchange, K+-K+ exchange, and pump r e v e r s a l . The relat i o n between K+ c o n c e n t r a t i o n and t h e r a t e o f c o n v e r s i o n of E l - N a t o E2-K l e d K a r l i s h et a l . (1978) t o conclude t h a t t h e a f f i n i t y of E l f o r K+ i o n s was l o w , and t h i s c o n c l u s i o n was s u p p o r t e d by s t u d i e s of t h e e f f e c t s of ATP and K+ on t h e e q u i l i b r i u m between E l and E 2 (Beaugg and Glynn, 1980; J d r g e n s e n and K a r l i s h , 1 9 8 0 ) . Recently K a r l i s h and P i c k ( 1 9 8 1 ) have produced i n d i r e c t , though r a t h e r s t r o n g , e v i d e n c e t h a t t h e K+ b i n d i n g s i t e s on E l are i n t r a c e l l u l a r . W e t h e r e f o r e s u s p e c t t h a t t h e equat i o n g i v e n above does i n f a c t r e p r e s e n t t h e mechanism by which K+ i o n s a r e moved inward o r outward a c r o s s t h e membrane by t h e sodium pump, though t h e r e i s s t i l l some r e a s o n f o r c a u t i o n f o r w e have n o t y e t been a b l e t o prove t h a t p h o s p h o r y l a t i o n of Ez-Rb+ by i n o r g a n i c phosp h a t e l e a d s t o t h e r e l e a s e of Rb'. Attempts t o p r o v e t h i s p o i n t by p a s s i n g t h e Rb+-containing enzyme through Sephadex loaded w i t h K + , Mg, and phosphate, p l a c e d j u s t above t h e r e s i n , m e t w i t h o n l y p a r t i a l success, p o s s i b l y because w e f a i l e d t o a c h i e v e c o n d i t i o n s i n which a l l of t h e enzyme would have been t r a n s i e n t l y p h o s p h o r y l a t e d .

VI.

OCCLUSION OF Na+ IONS

I n 1 9 7 1 , Glynn and Hoffman p o i n t e d o u t t h a t t h e dependence of Na+-Na+ exchange on ADP and i t s i n h i b i t i o n by oligomycin c o u l d both be e x p l a i n e d by supposing t h a t , i n t h e c o u r s e of t h e exchange, Na+ i o n s become temporar i l y t r a p p e d i n a form of phosphoenzyme t h a t can r e l e a s e Na+ t o t h e i n t e r i o r o n l y a f t e r t r a n s f e r r i n g i t s phospho group t o ADP, and c a n r e l e a s e Na+ t o t h e e x t e r i o r only a f t e r t h e o l i g o m y c i n - s e n s i t i v e c o n v e r s i o n of t h e phosphoenzyme from t h e E l - P form t o t h e E2-P f o r m . In the l a s t few weeks, w e have been u s i n g o u r r a p i d ionexchange t e c h n i q u e t o t r y t o determine whether E l - P d o e s , i n f a c t , c o n t a i n occluded Na+ i o n s . T e s t i n g f o r Na+ o c c l u s i o n i s i n t r i n s i c a l l y more d i f f i c u l t t h a n t e s t i n g f o r K+ o c c l u s i o n ; whereas t h e

ROLE OF OCCLUDED-ION FORMS OF Na,K-ATPase

635

change from E2-K ( t h e h y p o t h e t i c a l o c c l u d i n g form) t o ( t h e h y p o t h e t i c a l r e l e a s i n g form) i s slow i n t h e absence of n u c l e o t i d e , t h e c o n v e r s i o n o f E l - P N a ( t h e h y p o t h e t i c a l o c c l u d i n g form) t o E2-PNa ( t h e h y p o t h e t i c a l r e l e a s i n g form) i s f a s t - - j u s t how f a s t b e i n g t h e s u b j e c t o f some c o n t r o v e r s y ( c f . Mardh, 1975; P l e s n e r e t al. , 1 9 8 1 ) . To l o o k f o r N a + o c c l u s i o n , it is t h e r e f o r e n e c e s s a r y t o b l o c k t h e c o n v e r s i o n of E -P t o E 2 - P , and we have a t t e m p t e d t o do t h i s by p r e i n c u i a t i n g t h e enzyme w i t h N-ethylmaleimide under c o n d i t i o n s t h a t l e a d t o t h e l o s s o f a b o u t 80% of t h e A T P a s e a c t i v i t y and a b o u t 5 0 % o f t h e p h o s p h o r y l a t a b l e s i t e s (see Beauge and Glynn, 1 9 7 9 a ) . Because it i s u n l i k e l y t h a t t h i s t r e a t ment b l o c k s t h e c o n v e r s i o n c o m p l e t e l y , it i s n e c e s s a r y t o l o o k f o r o c c l u s i o n s h o r t l y a f t e r t h e enzyme i s f i r s t phosphorylated. W e t h e r e f o r e u s e t h e Sephadex method t o a r r a n g e t h a t t h e enzyme i s exposed t o ATP f o r o n l y a b r i e f p e r i o d ( a b o u t 0 . 2 sec) b e f o r e i t e n c o u n t e r s t h e r e s i n . This procedure a l s o has t h e advantage t h a t desp i t e t h e h i g h c o n c e n t r a t i o n o f enzyme, l i t t l e ADP i s produced, and t h i s i s i m p o r t a n t because dephosphorylat i o n o f E l - P by ADP would release any N a + t h a t w a s ocI f t h e hypothesis t h a t El-P occludes Na+ ions cluded. i s c o r r e c t , t h e enzyme s h o u l d c a r r y N a + i o n s t h r o u g h t h e columns o n l y under c o n d i t i o n s t h a t a l l o w E 1 P t o be formed and t h a t p r e v e n t b o t h i t s c o n v e r s i o n t o E2-P and i t s d e p h o s p h o r y l a t i o n by ADP. The enzyme s h o u l d , t h e r e f o r e , c a r r y o c c l u d e d N a + t h r o u g h t h e columns o n l y i f ( i ) it has been p r e t r e a t e d so a s t o b l o c k t h e c o n v e r s i o n o f E l - P t o Ez-P, ( i i ) c o n d i t i o n s are s u i t a b l e f o r phosp h o r y l a t i o n ( i . e . , ATP and Mg are b o t h p r e s e n t ) , and ( i i i ) ADP i s n o t p r e s e n t i n more t h a n v e r y low concent r a t i o n s . F i g u r e 2 shows t h e r e s u l t s of f o u r e x p e r i ments, done under s l i g h t l y d i f f e r e n t c o n d i t i o n s . A l l show t h a t e x t r a N a + i s c a r r i e d t h r o u g h t h e columns when ATP i s p r e s e n t , and t h e l a s t e x p e r i m e n t a l s o shows t h a t ADP a t a c o n c e n t r a t i o n t o o low t o a l l o w e f f e c t i v e compet i t i o n w i t h ATP c a n n e v e r t h e l e s s p r e v e n t t h e e f f e c t of ATP. F u r t h e r e x p e r i m e n t s show t h a t r e s u l t s l i k e t h e s e c a n n o t be o b t a i n e d i n t h e a b s e n c e of Mg o r i f t h e p r e t r e a t m e n t w i t h N-ethylmaleimide i s o m i t t e d . The o b v i o u s weakness o f t h e s e e x p e r i m e n t s i s t h a t t h e amount of “ATP-dependent Na+” t h a t i s c a r r i e d t h r o u g h t h e columns i s o n l y a s m a l l f r a c t i o n of t h e amount t h a t one would e x p e c t i f a b o u t 50% o f t h e enzyme were p h o s p h o r y l a t e d t o E l - P and most of t h a t E l - P s u r v i v e d t o emerge a t t h e bottom o f t h e r e s i n . F o r t u n a t e l y f o r t h e h y p o t h e s i s , i t t u r n s o u t t h a t when enzyme i s p a s s e d t h r o u g h Sephadex, u s i n g a f l o w r a t e and condit i o n s s i m i l a r t o t h o s e used i n the experiments of Fig. 2 , El-K

636

I. M. GLYNN AND D.E. RICHARDS

200 -

200,

i?

F

bQ

L

aJ

Q

0

Z

0

z

-

E

-0 E

Q

Q

0-

0

ATP

ATP (100pM) (100yM)

200

i? L aJ

€

Cl 0

Z

-

E

a

-

DP

0 ATP

(lOOpM1

F i g . 2 . Four e x p e r i m e n t s s h o w i n g t h a t ATP a l l o w s the NEMt r e a t e d e n z y m e t o c a r r y e x t r a ( p r e s u m a b l y o c c l u d e d ) Na+ t h r o u g h the resin columns. NEM-treated e n z y m e , s u s p e n d e d i n m e d i a c o n t a i n i n g 1 0 0 mM T r i s b u f f e r (pH 7 . 4 ) , 1 . 5 mM MqCl2, 0.5 mM E D T A , and 200 p M 22NaC1 a t room t e m p e r a t u r e , was f o r c e d f i r s t t h r o u g h S e p h a d e x l o a d e d w i t h ATP or ADP a t the c o n c e n t r a t i o n s shown i n the f i g u r e and then i m m e d i a t e l y t h r o u g h Dowex 50W i n the Na form. T h e e f f l u e n t s w e r e a n a l y z e d f o r r a d i o a c t i v i t y and p r o t e i n content. Each column i n the f i g u r e r e p r e s e n t s the mean (+SE) o f f o u r d e t e r m i n a tions.

ROLE OF OCCLUDED-ION FORMS OF Na,K-ATPase

637

o n l y a s m a l l f r a c t i o n of t h e enzyme becomes phosphorylat e d . I f w e t a k e t h i s i n t o a c c o u n t , t h e amount of occ l u d e d N a + t h a t w a s found i n t h o s e e x p e r i m e n t s i s of t h e e x p e c t e d o r d e r of magnitude. W e are n o t y e t i n a p o s i t i o n t o t a l k about stoichiometry, but we can say t h a t t h e r e i s a s t r o n g p r i m a f a c i e case t h a t El-P c a n o c c l u d e Na+ ions. F u r t h e r m o r e , s i n c e t h e r e i s a g r e a t d e a l of i n d i r e c t evidence t h a t it i s i n t r a c e l l u l a r N a + binding a t h i g h - a f f i n i t y s i t e s t h a t promotes t h e phos h o r y l a t i o n of E l by ATP, and t h a t it i s e x t r a c e l l u l a r N a p b i n d i n g a t l o w - a f f i n i t y s i t e s t h a t promotes t h e c o n v e r s i o n o f E2-P t o E l - P , i t seems t h a t t h e two p r o b a b l e r o u t e s t o t h e occluded-Na+ form can a l s o be c o u p l e d back t o back t o produce a sequence of r e a c t i o n s c a p a b l e of t r a n s p o r t i n g i o n s a c r o s s t h e membrane.

REFERENCES

Beaug6, L. A . , and Glynn, I. M. ( 1 9 7 9 a ) . Sodium i o n s , a c t i n g a t h i g h - a f f i n i t y e x t r a c e l l u l a r s i t e s , i n h i b i t sodium-ATPase act i v i t y of t h e sodium pump b y s l o w i n g d e p h o s p h o r y l a t i o n . J. P h y s i o l . (London) 289, 17-31. + Beaug6, L. A . , and Glynn, I . M. (197933). O c c l u s i o n o f K i o n s i n t h e u n p h o s p h o r y l a t e d sodium pump. N a t u r e (London) 280, 510-512. Beaug6, L. A . , and Glynn, I. M. ( 1 9 8 0 ) . The e q u i l i b r i u m between t h e d i f f e r e n t c o n f o r m a t i o n s of t h e u n p h o s p h o r y l a t e d sodium pump: e f f e c t s o f ATP and o f potassium i o n s , and t h e i r relevance t o potassium t r a n s p o r t . J . P h y s i o l . (London) 299, 367-383. B l o s t e i n , R . , a n d Chu, L. ( 1 9 7 7 ) . S i d e d n e s s o f (sodium, potass i u m ) - a d e n o s i n e t r i p h o s p h a t a s e o f i n s i d e - o u t red c e l l memb r a n e vesicles. J. B i o l . C h e m . 252, 3035-3043. Glynn, I . M., a n d Hoffman, J. F. ( 1 9 7 1 ) . N u c l e o t i d e r e q u i r e m e n t s f o r sodium-sodium exchange c a t a l y s e d by t h e sodium pump i n human r e d c e l l s . J . P h y s i o l . (London) 218, 239-256. Glynn, I . M . , a n d R i c h a r d s , D. E. ( 1 9 8 0 ) . F a c t o r s a f f e c t i n g t h e release o f o c c l u d e d rubidium i o n s from t h e sodium pump. J . P h y s i o l . (London) 3 0 8 , 58P. Glynn, I. M . , and R i c h a r d s , D. E . ( 1 9 8 1 ) . Two r o u t e s t o t h e occluded-K+ form o f t h e sodium pump. J. P h y s i o l . (London) 313, 31P. Glynn, I . M. , K a r l i s h , S. J. D., and Yates, D. W. ( 1 9 7 9 ) . The u s e o f formycin n u c l e o t i d e s t o i n v e s t i g a t e t h e mechanism In "Na,K-ATPase: S t r u c t u r e and K i n e t i c s " o f Na,K-ATPase. (J. C. Skou and J. G . Nbrby, e d s . ) , p p . 101-113. Academic P r e s s , N e w York.

638

I. M. GLYNNAND D. E. RICHARDS

Jpkgensen, P. L. (1974). P u r i f i c a t i o n and c h a r a c t e r i z a t i o n of 111. P u r i f i c a t i o n from t h e o u t e r medulla (Na+ + K+)-ATPase. of mammalian kidney a f t e r s e l e c t i v e removal of membrane Biochim. Biophys. compounds by sodium dodecyl s u l f a t e . A c t a 356, 36-52. Jfkgensen, P. L . , and K a r l i s h , S. J. D. (1980). D e f e c t i v e conform a t i o n a l r e s p o n s e i n s e l e c t i v e l y t r y p s i n i z e d ( N a + + K+-ATPase s t u d i e d w i t h tryptophan f l u o r e s c e n c e . B i o c h i m . B i o p h y s . A c t a 5 9 7 , 305-317. K a r l i s h , S. J. D., and P i c k , U. (1981). Sidedness o f t h e e f f e c t s of sodium and potassium i o n s on t h e conformational s t a t e of t h e sodium-potassium pump. J. P h y s i o l . (London) 3 1 2 , 505529. K a r l i s h , S. J. D . , and Yates, D. W. (1978). Tryptophan f l u o r escence of (Na+ + K+)-ATPase a s a t o o l f o r s t u d y of t h e enzyme mechanism. B i o c h i m . B i o p h y s . A c t a 5 2 7 , 115-130. K a r l i s h , S. J. D., Yates, D. W., and Glynn, I. M. (1978). Conform a t i o n a l t r a n s i t i o n s between Na+-bound and K+-bound forms of (Na+ + K+)-ATPase, s t u d i e d w i t h formycin n u c l e o t i d e s . B i o c h i m . B i o p h y s . A c t a 5 2 5 , 252-294. Msrdh, S. (1975) Bovine b r a i n Na+,K+-stimulated ATP phosphohydrol a s e s t u d i e d by a rapid-mixing technique. K+-stimulated l i b e r a t i o n of [32P]orthophosphate from [32P]phosphoenzyme and r e s o l u t i o n of t h e dephosphorylation i n t o t w o p h a s e s . B i o c h i m . B i o p h y s . A c t a 391, 448-463. P l e s n e r , I. W . , P l e s n e r , L . , Nqh-by, J. G . , and Klodos, I. (1981). The s t e a d y - s t a t e k i n e t i c mechanism o f ATP h y d r o l y s i s c a t a l y s e d by membrane-bound (Na+ + K+)-ATPase from ox b r a i n . 111. A minimal model. B i o c h i m . B i o p h y s . A c t a 6 4 3 , 483-494. P o s t , R . L., Hegyvary, C . , and K u m e , S . (1972). A c t i v a t i o n by adenosine t r i p h o s p h a t e i n t h e p h o s p h o r y l a t i o n k i n e t i c s o f sodium and potassium i o n t r a n s p o r t adenosine t r i p h o s p h a t a s e . J. B i o l . Chem. 2 4 7 , 6530-6540.

.

CURRENT TOPICS IN MEMBRANES AND TRANSPORT. VOLUME 19

Na and K Fluxes Mediated by ATP-Free and ATP-Activated Na,K-ATPase in Liposomes BEATRICE M.A"ER Depanment of Pharmacology Centre Medical Universiraire Geneva, Switzerland

I.

INTRODUCTION

The ATP-activated N a , K - A T P a s e i s c a p a b l e o f s u s t a i n i n g N a : K exchange a f t e r p u r i f i c a t i o n and r e c o n s t i t u t i o n i n p h o s p h a t i d y l c h o l i n e liposomes ( H i l d e n and Hokin, 1975; Sweadner and G o l d i n , 1975; Anner et a i . , 1 9 7 7 ) . However, l i t t l e i s known a b o u t t h e e f f e c t of t h i s i m p o r t a n t transmembranous p r o t e i n on t h e " p a s s i v e " N a and K f l u x e s . Liposomes p r e s e n t a unique t o o l f o r s u c h an i n v e s t i g a t i o n b e c a u s e t h e transmembranous c a t i o n f l u x e s i n pure l i p i d (Na,K-ATPase-free) liposomes can be comp a r e d t o t h e c a t i o n f l u x e s i n liposomes c o n t a i n i n g e i t h e r " r e s t i n g " (ATP-free) o r ATP-activated N a , K ATPase. T h e r e s u l t s p r e s e n t e d h e r e i n , i n agreement w i t h e a r l i e r work (Anner, 1 9 8 0 , 1 9 8 1 a ) , i l l u s t r a t e t h a t t h e r e c o n s t i t u t e d Na,K-ATPase i n c r e a s e s t h e c a t i o n f l u x e s a c r o s s t h e liposome membrane, t h e r e s u l t i n g K f l u x being 2 t o 3 t i m e s faster than t h e N a flux. 639

Copyright 0 1983 by Academic Press. Inc. All rights of repduction in any form R S C N ~ . ISBN 0-12-153319-0

640

BEATRICE M. ANNER

600

40 0

200 E

0 .z

c

0

g 1000 In a In

2 E

800

01

-0 c y)

01

600

5In 0

n

400

.-C

d U

e 0

I

200

In

c

.-0 U 2

0

0

2 4 6 Incubation time (hours)

24 25

166

F i g . 1 . Na ( a ) and K ( b ) f l u x e s m e d i a t e d b y A T P - f r e e and A T P - a c t i v a t e d Na ,K-ATPase. Liposomes were prepared a s d e s c r i b e d i n Section 11 i n a s o l u t i o n c o n t a i n i n g 50 mM NaCl, 50 mM KCI, 50 mM choline c h l o r i d e , 30 mM i m i d a z o l e , 5 mM MgC12, 1 mM c y s t e i n e , 1 mM EDTA (pH 7 . l o ) . I s o t o p e f l u x e s w e r e measured a s d e s c r i b e d i n Section 11 i n l i p o s o m e s w h i c h w e r e p r e p a r e d w i t h p u r e (Na ,K-ATPase-free) p h o s p h a t i d y l c h o l i n e ( 0 ) or w i t h p h o s p h a t i d y l choline p l u s Na,K-ATPase ( 0 ,0 ) " P a s s i v e " ( 0 ) f l u x e s (-ATP) and net ( 0 ) f l u x e s (+ATP) w e r e d e t e r m i n e d f o r ( a ) [ 8 6 R B ] K and ( b ) [22Na]Na. Segment I i n d i c a t e s the i n t e r n a l s p a c e o f the t r a n s p o r t - a c t i v e l i p o s o m e s ( a b o u t 600 n m o l e s ions/ml s u s p e n s i o n ) . S e g m e n t s I + II i n d i c a t e the l a b e l i n g o f the t o t a l i n t e r n a l Na pool (100 mM) w h i c h i s p r e s e n t a f t e r t h e Na:K e x c h a n g e d o n e by

.

Na AND K FLUXES MEDIATED IN LIPOSOMES

11.

641

METHODS

Liposomes were prepared with egg phosphatidylcholine (Sigma type 111-E) by the cholate dialysis method (Hilden and Hokin, 1975; Sweadner and Goldin, 19751, which has been shown to yield a homogeneous preparation of single-walled 90-nm vesicles (Skriver e t a l . , 1980). Functional Na,K-ATPase was incorporated into the liposome membrane by adding cholate-solubilized Na,K-ATPase to the phosphatidylcholine cholate solution as previously described (Anner, 1980) and transmembranous [22Na]Na and [86Rb]K fluxes were determined by a microversion of the gel-filtration method (Anner, 1981a).

111.

RESULTS AND DISCUSSION

Figure 1 illustrates that three rates of transmembranous Na and K fluxes can be distinguished: (1) a slow rate (days) across pure phosphatidylcholine membranes, (2) an intermediate rate (hours) across membranes containing resting (ATP-free) Na,K-ATPase, and ( 3 ) a fast rate (minutes) across membranes containing ATP-activated Na,K-ATPase. The presence of reconstituted Na,K-ATPase molecules increases the Na flux by a factor of about 15 and the K flux by a factor of about 40. External ATP, by activating the inside-out oriented pump molecules, replaces the internal K pool by Na. The rate of these net fluxes is about 100 times faster than Na and about 40 times faster for K than the rate of the passive fluxes (Anner, 1981a). A transport model including the net and the passive fluxes mediated by the Na,K-ATPase in liposomes has been proposed (Anner, 1981b).

( F i g . 1, c o n t i n u e d ) the a c t i v a t e d i n s i d e - o u t pump m o l e c u l e s . T h e l a b e l i n g i s doubled (segment I I / s e g m e n t I = 2 ) a f t e r the a d d i t i o n o f ATP b e c a u s e the i n t e r n a l K p o o l ( 5 0 mM) i s , f i r s t , r e p l a c e d b y an e x t e r n a l [22Na]Na p o o l (50 mM) a n d , s e c o n d , the 50 mM " c o l d " l i p o s o m e s d u r i n g d i a l y s i s ( 5 0 mM) Na p o o l w h i c h was i n c l u d e d i s exchanged for e x t e r n a l b y a Na:Na e x c h a n g e p r o c e s s , y i e l d i n g 1 0 0 mM i n t e r n a l [ compared t o 50 mM i n the a b s e n c e of ATP.

642

BEATRICE M. ANNER

The question whether an ion channel located in the center of the membrane-spanning polypeptide or whether a region in the phospholipid bilayer surrounding the Na,K-ATPase molecule mediates the Na,K fluxes is presently being investigated. Regardless of the precise molecular mechanism, the fact that each Na,K-ATPase molecule has a definite capacity to increase the membrane permeability more for K ions than for Na ions may be relevant to excitability and other physiological processes.

REFERENCES

Anner, €3. M. (1980). R a t i o of Na:K t r a n s p o r t i n r e c o n s t i t u t e d B i o c h e m . B i o p h y s . R e s . Commun. 9 4 , sodium pump v e s i c l e s . 1233-1241. Anner, B . M. (1981a). A K - s e l e c t i v e K-channel formed by Na,KB i o c h e m . I n t . 2, 366-371. ATPase i n liposomes. Anner, B. M. (1981b). A t r a n s p o r t model f o r t h e Na,K-ATPase i n liposomes i n c l u d i n g t h e Na,K-channel f u n c t i o n . B i o s c i . R e p . 1, 555-560. Anner, B. M., Lane, L. K . , Schwartz, A . , and P i t t s , B. J . R. (1977). A r e c o n s t i t u t e d Na,K-pump i n liposomes c o n t a i n i n g p u r i f i e d Na,K-ATPase from kidney medulla. B i o c h i m . B i o p h y s . A c t a 467, 340-345. Hilden, S., and Hokin, L. E. (1975). A c t i v e potassium t r a n s p o r t coupled t o a c t i v e sodium t r a n s p o r t i n v e s i c l e s r e c o n s t i t u t e d from p u r i f i e d sodium and potassium i o n - a c t i v a t e d adenosine t r i p h o s p h a t a s e from t h e r e c t a l g l a n d o f Squalus a c a n t h i a s . J. B i o l . C h e m . 2 5 0 , 6296-6303. S k r i v e r , E . , Maunsbach, A. B . , Anner, B. M . , and J!drgensen, P. L. ( 1 9 8 0 ) . E l e c t r o n microscopy o f p h o s p h o l i p i d v e s i c l e s reCell Biol. c o n s t i t u t e d w i t h p u r i f i e d r e n a l Na,K-ATPase. I n t . R e p . 4 , 585-591. Sweadner, K. J . , and Goldin, S. M. (1975). R e c o n s t i t u t i o n o f act i v e i o n t r a n s p o r t by t h e sodium and potassium ions t i m u l a t e d adenosine t r i p h o s p h a t a s e from canine b r a i n . J . B i o l . C h e m . 2 5 0 , 4022-4024.

CURRENT TOPICS IN MEMBRANES AND TRANSWRT, VOLUME 19

Sidedness of Cations and ATP Interactions with the Sodium Pump L. BEAUGEI Division de Biofisica Instituto de lnvestigacidn Midica Mercedes y Manin Ferreyra Crirdoba. Argentina

R. DIPOLO Cenrro de BiojTsica y Bioqu’mica Instituto Venezolanode Investigaciones cienr$cas Caracas, Venezuela

I.

INTRODUCTION

The i n t e r n a l d i a l y s i s t e c h n i q u e a p p l i e d t o s q u i d g i a n t axons p e r m i t s t h e c o n t r o l o f t h e i n t e r n a l and ext e r n a l environment of t h e c e l l f o r c a t i o n s , n u c l e o t i d e s , and m e t a b o l i t e s w i t h o u t d i s r u p t i o n o f t h e i n t r a c e l l u l a r o r g a n e l l e s . Experiments were performed w i t h t h i s t e c h n i q u e i n o r d e r t o s t u d y t h e s i d e d n e s s of t h e i n t e r a c t i o n s of t h e N a pump w i t h ATP and monovalent c a t i o n s .

11.

RESULTS AND D I S C U S S I O N

The f o l l o w i n g r e s u l t s were o b t a i n e d : 1. With 310 mM K i t 70 mM N a i l and 1 0 mM K sea water, a b o u t 97% of Na e f f l u x w a s ATP-dependent. The e f f l u x of N a w a s s t i m u l a t e d by ATP w i t h a ~ 0 . 5 of a b o u t 200 pM. 643

Copyright 0 1983 by Academic Press, Inc. All rights of reproduction in any form r e ~ e ~ e d . ISBN 0-12-1533194

L. BEAUGE AND R. DiPOLO

644

2.

A r e d u c t i o n i n t h e ATP c o n c e n t r a t i o n from 3-m

!JM reduced t h e maximal r a t e of KO-stimulated Na e f f l u x about 8 - f o l d . A t t h e same t i m e , t h e a p p a r e n t a f f i n i t y f o r e x t e r n a l K a s Na pump a c t i v a t o r was &c r e a s e d by a s i m i l a r f a c t o r . 3 . With 3-5 m~ ATP t h e o r d e r of e f f e c t i v e n e s s a s e x t e r n a l Na pump a c t i v a t o r followed t h e sequence K > NH4 > Rb. With 30-50 V M ATP t h e sequence was NH4 > > K > > Rb. The r e s u l t s were n o t a f f e c t e d by t h e Nai/Ki c o n c e n t r a t i o n r a t i o . 4 . With 3 mM ATP and 70 mM N a i removal of i n t e r n a l K had no e f f e c t on t h e ATP-dependent Na e f f l u x . When t h e ATP c o n c e n t r a t i o n was 30-50 U M , o r t h e Nai concen, of K i r e v e r s i b l y i n c r e a s e d t r a t i o n was 5-10 m ~ removal t h e ATP-dependent e f f l u x o f Na. The l a r g e s t i n c r e a s e w a s observed when b o t h ATP N a i were s i m u l t a n e o u s l y reduced. These e f f e c t s were a b o l i s h e d by 1 0 - 4 M o u a b a i n These r e s u l t s can be e x p l a i n e d by t h e f o l l o w i n s scheme c o n s i s t i n g of a sequence of c o n s e c u t i v e biochemic a l e v e n t s l e a d i n g t o a Ko-stimulated Na e x t r u s i o n and ATP h y d r o l y s i s (see Beauge and DiPolo, 1 9 8 1 ) : mM t o 30-50

KS

k2

k3

k4

\L pi

Nai

E1-Na where k3 = k;

(~+K,~,/[ATP])-'

(see Beauge and Glynn, 1 9 8 0 ) . I n t h i s model t h e occluded enzyme-K conformation E,(K) (see P o s t e t al., 1 9 7 2 ) can be formed by t h e i n t e4r'a c' t i o n of e x t e r n a l k b e f o r e d e p h o s p h o r y l a t i o n and by i n t e r n a l K b e f o r e r e p h o s p h o r y l a t i o n . The i n c r e a s e d e f f e c t i v e n e s s of N H 4 o v e r K and Rb and of K o v e r Rb a s e x t e r n a l pump a c t i v a t o r a t low ATP c o n c e n t r a t i o n s i n d i c a t e d t h a t t h e same p a t t e r n s e e n on t h e l e v e l s of

CATIONS,ATP, AND Na PUMP CONFORMATIONS

645

phosphoenzyme ( P o s t e t a l . , 1 9 7 2 ) i s due t o t h e e f f e c t s o f t h e s e c a t i o n s a t e x t r a c e l l u l a r s i t e s . The f a c t t h a t t h i s p a t t e r n w a s n o t a f f e c t e d by t h e N a i / K i r a t i o sugg e s t s t h a t t h e o c c l u d i n g enzyme d o e s n o t r e a c t w i t h N a i or Ki. I n a d d i t i o n , K i o n s have a n i n h i b i t o r y a c t i o n on t h e N a pump by i n t e r a c t i n g w i t h i n t r a c e l l u l a r s i t e s . The magnitude of t h a t K i i n h i b i t i o n depends on t h e conc e n t r a t i o n o f b o t h ATP and N a i ( K i r e d u c e s t h e a p p a r e n t a f f i n i t y o f t h e N a pump f o r A T P ) . F o r t h e r e s t r i c t e d case o f z e r o i n t e r n a l K and P i (which s i m p l i f i e s t h e c a l c u l a t i o n s b u t d o e s n o t change t h e g e n e r a l c o n c l u s i o n s ) t h e r a t e e q u a t i o n from Scheme (1 becomes k 2 [ t o t a l E] (3)

v = + A

where k(k-3+k4) A

=

l

+

k3k4

k2

+-

k4

(Note t h a t a s k3 i n c r e a s e s t o g e t h e r w i t h ATP, it f o l l o w s t h a t A i s reduced when ATP i n c r e a s e s . ) The model p r e d i c t s t h a t as ATP c o n c e n t r a t i o n i s reduced, t h e maximal r a t e o f pumping i s a l s o r e d u c e d , I n adwhereas t h e a p p a r e n t a f f i n i t y f o r KO i n c r e a s e s . d i t i o n , t h e r e are i m p o r t a n t k i n e t i c consequences obs e r v e d i n t h e reciprocal p l o t s of t h e d a t a (see F i g . 1 ) . ( i ) F o r two s u b s t r a t e s i t e s ( n = 2 ) t h e p l o t s / v l / n v e r s u s s i s o n l y l i n e a r when A = 1, a t r i v i a l case even f o r h i g h ATP c o n c e n t r a t i o n s ; when A > 1 t h e p l o t becomes ( i i ) A = 5 and b e n t upward a t low s c o n c e n t r a t i o n s . n = 3 g i v e a p l o t which e x p e r i m e n t a l l y would be i n d i s t i n g u i s h a b l e from a s t r a i g h t l i n e , even when o n l y two i n d e p e n d e n t and e q u a l s u b s t r a t e s i t e s are c o n s i d e r e d . This i n d i c a t e s t h a t s t r a i g h t l i n e s i n reciprocal p l o t s f o r complex k i n e t i c s y s t e m s can l e a d t o wrong conclus i o n s a b o u t t h e number of s i t e s i n v o l v e d and t h e t r u e a f f i n i t y of t h o s e s i t e s f o r t h e s u b s t r a t e o r a c t i v a t o r .

646

L. BEAUGP AND R. DiPOLO

v=

V

(5)

A

a

b

c

n

2

2

3

A

1

5

5

V

1

(*)’

Ks

’

Kt

2.5

2

+

l

V=l

K=l

b

0.26 0.23

F i g . 1 . R e c i p r o c a l p l o t o f the k i n e t i c e q u a t i o n ( 3 ) f o r a h y p o t h e t i c a l c a s e of t w o e q u i v a l e n t s u b s t r a t e b i n d i n g s i t e s w i t h e q u a l a f f i n i t i e s a s a f u n c t i o n o f A (see E q . ( 4 ) ) . V i s the m a x i mal r a t e ; v is the o b s e r v e d r a t e ; K i s the t r u e d i s s o c i a t i o n CORs t a n t o f the s u b s t r a t e - s i t e c o m p l e x . Inset i n the f i g u r e a r e t h e c a l c u l a t e d v a l u e s of K, f o r n = 2 , A = 1 ( a ) and n = 3 , A = 5 ( c ) . T h e K0.5 v a l u e s f o r A = 1 and 5 a r e a l s o g i v e n . Note t h a t d e s p i t e the f a c t there a r e o n l y t w o s i t e s , n = 3 g i v e s a s t r a i g h t - l i n e p l o t when A = 5 (c).

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REFERENCES

Beaug6, L . , and DiPolo, R. ( 1 9 8 1 ) . The e f f e c t s o f ATP on t h e i n t e r a c t i o n s between monovalent c a t i o n s and t h e sodium pump i n d i a l y z e d s q u i d axons. J . Physiol. (London) 314, 457-480. Beauge', L., and Glynn, I. M. (1980). The e q u i l i b r i u m between d i f f e r e n t c o n f o r m a t i o n s of t h e unphosphorylated N a pump: E f f e c t o f ATP and K i o n s and t h e i r r e l e v a n c e t o K t r a n s p o r t . J. Physiol. (London) 299, 367-383. P o s t , R . L . , Hegyvary, C . , and K u m e , S . ( 1 9 7 2 ) . A c t i v a t i o n by a d e n o s i n e t r i p h o s p h a t e i n t h e p h o s p h o r y l a t i o n k i n e t i c s of sodium and p o t a s s i u m i o n t r a n s p o r t a d e n o s i n e t r i p h o s p h a t a s e . J. Bio1. Chem. 247, 6530-6540.

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CURRENT TOPICS IN MEMBRANES AND TRANSPORT, VOLUME 19

Sidedness of Sodium Interactions with the Sodium Pump in the Absence of K+ RHONA BLOSTEIN' Departments of Biochemisry and Erperimental Medicine McGiN University, and the Division of Hematology Royal Victoria Hospital Montreal, Quebec, Canada

I.

INTRODUCTION

T h i s d i s c u s s i o n i s concerned w i t h t h e e f f e c t s of r e a c t i o n sequence i n r e l a t i o n t o t h e mechanism of o u a b a i n - s e n s i t i v e N a + t r a n s p o r t . I n p r e v i o u s s t u d i e s u s i n g i n s i d e - o u t v e s i c l e s o f human red c e l l membranes, w e have shown t h a t N a + - s t i m u l a t e d p h o s p h o r y l a t i o n of t h e enzyme o c c u r s w i t h less t h a n micromolar amounts of ATP and r e q u i r e s N a + a t o n l y t h e c y t o p l a s m i c s u r f a c e . Although e x t r a v e s i c u l a r N a + (Nacyt, N a + a t t h e c y t o p l a s m i c s u r f a c e ) a t v e r y low c o n c e n t r a t i o n (10.2 m M ) s t i m u l a t e s ATP h y d r o l y s i s , h i g h e r amounts a r e needed f o r N a + t r a n s l o c a t i o n from t h e c y t o p l a s m i c t o t h e e x t r a c e l l u l a r s i d e of t h e membrane (Blostein, 1979). I t w a s suggested t h a t a transl o c a t i o n s t e p , e . g . , t r a n s i t i o n from o n e form o f phosphoenzyme t o a n o t h e r , El-P -+ E 2 - P , r e q u i r e s a l l ( t h r e e ? ) Na+ b i n d i n g s i t e s t o be s a t u r a t e d . N a + on t h e N a , K - A T P a s e

'Present a d d r e s s : Research I n s t i t u t e , Montreal Hospital , Montreal, Canada. 649

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S e v e r a l o t h e r p r o p e r t i e s of Na+-stimulated A T P a s e (Na-ATPase) s u b s t a n t i a t e t h e c o n c l u s i o n t h a t t h i s aci s t h e enzymic b a s i s f o r ATP-dependent t r a n s p o r t i n t h e absence of K+. Thus, a t low c o n c e n t r a t i o n s of ATP, L i + and Rb+ ( o r K + ) a f f e c t Na+ t r a n s p o r t and Na+-ATPase s i m i l a r l y : i n t r a v e s i c u l a r L i + ( L i e x t r Li+ a t t h e normal e x t r a c e l l u l a r s u r f a c e ) s t i m u l a t e s , whereas i n t r a v e s i c u l a r Rb+ (Rbext) o r K+ i n h i b i t s b o t h a c t i v i t i e s . These e f f e c t s are c o n s i s t e n t w i t h t h e i r K+-like e f f e c t s on t h e sequence E2-P f K+ + K * E 2 +K.El-+-El + K+ whereby r e l e a s e of Rb+ ( o r K+) , b u t n o t L i + , from t h e enzyme i s r a t e - l i m i t i n g and i s acc e l e r a t e d only w i t h ATP bound t o a l o w - a f f i n i t y s i t e . I n a d d i t i o n , i n t r a v e s i c u l a r Na+ (NaeVt, Na+ a t t h e e x t r a c e l l u l a r s u r f a c e ) modulates b o t h a c t i v i t i e s i n a s i m i l a r , b i p h a s i c manner as f o l l o w s : u s i n g r e d c e l l g h o s t s , Glynn and K a r l i s h ( 1 9 7 6 ) showed t h a t e x t r a c e l l u l a r Na+, a t l o w c o n c e n t r a t i o n , i n h i b i t s Na+-ATPase a s w e l l as t h e a s s o c i a t e d ATP h y d r o l y s i s , Using i n s i d e o u t v e s i c l e s , w e showed t h a t t h e i n h i b i t o r y e f f e c t of low c o n c e n t r a t i o n s of Naext i s a s s o c i a t e d w i t h a dec r e a s e i n a p p a r e n t t u r n o v e r of phosphoenzyme (E-P) ( B l o s t e i n e t al., 1 9 7 9 ) ; t h i s i s i n c o n t r a s t t o e f f e c t s of Kext, Rbext, L i e x t , o r h i g h e r l e v e l s of N a e x t r a l l of which i n c r e a s e t h e a p p a r e n t t u r n o v e r of phosphoenzyme, whether o r n o t t h e y a c t i v a t e ( L i e x t , 2 25 mM Naext) o r i n h i b i t o v e r a l l pump and A T P a s e a c t i v i t y (Rbextr Kextr I 5 m~ Naext)

k';"kkT

11.

METHODS AND RESULTS

To g a i n i n s i g h t i n t o t h e q u e s t i o n of whether t h e r e s t o r a t i o n of ATP h y d r o l y s i s by r e l a t i v e l y h i g h l e v e l s of Naext was a s s o c i a t e d w i t h Na t r a n s p o r t , w e measured Na+ e f f l u x (normal i n f l u x ) a s w e l l as i n f l u x (normal e f f l u x ) u s i n g i n s i d e - o u t membrane v e s i c l e s . I t w a s observed t h a t t h e sodium pump can o p e r a t e i n a mode i n which i n f l u x and e f f l u x a r e a s s o c i a t e d w i t h ATP h y d r o l y s i s ( L e e and B l o s t e i n , 1 9 8 0 ) . D e s p i t e v a r i a t i o n i n a b s o l u t e a c t i v i t i e s of d i f f e r e n t p r e p a r a t i o n s , i n each c a s e , v a l u e s f o r b o t h f l u x e s a r e of s i m i l a r magn i t u d e when b o t h a r e measured i n t h e same way (22Na retained i n Millipor e- f ilter ed v e s i c l e s ) . This r e s u l t i s c o n s i s t e n t w i t h roughly one-for-one exchange. Moreover, w e have a s c e r t a i n e d t h a t 22Na e f f l u x i s n o t due t o ADP formed d u r i n g t h e c o u r s e of t h e experiment.

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Na-Na exchange i n t h e absence of ADP c a n be d i s t i n g u i s h e d from ADP-sensitive exchange i n t h e f o l l o w i n g way. Vanadate i n h i b i t s ATP-dependent Na+ i n f l u x i n both t h e p r e s e n c e and absence of Kext (normal N a - K exc h a n g e ) , a l t h o u g h t o a lesser e x t e n t w i t h r e l a t i v e l y high l e v e l s of Naext ( c f . Beauge et al., 1 9 8 0 ) . Howe v e r , w i t h 50 m~ Naext, it i s observed t h a t ADP-sensit i v e Na-Na exchange, measured a s ADP-sensitive 22Na i n f l u x , i s v i r t u a l l y i n s e n s i t i v e t o vanadate c o n c e n t r a t i o n s which p a r t i a l l y i n h i b i t Na-Na exchange i n t h e abs e n c e o f ADP. Na-Na exchange i n t h e absence of K+ and ADP may r e f l e c t exchange of Naext f o r Nac t i n a manner a n a l o gous t o t h e exchange of Kext f o r 8 a c y t , whereby NaeXt a c t s a t Kext s i t e s . However, t h e p o s s i b i l i t y remains sites, i n t h a t Naext a c t s a t s i t e s d i f f e r e n t from Ke,t p a r t i c u l a r , t h e Na+ d i s c h a r g e s i t e s ; N a e x t would t h e n s t i m u l a t e r e v e r s a l of E l - P + E 2 - P (see scheme below) i n f a v o r of E l - P which, i n t h e absence of ADP, i s hydrolyzed. ATP

I n f a v o r of t h e l a t t e r i n t e r p r e t a t i o n i s t h e remarkably s i m i l a r r e s p o n s e of ADP-ATP exchange, r e p o r t e d by Kaplan and H o l l i s ( 1 9 8 0 1 , and Na+-ATPase, a s d e s c r i b e d by Glynn and K a r l i s h ( 1 9 7 6 1 , t o changes i n Naext c o n c e n t r a t i o n . On t h e o t h e r hand, i f Naext a t h i g h l e v e l s s t i m u l a t e s h y d r o l y s i s v i a E l - P , i t may be argued t h a t t h e r a t e of t h e o v e r a l l ATPase could t h e n proceed a t a r a t e a t l e a s t as g r e a t a s t h a t v i a t h e "normal" r o u t e through E2-P. To t h e c o n t r a r y , w e o b s e r v e t h a t , under t h e p r e s e n t cond i t i o n s f o r o b s e r v i n g Na e f f l u x coupled t o Na-ATPase (5 m M N a c y t ) , oligomycin, which s t i m u l a t e s Na+-depend e n t ADP-ATP exchange and i s b e l i e v e d t o i n h i b i t t h e c o n v e r s i o n of E l - P t o E z - P , r e d u c e s ATP h y d r o l y s i s t o a r a t e less t h a n t h a t observed when h i g h l e v e l s of Naext are present.

ACKNOWLEDGMENT

This work was supported by The Medical Research Council of Canada.

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REFERENCES Beaugg, L. A . , C a v i e r e s , J. J . , Glynn, I. M., and Grantham, J . J . (1980). The e f f e c t s o f vanadate on t h e f l u x e s o f sodium and potassium i o n s through t h e sodium pump. J. P h y s i o l . (London) 301, 7-23. B l o s t e i n , R . (1979). S i d e - s p e c i f i c a c t i o n of sodium o n ( N a , K ) ATPase. J. B i o l Chem. 254, 6673-6677. B l o s t e i n , R . , Pershadsingh, H. A . , Drapeau, P . , and Chu, L. (1979). S i d e - s p e c i f i c i n t e r a c t i o n s o f a l k a l i c a t i o n i n t e r a c t i o n s w i t h Na,K-ATPase. I n "Na+,K+-ATPase: S t r u c t u r e and K i n e t i c s " (J. C. Skou and J. C. N$rby, e d s . ) , pp. 223245. Academic P r e s s , New York. Glynn, I. M., and K a r l i s h , S. J. D. (1976). ATP h y d r o l y s i s assoc i a t e d w i t h a n uncoupled sodium f l u x through t h e sodium pump: Evidence f o r an a l l o s t e r i c e f f e c t o f ATP and e x t r a c e l l u l a r sodium. J. P h y s i o l . (London) 256, 463-496. Kaplan, J. H . , and H o l l i s , R. J. (1980). E x t e r n a l N a dependence of o u a b a i n - s e n s i t i v e ATP:ADP exchange i n i t i a t e d by photol y s i s of i n t r a c e l l u l a r caged-ATP i n human r e d c e l l g h o s t s . N a t u r e (London) 288, 587-589. Lee, K. H . , and B l o s t e i n , R . (1980). Red c e l l sodium f l u x e s c a t a l y z e d by t h e sodium pump i n t h e absence o f K+ and ADP. N a t u r e (London) 285, 330-339.

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CURRENT TOPICS IN MEMBRANES AND TRANSPORT, VOLUME 19

Magnesium Dependence of Sodium Pump Mediated Sodium Transport in Intact Human Red Cells P. W. FLATMANAND

I/. L. LEW

Department of Physiology University of Edinburgh Medical School Edinburgh, England and Physiological Laboratory Cambridge, England

I.

INTRODUCTION

Magnesium i s a n e c e s s a r y c o f a c t o r f o r N a , K - A T P a s e a c t i v i t y and f o r many of t h e p a r t i a l r e a c t i o n s of t h e sodium pump (Glynn and K a r l i s h , 1 9 7 5 ) . However, t h e magnesium dependence of i o n t r a n s p o r t t h r o u g h t h e pump h a s n o t been s t u d i e d i n d e t a i l due t o problems i n cont r o l l i n g and measuring t h e c o n c e n t r a t i o n of i o n i z e d magnesium i n s i d e t h e c e l l where t h e magnesium b i n d i n g s i t e on t h e pump i s s i t u a t e d . R e c e n t l y w e developed methods f o r measuring and s e l e c t i v e l y a l t e r i n g t h e magnesium c o n t e n t of r e d c e l l s u s i n g t h e i o n o p h o r e A 2 3 1 8 7 (Flatman and Lew, 1 9 8 0 ) . W e now r e p o r t e x p e r i m e n t s i n which w e have used t h i s method t o examine t h e magnesium dependence of sodium pump-mediated N a - K and N a - N a exchange i n i n t a c t human r e d c e l l s .

653

Copyright 0 1983 by Academic Press, Inc. All rights of reproductionin any form reserved. ISBN 0-12-153319-0

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11.

MATERIALS AND METHODS

Red c e l l s l o a d e d w i t h 2 4 N a were i n c u b a t e d w i t h c o n s t a n t s t i r r i n g , a t 37OC and 5-10% h e m a t o c r i t , i n media c o n t a i n i n g a t l e a s t 10 mM K f o r Na-X exchange s t u d i e s o r no added K f o r N a - N a exchange s t u d i e s . A l l media c o n t a i n e d a t l e a s t 75 mM N a , 1 0 mM T r i s o r HEPES (pH 7.7 a t 37OC), 1 0 mM i n o s i n e as s u b s t r a t e , 0.01-0.05 mM EGTA t o c h e l a t e contaminant C a , and varyi n g c o n c e n t r a t i o n s o f MgC12 o r MgC12/EDTA b u f f e r s . A s m a l l q u a n t i t y o f A23187 s t o c k s o l u t i o n (1 m g / m l i n e t h a n o l , 1 . 9 m M ) was added t o t h e s u s p e n s i o n s t o g i v e a f i n a l c o n c e n t r a t i o n o f 3-10 P M . The ionophore makes t h e c e l l membrane v e r y permeable t o magnesium, r e s u l t i n g i n t h e r a p i d e q u i l i b r a t i o n of i o n i z e d magnesium a c r o s s t h e membrane u n t i l t h e f o l l o w i n g r e l a t i o n s h i p holds :

2+ m 2+ m where [Mg ] i t [Mg l o , [ C l ] i r and [ C l l 0 are t h e e q u i l i b r i u m c o n c e n t r a t i o n s o f i n t e r n a l i o n i z e d Mg, e x t e r n a l i o n i z e d Mg, i n t e r n a l C 1 , and e x t e r n a l C 1 , r e s p e c t i v e l y . The l a t t e r t h r e e c o n c e n t r a t i o n s were measured and t h e c o n c e n t r a t i o n of i n t e r n a l i o n i z e d magnesium w a s c a l c u l a t e d from t h e above e q u a t i o n . I n t h e p r e s e n c e of A23187 t h e c o n c e n t r a t i o n o f magnesium r e a c h e d t h e e q u i l i b r i u m l e v e l w i t h i n 2 0 min and w a s t h e n m a i n t a i n e d a t t h i s l e v e l f o r a t l e a s t t h e n e x t 4 0 min. The e f f l u x o f 24Na w a s measured, w i t h and w i t h o u t 1 mkf o u a b a i n i n t h e medium, o v e r a 40-min p e r i o d when Mg w a s a t e q u i l i b r i u m . The pump-mediated f l u x e s were d e t e r m i n e d as t h e ouabains e n s i t i v e components of N a e f f l u x . The d a t a w e r e a n a l y z e d a f t e r p l o t t i n g t h e ouabains e n s i t i v e e f f l u x r a t e c o n s t a n t s ( o . s . k e ) as f u n c t i o n s of i n t r a c e l l u l a r i o n i z e d Mg. Even a t v e r y l o w i o n i z e d Mg c o n c e n t r a t i o n s ( < 5 x 10-7 M ) t h e r e were s i g n i f i c a n t l e v e l s of pump-mediated Na-K ( o . s . ke = 0.012 ? 0.003 hr-1) and N a - N a exchange (0,s. k e = 0.010 5 0 . 0 0 4 h r y l ) . These l e v e l s of a c t i v i t y were n o t a f f e c t e d by changes i n t h e c o n c e n t r a t i o n of i o n i z e d Mg i n t h e submicromolar r a n g e . When t h e c o n c e n t r a t i o n o f i o n i z e d Mg was i n c r e a s e d above 2 v M , b o t h Na-K and N a - N a exchange were stimulated. I f it i s assumed t h a t Mg b i n d s t o a s i n g l e s i t e on t h e enzyme t o promote t r a n s p o r t and t h a t Michaelis-Menten k i n e t i c s are obeyed, t h e n Mg s t i m u l a t e s Na-K exchange w i t h = 45 v M and s t i m u l a t e s N a - N a

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exchange w i t h a Ko.5 = 9 LIM. Although t h i s s i m p l e model d e s c r i b e s t h e Na-Na exchange d a t a w e l l , a b e t t e r d e s c r i p t i o n of N a - K exchange data is o b t a i n e d by assuming t h a t t h e r e i s a s m a l l component of exchange which i n c r e a s e s l i n e a r l y w i t h i n c r e a s i n g i o n i z e d Mg as w e l l a s t h e s a t u r a t i n g component. I n t h i s case t h e ~ 0 . 5o f t h e s a t u r a t i n g component i s 30 P M . When t h e c o n c e n t r a t i o n o f i o n i z e d Mg was i n c r e a s e d above 0.8 mM, b o t h modes of t r a n s p o r t t h r o u g h t h e pump were i n h i b i t e d , w i t h N a - N a exchange b e i n g more s t r o n g l y i n h i b i t e d t h a n N a - K exchange. N a - N a exchange w a s reduced t o h a l f maximal a c t i v i t y when t h e c o n c e n t r a t i o n o f i o n i z e d Mg w a s 2 . 7 m M , whereas Na-K exchange w a s reduced t o h a l f maximal a c t i v i t y by 7.7 mM Mg. A l l t h e e f f e c t s o f Mg d e s c r i b e d above w e r e shown t o o c c u r a t t h e i n n e r s u r f a c e of t h e membrane s i n c e a l t e r i n g t h e e x t e r n a l c o n c e n t r a t i o n from 1 0 - 7 t o 5 x l O - 3 ~ i n t h e a b s e n c e of A23187 d i d n o t a f f e c t pump a c t i v i t y . When t h e e x t e r n a l i o n i z e d Mg c o n c e n t r a t i o n w a s a b o u t 0 . 1 5 mM, t h e a d d i t i o n of A23187 d i d n o t a f f e c t e i t h e r t h e i n t e r n a l i o n i z e d Mg c o n c e n t r a t i o n ( n o r m a l l y a b o u t 0 . 4 m~ i n oxygenated c e l l s , see Flatman and Lew, 1980) o r t h e r a t e s of N a - K and N a - N a exchange measured b e f o r e and a f t e r ionophore a d d i t i o n s . T h i s s u g g e s t s t h a t A23187 p e r s e does n o t a f f e c t pump a c t i v i t y . S i n c e Mg is i m p o r t a n t as a c o f a c t o r f o r many g l y c o l y t i c enzymes, i t i s n o t s u r p r i s i n g t o f i n d t h a t changes i n Mg r e s u l t e d i n changes i n c e l l c o n c e n t r a t i o n s of ATP and ADP. When i o n i z e d Mg w a s reduced t o 5 x 10-7 M I t h e c o n c e n t r a t i o n o f ATP f e l l by 30% i n 1 h r , and t h a t o f ADP i n c r e a s e d by 2 5 - 5 0 % . I n c r e a s i n g t h e c o n c e n t r a t i o n of i o n i z e d Mg above t h e p h y s i o l o g i c a l l e v e l ( 0 . 4 mM) c a u s e d a s m a l l rise i n ATP c o n t e n t and a s m a l l f a l l i n ADP c o n t e n t . C e l l s i n which N a - N a exchange w a s b e i n g s t u d i e d a l s o showed an i n c r e a s e d N a c o n t e n t a t low Mg l e v e l s (below 0 . 1 m M ) due t o a n i n f l u x of Na t h r o u g h t h e i o n o p h o r e under t h e s e c o n d i t i o n s . These changes p r o b a b l y l e d t o an o v e r e s t i m a t e of ~ 0 . 5 f o r Na-K exchange (which v a r i e s w i t h ATP c o n c e n t r a t i o n ) and a n u n d e r e s t i m a t e o f t h e ~ 0 . 5 f o r N a - N a exchange (which v a r i e s w i t h ADP c o n c e n t r a t i o n ) . Thus, t h e t r u e ~ 0 . 5 f o r e a c h o f t h e two r e a c t i o n s i s p r o b a b l y c l o s e r t h a n t h o s e r e p o r t e d above ( f o r f u l l d i s c u s s i o n , see Flatman and Lew, 1 9 8 1 ) .

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111.

DISCUSSION

I t w a s s u r p r i s i n g t o f i n d t h e pump s t i l l a c t i v e a t very low Mg c o n c e n t r a t i o n s . T h i s , t o g e t h e r w i t h t h e f i n d i n g t h a t r e d cells a l s o b i n d t i g h t l y 0.03-0.1 m m o l e / l i t e r c e l l s Mg a t t h e s e low i o n i z e d Mg l e v e l s (see Flatman and Lew, 19801, s u g g e s t s e i t h e r t h a t some Mg i s t r a p p e d on t h e pump's b i n d i n g s i t e , g i v i n g t h e pump r e s i d u a l a c t i v i t y , o r t h a t A23187 i s heterogeneo u s l y d i s t r i b u t e d among t h e c e l l s . The l a t t e r c o u l d r e s u l t i n some c e l l s c o n t a i n i n g much more Mg t h a n t h e a v e r a g e and t h e s e c e l l s would pump sodium a t a s i g n i f i cant rate. The s t i m u l a t i o n of N a - K and N a - N a exchange by i o n i z e d Mg c o n c e n t r a t i o n s between 0 . 0 0 2 and 0.8 mM probably r e f l e c t s t h e Mg r e q u i r e m e n t f o r pump phosphorylation. S i n c e t h i s s t e p i s common t o b o t h Na-K and N a - N a exchange, s i m i l a r Mg r e q u i r e m e n t s might be p r e d i c t e d f o r those reactions. The e x p e r i m e n t s showed, however , t h a t more Mg w a s r e q u i r e d t o s t i m u l a t e N a - K exchange ( ~ 0 . 5= 30-45 P M ) t h a n to s t i m u l a t e N a - N a exchange (K0.5 = 9 y M ) . The d i f f e r e n t Mg r e q u i r e m e n t s c o u l d , i n p a r t , be e x p l a i n e d by t h e changes i n n u c l e o t i d e concent r a t i o n s e e n a t low Mg c o n c e n t r a t i o n s . A s m a l l d i f f e r e n c e i n Mg r e q u i r e m e n t s i s p r e d i c t e d , however, i f it i s assumed t h a t t h e p h o s p h o r y l a t e d pump enzyme (E2-P) can r e v e r t t o t h e n a t i v e form ( E l ) f a s t e r d u r i n g Na-K exchange t h a n d u r i n g N a - N a exchange. Thus, i n t h e s t e a d y s t a t e , more enzyme would be i n t h e E l form d u r i n g N a - K exchange t h a n d u r i n g N a - N a exchange. S i n c e Mg b i n d s t o t h e n a t i v e ( E l ) form t o promote phosp h o r y l a t i o n , more Mg would a p p a r e n t l y be needed t o s t i m u l a t e Na-K exchange t h a n t o s t i m u l a t e N a - N a exchange, as w a s found. The i n h i b i t o r y e f f e c t s of e x c e s s Mg a r e p r o b a b l y due t o t h e s t a b i l i z a t i o n of E 2 forms of t h e enzyme, which would r e d u c e t h e r a t e of t u r n o v e r o f t h e pump. An a t t r a c t i v e h y p o t h e s i s i s t h a t e x c e s s Mg c o n v e r t s E2-P t o a form which cannot be d e p h o s p h o r y l a t e d by K o r ADP--the i n s e n s i t i v e phosphoenzyme ( E z - P i , see P o s t et al., 1975; Forgac, 1 9 8 0 ) . T h i s h y p o t h e s i s e x p l a i n s why N a - N a exchange i s more s t r o n g l y i n h i b i t e d by e x c e s s Mg t h a n N a - K exchange. I n t h e s t e a d y s t a t e , t h e l e v e l of E2-P ( t h e p r e c u r s o r bo E 2 - P i ) i s h i g h e r d u r i n g Na-Na exchange t h a n d u r i n g N a - K exchange. Thus, f o r any g i v e n Mg c o n c e n t r a t i o n , more enzyme i s c o n v e r t e d t o t h e i n s e n s i t i v e form d u r i n g Na-Na exchange t h a n d u r i n g N a - K exchange. A l t e r n a t i v e l y t h e e x t r a i n h i b i t i o n of N a - N a exchange may be

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due t o Mg i n h i b i t i n g t h e r e a c t i o n s which b r i n g N a i n t o t h e c e l l d u r i n g Na-Na exchange and which are n o t i n volved i n N a - K exchange.

ACKNOWLEDGMENT

W e would l i k e t o thank t h e MRC and Wellcome T r u s t f o r financ i a l s u p p o r t and t h e L i l l y Research Centre Ltd. f o r a g i f t o f A23187.

REFERENCES

Flatman, P. W., and Lew, V. L. (1980). Magnesium b u f f e r i n g i n i n t a c t human r e d blood c e l l s measured using t h e ionophore A23187. J. Physiol. ( L o n d o n ) 305, 13-30. Flatman, P. W., and Lew, V. L. (1981). The magnesium dependence of sodium-pump-mediated sodium-potassium and sodium-sodium exchange i n i n t a c t human red c e l l s . J. Physiol. ( L o n d o n ) 315, 421-446. Forgac, M. D. (lq60) C h a r a c t e r i z a t i o n of a Mg2+-stabilized s t a t e of t h e (Na f K ) - s t i m u l a t e d adenosine t r i o p h o s p h a t a s e using a f l u o r e s c e n t r e p o r t e r group. J. Biol. Chem. 2 5 5 , 1547-1553. Glynn, I. M . , and K a r l i s h , S. J . D. (1975). The sodium pump. A n n u . Rev. Physiol. 3 7 , 13-55. P o s t , R L . , Toda, G . , and Rogers, F. N. (1975). Phosphorylation by i n o r g a n i c phosphate of sodium p l u s potassium ion t r a n s p o r t adenosine t r i p h o s p h a t a s e . J. B i o l . Chem. 2 5 0 , 691-701.

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CURRENT TOPICS IN MEMBRANES AND TRANSPORT, VOLUME 19

K+-IndependentActive Transport of Na+ by Na,K-ATPase MICHAEL FORGAC AND GILBERT CHIN Depanment of Biochemistry and Molecular Biology Harvard Universiry Cambridge, Massachusetts

I.

INTRODUCTION

The N a , K - A T P a s e f u n c t i o n s i n v i v o t o c o u p l e a c t i v e t r a n s p o r t of Na+ and Kt. a c r o s s t h e plasma membrane t o ATP h y d r o l y s i s . I n t h e absence o f K+, a ouabaini n h i b i t e d Na-ATPase a c t i v i t y i s o b s e r v e d and one m i g h t a s k whether t h i s a c t i v i t y i s c o u p l e d t o N a + t r a n s p o r t . P r e v i o u s e x p e r i m e n t s o f Glynn and K a r l i s h (1976) have shown a n ATP-dependent, o u a b a i n - i n h i b i t e d N a + e f f l u x from r i g h t - s i d e o u t e r y t h r o c y t e g h o s t s suspended i n medium l a c k i n g N a + and K+. However, s i n c e N a + i s movi n g down i t s c o n c e n t r a t i o n g r a d i e n t , s u c h a f l u x does not necessarily represent active transport. I n an attempt t o d e t e r m i n e whether N a + can be pumped a g a i n s t a c o n c e n t r a t i o n g r a d i e n t i n t h e a b s e n c e of K + , w e have examined N a + t r a n s p o r t by t h e N a , K - A T P a s e r e c o n s t i t u t e d i n t o a r t i f i c i a l p h o s p h o l i p i d v e s i c l e s by t h e c h o l a t e d i a l y s i s p r o c e d u r e o f Goldin ( 1 9 7 7 ) .

659

Copyright 0 1983 by Academic Press,Inc. All rights of reproductionin any form R S C N ~ . ISBN 0-12-153319-0

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F i g . 1 . Active Na t r a n s p o r t b y the Na-ATPase. Reconstit u t e d vesicles p r e p a r e d i n the a b s e n c e of I@ w e r e e q u i l i b r a t e d f o r 3 d a y s a t 37OC w i t h 22Naf either i n the a b s e n c e ( 0 ,A, Ij or p r e s e n c e ( 0 ) of 200 vlcl o u a b a i n . A f t e r e q u i l i b r a t i o n , either 5 mM MgCl2 a l o n e ( A ) or w i t h 2 mM ATP ( ,0 ) or 1 mM AMP-PNP ( W ) was a d d e d , vesicles w e r e i n c u b a t e d a t 23OC and 5 0 p 1 a l i q u o t s w e r e removed a t the i n d i c a t e d t i m e s and a n a l y z e d for t r a p p e d 22Na+ b y p a s s a g e over a S e p h a d e x G-50 c o l u m n .

11.

RESULTS AND DISCUSSION

I n o r d e r t o measure a c t i v e t r a n s p o r t of N a + by t h e Na K-ATPase w i t h o u t i n t e r f e r e n c e from ATP-stimulated Naln 4 f. NaAUt exchange, v e s i c l e s were e q u i l i b r a t e d w i t h 22Na+ by i n c u b a t i o n f o r 3 days a t 37OC. A d d i t i o n of Mg2+ and ATP t o vesicles e q u i l i b r a t e d by t h i s p r o c e d u r e r e s u l t e d i n an i n c r e a s e i n t h e amount of 22Na+ t r a p p e d ( F i g . 1 ) . T h i s Na+ uptake w a s dependent on ATP hydrol y s i s ( n o t s u p p o r t e d by AMP-PNP) and w a s i n h i b i t e d by ouabain added a t t h e s t a r t 05 t h e e q u i l i b r a t i o n p e r i o d .

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S i n c e N a + w a s i n i t i a l l y p r e s e n t a t t h e same c o n c e n t r a t i o n on b o t h s i d e s o f t h e membrane, t h i s u p t a k e r e p r e s e n t s n e t t r a n s p o r t of N a + a g a i n s t a c o n c e n t r a t i o n g r a d i e n t . To d e t e r m i n e whether t h e o b s e r v e d N a + u p t a k e c o u l d b e e x p l a i n e d i n t e r m s of c o u p l e d N a + / K + exchange a t t h e endogenous l e v e l s o f K + o r NH4+ i o n s ( a s measured by a t o m i c a b s o r p t i o n and amino a c i d a n a l y s i s , r e s p e c t i v e l y ) , N a + t r a n s p o r t was measured a t 2 and 4 t i m e s these levels. 2 2 N a + u p t a k e w a s i n d e p e n d e n t o f K+ and NH4+ i o n s i n t h i s c o n c e n t r a t i o n r a n g e (510 UM). Measurement o f Na-ATPase a c t i v i t y gave a s t o i c h i o metry f o r t r a n s p o r t of 0.5 mole N a + / m o l e ATP. When v e s i c l e s w e r e e q u i l i b r a t e d under t h e same c o n d i t i o n s e x c e p t i n t h e p r e s e n c e of 2 0 mM K C 1 , ATP-dependent 22Na+ u p t a k e o c c u r r e d w i t h a s t o i c h i o m e t r y of 2 . 9 moles N a + / mole ATP, i n d i c a t i n g t h a t t h e lower s t o i c h i o m e t r y obs e r v e d f o r K+-independent N a + t r a n s p o r t w a s n o t due t o some m o d i f i c a t i o n of t h e enzyme i n t r o d u c e d d u r i n g rec o n s t i t u t i o n o r e q u i l i b r a t i o n . One p o s s i b l e e x p l a n a t i o n of t h i s s t o i c h i o m e t r y i s t h a t N a + i o n s ( e i t h e r 2 o r 3 ) b i n d t o t h e s i t e s which normally b i n d K + , t h u s g i v i n g rise t o e i t h e r a 3 f o r 2 o r a 3 f o r 3 Na+ exchange. T h i s model would p r e d i c t t h a t t h e s t o i c h i o m e t r y of N a + t r a n s p o r t should d e c r e a s e as t h e N a + c o n c e n t r a t i o n i s r a i s e d and a t h i r d N a + i o n i s f o r c e d o n t o t h e K + s i t e s . T h i s p r e d i c t i o n h a s been v e r i f i e d by measurement of t h e N a + dependence o f t h e s t o i c h i o m e t r y of K+-independent N a + t r a n s p o r t (Fig. 2 ) . Measurement of 36Cl' t r a p p i n g d u r i n g a c t i v e N a + u p t a k e i n d i c a t e d t h a t a p p r o x i m a t e l y e q u i v a l e n t amounts of N a + and C1' w e r e accumulated. The d r i v i n g force f o r t h i s 36Cl- u p t a k e w a s p o s t u l a t e d t o be t h e membrane pot e n t i a l g e n e r a t e d d u r i n g N a + u p t a k e . To t e s t t h i s hyp o t h e s i s , t h e membrane p o t e n t i a l w a s measured by [~H]triphenylmethylphosphoniumi o n d i s t r i b u t i o n ( s t a n d a r d i z e d u s i n g known membrane p o t e n t i a l s g e n e r a t e d w i t h K+ g r a d i e n t s i n t h e p r e s e n c e of v a l i n o m y c i n ) . A membrane p o t e n t i a l o f 50 mV ( p o s i t i v e i n s i d e ) w a s obs e r v e d d u r i n g N a + u p t a k e . Our r e s u l t s t h u s s u g g e s t t h a t t h e N a , K - A T P a s e i s c a p a b l e o f a K+-independent, e l e c t r o g e n i c t r a n s p o r t of N a + a g a i n s t a c o n c e n t r a t i o n gradient.

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F i g . 2. Na d e p e n d e n c e of the s t o i c h i o m e t r y of K + - i n d e p e n d e n t N a u p t a k e . V e s i c l e s were e q u i l i b r a t e d w i t h 22Na+ i n the p r e s e n c e of the i n d i c a t e d c o n c e n t r a t i o n s of N a C l . A f t e r e q u i l i b r a t i o n , ATP-dependent 22Na+ t r a n s p o r t was m e a s u r e d a t 23OC a s d e s c r i b e d i n F i g . 1 and ATP h y d r o l y s i s w a s m e a s u r e d b y release o f 32Pi f r o m "y-32PIATP.

K+-INDEPENDENTACTIVE TRANSPORT OF Na+

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ACKNOWLEDGMENTS

W e t h a n k Guido G u i d o t t i ( i n whose l a b o r a t o r y t h i s work w a s done) f o r h i s g u i d a n c e , as w e l l as S t a n l e y Goldin, R o b e r t Quirk, and Michael H o f o r t h e i r h e l p f u l d i s c u s s i o n s and t e c h n i c a l assistance. T h i s work w a s s u p p o r t e d by NIH G r a n t HL 08893 and NSF G r a n t PCM 7 8 4 4 3 6 4 .

REFERENCES

Glynn, I. M . , and K a r l i s h , S. J. D. ( 1 9 7 6 ) . J . Physiol. (London) 256, 465-496. Goldin, S. M. ( 1 9 7 7 ) . J . Biol. Chem. 252, 5630-5642.

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CURRENT TOPICS M MEMBRANES AND TRANSPORT, VOLUME 19

ADP-ATP Exchange in Internally Dialyzed Squid Giant Axons PAUL DE WEER, GERDA E. BREIWIESER, AND H. GILBERT SMITH Department of Physiology and Biophysics Washington Universio School of Medicine St. Louis, Missouri

BRIAN G. KENNEDY' Department of Physiology Yale University Scltwl of Medicine New Haven, Connecticut

I.

MATERIALS AND METHODS

E x p e r i m e n t s were d e s i g n e d t o t e s t w h e t h e r t h e sodium pump o r N a , K - A T P a s e o f i n t e r n a l l y d i a l y z e d s q u i d g i a n t a x o n s c a n c a t a l y z e ADP-ATP exchange u n d e r c o n d i t i o n s where o u a b a i n - s e n s i t i v e N a - N a exchange i s known t o take place. Sodium-sodium exchange i s e l e c t r o n e u t r a l ( G a r r a h a n and Glynn, 1 9 6 7 a ; Abercrombie and D e Weer, 19781, r e q u i r e s ADP ( D e Weer, 1 9 7 0 ; Glynn and Hoffman, 1971) a s w e l l as ATP ( C a v i e r e s and Glynn; 1 9 7 9 1 , b u t d o e s n o t h y d r o l y z e t h e l a t t e r ( G a r r a h a n and Glynn, 1 9 6 7 b ) . S i n c e one of t h e r e a c t i o n s c a t a l y z e d by membrane-bound Na,K-ATPase i s a sodium-dependent ADP-ATP exchange (Skou, 1 9 6 0 1 , i t h a s been p r o p o s e d ( D e Weer, 1970; Glynn and Hoffman, 1 9 7 1 ) t h a t N a - N a exchange and ADP-ATP exchange a r e m a n i f e s t a t i o n s o f a s i n g l e molecular operation. The i n t e r n a l d i a l y s i s t e c h n i q u e of B r i n l e y and M u l l i n s (1967) w a s employed. G i a n t a x o n s of L o l i g o p e a l e i ( d i a m e t e r 1: 5 0 0 p m ) were b a t h e d i n p o t a s s i u m ' P r e s e n t a d d r e s s : Department of B i o c h e m i s t r y and M o l e c u l a r Biology, Lhiversity of T e x a s M e d i c a l S c h o o l , H o u s t o n , T e x a s . 665

Copyright 0 1983 by Academic Press, Inc. All rights of reproductionin any form reserved.

ISBN &IZ-l533194

666

PAUL DE WEER et el.

f r e e a r t i f i c i a l seawater and p e r f u s e d i n t e r n a l l y v i a a c e l l u l o s e a c e t a t e c a p i l l a r y t u b e ( d i a m e t e r = 100-150 p m ) running down t h e c e n t e r of t h e axon. The c e l l u l o s e acet a t e c a p i l l a r y had been r e n d e r e d porous t o i o n s and molecules of up t o 1 0 0 0 d a l t o n s i n i t s middle 1 5 mm reg i o n by exposure t o a l k a l i . The i n t e r n a l e r f u s a t e cont a i n e d , b e s i d e s g l y c i n e , g l u t a m a t e , C1-, K , and HEPES b u f f e r : 1 0 0 m Na+, 5 mM ATP, 5 m M ADP, and 1 5 mM Mg; L-arginine was a b s e n t . The flow r a t e of t h e i n t e r n a l p e r f u s a t e was a b o u t 1 . 2 pl/min, r e s u l t i n g i n an ADP del i v e r y r a t e ( t o t h e c a p i l l a r y ) of a b o u t 50 pmoles/sec, and a l i n e a r flow r a t e of 2.5 mm/sec, g i v i n g a 6-sec dwell t i m e i n t h e porous r e g i o n . N u c l e o t i d e s and nuc l e o s i d e s i n t h e e f f l u e n t from t h e d i a l y s i s c a p i l l a r y were s e p a r a t e d by P E I - c e l l u l o s e t h i n - l a y e r chromatography. E s s e n t i a l l y a l l [14C]ADP o r ATP p r e s e n t e d t o t h e axon was r e c o v e r e d a s ATP, ADP, AMP, o r adenosine. The p e r m e a b i l i t y of t h e porous r e g i o n i n s i t u t o ATP and ADP was c a l c u l a t e d from t h e r a t e c o e f f i c i e n t of washout of [14C]ATP o r ADP from an axon e q u i l i b r a t e d with t h e s e l a b e l s . From t h i s i t was c a l c u l a t e d t h a t about 1 5 % of t h e n u c l e o t i d e d e l i v e r e d t o t h e c a p i l l a r y exchanges w i t h t h e axoplasm; t h e remainder e x i t s from t h e c a p i l l a r y (see F i g . 1 ) . The sodium pump of axons p e r f u s e d under t h e above c o n d i t i o n s engages i n Na-Na exchange ( D e Weer e t a l . , 1 9 7 9 ) . S i n c e b o t h NEM and oligomycin enhance ADP-ATP exchange (Fahn et a l . , 1966; B l o s t e i n , 1 9 7 0 1 , t h e i r e f f e c t on t h e s q u i d axon sodium pump was i n v e s t i g a t e d . Oligomycin, which b l o c k s Na-Na exchange i n e r y t h r o c y t e s (Garrahan and Glynn, 1 9 6 7 b ) , had no such e f f e c t when NEM a p p l i e d i n t r a a p p l i e d i n t r a c e l l u l a r l y a t 10-5 M . c e l l u l a r l y a t 5 m~ i n h i b i t e d o u a b a i n - s e n s i t i v e Na-K and Na-Na exchange, and r e n d e r e d t h e membrane l e a k y t o sodium i o n s . When axons were p e r f u s e d w i t h s o l u t i o n s c o n t a i n i n g [14c]ADP, up t o 8 % of t h e l a b e l w a s recovered a s *ATPI s u g g e s t i n g t h a t a t l e a s t h a l f of t h e *ADP t h a t r e a c h e s t h e axoplasm i s c o n v e r t e d t o ATP b e f o r e it r e e n t e r s t h e porous c a p i l l a r y . A d d i t i o n s of cyanide ( 2 m M ) , iodoa c e t a t e ( 2 m M ) , and a t r a c t y l o s i d e (50 U M ) , which s h o u l d block m e t a b o l i c r e c o n v e r s i o n of ADP t o ATP, and diadenos i n e pentaphosphate (0.5 m M ) , which b l o c k s a d e n y l a t e k i n a s e , brought t h e r e c o v e r y of l a b e l a s *ATP down t o 1% o r less. T h i s d e m o n s t r a t e s t h a t o u r p r o c e d u r e d e t e c t s p r e d i c t a b l e changes i n [ 14C]ATP f o r m a t i o n , and t h a t , w i t h t h e l i s t e d i n h i b i t o r s p r e s e n t , a b o u t 1/15 of t h e "ADP r e a c h i n g t h e axoplasm o r 1 pmole/sec, is recapt u r e d by t h e c a p i l l a r y a s 'ATP. T h i s c o u l d , of c o u r s e , mean t h a t ATP , i n c l u d i n g f r e s h l y produced [ 14C]ATP , i s

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nucleotide flow 100 pmol /sec

F i g . 1 . S c h e m a t i c summary of e x c h a n g e s b e t w e e n i n t e r n a l p e r f u s i o n c a p i l l a r y and a x o p l a s m , both c o n t a i n i n g 1 5 mM e a c h of ATP and ADP i n the s t e a d y s t a t e . F u r t h e r d e s c r i p t i o n i s cont a i n e d i n the t e x t .

(relconverted to ADP at a very high rate. We checked this possibility by perfusing axons with [14C]ATP as the label, and found that ATP + ADP conversion in a 15-mm stretch of axon amounted to less than 8 pmoles/ sec (see Fig. 1). If the Na-Na exchange say 2.25 pmoles/cm2 sec were concomitant with ADP-ATP exchange (3:l stoichiometry), then the sodium pumps present on an axon 15 mm long and 0.5 mm across would produce 2 pmoles *ATP from *ADP every second. About 1/3 to 1/2 would be reconverted to *ADP and the remainder would be captured by the porous capillary (Fig. 1). That is, we should expect to detect ouabain-sensitive production of *ATP at a rate of 1.0-1.3 pmoles/sec in our system. In fact, we never found as much as 2.0.5 pmole/sec, the limit of our sensitivity.

-

PAUL DE WEER eta/.

668

11.

RESULTS AND DISCUSSION

W e i n t e r p r e t t h i s f i n d i n g t o mean t h a t , under o u r e x p e r i m e n t a l c o n d i t i o n s , t h e sodium-carrying enzyme can skpttle Na+ s e v e r a l times a c r o s s t h e membrane bef o r e ATP i s r e l e a s e d . T h i s r e q u i r e s t h a t , i n t h e s q u i d axon enzyme, a d d i t i o n of ATP and Nai be o r d e r e d , n o t random:

ATP

ADP

-E%P*ADP(Na)

NaO

t

P-NaoG=E%P

Support f o r o r d e r e d r e l e a s e of ADP and Nao i n t h e r e d blood c e l l enzyme has been p r e s e n t e d by Glynn and Hoffman ( 1 9 7 1 ) . I t i s h i g h l y u n l i k e l y t h a t t h e f a i l u r e t o r e l e a s e *ATP i s due t o t h e h i g h [Na]i i n o u r e x p e r i ments s i n c e , i n f a c t , h i g h l e v e l s of sodium are known t o s t i m u l a t e ADP-ATP exchange i n kidney membranes (Beau94 and Glynn, 1 9 7 9 ) . R a t h e r , it a p p e a r s t h a t t h e a d d i t i o n of Nai must be preceded by t h e o r d e r e d a d d i t i o n of f i r s t ATP, t h e n Mg: ATP

M9

Nai E*ATPMg*Na,-.

2

--

High l e v e l s of M g would a l l o w Na-Na exchange t o t a k e p l a c e , y e t i n h i b i t t h e r e l e a s e of "ATP formed from "ADP. I n h i b i t i o n of ADP-ATP exchange by h i g h [Mg] i s w e l l documented (Fahn et a l . , 1 9 6 6 ; Robinson, 1 9 7 6 ; Beauge and Glynn, 1 9 7 9 ) .

ACKNOWLEDGMENT

Supported by NIH Grant NS 11223.

ADP - ATP EXCHANGE IN SQUID GIANT AXONS

669

REFERENCES Abercrombie, R. F., and De Weer, P. (1978). Electric current generated by squid giant axon sodium pump: External K and internal ADP effects. Am. J. Physiol. 235, C63-C68, Beaugd, L. A., and Glynn, I. M. (1979). Sodium ions, acting at high-affinity extracellular sites, inhibit sodium-ATPase activity of the sodiun pump by slowing dephosphorylation. J . Physiol. (London) 289, 17-31. Blostein, R. (1970). Sodium activated adenosine triphosphatase activity of the erythrocyte membrane. J. Biol. Chem. 245, 270-275. Brinley, F. J., Jr., and Mullins, L. J. (1967). Sodium extrusion by internally dialyzed squid axons. J . Gen. Physiol. 50, 2303-2331. Cavieres, J. D., and Glynn, I. M. (1979). Sodium-sodium exchange through the sodium pump: The roles of ATP and ADP. J . Physiol . (London) 297, 637-645. De Weer, P. (1970). Effects of intracellular adenosine-5'-diphosphate and orthophosphate on the sensitivity of sodium efflux from squid axon to external sodium and potassium. J. Gen. Physiol. 56, 583-620. De Weer, P., Kennedy, B. G., and Abercrombie, R. F. (1979). Relationship between the Na:K exchanging and Na:Na exchanging modes of operation of the sodium pump. In "Na,K-ATPase: Structure and Kinetics" (J. C. Skou and J. G. Ndrby, eds.), pp. 504-515. Academic Press, New York. Fahn, S., Hurley, M. R., Koval, G. J., and Albers, R. W. (1966). Sodium-potassium-activated adenosine triphosphatase of Electrophorus electric organ. 11. Effects of N-ethylmaleimide and other sulfhydryl reagents. J . Biol. Chem. 241, 1890-1895. Garrahan, P. J., and Glynn, I. M. (1967a). The behaviour of the sodium pump in red cells in the absence of external potassium. J. Physiol. (London) 192, 159-174. Garrahan, P. J., and Glynn, I. M. (1967b). The Stoicheiometry of the sodium pump. J . Physiol. (London) ,192, 217-235. Glynn, I. M., and Hoffman, J. F. (1971). Nucleotide requirements for sodium-sodium exchange catalysed by the sodium pump in human red blood cells. J. Physiol. (London) 218, 239-256. Robinson, J. D. (1976). The (Na++K+)-dependent ATPase. Mode of inhibition of ADP/ATP exchange activity by MgC12. Biochim. Biophys. Acta 440, 711-722. ++ + Na+-actiSkou, J. C. (1960). Further investigation on a Mg vated adenosinetriphosphatase, possibly related to the active, linked transport of Na' and K+ across the nerve membrane. Biochim. Biophys. A c t a 42, 6-23.

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CURRENT TOPICS IN MEMBRANES A N D TRANSPORT, VOLUME 19

Sodium Pump-CatalyzedATP-ADP Exchange in Red Blood Cells: The Effects of lntracellular and Extracellular Na and K Ions JACK H.KAPLAN Department of Physiology University of Pennsylvania Philadelphia, Pennsylvania

I.

INTRODUCTION

When human r e d b l o o d c e l l s a r e i n c u b a t e d i n K-free media c o n t a i n i n g N a i o n s , a n o u a b a i n - s e n s i t i v e exchange of i n t r a c e l l u l a r and e x t r a c e l l u l a r N a i o n s i s o b s e r v e d ( G a r r a h a n and Glynn, 1 9 6 7 a ) . S u b s e q u e n t s t u d i e s o n t h i s t r a n s p o r t mode of t h e N a pump have e s t a b l i s h e d t h a t t h e N a - N a exchange r a t e i s i n c r e a s e d by e l e v a t e d l e v e l s o f ADP, i s i n d e p e n d e n t of ATP i n t h e r a n g e 3 0 0 U M t o 1 5 0 0 U M (Glynn and Hoffman, 1 9 7 1 ) , and w i l l n o t t a k e p l a c e i n r e s e a l e d g h o s t s c o n t a i n i n g ADP b u t n o t ATP (Cavieres and Glynn, 1 9 7 9 ) . Although ATP i s r e q u i r e d f o r t h e Na pump t o s u p p o r t Na-Na e x c h a n g e , t h e r e i s a p p a r e n t l y no n e t h y d r o l y s i s o f ATP a s s o c i a t e d w i t h t h e t r a n s p o r t ( G a r r a h a n and Glynn, 1 9 6 7 ~ ) . These o b s e r v a t i o n s s u p p o r t t h e s u g g e s t i o n t h a t t h e biochemic a l e v e n t s c a t a l y z e d by t h e Na pump p r o t e i n w h i l e m e d i a t i n g N a - N a exchange are t h e r e v e r s i b l e p h o s p h o r y l a t i o n of t h e p r o t e i n by ATP and i t s d e p h o s p h o r y l a t i o n by ADP (Glynn and Hoffman, 1 9 7 1 ) . The b i o c h e m i c a l 671

Copyright 0 1983 by Academic Press, Inc. All nghts of reproduction in any form r e ~ e ~ e d . ISBN 0-12-153319-0

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JACK H. KAPLAN

r e a c t i o n , ATP-ADP exchange, was f i r s t o b s e r v e d i n p a r t i a l l y p u r i f i e d Na,K-ATPase enzyme by Fahn e t a l . ( 1 9 6 6 ) and l a t e r work h a s d e m o n s t r a t e d t h e complex N a dependence of t h e r e a c t i o n i n enzyme from p i g k i d n e y (Beau96 and Glynn, 1 9 7 9 ) , dog k i d n e y (Kaplan e t a l . , 1 9 8 1 ) , and r a t b r a i n (Wildes e t a l . , 1 9 7 3 ) , and i n human r e d b l o o d c e l l membranes (Kaplan and H o l l i s , 1 9 8 0 ) . The s h a p e of t h e c u r v e r e l a t i n g N a c o n c e n t r a t i o n t o ATP-ADP exchange r a t e i n t h e s e " u n s i d e d " p r e p a r a t i o n s i s composed p r e d o m i n a n t l y of t h r e e r e g i o n s . I n t h e reg i o n c o m p r i s i n g 0 t o a b o u t 3 m N a t h e exchange r a t e i n c r e a s e s with N a c o n c e n t r a t i o n , i n t h e region comprising 3 mM N a t o a b o u t 10 mM N a t h e r a t e i s p r o g r e s s i v e l y i n h i b i t e d by i n c r e a s i n g Na c o n c e n t r a t i o n s , and f i n a l l y i n t h e r e g i o n c o m p r i s i n g 1 0 mM Na t o 1 5 0 mM Na t h e r a t e i n c r e a s e s w i t h N a c o n c e n t r a t i o n , so t h a t by 1 5 0 mM N a t h e exchange r a t e i s a p p r o x i m a t e l y 2- t o 3 - f o l d h i g h e r t h a n a t 3 mM Na. The o b j e c t i v e of o u r c u r r e n t s t u d i e s i s t o c h a r a c t e r i z e t h e s i d e d n e s s of t h e s e N a a c t i v a t i n g (and i n h i b i t i n g ) e f f e c t s and examine t h e v a l i d i t y o f t h e hyp o t h e s i s t h a t ATP-ADP exchange and Na-Na exchange are c o u p l e d s i m u l t a n e o u s b i o c h e m i c a l and t r a n s p o r t i n g reactions.

11.

MATERIALS AND METHODS

The e x p e r i m e n t a l s y s t e m w e have employed i s t h e r e s e a l e d human r e d c e l l g h o s t p r e p a r e d by r e v e r s i b l e h y p o t o n i c l y s i s . The p r o c e d u r e u s e d t o p r e p a r e t h e res e a l e d g h o s t s employs a g e l f i l t r a t i o n t e c h n i q u e d e s cribed previously. The a d v a n t a g e of t h i s a p p r o a c h o v e r e a r l i e r p r o c e d u r e s i s t h a t v e r y h i g h d i l u t i o n s of c y t o p l a s m i c enzymes and s u b s t r a t e s are p o s s i b l e w i t h o u t u s i n g m u l t i p l e hemolysis s t e p s o r i n c o n v e n i e n t l y l a r g e volumes of hemolyzing s o l u t i o n s . I n o r d e r t o be a b l e t o measure t h e r a t e of t h e ATP-ADP exchange r e a c t i o n , it i s a l s o n e c e s s a r y t o be a b l e t o measure t h e r a t e o f a p p e a r a n c e of [ 3 H ] A T P from [3H]ADP i n t h e r e s e a l e d g h o s t s y s t e m . The major d i f f i c u l t y i n making t h e s e measurements h a s been i n i n i t i a t i n g t h e r e a c t i o n a f t e r p r e p a r a t i o n of t h e g h o s t s . Without a means of i n i t i a t i n g t h e r e a c t i o n , i s o t o p i c e q u i l i b r a t i o n and ATP d e g r a d a t i o n d u r i n g g h o s t r e s e a l i n g a t 37" would i n v a l i d a t e t h e measurements. I n o r d e r t o i n i t i a t e t h e i n t r a c e l l u l a r r e a c t i o n , w e have u t i l i z e d t h e p h o t o l a b i l e p r e c u r s o r of A T P , caged ATP. T h i s compound i s a s t a b l e e s t e r o f ATP b e a r i n g a 2 - n i t r o b e n z y l m o i e t y on t h e t e r m i n a l p h o s p h a t e

Na PUMP-CATALYZEDATP - ADP EXCHANGE

673

of ATP (Kaplan et a l . , 1 9 7 8 ) . Following a b r i e f p u l s e of l i g h t a t 350 nm, free ATP i s r e l e a s e d a s t h e t e r m i n a l p h o s p h a t e - e s t e r bond i s p h o t o l y t i c a l l y c l e a v e d . B r i e f l y , t h e p r o c e d u r e f o r measuring t h e r a t e o f t h e ATP-ADP exchange r e a c t i o n i n resealed red b l o o d c e l l g h o s t s c o n s i s t s o f p r e p a r i n g t h e g h o s t s by g e l f i l t r a t i o n and i n c o r p o r a t i n g i n t o t h e i n t r a c e l l u l a r compartment caged ATP, [ 3H]ADP, d i a d e n o s i n e p e n t a p h o s p h a t e ( A P s A ) , a n a d e n y l a t e k i n a s e i n h i b i t o r , b u f f e r , and app r o p r i a t e l e v e l s of MgC12 and N a + K s a l t s . The g h o s t s are t h e n suspended i n b u f f e r e d s o l u t i o n s c o n t a i n i n g N a o r K s a l t s and p h o t o l y z e d a t 350 nm f o r 30 sec. Samples are t h e n t a k e n a t 30-sec i n t e r v a l s and quenched i n c o l d a c i d , and t h e n u c l e o t i d e s a r e s e p a r a t e d by t h i n l a y e r chromatography. The r a t e of a p p e a r a n c e of [3H]ATP c a n t h e n b e o b t a i n e d . The d i f f e r e n c e between t h e s e r a t e s i n t h e p r e s e n c e and a b s e n c e o f 1 0 - 4 M o u a b a i n i s t a k e n a s t h e N a pump-mediated component (Kaplan and H o l l i s , 1980). W e have p r e v i o u s l y shown t h a t e x t e r n a l N a i n h i b i t s

t h e ATP-ADP exchange r e a c t i o n i n t h e r e g i o n 0-5 m~ N a and t h a t i n c r e a s i n g N a c o n c e n t r a t i o n s from 5 mM t o 150 m~ N a s t i m u l a t e t h e r e a c t i o n r a t e i n a r o u g h l y l i n e a r f a s i o n (Kaplan and H o l l i s , 1 9 8 0 ) . I n o u r c u r r e n t s t u d i e s w e have e x t e n d e d t h e s e o b s e r v a t i o n s t o show t h a t i n t e r n a l N a a c t i v a t e s t h e r e a c t i o n and s a t u r a t e s a t conc e n t r a t i o n s o f 2 mM o r less. No e v i d e n c e w a s o b t a i n e d f o r any i n h i b i t o r y e f f e c t s o f i n t r a c e l l u l a r N a . These d a t a a l l o w u s t o unambiguously a s s i g n e a c h o f t h e t h r e e p h a s e s of t h e c u r v e r e l a t i n g N a c o n c e n t r a t i o n t o ATP-ADP exchange r a t e t o i n t e r n a l o r e x t e r n a l a s p e c t s of t h e N a pump p r o t e i n . W e have a l s o examined t h e e f f e c t s o f ext r a c e l l u l a r K i o n s on t h e r a t e of t h e exchange r e a c t i o n . I n F i g . 1, t h e r e s u l t s o f s u c h a n e x p e r i m e n t are pres e n t e d . E x t r a c e l l u l a r K ( K o ) i n h i b i t s t h e ATP-ADP exchange r e a c t i o n w i t h an a p p a r e n t a f f i n i t y o f a b o u t 1 mM --a v a l u e i n c l o s e agreement w i t h t h e a p p a r e n t a f f i n i t y f o r KO i n i n h i b i t i n g N a - N a exchange and a c t i v a t i n g Na-K exchange (Garrahan and Glynn, 1967b). E x t r a c e l l u l a r L i ( L i o ) a l s o i n h i b i t s t h e ATP-ADP exchange r a t e , w i t h a much lower a f f i n i t y t h a n KO. ~ 0 . 5f o r L i 0 i s a b o u t 50 m ~ . I n t e r n a l K ( K i ) had l i t t l e e f f e c t on t h e r a t e o f ATP-ADP exchange, i n t h e c o n c e n t r a t i o n r a n g e where it i n h i b i t s a t t h e outside surface.

674

JACK H. KAPLAN

0'

2

4

6

8

10

[KI, Fig. 1.

111.

CONCLUSIONS

T o what e x t e n t do t h e s e r e s u l t s s u p p o r t t h e hyp o t h e s i s t h a t ATP-ADP exchange o c c u r s w h i l e t h e pump i s c a r r y i n g o u t a 1:l exchange of Nai f o r Na,? The e f f e c t s of N a o show t h e same b i p h a s i c c u r v e a s t h a t o b t a i n e d when t h e e f f e c t of Nao on o u a b a i n - s e n s i t i v e N a e f f l u x i s s t u d i e d i n human r e d c e l l s . Probably t h e n t h e same s i t e s a r e i n v o l v e d i n s t i m u l a t i o n o f Na e f f l u x and s t i m u l a t i o n of ATP-ADP exchange. The e f f e c t s of KO a l so s u p p o r t t h i s c o n c l u s i o n . The s i t e s which i n h i b i t Na-Na exchange (and s t i m u l a t e Na-K exchange) a l s o i n h i b i t ATP-ADP exchange. I t seems l i k e l y t h e n t h a t Na e n t r y i s coupled t o p h o s p h o r y l a t i o n of ADP by Na pump phosphoprotein. However, t h e o b s e r v a t i o n of ATP-ADP exchange i n t h e absence of e x t r a c e l l u l a r Na i o n s (Xaplan and H o l l i s , 1980) i m p l i e s t h a t Na e n t r y i s n o t a p r e r e q u i s i t e f o r t h e t r a n s p h o s p h o r y l a t i o n r e a c t i o n and t h a t t h e p r o c e s s e s may n o t be t i g h t l y coupled.

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675

ACKNOWLEDGMENTS

I am g r a t e f u l t o Richard J. H o l l i s and Linda J . Kenney f o r e x c e l l e n t t e c h n i c a l a s s i s t a n c e . The work was supported by N I H Grant HL-28457.

REFERENCES

Beaug6, L. A . , and Glynn, I . M. (1979). Sodium i o n s , a c t i n g higha f f i n i t y e x t r a c e l l u l a r s i t e s , i n h i b i t sodium-ATPase a c t i v i t y of t h e sodium pump by slowing dephosphorylation. J. Physiol. (London) 289, 17-31. Cavieres, J. D . , and Glynn, I . M. (1979). Sodium-sodium exchange through t h e sodium pump: The r o l e s o f ATP and ADP. J. Physiol. (London) 297, 637-645. Fahn, S . , Koval, G . J . , and Albers, R . L. (1966). Sodium-potassiuma c t i v a t e d adenosine t r i p h o s p h a t a s e of Electrophorus e l e c t r i c organ. J. Biol. C h e m . 241, 1882-1889. Garrahan, P. J , and Glynn, I. M. (1967a). The behavior of t h e sodium pump i n r e d c e l l s i n t h e absence of e x t e r n a l potassium. J . Physiol. (London) 192, 159-174. Garrahan, P. J . , and Glynn, I . M. (1967b). F a c t o r s a f f e c t i n g t h e r e l a t i v e magnitudes of t h e sodium:potassium and sodium:sodium exchanges c a t a l y s e d by t h e sodium pump. J. Physiol. (London) 192, 189-216. Garrahan, P. J . , and Glynn, I. M. ( 1 9 6 7 ~ ) . The s t o i c h i o m e t r y of t h e sodium pump. J. Physiol. 192, 217-235. Glynn, I. M., and Hoffman, J . F. (1971). Nucleoti.de requirements f o r sodium-sodium exchange c a t a l y s e d by t h e sodium pump i n human r e d c e l l s . J. Physiol. (London) 218, 239-256. Kaplan, J. H . , and H o l l i s , R. J. (1980). E x t e r n a l Na dependence of ouabain-sensitive ATP-ADP exchange i n i t i a t e d by p h o t o l y s i s o f Nature i n t r a c e l l u l a r caged-ATP i n human red c e l l ghosts. (London) 288, 587-589. Kaplan, J. H . , Forbush, B . , 111, and Hoffman, J . F. (1978). Rapid p h o t o l y t i c r e l e a s e of adenosine-51-triphosphate from a prot e c t e d analog: U t i l i z a t i o n by t h e Na-K pump of human r e d blood c e l l s . Biochemistry 17, 1929-1935. Kaplan, J . H . , H o l l i s , R. J . , and Mone, M. D . (1981). The regulat i o n o f Na pump-mediated ATP:ADP exchange by e x t r a c e l l u l a r Na i o n s . A d v . Physiol. Sci., Proc. I n t . Congr. , 28th, 1980, Vol. 6, pp. 293-298. Wildes, R. A . , Evans, H . J . , and Chiu, J . (1973). E f f e c t s of cat i o n s on t h e adenosine diphosphate-adenosine t r i p h o s p h a t e exchange r e a c t i o n c a t a l y z e d by r a t b r a i n microsomes. Biochim. Biophys. A c t a 307, 162-168.

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CURRENT TOPICS IN MEMBRANES AND TRANSPORT, VOLUME 19

Ouabain-Sensitive ATP-ADP Exchange and Na-ATPase of Resealed Red Cell Ghosts J. D.CAVIERES Physiological Laboratory University of Cambridge Cambridge, England

I.

INTRODUCTION

The sodium-sodium exchange that the sodium pump catalyzes in the absence of extracellular K with negligible associated hydrolysis of ATP, presumably represents a reversal of the uphill Na efflux seen in physiological conditions (Garrahan and Glynn, 1967; Baker et a l . , 1969). Sodium-sodium exchange is activated by ADP (Glynn and Hoffman, 1971) and it has been suggested that just as the Na efflux through the pump appears to be associated with the phosphorylation of the enzyme by ATP, the Na influx would be concomitant with the rephosphorylation of ADP by the phosphoenzyme (Garrahan and Glynn, 1967). This implied that, besides ADP, Na-Na exchange would need ATP, a requirement that has recently been demonstrated (Cavieres and Glynn, 1979). However, there are alternative explanations and one of these is that the carrier is really a form of the phosphoenzyme with bound ADP. 677

Copyrighr 0 1983 by Academic Press. Inc. All rightsof reproductionin any form R S ~ N C ~ . ISBN 0-12-153319-0

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One way of testing these possibilities was to find out whether the reversible phosphorylation required both internal and external Na as activators, as would be the case if transport and biochemical reactions were coupled. We knew that, besides Mg, the phosphorylation of broken membranes by ATP needed Na at high-affinity sites (Post et a l . , 19651, and these were most likely to be the high-affinity internal Na sites involved in Na-Na exchange (Garay and Garrahan, 1973). The ATP-ADP exchange activity of broken membranes--a consequence of the reversible phosphorylation--was also activated by Na ions acting at high-affinity sites (Fahn et al., 19661, but it had been so far impossible to measure ATP-ADP exchange mediated by Na,K-ATPase in cells with an intact or resealed membrane and to decide whether external Na was also required. One reason was that the cells contained enough adenylate kinase to equilibrate in a few seconds the nucleotide label of the resulting ghosts. This difficulty was overcome by combining the use of a potent inhibitor of adenylate kinase (PlIP5-di(adenosine-5'-)pentaphosphate or Ap5A) with a great dilution of the red cell adenylate kinase that could be achieved by improving the technique to make resealed ghosts. With the standard procedure, the various components are loaded into the cells by introducing them in the lysing solution. However, extensive dilution of the original cell's contents can be obtained by lysis in a large volume of hypotonic solution followed by centrifugation and resuspension of the membranes in a small volume of fresh solution containing the expensive ingredients, provided the temperature is kept close to O°C to avoid spontaneous resealing of the membranes (Cavieres and Glynn, 1979).

11.

RESULTS AND DISCUSSION

But there is another difficulty with resealed ghosts. If [3H]ADP is added to the suspension and the ghosts are then resealed and washed, ATP-ADP exchange will have already reached equilibrium by the time that they are ready for use. What probably happens is that any systems catalyzing ATP-ADP exchange must be at work in the suspension during the incubation period at 37OC necessary to reseal the membranes. To prevent this, it was decided to chelate Mg2+ (at 0.2 mM from the moment of lysis) with 5 mM EDTA added just before resealing and together with 1.25 mM [3H]ADPI 0.5 m~ [ Y ~ ~ P I A T P and ,

ATP - ADP EXCHANGE, Na-ATPase,AND EXTERNAL Na

679

8-10 m N a . Low " i n i t i a l " [ 3HIATP l e v e l s c o u l d t h u s be o b t a i n e d , though a t t h e expense of r e d u c i n g t h e y i e l d of r e s e a l e d g h o s t s . To s t a r t ATP-ADP exchange, t h e washed g h o s t s were warmed up t o 37OC and Mg2+ w a s a l lowed t o e n t e r w i t h t h e a i d of t h e d i v a l e n t - c a t i o n ionop h o r e A23187. ATP-ADP exchange seems t o be o p t i m a l a t 20-30 p M Mg2+, h i g h e r c o n c e n t r a t i o n s b e i n g i n h i b i t o r y (Fahn et a l . , 1 9 6 6 ) . To a v o i d h a v i n g i n t e r n a l Mg2+ s t i l l changing w h i l e measuring ATP-ADP exchange, Mg2+ w a s f i r s t allowed t o move q u i c k l y i n t o t h e c e l l s by s e t t i n g t h e e x t e r n a l Mg2+ c o n c e n t r a t i o n much h i g h e r t h a n t h e optimum above. T h i s w a s f o l l o w e d by a " c h a s e " w i t h a Mg b u f f e r when t h e e x t e n t o f t h e i n f l u x was a b o u t r i g h t t o a c h i e v e t h e d e s i r e d Mg2+ i n s i d e t h e g h o s t s . I n p r e l i m i n a r y e x p e r i m e n t s t o measure Mg i n f l u x i n t h e s e g h o s t s , t h a t c o n d i t i o n w a s g i v e n a t a b o u t 1 min a f t e r t h e a d d i t i o n o f 10-15 P M A23187 i n t h e p r e s e n c e of 0.5-2 m ext e r n a l MgC12. ATP happens t o b e a manageable b u f f e r i n t h e Mg2+ r a n g e f o r t h e s e e x p e r i m e n t s and it w a s used a t a c o n c e n t r a t i o n o f 5 mM p l u s 0.5 mM MgC12 (Mg2+ = 1 0 p M ) . The c o n t e n t i o n t h e n i s t h a t t h e MgATP b u f f e r f i x e s Mgi+, w h i l e Mgi+ c o n t r o l s Mgf+ by e q u i l i b r a t i o n t h r o u g h t h e ionophore. The p r o c e d u r e w a s t h e n a p p l i e d t o s u s p e n s i o n s o f ghosts containing t h e radioactive nucleotides. J u s t aft e r a d d i n g t h e e x t e r n a l MgATP b u f f e r , e a c h s u s p e n s i o n was d i v i d e d i n t o one p o r t i o n w i t h and one w i t h o u t 1 rn o u a b a i n which were t h e n sampled a t 1-min i n t e r v a l s . The samples were s t o p p e d by b o i l i n g , t h e n u c l e o t i d e s and P i m i x t u r e r e s o l v e d by t h i n - l a y e r chromatography, and t h e 3H and 32P c o n t e n t of each f r a c t i o n e x p r e s s e d as p e r c e n t of t h e t o t a l r e s p e c t i v e i s o t o p e i n t h e sample. To obt a i n t h e ATPase r a t e , s t r a i g h t l i n e s w e r e f i t t e d t o t h e p l o t s of 32Pi release v e r s u s t i m e . To e x t r a c t t h e ATP-ADP exchange r a t e , t h e % [3H]ATP d a t a w a s p l o t t e d i n s e m i l o g a r i t h m i c form f o r approach t o e q u i l i b r i u m (1 y/y,), where w a s c o r r e c t e d f o r e v e r y p o i n t by t h e e x t e n t of [y-3yP] ATP h y d r o l y s i s d e t e r m i n e d simult a n e o u s l y . Good s t r a i g h t l i n e s c o u l d be f i t t e d and t h i s i s t a k e n t o c o n f i r m t h a t t h e c o n d i t i o n s € o r t h e exchange were c o n s t a n t . The r e s u l t s c a n be summarized as f o l l o w s : (1) The o u a b a i n - s e n s i t i v e f r a c t i o n o f t h e ATP-ADP exchange i n 1 4 0 m N a medium r e p r e s e n t s 20-35% o f t h e t o t a l . On a v e r a g e , it amounts t o 0.78 m m o l e / l i t e r g h o s t s p a c e / h r ( r a n g e = 0.28-2.0). ( 2 ) The pump-mediated ATP-ADP exchange i n g h o s t s of a b a t c h i s g r e a t e r when t h e y are suspended i n 1 4 0 mM N a C l t h a n when suspended i n 1 4 0 mM c h o l i n e c h l o r i d e , t h e Na/choline r a t i o b e i n g 3.7, 1 0 , > 2 0 , 2 , and 2.5 f o r f i v e

-

680

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e x p e r i m e n t s . The average o u a b a i n - s e n s i t i v e ATP-ADP exchange found i n Na-free medium i s 0 . 0 7 9 mmole/liter g h o s t space/hr ( r a n g e = - 0 - 0 . 1 7 9 ) . The observed s t i m u l a t i o n of ATP-ADP exchange by high e x t e r n a l Na i s i n agreement w i t h t h e r e s u l t s o b t a i n e d by Kaplan and H o l l i s (1980) a t much lower n u c l e o t i d e c o n c e n t r a t i o n s . ( 3 ) High e x t e r n a l Na i n h i b i t s t h e o u a b a i n - s e n s i t i v e ATPase a c t i v i t y of t h e same g h o s t s . A t z e r o e x t e r n a l Na, t h e a v e r a g e o u a b a i n - s e n s i t i v e ATPase a c t i v i t y i s 0.056 mmole/liter g h o s t s p a c e / h r ( r a n g e = 0.038-0.073), whereas a t 1 4 0 mM N a it i s 0 , 0 0 9 m m o l e / l i t e r g h o s t The Na/choline r a t i o s a r e space/hr ( r a n g e = 0 - 0 . 0 2 4 ) . 2 , -0, -0, 0.28, and 0.25 f o r t h e same e x p e r i m e n t s . ( 4 ) The r a t i o of ATP-ADP exchange t o ATPase under o p t i m a l c o n d i t i o n s ( h i g h and z e r o e x t e r n a l N a , r e s p e c t i v e l y ) i s 9.8, 7 . 4 , 37, 6 . 5 , and 6 . 2 f o r t h e f i v e experiments. C o n s i d e r i n g t h a t , a l t h o u g h s m a l l , t h e r e i s some o u a b a i n - s e n s i t i v e ATP-ADP exchange a t z e r o e x t e r n a l Na, t h e most l i k e l y i n t e r p r e t a t i o n of t h e s t i m u l a t i o n by 1 4 0 m M e x t e r n a l Na i s t h a t a l a r g e p r o p o r t i o n , b u t n o t a l l , of t h e phosphoenzyme i s i n a form which cannot r e a c t w i t h ADP and t h a t Na b i n d i n g a t e x t e r n a l s i t e s promotes t h e c o n v e r s i o n of t h i s form i n t o t h e ADPr e a c t i v e form. These a r e q u i t e l i k e l y t o be t h e Ks e n s i t i v e (E2-P) and ADP-sensitive ( E l - P ) phosphoenzyme forms proposed by P o s t e t al. ( 1 9 6 9 1 , where t h e back r e a c t i o n from E2-P o c c u r s a t only h i g h Na c o n c e n t r a t i o n s ( P o s t e t a l . , 1 9 7 4 ) . The i n h i b i t i o n of Na-ATPase by h i g h e x t e r n a l sodium, which a p p a r e n t l y c o n t r a d i c t s t h e f i n d i n g s of Glynn and K a r l i s h ( 1 9 7 6 1 , can be e x p l a i n e d by t h e same scheme s i n c e t h e r e l a t i v e l y h i g h ADP concent r a t i o n i n t h e p r e s e n t experiments ( n i l i n t h e i r s ) s h o u l d d i s p l a c e t h e e q u i l i b r i u m back, f u r t h e r away from E2-P. A t z e r o e x t e r n a l Na, i n t u r n , t h e spontaneous dep h o s p h o r y l a t i o n of E2-P (Na-ATPase) would b e f a s t enough t o r e p l e n i s h t r a n s i e n t l y t h e enzyme forms t h a t p a r t i c i p a t e i n ATP-ADP exchange and t h u s a l l o w transphosphoryl a t i o n t o proceed a t t h e low r a t e observed. These f i n d i n g s a r e a l l c o n s i s t e n t w i t h t h e i d e a of a l i n k a g e between Na-Na and ATP-ADP exchange. F u r t h e r more, t h e y show t h a t i n K-free systems t h e r a t e of rev e r s i b l e p h o s p h o r y l a t i o n ( i n 1 4 0 mM Na medium) can be one o r d e r of magnitude g r e a t e r t h a n t h e o v e r a l l t u r n o v e r r a t e ( i n Na-free medium). T h i s i s m t r r e t h a n enough t o accommodate Na-Na exchange, which can a t t a i n r a t e s s e v e r a l b u t less t h a n 1 0 t i m e s f a s t e r t h a n t h a t of uncoupled Na e f f l u x ( t h e pump mode a s s o c i a t e d w i t h t h e Na-ATPase a c t i v i t y i n Na-free medium, Glynn and K a r l i s h , 1 9 7 6 ) , i f t h e u s u a l s t o i c h i o m e t r y of 3 Na/cycle a l s o

ATP - ADP EXCHANGE, Na-ATPase,AND EXTERNAL Na

o b t a i n s h e r e . From t h e f o r e g o i n g d i s c u s s i o n , c l e a r t h a t Na-Na and ATP-ADP exchanges s h o u l d p a r t i a l l y o v e r l a p . Simultaneous measurements exchanges s h o u l d h e l p t o d e c i d e how t i g h t t h e is.

681

it i s only of b o t h linkage

ACKNOWLEDGMENTS

Supported by g r a n t s from t h e Medical Research C o u n c i l a n d The Royal S o c i e t y . The A23187 w a s a g i f t from t h e L i l l y Research C e n t r e Ltd. ( E n g l a n d ) .

REFERENCES

Baker, P. F., B l a u s t e i n , M. P . , Keynes, R. D., Manil, J . , Shaw, T. I . , and S t e i n h a r d t , R . A. ( 1 9 6 9 ) . J. Physiol. ( L o n d o n ) 2 0 0 , 459-496. C a v i e r e s , J . D., and Glynn, I. M. ( 1 9 7 9 ) . J. Physiol. ( L o n d o n ) 2 9 7 , 637-645. Fahn, S., Koval, G. J., and A l b e r s , R. W. ( 1 9 6 6 ) . J. Biol. Chem. 2 4 1 , 1882-18a9. Garay, R. P., and Garrahan, P. J. ( 1 9 7 3 ) . J. Physiol. ( L o n d o n ) 2 3 1 , 297-325. Garrahan, P. J . , and Glynn, I. M. ( 1 9 6 7 ) . J. Physiol. ( L o n d o n ) 1 9 2 , 189-216. Glynn, I. M . , and Hoffman, J. F. (1971). J. Physiol. ( L o n d o n ) 2 1 8 , 239-256. Glynn, I . M., and K a r l i s h , S. J. D. ( 1 9 7 6 ) . J. Physiol. ( L o n d o n ) 2 5 6 , 465-496. Kaplan, J. H . , and H o l l i s , R. J. ( 1 9 8 0 ) . Nature ( L o n d o n ) 2 8 8 , 587-589. P o s t , R. L . , Sen, A. K . , and R o s e n t h a l , A. S. ( 1 9 6 5 ) . J. Biol. Chem. 2 4 0 , 1437-1445. P o s t , R. L . , Kume, S., Tobin, T . , O r c u t t , B . , and Sen, A. K. ( 1 9 6 9 ) . J. Gen. Physiol. 5 4 , 306s-326s. P o s t , R. L . , T a n i g u c h i , K . , and Toda, G. (1974). A n n . N.Y. A c a d . Sci. 2 4 2 , 80-91.

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CURRENT TOPICS IN MEMBRANES AND TRANSPORT, VOLUME 19

Effect of Internal Adenine Nucleotides on Sodium Pump-Catalyzed Na-Na and Na-K Exchanges BRIAN G. KENNEDY, WRM LUNN, AND JOSEPH F. HOFFMAN Depanmetu of Physiology Yale University School of Medicine New Haven, Connecticut

I.

INTRODUCTION

I t h a s been c l e a r l y shown t h a t t h e sodium pump o f animal c e l l s can c a t a l y z e s e v e r a l exchange r e a c t i o n s o t h e r t h a n t h e normal Na-K exchange (see Glynn and K a r l i s h , 1975, f o r r e f e r e n c e s ) . Here w e w i l l b e conc e r n e d w i t h two of t h e s e r e a c t i o n s : N a - N a and N a - K exchanges. Both modes o f exchange have been w e l l c h a r a c t e r i z e d a s a f u n c t i o n of i n t e r n a l and e x t e r n a l c a t i o n a f f i n i t i e s (Robinson and F l a s h n e r , 1 9 7 9 ) . A l though n u c l e o t i d e e f f e c t s have been e x t e n s i v e l y s t u d i e d u s i n g v a r i o u s p r e p a r a t i o n s of i s o l a t e d A T P a s e , it h a s proven d i f f i c u l t t o examine t h e n u c l e o t i d e dependence o f t h e sodium pump i n an i n t a c t system. T o p r o v i d e a d e t a i l e d k i n e t i c and m o l e c u l a r mechanism f o r pump f u n c t i o n , b o t h c a t i o n a f f i n i t i e s and n u c l e o t i d e dependence must be examined i n a s i d e d s y s t e m c a p a b l e of v e c t o r i a l t r a n s p o r t . u s i n g r e s e a l e d human r e d c e l l g h o s t s , w e have e s t a b l i s h e d a system where t h e n u c l e o t i d e medium b a t h i n g t h e i n n e r f a c e o f t h e sodium pump 683

Copynght 0 1983 by Acddemc Press,Inc All nghts of reproduction in any form reserved ISBN 0-12-1533190

BRIAN G. KENNEDY eta/.

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can be controlled. S p e c i f i c a l l y , Na-K and N a - N a exchanges have been examined as a f u n c t i o n o f ATP concent r a t i o n a t f i x e d ADP c o n c e n t r a t i o n and v i c e versa.

11.

FC3SULTS AND DISCUSSION

Red c e l l s from f r e s h l y drawn blood were hemolyzed twice, producing an o v e r a l l d i l u t i o n o f t h e i n t r a c e l l u l a r c o n t e n t s of a b o u t 3500-fold. E i t h e r of two reg e n e r a t i n g s y s t e m s , i n c o r p o r a t e d d u r i n g t h e second hem o l y s i s , were employed t o m a i n t a i n v a r y i n g ATP-ADP mixt u r e s . The phosphocreatine/creatine k i n a s e system maint a i n s ATP i n t h e p r e s e n c e of low ADP c o n c e n t r a t i o n s . The phosphoarginine/arginine k i n a s e system c a n b e used t o b u f f e r s i g n i f i c a n t l y h i g h e r ADP l e v e l s , i n t h e p r e s e n c e of a s t a b l e background ATP c o n c e n t r a t i o n . U s i n g t h e s e s y s t e m s , w e have been a b l e t o set and m a i n t a i n ATP/ADP r a t i o s from 0 . 2 t o 2 0 0 . Ouabain-sensitive N a e f f l u x w a s measured i n a 150 mM NaC1/15 mM KC1 medium as t h e i n d e x of N a - K exchange. N a - N a exchange w a s measured a s o u a b a i n - s e n s i t i v e N a e f f l u x i n t o a K-free medium (Glynn and Hoffman, 1 9 7 1 ) . N u c l e o t i d e l e v e l s i n s i d e t h e g h o s t s were always a s s a y e d i n p a r a l l e l w i t h the e f f l u x determinations. W e found N a - K exchange t o b e a s a t u r a t i n g f u n c t i o n of i n t e r n a l ATP, w i t h h a l f - s a t u r a t i o n a t a b o u t 250 p M ATP. T h i s a f f i n i t y i s v e r y c l o s e t o t h a t o b s e r v e d by Glynn and K a r l i s h (1976) a t t h e s o - c a l l e d l o w - a f f i n i t y s i t e . The e f f e c t of ADP on N a - K exchange w a s a l s o determined. With ATP h e l d c o n s t a n t ( a t a b o u t 4 0 0 P M ) e l e v a t i o n of ADP c o n c e n t r a t i o n up t o 1500 V M produced a b o u t a 4 0 % d e c r e a s e i n N a - K exchange. The i n h i b i t i o n by ADP w a s dependent on t h e ATP c o n c e n t r a t i o n , s i n c e t h e ADP e f f e c t was less marked when t e s t e d i n t h e p r e s e n c e of a h i g h e r ATP l e v e l . D e Weer e t a l . (1979) r e p o r t e d t h a t N a - K exchange w a s n o t s i g n i f i c a n t l y i n h i b i t e d by ADP c o n c e n t r a t i o n s h i g h enough t o a c t i v a t e N a - N a exchange. A s t r a i g h t f o r ward k i n e t i c model was d e s c r i b e d which mimicked t h i s behavior. I t s h o u l d be n o t e d t h a t t h e r e s u l t s r e p o r t e d h e r e are n o t a t v a r i a n c e w i t h t h i s work. The D e Weer et a l . (1979) model w a s e x t e n d e d by making t h e s t i m u l a t i o n of Na-K exchange by ATP e x p l i c i t i n t h e model. T h i s r e v i s e d model d i d p r e d i c t moderate i n h i b i t i o n 6f N a - K exchange by ADP a t low ATP c o n c e n t r a t i o n s , and f u r t h e r predicted t h a t t h e i n h i b i t i o n should decrease a t h i g h e r ATP c o n c e n t r a t i o n s .

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N a - N a exchange depends on t h e p r e s e n c e o f ADP ( D e Weer, 1970; Glynn and Hoffman, 1 9 7 1 ) . However, t h e p r e c i s e r e l a t i o n s h i p between ADP c o n c e n t r a t i o n and exchange r a t e h a s n o t been q u a n t i f i e d . T o d e t e r m i n e t h i s r e l a t i o n s h i p , ATP w a s h e l d c o n s t a n t a t a b o u t 6 0 0 p M and ADP v a r i e d from 5 0 t o 2 0 0 0 pM. N a - N a exchange w a s a s a t u r a t i n g f u n c t i o n o f ADP, w i t h half-maximal a c t i v a t i o n o b s e r v e d a t a b o u t 350 P M ADP. W e were a b l e t o examine t h e ADP a c t i v a t i o n a t a b o u t 4 0 0 , 6 0 0 , and 1 0 0 0 p M ATP. Over t h i s r a n g e , ADP a c t i v a t i o n w a s i n d e p e n d e n t o f t h e ATP c o n c e n t r a t i o n . A s i n d i c a t e d above, w e a r e a b l e t o s e t a wide r a n g e o f ATP/ADP r a t i o s u s i n g t h e two r e g e n e r a t i n g s y s t e m s . W e have begun t o examine c a t i o n i n t e r a c t i o n s w i t h t h e pump, as a f u n c t i o n of t h e ATP/ADP r a t i o . I n t h e p r e s e n c e o f s a t u r a t i n g e x t e r n a l K ( 2 0 mM) and w i t h t h e i n t e r n a l ATP/ADP r a t i o clamped a t a b o u t 0 . 5 , removal of e x t e r n a l N a produced a 50% i n c r e a s e i n ouabains e n s i t i v e N a e f f l u x . A t h i g h e r ATP/ADP r a t i o s t h e s t i m u l a t i o n w a s less marked. T h i s i s i n l i n e w i t h o t h e r s t u d i e s s i n c e Sachs (1970) o b s e r v e d l i t t l e e f f e c t of ext e r n a l N a removal, i n t h e p r e s e n c e of h i g h e x t r a c e l l u l a r K , on o u a b a i n - s e n s i t i v e N a e f f l u x i n f r e s h r e d c e l l s (which p o s s e s s a r e l a t i v e l y h i g h ATP/ADP r a t i o ) . Thus, t h e e f f e c t o f e x t e r n a l N a on t h e pump is m o d i f i e d by t h e i n t e r n a l ATP/ADP r a t i o . Measurements of ouabains e n s i t i v e N a and K i n f l u x e s s h o u l d shed l i g h t on t h e mechanism of t h i s e f f e c t .

I 11.

CONCLUS I O N S

I n summary, ATP a c t i v a t e d Na-K exchange, w h e r e a s ADP i n h i b i t e d N a - K exchange and a c t i v a t e d Na-Na exchange. The n u c l e o t i d e a f f i n i t i e s o b s e r v e d h e r e , i n a n i n t a c t t r a n s p o r t i n g system, a r e c o n s i s t e n t w i t h t h o s e observed by Hexum e t a l . ( 1 9 7 0 ) and Robinson (1976) i n microsomal A T P a s e p r e p a r a t i o n s , and by Glynn and K a r l i s h ( 1 9 7 6 ) i n r e d c e l l s . Examination o f n u c l e o t i d e i n t e r a c t i o n s w i t h t h e sodium pump, i n a s i d e d system c a p a b l e o f n e t t r a n s p o r t , s h o u l d p r o v i d e i n s i g h t i n t o t h e mechanism underl y i n g pump f u n c t i o n .

BRIAN G . KENNEDY etel,

686

ACKNOWLEDGMENT

Supported by USPHS, N I H g r a n t s HL-09906 and AM-17433.

REFERENCES

(1970). E f f e c t s of i n t r a c e l l u l a r adenosine-5'diphosphate and orthophosphate on t h e s e n s i t i v i t y of sodium e f f l u x from s q u i d axon t o e x t e r n a l sodium and potassium. J . Gen. P h y s i o l . 56, 583-620. D e Weer, P . , Kennedy, B. G . , and Abercrombie, R. F. (1979). Rel a t i o n s h i p between t h e Na:K exchanging and N a : N a exchanging modes of o p e r a t i o n of t h e sodium pump. I n "Na,K-ATPase: S t r u c t u r e and K i n e t i c s " (J. C. Skou and J. G. Ndrby, e d s . ) , pp. 503-515. Academic P r e s s , New York. Glynn, I . M . , and Hoffman, J. F. (1971). Nucleotide requirements f o r sodium-sodium exchange c a t a l y s e d by t h e sodium pump i n human r e d c e l l s . J . P h y s i o l . (London) 2 1 8 , 239-256. Glynn, I. M., and K a r l i s h , S . J. D. (1975). The sodium pump. Annu. Rev. P h y s i o l . 37, 13-55. Glynn, I . M . , and K a r l i s h , S. J. D. (1976). ATP h y d r o l y s i s ass o c i a t e d w i t h an uncoupled sodium f l u x through t h e sodium pump: Evidence f o r a l l o s t e r i c e f f e c t s of i n t r a c e l l u l a r ATP and e x t r a c e l l u l a r sodium. J . P h y s i o l . (London) 256, 465496. 1970). K i n e t i c Hexum, T., Samson, F. E . , and H i m e s , R. H . Biochim. Biophys. s t u d i e s of membrane (Na-K-Mg)-ATPase. Acta 212, 322-331. Robinson, J. D. (1976). S u b s t r a t e s i t e s of t h e (Na+K)-dependent ATPase. B i o c h i m . B i o -~ p h y s . Acta 4 2 9 , 006-1019. Robinson, J D. , and F l a s h n e r , M. S. (1979). The (Na+K) - a c t i v a t e d ATPase Enzymatic and t r a n s p o r t p r o p e r t i e s . Biochim. B i o p h y s . Acta 5 4 9 , 145-176. Sachs, J. R. (1970). Sodium movements i n t h e human r e d blood c e l l . J. Gen. P h y s i o l . 56, 322-341. D e Weer, P.

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CURRENT TOPICS IN MEMBRANES AND TRANSPORT, VOLUME 19

NalK Pump in Insideout Vesicles Utilizing ATP Synthesized at the Membrane ROBERT W. MERCER,' BEVEmEYE. FARQUHARSON, AND PHILIP B. DUNHAM Department of Biology Syrucuse University Syracuse, New York

I.

INTRODUCTION

The Na/K pump in human red cells is fueled by ATP synthesized during glycolysis. In human red cells it has been suggested that two glycolytic enzymes, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and phosphoglycerate kinase (PGK), are associated with the Na/K pump (Schrier, 1966; Parker and Hoffman, 1967). With orthophosphate (Pi) and NAD, GAPDH converts glyceraldehyde-3-phosphate (GAP) to 1,3-diphosphoglycerate (1,3-DPG). PGK then catalyzes the transfer of a phosphate group from 1,3-DPG to ADP to form ATP. It has also been suggested that the ATP synthesized by membrane-bound GAPDH and PGK is compartmentalized in a "membrane-pool" which is used preferentially by the Na/K pump (Okonkwo e t a ] . , 1975; Proverbio and Hoffman,, 1977). ETesent a d d r e s s : D e p a r t m e n t of Human Genetics , Y a l e Univers i t y School of Medicine, New Haven, CT 06510. 687

Copyright 0 1983 by Academic Press, Inc. All rights of reproduction in any form resewed. ISBN 0-12-153319-0

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ROBERT W. MERCER eta/.

MATERIALS, METHODS, AND RESULTS

We have reexamined t h i s q u e s t i o n hoping t o demons t r a t e d i r e c t l y t h a t g l y c o l y t i c enzymes s y n t h e s i z e ATP and d e p o s i t it i n t o a membrane-associated compartment from which it i s used by t h e N a / K pump. I n s i d e - o u t v e s i c l e s ( I O V s ) p r e p a r e d from human r e d c e l l s p r o v i d e a d i r e c t approach t o t h e problem w i t h o u t some o f t h e d i f f i c u l t i e s and u n c e r t a i n t i e s which a t t e n d t h e use of i n t a c t c e l l s and r e s e a l e d g h o s t s . Moreover, t h e p e r m e a b i l i t y of t h e v e s i c l e s t o c a t i o n s i s such t h a t t r a n s p o r t t h r o u g h t h e N a / K pump can be measured conv e n i e n t l y . F o r example, w i t h K a t t h e i n t r a v e s i c u l a r ( e x t r a c e l l u l a r ) membrane s u r f a c e , ATP s t i m u l a t e s N a transport i n t o IOVs. T h i s u p t a k e i s mediated by t h e N a / K pump s i n c e it i s c o m p l e t e l y i n h i b i t e d by t h e pump i n h i b i t o r s t r o p h a n t h i d i n . With I O V s it s h o u l d be poss i b l e t o d e t e r m i n e t h e r e l a t i o n s h i p between t h e N a / K pump and ATP d e r i v e d from t h e GAPDH-PGK pathway. I n I O V s , i f t h e g l y c o l y t i c enzymes GAPDH and PGK s u p p l y ATP t o t h e pump v i a a membrane-associated comp a r t m e n t , t h e n N a t r a n s p o r t s h o u l d be s t i m u l a t e d when t h e enzymes a r e s y n t h e s i z i n g ATP, and t h i s ATP, i f compartmentalized, s h o u l d be i n a c c e s s i b l e t o a g e n t s i n t h e medium which degrade it. W e found t h a t t h e subs t r a t e s (ADP, N A D , P i ! and GAP) f o r GAPDH and P G K , w i t h o u t added ATP, s t i m u l a t e d N a t r a n s p o r t i n t o I O V s . Hexokinase p l u s glucose--agents t h a t promote t h e b r e a k down of ATP, w h i l e p r e v e n t i n g t h e s t i m u l a t i o n o f N a t r a n s p o r t by exogenous ATP--did n o t i n h i b i t t h e s t i m u l a t i o n of t r a n s p o r t by t h e s u b s t r a t e s o f GAPDH and P G K . These r e s u l t s c o n f i r m t h a t membrane-bound GAPDH and PGK s u p p l y ATP d i r e c t l y t o t h e N a / K pump w i t h o u t accessib i l i t y o f t h e ATP t o t h e e x t r a v e s i c u l a r medium. The i m p o r t a n c e of GAPDH i n s u p p l y i n g ATP t o t h e pump w a s v e r i f i e d u s i n g I O V s i n which GAPDH had been removed from t h e membrane. I n t h e s e v e s i c l e s t h e subs t r a t e s f o r GAPDH and PGK d i d n o t promote Na t r a n s p o r t . However, exogenous ATP s t i m u l a t e d N a t r a n s p o r t , i n d i c a t i n g t h a t t h e Na/K pump i t s e l f w a s u n a f f e c t e d by removal of GAPDH. I t h a s a l s o proved p o s s i b l e t o r e c o n s t i t u t e t h e membrane assembly i n v e s i c l e s d e p l e t e d of GAPDH. I n preliminary experiments, depleted v e s i c l e s incubated w i t h r a b b i t muscle GAPDH ( 3 0 min, 0 . 1 mg/ml) w e r e a b l e t o b i n d exogenous GAPDH; t h i s GAPDH was a c t i v e s i n c e t h e substrates f o r GAPDH and PGK were a g a i n a b l e t o promote N a t r a n s p o r t t h r o u g h t h e pump. T h e r e f o r e , GAPDH r e a d i l y r e b i n d s t o t h e a p p r o p r i a t e s i t e s (as y e t

Na/K PUMP IN VESICLES UTILIZING ATP

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Fig. 1. (A) Time c o u r s e o f i n c o r p o r a t i o n o f 32P-labeled o r t h o p h o s p h a t e i n t o m e m b r a n e - a s s o c i a t e d ATP. V e s i c l e s c o n t a i n i n g choline C1 ( 2 0 m M ) , MgCl2 ( 1 mM) a n d T r i s - g l y c y l g l y c i n e ( 2 . 5 mM) (pH 7 . 4 ) w e r e s u s p e n d e d i n a m e d i u m of the s a m e c o m p o s i t i o n , which a l s o c o n t a i n e d g l y c e r a l d e h y d e 3 - p h o s p h a t e ( 2 mM), NAD ( 4 mM), ADP (1 m M ) , a n d 3 2 P - l a b e l e d o r t h o p h o s p h a t e ( 0 . 5 mM; 2 0 0 p C i / m l ) . The vesicles w e r e i n c u b a t e d f o r the t i m e s d e s i r e d , a n d then w e r e w a s h e d a n d e x t r a c t e d f o r [ 3 a P ] A T P . (B) U t i l i z a t i o n o f m e m b r a n e b o u n d ATP b y the N a / K p u m p o f I O V s w h i c h w e r e s i m u l t a n e o u s l y t r a n s p o r t i n g Na a n d s y n t h e s i z i n g 32P A T P . V e s i c l e s a n d c o n d i t i o n s w e r e i d e n t i c a l t o those d e s c r i b e d i n F i g . l A e x c e p t vesicles a n d A f t e r 4 5 m i n the vesicles w e r e s u s m e d i u m c o n t a i n i n g K C l ( 5 mM). p e n d e d i n a s i m i l a r m e d i u m , e x c e p t t h e c o n c e n t r a t i o n s o f ADP, GAP, NAD, P i , and choline were r e d u c e d b y h a l f ; Na w a s a d d e d t o a f i n a l c o n c e n t r a t i o n o f 10 mM. T o some a l i q u o t s , s t r o p h a n t h i d i n ( 1 p M ) w a s a l s o a d d e d a s i n d i c a t e d . A t v a r i o u s times s a m p l e s o f vesicles w e r e r e m o v e d , w a s h e d , a n d ATP e x t r a c t e d . ( C ) U t i l i z a t i o n of membrane-bound ATP b y t h e N a / K pump of I O V s i n w h i c h [ 3 2 P ] A T P s y n thesis w a s s t o p p e d a t the s a m e t i m e the pump w a s a c t i v a t e d . T h e e x p e r i m e n t w a s c a r r i e d o u t the s a m e w a y a s i n F i g . 1B e x c e p t t h a t w h e n the f l u x w a s i n i t i a t e d b y a d d i n g N a , the s p e c i f i c a c t i v i t y o f 3 2 P - l a b e l e d o r t h o p h o s p h a t e w a s r e d u c e d . 4 0 - f o l d b y d i l u t i o n of the m e d i u m a n d b y a d d i t i o n of P i t o 10 mM, t h e r e b y s t o p p i n g t h e s y n t h e s i s of [ 3 2 P ] A T P ( b u t not of u n l a b e l e d A T P ) .

unidentified), and PGK is not removed by the procedure that elutes GAPDH. In glycolysis the inorganic phosphate that is incorporated into 1,3-DPG becomes the y-phosphate of ATP formed in the PGK reaction. It therefore seemed possible to verify the existence of the compartmentalized ATP by labeling it with 32P-labeled orthophosphate.

ROBERT W. MERCER eta/.

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The r e s u l t s of t h e s e e x p e r i m e n t s a r e shown i n F i 1. I O V s w e r e i n c u b a t e d i n Na-free media c o n t a i n i n g 1 2 P l a b e l e d o r t h o p h o s p h a t e , NAD, and GAP, w i t h and w i t h o u t ADP. A t t h e t i m e s i n d i c a t e d t h e v e s i c l e s were washed and t h e membrane-bound o r g a n i c p h o s p h a t e , which w a s shown c h r o m a t o g r a p h i c a l l y t o be ATP, d e t e r m i n e d . A s shown i n F i g . l A , i n c o r p o r a t i o n of 3 2 P - l a b e l e d o r t h o p h o s p h a t e i n t o ATP was r a p i d and c o m p l e t e w i t h i n 2 min. T h i s l a b e l e d ATP, i f a c c e s s i b l e t o t h e Na/K pump, s h o u l d be u t i l i z e d when t h e pump i s o p e r a t i n g . I n t h e experim e n t a l r e s u l t s shown i n F i g . lB, v e s i c l e s i n Na-free medium c o n t a i n i n t h e GAPDH-PGK s u b s t r a t e s were a l l o w e d t o i n c o r p o r a t e 33P i n t o membrane-bound ATP t o a cons t a n t l e v e l a s i n F i g . 1 A . Then N a w a s added ( z e r o t i m e ) , promoting u t i l i z a t i o n of [32P]ATP by t h e Na/K pump, and a d e c r e a s e i n membrane-bound [32P]ATP t o a l e v e l r e p r e s e n t i n g t h e b a l a n c e between r a t e s o f u t i l i z a t i o n and r e s y n t h e s i s of A T P . The d e c r e a s e i n bound ATP c a u s e d by N a w a s b l o c k e d by t h e a d d i t i o n of s t r o p h a n t h i d i n , c o n f i r m i n g t h a t t h e d e c r e a s e w a s m e d i a t e d by t h e pump. F i g u r e 1 C shows a s i m i l a r e x p e r i m e n t e x c e p t t h a t an e x c e s s o f u n l a b e l e d P i was added w i t h t h e Na, p r e v e n t i n g f u r t h e r s y n t h e s i s of l a b e l e d ATP. The s t r o p h a n t h i d i n - i n h i b i t a b l e d e c l i n e i n membrane-associated ATP was more r a p i d t h a n i n F i g . 1B (where s y n t h e s i s of ATP c o n t i n u e d ) . Under c o n d i t i o n s i n which s y n t h e s i s of l a b e l e d ATP was p r e v e n t e d , n e a r l y all t h e ATP a s s o c i a t e d w i t h t h e membrane w a s u t i l i z e d by t h e pump.

111.

CONCLUSIONS These s t u d i e s i n d i c a t e t h a t t h e g l y c o l y t i c enzymes

GAPDH and PGK bound t o t h e r e d c e l l membrane s y n t h e s i z e ATP and d e p o s i t i t i n a membrane-associated compartment from which i t i s used by t h e N a / K pump. The major p o i n t s

of e v i d e n c e a r e t h e f o l l o w i n g : (1) A c t i v e Na t r a n s p o r t i n t o I O V s i s promoted by a d d i t i o n of t h e s u b s t r a t e s f o r GAPDH and P G K , w i t h o u t added ATP. ( 2 ) The GAPDH-PGK complex s y n t h e s i z e s A T P , which remains a s s o c i a t e d w i t h t h e membrane. ( 3 ) The membrane-associated ATP m u s t b e compartmentalized because it i s i n a c c e s s i b l e t o degrad a t i o n c a t a l y z e d by h e x o k i n a s e . ( 4 ) The membrane-associated ATP f u e l s t h e pump s i n c e i t s l e v e l i s r e d u c e d by t h e a d d i t i o n of N a , and t h i s r e d u c t i o n i s i n h i b i t e d by s t r o p h a n t h i d i n .

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A f u l l r e p o r t of some of t h e s e r e s u l t s h a s been p u b l i s h e d (Mercer and Dunham, 1 9 8 1 ) .

REFERENCES

Mercer, R. W . , a n d Dunham, P. B. ( 1 9 8 1 ) . Membrane-bound ATP f u e l s t h e Na/K pump: S t u d i e s on membrane-bound g l y c o l y t i c enzymes on i n s i d e o u t v e s i c l e s from human r e d c e l l membranes. J . Gen. P h y s i o l . 78, 547-568. Okonkwo, P. O . , Longenecker, G . , and A s k a r i , A. ( 1 9 7 5 ) . S t u d i e s o n t h e mechanism o f t h e r e d c e l l metabolism by c a r d i a c g l y c o s i d e s . J . P h a r m a c o l . E x p . T h e r . 1 9 4 , 244-254. P a r k e r , J. C . , a n d Hoffman, J . F. ( 1 9 6 7 ) . T h e role o f membrane p h o s p h o g l y c e r a t e k i n a s e i n t h e c o n t r o l o f g l y c o l y t i c r a t e by a c t i v e c a t i o n t r a n s p o r t i n human r e d b l o o d cells. J . G e n . P h y s i o l . 5 0 , 893-916. P r o v e r b i o , F . , and Hoffman, J. F. ( 1 9 7 7 ) . Membrane compartmentali z e d ATP and i t s p r e f e r e n t i a l u s e b y t h e Na,K-ATPase o f human r e d c e l l g h o s t s . J . Gen. P h y s i o l . 69, 605-632. S c h r i e r , S . L. ( 1 9 6 6 ) . O r g a n i z a t i o n of enzymes i n human e r y t h r o c y t e membranes. Am. J. P h y s i o l . 2 1 0 , 139-145.

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CURRENT TOPICS IN MEMBRANES AND TRANSPORT, VOLUME 19

Anion-Coupled Na Efflux Mediated by the NdK Pump in Human Red Blood Cells S.DISSING AND JOSEPH F. HOFFMAN Department of Physiology Yale University School of Medicine New Haven, Connecticut

I.

INTRODUCTION

Previous work has shown that the Na/K pump in human red blood cells is electrogenic, that is, that the pump transfers net charge across the membrane (Hoffman et a l . , 1979). Monitoring the cell's membrane poten~ means of the fluorescent dye technique [as tial ( E by described by Hoffman and Laris (19741, with the dye DiS-C3(5)], a hyperpolarization is observed when K is added to a cell suspension which is reversed by the addition of ouabain. The net transfer of charge which gives rise to this hyperpolarization under the conditions of our experiments (Na-loaded, SO4-equilibrated, and DIDS-treated cells) is 2 mM Na/liter cells/hr since the total ouabain-sensitive Na efflux is 6 mM/liter cells/hr and we assume a stoichiometry of 3:2 for Nai exchanged for KO.

693

Copyright 0 1983 by Academic Press, Inc. All rights of reproduction in any form reserved.

ISBN 0-12-1533190

S. DlSSlNG AND J. F. HOFFMAN

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11.

RESULTS AND DISCUSSION

When r e d c e l l s are suspended i n a medium f r e e of N a and K , a n o u a b a i n - s e n s i t i v e e f f l u x of N a h a s been

shown t o o c c u r (Garrahan and Glynn, 1 9 6 7 ) . Under o u r e x p e r i m e n t a l c o n d i t i o n s , t h i s f l u x amounts t o 1 m~ Na/liter c e l l s / h r . T h i s N a e f f l u x i s s t i l l observed when SO4 i s s u b s t i t u t e d f o r C 1 on b o t h s i d e s of t h e membrane. W e t e s t e d whether o r n o t t h i s pump e f f l u x of N a i s e l e c t r o g e n i c by measuring E~ a s ouabain i s added t o t h e c e l l s u s p e n s i o n . I f t h i s pump f l u x i s elect r o g e n i c , it i s l a r g e enough t o be d e t e c t e d s i n c e i t should give rise t o a hyperpolariz a t i o n h a l f t h e s i z e of t h a t seen when o u a b a i n i s added t o a c e l l s u s p e n s i o n c o n t a i n i n g 5 m M KO. However, w e c o u l d d e t e c t no change i n E~ a f t e r t h e a d d i t i o n of ouabain. T h i s r e s u l t i n d i c a t e s t h a t t h e pump e f f l u x of N a t h a t o c c u r s when c e l l s are p l a c e d i n a medium f r e e of N a and K is e l e c t r o n e u t r a l and n o t e l e c t r o g e n i c . T h i s o u a b a i n - s e n s i t i v e e f f l u x of N a i s n o t a f f e c t e d by t h e p r e s e n c e of DiS-C3(5). I f t h i s o u a b a i n - s e n s i t i v e e f f l u x of N a i s e l e c t r o n e u t r a l , t h e n t h e q u e s t i o n c a n be raised whether a counterexchange of p r o t o n s o r a c o t r a n s p o r t of a n i o n s i s a s s o c i a t e d w i t h t h e N a e f f l u x . W e were u n a b l e t o det e c t any movement of p r o t o n s a s s o c i a t e d w i t h t h e N a e f f l u x i n C1- o r S O - s u b s t i t u t e d , DIDS-treated c e l l s s u s pended i n an unbu f e r e d medium c o n t a i n i n g e i t h e r i s o t o n i c c h o l i n e c h l o r i d e o r MgS04. The c e l l s were r i d of b i c a r b o n a t e by g a s s i n g t h e s u s p e n s i o n s w i t h N 2 ; g l y c o l y s i s w a s i n h i b i t e d by a d d i t i o n of 2 m~ iodoacetamide t o p r e v e n t t h e f o r m a t i o n of p r o t o n s . No o u a b a i n - s e n s i t i v e change i n t h e medium pH was observed o v e r a 20-min p e r i o d i n a 4 0 % c e l l s u s p e n s i o n a t 37OC, even though t h e system w a s s e n s i t i v e enough t o d e t e c t a change i f a counterexchange of p r o t o n s had o c c u r r e d . W e t h e n s t u d i e d whether o r n o t a movement of S O 4 ions i s associated with the ouabain-sensitive e f f l u x of W e measured 35SO4 N a ( i n a medium f r e e of N a o and KO). e f f l u x from S O q - e q u i l i b r a t e d , DIDS-treated r e d c e l l s suspended i n 95 m~ (Tris)z-SOq (pH 7 . 2 ) a t 37OC. The a d d i t i o n of o u a b a i n t o t h e c e l l s u s p e n s i o n d e c r e a s e d t h e r a t e c o n s t a n t f o r SO4 e f f l u x ( f o r i n s t a n c e , i n one e x p e r i m e n t ) from 0 . 0 0 4 7 f 0 , 0 0 0 7 t o 0 . 0 0 2 7 0.0007 hr-1 ( t h e s t a n d a r d d e v i a t i o n i s g i v e n ; n = 1 2 ) . T h i s t y p e of o u a b a i n - s e n s i t i v e e f f l u x of SO4 i s a l s o observed when c e l l s are suspended i n MgS04 b u f f e r e d w i t h HEPES. N e v e r t h e l e s s , t h i s magnitude of o u a b a i n - s e n s i t i v e S O 4 e f f l u x v a r i e s from a b o u t o n e - t h i r d t o one-half t h e o u a b a i n - s e n s i t i v e N a e f f l u x . Presumably, t r a c e amounts

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ANION-COUPLED Na EFFLUX IN HUMAN RED BLOOD CELLS

695

of b i c a r b o n a t e i o n s a re i n v o l v e d i n t h e v a r i a b i l i t y of t h i s s t o i c h i o m e t r y and t h i s and o t h e r f a c t o r s a r e s t i l l u n d e r s t u d y . T h i s o u a b a i n - s e n s i t i v e SO4 e f f l u x i s n o t o b s e r v e d when t h e medium c o n t a i n s e i t h e r 5 m Nao o r 1 0 m M KO--conditions known t o i n h i b i t t h e o u a b a i n s e n s i t i v e N a e f f l u x ( G a r r a h a n and Glynn, 1 9 6 7 ) . These r e s u l t s i n d i c a t e t h a t a n i o n s accompany t h e pump e f f l u x of N a t h a t o c c u r s i n t h e a b s e n c e of N a o and KO. When s m a l l amounts ( e . g . , 5 mM) o f C 1 a r e p r e s e n t on t h e i n s i d e o f S O q - e q u i l i b r a t e d , DIDS-treated c e l l s t h e o u a b a i n - s e n s i t i v e e f f l u x o f SO4 i s i n h i b i t e d , i n d i c a t i n g t h a t t h e s e l e c t i v i t y of t h e Na e f f l u x s y s t e m e v i d e n t l y p r e f e r s C 1 o v e r SO4. Although t h e f o r e g o i n g r e s u l t s est a b l i s h t h a t t h e N a / K pump can o p e r a t e a s a n e u t r a l s a l t pump, t h e f u n c t i o n a l s i g n i f i c a n c e of t h i s t y p e of t r a n s p o r t has y e t t o be defined.

ACKNOWLEDGMENT

T h i s work w a s s u p p o r t e d by N I H G r a n t s HL09906 and AM05644.

REFERENCES

G a r r a h a n , P. J . , and Glynn, I . M. ( 1 9 6 7 ) . The s e n s i t i v i t y o f t h e J . Physiol. (London) 192, 175sodium pump t o e x t e r n a l N a . 188. Hoffman, J. F . , and L a r i s , P. C . ( 1 9 7 4 ) . D e t e r m i n a t i o n of membrane p o t e n t i a l s i n human and Amphiuma red b l o o d c e l l s by means o f a f l u o r e s c e n t probe. J. Physiol. (London) 239, 519-552. Hoffman, J. F . , Kaplan, J. H . , a n d C a l l a h a n , T. J . ( 1 9 7 9 ) . The N a : K pump i n r e d c e l l s is e l e c t r o g e n i c . F e d . Proc. 3 8 , 2440-2441.

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CURRENT TOPICS IN MEMBRANES ANDTRANSPORT, VOLUME 19

Effect of Trypsin Digestion on the Kinetic Behavior of the Na/K Pump in Intact Erythrocytes DONNA L. KROPP Department of Physiology UMDNJ-New Jersey Medical School Newark, New Jersey

I.

INTRODUCTION

T r y p s i n h a s been used r a t h e r e x t e n s i v e l y as a t o o l € o r e l u c i d a t i n g t h e s t r u c t u r e of Na,K-ATPase and i t s r e l a t i o n s h i p t o t h e f u n c t i o n o f t h i s enzyme ( J g k g e n s e n , 1975; K o e p s e l l , 1 9 7 9 ) . S i n c e t h e s e s t u d i e s h a v e u s e d microsomal p r e p a r a t i o n s o f t h e enzyme, i t i s d i f f i c u l t t o r e l a t e t h e r e s u l t s t o t h e f u n c t i o n o f t h e N a / K pump i n t h e t r a n s p o r t of Na and K i n i n t a c t c e l l s . W e h a v e t h e r e f o r e exposed t h e e x t e r n a l s u r f a c e of i n t a c t r e d c e l l s t o t r y p s i n and have examined t h e r e s u l t i n g c h a n g e s i n t h e k i n e t i c p r o p e r t i e s o f t h e pump.

697

Copyright 0 1983 by Academic Press, Inc. All rights ofreproduction in any form r e ~ e ~ e d . lSBN 0-12-1533194

DONNA L. KROPP

698

11.

MATERIALS AND METHODS

For t h e t r e a t m e n t w i t h t r y p s i n w e s o u g h t condit i o n s t h a t removed band I11 p r o t e i n s (which i n c l u d e t h e N a / K pump) e s s e n t i a l l y c o m p l e t e l y b u t had no e f f e c t on bands I and I1 p r o t e i n s ( l o c a t e d on t h e i n t e r n a l s u r f a c e of t h e r e d c e l l ) , a s d e t e c t e d by s u b s e q u e n t p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s . Human r e d c e l l s w e r e o b t a i n e d by v e n i p u n c t u r e u s i n g h e p a r i n as an a n t i c o a g u l a n t . C e l l s were removed from t h e plasma by t h r e e washes i n i s o t o n i c MgC12 and t h e n two washes i n a medium c o m p r i s i n g 95% i s o t o n i c s u c r o s e and 5 % i s o t o n i c T r i s - C 1 (pH 7 . 4 ) . C e l l s were t h e n resuspended a t 5 0 % hematocrit i n t h e sucrose-Tris solution containing t r y p s i n a t a f i n a l c o n c e n t r a t i o n of 2 m g / m l c e l l s u s p e n s i o n . T r y p s i n w a s o m i t t e d from t h e c o n t r o l c e l l s u s p e n s i o n s . The low i o n i c s t r e n g t h of t h e s u c r o s e T r i s s o l u t i o n f a c i l i t a t e d t h e d e g r a d a t i o n o f band I11 p r o t e i n s . The c e l l s u s p e n s i o n s w e r e i n c u b a t e d a t 37OC f o r 6 0 min. T r y p s i n w a s removed by s i x washes i n i s o t o n i c MgCl2 c o n t a i n i n g 20 mg% b o v i n e s e r u m albumin. No i n h i b i t o r of t r y p s i n w a s used. The washed c e l l s were t h e n resuspended a t 5% h e m a t o c r i t i n p h o s p h a t e b u f f e r e d s a l i n e c o n t a i n i n g PO4 ( 2 0 m ) , Mg ( 1 . 0 m M ) , g l u c o s e ( 5 mM) , a d e n i n e ( 3 mM) , and i n o s i n e ( 2 mM) N a and K were a d j u s t e d a s needed and i s o t o n i c i t y w a s m a i n t a i n e d w i t h c h o l i n e c h l o r i d e . T h i s s u s p e n s i o n was i n c u b a t e d a t 37'C f o r 6 0 min and t h e n o v e r n i g h t a t 4OC. The c e l l s were t h e n washed t h r e e t i m e s i n i s o t o n i c MgC12 c o n t a i n i n g 2 0 mg% albumin and were t h e n r e a d y f o r u s e . For some of t h e s t u d i e s , i n t r a c e l l u l a r c o n c e n t r a t i o n s of N a and K were a l t e r e d p r i o r t o e x p o s u r e t o t r y p s i n , by i n c u b a t i n g t h e c e l l s i n b u f f e r e d s a l i n e of t h e d e s i r e d c o m p o s i t i o n c o n t a i n i n g 0 . 1 mM p-chloromerc u r i b e n z e n e s u l f o n a t e f o r 36 h r w i t h two changes. C e l l s were t h e n r e s e a l e d w i t h d i t h i o t h r e i t o l (Sachs et a l . , 1974). C h o l i n e c h l o r i d e r e p l a c e d Na and K t o maintain isotonicity. Using o u a b a i n - s e n s i t i v e ( 1 0 - 4 bf) K i n f l u x (Sachs e t a l . , 1 9 7 4 ) , w e examined t h e i n t e r a c t i o n o f b o t h ext r a c e l l u l a r N a ( N a o ) and K ( K O ) and i n t r a c e l l u l a r N a ( N a i ) and K ( K i ) w i t h t h e N a / K pump. Our r e s u l t s show t h a t t r y p s i n d i g e s t i o n had no o b s e r v a b l e e f f e c t on t h e e x t r a c e l l u l a r a s p e c t of t h e Na/X pump. N e i t h e r Vmax nor ~ 0 . 5 f o r e i t h e r KO o r N a o was s i g n i f i c a n t l y d i f f e r e n t from t h o s e o f t h e c o n t r o l . F u r t h e r m o r e , t h e i n t e r a c t i o n o f N a a t t h e i n t e r n a l a s p e c t of t h e pump a l so appeared n o t t o be i n f l u e n c e d by t r y p s i n d i g e s t i o n (see Table I ) .

.

EFFECTOF TRYPSIN ON PUMP BEHAVIOR

TABLE I .

699

K i n e t i c P r o p e r t i e s of t h e Pump f o r I n t e r n a l K and I n t e r n a l Naa

Na i (mM/liter c e l l s )

V

max (mM/liter c e l l s / h r )

K

0.5 (mM/liter c e l l s )

KO

(mM)

Na

0

(m)

For I n t e r n a l K 1.7

Trypsin Control

0.820 ( p < 0.05) 0.620

7.4

Trypsin Control

0.798 0.634

34

Trypsin Control

Ki (mM/liter c e l l s )

57.0 10.0

1.56

0.081

( p < 0.05)

1.99 0.933

2.33

0.754

0.848 ( p < 0.05) 0.662

6.82 0.779

1.73

0.910

V

max (mM/liter c e l l s / h r )

K0.5 (mM/liter c e l l s )

KO

Na

0

(IW) ( m ~ )

For I n t e r n a l N a 1.0

Trypsin Control

0.8 1.02

(N.S.)

11.0 9.5

1.56

0.091

7.0

Trypsin Control

1.03 1.14

(N.S.)

7.07 8.65

1.87

0.071

aOuabain-sensitive K i n f l u x was determined a s a function o f the i n t r a c e l l u l a r concentration o f K ( t o p ) and Na (bottom), a t a constant concentration o f the other cation. The i n t r a c e l l u l a r concentration of the cation held constant and the external concentrat i o n s of K and Na of each experiment are given i n the t a b l e . Values f o r Vmax and K0.5 were calculated by the l e a s t squares method from the reciprocal o f K i n f l u x a s a function of the reciprocal of the i n t r a c e l l u l a r concentration o f e i t h e r K or N a .

When K i n f l u x w a s examined as a f u n c t i o n of K i i n t h e a b s e n c e of N a j , t h e c o n t r o l pump o p e r a t e d a s a K/K exchanger, demonstrating k i n e t i c s t y p i c a l of a f i r s t o r d e r r a t e p r o c e s s . The t r y p s i n - t r e a t e d pump o p e r a t e d a t a c o n s i d e r a b l y s l o w e r r a t e t h a n t h e c o n t r o l , showed some e v i d e n c e of c o o p e r a t i v i t y a t low ( < l o m M / l i t e r c e l l s ) K i c o n c e n t r a t i o n s , and d i d n o t s a t u r a t e o v e r t h e r a n g e of K i w e examined ( 0 - 8 0 mM/liter c e l l s ) . The a f f i n i t y of t h e pump f o r K i w a s reduced t o < 2 0 % of t h a t of t h e c o n t r o l . A t K i c o n c e n t r a t i o n s lower t h a n

700

DONNA L. KROPP

7 m w / l i t e r c e l l s t h e r e w a s no o u a b a i n - s e n s i t i v e K i n f l u x . These two f a c t o r s may be r e s p o n s i b l e f o r t h e suggestion of c o o p e r a t i v i t y a f t e r t r y p s i n treatment. S i m i l a r e x p e r i m e n t s were performed a t h i g h e r conc e n t r a t i o n s of N a i and ~ 0 . 5 and Vmax were c a l c u l a t e d ( T a b l e I ) . A t a l l v a l u e s of N a i , VmaX w a s a b o u t 30% h i g h e r i n t h e t r y p s i n - t r e a t e d pumps and d i d n o t change a s Nai was i n c r e a s e d . T r y p s i n s u b s t a n t i a l l y i n c r e a s e d t h e ~ 0 . 5b u t i t s r e l a t i o n s h i p t o N a i was i n c o n s i s t e n t ( T a b l e I ) . I n c r e a s i n g N a i appeared t o r e d u c e ~ 0 . 5 f o r Ki i n t h e c o n t r o l pumps.

111.

DISCUSSION

Trypsin thus increases ouabain-sensitive K i n f l u x dependent on Ki a l t h o u g h i t r e d u c e s t h e a f f i n i t y of t h e On t h e o t h e r hand, t r y p s i n h a s n o pump f o r i n t e r n a l K . s i g n i f i c a n t e f f e c t on t h e i n t e r a c t i o n of i n t e r n a l N a w i t h t h e pump. I t a l s o h a s no a p p a r e n t e f f e c t on t h e i n t e r a c t i o n of e x t e r n a l K o r N a w i t h t h e pump. Treatment o f human r e d c e l l s w i t h t r y p s i n r e s u l t s i n d e g r a d a t i o n of t h e N a / K pump (and a l l o t h e r band I11 p r o t e i n s ) i n t o two smaller-molecular-weight p e p t i d e s (Kropp and R u b i n s t e i n , 1 9 8 1 ) . S i n c e t h e s p e c t r i n prot e i n s , which a r e l o o s e l y a s s o c i a t e d w i t h t h e i n t e r n a l s u r f a c e of t h e membrane, are n o t a f f e c t e d by t r y p s i n , w e conclude t h a t t r y p s i n a t t a c k s t h e N a / K pump from t h e external surface only. T h a t t r y p s i n a f f e c t s o n l y t h e i n t e r a c t i o n of i n t e r n a l K and n o t t h a t o f i n t e r n a l N a w i t h t h e pump i s cons i s t e n t w i t h N a and K b i n d i n g t o d i f f e r e n t s i t e s on t h e pump. W e s p e c u l a t e t h a t t h e p r o p e r t i e s o f t h e K b i n d i n g s i t e a r e d e t e r m i n e d by t h e e x t e r n a l s t r u c t u r e o f t h e pump, whereas t h e p r o p e r t i e s of t h e N a b i n d i n g s i t e a r e a f u n c t i o n of t h e i n t e r n a l s t r u c t u r e of t h e pump. The p r o p e r t i e s o f t h e N a binding s i t e are t h e r e f o r e n o t i n f l u e n c e d by t r y p s i n . When t h e pump p r o t e i n i s i n t h e c o n f i g u r a t i o n w i t h t h e K b i n d i n g s i t e exposed e x t e r n a l l y , t h e p r o p e r t i e s o f t h i s s i t e a p p a r e n t l y are a l s o n o t a f f e c t e d by t r y p s i n . When t h e pump enzyme undergoes a c o n f i g u r a t i o n a l change and t h e K s i t e i s b r o u g h t t o t h e i n t e r n a l s u r f a c e of t h e c e l l , t h e p r o p e r t i e s o f t h e K s i t e i n t h i s c o n f o r m a t i o n of t h e pump have been a l t e r e d by t r y p s i n . The o b s e r v a t i o n t h a t a f t e r t r e a t m e n t w i t h t r y p s i n , i n t e r n a l K i n c r e a s e s t h e maximum r a t e o f K i n f l u x sugg e s t s t h a t t h e pump may be o p e r a t i n g as a K/K exchange

EFFECT OF TRYPSIN ON PUMP BEHAVIOR

701

pump r a t h e r t h a n , o r p e r h a p s i n a d d i t i o n t o , a N a / K exchange pump. There does a p p e a r t o b e a r e q u i r e m e n t for i n t e r n a l K o r a t l e a s t a minimum l e v e l o f i n t e r n a l N a + K c o n c e n t r a t i o n i n o r d e r t o have a ouabains e n s i t i v e K i n f l u x . However, t h i s may w e l l be a consequence of t h e v e r y l o w a f f i n i t y of t h e pump f o r i n t e r n a l K.

ACKNOWLEDGMENT

T h i s work w a s s u p p o r t e d by USPHS g r a n t HL-20711, NIH.

REFERENCES

J d r g e n s e n , P. L. (1975). B i o c h i r n . B i o p h y s . A c t a 4 0 1 , 399-415. K o e p s e l l , H . (1979). J. Mernbr. B i o l . 48, 69-94, Kropp, D. L., and R u b i n s t e i n , H . (1981). F e d . P r o c . , F e d . Am. SOC. E x p . B i o l . 4 0 , 384. S a c h s , J . R . , Ellory, J. C . , Kropp, D. L., Dunham, P. B., and Hoffman, J. F. (1974). J. Gen. P h y s i o l . 6 3 , 389-414.

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CURRENT TOPICS IN MEMBRANES AND TRANSPORT, VOLUME 19

Sodium Movement and ATP Hydrolysis in Basolateral Plasma Membrane Vesicles from Proximal Tubular Cells of Rat Kidney F. PROVERBIO, T. PROVERBIO, AND R. MARtN Insrituro Venezolano de lnvestigaciones Cienrijicas

Caracas. Venezuela

I.

INTRODUCTION

C e l l s of s e v e r a l mammalian k i d n e y c o r t e x e x t r u d e Na+ i n two ways: (1) N a + i s e x t r u d e d and r e p l a c e d by K+. T h i s mode r e q u i r e s t h e presence of K+ i n t h e ext e r n a l medium, i s t o t a l l y i n h i b i t e d by o u a b a i n , and i s o n l y p a r t i a l l y i n h i b i t e d by e t h a c r y n i c a c i d . ( 2 ) Na+ i s e x t r u d e d accompanied by C 1 - and water. T h i s mode d o e s n o t r e q u i r e t h e p r e s e n c e of K+ i n t h e e x t e r n a l medium, i s i n s e n s i t i v e t o o u a b a i n , and i s t o t a l l y i n h i b i t e d by e t h a c r y n i c a c i d (Whittembury and P r o v e r b i o , 1 9 7 0 ; Munday e t a l . , 1 9 7 1 ) . T h e r e have been d e s c r i b e d two Na'-stimulated A T P a s e a c t i v i t i e s i n plasma memb r a n e s of r e n a l t i s s u e ( P r o v e r b i o e t a l . , 1975; Prov e r b i o and d e l C a s t i l l o , 1 9 8 1 ) . The c h a r a c t e r i s t i c s of t h e s e two N a + - s t i m u l a t e d A T P a s e a c t i v i t i e s p a r a l l e l t h e c h a r a c t e r i s t i c s of t h e two ways of N a + e x t r u s i o n . I n t h e p r e s e n t work w e s t u d i e d t h e N a + movement a c r o s s v e s i c l e s p r e p a r e d w i t h b a s o l a t e r a l plasma memb r a n e s from r a t k i d n e y t u b u l a r c e l l s , a s w e l l as t h e e n e r g y r e q u i r e m e n t s of t h e s e movements.

703

Copyright 0 1983 by Academic Press. Inc All rights of reproduction in any form reserved. ISBN 0-12-153319-0

704

F. PROVERB10etal.

c

0

OI

4

6-

30

4-

P

Mg2+ t No+

E

a.

2-

0

5

10

15

20

25

30

time (minutes 1

Fig. 1. Time course of Na' incorporation by inside-out v e s i c l e s . The values are the mean +SE (n = 10).

11.

RESULTS AND CONCLUSIONS

B a s o l a t e r a l plasma membrane-enriched f r a c t i o n s were p r e p a r e d a s a l r e a d y d e s c r i b e d ( P r o v e r b i o and d e l C a s t i l l o , 1 9 8 1 ) . The p r e p a r a t i o n medium c o n t a i n e d A l l the 250 mM s u c r o s e and 2 0 mM T r i s - H C 1 (pH 7 . 2 ) . p r e p a r a t i o n s were t e s t e d f o r t h e p r e s e n c e of v e s i c l e s by d e t e r m i n i n g t h e i r ATPase a c t i v i t y and [3H]ouabain b i n d i n g b e f o r e and a f t e r t r e a t i n g t h e membranes w i t h 0 . 0 6 % DOC and 2 mM EDTA f o r 30 min a t 23OC. I t w a s found t h a t o u r p r e p a r a t i o n s comprised about 75% i n s i d e out vesicles. F i g u r e 1 shows t h e Na+ i n c o r p o r a t i o n i n v e s i c l e s i n c u b a t e d a t 37'C, a t d i f f e r e n t t i m e s , . i n s e v e r a l media. There i s a p a s s i v e e n t r y of Na+, which r e a c h e s a s t e a d y s t a t e a f t e r a b o u t 15 min of i n c u b a t i o n , i n a l l t h e cond i t i o n s i n d i c a t e d i n t h e f i g u r e . I f a f t e r 15 min ATP i s added t o t h e medium c o n t a i n i n g Mg2+ + Na+, t h e v e s i c l e s accumulate s i g n i f i c a n t l y more Na+. An even h i g h e r Na+ i n c o r p o r a t i o n i s achieved upon t h e a d d i t i o n of ATP, i f t h e v e s i c l e s are i n c u b a t e d i n a medium w i t h Mg2+ + Na+ + K+ + valinomycin. F i g u r e 2 shows t h e e f f e c t of e t h a c r y n i c a c i d (EA) i n t h e medium or of ouabain i n s i d e t h e v e s i c l e s on t h e Na+ i n c o r p o r a t i o n by v e s i c l e s i n c u b a t e d a t 37OC f o r

705

SODIUM MOVEMENT AND ATP HYDROLYSIS

c

6-

a No'

5-

=

Mg2++NaT+ATP Mg2*+Na*+ K*+ATP

4-

2 Q f

3-

\

t

zIn

2-

a

I -

0

z?

0-

Cont ro I

t

2mM EA

+ 7 m M Ouab. (inside 1

F i g . 2 . E f f e c t of e t h a c r y n i c a c i d or o u a b a i n on the Na" i n c o r p o r a t i o n . The v a l u e s are mean L S E . T h e v a l u e s o f n a r e given i n parentheses.

5 min i n d i f f e r e n t media, as i n d i c a t e d i n t h e f i g u r e . The p a s s i v e e n t r y o f N a + ( w h i t e columns) i s i n s e n s i t i v e t o EA and t o o u a b a i n . The MgATP-dependent N a + i n c o r p o r a t i o n ( i n c r e m e n t showed by t h e c o n t r o l h a t c h e d column) i s t o t a l l y i n h i b i t e d by EA and i s i n s e n s i t i v e t o o u a b a i n . The Mg ATP-K-dependent N a + i n c o r p o r a t i o n ( f u r t h e r i n c r e m e n t showed by t h e c o n t r o l b l a c k column) i s i n s e n s i t i v e t o EA and i s i n h i b i t e d by o u a b a i n . S i m i l a r r e s u l t s showed t h e h y d r o l y s i s o f ATP d u r i n g t h e same e x p e r i m e n t s . I n t h e p r e s e n c e of N a + , t h e r e i s a s i n i f i c a n t i n c r e m e n t o f t h e ATP h y d r o l y z e d by t h e Mg3+-ATPase, which i s o u a b a i n i n s e n s i t i v e and t o t a l l y I n t h e p r e s e n c e of N a + + K+, t h e r e i s i n h i b i t e d by EA. a f u r t h e r i n c r e m e n t of t h e ATP h y d r o l y z e d , w h i c h i s tot a l l y i n h i b i t e d by o u a b a i n and i s i n s e n s i t i v e t o EA. These r e s u l t s r e p r e s e n t s t r o n g e v i d e n c e t h a t t h e two ways of N a + movement a r e due t o t h e work of two d i f f e r e n t N a + pumps.

F.PROVERB10 eta/.

706

ACKNOWLEDGMENTS

W e thank Mrs. Anita I s t o k and M i s s D. Otero f o r t h e i r h e l p .

REFERENCES

Munday, K. A . , Parsons, B. J . , and Poat, J . A. (1971). The e f f e c t o f a n g i o t e n s i n on c a t i o n t r a n s p o r t by r a t kidney c o r t e x s l i c e s . J. P h y s i o l . (London) 215, 269-282. Proverbio, F , and d e l C a s t i l l o , J. (1981). Na+-stimulated ATPase a c t i v i t i e s i n kidney basal-1.ateral plasma membranes. B i o c h i m . B i o p h y s . Acta 6 4 6 , 99-108. Proverbio, F. , Condrescu-Guidi , M. , and Whittembur G. (1975) Ouabain-insensitive Na+ s t i m u l a t i o n of an Mg"-dependent B i o c h i m . B i o p h y s . Acta 394, 281ATPase i n kidney t i s s u e . 292. Whittembury, G . , and Proverbio, F. (1970). Two modes of Na+ ext r u s i o n i n c e l l s from guinea-pig kidney c o r t e x s l i c e s . P f l t f g e r s A r c h . 3 1 6 , 1-25.

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CURRENT TOPICS IN MEMBRANES ANDTRANSPORT. VOLUME 19

Stoichiometry of the Electrogenic Na Pump in Barnacle Muscle: Simultaneous Measurement of Na Efflux and Membrane Current M. T. NELSON'AND W. J . LEDERER Deparhnent of Physiology Universiryof Maryland School of Medicine Baltimore, Maryland

I.

INTRODUCTION

T h e Na,K-ATPase i s an enzyme l o c a t e d i n t h e plasma membrane of c e l l s and f u n c t i o n s t o t r a n s p o r t sodium o u t o f t h e c e l l and p o t a s s i u m i n t o t h e c e l l . F l u x s t u d i e s have i n d i c a t e d t h a t a p p r o x i m a t e l y 3 moles of Na i o n s are t r a n s p o r t e d o u t of r e d b l o o d c e l l s f o r e v e r y mole of ATP h y d r o l y z e d (Garrahan and Glynn, 1 9 6 7 ) . Garrahan and Glynn a l s o i n v e s t i g a t e d o u a b a i n - s e n s i t i v e N a e f f l u x and o u a b a i n - s e n s i t i v e K i n f l u x and d e m o n s t r a t e d t h a t t h e r a t i o of t h e s e two o u a b a i n - s e n s i t i v e f l u x e s w a s between 2:l and 3:2 f o r N a : K f o r t h e c o u n t e r t r a n s p o r t o f t h e s e two c a t i o n s . The s t o i c h i o m e t r i c i n e q u a l i t y o f N a + and

'Present a d d r e s s : F a k u l t X t f i r B i o l o g i e , U n i v e r s i t a t K o n s t a n z , 0-7750 K o n s t a n z , F e d e r a l R e p u b l i c of G e r m a n y . 2 A d d r e s s a l l c o r r e s p o n d e n c e and r e p r i n t r e q u e s t s t o W. J . L e d e r e r i n B a l t i m o r e . 707

Copyright 0 1983 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-153319-0

708

M. T. NELSON AND W. J. LEDERER

+

t r a n s p o r t h a s been s u p p o r t e d i n numerous s u b s e q u e n t f l u x s t u d i e s and many e l e c t r i c a l s t u d i e s . A h y p e r p o l a r i z a t i o n o f t h e c e l l membrane accompanies a n i n c r e a s e d t u r n o v e r r a t e of t h e enzyme, as would be e x p e c t e d from t h e above s t o i c h i o m e t r y . Such e l e c t r o g e n i c i t y of sodium and p o t a s s i u m t r a n s p o r t by t h e N a pump h a s been used r e c e n t l y t o estimate t h e s t o i c h i o m e t r y o f t h e N a pump by measuring s i m u l t a n e o u s l y t h e change i n c u r r e n t and t h e change i n i n t r a c e l l u l a r Na a c t i v i t y i n s n a i l n e u r o n s (Thomas, 1 9 6 9 ) and i n c a r d i a c P u r k i n j e f i b e r s ( E i s n e r e t a l . , 1981) when t h e N a pump a c t i v i t y h a s been a l t e r e d . Such s t u d i e s assume a f i x e d r e l a t i o n s h i p between t h e amount o f N a t r a n s p o r t e d and t h e measured i n t r a c e l l u l a r Na a c t i v i t y . The more d i r e c t approach used by Cooke e t a l . (1974) h a s a l s o been used i n t h e e x p e r i ments p r e s e n t e d here. Cooke e t a l . i n j e c t e d N a (and 2 4 N a ) i o n t o p h o r e t i c a l l y and measured N a e f f l u x from a n A p l y s i a neuron w h i l e t h e c e l l w a s v o l t a g e clamped e i t h e r i n t h e p r e s e n c e or a b s e n c e o f s t r o p h a n t h i d i n . C o r r e l a t i n g t h e o u a b a i n - s e n s i t i v e c u r r e n t and f l u x c h a n g e s , t h e y e s t i m a t e d a N a pump s t o i c h i o m e t r y ( N a : K ) of 2 . 9 t o 1 . 7 . T h i s r a n g e o f v a l u e s i s much l a r g e r t h a n t h e r a n g e rep o r t e d by Thomas o f 1 . 5 t o 1 . 3 , whereas i t overlaps t h e h i g h e r v a l u e s r e p o r t e d by Garrahan and Glynn (1967) and E i s n e r et al. ( 1 9 8 1 ) . I n t h e p r e s e n t e x p e r i m e n t s on b a r n a c l e muscle s i n g l e c e l l s , w e have measured N a e f f l u x and membrane c u r r e n t s i m u l t a n e o u s l y i n t h e manner of Cooke e t a l . b u t have c o n t r o l l e d i n t r a c e l l u l a r c o n s t i t u e n t s by means of a p e r f u s i o n method. O u r r e s u l t s a r e i n agreement w i t h t h e s t o i c h i o m e t r y o f sodium and p o t a s sium t r a n s p o r t by t h e Na,K-ATPase determined by Garrahan and Glynn (1967) and E i s n e r e t a l . ( 1 9 8 1 ) . K

11.

METHODS A s i n g l e muscle c e l l from t h e g i a n t b a r n a c l e

was p e r f u s e d w i t h an i n t e r n a l s o l u t i o n c o n t a i n i n g (among o t h e r c o n s t i t u e n t s ) ATP, a n ATP reg e n e r a t i n g s y s t e m , and 2 2 N a as h a s been d e s c r i b e d by Nelson and B l a u s t e i n ( 1 9 8 0 ) . The s i g n i f i c a n t d i f f e r ence between t h e p r e p a r a t i o n a s d e s c r i b e d by Nelson and B l a u s t e i n (1980) and t h e methods used f o r t h e s e e x p e r i ments i s t h e i n t e r n a l , a x i a l p l a t i n i z e d - p l a t i n u m w i r e ( t o p a s s c u r r e n t ) and s t a n d a r d voltage-clamp e l e c t r o n ics. Balanus nubilis

STOICHIOMETRY OF ELECTROGENIC Na PUMP IN BARNACLES

709

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F i g . 1 . O u a b a i n - d e p e n d e n t current and Na e f f l u x i n b a r n a c l e A t a h o l d i n g p o t e n t i a l of -39.2 mV, o u a b a i n (10-4 M ) was a p p l i e d a s i n d i c a t e d b y the s o l i d b a r i n the f i g u r e . C o n d u c t a n c e ( 0 ) was m e a s u r e d b y a p p l y i n g 2.26-mV d e p o l a r i z i n g p u l s e s f o r 4 sec once every 30 sec. T h e o u a b a i n - d e p e n d e n t f a l l i n Na e f f l u x ( ) i s shown t o be a c c o m p a n i e d b y a f a l l i n o u t w a r d current ( X ) T e t r a e t h y l a m m o n i u m ( T E A ) was p r e s e n t i n the i n t e r n a l p e r f u s a t e (50 mM) and 22Na was u s e d a s the Na t r a c e r .

muscle.

.

111.

RESULTS AND D I S C U S S I O N

The methods u s e d i n t h e s e e x p e r i m e n t s a l l o w t h r e e (1) p e r t i n e n t v a r i a b l e s t o be measured c o n t i n u o u s l y : N a e f f l u x , ( 2 ) membrane c u r r e n t a t a f i x e d h o l d i n g pot e n t i a l , and ( 3 ) membrane c o n d u c t a n c e . F i g u r e l shows

710

M. T. NELSON AND W. J. LEDERER

an e x p e r i m e n t d u r i n g which t h e membrane p o t e n t i a l w a s clamped t o -39 mV. Throughout t h e e x p e r i m e n t , conduct a n c e w a s measured c o n t i n u o u s l y by a p p l y i n g 2.26-mV dep o l a r i z a t i o n s f o r 4 sec e v e r y 30 sec. F i g u r e 1 shows t h a t upon a p p l y i n g 1 0 - 4 M o u a b a i n t o t h e s u p e r f u s i n g sol u t i o n , there w a s a decrease i n N a e f f l u x of about 42 pmoles/sec and a d e c r e a s e o f outward c u r r e n t o f a b o u t 2 VA. [Note t h a t I / F = ( 2 x 1 0 - 6 C / s e c ) / ( l 0 5 C/Eq) = 20 pEq/sec.] T h i s r e s u l t i n d i c a t e s t h a t a b o u t 0.48 of t h e N a e f f l u x t h a t i s o u a b a i n - s e n s i t i v e a p p e a r s t o move as uncompensated c h a r g e . The t o t a l membrane conductance was n o t d i f f e r e n t a t t h e end o f t h e e x p e r i m e n t compared t o i t s v a l u e a t t h e b e g i n n i n g o f t h e e x p e r i m e n t . Between 35 min and 50 min t h e r e was a b o u t a 0 . 1 mS change i n conductance w i t h o u t any obvious e f f e c t on t h e N a e f f l u x . During t h e c o u r s e of t h i s e x p e r i m e n t , t h e i n t e r n a l p e r f u s a t e c o n t a i n e d 50 mM tetraethylammonium (TEA) t o r e d u c e qK. The p r e s e n c e of t h i s potassium-channel b l o c k e r had no d i s c e r n i b l e e f f e c t o n t h e c a l c u l a t e d e l e c t r o g e n i c f r a c t i o n of o u a b a i n - s e n s i t i v e N a e f f l u x when compared t o e x p e r i m e n t s performed i n t h e absence of i n t e r n a l TEA. I n f o u r e x p e r i m e n t s t h i s f r a c t i o n w a s 0 .3 9 f 0 . 0 4 . I n summary, t h e i n t e r n a l l y p e r f u s e d b a r n a c l e m u s c l e c e l l h a s been used t o estimate t h e s t o i c h i o m e t r y of t h e N a , K - A T P a s e under c o n d i t i o n s of c o n t r o l l e d i n t e r n a l and e x t e r n a l environments o f t h e c e l l membrane. C o n t r o l l i n g f o r a l t e r a t i o n s i n t o t a l membrane conductance and potassium-channel c o n d u c t a n c e , w e h a v e found t h a t 1.5-2.0 N a i o n s are t r a n s p o r t e d o u t o f t h e c e l l f o r every K ion transported i n t o t h e cell. Besides confirming t h e r e s u l t s of earlier i n v e s t i g a t i o n s i n t o t h e stoichiome t r y of t h e Na pump and besides d e v e l o p i n g a method f o r a d d i t i o n a l q u a n t i t a t i v e f l u x and c u r r e n t s t u d i e s of t h e Na,K-ATPase, t h i s work d e m o n s t r a t e s t h e u t i l i t y o f f l u x and c u r r e n t s t u d i e s i n g e n e r a l u s i n g t h i s p r e p a r a t i o n (see L e d e r e r and Nelson, 1 9 8 1 ) .

ACKNOWLEDGMENTS We would l i k e t o thank M. P. B l a u s t e i n for h i s s u p p o r t , encouragement, and advice throughout t h e course of t h e s e experiments. D r . E . Santiago helped w i t h t h e d i s s e c t i o n s and w i t h trouble-shooting equipment problems. D r s . L. Goldman and L. Horn helped us prepare t h e p l a t i n i z e d - p l a t i n u m e l e c t r o d e s . This work has been supported by NSF and MDA g r a n t s to M. P. B l a u s t e i n . M. T . Nelson had a r e s e a r c h fellowship supported by t h e Maryland

STOICHIOMETRY OF ELECTROGENIC Na PUMP IN BARNACLES

71 1

A f f i l i a t e o f t h e American Heart A s s o c i a t i o n . A d d i t i o n a l s u p p o r t h a s been p r o v i d e d by t h e N I H (HL-25675) and by a B a s i l O'Connor G r a n t o f t h e N a t i o n a l F o u n d a t i o n f o r t h e March o f D i m e s t o W. J. L e d e r e r , who i s a n e s t a b l i s h e d i n v e s t i g a t o r of t h e American Heart A s s o c i a t i o n and i t s Maryland S t a t e A f f i l i a t e .

REFERENCES

Cooke, I . M., L e b l a n c , G . , and Tauc, L. ( 1 9 7 4 ) . Sodium pump s t o i c h i o m e t r y i n A p l y s i a n e u r o n e s f r o m s i m u l t a n e o u s c u r r e n t and tracer measurements. Nature (London) 251, 254-256. E i s n e r , D. A., L e d e r e r , W. J . , and Vaughan-Jones, R. D. ( 1 9 8 1 ) . The dependence o f sodium pumping and t e n s i o n o n i n t r a c e l l u l a r sodium a c t i v i t y i n v o l t a g e - c l a m p e d s h e e p P u r k i n j e f i b r e s . J. P h y s i o l . (London) 317, 163-187. G a r r a h a n , P . , and Glynn, I. M. ( 1 9 6 7 ) . The s t o i c h i o m e t r y o f t h e sodium pump. J. P h y s i o l . (London) 1 9 2 , 217-235. L e d e r e r , W. J . , a n d N e l s o n , M. T . ( 1 9 8 1 ) . C u r r e n t a s s o c i a t e d w i t h Nao-dependent C a e f f l u x i n b a r n a c l e muscle c e l l s . J. P h y s i o l . (London) ( i n p r e s s ) N e l s o n , M. T . , and B l a u s t e i n , M. P. ( 1 9 8 0 ) . P r o p e r t i e s o f sodium pumps i n i n t e r n a l l y p e r f u s e d b a r n a c l e m u s c l e f i b e r s . J. Gen. P h y s i o l . 7 5 , 183-206. Thomas, R. C . ( 1 9 6 9 ) . Membrane c u r r e n t and i n t r a c e l l u l a r sodium c h a n g e s i n a s n a i l neurone d u r i n g e x t r u s i o n of i n j e c t e d sodium. J. P h y s i o l . (London) 201, 495-514.

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Part VIII

Biosynthesis, Multiple Forms, and Immunology

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CURRENT TOPICS IN MEMBRANES AND TRANSPORT, VOLUME 19

Regulation of Na,K-ATPase by Its Biosynthesis and Turnover NORMAN J. KARIN AND JOHN S. COOK The University of Tennessee- Oak Ridge Graduate School of Biomedical Sciences and The Biology Division Oak Ridge National Laboratory Oak Ridge, Tennessee

I.

INTRODUCTION

The r e g u l a t i o n o f a l k a l i c a t i o n t r a n s p o r t i n a c e l l o r t i s s u e i s i n g e n e r a l b r o u g h t a b o u t by t h e i n t e r a c t i o n o f two p r o c e s s e s : m o d u l a t i o n of t h e number of c o p i e s o f Na,K-ATPase and modulation o f t h e a c t i v i t y of e x i s t i n g c o p i e s by a p p r o p r i a t e l i g a n d s and subs t r a t e s . The l a t t e r s o r t of c o n t r o l h a s been w i d e l y i n v e s t i g a t e d and i s d i s c u s s e d i n many a r t i c l e s i n t h i s volume. With r e g a r d t o t h e former p r o c e s s , t h e quest i o n s o f how many c o p i e s a r e t h e r e and what f a c t o r s i n f l u e n c e t h i s number are t h e p r i n c i p a l themes o f t h e overview p r e s e n t e d i n t h i s a r t i c l e . Throughout i t w i l l be i m p l i c i t , and o c c a s i o n a l l y e x p l i c i t , t h a t t i m e i s a n i m p o r t a n t c o n s i d e r a t i o n . W e have found i t c o n v e n i e n t t o d i s t i n g u i s h between s h o r t - and long-term r e g u l a t i o n of Na,K-ATPase a c t i v i t y . A well-known example o f t h e former i s a n e p i t h e l i a l c e l l i n t o which an a c c e l e r a t e d Na+ i n f l u x from t h e a p i c a l s u r f a c e e l e v a t e s c e l l Na+; t h e r e s p o n s e is a n enhanced a c t i v i t y of t h e N a , K - A T P a s e 713

Copyright 0 1983 by Academic Press, Inc. All nghts of reproduction in any form reserved. ISBN 0-12-153319-0

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a t t h e b a s o l a t e r a l s u r f a c e s and e x t r u s i o n of N a + from t h e o p p o s i t e s i d e of t h e c e l l , y i e l d i n g n e t t r a n s e p i t h e l i a l Na' t r a n s p o r t . There a r e o t h e r e x p e r i m e n t a l and p h y s i o l o g i c a l c o n d i t i o n s i n which e l e v a t i o n of c e l l Na+ o r o t h e r compounds i n t e r a c t i n g w i t h t h e enzyme l e a d s t o an immediate response by s t i m u l a t i n g e x i s t i n g enzyme. I n t h e long term, however, such a r e s p o n s e may be i n a d e q u a t e t o s a t i s f y needs. Maintaining t h e enzyme a c t i v i t y a t a l e v e l approaching i t s maximum r e d u c e s t h e f l e x i b i l i t y of t h e enzyme i n responding t o f u r t h e r demands, whereas modulating t h e number of c o p i e s may a l l e v i a t e t h i s l i m i t a t i o n . To do t h i s r e q u i r e s t i m e ; hence t h e n o t i o n of long-term r e g u l a t i o n . E x p l o r a t i o n of t h i s q u e s t i o n r e q u i r e s s t u d i e s on b i o s y n t h e s i s and d e g r a d a t i o n ( o r t u r n o v e r ) of t h e enzyme, and work i n t h e f i e l d s of development and d i f f e r e n t i a t i o n , endocrinology, c e l l c y c l e k i n e t i c s , t i s s u e c u l t u r e systems, a s w e l l as whole animal physiology a r e r e l e v a n t and a r e b r i e f l y o u t l i n e d h e r e . Most commonly, o n l y d a t a on t h e s p e c i f i c a c t i v i t y of t h e Na,K-ATPase o r numbers of ouabain b i n d i n g s i t e s a r e a v a i l a b l e , and even though t h e s e a r e a c t i v i t y d a t a , t h e y a r e o f t e n t a k e n a s a r e f l e c t i o n of t h e number of f u n c t i o n a l In c o p i e s of Na,K-ATPase p e r c e l l o r u n i t of t i s s u e . general w e s h a l l accept t h i s assessment, b u t t h e t r u t h remains t h a t n o t u n t i l t h e enzyme i s i s o l a t e d and quant i t a t e d i n each i n s t a n c e can w e be c e r t a i n . There a r e a few, b u t o n l y a few, cases where t h i s i d e a l h a s been achieved. The p r e s e n t overview i s by no means e x h a u s t i v e b u t f o c u s e s on a few b e s t - s t u d i e d c a s e s . I t i s o u r i n t e n t t o emphasize h e r e t h a t t h e Na,K-ATPase, whose enzymatic p r o p e r t i e s have been so e x t e n s i v e l y s t u d i e d i n t h e t e s t tube and whose t r a n s p o r t p r o p e r t i e s have been so d e e p l y e x p l o r e d i n r e l a t i v e l y s h o r t - t e r m experiments w i t h c e l l s and t i s s u e s , i s a l s o a v e r y dynamic p r o t e i n , c o n s t a n t l y being s y n t h e s i z e d and t u r n e d over i n many t i s s u e s and s u s c e p t i b l e t o up and down r e g u l a t i o n i n number of c o p i e s i n response t o a v a r i e t y of environmental stimuli.

11.

A.

BIOSYNTHESIS AND TURNOVER BIOSYNTHESIS

I n comparison t o enzymological and p h y s i o l o g i c a l i n f o r m a t i o n on Na,K-ATPase, r e l a t i v e l y l i t t l e i s known a b o u t t h e enzyme's b i o s y n t h e s i s . Membrane and s e c r e t o r y

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p r o t e i n s are g e n e r a l l y b e l i e v e d t o be s y n t h e s i z e d i n t h e rough endoplasmic r e t i c u l u m ( R E R ) , b e i n g c o t r a n s l a t i o n a l l y i n s e r t e d p a r t i a l l y (membrane p r o t e i n s ) o r c o m p l e t e l y ( s e c r e t o r y p r o t e i n s ) i n t o t h e lumen of t h a t o r g a n e l l e ( P a l a d e , 1 9 7 5 ) . Here, t h e s m a l l hydrophobic s i g n a l sequence ( B l o b e l and D o b b e r s t e i n , 1975) i s removed and common o l i g o s a c c h a r i d e p r e c u r s o r s a r e t r a n s f e r r e d t o t h e p e p t i d e backbone. RER-derived v e s i c l e s c o n t a i n i n g t h e s e p r o t e i n s are t h e n s h u t t l e d t o t h e G o l g i a p p a r a t u s where f u r t h e r p r o c e s s i n g o c c u r s , such as o l i g o s a c c h a r i d e trimming and t e r m i n a l g l y c o s y l a t i o n , and s e l e c t i v e p r o t e o l y s i s from l'pro'' t o m a t u r e forms (Rothman, 1 9 8 1 ) . These p r o t e i n s a r e t h e n s o r t e d v i a some unknown p r o c e s s and t r a n s f e r r e d t o s p e c i f i c o r g a n e l l e s ( S a b a t i n i e t a l . , 1982) o r , i n t h e case o f s e c r e t o r y p r o d u c t s , e x p o r t e d o u t of t h e c e l l by exocyt o s i s . The t r a n s l o c a t i o n and c o v a l e n t m o d i f i c a t i o n o f the p r o t e i n s accounts f o r a several-hour lag o r " t r a n s i t " t i m e between t r a n s l a t i o n and t h e a r r i v a l a t t h e i r funct i o n a l d e s t i n a t i o n (Devreotes and Fambrough, 1 9 7 6 ) . C h u r c h i l l and Hokin ( 1 9 7 9 ) a n a l y z e d t h e b i o s y n t h e s i s and i n s e r t i o n o f N a , K - A T P a s e s u b u n i t s i n t o e e l elect r o p l a x membranes i n v i t r o . Sachs o r g a n s were i n c u b a t e d w i t h [ 3H] v a l i n e , N a , K-ATPase w a s i s o l a t e d , and t h e subu n i t s w e r e e l e c t r o p h o r e t i c a l l y a n a l y z e d . The a u t h o r s obs e r v e d a 2.5- t o 3-hr l a g p e r i o d b e f o r e r a d i o a c t i v i t y was d e t e c t e d i n t h e i s o l a t e d enzyme, w i t h s u b s e q u e n t l i n e a r i n c o r p o r a t i o n i n t o b o t h t h e a- and @ - s u b u n i t s . The r a t i o o f s p e c i f i c r a d i o a c t i v i t y f o r t h e t w o s u b u n i t s was n e a r l y u n i t y a t a l l sampling t i m e s . These r e s u l t s were i n t e r p r e t e d t o mean t h a t holoenzyme assembly is coo r d i n a t e d and o c c u r s 2-3 h r a f t e r d e n o v o s y n t h e s i s o f t h e i n d i v i d u a l s u b u n i t s . The congruency of t h e l a g p e r i o d f o r t h e incorporation of r a d i o a c t i v e l a b e l i n t o t h e n o n g l y c o s y l a t e d ( o r p o o r l y g l y c o s y l a t e d ) a- and h e a v i l y g l y c o s y l a t e d 8-subunits i n d i c a t e d t o t h e a u t h o r s e i t h e r assembly of t h e enzyme p r i o r t o g l y c o s y l a t i o n o r t h a t t h e a v a i l a b i l i t y of t h e s m a l l s u b u n i t i s r a t e limiting. S i m i l a r l y , when p r o t e i n s y n t h e s i s i s i n h i b i t e d by c y c l o h e x i m i d e , a 1-1/2-hrdelay i s s e e n b e f o r e t h e s p e c i f i c r a d i o a c t i v i t y decreased i n e i t h e r subunit, p o i n t i n g t o c o n c e r t e d assembly. T h a t t h e r a t e of i n c o r p o r a t i o n of l a b e l i s e q u a l i n b o t h s u b u n i t s i m p l i e s a s i m i l a r r a t e of t u r n o v e r . Turnover of t h e enzyme w i l l be d i s c u s s e d i n d e t a i l i n t h e f o l l o w i n g s e c t i o n . S a b a t i n i ' s group h a s r e p o r t e d a t t h i s m e e t i n g (Sherman e t a i . , t h i s volume) some unexpected p r o p e r t i e s o f N a , K - A T P a s e b i o s y n t h e s i s . Using a c e l l - f r e e t r a n s l a t i o n s y s t e m , t h e y found t h a t , a l t h o u g h t h e 8-subunit i s s y n t h e s i z e d on membrane-bound polysomes, i t s i n s e r t i o n

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d o e s n o t a p p e a r t o i n v o l v e t h e c l e a v a g e of a s i g n a l p e p t i d e . The g l y c o s y l a t i o n k i n e t i c s i n d i c a t e t h a t t h e B p r o t e i n i s c o v a l e n t l y modified i n t h e Golgi a p p a r a t u s , a s i s t r u e f o r o t h e r membrane and s e c r e t o r y g l y c o p r o t e i n s . More s u r p r i s i n g y e t were t h e i r f i n d i n g s r e g a r d i n g t h e b i o s y n t h e s i s and i n s e r t i o n of t h e a - s u b u n i t . They found i t t o be s y n t h e s i z e d in v i t r o o n l y by f r e e , n o t bound, polysomes and t h a t i n MDCK c e l l s ( a c u l t u r e d e p i t h e l i a l l i n e ) i t m a n i f e s t s i t s e l f i n t h e plasma memb r a n e w i t h o u t e v e r a p p e a r i n g i n t h e RER. Accordingly, Sherman e t a l . ( t h i s volume) found no change i n t h e e l e c t r o p h o r e t i c m o b i l i t y o f t h e a - s u b u n i t when t r a n s l a t i o n was c a r r i e d o u t i n t h e p r e s e n c e of t u n i c a m y c i n , a s p e c i f i c i n h i b i t o r of p r o t e i n g l y c o s y l a t i o n . That t h i s s u b u n i t t h u s a p p e a r s t o be w i t h o u t a s p a r a g i n e - l i n k e d o l i g o s a c c h a r i d e s i s i n agreement w i t h t h e d e s c r i b e d p a t h way which b y p a s s e s t h e c l a s s i c s i t e s of g l y c o s y l a t i o n . I t c a n b e c o n c l u d e d from t h e s e r e s u l t s t h a t t h e pathway of s y n t h e s i s of Na,K-ATPase d e v i a t e s i n s e v e r a l ways from most o t h e r membrane p r o t e i n s . Cytoplasmic s y n t h e s i s of membrane components i s n o t , however, w i t h o u t p r e c e d e n t , p a r t i c u l a r l y f o r some membrane p r o t e i n s of m i t o c h o n d r i a and c h l o r o p l a s t s . T h e o r i e s are p r e s e n t l y b e i n g p r o pounded i n v o l v i n g i n s e r t i o n of membrane p r o t e i n s v i a t r a n s i e n t hydrophobic conformations, allowing spontaneo u s i n t e r c a l a t i o n i n t o l i p i d domains (Wickner, 1 9 8 0 ; Engelman and S t e i t z , 1 9 8 1 ) . A l s o , l i k e t h e N a , K - A T P a s e @ - s u b u n i t , t h e i n s e r t i o n o f a number of o t h e r membrane proteins appears not t o involve p r o t e o l y t i c processing (Sabatini et a l . , 1982). McDonough e t a l . ( t h i s volume) have a l s o u s e d c e l l f r e e t r a n s l a t i o n t o c h a r a c t e r i z e t h e two s u b u n i t s and found t h a t t h e a - s u b u n i t i s t r a n s l a t e d from a mRNA of 22-28 S , t h e c o r r e c t s i z e c l a s s f o r t h e g e n e r a t i o n of a 96,000-dalton p r o t e i n . T h i s i n d i c a t e s t h a t t h e a- and % - s u b u n i t s a r e n o t p r o t e o l y t i c a l l y c l e a v e d from a common p r e c u r s o r . Several r e p o r t s a t t h i s meeting d e a l t w i t h another a s p e c t of Na,K-ATPase which r e q u i r e s e l u c i d a t i o n i n terms of b i o s y n t h e s i s , namely t h e p r e s e n c e of two t i s s u e - s p e c i f i c forms of t h e a - s u b u n i t (Sweadner, 1979 and t h i s v o l u m e ) . Sweadner d e s c r i b e d a form of c a t a l y t i c s u b u n i t ( a( + ) ) found s p e c i f i c a l l y i n m y e l i n a t e d axons from b r a i n which i s a b o u t 2 0 amino a c i d s l a r g e r t h a n t h a t of u n m y e l i n a t e d n e u r o n s o r k i d n e y t i s s u e . A f u n c t i o n a l s i g n i f i c a n c e h a s been s u g g e s t e d i n t h a t t h e s e p r o t e i n s d i f f e r i n t h e i r a f f i n i t i e s f o r c a r d i a c glycos i d e s and may b e i n v o l v e d i n t h e enzyme's r e g u l a t i o n by p u t a t i v e endogenous compounds w i t h g l y c o s i d e - l i k e activity. S c h e l l e n b e r g e t a l . ( t h i s volume) have found

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t h a t t h e a and a ( + ) p r o t e i n s a r e immunologically c r o s s reactive. T h i s a g r e e s w i t h t h e i r s i m i l a r i t y o f Clevel a n d p e p t i d e maps (Sweadner, t h i s volume) i n i n d i c a t i n g p r o b a b l e sequence homology between t h e two forms. I t i s c o n c e i v a b l e t h a t b o t h forms a r i s e from a common mRNA with p o s t t r a n s l a t i o n a l modification accounting f o r the It is a l s o relevant here t o observed s i z e d i f f e r e n c e . n o t e t h a t some immunological c r o s s - r e a c t i v i t y h a s been observed between a- and 8 - s u b u n i t s t h a t c a n n o t be acc o u n t e d f o r by c o n t a m i n a t i o n ( B a l l et a l . , t h i s v o l u m e ) , implying some s t r u c t u r a l homology. I n p o l a r i z e d e p i t h e l i a l cells Na,K-ATPase is g e n e r a l l y c o n s i d e r e d t o reside e x c l u s i v e l y i n t h e basol a t e r a l membrane ( S t i r l i n g , 1972; E r n s t and M i l l s , 1 9 8 0 ) . Geering et a l . (1981) have used c o l l o i d a l g o l d - d e r i v a t i z e d a n t i b o d i e s t o N a , K - A T P a s e s u b u n i t s t o f o l l o w by e l e c t r o n microscopy t h e i r i n s e r t i o n i n t o t h e plasma memb r a n e s o f MDCK c e l l s . They found t h e t r a c e r e x c l u s i v e l y i n t h e b a s o l a t e r a l surface, indicating direct i n s e r t i o n i n t o t h i s membrane. I n s e r t i o n d i r e c t l y i n t o t h e enzyme's f u n c t i o n a l s i t e c a n n o t be assumed i n l i g h t of o b s e r v a t i o n s t h a t a m i n o p e p t i d a s e , a l u m i n a l membrane m a r k e r , i s f i r s t i n s e r t e d a t areas of c e l l - c e l l c o n t a c t , t h e n d i f f u s e s l a t e r a l l y t o t h e a p i c a l s u r f a c e (Louvard, 1 9 8 0 ) . A s i m i l a r phenomenon w a s d e s c r i b e d f o r N a , K - A T P a s e by S p e c h t ( t h i s volume) i n hamster o p t i c n e r v e . By m o n i t o r i n g t h e appearance of r a d i o l a b e l e d enzyme i n n e r v e endi n g s , s h e concluded t h a t Na,K-ATPase was s y n t h e s i z e d i n t h e c e l l body and t r a n s f e r r e d r a p i d l y t o t h e n e r v e endi n g s which c a n n o t s y n t h e s i z e p r o t e i n . The complete pathway o f s y n t h e s i s f o r t h i s enzyme i s n o t y e t w e l l d e f i n e d . The d e t e r m i n a n t s which g u i d e t h e individual subunits t o t h e i r functional location repres e n t one o f t h e more i m p o r t a n t q u e s t i o n s i n p r e s e n t - d a y c e l l b i o l o g y . Na,K-ATPase, w i t h i t s v a r i e d s t r u c t u r a l and s y n t h e t i c p r o p e r t i e s , r e p r e s e n t s an i n t e r e s t i n g s y s t e m w i t h which t o s t u d y membrane assembly. The o t h e r s i d e o f t h e c o i n , namely d e g r a d a t i o n ( t u r n o v e r ) , i s d i s cussed i n t h e following section. B.

TURNOVER S T U D I E S I N CULTURED C E L L S

Once s y n t h e s i z e d and i n s e r t e d i n t h e c e l l membrane, t h e N a , K - A T P a s e i s n o t a permanent r e s i d e n t i n i t s f u n c t i o n a l l o c a t i o n . A s w i t h most i f n o t a l l c e l l p r o t e i n s , t h e enzyme i s s u b j e c t t o t u r n o v e r . The s u r f a c e d e n s i t y of t h e enzyme f o r c e l l s i n t h e s t e a d y s t a t e , where t h e r a t e s of s y n t h e s i s and t u r n o v e r a r e e q u a l , i s t h e res u l t a n t o f t h e two p r o c e s s e s and may be d e s c r i b e d by

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71a

= ksyn/kto

where E i s t h e number of s u r f a c e enzymes p e r c e l l , k i s t h e z e r o - o r d e r r a t e of s y n t h e s i s , and kto i s t h e syn f i r s t - o r d e r r a t e of t u r n o v e r o r removal from t h e s u r f a c e . Turnover s e r v e s two f u n c t i o n s o f advantage t o t h e c e l l . F i r s t , t h e c o n s t a n t r e p l a c e m e n t of t h e enzyme c l e a r s t h e s u r f a c e of m o l e c u l e s t h a t may have been d e n a t u r e d o r i n a c t i v a t e d f o r any r e a s o n , a p r o c e s s t h a t may be regarded as a r e p a i r function. Second, w i t h b o t h synt h e t i c and d e g r a d a t i v e pathways i n place, m o d i f i c a t i o n of e i t h e r o r b o t h i n r e s p o n s e t o c h a n g i n g e n v i r o n m e n t a l o r hormonal s t i m u l i can change t h e number o f f u n c t i o n a l m o l e c u l e s a t t h e c e l l s u r f a c e . The r o l e of t u r n o v e r i n r e s p o n s e t o hormonal s t i m u l i i s c o n s i d e r e d i n S e c t i o n IV C u l t u r e d c e l l s make c o n v e n i e n t and c l e a n model s y s t e m s f o r s t u d y i n g t u r n o v e r , and i n o u r l a b o r a t o r y w e have u s e d c l o n e d HeLa-S3 c e l l s e x t e n s i v e l y f o r t h i s p u r pose. The number o f Na,K-ATPase molecules ( E ) , a s measured by t h e number o f ouabain-binding s i t e s , is app r o x i m a t e l y 1 0 6 p e r c e l l i n l o g a r i t h m i c a l l y growing p o p u l a t i o n s . T h i s i s , of c o u r s e , a mean v a l u e , t h e numbers b e i n g a b o u t 0.7 x 1 0 6 immediately a f t e r c y t o k i n e s i s , i n c r e a s i n g t o a b o u t 1 . 3 x 1 0 6 immediately b e f o r e t h e n e x t d i v i s i o n (Cook e t a l . , 1 9 7 6 ) . I f one assumes t h a t t h e s u r f a c e area i n c r e a s e s p r o p o r t i o n a t e l y t o c e l l volume d u r i n g t h e c e l l c y c l e , t h e s u r f a c e d e n s i t y o f Na,K-ATPase a p p e a r s t o remain a p p r o x i m a t e l y c o n s t a n t d u r i n g growth of HeLa c e l l s , a l t h o u g h i n t r a n s f o r m e d hamster f i b r o b l a s t s i t h a s been c l a i m e d t h a t t h e s u r f a c e d e n s i t y r e a c h e s a l a r g e peak v a l u e i n G 2 (Graham et al., 1973) R e s u l t s somewhat s i m i l a r t o t h o s e f o r HeLa c e l l s have been o b t a i n e d by Mummery et al. (1981) w i t h sync h r o n i z e d mouse neuroblastoma c e l l s i n which Na,K-ATPase p e r c e l l , a s s a y e d on whole c e l l homogenates, f e l l a l i t t l e more t h a n 2 - f o l d on d i v i s i o n and s u b s e q u e n t l y i n c r e a s e d a g a i n i n a more o r less r e g u l a r f a s h i o n t h r o u g h The a s s a y i s presumably a measure o f t h e number of G2. c o p i e s of t h e enzyme p e r c e l l . Of s p e c i a l i n t e r e s t i n t h i s s t u d y are t h e p a r a l l e l o b s e r v a t i o n s on c e l l K+ cont e n t , K+ l e a k f l u x e s , o u a b a i n - s e n s i t i v e K+ f l u x e s , and membrane p o t e n t i a l i n i n t a c t c e l l s . These p a r a m e t e r s undergo w e l l - d e f i n e d c y c l i c f l u c t u a t i o n s w i t h a peak of o u a b a i n - s e n s i t i v e K+ i n f l u x j u s t p r i o r t o t h e Gl/S t r a n s i t i o n , a l t h o u g h t h e r e i s no c o r r e s p o n d i n g peak i n t o t a l Na,K-ATPase a c t i v i t y i n c e l l homogenates. I n h i b i t i o n of K+ u p t a k e by o u a b a i n a t t h i s p o i n t i n t h e c e l l c y c l e i s p a r t i c u l a r l y e f f e c t i v e i n i n h i b i t i n g DNA s y n t h e s i s . These r e s u l t s p o i n t up t h e i m p o r t a n c e o f s h o r t - t e r m

.

.

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719

physiological regulators, i n addition t o the surface d e n s i t y of t h e enzyme, i n t h e g r o w t h - a s s o c i a t e d c o n t r o l of Na-K t r a n s p o r t . T h e a u t h o r s p r o p o s e , f o r example, t h a t a n i n c r e a s e d Na+-H+ exchange i n G 1 may l e a d t o an increased i n t r a c e l l u l a s Na+ t h a t stimulates the a c t i v i t y of t h e Na,K-ATPase. The mechanisms of such t i m e dependent i n t e r a c t i o n s i n t h e r e g u l a t i o n of t r a n s p o r t a r e n o t y e t e n t i r e l y worked o u t . I n s o f a r a s t h e r e g u l a t i o n of t h e s u r f a c e d e n s i t y of t h e enzyme i s concerned, t h e s y n t h e t i c r a t e i n growi n g p o p u l a t i o n s must be e q u a l t o t h e sum of b o t h t u r n o v e r and n e t s y n t h e s i s a s s o c i a t e d w i t h growth. Three methods, g i v i n g s u b s t a n t i a l l y i d e n t i c a l r e s u l t s , have been used t o s t u d y t u r n o v e r of Na,K-ATPase i n HeLa c e l l s . One i s t h e d e t e r m i n a t i o n of t h e r a t e of i n t e r n a l i z a t i o n of t h e s p e c i f i c l i g a n d [3H]ouabain. The method assumes t h a t t h e r a t e of e n d o c y t o s i s of t h e l i g a n d c o r r e s p o n d s t o t h e r a t e of e n d o c y t o s i s of t h e enzyme, and t h a t t h i s r a t e i s a measure of t h e t u r n o v e r a s s o c i a t e d r a t e of c l e a r a n c e of t h e t r a n s p o r t e r from t h e c e l l s u r f a c e . I f HeLa c e l l s a r e grown i n s u b l e t h a l conc e n t r a t i o n s of t h e g l y c o s i d e ( e . g . , 2 x 10-8 M , which i s about e q u a l t o b o t h t h e kd and k i of t h e d r u g ) , t h e i n t e r n a l i z a t i o n r a t e c a l c u l a t e d from t h e assumptions g i v e n i s 3 sets of s u r f a c e Na,K-ATPase p e r g e n e r a t i o n (Cook e t al., 1 9 8 2 ) . The experiment i s n o t e n t i r e l y s a t i s f a c t o r y , however, because even i n t h i s low c o n c e n t r a t i o n of ouabain c e l l growth i s slowed t o a b o u t h a l f i t s normal rate. The second method circumvents t h e t o x i c e f f e c t s of long-term exposure t o g l y c o s i d e . I n t h i s t e c h n i q u e , t h e c e l l s a r e p u l s e - l a b e l e d w i t h [3H]ouabain and resuspended i n f r e s h d r u g - f r e e medium. I t i s n e c e s s a r y i n t h i s c a s e t o a l l o w f o r t h e f a c t t h a t o u a b a i n d i s s o c i a t e s from i t s b i n d i n g s i t e w i t h a h a l f - t i m e of a b o u t 5 h r i n HeLa c e l l s . A model h a s been c o n s t r u c t e d f o r t h e r e l e a s e of pulse-bound ouabain from t h e s e c e l l s . T h e model assumes t h a t ouabain on t h e s u r f a c e e i t h e r simply d i s s o c i a t e s o r i s removed by i n t e r n a l i z a t i o n ( t u r n o v e r ) , and t h a t t h e i n t e r n a l i z e d b u t o t h e r w i s e unmetabolized ouabain i s subs e q u e n t l y r e l e a s e d from t h e c e l l s by e x o c y t o s i s from t h e lysosomal compartment (Cook et a l . , 1 9 7 6 , 1982; W i l l e t al., 1 9 7 7 ; P o l l a c k et a l . , 1 9 8 2 ) . Ouabain r e l e a s e d a t a f i t t o t h i s model g i v e a h a l f - t i m e f o r i n t e r n a l i z a t i o n of about 5 h r , o r a r a t e c o n s t a n t of a b o u t 3 p e r generat i o n i n c e l l s growing w i t h a p o p u l a t i o n d o u b l i n g t i m e of 24 h r . The a d v a n t a g e s of t h i s approach a r e t h a t t h e doubling t i m e i s n o t s e r i o u s l y p e r t u r b e d by t h e p u l s e l a b e l and t h a t t h e measurements may be made on i n t a c t c e l l s . C o r r e l a t i v e s t u d i e s on f r a c t i o n a t e d c e l l s q u a n t i t a t i v e l y s u p p o r t t h e model, and show t h a t t h e combined

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p r o c e s s e s of d i s s o c i a t i o n and i n t e r n a l i z a t i o n c l e a r t h e plasma membrane of ouabain w i t h a h a l f - t i m e of less t h a n 3 h r ( W i l l e t a l . , 1 9 7 7 ) . I n o t h e r words, t u r n o v e r p l a y s a s i g n i f i c a n t r o l e i n t h e r e c o v e r y of HeLa c e l l s from o u a b a i n i n t o x i c a t i o n . S i m i l a r l y , A i t o n et a l . ( 1 9 8 1 ) have observed a c y c l o h e x i m i d e - s e n s i t i v e r e c o v e r y of pump s i t e s i n b o t h HeLa and embryonic c h i c k h e a r t c e l l s a f t e r c h r o n i c exposure of t h e c e l l s t o low c o n c e n t r a t i o n s of u n l a b e l e d ouabain. They i n t e r p r e t t h e r e c o v e r y a s t h e s y n t h e s i s of new s i t e s . I n t e r e s t i n g l y , t h e r e c o v e r y i n t h e i r HeLa c e l l s has a h a l f - t i m e of 5-6 h r . I t i s w e l l known t h a t l i g a n d b i n d i n g may a f f e c t t u r n o v e r r a t e s and t h e r e f o r e a t h i r d method was developed t o measure Na,K-ATPase t u r n o v e r w i t h o u t t h e u s e of ouabain. The method was a d e n s i t y l a b e l t e c h n i q u e , i n t r o d u c e d by F i l n e r and Varner ( 1 9 6 7 ) and adapted f o r study of t u r n o v e r of t h e a c e t y l c h o l i n e r e c e p t o r by Fambrough and h i s a s s o c i a t e s (Devreotes and Fambrough, 1 9 7 6 ; Gardner and Fambrough, 1 9 7 9 ) . L. R. P o l l a c k i n o u r l a b o r a t o r y i s o l a t e d HeLa plasma membranes, and s p e c i f i c a l l y l a b e l e d t h e Na,K-ATPase c a t a l y t i c s u b u n i t by p h o s p h o r y l a t i o n w i t h [32P] ATP i n a Na+-containing r e a c t i o n m i x t u r e . When such membranes were d i s p e r s e d i n sodium dodecyl s u l f a t e (SDS) and analyzed by g e l e l e c t r o p h o r e s i s , he found a 32P-labeled p r o t e i n w i t h mol e c u l a r weight 93,000, whereas no l a b e l e d peaks were found a f t e r l a b e l i n g i n a Na+-free, K+-containing r e a c t i o n m i x t u r e . By t h i s and o t h e r c r i t e r i a , t h i s peak was t a k e n t o be t h e c a t a l y t i c s u b u n i t o f Na,K-ATPase. P o l l a c k t h e n grew HeLa c e l l s f o r s e v e r a l g e n e r a t i o n s i n medium c o n t a i n i n g 13C-labeled amino a c i d s . A f t e r t h e c e l l s had come t o a s t e a d y s t a t e i n t h e d e n s i t y l a b e l , he resuspended them i n medium c o n t a i n i n g normal [12C]l a b e l e d amino a c i d s . A t i n t e r v a l s , he removed a l i q u o t s , and a g a i n i s o l a t e d and s p e c i f i c a l l y p h o s p h o r y l a t e d t h e membranes. A t t h e Same t i m e he i s o l a t e d membranes from c o n t r o l c e l l s and phosphorylated them w i t h [33P]ATP. These membranes were mixed, s o l u b i l i z e d i n (SDS), and c e n t r i f u g e d i n metrizamide-D20 g r a d i e n t s . Among a l l o t h e r p r o t e i n s on t h e s e g r a d i e n t s , t h e s e p a r a t i o n o f t h e two r a d i o a c t i v e i s o t o p e s marked t h e d e n s i t y s h i f t of t h e 13C-labeled N a ,K-ATPase c a t a l y t i c s u b u n i t . A f t e r r e s u s p e n s i o n and growth of t h e 13C-labeled c e l l s i n normal medium, t h e d e n s i t y s h i f t of t h e s u b u n i t became s m a l l e r w i t h t i m e and approached t h e c o n t r o l d e n s i t y . By quant i t a t i n g t h e s e d e n s i t y changes, P o l l a c k w a s a b l e t o show t h a t 13C-labeled c a t a l y t i c s u b u n i t was d e c r e a s i n g i n t h e membrane f r a c t i o n 4 times f a s t e r t h a n c o u l d be accounted f o r by d i l u t i o n due t o n e t growth. H e concluded t h a t i n

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e a c h g e n e r a t i o n t h e c e l l s s y n t h e s i z e d 4 sets o f catal y t i c s u b u n i t s and t u r n e d o v e r 3 , a r e s u l t t h a t c o r responded c l o s e l y t o o u r p r e v i o u s estimates from t h e ouabain i n t e r n a l i z a t i o n data (Pollack e t a l . , 1981b). The r e s u l t t h u s a l s o s u b s t a n t i a t e d o u r e a r l i e r assumpt i o n t h a t l i g a n d binding does n o t p e r t u r b t h e turnover r a t e of t h i s p a r t i c u l a r p r o t e i n . Given t h e s e mean v a l u e s and t h e measurement of 106 ouabain-binding sites p e r c e l l , w e calculate t h a t t h e N a , K - A T P a s e s u b u n i t i s s y n t h e s i z e d a t a mean r a t e of 2800 m o l e c u l e s / c e l l / m i n , and t u r n e d o v e r a t a r a t e o f 2100 molecules/cell/min. I n h i s a n a l y s i s of d e n s i t y - l a b e l e d H e L a membranes, P o l l a c k made two o t h e r i m p o r t a n t o b s e r v a t i o n s . F i r s t , a f t e r r e s u s p e n s i o n o f 13C-labeled c e l l s i n normal medium, t h e d e c r e a s e i n d e n s i t y - l a b e l e d c a t a l y t i c subu n i t i n t h e membranes w a s n o t d e t e c t a b l e f o r 4 h r , a l This delay i s though i t w a s t h e r e a f t e r f i r s t - o r d e r . a s c r i b e d t o t h e t r a n s i t t i m e from t h e s i t e o f s y n t h e s i s w i t h i n t h e c e l l t o t h e f i n a l i n s e r t i o n of new enzyme i n t h e c e l l s u r f a c e , and i s s i m i l a r t o t h e t r a n s i t t i m e s o f a b o u t 2.5 h r o b s e r v e d by C h u r c h i l l and Hokin (1979) i n t h e s y n t h e s i s of Na,K-ATPase i n e l e c t r o p l a x . During t h e t r a n s i t t i m e , enzyme t h a t w a s s y n t h e s i z e d w h i l e t h e c e l l s were i n 13C-labeled medium c o n t i n u e s i n i t s p a s s a g e t o t h e membrane and m a i n t a i n s t h e d e n s i t y l a b e l a t t h e s u r f a c e . N o r m a l d e n s i t y p r o t e i n does n o t b e g i n t o d i l u t e t h e h i g h - d e n s i t y l a b e l u n t i l i t c o m p l e t e s i t s own 4 hr transit. P o l l a c k ' s second a d d i t i o n a l o b s e r v a t i o n i s t h a t t h e t u r n o v e r rates of membrane p r o t e i n s are c l e a r l y h e t e r o geneous. Twenty-four h o u r s a f t e r r e s u s p e n s i o n of d e n s i t y - l a b e l e d c e l l s i n normal medium, t h e d e n s i t y s h i f t i n t h e Na,K-ATPase c a t a l y t i c s u b u n i t w a s v i r t u a l l y i n d e t e c t a b l e (less t h a n 5% of t h e i n i t i a l ) . But b mass s p e c t r o m e t r y of t h e membranes, n e a r l y h a l f of t h e s p e c i f i c a c t i v i t y of a l l p r o t e i n s w a s s t i l l t h e r e , an amount t h a t c o u l d be a c c o u n t e d f o r l a r g e l y by d i l u t i o n due t o growth. O t h e r s (Huang e t a l . , 1 9 7 3 ) , u s i n g s u r f a c e i o d i n a t i o n t e c h n i q u e s , have shown t h a t t h e m a j o r i t y of H e L a membrane p r o t e i n s t u r n o v e r w i t h a v e r y l o n g h a l f - t i m e , o n t h e o r d e r of 70 h r . The mass-spectrometry d a t a a r e i n agreement w i t h t h i s , whereas t h e s p e c i f i c p h o s p h o r y l a t i o n d a t a show t h a t t h e Na,K-ATPase s u b u n i t i s among t h e m i n o r i t y of s u r f a c e p r o t e i n s t h a t t u r n o v e r a t a much f a s t e r r a t e . Given t h e importance of a l k a l i c a t i o n t r a n s p o r t t o c e l l growth and s u r v i v a l , r a p i d t u r n o v e r may be f u n c t i o n a l l y s i g n i f i c a n t i n a s s u r i n g t h e maintenance of Na,K-ATPase a c t i v i t y i n good r e p a i r .

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C.

PATHWAYS F O R T U R N O V E R

There is l i t t l e d i r e c t e v i d e n c e concerning t h e pathway, much less t h e r e g u l a t o r y mechanism, f o r t h e removal o f Na,K-ATPase from t h e c e l l s u r f a c e i n t u r n o v e r . Since t h e i n t e r n a l i z a t i o n of surface-bound ouab a i n h a s t h e same k i n e t i c s a s t h e t u r n o v e r of 13Cl a b e l e d c a t a l y t i c s u b u n i t , it may be presumed t h a t t h e u p t a k e of t h e s p e c i f i c l i g a n d i s a m a r k e r f o r t h e i n t e r n a l i z a t i o n of t h e enzyme i t s e l f . With t h i s presumpt i o n , w e have followed by f r a c t i o n a t i o n s t u d i e s t h e i n t e r n a l i z a t i o n of [3H]ouabain i n HeLa c e l l s (Cook e t a l . , 1 9 8 2 ) . An added advantage t o t h i s approach i s t h a t ouabain i s n o t m e t a b o l i z e d by t h e s e c e l l s ( W i l l e t al., 1 9 7 7 ) . A s e x p e c t e d , immediately a f t e r a p u l s e l a b e l , a l l o f t h e r a d i o a c t i v e g l y c o s i d e i s found a s s o c i a t e d w i t h t h e membrane f r a c t i o n . With t i m e , t h e l a b e l moves i n t o a p a r t i c u l a t e s u b c e l l u l a r f r a c t i o n where it i s cod i s t r i b u t e d w i t h 8-hexosaminidase, a lysosomal marker. N o s i g n i f i c a n t amount of l a b e l i s found a s s o c i a t e d w i t h markers f o r o t h e r s u b c e l l u l a r o r g a n e l l e s . T h i s a n a l y s i s , done w i t h p r e p a r a t i v e s u c r o s e g r a d i e n t s by c o n v e n t i o n a l methods, was s u b s t a n t i a t e d by more q u a n t i t a t i v e a n a l y t i c a l methods comparing t h e s e n s i t i v i t y t o s h e a r ( i n a Dounce homogenizer) and t o osmotic shock of b o t h i n t e r n a l i z e d ouabain and 8-hexosaminidase. Again t h e two markers were c o d i s t r i b u t e d . The s h e a r s e n s i t i v i t y was found t o be f i r s t - o r d e r , s u g g e s t i n g t h a t t h e two markers were i n a s i n g l e compartment. When t h e c u r v e f o r t h e r e l e a s e of t h e markers by s h e a r was e x t r a p o l a t e d t o z e r o s h e a r , it was found t h a t 1 0 0 % of e a c h m a r k e r was w i t h i n t h e p a r t i c u l a t e compartment. I n o t h e r words, t h e r e w a s no e v i d e n c e t h a t any f r e e ouabain e n t e r s t h e c y t o s o l . Ouabain r e l e a s e d from i t s i n t e r n a l p a r t i c u l a t e Compartment by osmotic shock i s f r e e ouabain and i s n o t bound t o a l a r g e r molecule. I n c o n t r a s t , o u a b a i n bound t o i s o l a t e d plasma membranes c a n n o t be r e l e a s e d by osmot i c shock. The c o n c l u s i o n from t h i s a n a l y s i s was t h a t Na,K-ATPase, w i t h i t s bound l i g a n d , i s i n t e r n a l i z e d probably by an e n d o c y t i c p r o c e s s , and t h a t t h e i n t e r n a l i z e d endosomes f u s e w i t h secondary lysosomes where t h e enzyme i s degraded and t h e o u a b a i n r e l e a s e d i n t o t h e i n t r a l y s o s o m a l s p a c e . E v e n t u a l l y t h e i n t e r n a l i z e d ouab a i n i s l o s t from t h e c e l l s by a mechanism t h a t i s app a r e n t l y e x o c y t o s i s ( P o l l a c k e t a 1 , 1982) I f t h e i n t e r n a l i z a t i o n i s i n d e e d by e n d o c y t o s i s , t h e r e should be an i n t e r n a l endosome compartment en r o u t e from t h e s u r f a c e t o t h e lysosomes. Our i n i t i a l a t t e m p t s t o l o c a t e such a compartment have n o t been s u c c e s s f u l (Cook e t al., 1 9 8 2 1 , s u g g e s t i n g e i t h e r t h a t

.

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s u c h a compartment i s v e r y s m a l l o r t h a t t h e t i m e req u i r e d f o r t r a n s i t from t h e s u r f a c e t o t h e lysosomes i s very short. This question is unresolved. I n t e r n a l i z a t i o n i s energy dependent. I f t h e cells a r e p o i s o n e d and t h e i r ATP d e p l e t e d by t r e a t m e n t w i t h NaN3 and 2-deoxyglucose, i n t e r n a l i z a t i o n i s t o t a l l y b l o c k e d . I n t h i s case, pulse-bound o u a b a i n s i m p l y d i s s o c i a t e s from t h e c e l l s u r f a c e w i t h k i n e t i c s i d e n t i c a l t o i t s d i s s o c i a t i o n from i s o l a t e d membranes (Cook e t a l . , 1976).

III.

REGULATION DEPLETION

IN

RESPONSE TO N a + LOADING AND K+

A s n o t e d above, w e d i s t i n g u i s h between s h o r t and long-term r e g u l a t i o n o f N a , K - A T P a s e a c t i v i t y i n t h e c e l l surface. I n t h e former w e i n c l u d e t h e m o d u l a t i o n s o f a c t i v i t y c a u s e d by p h y s i o l o g i c a l s u b s t r a t e s , such a s i n t r a c e l l u l a r N a + and ATP, t h e i n t e r a c t i o n s of i n t r a c e l l u l a r K+ a t t h e Na+ b i n d i n g s i t e (Sachs, 1981) , C a 2 + dependent c y c l i c GMP e f f e c t s ( S t e w a r t and Sen, 19811, and any o t h e r f a c t o r s t h a t may a f f e c t t h e pumping r a t e of t h e enzyme. Well-known examples of s h o r t - t e r m regul a t i o n a r e t h e enhanced pump r a t e p e r s i t e i n c e l l s t h a t have been Na+-loaded by t r e a t m e n t w i t h c o l d ( P o s t and J o l l y , 1957; P o l l a c k e t a l . , 1981a1, monensin (Smith and A u s t i c , 19801, o r i n r e s p o n s e t o mitogen s t i m u l a t i o n (Rozengurt and Mendoza, 1 9 8 0 ) . S i n c e t h e c y t o s o l i c Na+ c o n c e n t r a t i o n i n most c e l l s i s n e a r t h e K~ f o r N a + stimul a t i o n o f t h e pump, any f a c t o r t h a t e l e v a t e s i n t r a c e l l u l a r N a + , i n c l u d i n g a l l o f t h e above, w i l l i n c r e a s e pump a c t i v i t y p r o v i d e d o t h e r f a c t o r s s u c h a s t h e ATP s u p p l y do n o t become l i m i t i n g . Long-term r e g u l a t i o n , which i s t h e p r i n c i p a l concern of t h i s d i s c u s s i o n , r e f e r s t o a change i n t h e a c t u a l number o f f u n c t i o n a l enzyme molec u l e s . Where t h e y have been measured, t h e p r o p e r t i e s of t h e up--or down-regulated enzyme a r e t h e same as t h e enzyme i n t h e c o n t r o l s t a t e , and c o n s e q u e n t l y t h e a c t i v i t i e s o f t h e Na,K-ATPase are s t i l l s u b j e c t t o t h e same f a c t o r s t h a t mediate short-term responses. A s d e s c r i b e d above, such s h o r t - t e r m m o d u l a t i o n app e a r s t o be r e s p o n s i b l e f o r t h e c e l l - c y c l e - a s s o c i a t e d f l u c t u a t i o n s i n pumping a c t i v i t y and K+ c o n t e n t o b s e r v e d i n neuroblastoma c e l l s (Mummery e t al., 1 9 8 1 ) .

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A.

S T U D I E S ON C U L T U R E D C E L L S

T e n y e a r s a g o , J . F . Lamb and h i s a s s o c i a t e s showed t h a t when a v a r i e t y o f c u l t u r e d c e l l s were grown i n media e i t h e r low i n K+ or c o n t a i n i n g s u b l e t h a l concent r a t i o n s of ouabain, t h e c e l l s responded w i t h an i n c r e a s e i n t h e numbers of o u a b a i n - b i n d i n g s i t e s and w i t h enhanced Na,K-ATPase a c t i v i t y and t r a n s p o r t c a p a c i t y (Lamb and McCall, 1 9 7 2 ; Boardman e t al., 1 9 7 2 ) . W e have conf i r m e d t h e s e o b s e r v a t i o n s (Cook e t a l . , 1 9 7 6 ; P o l l a c k e t al., 1 9 8 1 a , b , 1 9 8 2 ) . B a s i c a l l y , what i s o b s e r v e d i s t h a t when c e l l s (HeLa i n o u r e x p e r i m e n t s ) a r e rown i n a medium w i t h K + a t o r j u s t below t h e K, f o r Kq s t i m u l a t i o n o f t h e pump, t h e c e l l s l o s e a f r a c t i o n of t h e i r i n t e r n a l K + and g a i n i n t r a c e l l u l a r N a + . I n t h e f i r s t few h o u r s of K + d e p l e t i o n , t h e number o f o u a b a i n - b i n d i n g s i t e s p e r c e l l c h a n g e s only s l i g h t l y , b u t t h e pump a c t i v i t y p e r s i t e r i s e s t o a b o u t 0 . 8 vmax when a s s a y e d und e r normal K+ c o n d i t i o n s . This appears t o be a c l a s s i c a l short-term response: enhanced a c t i v i t y p e r s i t e a s a consequence o f t h e e l e v a t e d i n t e r n a l N a + . Although t h e c e l l s might s u r v i v e i n d e f i n i t e l y i n t h i s way, t h e t r a n s p o r t system i s o p e r a t i n g so c l o s e t o i t s maximum c a p a c i t y t h a t there i s very l i t t l e f l e x i b i l i t y f o r response t o worsening c o n d i t i o n s . A f t e r a n o t h e r 20-30 h r i n lowK+ medium, i t i s found t h a t t h e number o f o u a b a i n b i n d i n g s i t e s p e r c e l l h a s d o u b l e d , and t h a t t h e t r a n s p o r t a c t i v i t y when a s s a y e d u n d e r s t a n d a r d c o n d i t i o n s i s back t o 0 . 6 Vmax, t h u s r e s t o r i n g f l e x i b i l i t y . By m a n i p u l a t i n g t h e medium Na+ a s w e l l a s K + , m a i n t a i n i n g o s m o l a r i t y w i t h s o r b i t o l , Lamb's group o b t a i n e d e v i d e n c e t h a t t h e c e l l s were r e s p o n d i n g t o an e l e v a t e d i n t r a c e l l u l a r Na+ r a t h e r t h a n t o a loss of c e l l K+ (Boardman e t al., 1974). Of more p h a r m a c o l o g i c and p o s s i b l y c l i n i c a l i n t e r e s t i s t h e r e s p o n s e t o growth i n low l e v e l s of o u a b a i n . The same k i n d of r e s p o n s e i s o b s e r v e d a s i n low K + ( P o l l a c k e t a l . , 1 9 8 1 a ) , and t h e r e i s no r e a s o n t o bel i e v e t h a t t h e c e l l s ' perception of t h e environmental s t r e s s i s d i f f e r e n t i n t h e two c a s e s . S i n c e t h e doser e s p o n s e c u r v e t o o u a b a i n i s s t e e p , making t h e o u a b a i n experiments d i f f i c u l t t o reproduce q u a n t i t a t i v e l y , and, s i n c e t h e u s e o f o u a b a i n a s a stress c o m p l i c a t e s i t s f u r t h e r u s e a s an a n a l y t i c a l t o o l , o u r e x p e r i m e n t s have been p r i m a r i l y c o n c e r n e d w i t h t h e low-K+ stress. T h i s long-term r e s p o n s e i s o b s e r v e d n o t o n l y by a n i n c r e a s e i n o u a b a i n b i n d i n g p e r c e l l . Lamb's g r o u p found p a r a l l e l i n c r e a s e s i n o u a b a i n - s e n s i t i v e e x t r u s i o n of N a + and i n Na,K-ATPase a c t i v i t y (Boardman e t a l . , 1 9 7 2 ) , and w e found p a r a l l e l i n c r e a s e s i n o u a b a i n -

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+

s e n s i t i v e Vmax f o r 86Rb u p t a k e and i n K - s e n s i t i v e phosp h o r y l a t i o n of i s o l a t e d membranes. The r e s p o n s e a p p e a r s t o be s p e c i f i c i n t h a t 5 ' - n u c l e o t i d a s e and K+-insensit i v e p h o s p h o r y l a t i o n are u n a l t e r e d i n membranes i s o l a t e d from s t r e s s e d c e l l s ( P o l l a c k e t a l . , 1 9 8 1 a ) , a l t h o u g h t h e Vmax f o r a - a m i n o i s o b u t y r i c a c i d t r a n s p o r t may i n c r e a s e i n p a r a l l e l w i t h t h a t f o r 4 2 K t r a n s p o r t ( J . s. Graves, p e r s o n a l communication). A l l of t h e measurements c i t e d above are c o n s i s t e n t w i t h t h e n o t i o n t h a t t h e amount of enzyme i s doubled i n the s t r e s s e d cells, but they a r e nevertheless a c t i v i t y measurements. The e x p o s u r e o f p r e e x i s t i n g c r y p t i c s i t e s i s n o t r u l e d o u t by them. Hansen e t a l . ( 1 9 7 9 ) have i n f a c t suggested, i n i n t e r p r e t i n g t h e i r s t u d i e s with deterg e n t e f f e c t s o n t h i s enzyme, t h a t s u c h e x p o s u r e o f c r y p t i c s i t e s o r p o s s i b l y changing s t o i c h i o m e t r i e s m i g h t p l a y a p h y s i o l o g i c a l r o l e i n changing a c t i v i t i e s . Poll a c k approached t h i s q u e s t i o n by measuring t h e d e t e r g e n t a c t i v a t i o n o f K + - s e n s i t i v e p h o s p h o r y l a t i o n i n membranes from c o n t r o l and long-term s t r e s s e d c e l l s . The deterg e n t a c t i v a t i o n w a s c l e a r l y demonstrable, b u t occurred t o t h e same e x t e n t i n b o t h p r e p a r a t i o n s . A c t i v a t i o n i n t h i s sense does n o t account f o r t h e r e s u l t s (Pollack et a 1 . , 1981a ,b ) Direct measurements on t u r n o v e r i n s t r e s s e d c e l l s , u s i n g t h e 1 3 C d e n s i t y l a b e l t e c h n i q u e d e s c r i b e d above , confirmed t h a t t h e l e v e l of enzyme p e r c e l l h a s i n c r e a s e d . A f t e r K+-depleted c e l l s had come i n t o a new s t e a d y s t a t e , w i t h t h e number of o u a b a i n - b i n d i n g s i t e s 2 . 2 t i m e s t h a t i n t h e c o n t r o l s b u t w i t h growth r a t e norm a l , t h e t u r n o v e r c o n s t a n t w a s found t o have d e c r e a s e d from 3 . 1 p e r g e n e r a t i o n i n t h e c o n t r o l s t o 1 . 3 p e r gener a t i o n i n t h e low-K+-medium. A s i s e v i d e n t from E q . (1), t h i s d e c r e a s e i n t u r n o v e r c o n s t a n t i s a d e q u a t e t o a c c o u n t f o r a l l of t h e i n c r e a s e i n N a , K - A T P a s e s u r f a c e d e n s i t y . The c a l c u l a t e d r a t e o f s y n t h e s i s i s v i r t u a l l y unchanged i n t h e s e c e l l s , b u t it i s n o t u n t i l t h e number of c o p i e s h a s doubled t h a t t h e t u r n o v e r r a t e a g a i n r e a c h e s a new s t e a d y s t a t e . The c o n c l u s i o n t h a t t h e number of c o p i e s p e r c e l l i s r e g u l a t e d by t u r n o v e r r a t h e r t h a n s y n t h e s i s was s u b s t a n t i a t e d i n experiments on t h e t r a n s i t i o n f r o m t h e stressed s t a t e back t o t h e c o n t r o l s t a t e . When t h e medium K+ w a s r e s t o r e d t o normal l e v e l s ( 5 . 5 m M ) , t h e c e l l K+ r e c o v e r e d w i t h i n m i n u t e s and i n f a c t , s i n c e t h e c e l l s had t w i c e t h e i r u s u a l t r a n s p o r t c a p a c i t y , K+ o v e r s h o t s l i g h t l y . With t h e stress r e l i e v e d , t h e number o f ouabain binding sites p e r c e l l s t a r t e d t o f a l l almost immediately. There w a s no t r a n s i t t i m e i n t h i s f a l l - an o b s e r v a t i o n c o n s i s t e n t w i t h t h e notion t h a t the turn-

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o v e r c o n s t a n t had r e t u r n e d t o i t s normal v a l u e and t h a t Na,K-ATPase was being removed from t h e s u r f a c e a t t h e h i g h e r c o n t r o l r a t e s . I f t h e r e g u l a t i o n were on t h e s i d e of s y n t h e s i s , it would have been expected t h a t t h e d e c r e a s e i n ouabain b i n d i n g s i t e s would n o t become app a r e n t u n t i l a l l of t h e membrane s y n t h e s i z e d a t t h e t i m e of K+ r e s t o r a t i o n had been through i t s normal p r o c e s s i n g and had been i n s e r t e d i n t o t h e c e l l s u r f a c e , b u t t h i s w a s n o t observed. The t r a n s i t i o n from t h e h i g h e r l e v e l of Na,K-ATPase surface concentration t o t h e contro l lev el w a s again 5 h r . B e r l i n and Schimke (1965) have shown t h a t h a l f t i m e s f o r such t r a n s i t i o n s correspond t o t h e t u r n o v e r h a l f - t i m e s i n t h e s t a t e t o which t h e t r a n s i t i o n i s movi n g . Since t h e s t a t e t o which t h e c e l l s a r e p r o g r e s s i n g i s the control s t a t e , the r a t e constant f o r the transit i o n should be t h e c o n t r o l t u r n o v e r c o n s t a n t . The h a l f t i m e of 5 h r observed i n t h i s t r a n s i t i o n i s t h u s a t h i r d measurement of t h i s v a l u e , and c o r r e s p o n d s c l o s e l y w i t h t h e p r e v i o u s t w o d e t e r m i n a t i o n s made by o t h e r means. B.

WHOLE ANIMAL STUDIES

Adaptive changes i n Na,K-ATPase a c t i v i t y and/or numb e r s of ouabain-binding s i t e s have been observed i n a v a r i e t y of t i s s u e s i n K+-depleted r a t s . I n one of t h e f i r s t such s t u d i e s , Chan and Sanslone ( 1 9 6 9 ) showed t h a t i n K+-starved animals t h e plasma K+ f e l l t o a b o u t h a l f i t s normal v a l u e w i t h i n a b o u t 2 weeks, and a t about t h e f i f t h week t h e Na,K-ATPase a c t i v i t y i n e r y t h r o c y t e memb r a n e s s t a r t e d t o r i s e . By t h e t e n t h week it was 50-100% h i g h e r t h a n i n t h e c o n t r o l s . I n t r a c e l l u l a r K+ d i d n o t change s i g n i f i c a n t l y . The r e s p o n s e was s p e c i f i c i n t h a t membrane a c e t y l c h o l i n e s t e r a s e was u n a f f e c t e d . The response w a s a l s o slowly r e v e r s i b l e a f t e r normal K+ was res t o r e d t o t h e d i e t . The s i g n a l t o which t h e bone marrow from which t h e s e c e l l s o r i g i n a l l y responded i s n o t known. Using a similar e x p e r i m e n t a l p r o t o c o l f o r K+ d e p l e t i o n , Garg et a l . , ( t h i s volume) i n v e s t i g a t e d t h e Na,KATPase a c t i v i t y i n 1 0 s e p a r a t e segments of t h e r a t nephron (Garg e t a l . , 1 9 8 1 ) . The o n l y one t o show s i g n i f i c a n t change w a s t h e c o r t i c a l c o l l e c t i n g d u c t , where t h e Na,K-ATPase a c t i v i t y f e l l t o a t h i r d of t h e c o n t r o l v a l u e . This segment i s a l s o f u n c t i o n a l l y i m p o r t a n t i n Na+ r e a b s o r p t i o n and K+ s e c r e t i o n , and t h e change a g a i n a p p e a r s t o be a d a p t i v e l y i m p o r t a n t i n combating t h e lowK+ stress. S k e l e t a l muscle K+ f a l l s d r a m a t i c a l l y i n K"-depleted r a t s , and w i t h it t h e number of ouabain-binding s i t e s i n

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t h i s t i s s u e (Ngfrgaard e t a l . , 1 9 8 1 ) . T h e number of ouabain-binding s i t e s a f t e r 8 weeks may f a l l t o a t h i r d o r less of t h e c o n t r o l v a l u e . The d e c r e a s e d pumping c a p a c i t y and concomitant l o s s of K+ from muscle a p p e a r t o a c t a s a r e s e r v o i r f o r K+, s e r v i n g t o m a i n t a i n normal e l e c t r o l y t e composition i n o t h e r t i s s u e s whose funct i o n may be more c r i t i c a l t o s u r v i v a l - - h e a r t , l i v e r , and b r a i n , i n which t h e K+ l o s s i s v e r y s m a l l . I t would be i n t e r e s t i n g t o know w h e t h e r , i n t h e f a c e of t h e d r a s t i c a l l y reduced plasma K+, t h e numbers of ouabain-bindi n g s i t e s i n t h e s e t i s s u e s had a c t u a l l y i n c r e a s e d . The p i c t u r e t h a t emerges from t h e s e s t u d i e s i s one of t i s s u e - s p e c i f i c a d a p t i v e r e s p o n s e s t h a t may i n v o l v e e i t h e r i n c r e a s e d o r d e c r e a s e d s p e c i f i c a c t i v i t y of Na,K-ATPase. There i s some s u g g e s t i o n (Ngfrgaard e t a l . , 1 9 8 1 ) t h a t t h e r e s p o n s e s may be hormonally m e d i a t e d , b u t t h i s i s s t i l l very unclear. Certainly t h e c e l l s i n cult u r e are n o t responding t o a change i n hormonal s t i m u l i , b u t t h e i n c r e a s e d Na,K-ATPase a c t i v i t y i n K+-starved c u l t u r e d c e l l s i s n o t s e e n u n t i l t h e e x t r a c e l l u l a r K+ i s reduced t o l e v e l s w e l l below t h o s e of t h e plasma i n K+d e p l e t e d animals. The two e x p e r i m e n t a l c o n d i t i o n s may n o t be d i r e c t l y comparable. Among t h e most d r a m a t i c p h y s i o l o g i c a l examples of r e s p o n s e s t o s a l t l o a d i n g and s a l t d e p r i v a t i o n a r e t h o s e t o be found i n t h e e x t r a r e n a l o r g a n s of animals moving between marine and f r e s h w a t e r environments, and a n i m a l s , e s p e c i a l l y marine b i r d s , i n g e s t i n g h i g h s a l t l o a d s i n t h e i r d i e t . A v a r i e t y of such o r g a n s ( g i l l s and r e c t a l glands i n f i s h , n a s a l glands i n b i r d s , lacrymal glands i n r e p t i l e s ) f u n c t i o n t o m a i n t a i n a plasma o s m o l a r i t y t h a t may be widely d i f f e r e n t from t h e environment o r below t h a t of i n g e s t e d f l u i d s . The comparative a s p e c t s of t h e s e o r g a n s have been r e c e n t l y reviewed ( K i r s c h n e r , 1 9 8 0 ; Peaker and L i n z e l l , 1 9 7 5 ) . Of p a r t i c u l a r i n t e r e s t t o t h i s a r t i c l e are t h e o b s e r v a t i o n s t h a t t h e amount of Na,K-ATPase i n t h e s e organs i s r e g u l a t e d i n r e s p o n s e t o changing environments. E p s t e i n e t a l . ( 1 9 6 7 ) observed t h a t t h e a c t i v i t y of t h e enzyme i s 7 - f o l d h i g h e r i n t h e g i l l s of t h e k i l l i f i s h , F u n d u l u s h e t e r o c l i t u s , a d a p t e d t o s e a w a t e r when compared t o t h e a c t i v i t y i n specimens adapted t o f r e s h w a t e r . Hypophysectomized F u n d u l u s I which a r e c a p a b l e of s u r v i v a l i n s e a w a t e r , showed only a b o u t h a l f of t h e r e s p o n s e . A t a b o u t t h e same t i m e , E r n s t e t a l . ( 1 9 6 7 ) demonstrated a s i m i l a r i n c r e a s e i n Na,K-ATPase i n t h e n a s a l g l a n d s of Pekin d u c k l i n g s g i v e n a regimen of N a C l i n t h e i r d r i n k i n g w a t e r . T h i s p r e p a r a t i o n h a s been p a r t i c u l a r l y w e l l s t u d i e d and, a s a p r o t o t y p e of s a l t - e x c r e t i n g o r g a n s , w i l l be t h e f o c u s of t h e remainder of t h i s s e c t i o n .

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The s e c r e t i o n of h y p e r t o n i c s a l i n e by t h i s gland has l o n g been known t o be under c h o l i n e r g i c c o n t r o l . S t e w a r t and Sen (1981) have proposed a model i n v o l v i n g t h e Ca2+-dependent , c y c l i c GMP-mediated a c t i v a t i o n of t h e Na,K-ATPase i n r e s p o n s e t o c h o l i n e r g i c s t i m u l a t i o n . As t h e a u t h o r s p o i n t o u t , t h i s i s a s h o r t - t e r m r e g u l a t i o n , and prolonged s a l t stress l e a d s t o b o t h hypert r o p h y of t h e organ and i n c r e a s e d s p e c i f i c a c t i v i t y of Na,K-ATPase. How t h e enzyme f u n c t i o n s i n c o n t r o l l i n g s e c r e t i o n i s a s e p a r a t e i s s u e , and f o r models of t h e mechanism t h e r e a d e r i s r e f e r r e d t o d i s c u s s i o n s by E l l i s et a l . ( 1 9 7 7 ) , E r n s t and Mills ( 1 9 7 7 ) , and Peaker (1978) E r n s t and E l l i s (1969) have d e s c r i b e d t h e c y t o l o g y of t h e n a s a l g l a n d . The c e l l s a r e grouped i n l o b u l e s w i t h a c e n t r a l lumen. The a p i c a l s u r f a c e , exposed t o t h e lumen, i s always r e l a t i v e l y s m a l l . With s a l t loadi n g , t h e weight of t h e g l a n d i n d u c k l i n g s doubled i n about 5 d a y s , i n c r e a s i n g i n mass a t a g r e a t e r r a t e t h a n t h e o v e r a l l growth of t h e b i r d . A t t h e same t i m e t h e r e was an e x t e n s i v e i n c r e a s e i n t h e s u r f a c e a r e a of t h e b a s o l a t e r a l membranes of t h e s e c r e t i n g c e l l s , l e a d i n g t o a v e r y complex p a t t e r n of i n v a g i n a t i o n s , e v a g i n a t i o n s , and i n t e r d i g i t a t i o n s between c e l l s . S i m u l t a n e o u s l y , t h e c e l l complement of m i t o c h o n d r i a was a l s o g r e a t l y i n c r e a s e d , many of t h e s e m i t o c h o n d r i a being found i n t h e f o l d s of t h e b a s o l a t e r a l membranes. I n l a t e r cytochemic a l s t u d i e s , K+-dependent p - n i t r o p h e n y l phosphatase act i v i t y and s p e c i f i c [3H]ouabain-binding s i t e s were t o be found o n l y on t h e b a s o l a t e r a l membranes; no such act i v i t i e s could be demonstrated on t h e a p i c a l s u r f a c e s of t h e s e c r e t i n g c e l l s ( E r n s t , 1 9 7 2 ; E r n s t and M i l l s , 1 9 7 7 ) There was a g r a d i e n t of ouabain-binding a c t i v i t y i n g l a n d s from s a l t - s t r e s s e d b i r d s , t h e g r e a t e s t a c t i v i t y being found deep i n t h e c e n t e r of t h e l o b u l e s . S i m i l a r r e s u l t s were d e s c r i b e d by H o s s l e r et a l . ( 1 9 7 8 ) . Concomitant w i t h t h i s e x t e n s i v e b i o g e n e s i s of basol a t e r a l membranes, and t o a lesser e x t e n t c e l l p r o l i f e r a t i o n , i s an i n c r e a s e i n Na,K-ATPase s p e c i f i c a c t i v i t y , which i n c r e a s e s 4- t o 5-fold i n a b o u t 9 days ( E r n s t e t a l . , 1 9 6 7 ; H o s s l e r et a l . , 1 9 7 8 ) . O f s p e c i a l i n t e r e s t i n both these s t u d i e s i s t h e observation t h a t the i n c r e a s e d membrane b i o g e n e s i s a p p e a r s t o i n v o l v e s p e c i f i c a l l y membranes r i c h i n Na,K-ATPase. Other membranebound enzymes, Mg-ATPase and 5 ' - n u c l e o t i d a s e , i n c r e a s e d t o a much s m a l l e r d e g r e e , i . e . , less t h a n 2-fold. These r e s p o n s e s were r e v e r s i b l e i n b o t h s t u d i e s c i t e d . When s a l t - s t r e s s e d d u c k l i n g s w e r e g i v e n o n l y freshwater, t h e Na,K-ATPase s p e c i f i c a c t i v i t y f e l l log a r i t h m i c a l l y t o c o n t r o l l e v e l s w i t h a h a l f - t i m e of app r o x i m a t e l y 5 d a y s . T h i s h a l f - t i m e may r e p r e s e n t t h e

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t u r n o v e r r a t e of n a s a l g l a n d membrane i n u n s t r e s s e d animals. S t e w a r t e t a l . ( 1 9 7 6 ) approached t h e q u e s t i o n o f whether t h e r e was i n d e e d more enzyme i n s a l t - s t r e s s e d a n i m a l s o r whether p r e e x i s t i n g enzyme was b e i n g a c t i vated. By i s o l a t i n g t h e c a t a l y t i c s u b u n i t , t h e y were a b l e t o show t h a t t h i s p r o t e i n i n c r e a s e d commensuratel y w i t h t h e a c t i v i t y o f t h e enzyme a f t e r s a l i n e t r e a t ment, and concluded t h a t t h e i n c r e a s e d a c t i v i t y w a s due t o d e n o v o s y n t h e s i s of t h e enzyme. I n a n e x t e n s i o n of t h i s s t u d y , t h i s group (Lingham e t a l . , 1980) measured p r o t e i n s y n t h e s i s i n s a l t g l a n d s l i c e s by a d o u b l e l a b e l t e c h n i q u e . A f t e r a s l i t t l e a s 2 4 h r of s a l i n e t r e a t m e n t of whole b i r d s , s l i c e s from t h e i s o l a t e d g l a n d s showed a p r e f e r e n t i a l i n c o r p o r a t i o n of l e u c i n e i n t o p r o t e i n s of M r 9 6 , 0 0 0 and 5 4 , 0 0 0 , t a k e n t o be t h e c a t a l y t i c and g l y Acc o p r o t e i n s u b u n i t s , r e s p e c t i v e l y , o f Na,K-ATPase. c e p t i n g t h i s i d e n t i f i c a t i o n of t h e p r o t e i n , t h i s s t u d y s u p p o r t s t h e n o t i o n t h a t Na,K-ATPase i s b e i n g more o r less s p e c i f i c a l l y induced i n r e s p o n s e t o t h e s a l t l o a d . A s n o t e d above, t h e e n d o c r i n e system may p l a y a n import a n t r o l e i n t h e i n d u c t i o n , b u t t h e d e t a i l s do n o t app e a r t o have been worked o u t . A d i f f i c u l t y i n q u a n t i t a t i v e biochemical s t u d i e s w i t h t h e s a l t g l a n d h a s been t h e poor v a s c u l a r i z a t i o n i n c o n t r o l a n i m a l s and t h e problem o f d i f f u s i o n of rad i o a c t i v e p r e c u r s o r s i n t o t h e complex t i s s u e of i s o l a t e d o r g a n s . A s i n t h e work of S e n ' s group (Lingham c t a l . , 1 9 8 0 ) , s l i c e s have been u s e f u l i n r e l a t i v e l y s h o r t - t e r m s t u d i e s . O t h e r u s e f u l a p p r o a c h e s have been t h e i s o l a t i o n o f d i s s o c i a t e d c e l l s (Hootman and E r n s t , 1980) o r of " m i n i l o b u l e s " t h a t c a n be m a i n t a i n e d i n o r g a n c u l t u r e f o r s e v e r a l d a y s (Mazurkiewicz and B a r r n e t t , 1 9 8 1 ) . The l a t t e r a u t h o r s , using s p e c i f i c immunoprecipitation techn i q u e s , have shown t h a t i n " m i n i l o b u l e s " from s a l t s t r e s s e d b i r d s up t o 1 0 % of i n c o r p o r a t e d l a b e l e d l e u c i n e c o u l d be found i n Na,K-ATPase. S i n c e t h e induced res p o n s e s a p p e a r t o p e r s i s t i n t i s s u e s i s o l a t e d from t h e donor b i r d , t h e s e approaches a r e v e r y p r o m i s i n g . A v e r y d e s i r a b l e g o a l , n o t y e t a c h i e v e d , i s t h e i n d u c t i o n of c o n t r o l glands i n v i t r o .

IV.

EFFECTS O F HOREIONES ON LONG-TERM REGULATION

N a , K - A T P a s e i s modulated by a v a r i e t y o f hormones, i n c l u d i n g o v a r i a n and a d r e n a l s t e r o i d s , p e p t i d e hormones, and n e u r o t r a n s m i t t e r s . I n s u l i n (Czech, 1977;

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Fehlman and F r e y c h e t , 19811, glucagon (Fehlman and F r e y c h e t , 19811, and v a s o p r e s s i n (Mendoza e t a l . , 1980) a p p e a r t o s t i m u l a t e t h e sodium pump by e l e v a t i n g t h e i n t r a c e l l u l a r N a + c o n c e n t r a t i o n ( a s do a v a r i e t y of o t h e r treatments, i n c l u d i n g serum, m i t o g e n i c l e c t i n s and tumor promotors; f o r a r e v i e w , see Rozengurt and Mendoza, 1 9 8 0 ) . Catecholamines, s p e c i f i c a l l y n o r e p i n e p h r i n e , enhances Na,K-ATPase a c t i v i t y (Swann e t a l . , 1981; a l s o Swann, t h i s volume) and have been s u g g e s t e d t o a c t by t h e c h e l a t i o n o f e i t h e r i r o n ( S c h a e f e r e t ai., 1974) o r v a n a d a t e ( C a n t l e y e t a l . , 19781, o r by a c y c l i c AMP-independent a d r e n e r g i c r e c e p t o r mechanism (Wu and P h i l l i s , 1 9 8 0 ) . A l l of t h e s e may be c l a s s i f i e d a s s h o r t - t e r m r e g u l a t o r y e f f e c t s . Only t h e t h y r o i d hormones and a d r e n a l s t e r o i d s have been i m p l i c a t e d i n modul a t i n g d i r e c t l y t h e b i o s y n t h e s i s of t h e enzyme; t h e s e are d e a l t w i t h h e r e i n more d e t a i l . A.

T H Y R O I D HORMONES

The t h y r o i d hormones t h y r o x i n e ( T 4 ) and t r i i o d o t h y r o n i n e (T3) have l o n g been known t o r e g u l a t e oxygen consumption ( Q 1 and, c o r r e s p o n d i n g l y , t h e r m o g e n e s i s Treatment o f e i t h e r i n homoiothermP8 v e r t e b r a t e s . e u t h y r o i d o r h y p o t h y r o i d r a t s w i t h T3 i n c r e a s e s Q O ~i n a v a r i e t y of t i s s u e s , namely l i v e r ( I s m a i l - B e i g i et a l . , 1 9 7 9 ; I s m a i l - B e i g i and Edelman, 1970, 1 9 7 1 , 1 9 7 4 ; Somjen e t a l . , 19811, kidney ( I s m a i l - B e i g i and Edelman, 1 9 7 1 ; Katz and Genant, 1 9 7 1 ) , and s k e l e t a l muscle (Asano et a l . , 1976; I s m a i l - B e i g i and Edelman, 19701, w h i l e having no e f f e c t o n b r a i n r e s p i r a t i o n (IsmailB e i g i and Edelman, 1 9 7 1 ) . Na,K-ATPase meets t h e c r i t e r i a f o r b e i n g t h e a c t i v e c e l l u l a r component o f h e a t g e n e r a t i o n , b e i n g u b i q u i t o u s i n t a r g e t c e l l s and a b l e t o h y d r o l y z e s u f f i c i e n t ATP t o a f f e c t r e s p i r a t i o n ( I s m a i l - B e i g i and Edelman, 1 9 7 0 ) . L a r g e l y due t o t h e e f f o r t s o f Edelman and h i s c o l l e a g e s , t h e enzyme h a s been i d e n t i f i e d as a major i n s t r u m e n t i n t h y r o i d hormone a c t i o n . A s i g n i f i c a n t p o r t i o n o f t h e hormone-induced i n c r e a s e i n 902 was found t o be s e n s i t i v c t o i n h i b i t i o n by o u a b a i n (Asano e t al., 1976; IsmailB e i g i et a l . , 1979; I s m a i l - B e i g i and Edelman, 1 9 7 0 , 1971 1 9 7 4 ; Somjen e t al., 1 9 8 1 ) . T h i s e f f e c t i s e s p e c i a l l y a p p a r e n t i n t h e l i v e r o f b o t h e u t h y r o i d and h y p o t h y r o i d r a t s where o u a b a i n p r e v e n t s g r e a t e r t h a n 90% o f t h e T 3 - s p e c i f i c i n c r e a s e i n oO2 ( I s m a i l - B e i g i and Edelman, 1 9 7 0 , 1 9 7 1 , 1 9 7 4 ) . The Na,K-ATPase-dependent i n c r e m e n t s of Q~~ i n r a t s k e l e t a l muscle and k i d n e y a r e less e v i dent.

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A n a l y s i s of o u a b a i n - s e n s i t i v e ATP h y d r o l y s i s i n these t i s s u e s h a s r e v e a l e d a patte-:n s i m i l a r t o t h a t o f

oxygen consumption. Na,K-ATPase a c t i v i t y , f o l l o w i n g t h y r o i d e c t o m y , i s d e c r e a s e d i n k i d n e y ( I s m a i l - B e i g i and Edelman, 1971; Katz and Lindheimer, 1973; Somjen e t a l . , 19811, l i v e r (Somjen et a l . , 19811, and s k e l e t a l muscle (Asano e t a l . , 1 9 7 6 ) . The b r a i n enzyme i s u n a f f e c t e d ( I s m a i l - B e i g i and Edelman, 1 9 7 1 ) . Exogenous T3, when administered t o e i t h e r hypothyroid o r euthyroid r a t s , i n c r e a s e s Na,K-ATPase a c t i v i t y i n t h e t a r g e t t i s s u e s . The n a t u r e of t h e change i n Na,K-ATPase remained t o be e l u c i d a t e d , i . e . , i n c r e a s e d a c t i v i t y o f t h e e x i s t i n g enzyme v i s - a - v i s i n c r e a s e d enzyme c o n c e n t r a t i o n . Asano et a l . ( 1 9 7 6 ) a n a l y z e d s k e l e t a l muscle from hypot h y r o i d r a t s and found t h a t T3 t r e a t m e n t e l i c i t e d an i n Lo e t c r e a s e i n Vmax(ATP) w i t h no change i n K m ( A T P ) a l . (1976) c h a r a c t e r i z e d a v a r i e t y of p a r a m e t e r s of Na,K-ATPase i n r e n a l t i s s u e from h y p o t h y r o i d r a t s , rep o r t i n g t h a t a p u l s e o f T3 i n c r e a s e d s u b s t a n t i a l l y t h e s p e c i f i c a c t i v i t y of microsomal N a , K - A T P a s e from t h e c o r t e x b u t i n c r e a s e d it o n l y s l i g h t l y i n microsomes from t h e m e d u l l a ; p a p i l l a r y t i s s u e w a s u n r e s p o n s i v e . With c h r o n i c T3 t r e a t m e n t o n l y t h e c o r t i c a l enzyme responded w i t h i n c r e a s e d a c t i v i t y . I n a n a n a l y s i s o f t h e k i n e t i c s o f r e n a l c o r t i c a l Na,K-ATPase i n h y p o t h y r o i d a n i m a l s t h e y found t h a t T3 i n c r e a s e d t h e vmax f o r ATP, Na+, and K+, w i t h o u t a concomitant a l t e r a t i o n i n t h e K~ f o r ATP o r t h e K ~ €o . r ~N a + and K+. Two o t h e r i n d i c a t o r s o f enzyme number, t3H]ouabain b i n d i n g and K+s e n s i t i v e p h o s p h o r y l a t i o n , were i n c r e a s e d by a magnitude similar t o t h e s p e c i f i c a c t i v i t y a f t e r T3 treatment. T3-induced i n c r e a s e s i n [3H]ouabain b i n d i n g were a l s o r e p o r t e d by L i n and Akera (1978) i n l i v e r and muscle, a s w e l l a s i n k i d n e y , and as w i t h a c t i v i t y measurements, b i n d i n g l e v e l s were unchanged i n b r a i n tissue. Although t h e s e d a t a imply an i n c r e a s e i n t h e numb e r of enzyme m o l e c u l e s , hormone-induced e x p o s u r e o f l a t e n t Na,K-ATPase i s a l s o p o s s i b l e . Evidence t h a t t h i s i s n o t o c c u r r i n g comes from o b s e r v a t i o n s t h a t T3-induced i n c r e a s e s i n [3H]ouabain b i n d i n g ( L i n and Akera, 1978) and o u a b a i n - s e n s i t i v e ATP h y d r o l y s i s (Lo and Lo, 1 9 7 9 ) are r e t a i n e d when a s s a y e d i n t h e p r e s e n c e of d e t e r g e n t s so a s t o p r o v i d e maximum a c c e s s i b i l i t y of l i g a n d o r subs t r a t e t o c r y p t i c s i t e s . Another p o s s i b i l i t y i s t h a t T3 i s e f f e c t i n g an i n c r e a s e i n a l l enzymes of t h e plasma membrane ( i . e . , i n d u c i n g a g e n e r a l i z e d membrane s y n t h e s i s ) . T h i s i s r u l e d o u t by t h e r e p o r t e d l a c k of T3 e f f e c t s on t h e o t h e r marker enzymes of t h e plasma membrane, s u c h a s Mg-ATPase ( I s m a i l - B e i g i e t a l . , 1 9 7 9 ; Ismail-

.

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B e i g i and Edelman, 1971; K a t z and Lindheimer, 1973; Lo et a 2 . , 1976; L o and Lo, 1981) , 5'-nucleotidase ( I s m a i l B e i g i and Edelman, 1971; K a t z and Lindheimer, 1 9 7 3 ) , and a d e n y l a t e c y c l a s e (Levey et al., 1 9 6 9 ) . To d e t e r m i n e t h e b a s i s f o r i n c r e a s e d enzyme concent r a t i o n , t h e s y n t h e s i s and t u r n o v e r r a t e s o f r e n a l cort i c a l Na,K-ATPase were d e t e r m i n e d . To t h i s e n d , r a t s were p l a c e d on a T 3 regimen which i n c r e a s e d t h e N a , K A T P a s e c o n c e n t r a t i o n t o a new s t e a d y - s t a t e l e v e l (Lo and Edelman, 1 9 7 6 ) . M e t a b o l i c a l l y d o u b l e - l a b e l e d N a , K )ATPase w a s p a r t i a l l y p u r i f i e d and a n a l y z e d by p o l y a c r y l amide g e l e l e c t r o p h o r e s i s (Lo and Edelman, 1976; Lo and Lo, 1 9 8 0 ) . The a - s u b u n i t w a s i d e n t i f i e d on t h e g e l s by K + - s e n s i t i v e p h o s p h o r y l a t i o n by 32P ATP (L o and Edelman, 1976; Lo and L o , 1980) and t h e a - s u b u n i t by e i t h e r PAS s t a i n i n g (Lo and Edelman, 1976) o r r e d u c t i o n w i t h [3H]NaBHq (Lo and Lo, 1 9 8 0 ) . The s u b u n i t bands were judged by Ferguson a n a l y s i s t o be homogeneous (Lo and Edelman, 1976)--a n e c e s s a r y p r o v i s i o n f o r t h e s e t u r n o v e r measurements. These e x p e r i m e n t s a l l o w e d d e t e r m i n a t i o n o f d e g r a d a t i o n r a t e c o n s t a n t s f o r e a c h s u b u n i t and revealed t h a t t h e degradation of n e i t h e r s u b u n i t w a s a f f e c t e d by T3 t r e a t m e n t , i n d i c a t i n g i n c r e a s e d s y n t h e s i s of b o t h s u b u n i t s (Lo and Lo, 1 9 8 0 ) . These d a t a w e r e s y s t e m a t i c a l l y shown t o be i n d e p e n d e n t of a r t i f a c t s due t o p o s s i b l e hormonal i n f l u e n c e on enzyme p u r i f i c a t i o n , r e c o v e r y , o r l a t e n c y ( L o and Lo, 1 9 8 1 ) . The o b s e r v e d e f f e c t s of T3 on b o t h Qo2 and N a , K A T P a s e were i n i t i a l l y a t t r i b u t e d t o d i r e c t hormonal act i o n on t a r g e t c e l l s ( I s m a i l - B e i g i and Edelman, 1970, 1 9 7 1 ) . T h i s view was c h a l l e n g e d i n l i g h t of e v i d e n c e by Katz and co-workers (Katz and Genant, 1971; Katz and Lindheimer, 1973) t h a t r e n a l Na,K-ATPase a c t i v i t y c o u l d be i n c r e a s e d i n h y p o t h y r o i d a n i m a l s by t r e a t m e n t s which i n c r e a s e N a + a b s o r p t i o n . Enzyme a c t i v i t y , d e c r e a s e d by ~ 2 5 %i n b o t h t h e c o r t e x and medulla by t h y r o i d e c t o m y , was r e s t o r e d t o c o n t r o l l e v e l s by p a r t i a l nephrectomy o r t h e a d m i n i s t r a t i o n o f m e t h y l p r e d n i s o l o n e ( K a t z and Lindheimer, 1 9 7 3 ) . I n f u s i o n of i s o t o n i c s a l i n e i n t o r a t s i n c r e a s e d b o t h Na,K-ATPase and Mg-ATPase i n microsomes from r e n a l medulla b u t n o t t h e c o r t e x ( K a t z and Genant, 1 9 7 1 ) . Furthermore, T3 added d i r e c t l y t o r e n a l microsomes i n c o n c e n t r a t i o n s up t o 10-5 M had no e f f e c t on Na,K-ATPase (Katz and Lindheimer, 1 9 7 3 ) . Taken t o g e t h e r , these data indicated t o the authors t h a t r e n a l Na,KA T P a s e was a l t e r e d i n h y p o t h y r o i d rats p r i m a r i l y by t u b u l a r sodium r e a b s o r p t i o n and o n l y s e c o n d a r i l y by T3. Many r e p o r t s have s u b s e q u e n t l y shown t h a t , w h i l e n o t a c t i n g d i r e c t l y on t h e p r o t e i n i t s e l f , T3 i n c r e a s e s Na,K-ATPase t h r o u g h d i r e c t i n t e r a c t i o n w i t h t a r g e t c e l l s .

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T h a t t h e enzyme i s a l t e r e d by t h y r o i d e c t o m y and T3 treatment i n nonrenal tissues--i.e., l i v e r (IsmailB e i g i e t al., 1979; I s m a i l - B e i g i and Edelman, 1 9 7 0 , 1971, 1974; Somjen e t a l . , 1981) and s k e l e t a l muscle (Asano et al., 1976; I s m a i l - B e i g i and Edelman, 1970)-i m p l i e s a mechanism i n d e p e n d e n t of r e n a l sodium a d s o r p tion. Whether T3 and sodium r e a b s o r p t i o n a f f e c t d i f f e r e n t regions of t h e kidney i s unclear. While Lo e t a 1 (1976) reported t h a t thyroid s t a t u s influenced c o r t i c a l Na,KA T P a s e t o a g r e a t e r e x t e n t t h a n m e d u l l a r y enzyme, e q u a l r e s p o n s e s t o thyroidectomy (Katz and Lindheimer, 1973) and T3 t r e a t m e n t ( S i l v a e t a l . , 1976) have been rep o r t e d . S a l i n e l o a d i n g seems t o i n c r e a s e Na,K-ATPase o n l y i n t h e r e n a l medulla (Katz and Genant, 1 9 7 1 ) , whereas p a r t i a l nephrectomy o r m e t h y l p r e d n i s o l o n e a l t e r s b o t h c o r t i c a l and m e d u l l a r y a c t i v i t y (Katz and Lindh e i m e r , 1973; S i l v a e t a ~ . , 1 9 7 6 ) . The c l e a r e s t e v i d e n c e f o r a d i r e c t a c t i o n o f T3 on i t s t a r g e t t i s s u e s comes from e x p e r i m e n t s where changes i n r e n a l Na,K-ATPase have been d i s s o c i a t e d from t u b u l a r sodium u p t a k e . S i l v a e t a l . (1976) found t h a t T3 adm i n i s t e r e d on a l t e r n a t e d a y s augmented Na,K-ATPase act i v i t y i n b o t h t h e c o r t e x and medulla o f t h e r a t k i d n e y . The same dosage f a i l e d t o a l t e r t h e g l o m e r u l a r f i l t r a t i o n r a t e (measured by i n u l i n c l e a r a n c e ) and sodium uptake. S i m i l a r l y , Lo and Lo ( 1 9 7 9 ) found t h a t plasma sodium l e v e l s were u n a l t e r e d i n r a t s a f t e r thyroidectomy and were t h e same i n h y p o t h y r o i d r a t s w i t h and w i t h o u t exogenous T3. They a l s o a n a l y z e d t h e t i m e c o u r s e of T3 a c t i o n i n h y p o t h y r o i d r a t s and found t h a t , a l t h o u g h Na,K-ATPase a c t i v i t y i n t h e r e n a l c o r t e x i n c r e a s e d p r o g r e s s i v e l y a t 24 and 48 h r , n e i t h e r t h e r a t e o f sodium f i l t r a t i o n nor i n u l i n clearance increased u n t i l 72 h r a f t e r t h e hormone dose. R o s s i e r and h i s c o l l e a g u e s (1979a,b) have r e p o r t e d t h a t t h y r o i d hormones a l o n e f a i l t o i n d u c e Na,K-ATPase i n v a r i o u s t o a d t i s s u e s , and s p e c u l a t e t h a t t h y r o i d involvement may be an a d a p t a t i o n r e l a t e d t o t h e r m o g e n e s i s found o n l y i n homoiothermic a n i m a l s . Geering e t a l . ( t h i s volume) r e p o r t e d , however, t h a t T3 and a l d o s t e r o n e a c t s y n e r g i s t i c a l l y i n t o a d b l a d d e r t o s t i m u l a t e t h e s y n t h e s i s of t h e Na,K-ATPase a - s u b u n i t , and t h a t a m i l o r i d e t r e a t m e n t does n o t a l t e r t h i s r e s u l t - - a n o t h e r c a s e of t h e d i s s o c i a t i o n o f sodium u p t a k e from hormonal r e g u l a t i o n of Na,K-ATPase. P o s s i b l y t h e most c o n v i n c i n g e v i d e n c e f o r d i r e c t T3 a c t i o n comes from I s m a i l - B e i g i e t a l . ( 1 9 7 9 ) , who found t h a t T3 s t i m u l a t e s b o t h N a , K - A T P a s e and r e s p i r a t i o n i n primary c u l t u r e s of h e p a t o c y t e s from h y p o t h y r o i d r a t s . A s w i t h s t u d i e s on l i v e r i n v i v o , % g o % of t h e Qo2

NORMAN J. KARIN AND JOHN S. COOK

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increase w a s s e n s i t i v e t o ouabain. These e f f e c t s were s e e n under serum-free c u l t u r e c o n d i t i o n s and were i n dependent o f i n s u l i n and c o r t i c o s t e r o n e . While T3 a t M e l i c i t e d a half-maximal e f f e c t on b o t h N a , K 8 x A T P a s e a c t i v i t y and Q n e a r l y 9 0 % o f t h e hormone w a s F u r t h e r , a b r i e f ( 4 h r ) excatabolized within 6 p o s u r e t o T3 i s s u f f i c i e n t t o e l i c i t a s i g n i f i c a n t i n crease i n Na,K-ATPase a c t i v i t y ( 7 5 % of t h a t s e e n a f t e r a 48-hr e x p o s u r e ) . These d a t a imply t h a t T3 may be n e c e s s a r y o n l y b r i e f l y and i n minute amounts t o i n i t i a t e a r e s p o n s e by t h e t a r g e t c e l l s . These hormonal e f f e c t s i n h e p a t o c y t e c u l t u r e s have n o t been o b s e r v e d i n est a b l i s h e d c e l l l i n e s , s u c h as r a t hepatoma c e l l s ( I s m a i l B e i g i et a l . , 1979; N . J . K a r i n and J. S. Cook, unpubl i s h e d o b s e r v a t i o n s ) o r f i b r o b l a s t i c and e p i t h e l i o i d c e l l s d e r i v e d from normal r a t kidney c e l l s ( N . J. K a r i n and J . S. Cook, u n p u b l i s h e d o b s e r v a t i o n s ) . I t i s conc e i v a b l e t h a t among t h e a l t e r a t i o n s i n growth p r o p e r t i e s n e c e s s a r y f o r a c e l l t o p r o l i f e r a t e i n v i t r o i s t h e loss of t h e sodium pump's r e s p o n s i v e n e s s t o t h y r o i d hormones. I n summary, t h e s e r e s u l t s c l e a r l y i n d i c a t e a d i r e c t a c t i o n of T3 on t a r g e t : c e l l s , r e s u l t i n g i n i n c r e a s e d n e t N a , K - A T P a s e s y n t h e s i s and s u b s e q u e n t l y h i g h e r enzyme act i v i t y . T h i s i s i n a c c o r d a n c e w i t h t h e c u r r e n t model o f T3 a c t i o n , i . e . , t h a t t h e hormone b i n d s t o membrane rec e p t o r s and e v e n t u a l l y r e a c h e s s p e c i f i c s i t e s i n t h e n u c l e u s , l e a d i n g t o i n c r e a s e d s y n t h e s i s of mRNA and p r o t e i n (Oppenheimer e t al., 1979; Somjen e t a l . , 1981; T a t a , 1 9 6 8 ) . Indeed, t h e d o s e dependence f o r s a t u r a b l e n u c l e a r b i n d i n g and i n c r e a s e d Na,K-ATPase a c t i v i t y were s i m i l a r i n magnitude i n b o t h l i v e r and k i d n e y , implying a q u a n t i t a t i v e r e l a t i o n s h i p between t h e two e v e n t s (Somjen et a l . , 1 9 8 1 ) .

%!:

B.

ADRENAL STEROIDS

I t i s w e l l e s t a b l i s h e d t h a t sodium u p t a k e through many t r a n s p o r t i n g e p i t h e l i a i s d e c r e a s e d i n a d r e n a l e c tomized a n i m a l s and t h a t t h e a d m i n i s t r a t i o n o f e i t h e r m i n e r a l o c o r t i c o i d s o r g l u c o c o r t i c o i d s can p a r t i a l l y o r t o t a l l y r e s t o r e normal t r a n s p o r t . Na,K-ATPase a c t i v i t y d e c l i n e s f o l l o w i n g adrenalectomy and i s r e s t o r e d by a d r e n a l s t e r o i d s ( C h i g n e l l and T i t u s , 1 9 6 6 ) , and h a s t h e r e f o r e been i m p l i c a t e d i n t h e hormonal a c t i o n on sodium u p t a k e . But d e s p i t e y e a r s of r e s e a r c h by many l a b o r a t o r i e s a c l e a r mechanism remains e l u s i v e . Complic a t i n g t h e r e s o l u t i o n of a r o l e f o r Na,K-ATPase a r e t h e many t y p e s and s o u r c e s of t i s s u e s t u d i e d , a s w e l l a s v a r i a t i o n s i n p r o t o c o l r e g a r d i n g t y p e s of f r a c t i o n a t i o n ,

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t h e p r e s e n c e o r a b s e n c e of d e t e r g e n t d u r i n g f r a c t i o n a t i o n and a s s a y , and hormone d o s a g e - - e s p e c i a l l y i n l i g h t of t h e p o s s i b l e occupancy of g l u c o c o r t i c o i d r e c e p t o r s by a l d o s t e r o n e (Marver, 1 9 8 0 ) . The remainder of t h i s s e c t i o n r e v i e w s work r e l a t i n g t o t h e a c t i o n of t h e s e hormones t o N a , K - A T P a s e b i o s y n t h e s i s . 1.

Mineralocorticoids

The s y n t h e s i s of b o t h RNA and p r o t e i n i s r e q u i r e d f o r a l d o s t e r o n e a c t i o n on sodium u p t a k e (Edelman e t ai., 1963; Law and Edelman, 1 9 7 8 a ) . I n a d d i t i o n , c e r t a i n of these proteins, those destined f o r the cell surface, require f a t t y acid production f o r t h e i r expression ( S c o t t e t a l . , 1 9 7 9 ) . R o s s i e r and co-workers, 1977; R o s s i e r , 19781, s t u d y i n g t h e e f f e c t s o f v a r i o u s RNA synt h e s i s i n h i b i t o r s on toad b l a d d e r , r e p o r t e d t h a t aldos t e r o n e i n d u c e s t h e s y n t h e s i s o f mRNA ( b o t h p o l y ( A ) ( + ) and p o l y ( A ) ( - ) d u r i n g t h e 30- t o 60-min l a t e n t p e r i o d between t h e a d m i n i s t r a t i o n of hormone and t h e o b s e r v e d p h y s i o l o g i c a l e f f e c t . P r e v e n t i o n of t h e i r s y n t h e s i s a b o l i s h e s t h e r e s p o n s e t o a l d o s t e r o n e . 3I-Deoxycytid i n e , a n i n h i b i t o r of rRNA s y n t h e s i s , d o e s n o t a l t e r hormone a c t i o n d u r i n g t h e f i r s t 3 h r , w i t h l a t e r e f f e c t s a t t r i b u t e d t o general t o x i c i t y . A l d o s t e r o n e s t i m u l a t e s t h e s y n t h e s i s of a d i s c r e t e s e t o f p r o t e i n s i n t o a d b l a d d e r , as d e t e r m i n e d by p o l y a c r y l a m i d e g e l a n a l y s i s of s i n g l e o r d o u b l e i s o t o p e l a b e l e d t i s s u e . S c o t t e t al. (1978, 1981) have separ a t e d m i t o c h o n d r i a - r i c h (MR) c e l l s from m i t o c h o n d r i a p o o r g r a n u l a r ( G ) c e l l s of t h e b l a d d e r mucosa and found t h a t t h e a l d o s t e r o n e r e s p o n s e i n v o l v e s MR c e l l s e x c l u s i v e l y . A n a l y s i s o f s u b c e l l u l a r f r a c t i o n s from hormonet r e a t e d and c o n t r o l MR c e l l s i n d i c a t e d t h e e x i s t e n c e o f d i s t i n c t s e t s of a l d o s t e r o n e - i n d u c e d p r o t e i n s (AIPs) i n t h e plasma membranes and c y t o s o l compartments. The f u n c t i o n a l i d e n t i t y of t h e s e p r o t e i n s c a n n o t be ascert a i n e d by e l e c t r o p h o r e t i c m o b i l i t y a l o n e and s u b s e q u e n t l y , these s t u d i e s n e i t h e r r u l e o u t nor v e r i f y Na,KA T P a s e a s a n AIP. I t i s p o s s i b l e t h a t a membranea s s o c i a t e d A I P of 1 1 0 , 0 0 0 d a l t o n s i s t h e a - s u b u n i t of Na,K-ATPase ( S c o t t e t a l . , 1 9 8 1 ) . Biochemical a n a l y s i s o f v a r i o u s a l d o s t e r o n e t r e a t e d t i s s u e s has revealed i n c r e a s e s i n the a c t i v i t y of s e v e r a l enzymes. Toad b l a d d e r and r e n a l c i t r a t e synt h a s e ( K i r s t e n e t a l . , 1968; Law and Edelman, 1978b) , c a r b o n i c a n h y d r a s e i n k i d n e y and i n t e s t i n a l mucosa (Voute e t al., 1975; S u z u k i , 19811, and i n t e s t i n a l MgIHCO3-ATPase ( S u z u k i , 1981) have a l l been i m p l i c a t e d i n t h e a l d o s t e r o n e r e s p o n s e . Cycloheximide p r e v e n t s t h e

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i n d u c t i o n o f t h e l a t t e r two enzymes ( S u z u k i , 1 9 8 1 ) , and a l d o s t e r o n e i n c r e a s e s b o t h t h e vmax ( b u t n o t t h e subs t r a t e K~ v a l u e s ) of c i t r a t e s y n t h a s e and t h e amount o f immunoreactive enzyme p r e s e n t (Law and Edelman, 1978a) These r e s u l t s i n d i c a t e s t e r o i d - i n d u c e d d e novo s y n t h e s i s of t h e above enzymes. There may be t i s s u e s p e c i f i c i t y i n t h a t c i t r a t e s y q t h a s e i s n o t induced i n t h e i n t e s t i n e ( W i l l e t a l . , 1981) , n o r i s i t s t i m u l a t e d i n t o a d b l a d d e r e p i t h e l i a i n c u l t u r e (Handler e t a l . , 1 9 8 1 ) . C a u t i o n s h o u l d t h e r e f o r e be e x e r c i s e d i n t h e u s e of t h e s e enzymes a s markers o f m i n e r a l o c o r t i c o i d a c t i o n . Ambiguity h a s c h a r a c t e r i z e d t h e r e s u l t s of e x p e r i ments d e s i g n e d t o a s s i g n a r o l e f o r N a , K - A T P a s e i n a l d o s t e r o n e a c t i o n (Marver, 1 9 8 0 ) . Knox and Sen r e p o r t e d i n 1 9 7 4 t h a t s y n t h e s i s of t h e a - s u b u n i t (and p o s s i b l y t h e $ - s u b u n i t ) w a s i n c r e a s e d i n t h e k i d n e y f o l l o w i n g adminis t r a t i o n o f h i g h d o s e s of a l d o s t e r o n e t o a d r e n a l e c t o m i z e d r a t s . Geering e t al. ( t h i s volume) have o b s e r v e d i n creases i n i m m u n o p r e c i p i t a b l e Na,K-ATPase s u b u n i t s f o l lowing a l d o s t e r o n e treatment: of t o a d b l a d d e r . It is int e r e s t i n g t o n o t e t h a t they a l s o reported a s y n e r g i s t i c e f f e c t of T3. Many i n v e s t i g a t o r s have a t t r i b u t e d t h e s t i m u l a t i o n of Na,K-ATPase t o t h e i n c r e a s e d sodium l o a d i n g which a l d o s t e r o n e e l i c i t s i n t a r g e t t i s s u e s (Doucet and Katz, 1981; Handler e t al., 1981; Jdrgensen, 1 9 6 9 ; W i l l e t a l . , 1980, 1 9 8 1 ) . The e v i d e n c e which f a v o r s t h i s secondary e f f e c t o f s t e r o i d s i s m a n i f o l d . T h e hormoneinduced a l t e r a t i o n i n sodium e x c r e t i o n i s o b s e r v e d w i t h i n 1-2 h r and may i n v o l v e a m i l o r i d e - s e n s i t i v e chann e l s ( W i l l e t al., 19811, whereas r e n a l Na,K-ATPase d o e s not i n c r e a s e u n t i l 4-5 h r l a t e r ( J d r g e n s e n , 1 9 6 9 ) . S t u d i e s w i t h d i s s e c t e d nephron segments have s i m i l a r l y f a i l e d t o r e v e a l any immediate ( 3 h r ) e f f e c t s of a l d o sterone on Na,K-ATPase t o c o i n c i d e w i t h t h e o b s e r v e d changes i n c a t i o n t r a n s p o r t (Doucet and K a t z , 1981) Also, Na,K-ATPase i s i n d u c e d by f e e d i n g r a t s a lowsodium d i e t ( l e a d i n g t o e l e v a t e d endogenous a l d o s t e r o n e l e v e l s ) ; the rapid reversion t o c o n t r o l l e v e l s following d i e t a r y sodium r e p l a c e m e n t i m p l i e s a s e c o n d a r y e f f e c t ( W i l l et a l . , 1 9 8 0 ) . Handler e t a l . (1981) f a i l e d t o o b s e r v e a n i n c r e a s e i n sodium-dependent ATP h y d r o l y s i s i n aldosterone-treated toad bladder e p i t h e l i a i n c u l t u r e . They d i d r e p o r t e l e v a t e d o u a b a i n b i n d i n g t o homogenates, b u t t h a t a m i l o r i d e a b o l i s h e d t h i s , i n d i c a t i v e o f a sodium e f f e c t on e x p r e s s i o n of Na,K-ATPase a c t i v i t y . Although it i s n o t known whether t h e work of Knox and Sen (1974) r e f l e c t s a d i r e c t o r s e c o n d a r y a c t i o n of a l d o s t e r o n e , t h e i n c r e a s e s i n Na,K-ATPase p r o t e i n obs e r v e d by G e e r i n g e t a l . ( t h i s volume) were i n s e n s i t i v e

.

.

Na,K-ATPaseBIOSYNTHESIS AND TURNOVER

737

t o t h e p r e s e n c e of a m i l o r i d e , implying a d i r e c t e f f e c t of t h e hormone. A r e c e n t r e p o r t by Garg et al. (1981) p r e s e n t e d e v i d e n c e f o r a mineralocorticoid-induced i n crease i n r e n a l Na,K-ATPase c o n c e n t r a t i o n . They comp a r e d t h e r a t e s o f Na+-dependent ATP h y d r o l y s i s and N a + t r a n s p o r t i n i s o l a t e d nephron segments from r a t s which were e i t h e r f e d a low-Na+ d i e t o r t r e a t e d w i t h deoxyc o r t i c o s t e r o n e (DOCA), a p o t e n t m i n e r a l o c o r t i c o i d . They found t h a t DOCA i n c r e a s e d N a , K - A T P a s e a c t i v i t y 6 - f o l d i n t h e c o r t i c a l c o l l e c t i n g d u c t , whereas low-Na+ stress c a u s e d o n l y a 2 - f o l d enhancement. I t i s import a n t h e r e t o n o t e t h a t t h e ATP h y d r o l y s i s a s s a y s were performed u n d e r c o n d i t i o n s which expose a l l c r y p t i c act i v i t y . T h i s may be c r i t i c a l i n t h a t enzyme l a t e n c y h a s been c i t e d as a s i g n i f i c a n t f a c t o r i n t h e p r e s e n t c o n f u s i o n r e g a r d i n g t h e mode of a d r e n a l s t e r o i d a c t i o n on e p i t h e l i a l t r a n s p o r t (Marver, 1 9 8 0 ) . A h i g h c o r r e l a t i o n between N a + t r a n s p o r t and N a , K - A T P a s e w a s o b s e r v e d i n b o t h t r e a t m e n t g r o u p s . The a u t h o r s a l s o m o n i t o r e d c e l l number by q u a n t i t a t i n g n u c l e i a f t e r s t a i n i n g t h e segments w i t h a c r i d i n e o r a n g e . They c o n c l u d e d t h a t t h e membrane d e n s i t y o f N a , K - A T P a s e m o l e c u l e s p e r c e l l w a s i n c r e a s e d by b o t h t r e a t m e n t s , b u t t o d i f f e r e n t d e g r e e s . T h a t DOCA augments t h e b a s o l a t e r a l s u r f a c e area o n l y 2- t o 3 - f o l d (Wade et a l . , 1979) i n d i c a t e s t h a t t h e 6 - f o l d enhancement o f Na,K-ATPase c a n n o t be a t t r i b u t e d o n l y t o g e n e r a l membrane s y n t h e s i s . Other p o s s i b l e mechanisms of s t e r o i d a c t i o n on cat i o n t r a n s p o r t have been proposed. The s t i m u l a t i o n of Na,K-ATPase h a s been a t t r i b u t e d t o i n c r e a s e d i n t r a c e l l u l a r ATP l e v e l s - - a consequence of t h e i n d u c t i o n of c i t r a t e s y n t h a s e ( K i r s t e n et a l . , 1968; Law and Edelman, 1978b; S u z u k i , 1981; Voute et a l . , 1975), a l t h o u g h t h i s outcome i s n o t u n i v e r s a l l y o b s e r v e d (Handler e t a l . , 1981; W i l l et ai., 1 9 8 1 ) . A l d o s t e r o n e h a s a l s o been rep o r t e d t o i n t e r a c t d i r e c t l y w i t h t h e enzyme p r o t e i n s themselves i n e r y t h r o c y t e g h o s t s , leading t o a 450% i n c r e a s e i n a c t i v i t y (Hamlyn and Duffy, 1 9 7 8 ) . 2.

G 1 ucoco r t i c o i d s

S e v e r a l s t u d i e s have i n d i c a t e d t h a t t h e a l d o s t e r o n e e f f e c t s on N a , K - A T P a s e are implemented t h r o u g h g l u c o c o r t i c o i d r e c e p t o r s . Aldosterone i n c r e a s e d Na,K-ATPase act i v i t y i n t h e proximal t u b u l e s of t h e r a t d e s p i t e a lack of m i n e r a l o c o r t i c o i d r e c e p t o r s and t h e p r e s e n c e of canrenone, an e f f e c t i v e b l o c k e r o f m i n e r a l o c o r t i c o i d r e c e p t o r s ( A p e r i a et al., 1 9 8 1 ) . I n t h e i n t e s t i n a l e p i t h e l i um, N a , K - A T P a s e s t i m u l a t i o n by a l d o s t e r o n e l a g s b e h i n d t h e i n c r e a s e i n sodium r e s o r p t i o n and i s l i k e l y to be a

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s e c o n d a r y e f f e c t of t h e hormone, whereas enzyme induct i n by dexamethasone, a p o t e n t s y n t h e t i c g l u c o c o r t i c o i d , p a r a l l e l s t h e sodium u p t a k e and i s p o s t u l a t e d t o be a d i r e c t r e s p o n s e t o hormone i n d u c t i o n ( W i l l e t a l . , 1 9 8 1 ) . P o s s i b l y t h e most c o n v i n c i n g e v i d e n c e f o r r e c e p t o r c r o s s occupancy i s t h a t of Rodriguez e t a l . (1981) , who d e t e r mined d o s e s o f a l d o s t e r o n e and dexamethasone which s t i m u l a t e d g l u c o n e o g e n e s i s and a l t e r e d u r i n a r y e l e c t r o l y t e excretion, respectively, without cross-reacting. Renal Na,K-ATPase w a s o n l y a f f e c t e d by dexamethasone; s t i m u l a t i o n by a l d o s t e r o n e was a c h i e v e d o n l y a t d o s e s which i n c r e a s e d g l u c o n e o g e n e s i s , i n d i c a t i n g occupancy of g l u c o c o r t i c o i d r e c e p t o r s . These r e s u l t s c o r r e l a t e w i t h o b s e r v a t i o n s by K a t z and E p s t e i n ( 1 9 6 7 ) of s t i m u l a t i o n by t h e g l u c o c o r t i c o i d m e t h y l p r e d n i s o l o n e of sodium-dependent ATP h y d r o l y s i s i n p a r a l l e l w i t h e l e v a t i o n s i n g l o m e r u l a r f i l t r a t i o n and r e n a l sodium r e a b s o r p t i o n . S i m i l a r l y , W i l l e t a l . (1981) r e p o r t e d i n c r e a s e d N a , K - A T P a s e i n t h e i n t e s t i n a l mucosa f o l l o w i n g dexamethasone t r e a t m e n t . I n agreement w i t h t h e d i r e c t hormonal i n d u c t i o n t h e o r i e s , t h e dexamethasone s t i m u l a t i o n of Na,K-ATPase can be e n t i r e l y accounted f o r by an i n c r e a s e i n t h e number of enzymes. Hormone t r e a t m e n t r e s t o r e s r e n a l N a , K - A T P a s e t o normal l e v e l s i n a d r e n a l e c t o m i z e d r a t s and r e s u l t s i n i n c r e a s e d K + - s e n s i t i v e p h o s p h o r y l a t i o n , a measure of enzyme numb e r l (Rodriguez and K l a h r , 1980; Sinha e t a l . , 1 9 8 1 ) . The Vmax v a l u e s f o r N a + , K f , and ATP are a l s o i n c r e a s e d , w i t h o u t changes i n s u b s t r a t e a f f i n i t i e s ( S i n h a e t a l . , 1 9 8 1 ) . The e l e v a t i o n on s p e c i f i c enzyme a c t i v i t y when normalized t o DNA c o n t e n t a l s o i n d i c a t e s t h e p r e s e n c e of more enzyme p e r c e l l ( S i n h a e t a l . , 1 9 8 1 ) . These d a t a c o r r e l a t e w i t h t h e s t i m u l a t i o n by dexamethasone of t h e i n c o r p o r a t i o n of [35S]methionine i n t o p r o t e i n i n t h e r e n a l medulla ( L a w and Edelman, 1 9 7 8 a ) . I n p o l a r i z e d e p i t h e l i a Na,K-ATPase i s g e n e r a l l y t h o u g h t t o be l o c a t e d i n t h e b a s o l a t e r a l plasma membrane. I t i s r e l e v a n t h e r e t o d i s c u s s o b s e r v a t i o n s by Kashqarian (1980) t h a t adrenal s t e r o i d s e l i c i t increases i n t h e sur-

' I t i s i n t e r e s t i n g t o note t h a t dexamethasone was r e p o r t e d t o increase Na,K-ATPase w i t h i n 2 hr of a d d i t i o n ( S i n h a e t a l . , 1 9 8 1 ) . P o l l a c k e t a l . ( 1 9 8 1 b ) a n d C h u r c h i l l a n d Hokin ( 1 9 7 9 ) have o b s e r v e d a t r a n s i t t i m e o f more than 2 hr f o r the n e w l y s y n t h e s i z e d a - s u b u n i t t o enter p l a s m a membranes. T h i s may i m p l y

h o r m o n a l r e g u l a t i o n b y a n i m m e d i a t e c h a n g e i n r a t e of. d e g r a d a t i o n , i n v o l v i n g no l a g , a s o p p o s e d t o the i n c r e a s e d s y n t h e t i c r a t e e l i c i t e d i n response t o T ( L o a n d Lo, 1 9 8 0 ) . 3

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f a c e area o f t h i s membrane and may have a r o l e i n c a t i o n transport. S u p p o r t f o r t h e s e o b s e r v a t i o n s comes from biochemical evidence t h a t g l u c o c o r t i c o i d s a l t e r N a , K A T P a s e w i t h o u t a f f e c t i n g enzymes l o c a l i z e d i n t h e b r u s h b o r d e r , namely s u c r a s e and a l k a l i n e p h o s p h a t a s e ( W i l l e t al., 1 9 8 1 ) . On t h e o t h e r hand, it i s w o r t h n o t i n g t h a t a d e n y l a t e c y c l a s e , a n o t h e r b a s o l a t e r a l membrane marker, i s unchanged by a d r e n a l s t e r o i d s (Handler et al., 1981; Hendler e t al., 1 9 7 2 ) .

V.

OBESITY

I n c e r t a i n p a t h o l o g i c a l s t a t e s t h e N a , K - A T P a s e cont e n t i n v a r i o u s t i s s u e s may be a l t e r e d . T h i s s e c t i o n c o n s i d e r s one s u c h d i s o r d e r - - o b e s i t y . Much o f t h e d a t a p e r t a i n i n g t o t h e Na,K-ATPase i n t h i s c o n d i t i o n may p o s s i b l y be e x p l a i n e d a s an a b n o r m a l i t y i n t h e enzyme's biosynthesis. Obese ( o b / o b ) mice were found by Lin et a l . (1978, 1 9 7 9 ) t o have lower l e v e l s o f s p e c i f i c o u a b a i n b i n d i n g i n s k e l e t a l muscle and l i v e r t h a n t h e i r l e a n c o u n t e r p a r t s . The d i s s o c i a t i o n c o n s t a n t s (Kd) were unchanged and t h e a u t h o r s a t t r i b u t e d t h e v a r i a t i o n t o a d e c r e a s e d number o f b i n d i n g s i t e s . The l e v e l o f b i n d i n g t o k i d n e y t i s s u e w a s t h e same i n b o t h g r o u p s . However, York e t a l . (1978) a l s o a n a l y z e d t i s s u e s from o b / o b mice and found d e c r e a s e d Na,K-ATPase a c t i v i t y i n kidney a s w e l l as l i v e r . The k i d n e y enzyme i n t h i s s t u d y d i d n o t approach c o n t r o l l e v e l s a f t e r NaI-treatment, diminishing t h e p o s s i b i l i t y of enzyme l a t e n c y a s t h e b a s i s of o b e s i t y - r e l a t e d a l t e r a t i o n s . Also i n t h i s s t u d y , mice whose o b e s i t y r e s u l t e d from a hypothalamic l e s i o n ( g o l d t h i o g l u c o s e (GTG) t r e a t e d ) d i d n o t show a l t e r e d enzyme l e v e l s i n t h e l i v e r , i n d i c a t i n g t h a t o b e s i t y p e r se i s n o t r e s p o n s i b l e f o r t h e a l t e r e d Na,K-ATPase a c t i v i t y . That s k e l e t a l muscle from m i c e which had been s u r g i c a l l y l e s i o n e d a t t h e hypothalamus e x h i b i t e d d e c r e a s e d o u a b a i n b i n d i n g was t a k e n a s e v i d e n c e o f f e w e r N a , K - A T P a s e molec u l e s (Vander Tuig e t al., 1 9 8 1 ) . The s o u r c e s of d i s crepancy among t h e s e r e p o r t s are u n c l e a r . An a n i m a l ' s t h y r o i d s t a t u s , a s p r e v i o u s l y d e s c r i b e d (see S e c t i o n I V , A ) , h a s a profound e f f e c t on t h e sodium pump c o n c e n t r a t i o n i n a number of t i s s u e s . T h i s i n t e r a c t i o n i s i m p o r t a n t i n b o t h energy metabolism (Bray, 1969) and c e l l u l a r t h e r m o g e n e s i s (Smith and Edelman, 1 9 7 9 ) -two e l e m e n t s which have been r e l a t e d t o t h e e t i o l o g y of o b e s i t y (Lin e t a l . , 1 9 7 8 ) . A l i n k h a s t h e r e f o r e been

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s o u g h t between o b e s i t y and t h y r o i d hormone r e g u l a t i o n While m o s t i n v e s t i g a t o r s a g r e e t h a t a of Na,K-ATPase. d e f e c t i n t h e sodium pump e x i s t s i n g e n e t i c o b e s i t y , t h e n a t u r e of t h e l e s i o n remains t o be p i n p o i n t e d . York et al. (1978) found l i v e r N a , K - A T P a s e o f o b / o b mice t o be i n s e n s i t i v e t o T3 t r e a t m e n t , whereas b o t h c o n t r o l ( l e a n ) and GTG-treated mice responded w i t h act i v i t y i n c r e a s e s . Another T 3 - s e n s i t i v e enzyme, g l y c e r o l - 3 - p h o s p h a t e dehydrogenase, w a s s t i m u l a t e d i n o b / o b mouse l i v e r , implying a s p e c i f i c a l t e r a t i o n i n The a u t h o r s s p e c u l a t e t h a t t i s s u e s t h e Na,K-ATPase. i n obese a n i m a l s respond t o t h y r o i d hormone w i t h t h e s y n t h e s i s of a f a u l t y Na,K-ATPase a n d , c i t i n g a r e p o r t of d e f e c t i v e p r o t e i n glycosylation i n obese m i c e (Chang et a1 ., 1975) , p o s t u l a t e a n o n f u n c t i o n a l B-subu n i t . It appears equally l i k e l y t h a t t h e d e f e c t i s t h e l a c k o f any Na,K-ATPase s y n t h e s i s i n r e s p o n s e t o T3. Bray e t a l . (1978) s t u d i e d t h e r e l a t i o n s h i p of t h y r o i d s t a t u s t o t h e sodium pump i n a number of t y p e s of o b e s i t y i n r o d e n t s . They r e p o r t e d t h a t c e r t a i n forms of o b e s i t y which a r e r e c e s s i v e l y i n h e r i t e d ( o b / o b , d b / d b ) a r e c h a r a c t e r i z e d by d e c r e a s e d l i v e r enzyme act i v i t y . When a n i m a l s t h a t are homozygous f o r t h i s t r a i t a r e made h y p o t h y r o i d and t h e n t r e a t e d w i t h T3, h e p a t i c Na,K-ATPase i s n o t i n d u c e d , a l t h o u g h g l y c e r o l - 3 - p h o s p h a t e dehydrogenase i s s t i m u l a t e d , i n agreement w i t h York e t a l . ( 1 9 7 8 ) . S i n c e o t h e r forms o f o b e s i t y , namely GTGinduced ( n o n g e n e t i c ) , y e l l o w o b e s e (dominant) , and t h e f a t t y r a t ( r e c e s s i v e ) , are n o t c h a r a c t e r i z e d by a l t e r e d sodium-dependent ATP h y d r o l y s i s , and Mendelian recess i v e s g e n e r a l l y i n v o l v e a d e f e c t i n a s i n g l e p r o t e i n (enz y m e ) , i t i s p o s s i b l e t h a t t h e l e s i o n o f o b / o b and d b / d b mice i s i n t h e Na,K-ATPase o r i t s i n d u c i b i l i t y by t h y r o i d hormones. I n d i r e c t c o n t r a s t t o t h e s e d a t a a r e t h e f i n d i n g s o f L i n e t a l . ( 1 9 7 8 ) , who r e p o r t e d i n c r e a s e d res p o n s i v e n e s s o f o b / o b mouse muscle and l i v e r t o t h y r o i d hormones, i n t h i s case t h y r o x i n e ( T 4 ) , w i t h t h e r e s u l t b e i n g a l a r g e r i n c r e a s e i n o u a b a i n r e c e p t o r number t h a n s e e n i n hormone-treated l e a n mice. The Kd f o r o u a b a i n was unchanged i n e i t h e r group of a n i m a l s . The r e a s o n s f o r t h e c o n t r a d i c t i o n may be m a n i f o l d s i n c e d i f f e r e n t hormones (T3 v e r s u s T 4 ) and d o s e s and d i f f e r e n t a s s a y s (ATP h y d r o l y s i s v e r s u s o u a b a i n b i n d i n g ) were used i n these studies. of p a r t i c u l a r c l i n i c a l i n t e r e s t are r e c e n t r e p o r t s of t h e e x p r e s s i o n of a N a , K - A T P a s e d e f e c t i n human obes i t y . DeLuise e t a l . (1980) drew wide and v a r i e d commentary a f t e r t h e i r r e p o r t t h a t o b e s e p a t i e n t s a s a group e x h i b i t low e r y t h r o c y t e o u a b a i n b i n d i n g and Rb+ u p t a k e , as w e l l a s g e n e r a l l y h i g h l e v e l s of i n t r a c e l l u l a r

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sodium, a l l i n d i c a t i v e o f fewer N a , K - A T P a s e m o l e c u l e s . The a u t h o r s found a good n e g a t i v e c o r r e l a t i o n between t h e number of o u a b a i n - b i n d i n g s i t e s p e r red c e l l and body w e i g h t . N o r b i a t o e t a l . (1981) r e p o r t e d h i g h e r t h a n normal serum p o t a s s i u m i n o b e s e p a t i e n t s f o l l o w i n g e x e r c i s e t h e r a p y , i n agreement w i t h a d e c r e a s e d sodium pump h y p o t h e s i s . K a j i (19811, however, c r i t i c i z e d t h e o r i g i n a l r e p o r t because of p o s s i b l e changes i n t h e c e l l s ' a f f i n i t y f o r ouabain, e i t h e r as a direct r e s u l t o f t h e o b e s e s t a t e o r t h e h i g h c y t o s o l i c sodium l e v e l , f a c t o r s t h a t were n o t a d e q u a t e l y c o n t r o l l e d . About a y e a r a f t e r t h e DeLuise group p u b l i s h e d t h e i r r e s u l t s , M i r et a l . (1981) r e p o r t e d t h a t t h e obese p a t i e n t s i n t h e i r s t u d y showed h i g h e r red c e l l sodium pump a c t i v i t y t h a n t h i n s u b j e c t s . S u b s e q u e n t l y , DeLuise and F l i e r (1982, and t h i s volume) have found i n e r y t h r o c y t e s from a morbidly o b e s e p a t i e n t markedly h i g h N a , K - A T P a s e a c t i v i t y ( 1 4 - f o l d h i g h e r t h a n c o n t r o l s ) and o u a b a i n b i n d i n g (18-fold h i g h e r ) , i n c o n t r a s t t o t h e i r p r e v i o u s d a t a . The r e d c e l l s d i d n o t have abnormally h i g h i n t r a c e l l u l a r sodium so t h a t t h e e l e v a t e d Na,KA T P a s e a c t i v i t y c o u l d n o t be e x p l a i n e d a s s t i m u l a t i o n o f t h e pump from t h e c y t o s o l s i d e . The a u t h o r s f e e l t h a t a n a l t e r e d a f f i n i t y f o r i o n s and o u a b a i n may be t h e p r i mary d e f e c t w i t h t h e i n c r e a s e d number o f pumps a seconda r y e f f e c t . Leukocytes from t h i s same p a t i e n t e x h i b i t e d normal sodium pump c h a r a c t e r i s t i c s , l e a d i n g t o s p e c u l a t i o n t h a t t h e enzyme may r e s u l t from a c t i v a t i o n of separ a t e genes i n d i f f e r e n t t i s s u e s . The red c e l l may n o t be a v a l i d i n d i c a t o r f o r t i s s u e s i n g e n e r a l a s e v i d e n c e d by t h e f i n d i n g t h a t e r y t h r o c y t e N a , K - A T P a s e i s decreased i n hyperthyroid p a t i e n t s , i n c o n t r a s t t o t h e augmentation s e e n i n many g l a n d u l a r t i s s u e s and muscle (see S e c t i o n I V , A ) . Bray e t al. (1981) found N a , K ATPase t o be h i g h e r t h a n c o n t r o l v a l u e s i n l i v e r samples from o b e s e p a t i e n t s . I n t h i s s t u d y a p o s i t i v e c o r r e l a t i o n between h e p a t i c N a , K - A T P a s e a c t i v i t y and body w e i g h t w a s found. The seemingly c o n t r a d i c t o r y r e s u l t s are r e m i n i s c e n t of t h e d i v e r g e n c e i n t i s s u e - s p e c i f i c res p o n s e s t o K+ d e p l e t i o n , a l t h o u g h t h e a d a p t i v e n a t u r e of t h e r e s p o n s e s a r e l e s s obvious i n o b e s i t y . Although human o b e s i t y i s u n l i k e t h e murine forms i n many p h y s i o l o g i c a l r e s p e c t s (Bray e t a l . , 19811, it may be s i m i l a r i n t h a t t h e v a r i a t i o n s e e n might r e f l e c t d i f f e r e n t forms o f o b e s i t y . A g e n e t i c b a s i s f o r human o b e s i t y h a s been s u g g e s t e d t o a c c o u n t f o r a n a p p a r e n t f a m i l i a l i n h e r i t a n c e p a t t e r n (DeLuise and F l i e r , 1 9 8 2 ) . More c o n v i n c i n g l y , Mott e t a l . ( t h i s volume) have d e s c r i b e d a l t e r e d red c e l l N a , K - A T P a s e i n a n e n t i r e populat i o n , t h e P i m a I n d i a n s , which i s c h a r a c t e r i z e d by t h e

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p r e v a l e n c e of o b e s i t y . A t h y r o i d involvement i n human o b e s i t y h a s been s u g g e s t e d (DeLuise et a l . , 1980) and may b e confirmed w i t h f u r t h e r i n v e s t i g a t i o n . F a u l t y , d i m i n i s h e d , o r o t h e r w i s e a l t e r e d enzyme biosynthesis i s a reasonable explanation f o r the observ a t i o n s i n t i s s u e s from o b e s e a n i m a l s , e s p e c i a l l y mice. To d e t e r m i n e i f t h i s h y p o t h e s i s i s v a l i d f o r r e d c e l l s , e x p e r i m e n t s w i l l have t o be c a r r i e d o u t on t h e i r marrow o r r e t i c u l o c y t e p r e c u r s o r s s i n c e t h e e r y t h r o c y t e its e l f i s unable t o s y n t h e s i z e p r o t e i n . Pathologies, l i k e genetic obesity, i n experimental a n i m a l s l e a d i n g t o a l t e r e d , Na,K-ATPase l e v e l s s h o u l d become u s e f u l t o o l s f o r u n r a v e l i n g t i s s u e - s p e c i f i c cont r o l s on t h e r e g u l a t i o n o f t h i s enzyme, a s have enzyme m u t a n t s i n many o t h e r s y s t e m s .

VI

.

CONCLUSION

I t h a s been r e c o g n i z e d f o r 25 y e a r s t h a t Na,KA T P a s e i s an e s s e n t i a l enzyme t h a t u n d e r l i e s many cel-

l u l a r f u n c t i o n s : c e l l volume r e g u l a t i o n i n t h e f a c e of i n i m i c a l Donnan f o r c e s , maintenance o f a d e q u a t e c e l l K+ t o s u p p o r t p r o t e i n s y n t h e s i s and growth, a n e l e c t r o l y t e d i s t r i b u t i o n t h a t i s t h e b a s i s o f e l e c t r i c a l act i v i t y i n specialized cells, secretory a c t i v i t y i n epit h e l i a , and a r e c e n t l y r e c o g n i z e d r o l e i n m i t o g e n i c s t i m u l a t i o n of c e l l s and p o s s i b l y i n t u m o r i g e n e s i s . Given t h i s b a t t e r y of f u n c t i o n s , it i s n o t s u r p r i s i n g t h a t i n t h e enzyme's e v o l u t i o n it h a s become a d a p t e d t o many l i g a n d s t h a t r e g u l a t e i t s a c t i v i t y . N e v e r t h e l e s s , t h e r a n g e of a c t i v i t y of a s i n g l e enzymatic u n i t i s limited. I n HeLa c e l l s , t h e t r a n s p o r t a s s o c i a t e d w i t h one ouabain-binding s i t e may v a r y from z e r o t o a b o u t 1 0 0 K+ i o n s / s e c . T h i s Vmax v a l u e seems t o be i n t h e r a n g e found i n o t h e r mammalian c e l l s . The i m p o s i t i o n of g r e a t e r demands t h a n t h i s f o r a l k a l i c a t i o n t r a n s p o r t may n e c e s s i t a t e more enzyme. Many systems a r e respons i v e t o such demands e i t h e r w i t h i n c r e a s e d s y n t h e s i s or reduced t u r n o v e r . But even i n t h e a b s e n c e o f changing c e l l u l a r r e q u i r e m e n t s , Na,K-ATPase, l i k e any p r o t e i n , i s n o t immortal b u t i s i n t h e long r u n s u b j e c t t o a v a r i e t y of s p e c i f i c and n o n s p e c i f i c i n a c t i v a t i n g e v e n t s . The a c t i v i t y of t h e enzyme a t t h e c e l l s u r f a c e ( e r y t h r o c y t e s e x c e p t e d ) a p p e a r s t o be m a i n t a i n e d i n good r e p a i r by c o n t i n u o u s s y n t h e s i s and t u r n o v e r , t h e fundamental p r o cesses on which t h e o t h e r a d a p t i v e r e s p o n s e s may be b a s e d . I n a d d i t i o n t o i t s c a t a l y t i c and t r a n s p o r t a c t i v i t i e s , Na,K-ATPase i s a h i g h l y dynamic m o l e c u l e .

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ACKNOWLEDGMENT

Research sponsored by t h e O f f i c e o f H e a l t h and Environmental R e s e a r c h , U.S. Department of Energy, under c o n t r a c t W-7405-eng-26 w i t h t h e Union Carbide C o r p o r a t i o n .

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A i t o n , J. F., Lamb, J. F., and Ogden, P. ( 1 9 8 1 ) . Down-regulation o f t h e sodium pump f o l l o w i n g c h r o n i c exposure o f H e L a c e l l s and c h i c k embryo h e a r t c e l l s t o o u a b a i n . B r . J . P h a r m a c o l . 7 3 , 333-340. Aperia, A . , Larsson, L . , and Z e t t e r s t r E m , R. ( 1 9 8 1 ) . Hormonal i n d u c t i o n o f Na-K-ATPase i n d e v e l o p i n g p r o x i m a l t u b u l a r c e l l s . Am. J . P h y s i o l . 241, F356-F360. Asano, Y . , L i b e m a n , U. A . , and Edelman, I. S. ( 1 9 7 6 ) . Thyroid t h e r m o g e n e s i s . R e l a t i o n s h i p s between Na+-dependent respirat i o n and Na+ + K+-adenosine t r i p h o s p h a t a s e a c t i v i t y i n r a t s k e l e t a l muscle. J . C l i n . I n v e s t . 5 7 , 368-379. B e r l i n , C. M . , and Schimke, R. T. ( 1 9 6 5 ) . The i n f l u e n c e o f t u r n o v e r r a t e s on t h e r e s p o n s e s o f enzymes t o c o r t i s o n e . Mol. P h a r m a c o l . 1 , 149-156. B l o b e l , G., and D o b b e r s t e i n , B. ( 1 9 7 5 ) . , T r a n s f e r of p r o t e i n s a c r o s s membranes. I . P r e s e n c e of p r o t e o l y t i c a l l y p r o c e s s e d and unprocessed n a s c e n t immunoglobulin l i g h t c h a i n s on membrane-bound ribosomes o f murine myeloma. J. C e l l Biol. 67, 835-851. Boardman, L . J . , Lamb, J. F., and M c C a l l , D. ( 1 9 7 2 ) . Uptake o f [3H]-ouabain and N a pump t u r n o v e r rates i n c e l l s c u l t u r e d i n o u a b a i n . J . P h y s i o l . ( L o n d o n ) 225, 619-635. P . , and Boardman, L. J . , H u e t t , M., Lamb, J . F., Newton, Polson, J. M. ( 1 9 7 4 ) . Evidence f o r t h e g e n e t i c c o n t r o l of t h e sodium pump d e n s i t y i n HeLa c e l l s . J. P h y s i o l . ( L o n d o n ) 241, 771-794. Bray, G. A. (1969). E f f e c t of d i e t and t r i i o d o t h y r o n i n e on t h e a c t i v i t y of sn-glycerol-3-phosphate dehydrcqenase and on t h e m e t a b o l i s m o f g l u c o s e and p y r u v a t e by a d i p o s e t i s s u e o f obese p a t i e n t s . J . C l i n . Invest. 48, 1413-1422. Bray, G . A. , York, D. A., and Yukimura, Y . ( 1 9 7 8 ) . A c t i v i t y o f ( N a + + K+)-ATPase i n t h e l i v e r o f a n i m a l s w i t h e x p e r i m e n t a l o b e s i t y . Life S c i . 22, 1637-1642. Bray, G . A . , K r a l , J . G . , and B j o r n t o r p , P. ( 1 9 8 1 ) . Hepatic sodium-potassium-dependent ATPase i n o b e s i t y . N. Engl J . M e d . 304, 1580-1582. C a n t l e y , L. C . , Ferguson, J. H., and K u s t i n , J. ( 1 9 7 8 ) . Norepinep h r i n e complexes and r e d u c e s vanadium (V) t o r e v e r s e v a n a d a t e i n h i b i t i o n o f t h e (Na+,K+)-ATPase. J . A m . Chern. SOC. 1 0 0 , 5210-5212.

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Lo, C . S., August, T. R . , Liberman, U. A., and Edelman, I. S. (1976) Dependence o f r e n a l ( N a + + K)' -adenosine t r i p h o s p h a t a s e on t h y r o i d s t a t u s . J. B i o l . Chem. 251, 7826-7833. LOuvard, D. (1980). Apical membrane aminopeptidase appears a t s i t e of c e l l - c e l l c o n t a c t i n c u l t u r e d kidney e p i t h e l i a l cells. P r o c . Natl. A c a d . S c i . USA 7 7 , 4132-4136. Marver, D. (1980). A l d o s t e r o n e a c t i o n i n t a r g e t e p i t h e l i a . V i t a m . Horm. ( N . Y . ) 38, 57-117. Mazurkiewicz, J. E . , and Barrnett, R. J. (1981). Organotypic c u l t u r e s of t h e a v i a n s a l t g l a n d : B i o s y n t h e s i s o f membrane proteins. J. C e l l S c i . 48, 75-88. Mendoza, S. A . , Wigglesworth, N. M . , and Rozengurt, E. (1980). V a s o p r e s s i n r a p i d l y s t i m u l a t e s N a e n t r y and Na-K pump act i v i t y i n q u i e s c e n t c u l t u r e s of mouse 3T3 c e l l s . J. C e l l . P h y s i o l . 105, 153-162. M i r , M. A., Charalambous, B. M., Morgan, K . , and Evans, P. J. (1981) E r y t h r o c y t e sodium-potassium-ATPase and sodium t r a n s p o r t i n o b e s i t y . N . E n g l . J. Med. 305, 1264-1268. Mummery, C. L . , B o o n s t r a , J . , Van Der Saag, P. T., and DeLaat, S. W. ( 1 9 8 1 ) . Modulation of f u n c t i o n a l and o p t i m a l ( N a + - K+)-ATPase a c t i v i t y d u r i n g the c e l l c y c l e of neurob l a s t m a cells. J. C e l l . P h y s i o l . 1 0 7 , 1-9. N o r b i a t o , G., Vago, T., B e v i l a c q u a , M., a n d B o s i s i o , E ( 1 9 8 1 ) . Red-cell sodium-potassium pump i n o b e s i t y . N Engl. J . Med. 304, 540Nfirgaard, A. , K j e l d s e n , K. , and Clausen, T. ( 1 9 8 1 ) . Potassium d e p l e t i o n d e c r e a s e s t h e number o f 3H-ouabain b i n d i n g s i t e s and t h e a c t i v e Na-K t r a n s p o r t i n skeletal muscle. N a t u r e (London) 293, 739-741. Oppenheimer, J. H., Dillman, W. H . , Schwartz, H. L., and Towle, H. C. (1979). Nuclear r e c e p t o r s and t h y r o i d hormone a c t i o n : A p r o g r e s s report. Fed. Am. SOC. Exp. B i o l . 38, 2154-2161. P a l a d e , G . (1975). I n t r a c e l l u l a r aspects o f t h e p r o c e s s of prot e i n s y n t h e s i s . S c i e n c e 189, 347-358. P e a k e r , M. ( 1 9 7 8 ) . S a l t g l a n d s i n marine b i r d s : What t r i g g e r s s e c r e t i o n and what makes them grow? In "Comparative Water, I o n s , and F l u i d Mechanics'' (K. SchmidtPhysiology: N i e l s e n , L. B o l i s , and S. H. P. Maddrell, e d s . ) , pp. 207212. Cambridge Univ. P r e s s , London/New York. P e a k e r , M., and L i n z e l l , J. L. ( 1 9 7 5 ) . " S a l t Glands i n B i r d s and Reptiles. " Cambridge Univ. P r e s s , London/New York. P o l l a c k , L. R., Tate, E. H . , and Cook, J . S. (1981a). Na+,K+ATPase i n HeLa c e l l s a f t e r prolonged growth i n low K+ o r ouabain. J. C e l l . P h y s i o l . 106, 85-97. P o l l a c k , L. R . , T a t e , E. H., and Cook, J. S. (1981b). Turnover and r e g u l a t i o n o f Na,K-ATPase i n HeLa c e l l s . Am. P h y s i o l . 2 4 1 , C173-C183. P o l l a c k , L. R., T a t e , E. H . , and Cook, J. S. (1982). R e g u l a t i o n by t u r n o v e r o f Na,K-ATPase i n HeLa c e l l s . In "Membranes i n Growth and Developnent" (J. F. Hoffman, G. G i e b i s c h , and L. E. Bolis, e d s . ) , Alan R. L i s s , I n c . , New York.

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P o s t , R. L., and J o l l y , P. C . ( 1 9 5 7 ) . The l i n k a g e o f sodium, p o t a s s i u m , and ammonium a c t i v e t r a n s p o r t across t h e human B i o c h i m . B i o p h y s . A c t a 2 5 , 118-128. e r y t h r o c y t e membrane. R o d r i g u e z , H. J . , and K l a h r , S. ( 1 9 8 0 ) . Mechanism o f t h e r e g u l a t o r y e f f e c t N a + on renal Na+K+ ATPase. C l i n . R e s . 2 8 , 460A. Rodriguez, H. J . , S i n h a , S . K . , S t a r l i n g , J . , and K l a h r , S . ( 1 9 8 1 ) . R e g u l a t i o n of r e n a l Na+-K+-ATPase i n t h e r a t by a d r e n a l s t e r o i d s . Am. J . P h y s i o l . 2 4 1 , F186-Fl95. Rossier, B. C. ( 1 9 7 8 ) . Role o f RNA i n t h e a c t i o n o f a l d o s t e r o n e on N a f t r a n s p o r t . J . Membr. B i o l . 4 0 , 187-198. ROsSier, B. C . , Wilce, P. A , , I n c i a r d i , J. F . , Yoshimura, F. K . , and Edelman, I. S. ( 1 9 7 7 ) . E f f e c t s o f 3 ' - d e o x y c y t i d i n e on rRNA s y n t h e s i s i n t o a d b l a d d e r : Analysis of response t o a l d o s t e r o n e . Am. J . P h y s i o l . 2 3 2 , C174-Cl79. Rossier, B. C. , R o s s i e r , M . , and Lo, C. S . ( 1 9 7 9 a ) . T h y r o x i n e and N a + t r a n s p o r t i n toad: R o l e i n t r a n s i t i o n from p o i k i l o - t o homeothermy. Am. J . P h y s i o l . 2 3 6 , C117-Cl24. Rossier, B. C . , G a g g e l e r , H.-P. , B r u n n e r , D. P. , K e l l e r , I. , and Rossier , M. (197933). T h y r o i d hormone-aldosterone i n t e r a c t i o n Am. J . P h y s i o l . 2 3 6 , on N a + t r a n s p o r t i n t o a d b l a d d e r . C125-Cl31. Rothman, J. E. ( 1 9 8 1 ) . The G o l g i a p p a r a t u s : Two o r g a n e l l e s i n Science 2 1 3 , 1212-1219. tandem. Rozengurt, E. , and Mendoza, S. ( 1 9 8 0 ) . Monovalent i o n f l u x e s and t h e c o n t r o l o f cell p r o l i f e r a t i o n i n c u l t u r e d f i b r o b l a s t s . A n n . N . Y . Acad. S c i . 3 3 9 , 175-190. S a b a t i n i , D. D., K r e i b i c h , G . , Morimoto, T . , and Adesnik, M. ( 1 9 8 2 ) . Mechanism f o r the i n c o r p o r a t i o n o f p r o t e i n s i n membranes and o r g a n e l l e s . J . C e l l B i o l . 9 2 , 1-22. S a c h s , J. R. (1981). M e c h a n i s t i c i m p l i c a t i o n s o f t h e p o t a s s i u m potassium exchange carried o u t by t h e sodium-potassium pump. J . P h y s i o l . ( L o n d o n ) 3 1 6 , 263-277. S c h a e f e r , A . , S e r e g i , A . , and K o m l o s , M . ( 1 9 7 4 ) . Ascorbic a c i d l i k e e f f e c t o f t h e s o l u b l e f r a c t i o n o f r a t b r a i n on a d e n o s i n e t r i p h o s p h a t a s e s and i t s r e l a t i o n t o c a t e c h o l a m i n e s and c h e l a t i n g agents. B i o c h e m . P h a r m a c o l 2 3 , 2257-2271. S c o t t , W. N . , R e i c h , I . M . , Brown, J . A., Jr., and Yang, C.-P. H. (1978) Comparison o f t o a d b l a d d e r a l d o s t e r o n e - i n d u c e d prot e i n s and p r o t e i n s s y n t h e s i z e d i n v i t r o u s i n g a l d o s t e r o n e i n d u c e d messenger RNA a s template. J. M e m b r . B i o l . 4 0 , 213-220. S c o t t , W. N . , R e i c h , I. M . , and Goodman, D. B. P. ( 1 9 7 9 ) . I n h i b i t i o n o f f a t t y acid s y n t h e s i s p r e v e n t s t h e i n c o r p o r a t i o n o f J . Biol Chem. a l d o s t e r o n e - i n d u c e d p r o t e i n s i n t o membranes. 2 5 4 , 4957-4959. S c o t t , W. N . , Yang, C.-P., S k i p s k i , I. A . , Cobb, M. H. , R e i c h , I. M. , and T e r r y , P. M. (1981) Aldosterone-induced s y n t h e s i s o f p r o t e i n s related t o sodium t r a n s p o r t i n the t o a d ' s u r i n a r y b l a d d e r . Ann. N.Y. A c a d . Sci. 3 7 2 , 15-29. S i l v a , P . , T o r r e t t i , J . , H a y s l e t t , J. P., and E p s t e i n , F . H. ( 1 9 7 6 ) . R e l a t i o n between Na-K-ATPase a c t i v i t y a n d r e s p i r a t o r y rate i n t h e r a t k i d n e y . Am. J. P h y s i o l . 230, 1432-1438.

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Sinha, S. K . , Rodriguez, H. J . , Hogan, W. C., and Klahr, S. (1981). Mechanisms o f a c t i v a t i o n o f renal ( N a + + K+)-ATPase i n t h e rat. E f f e c t s of a c u t e and c h r o n i c a d m i n i s t r a t i o n of dexamethasone. Biochirn. Biophys. Acta 641, 20-35. Smith, J. B., and A u s t i c , R. E. (1980). A c t i v a t i n g t h e Na-K pump with monensin i n c r e a s e s a m i n o i s o b u t y r i c a c i d uptake by mouse f i b r o b l a s t s . Biochern. Biophys. R e s . Commun. 93, 392398. Smith, T. J., and Edelman, I. S. (1979). The r o l e of sodium Fed. Am. SOC. Exp. Biol. t r a n s p o r t i n t h y r o i d thermogenesis. 38, 2150-2153. Somjen, D., I s m a i l - B e i g i , F., and E d e h a n , I. S. (1981). Nuclear b i n d i n g of T3 and e f f e c t s on Qo,, Na-K-ATPase, and a-GPDH i n l i v e r and kidney. Am. J. P h y s i o l . 240, E146-El54. S t e w a r t , D. J . , and Sen, A. K. (1981). R o l e of c y c l i c GMP i n c h o l i n e r g i c a c t i v a t i o n of Na-K pump i n duck s a l t g l a n d . Am. J . P h y s i o l . 240, 207-214. S t e w a r t , D. J., Semple, E. W . , S w a r t , G. T., and Sen, A. K. (1976). I n d u c t i o n o f t h e c a t a l y t i c p r o t e i n o f ( N a + + K+)-ATPase i n t h e s a l t g l a n d of t h e duck. Biochim. Biophys. Acta 419, 1 50-163. S t i r l i n g , C. E. (1972). Radiographic l o c a l i z a t i o n of sodium pump s i t e s i n r a b b i t i n t e s t i n e . J . C e l l B i o l . 53, 704-714. 2+ Suzuki, S. (1981). Carbonic anhydrase, Mg2+-HC03'-ATPase and Mg Na+-K+-ATPase i n r a t i n t e s t i n a l mucosa: E f f e c t s of a d r e n a l ectomy and a l d o s t e r o n e a d m i n i s t r a t i o n . J . S t e r o i d Biochern. 14, 449-456. Swann, A. C , Crawley, J. N . , Grant, S. J., and Maas, J. W. (1981). Nonadrenergic s t i m u l a t i o n i n vivo i n c r e a s e s ( N a + , K + ) adenosine t r i p h o s p h a t a s e a c t i v i t y . L i f e Sci. 28, 251-256. Sweadner, K. J. 11979). Two molecular forms of (Na' + K+) s t i m u l a t e d ATPase i n b r a i n . S e p a r a t i o n and d i f f e r e n c e i n a f f i n i t y f o r s t r o p h a n t h i d i n . J. B i o l . Chern. 254, 6060-6067. T a t a , J. R. (1968). Co-ordinated formation o f membranes and bios y n t h e t i c a c t i v i t y d u r i n g growth and development. BBA L i b r . 11, 222-235. Vander Tuig, J. G . , Flynn, A. M . , and Romsos, D. R. (1981). Ventromedial hypothalamic l e s i o n s reduce t h e number of Na+, K+ATPase enzyme units i n s k e l e t a l muscle of weanling rats. Proc. SOC. E x p . B i o l . Med. 1 6 7 , 475-479. Voute, C. L. , Thummel, J. , and Brenner, M. (1975). Aldosterone eff e c t i n t h e e p i t h e l i u m of t h e f r o g skin--a new s t u d y a b o u t an o l d enzyme. J . S t e r o i d Biochern. 6 , 1175-1179. Wade, J. B . , O ' N e i l , R. G . , P r y o r , J . L . , and Boulpaep, E. L. (1979). Modulation o f c e l l membrane a r e a i n r e n a l c o l l e c t i n g t u b u l e s by c o r t i c o s t e r o i d hormones. J . C e l l B i o l . 81, 439445. Wickner, W. (1980). Assembly of p r o t e i n s i n t o membranes. S c i e n c e 210, 861-868. W i l l , P. C . , Longworth, J. W., Brake, E. T . , and Cook, J. S. ( 1 9 7 7 ) . Analysis o f i n t r a c e l l u l a r drug (ouabain) s e q u e s t r a t i o n as a mechanism o f d e t o x i f i c a t i o n . Mol. P h a m a c o l . 1 3 , 161-177.

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W i l l , P. C . , Lebowitz, J. L . , and Hopfer, U. ( 1 9 8 0 ) . I n d u c t i o n of m i l o r i d e - s e n s i t i v e sodium t r a n s p o r t i n t h e r a t c o l o n by m i n e r a l o c o r t i c o i d s . Am. J. P h y s i o l . 2 3 8 , F261-F268. W i l l , P. C . , D e L i s l e , R. C . , C o r t r i g h t , R. N . , and Hopfer, U. ( 1 9 8 1 ) . I n d u c t i o n of m i l o r i d e - s e n s i t i v e sodium t r a n s p o r t Ann. N.Y. A c a d . S c i . i n t h e i n t e s t i n e s by a d r e n a l s t e r o i d s . 372, 64-78. Wu, P. H . , and P h i l l i s , J. W. ( 1 9 8 0 ) . C h a r a c t e r i z a t i o n of r e c e p t o r mediated c a t e c h o l a m i n e a c t i v a t i o n o f r a t b r a i n c o r t i c a l N a + K+-ATPase. Int. J . B i o c h e m . 1 2 , 353-359. York, D. A . , Bray, G . A., and Yakimura, Y. (1978). An enzymatic d e f e c t i n t h e o b e s e (ob/ob) mouse: Loss of t h y r o i d - i n d u c e d sodium- and potassium-dependent a d e n o s i n e t r i p h o s p h a t a s e Proc. N a t l . A c a d . S c i . USA 7 5 , 477-481.

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CURRENT TOPICS IN MEMBRANES AND TRANSPORT, VOLUME 19

Biosynthesis of the Na,K-ATPase in MDCK Cells J . SHERMAN, T. MORIMOTO,

AND D.D.SABATIIW

Department of Cell Biology New York University School of Medicine New York, New York

I.

INTRODUCTION

P l a s m a membrane p r o t e i n s m e d i a t e t h e i n t e r a c t i o n s of c e l l s w i t h t h e i r environment and p l a y a c e n t r a l r o l e i n r e g u l a t i n g t h e composition of t h e i n t r a c e l l u l a r m i l i e u . B i o g e n e t i c mechanisms which govern t h e i n c o r p o r a t i o n o f s p e c i f i c p o l y p e p t i d e s i n t o plasma membranes and d e t e r m i n e t h e i r d i s p o s i t i o n w i t h r e s p e c t t o t h e p h o s p h o l i p i d b i l a y e r a r e , t h e r e f o r e , of p a r t i c u l a r i n terest t o c e l l b i o l o g i s t s . The plasma membrane A T P a s e , which i n e u k a r y o t i c c e l l s e x t r u d e s Na+ and e s t a b l i s h e s t h e h i g h i n t r a c e l l u l a r K+ c o n c e n t r a t i o n , c o n s i s t s of c a t a l y t i c o r a( 1 0 0 , 0 0 0 d a l t o n s ) and g l y c o p r o t e i n o r B- ( a b o u t 6 0 , 0 0 0 d a l t o n s ) s u b u n i t s ( c f . Sweadner and G o l d i n , 1 9 8 0 ; Stekhoven and B o n t i n g , 1981) which are t h o u g h t t o form a l a r g e i n t e g r a l membrane p r o t e i n complex. The a-subu n i t a p p e a r s t o s p a n t h e membrane, s i n c e it c o n t a i n s a o u a b a i n - b i n d i n g s i t e which i s exposed on t h e e x t r a c e l l u l a r s u r f a c e ( P e r r o n e and B l o s t e i n , 1973; Ruoho and Kyte, 753

Copynght 0 1983 by Academic Press, Inc. All rights of reproduction inany form reserved. ISBN 012-153319-0

J. SHERMAN eta/.

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1 9 7 4 ) and an amino a c i d r e s i d u e a c c e s s i b l e t o phosphoryl a t i o n on t h e c y t o p l a s m i c s i d e of t h e membrane (Whittam, 1 9 6 2 ; Uesugi e t al., 1 9 7 1 ) . The e x a c t d i s p o s i t i o n of t h e @ - s u b u n i tw i t h r e s p e c t t o t h e p h o s p h o l i p i d b i l a y e r has n o t been e s t a b l i s h e d , a l t h o u g h t h i s p o l y p e p t i d e a l s o can be l a b e l e d by a o u a b a i n a n a l o g a p p l i e d t o t h e c e l l s u r f a c e ( H a l l and Ruoho, 1 9 8 0 ) . C e l l s of r e n a l e p i t h e l i a , which a r e a c t i v e l y engaged i n i o n t r a n s p o r t , c o n t a i n h i g h c o n c e n t r a t i o n s of t h e Na,K-ATPase and t h e r e f o r e p r o v i d e a u s e f u l system f o r s t u d i e s on t h e b i o s y n t h e s i s of t h i s enzyme. The ATPase i s c o n f i n e d t o t h e b a s o l a t e r a l domains of t h e plasma membranes ( F u j i t a e t a l . , 1 9 7 2 ; Kyte, 1 9 7 6 1 , and t h i s r e s t r i c t e d l o c a l i z a t i o n of t h e enzyme i s thought t o p l a y an i m p o r t a n t r o l e i n e s t a b l i s h i n g t h e f u n c t i o n a l p o l a r i z a t i o n of t h e c e l l s . I n t h i s a r t i c l e w e r e p o r t s t u d i e s on t h e b i o s y n t h e sis of t h e ATPase c a r r i e d o u t employing t h e MDCK e p i t h e l i a l c e l l l i n e , which e x h i b i t s many of t h e s t r u c t u r a l and f u n c t i o n a l p r o p e r t i e s of p o l a r i z e d r e n a l e p i t h e l i a (Misfeldt e t a l . , 1976; Cereijido e t a l . , 1 9 7 8 ) , includi n g t h e development of j u n c t i o n a l complexes, which s e a l i n t e r c e l l u l a r s p a c e s and d e f i n e luminal and b a s o l a t e r a l c e l l s u r f a c e domains.

11.

METHODS

The p r o c e d u r e of J6rgensen ( 1 9 7 4 ) was used t o i s o l a t e t h e Na,K-ATPase from dog kidney. The i n d i v i d u a l s u b u n i t s were s e p a r a t e d by p r e p a r a t i v e sodium dodecyl s u l f a t e - p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s (SDS-PAGE) and i n j e c t e d i n t o r a b b i t s t o p r e p a r e a n t i b o d i e s which were p u r i f i e d by a f f i n i t y chromatography t o t h e immob i l i z e d enzyme. A.

C U L T U R E D CELLS

MDCK c e l l s were grown a t 37OC i n d i s p o s a b l e p l a s t i c r o l l e r b o t t l e s w i t h E a g l e ' s m i n i m a l e s s e n t i a l medium (Gibco) c o n t a i n i n g 1 0 % c a l f serum. Monolayers were h a r v e s t e d by s c r a p i n g i n a phosphate-buffered s a l i n e s o l u t i o n , and c e l l s w e r e broken by homogenization i n 0 . 5 M s u c r o s e w i t h 50 s t r o k e s of a t i g h t - f i t t i n g Dounce homog e n i z e r . Rough microsomes and f r e e polysomes w e r e isol a t e d a s d e s c r i b e d elsewhere (Feldman e t a l . , 1 9 8 2 ) .

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F o r i n v i v o l a b e l i n g o f MDCK c e l l s , 6 0 mM d i s h e s of l i g h t l y c o n f l u e n t MDCK c e l l s were p r e i n c u b a t e d 15-30 min i n 2 m l o f s e r u m - f r e e , m e t h i o n i n e - f r e e RPMI medium ( G i b c o ) b e f o r e t h e a d d i t i o n o f 62.5 v C i / m l [35S]methionine (New England N u c l e a r ) . Labeled c e l l s were h a r v e s t e d , a f t e r removal of t h e l a b e l i n g medium and t h r e e washings o f t h e monolayer w i t h p h o s p h a t e - b u f f e r e d s a l i n e , by s c r a p i n g i n t o 1 m l / p l a t e of 1 0 mM T r i s - H C 1 (pH 7 . 4 ) , 1 0 mM KC1, 1 mM MgC12, c o n t a i n i n g 0.5% w/v T r i t o n X-100. The l y s e d c e l l s were homogenized by f o u r s t r o k e s o f a Dounce homogenizer, and t h e n u c l e i were removed by 1-min c e n t r i f u g a t i o n i n an Eppendorf c e n t r i f u g e . TCA p r e c i p i t a t e s o f t h e p o s t n u c l e a r s u p e r n a t a n t s were s o l u b i l i z e d and p r o c e s s e d f o r i m m u n o p r e c i p i t a t i o n as d e s c r i b e d below. B.

IMMUNEPRECIPITATION AND S D S - P A G E

A T P a s e p o l y p e p t i d e s l a b e l e d i n v i v o or s y n t h e s i z e d i n v i t r o were r e c o v e r e d by i n d i r e c t i m m u n o p r e c i p i t a t i o n from SDS-solubilized samples employing p r o t e i n A Sephar o s e . TCA p r e c i p i t a t e s o f l a b e l e d c e l l s w e r e S Q l u b i l i z e d i n 1 M T r i s and SDS ( 1 0 0 mM T r i s (pH 8 . 5 ) and 2 % S D S f i -

nal c o n c e n t r a t i o n ) and b o i l e d f o r 2 min. T r a n s l a t i o n m i x t u r e s w e r e s i m i l a r l y t r e a t e d w i t h SDS and h e a t e d . A l l samples were d i l u t e d 4 - f o l d w i t h a b u f f e r c o n t a i n i n g 150 m M NaC1, 50 mM T r i s - H C 1 (pH 7 . 4 1 , 5 mM EDTA, 2.5% w/v T r i t o n X-100, and 1 0 0 u n i t s / m l t r a s y l o l , and a s u i t a b l e amount o f a f f i n i t y - p u r i f i e d c a t a l y t i c o r g l y c o p r o t e i n IgG w a s added. The i m m u n o p r e c i p i t a t i o n m i x t u r e s were i h c u b a t e d f o r 2 h r a t room t e m p e r a t u r e , o r o v e r n i g h t a t 4 O C , and t h e n 2 h r a t room t e m p e r a t u r e w i t h p r o t e i n A Sepharose 4 B (Pharmacia , Bromma , Sweden) t o a d s o r b t h e a n t i b o d y - a n t i g e n complexes. A f t e r r e p e a t i n g 'washings o f t h e Sepharose b e a d s , t h e a d s o r b e d p r o t e i n s were s o l u b i l i z e d by h e a t i n g f o r 2 min a t 1 0 0 O C i n 5% SDS c o n t a i n i n g 50 mM T r i s - H C 1 (pH 8 . 5 ) , 5 mM EDTA, and 1 0 mM d i t h i o Samples were a n a l y z e d by e l e c t r o p h o r e t h r e i t o l (DTT) s i s on SDS-polyacrylamide g e l s ( 1 0 % o r 6-12% g r a d i e n t ) f o l l o w e d by f l u o r o g r a p h y o f d r i e d g e l s u s i n g Kodak (XR-5) f i l m (Laskey and M i l l s , 1 9 7 5 ) .

.

C.

PREPARATION O F mRNA

T o t a l RNA was e x t r a c t e d from c u l t u r e d c e l l s u s i n g g u a n i d i n e h y d r o c h l o r i d e ( p r a c t i c a l g r a d e , Sigma) (Cox , 1 9 6 8 ) . mRNA w a s p u r i f i e d by two c y c l e s of oligo-dT

J. SHERMANeta/.

756

c e l l u l o s e ( C o l l a b o r a t i v e Research, Waltham, Massac h u s t t s ) chromatography (Aviv and Leder, 1 9 7 2 ) . D.

In v i t r o P R O T E I N S Y N T H E S I S

Poly(A) + mRNA (0.10 OD 6 /50 111 t r a n s l a t i o n v o l ume) o r polysomes ( 1 . 0 OD260758 p 1 t r a n s l a t i o n volume) were used t o program n u c l e a s e - t r e a t e d r a b b i t r e t i c u l o c y t e l y s a t e s (Pelham and Jackson, 1 9 7 6 ) o r wheat germ e x t r a c t s (Roman e t a l . , 1976) which were i n c u b a t e d a t 28' o r 25OC, r e s p e c t i v e l y . [35S]Methionine used i n t h e t r a n s l a t i o n s w a s o b t a i n e d from N e w England Nuclear. E.

SPECIAL MATERIALS

Tunicamycin was a g i f t from D r . F. Tomita (Kyowa Hakko Kogyo Co., L t d . , Tokyo, J a p a n ) . Monensin was g e n e r o u s l y s u p p l i e d by E l i L i l l y Co., I n d i a n a p o l i s , Indiana.

111.

RESULTS

In v i t r o t r a n s l a t i o n experiments w i t h f r e e and bound polysomes o b t a i n e d from MDCK c e l l s were c a r r i e d o u t t o d e t e r m i n e t h e s u b c e l l u l a r s i t e of s y n t h e s i s of t h e ATPase p o l y p e p t i d e s . I t was found t h a t bound, b u t n o t f r e e , polysomes s y n t h e s i z e d a 3 8 , 0 0 0 M y p o l y p e p t i d e which was immunoprecipitated w i t h a n t i b o d i e s a g a i n s t t h e B-subunit ( F i g . l a and b ) . The e l e c t r o p h o r e t i c mob i l i t y of t h i s p o l y p e p t i d e was much g r e a t e r t h a n t h a t of t h e mature 8-subunit ( 6 0 , 0 0 0 M ~ ) , which w a s immunop r e c i p i t a t e d from c e l l s l a b e l e d f o r 4 h r ( F i g . l e ) , b u t was i n d i s t i n g u i s h a b l e i n s i z e from t h e primary t r a n s l a t i o n p r o d u c t r e c o v e r e d from t r a n s l a t i o n m i x t u r e s programmed w i t h t o t a l mRNA from MDCK c e l l s ( F i g . l c ) . The f i n d i n g t h a t t h e 8-subunit of t h e ATPase i s s y n t h e s i z e d i n bound polysomes i m p l i e s t h a t t h e polypept i d e i s c o t r a n s l a t i o n a l l y i n s e r t e d i n t o t h e endoplasmic r e t i c u l u m (ER) membranes. To determine i f t h i s i n s e r t i o n i s accompanied by t h e concomitant c l e a v a g e of an amino t e r m i n a l i n s e r t i o n s i g n a l , t h e e l e c t r o p h o r e t i c m o b i l i t y of t h e primary t r a n s l a t i o n p r o d u c t was compared t o t h a t of t h e p o l y p e p t i d e s y n t h e s i z e d i n c e l l s t r e a t e d w i t h tunicamycin, a drug which i n h i b i t s c o t r a n s l a t i o n a l

BIOSYNTHESIS OF Na,K-ATPase IN MDCK CELLS

a

b

c

757

d

e

F i g . 1 . S y n t h e s i s o f the 6 - s u b u n i t o f the Na,K-ATPase on membrane-bound p o l y s o m e s . I m m u n o p r e c i p i t a t e s f r o m r e t i c u l o c y t e l y s a t e s programmed w i t h f r e e ( a ) and bound ( b ) p o l y s o m e s and tot a l mRNA ( c ) from MDCK c e l l s w e r e compared t o the g l y c o p r o t e i n s u b u n i t i s o l a t e d f r o m MDCK c e l l s l a b e l e d w i t h [ 3 5 S ] m e t h i o n i n e ( 1 2 5 p C i / p l a t e ) i n the p r e s e n c e ( a ) o r a b s e n c e ( e ) o f t u n i c a m y c i n ( 3 p g / m l , 2-hr p r e t r e a t m e n t ) . S a m p l e s were a n a l y z e d b y electrop h o r e s i s on a 1 0 % p o l y a c r y l a m i d e g e l .

g l y c o s y l a t i o n ( T r a c z and Lampen, 1975; S t r u c k and L e n n a r z , 1 9 7 7 ) . N o d i f f e r e n c e in a p p a r e n t m o l e c u l a r w e i g h t w a s d e t e c t e d ( F i g . I d ) , which s u g g e s t s t h a t , u n l i k e m o s t s e c r e t o r y and several membrane p r o t e i n s ( c f . K r e i b i c h e t a l . , 1980a; E m r e t a l . , 1980; L o d i s h

758

J. SHERMAN et el.

F i g . 2 . Co- and p o s t t r a n s l a t i o n a l m o d i f i c a t i o n s o f the 8-subunit. Dishes of l i g h t l y c o n f l u e n t MDCK c e l l s w e r e p r e i n c u b a t e d w i t h either no d r u g (control) I t u n i c a m y c i n (3 v g / m l 2 hr) , or mnensin M, 1 h r ) i n c o m p l e t e medium. The preincubation medium was r e p l a c e d w i t h l a b e l i n g medium, and f o l l o w i n g a 30-min i n c u b a t i o n , [ 3 5 S ] m e t h i o n i n e was added t o e a c h p l a t e . T h e monol a y e r s were h a r v e s t e d 2 hr l a t e r , and S D S - s o l u b i l i z e d TCA p r e c i p i t a t e s o f the p o s t n u c l e a r s u p e r n a t a n t s w e r e a s s a y e d for l a b e l e d 8 - s u b u n i t b y i m m u n o p r e c i p i t a t i o n and e l e c t r o p h o r e s i s on a 6-12% p o l y a c r y l a m i d e g e l : ( a ) control , ( b ) t u n i c a m y c i n , (c) monensin.

1 9 8 1 ) , t h e s m a l l s u b u n i t of t h e A T P a s e does n o t c o n t a i n a t r a n s i e n t amino t e r m i n a l i n s e r t i o n s i g n a l . Had a s i g n a l been removed from t h e 8-subunit s y n t h e s i z e d i n c e l l s t r e a t e d w i t h tunicamycin, a d e t e c t a b l e increase i n t h e e l e c t r o p h o r e t i c m o b i l i t y of t h i s r e l a t i v e s m a l l p o l y p e p t i d e would have r e s u l t e d . I t a p p e a r s , t h e r e f o r e , t h a t t h e 8-subunit should be added t o t h e growing l i s t of membrane p r o t e i n s t h a t a r e s y n t h e s i z e d i n bound polysomes, b u t a r e n o t p r o c e s s e d p r o t e o l y t i c a l l y during t h e i r cotr an slatio n al i n s e r t i o n et al.,

BIOSYNTHESIS OF Na,K-ATPaseIN MDCK CELLS

759

i n t o t h e membranes ( B o n a t t i and B l o b e l , 1979; Chyn e t a l . , 1979; S c h e c h t e r et a l . , 1979; Bar-Nun e t a l . , 1980; c f . K r e i b i c h e t a1 1980b; Okada e t a l . , 1 9 8 2 ) . A n a l y s i s of 3 S & l a b e l e d p o l y p e p t i d e s s y n t h e s i z e d i n normal and t u n i c a m y c i n - t r e a t e d c e l l s a l s o d e m o n s t r a t e d t h a t e x t e n s i v e co- and p o s t t r a n s l a t i o n a l g l y c o s y l a t i o n of t h e 8 - s u b u n i t a c c o u n t s f o r t h e l a r g e d i f f e r e n c e i n app a r e n t m o l e c u l a r w e i g h t between t h e p r i m a r y t r a n s l a t i o n p r o d u c t and t h e mature s u b u n i t . I n b r i e f l y l a b e l e d MDCK c e l l s (Fig. 2a) it w a s p o s s i b l e t o i d e n t i f y , i n a d d i t i o n t o t h e mature glycoprotein ( 6 0 , 0 0 0 M r ) , a polypeptide (45 ,0 0 0 M r ) which r e p r e s e n t e d an i n t e r m e d i a t e s t a g e i n t h e g l y c o s y l a t i o n p r o c e s s . T h i s p r o d u c t must r e s u l t from t h e c o t r a n s l a t i o n a l t r a n s f e r of o l i g o s a c c h a r i d e s t o t h e n a s c e n t c h a i n , s i n c e it w a s n o t p r e s e n t i n t h e tunicamyc i n - t r e a t e d c e l l s , which c o n t a i n e d o n l y an u n g l y c o s y l a t e d p o l y p e p t i d e o f M r 38,000 ( F i g . 2 b ) . Direct e v i d e n c e f o r t h e c o t r a n s l a t i o n a l g l y c o s y l a t i o n o f t h e B-subunit w a s o b t a i n e d i n c o l l a b o r a t i o n w i t h D r . D. Colman u s i n g t r a n s l a t i o n s y s t e m s programmed w i t h t o t a l r a t b r a i n mRNA and supplemented w i t h microsomal membranes. Under t h e s e cond i t i o n s a membrane a s s o c i a t e d p o l y p e p t i d e of M r 45,000 was o b t a i n e d . The second g l y c o s y l a t i o n s t a g e d u r i n g t h e m a t u r a t i o n of t h e 8 - s u b u n i t , which i n c r e a s e d t h e a p p a r e n t m o l e c u l a r w e i g h t compared t o t h a t c h a r a c t e r i s t i c o f t h e m a t u r e p o l y p e p t i d e ( M 6~ 0 , 0 0 0 ) , a p p e a r s t o t a k e p l a c e i n t h e G o l g i a p p a r a t u s , s i n c e i t w a s p a r t i a l l y b l o c k e d by t r e a t ment o f c e l l s w i t h t h e sodium ionophore monensin ( F i g . 2 c ) . This drug is thought t o impair i n t r a c e l l u l a r t r a f f i c o f s e c r e t o r y ( T a r t a k o f f and V a s s a l l i , 1978; Uchida e t a l . , 1980) and membrane p r o t e i n s (Johnson and S c h l e s i n g e r , 1980; Ktitiriainen et a l . , 1980; S t r o u s and L o d i s h , 1980) a f t e r t h e y e x i t from t h e ER, b u t b e f o r e t h e y r e a c h t h e plasma membrane. The c o n c l u s i o n t h a t t h e 45,000 M~ p o l y p e p t i d e det e c t e d i n MDCK c e l l s r e p r e s e n t s an immature m i C r O S O m a 1 form o f t h e f3-subunit, which must t r a v e r s e t h e G o l g i app a r a t u s f o r i t s m a t u r a t i o n and t r a n s f e r t o t h e plasma membrane, has been s u b s t a n t i a t e d by p u l s e c h a s e e x p e r i ments i n which t h e d i s t r i b u t i o n of newly s y n t h e s i z e d $ - s u b u n i t w a s examined i n s u b c e l l u l a r f r a c t i o n s o f MDCK c e l l 9 l a b e l e d w i t h 35s m e t h i o n i n e . Five minutes a f t e r a d m i n i s t r a t i o n of t h e l a b e l , a 45,000 M r . 8 - s u b u n i t p o l y p e p t i d e was d e t e c t e d i n p u r i f i e d rough microsomes. T h i r t y m i n u t e s l a t e r , however, t h i s p o l y p e p t i d e w a s no l o n g e r p r e s e n t i n t h i s f r a c t i o n , a s e x p e c t e d of a p r o d u c t i n t r a n s i t t o a d i f f e r e n t c e l l u l a r l o c a t i o n , presumably t h e plasma membrane. A t t h a t t i m e t h e 45,000 M r p o l y p e p t i d e w a s s t i l l d e t e c t e d i n a f r a c t i o n of smooth memb r a n e s where a t a b o u t 4 5 min t h e m a t u r e form w a s found.

760

J. SHERMAN eta/.

ur

a

b

F i g . 3 . In vivo and i n v i t r o s y n t h e s i s o f the a - s u b u n i t o f the Na,K-ATPase. The a - s u b u n i t o f the ATPase was i m m u n o p r e c i p i t a t e d from MDCK c e l l s l a b e l e d f o r 30 m i n w i t h [ 3 5 S ] m e t h i o n i n e i n the a b s e n c e ( a ) or p r e s e n c e (b) o f t u n i c a m y c i n . These i n vivo s y n t h e s i z e d p o l y p e p t i d e s were compared t o those o b t a i n e d from r e t i c u l o c y t e l y s a t e s programmed w i t h f r e e ( c ) and bound (a) p o l y somes p r e p a r e d from MDCK c e l l s . An i m m u n o p r e c i p i t a t e d p r o d u c t o f t o t a l p o l y s o m e s i s shown i n l a n e ( e ) . T r a n s l a t i o n s of MDCK t o t a l mRNA i n a r e t i c u l o c y t e l y s a t e (f) and wheat germ l y s a t e s ( 9 ) and ( h ) y i e l d e d s i m i l a r r e s u l t s . S a m p l e s w e r e a n a l y z e d on a 10% g e l .

S t u d i e s on t h e b i o s y n t h e s i s of t h e A T P a s e a - s u b u n i t were a l s o c a r r i e d o u t w i t h c u l t u r e d MDCK c e l l s , a s w e l l as c e l l - f r e e s y s t e m s programmed w i t h polysomes o r p u r i f i e d mRNA. The a - s u b u n i t immunoprecipitated from c e l l s l a b e l e d w i t h [35S]methionine f o r 30 min had t h e same s i z e a s t h e m a t u r e s u b u n i t p u r i f i e d from dog k i d n e y . A p o l y p e p t i d e of t h e same m o b i l i t y w a s o b t a i n e d from t u n i c a m y c i n - t r e a t e d c e l l s ( F i g . 3a and b) a s e x p e c t e d f o r a product t h a t does n o t c o n t a i n asparagine-linked oligosaccharide chains.

BIOSYNTHESIS OF Na.K-ATPase IN MDCK CELLS

76 1

A s was t h e case f o r t h e 8-subunit t h e newly s y n t h e s i z e d a - s u b u n i t l a b e l e d d u r i n g s h o r t (5-min) p u l s e s w i t h [35S]methionine w a s found i n t r a c e l l u l a r l y i n a s s o c i a t i o n w i t h rough microsomal membranes, b e f o r e i t a p p e a r e d i n a f r a c t i o n o f smooth membranes which c o n t a i n e d plasma memb r a n e f r a g m e n t s . A d i r e c t d e t e r m i n a t i o n o f t h e s i t e of s y n t h e s i s of t h e a - s u b u n i t w a s hampered by d i f f i c u l t i e s i n o b t a i n i n g a n i n v i t r o t r a n s l a t i o n p r o d u c t of t h e exp e c t e d s i z e , when f r e e o r bound polysomes ( F i g . 3c and d ) o r p u r i f i e d mRNAs ( F i g . 3 f , g , and h ) from MDCK c e l l s were u s e d t o program r e t i c u l o c y t e o r wheat germ t r a n s l a t i o n s y s t e m s . Although o c c a s i o n a l l y a 1 0 0 , 0 0 0 Mr-pyod u c t w a s r e c o v e r e d ( F i g . 3e and h ) , an i m m u n o p r e c i p i t a b l e p r o d u c t of a p p r o x i m a t e l y 85,000 M~ w a s g e n e r a l l y o b t a i n e d and f r e q u e n t l y t h e y i e l d o f t h i s p r o d u c t w a s h i g h e r when It appears f r e e r a t h e r t h a n bound polysomes were used. t h a t t h i s p r o d u c t r e s u l t e d from i n c o m p l e t e t r a n s l a t i o n r a t h e r t h a n from p r o t e o l y t i c d e g r a d a t i o n , as it w a s a l s o found when a combination o f p r o t e a s e i n h i b i t o r s were added t o t h e t r a n s l a t i o n s y s t e m s . R e c e n t l y , i n c o l l a b o r a t i o n with,N. Nabi, w e have found t h a t i n c o n t r a s t t o t h e s i t u a t i o n w i t h polysomes from MDCK c e l l s , i n v i t r o t r a n s l a t i o n o f b r a i n polysomes y i e l d s a p r o d u c t of t h e s i z e e x p e c t e d f o r t h e l a r g e s t ( a + ) o f t h e two forms of t h e a - s u b u n i t c h a r a c t e r i s t i c of b r a i n A T P a s e (Sweadner, 1 9 7 9 ) . I n t h i s i n s t a n c e t h e t r a n s l a t a b l e mRNA w a s o n l y c o n t a i n e d i n bound polysomes. Taken t o g e t h e r , o u r o b s e r v a t i o n s s u g g e s t t h a t , a l t h o u g h a s i z a b l e f r a c t i o n of mRNA f o r a - s u b u n i t may be found i n f r e e polysomes o f MDCK c e l l s where p a r t i a l s y n t h e s i s may t a k e p l a c e , i n s e r t i o n of t h e p o l y p e p t i d e i n t o t h e ER membrane a c t u a l l y i s a c o t r a n s l a t i o n a l e v e n t . Using i n v i t r o systems c o n t a i n i n g microsomal memb r a n e s w e are c u r r e n t l y a t t e m p t i n g t o e l u c i d a t e t h e sequence o f e v e n t s i n v o l v e d i n t h e s y n t h e s i s and i n c o r p o r a t i o n o f t h e a - s u b n i t i n t o t h e membrane and t h e s i t e and mode of a s s o c i a t i o n of t h e two s u b u n i t s i n t o a f u n c t i o n a l complex. The MDCK c u l t u r e c e l l system a l s o a p p e a r s t o p r o v i d e a s u i t a b l e model t o s t u d y t h e mechanisms t h a t est a b l i s h and m a i n t a i n t h e r e s t r i c t e d l o c a l i z a t i o n o f t h e enzyme t o t h e b a s o l a t e r a l domains of e p i t h e l i a l c e l l s and thus determine t h e i r functional p o l a r i t y .

ACKNOWLEDGMENT

W e thank M r . B i l l Dolan, M s . Susan Malamet, and M s . Harriet S n i t k i n € o r t i s s u e c u l t u r e work. We are g r a t e f u l to M r s . Myrna Chung, M r . B r i a n Z i e t l o w , and Ms. Jody C u l k i n € o r a s s i s t a n c e i n t h e The work w a s s u p p o r t e d by N I H g r a n t s p r e p a r a t i o n of t h e m a n u s c r i p t . t o D r . S a b a t i n i AG 01461, GM 2 0 2 7 7 , and AG 00378.

762

J. SHERMAN eta/.

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Stekhoven, F. S., and Bonting, S. L. (1981). T r a n s p o r t adenosine t r i p h o s p h a t a s e s : p r o p e r t i e s and f u n c t i o n s . P h y s i o l . R e v . 6 1 , 1-76. S t r o u s , E. J. A. M . , and Lodish, M. F. (1980). I n t r a c e l l u l a r t r a n s p o r t of s e c r e t o r y and membrane p r o t e i n s i n hepatoma Cell 22, c e l l s i n f e c t e d by v e s i c u l a r s t o m a t i t i s v i r u s . 709-717. S t r u c k , D. K . , and Lennarz, W. J. (1977). Evidence f o r t h e p a r t i cipation of saccharide-lipids i n the synthesis of the oligos a c c h a r i d e c h a i n o f ovalbumin. J. B i o l . Chem. 2 5 2 , 10071113. Sweadner, K. J. (1979). Two m o l e c u l a r forms of ( N a + + K+)J . B i o l . Chem. 2 5 4 , 6060-6067. s t i m u l a t e d ATPase i n b r a i n . Sweadner, K. J . , and Goldin, S. M. (1980). Active t r a n s p o r t of sodium and potassium i o n s . N. E n g l . J . Med. 302, 777-783. T a r t a k o f f , A . , and V a s s a l l i , P. (1978). Comparative s t u d i e s of J. Cell i n t r a c e l l u l a r transport of secretory proteins. B i o l . 7 9 , 694-707. Tracz, J. S . , and Lampen, 0. (1975). Tunicamycin i n h i b i t i o n of p o l y i s o p r e n y l N-acetylglucosaminyl phosphate formation i n B i o c h e m . B i o p h y s . R e s . Commun. 6 5 , c a l f l i v e r microsomes. 248-257. uchida, N . , Smilowitz, H . , Ledger, P. W., and Tanzer, M. L. (1980). K i n e t i c s t u d i e s of t h e i n t r a c e l l u l a r t r a n s p o r t of p r o c o l l a g e n and f i b r o n e c t i n i n human f i b r o b l a s t s - - E f f e c t s of t h e monoval e n t ionophore monensin. J . B i o l . Chem. 2 5 5 , 8638-8644. Uesugi, S . , Dulak, N . C . , Dixon, J. F . , Hexum, T D . , Dahl, J. L . , Perdue, J. F., and Hokin, L. E. (1971). S t u d i e s on t h e c h a r a c t e r i z a t i o n of t h e sodium-potassium t r a n s p o r t adenosine J. B i o l . Chem. 2 4 6 , 531-543. triphosphatase. Whittam, R. (1962). The asymmetrical s t i m u l a t i o n o f a membrane adenosine t r i p h o s p h a t a s e i n r e l a t i o n t o a c t i v e c a t i o n t r a n s port. B i o c h e m . J . 8 4 , 110-118.

CURRENT TOPICS IN MEMBRANES AND TRANSPORT. VOLUME 19

Possible Functional Differences between the Two Na,K-ATPases of the Brain KATHLEEN J. SWEALINER' Depanment of Neurobiology Harvard Medical School Boston, Massachusetts

I.

INTRODUCTION

Na,K-ATPase a c t i v i t y i s p r e s e n t i n high concentrat i o n s i n t h e b r a i n , where i t h a s s e v e r a l i m p o r t a n t r o l e s i n t h e complex and f i n e l y t u n e d c o n t r o l o f t h e i o n i c environment. I t m a i n t a i n s t h e i o n g r a d i e n t s which are t h e d r i v i n g f o r c e f o r t h e a c t i o n p o t e n t i a l and f o r N a dependent a c t i v e t r a n s p o r t s y s t e m s f o r Ca e f f l u x , neurot r a n s m i t t e r u p t a k e , and n u t r i e n t u p t a k e . I t i s p r e s e n t i n g l i a l c e l l s , which may have a c r u c i a l r o l e i n t h e upt a k e and c l e a r a n c e o f e x t r a c e l l u l a r K . And b e c a u s e it i s e l e c t r o g e n i c , i t s a c t i v i t y has a d i r e c t e f f e c t on membrane p o t e n t i a l i n n e r v e s , which a f f e c t s s y n a p t i c i n t e g r a t i o n , and which h a s been proposed t o have a r o l e i n

'Present a d d r e s s : N e u r o s u r g i c a l R e s e a r c h , M a s s a c h u s e t t s G e n e r a l H o s p i t a l and D e p a r t m e n t of P h y s i o l o g y , Harvard M e d i c a l School, Boston, M a s s a c h u s e t t s . 765

Copyright 0 1983 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-153319.0

KATHLEEN J. SWEADNER

766

s e n s o r y a d a p t a t i o n and pacemaker modulation a s w e l l (reviewed i n DeWeer, 1 9 7 5 ) . There are i n d i c a t i o n s t h a t t h e m a n i f e s t a c t i v i t y of t h e Na,K-ATPase c a n be regul a t e d , and i n t h e nervous system, s u c h r e g u l a t i o n h a s been proposed a s a g e n e r a t o r o f slow i n h i b i t o r y synapt i c p o t e n t i a l s and a s a modulator of t r a n s m i t t e r release. Attempts have been made t o l o c a l i z e t h e N a , K A T P a s e i n t h e b r a i n by b i o c h e m i c a l , h i s t o l o g i c a l , and immunocytochemical t e c h n i q u e s , and t h e b e s t e v i d e n c e i n d i c a t e s t h a t it i s p r e s e n t i n h i g h e s t concentrations i n neurons and a s t r o c y t e s . Immunocytology w i t h t h e peroxidase/antiperoxidase t e c h n i q u e h a s d e m o n s t r a t e d t h e p r e s e n c e o f Na,K-ATPase i n t h e membranes o f g l i a , and a p p a r e n t l y c l u s t e r e d a t t h e nodes o f Ranvier i n m y e l i n a t e d n e r v e (Wood et al., 1 9 7 7 ; Schwartz et a l . , 1981). The p r e s e n c e of two d i f f e r e n t N a , K - A T P a s e s i n t h e b r a i n w a s f i r s t s u s p e c t e d from t h e h e t e r o g e n e i t y i n t h e b i n d i n g k i n e t i c s f o r o u a b a i n . The k i n e t i c d a t a were i n t e r p r e t e d a s e v i d e n c e f o r t w o t y p e s o f N a, K -A T Pases, b u t l a t e r f o r t w o i n t e r c o n v e r t i b l e , n e g a t i v e l y coopera t i v e b i n d i n g s i t e s on t h e same enzyme ( T a n i g u c h i and I i d a , 1 9 7 2 ; Erdmann and Schoner, 1 9 7 3 ) . Marks and Seeds (1978) showed t h a t r e a g g r e g a t e b r a i n c e l l c u l t u r e s had complex ouabain-binding k i n e t i c s , whereas monolayer c u l t u r e s ( i n which few n e u r o n s s u r v i v e ) had s i m p l e k i n e t i c s , which s u g g e s t e d t h a t t h e c o m p l e x i t y had a c e l l u l a r r a t h e r t h a n k i n e t i c o r i g i n . Urayama and Nakao (1979) a l s o came t o t h e c o n c l u s i o n t h a t t h e d a t a were best f i t by t h e e x i s t e n c e o f two N a , K - A T P a s e s , based on s t u d i e s of t h e e f f e c t s of t h e K i o n and of t h e s e n s i t i v i t y t o N-ethylmaleimide. T h e r e i s e v i d e n c e now f o r t h e e x i s t e n c e o f two d i f f e r e n t N a , K - A T P a s e s i n t h e b r a i n (Sweadner, 1979; P e t r a l i and S u l a k h e , 19801, b r i n e shrimp n a u p l i i ( P e t e r s o n et a l . , 1978) , t h e l e n s ( N e v i l l e et a l . , 1978; Takemoto et a l . , 19811, and p o s s i b l y i n a d i p o s e c e l l s (Resh et a 1 1980). The two N a , K - A T P a s e s from mammalian b r a i n have b e e n s e p a r a t e d , and t h e i r c a t a l y t i c s u b u n i t s are found t o have s t r u c t u r a l d i f f e r e n c e s a s w e l l a s d i f f e r e n c e s i n t h e i r a f f i n i t i e s f o r cardiac g l y c o s i d e s (Sweadner, 1979) A f i r s t i n d i c a t i o n o f d i f f e r e n t p h y s i o l o g i c a l r o l e s comer from t h e o b s e r v a t i o n t h a t t h e y a r e a s s o c i a t e d w i t h d i f f e r e n t k i n d s o f c e l l s : one i s found i n a s t r o c y t e s and i n unmyelinated s y m p a t h e t i c n e u r o n s , whereas t h e o t h e r i c found i n axolemma from m y e l i n a t e d n e r v e . G e n t l e t i s s u e f r a c t i o n a t i o n i s s u f f i c i e n t t o s e p a r a t e t h e two N a , K A T P a s e s from b r a i n , a n e s s e n t i a l f i r s t s t e p i n t h e molec u l a r d i s s e c t i o n of t h e i r t r a n s p o r t f u n c t i o n .

.,

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TWO Na.K-ATPasesOF THE BRAIN

11.

SEPARATION O F THE TWO N a , K - A T P a s e s

OF THE BRAIN

The e x i s t e n c e o f two forms o f t h e enzyme was f i r s t d i s c o v e r e d by t h e a p p e a r a n c e of two bands a t t h e molecul a r w e i g h t of t h e a - s u b u n i t on h i g h - r e s o l u t i o n S D S g e l s (Sweadner, 1 9 7 9 ) . These were b o t h i d e n t i f i e d a s Na,KA T P a s e by t h e i r s p e c i f i c N a - s t i m u l a t e d , K-discharged p h o s p h o r y l a t i o n from [32P]ATP. One of t h e two forms h a s t h e same e l e c t r o p h o r e t i c m o b i l i t y a s t h e a - s u b u n i t of t h e p u r i f i e d k i d n e y N a , K - A T P a s e , and i s c a l l e d a . The o t h e r form h a s a s l o w e r m o b i l i t y , e q u i v a l e n t t o t h e m o b i l i t y e x p e c t e d if t h e p o l y p e p t i d e c h a i n were l a r g e r by 2 0 amino a c i d s . Because i t a p p e a r s t o be l a r g e r , it i s c a l l e d a ( + ) . The number of r e a c t i v e s u l f h y d r y l groups on t h e a and a ( + ) bands was measured by r e a c t i o n it w a s found t h a t a ( + ) w i t h ~ - [ ~ H ] e t h y l m a l e i m i d eand , h a s two more s u l f h y d r y l s t h a n a under t h e c o n d i t i o n s used. This i s c o n s i s t e n t w i t h t h e hypothesis t h a t a ( + ) h a s an e x t r a s t r e t c h of amino a c i d s , b u t d o e s n o t p r o v e it: a p o s t t r a n s l a t i o n a l modification such as glycosylat i o n might change t h e p r o t e i n ' s c o n f o r m a t i o n a n d expose more s u l f h y d r y l s t o t h e r e a g e n t . The s e n s i t i v i t i e s of t h e two forms t o t r y p t i c d i g e s t i o n and c r o s s - l i n k i n g were a l s o found t o be d i f f e r e n t , b u t t h e y had s i m i l a r C l e v e l a n d p e p t i d e maps, which i n d i c a t e s t h a t t h e i r s t r u c t u r e s are c l o s e l y r e l a t e d . S e p a r a t i o n o f t h e two N a , K - A T P a s e s w a s a c h i e v e d by f r a c t i o n a t i n g b r a i n t i s s u e i n t o axolemmal and g l i a l p r e p a r a t i o n s . Axolemma from m y e l i n a t e d n e r v e w a s p r e p a r e d by t h e method o f D e V r i e s (19811, and was found t o c o n t a i n o n l y t h e a ( + ) form. T h i s o b s e r v a t i o n w a s conf i r m e d by P e t r a l i and Sulakhe (19801, who r e p o r t e d t h e same g e l p a t t e r n . A s t r o c y t e s were p r e p a r e d by p r i m a r y c e l l c u l t u r e from n e o n a t a l r a t b r a i n (Cummins and G l o v e r , 1 9 7 8 ) , and w e r e found t o c o n t a i n o n l y t h e a form (Sweadner, 1 9 7 9 ) . These t e c h n i q u e s are u s e f u l f o r p r e p a r i n g t h e two enzymes f o r b i o c h e m i c a l a n a l y s i s , b u t are n o t proof of a n e u r o n a l v e r s u s g l i a l d i s t r i b u t i o n of t h e t w o forms i n b r a i n . Because t h e axon i s a h i g h l y s p e c i a l i z e d s t r u c t u r e , t h e axolemma p r e p a r a t i o n i s v e r y p o o r i n s y n a p t i c and n e u r o n a l p e r i k a r y a l membrane, and so i s n o t n e c e s s a r i l y r e p r e s e n t a t i v e o f a l l n e u r a l memb r a n e . S y m p a t h e t i c n e u r o n s were found t o e x p r e s s o n l y t h e a form, and p r e p a r a t i o n s of synaptosomes c o n t a i n e d b o t h forms. Synaptosome p r e p a r a t i o n s a r e p r o b a b l y h e a v i l y c o n t a m i n a t e d w i t h membrane from b o t h axons and g l i a , and so t h e l a s t r e s u l t i n p a r t i c u l a r i s u n i n t e r p r e t a b l e . Only a n a t o m i c a l t e c h n i q u e s w i l l g i v e a s a t i s f a c t o r y answer t o t h e q u e s t i o n of t h e r e a l c e l l u l a r d i s t r i b u t i o n o f t h e t w o forms.

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F i g . 1 . S t r o p h a n t h i d i n i n h i b i t i o n o f r a t Na,K-ATPases. ATPase a c t i v i t y was a s s a y e d i n the p r e s e n c e o f 140 mM Na, 20 mM K, 3 mM Mg, and 3 mM ATP, w i t h and w i t h o u t d i f f e r e n t c o n c e n t r a t i o n s o f s t r o p h a n t h i d i n ( S i g m a ) , and the r e a c t i o n was s t o p p e d a f t e r 8 min, a s i n Sweadner ( 1 9 7 9 ) . S t r o p h a n t h i d i n was d i l u t e d 5 0 - f o l d i n t o the enzyme r e a c t i o n m i x t u r e from stock s o l u t i o n s i n d i m e t h y l f o r m a m i d e , a n d d i m e t h y l f o r m a m i d e was added t o the controls. ( 0 )Axolemma; ( A ) a s t r o c y t e ; ( m ) k i d n e y ; ( 0 ) s y m p a t h e t i c n e u r o n s . Strophanthidin-insensitive (Mg-ATPase) a c t i v i t y h a s been s u b t r a c t e d f r o m the d a t a shown. Reproduced w i t h p e r m i s s i o n f r o m the J o u r n a l o f B i o l o g i c a l C h e m i s t r y .

Evidence was p r e s e n t e d i n t h e same p a p e r (Sweadner, 1 9 7 9 ) t h a t t h e two forms have d i f f e r e n t a f f i n i t i e s f o r

cardiac glycosides. In the r a t these a f f i n i t i e s d i f f e r by t h r e e o r d e r s of magnitude, t h e a form found i n kidney and c a r d i a c t i s s u e b e i n g t h e l o w - a f f i n i t y form, and t h e a ( + ) form found i n t h e b r a i n b e i n g t h e higha f f i n i t y form. The c a r d i a c g l y c o s i d e a f f i n i t i e s of t h e two forms of Na,K-ATPase i n b r a i n were measured f i r s t by t h e i n h i b i t i o n of c o v a l e n t p h o s p h o r y l a t i o n of t h e enzyme by [32P]ATP, and l a t e r t h e r e s u l t w a s confirmed when t h e two enzymes had been s e p a r a t e d i n f u l l y a c t i v e form. The a f f i n i t i e s of t h e two s e p a r a t e d forms, measured under t h e c o n d i t i o n s of t h e ATPase a s s a y , a r e shown i n F i g . 1. The d i f f e r e n c e i n a f f i n i t i e s may be i m p o r t a n t i f t h e r e i s an endogenous analog f o r t h e c a r d i a c g l y c o s i d e s analogous t o e n k e p h a l i n . S e v e r a l g r o u p s have r e p o r t e d i n h i b i t i o n of ATPase a c t i v i t y by p e p t i d e s

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and c a r d i a c g l y c o s i d e - l i k e endogenous f a c t o r s from mammalian b r a i n ( B a t t a i n i and P e t e r k o f s k y , 1 9 8 0 ; Haupert and Sancho, 1979; Fishman, 1 9 7 9 ; L i c h t s t e i n and Samuelov, 1 9 8 0 ) . If t h e s e prove t o be of p h y s i o l o g i c a l s i g n i f i c a n c e , t h e f a c t t h a t t h e two b r a i n Na,K-ATPases have c a r d i a c g l y c o s i d e b i n d i n g s i t e s w i t h d i f f e r e n t a f f i n i t i e s suggests d i f f e r e n t i a l regulation.

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The i n i t i a l r e p o r t s of Haupert and Sancho ( 1 9 7 9 ) and Fishman ( 1 9 7 9 ) d e s c r i b e d an a c t i v i t y i n e x t r a c t s of bovine hypothalamus o r g u i n e a p i g b r a i n which competed w i t h ouabain f o r b i n d i n g t o t h e Na,K-ATPase, and which i n h i b i t e d Na and K t r a n s p o r t and Na,K-ATPase a c t i v i t y . I t was proposed t h a t t h i s might be an endogenous regul a t o r of t h e sodium pump. The a c t i v i t y was o b t a i n e d by making acetone/HCl e x t r a c t s of b r a i n of t h e s o r t used t o i s o l a t e p e p t i d e s , p a r t i a l l y d e i o n i z i n g t h e ext r a c t , and chromatographing it on columns of Sephadex G 1 0 o r G25. I n b o t h c a s e s t h e a c t i v i t y was found i n a peak r a t h e r c l o s e t o t h e s a l t peak i n t h e column e f f l u e n t . These r e s u l t s were confirmed by L i c h t s t e i n and Samuelov ( 1 9 8 0 1 , who, i n a d d i t i o n , showed t h a t t h e a c t i v i t y was n o t p r e c i p i t a t e d by t r i c h l o r o a c e t i c a c i d . The r e p o r t s s t i l l l e f t open t h e q u e s t i o n of whether t h e fact o r was indeed an o r g a n i c m o l e c u l e , d i f f e r e n t from p r e v i o u s l y r e p o r t e d h e a t - s t a b l e , d i a l y z a b l e i n h i b i t o r y fact o r s from b r a i n s u p e r n a t a n t s and c e r e b r o s p i n a l f l u i d ( S c h a e f e r et a l . , 1 9 7 2 , 1 9 7 4 ; Rozhanets e t al., 1 9 7 9 ) . S o l u b l e f a c t o r s have a l s o been r e p o r t e d t o enhance t h e i n h i b i t i o n of Na,K-ATPase by p r o s t a g l a n d i n E l ( G i l b e r t and Wyllie, 1 9 7 9 ) , and t o enhance t h e a c t i v a t i o n of Na,KATPase by c a t e c h o l a m i n e s (Rodriguez de Lores Arnaiz and M i s t r o r i g o de Pachecho, 1 9 7 8 ) . The p o s s i b i l i t y t h a t t h e f a c t o r h a s a p h y s i o l o g i c a l l y i m p o r t a n t r o l e i n regul a t i n g t h e t w o Na,K-ATPases a t t h e i r c a r d i a c g l y c o s i d e b i n d i n g s i t e s was s u f f i c i e n t j u s t i f i c a t i o n t o w a r r a n t r e p e a t i n g t h e r e s u l t s , however, and measure t h e i r a f f i n i t i e s on t h e axolemma and a s t r o c y t e Na,K-ATPases. The e x t r a c t w a s f r a c t i o n a t e d a c c o r d i n g t o Fishman ( 1 9 7 9 ) . Rather t h a n i n h i b i t i o n of ouabain b i n d i n g o r of a c t i v e K t r a n s p o r t i n i n t a c t c e l l s , t h e i n h i b i t i o n of strophanthidin-sensitive ATPase a c t i v i t y w a s measured. The r e s u l t was t h a t a s u b s t a n c e was found i n e x t r a c t s of r a t b r a i n t h a t i n h i b i t e d Na,K-ATPase a c t i v i t y , and i t appeared t o i n h i b i t axolemma and a s t r o c y t e Na,K-ATPases

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F i g . 2 . I n h i b i t i o n o f a x o l e m a and a s t r o c y t e Na,K-ATPases b y a b r a i n e x t r a c t f a c t o r . Strophanthidin-sensitive a x o l e m a ( 0 1 and a s t r o c y t e ( a ) ATPase a c t i v i t i e s were a s s a y e d a s i n F i g . 1 , i n the p r e s e n c e a n d a b s e n c e o f b r a i n e x t r a c t f a c t o r . T h e d e i o n i z e d e x t r a c t was n e u t r a l i z e d w i t h T r i s b a s e , w h e r e a s the a s h e d e x t r a c t d i d not r e q u i r e n e u t r a l i z a t i o n . A t the h i g h e s t c o n c e n t r a t i o n u s e d , 2 1.11 of c o n c e n t r a t e d e x t r a c t w e r e d i l u t e d i n t o 50 1.11 of r e a c t i o n m i x t u r e ; t h i s i s c a l c u l a t e d t o be r o u g h l y the c o n c e n t r a t i o n o f f a c t o r p r e s e n t i n b r a i n . (A) Deionized e x t r a c t ; ( B ) a s h e d , deionized e x t r a c t .

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E f f e c t o f 2 - m e r c a p t o e t h a n o l on e x t r a c t a c t i v i t y . was a s s a y e d a t d i f f e r e n t c o n c e n t r a t i o n s o f a s h e d e x t r a c t f a c t o r i n the p r e s e n c e (e ) and a b s e n c e ( 0 ) of 5 mM 2 - m e r c a p t o e t h a n o l , ( B ) Axolemma Na,K-ATPase was a s s a y e d i n d i f f e r e n t c o n c e n t r a t i o n s o f 2 - m e r c a p t o e t h a n o l i n the p r e s e n c e ( 8 ) or a b s e n c e ( 0 )o f a 0.03 d i l u t i o n o f a s h e d e x t r a c t f a c t o r . Fig. 3 .

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KATHLEEN J. SWEADNER

with d i f f e r e n t a f f i n i t i e s (Fig. 2A). I n h i b i t i o n i s expressed as a percentage of t h e t o t a l strophanthidins e n s i t i v e ATPase a c t i v i t y t o normalize f o r t h e d i f f e r e n t s p e c i f i c a c t i v i t i e s of t h e u n p u r i f i e d axolemma and a s t r o c y t e membrane p r e p a r a t i o n s . The axolemma p r e p a r a t i o n had a s p e c i f i c a c t i v i t y of 89.6 umoles/hr/mg p r o t e i n , wherea s t h e a s t r o c y t e p r e p a r a t i o n had a s p e c i f i c a c t i v i t y of 15.7. The e x t r a c t had been d e i o n i z e d by t r e a t m e n t w i t h a mixed-bed ion-exchange r e s i n (AG501-X8) i n 4 M p y r i d i n e a c e t a t e , a s d e s c r i b e d by Fishman (19791, and t h a t t h i s s t e p removed most& t h e i n o r g a n i c phosphate w a s confirmed i n t h e p r e s e n t work: t h e r e was less t h a n 1 mbi i n o r g a n i c phosphate i n t h e a s s a y r e a c t i o n m i x t u r e a t t h e h i g h e s t c o n c e n t r a t i o n of e x t r a c t used ( d a t a n o t shown). To r u l e o u t c o n t a m i n a t i o n by heavy metals, p a r t i c u l a r l y v e n a d a t e , however, t h e e x t r a c t was ashed by h e a t i n g it a t 6 O O O C o v e r n i g h t i n a m u f f l e f u r n a c e . The i n h i b i t o r y a c t i v i t y w a s n o t a b o l i s h e d by t h i s treatment ( F i g . 2B), and so must be due t o an i n o r g a n i c molecule. Several experiments i n d i c a t e t h a t t h e f a c t o r i s n o t v a n a d a t e , t h e i o n found t o be an e f f e c t i v e i n h i b i t o r o f Na,K-ATPase (reviewed i n Simons, 1 9 7 9 ) , b u t i t s i d e n t i t y i s n o t y e t known. F i r s t , i t s e f f e c t i s n o t a n t a g o n i z e d by 1 mM n o r e p i n e p h r i n e under t h e c o n d i t i o n s u s e d ( d a t a n o t shown). Second, i t s e f f e c t i v e n e s s i s i n f l u e n c e d by whether o r n o t it i s a s s a y e d i n t h e p r e s e n c e of a reducing a g e n t , 2-mercaptoethanol ( F i g . 3A). A t a . f i x e d c o n c e n t r a t i o n of e x t r a c t s u f f i c i e n t t o g i v e p a r t i a l i n h i b i t i o n of A T P a s e a c t i v i t y ( t h e axolemma Na,K-ATPase i n F i g . 3 1 , low c o n c e n t r a t i o n s of 2-mercaptoethanol (0.3 mM) enhance t h e i n h i b i t o r y a c t i v i t y , whereas h i g h e r concent r a t i o n s block it. 2-Mercaptoethanol a l o n e h a s no e f f e c t on enzyme a c t i v i t y ( F i g . 3B). The e f f e c t s of v a n a d a t e ( F i s h e r S c i e n t i f i c ) were assayed under t h e same c o n d i t i o n s . The b i n d i n g of v a n a d a t e t o t h e enzyme i s slow, and s i n c e s h o r t a s s a y p e r i o d s were used (8 min) , i n c l u d i n g t h e i n i t i a l r a t e of i n h i b i t i o n , v e r y h i q h c o n c e n t r a t i o n s ( 1 0 0 p M ) were r e q u i r e d t o see s i g n i f i c a n t i n h i b i t i o n ( F i g . 4 A ) . 2-Mercaptoethanol ( o v e r t h e same range of c o n c e n t r a t i o n s t h a t w a s found t o f i r s t enhance and t h e n a n t a g o n i z e t h e i n h i b i t i o n by t h e b r a i n e x t r a c t f a c t o r ) had no e f f e c t on t h e i n h i b i t i o n by v a n a d a t e ( F i g . 4B). The e f f e c t of 2-mercaptoethanol on t h e i n h i b i t i o n of t h e Na,K-ATPase by t h e b r a i n e x t r a c t f a c t o r c o u l d b e due e i t h e r t o changes i n t h e o x i d a t i o n s t a t e of t h e f a c t o r i t s e l f o r t o t h e b r e a k i n g of d i s u l f i d e bonds on t h e enzyme. The exposure of s u l f h y d r y l groups m i g h t enhance s e n s i t i v i t y t o a heavy metal such as mercury, f o r example. The f a c t o r s d e s c r i b e d by S c h a e f e r and c o l -

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l e a g u e s 11972, 1 9 7 4 ) and by Rodriguez de Lores Arnaiz and M i s t r o r i g o de Pachecho ( 1 9 7 8 ) , however, a l s o showed evidence of b e i n g r e o x i d i z a b l e . The f a c t o r s e e n by S c h a e f e r et a l . ( 1 9 7 4 ) was a n t a g o n i z e d by c h e l a t o r s i n c l u d i n g n o r e p i n e p h r i n e , and was mimicked by a s c o r b a t e . The f a c t o r s e e n by Rodriguez de Lores Arnaiz and M i s t r o r i g o de Pachecho (1978) d i d n o t i n h i b i t t h e Na,K-ATPase when it was f r e s h l y p r e p a r e d , b u t d i d i n h i b i t a f t e r storage overnight frozen, suggesting sensit i v i t y t o atmospheric oxidation. I n i t s presence n o r e p i n e p h r i n e s t i m u l a t e d Na,K-ATPase a c t i v i t y . A l l of t h e s e o b s e r v a t i o n s e x c e p t t h e d a t a i n F i g . 4 a r e c o n s i s t e n t w i t h t h e p r e s e n c e of v a n a d a t e i n t h e b r a i n e x t r a c t s . Vanadate has been shown t o b e reduced t o vanadyl i o n by a s c o r b a t e , n o r e p i n e p h r i n e , and g l u t a t h i o n e ( C a n t l e y et a l . , 1 9 7 7 , 1978; Macara e t al., 1980: Adam-Vizi et a l . , 1 9 8 1 ) , and t h e vanadyl i o n does n o t i n h i b i t Na,K-ATPase a c t i v i t y . Vanadyl, i n f a c t , i s p r o b a b l y t h e form of most vanadium s t o r e d i n t i s s u e s w i t h h i g h l e v e l s of e i t h e r a s c o r b a t e o r g l u t a t h i o n e . I t i s r e a d i l y o x i d i z e d t o vanadate when exposed t o a i r a t n e u t r a l pH (Macara et al., 1 9 8 0 ) . One might e x p e c t f r e s h p r e p a r a t i o n s c o n t a i n i n g vanadyl i o n t o i n c r e a s e i n t h e i r a b i l i t y t o i n h i b i t t h e Na,K-ATPase a s t h e y s t a n d exposed t o a i r , a l t h o u g h b i n d i n g of vanadyl t o p r o t e i n has been r e p o r t e d t o r e t a r d t h i s p r o c e s s ( C a n t l e y and Aisen, 1 9 7 9 ) . The l a c k of e f f e c t of 2mercaptoethanol on t h e i n h i b i t i o n o f t h e Na,K-ATPase by vanadate s e e n i n F i g . 4 remains t o be e x p l a i n e d , howe v e r : it i s p o s s i b l e t h a t t h e r e d u c i n g p o t e n t i a l of 2mercaptoethanol i s n o t h i g h enough, o r t h a t t h e format i o n o f vanadate polymers i n s o l u t i o n s of e x c e s s vanad a t e (Simons, 1 9 7 9 ) may r e t a r d r e d u c t i o n . The d i f f e r e n c e i n t h e a p p a r e n t a f f i n i t i e s of t h e a s t r o c y t e and axolemma Na,K-ATPases f o r t h e b r a i n ext r a c t f a c t o r may g i v e i t some p h y s i o l o g i c a l , o r a t l e a s t t o x i c o l o g i c a l , i n t e r e s t . The experiments were done on c r u d e membrane p r e p a r a t i o n s , however, i n which t h e strophanthidin-sensitive ATPase a c t i v i t y of t h e a s t r o c y t e membrane p r e p a r a t i o n s does n o t d i s p l a y normal a c t i v a t i o n by Na and K (see t h e n e x t s e c t i o n ) . S i n c e p u r i f i c a t i o n of t h e two Na,K-ATPases does n o t a l t e r t h e i r a f f i n i t i e s f o r s t r o p h a n t h i d i n , p u r i f i e d enzymes s h o u l d be used t o r e e v a l u a t e t h e i r a f f i n i t i e s f o r t h e b r a i n ext r a c t factor.

TWO Na,K-ATPases OF THE BRAIN

IV.

775

STROPHANTHIDIN-SENSITIVE, ACTIVITY

ION-INDEPENDENT A T P a s e

An e n t i r e l y u n r e l a t e d p o s s i b l e mechanism o f N a , K A T P a s e r e g u l a t i o n w a s s u g g e s t e d by measurements o f t h e a f f i n i t i e s of t h e two enzymes f o r N a and K . The a ( + ) (axolemma) form o f t h e N a , K - A T P a s e showed normal a f f i n i t i e s f o r Na and K b o t h b e f o r e and a f t e r p u r i f i c a t i o n by sodium d o d e c y l s u l f a t e (SDS) e x t r a c t i o n , b u t t h e same w a s n o t t r u e o f t h e a ( a s t r o c y t e ) form. I n s t e a d , t h e unexp e c t e d o b s e r v a t i o n w a s made t h a t t h e a s t r o c y t e N a , K - A T P a s e a p p e a r e d t o be p a r t i a l l y a c t i v e i n t h e a b s e n c e of added N a and K when a s s a y e d i n c r u d e membrane p r e p a r a t i o n s . The c e l l s and membranes were e x t e n s i v e l y washed w i t h suc r o s e , Tris-ethylenediaminetetraacetic a c i d (EDTA) b o t h b e f o r e and a f t e r homogenization. Contaminating N a and K c o n c e n t r a t i o n s i n t h e f i n a l a s s a y m i x t u r e , measured by f l a m e photometry, were 140-380 U M N a and 1 0 - 2 0 U M K , two o r d e r s o f magnitude lower t h a n t h e normal a f f i n i t i e s of t h e enzyme f o r e a c h o f t h e s e c a t i o n s . The p e r c e n t a g e of t h e t o t a l strophanthidin-sensitive a c t i v i t y t h a t i s i n d e p e n d e n t of added N a and K v a r i e d f r o m p r e p a r a t i o n t o p r e p a r a t i o n ; t h e mean f o r a s t r o c y t e s was 8 6 . 1 2 1 6 . 3 ( S D n = 55) f o r N a and 70.0 9.2 ( n = 5 ) f o r K , whereas comparable v a l u e s f o r axolemma w e r e 4.8 f 2 . 4 ( n = 5 ) f o r N a and 7 . 4 5.4 ( n = 2 ) f o r X . Fragments o f membrane o f t e n v e s i c u l a r i z e d u r i n g hom o g e n i z a t i o n , and might t r a p i o n s i n s i d e . To t e s t whether t r a p p e d Na i s r e s p o n s i b l e f o r t h e Na-independent a c t i v i t y , f r e s h p r e p a r a t i o n s of a s t r o c y t e membrane w e r e t r e a t e d w i t h a v a r i e t y of a g e n t s known t o d i s r u p t t h e membrane o r make i t l e a k y t o i o n s , i n c l u d i n g monensin, a l a m e t h i c i n , A23187, s o n i c a t i o n i n a b a t h s o n i c a t o r , l y s o p h o s p h a t i d y l c h o l i n e and p h o s p h a l i p a s e A 2 , SDS ( a t conc e n t r a t i o n s lower t h a n t h o s e needed t o p u r i f y t h e enzyme), and Tween 8 0 . None of t h e s e a g e n t s c a u s e d a s i g n i f i c a n t d e c r e a s e i n t h e p r o p o r t i o n o f Na-independent a c t i v i t y (Table I ) , L y s o p h o s p h a t i d y l c h o l i n e and Tween 80 a t c o n c e n t r a t i o n s h i g h enough t o c l a r i f y t h e membrane s u s p e n s i o n caused c o n s i d e r a b l e l o s s of t o t a l a c t i v i t y , b u t what a c t i v i t y remained w a s N a i n d e p e n d e n t . The cons i s t e n t l a c k of e f f e c t of s u c h v a r i e d t r e a t m e n t s a r g u e s s t r o n g l y t h a t t r a p p e d N a i s n o t t h e r e a s o n t h a t t h e enzyme i s a c t i v e i n t h e absence o f added N a . E x t r a c t i o n o f t h e membranes w i t h enough SDS t o b r i n g a b o u t a 5- t o 6 - f o l d p u r i f i c a t i o n of t h e enzyme (Sweadner, 1978) a p p e a r s t o c o n v e r t t o Na,K-independent a c t i v i t y t o a Na,K-dependent a c t i v i t y l i k e t h a t of axolemma, accompanied by t h e loss o f ~ 5 0 %of t h e t o t a l _+

+_

KATHLEEN J. SWEADNER

776

TABLE I.

E f f e c t o f Membrane D i s r u p t i o n on t h e M a n i f e s t a t i o n of Na+-Independent ATPase A c t i v i t y a %

Independent Na+

Preincubation condition

% Recovery

Control M I 22', 30 min MOnensin, Monensin, 10-3 M I 22', 30 min Alamethicin, M I 22', 30 rnin M I 22', 30 minb Alamethicin, A23187, 2 x 10-5 M I 22', 30 rnin

100 102 60.8 112 42 111

Control Sonication

100 90

88.8 88.8

Control Lyso-PCIc 0.1%, 22', 1 5 minb Lyso-PCIc 1.0%, 22', 1 5 minb Phospholipase A 2 (0.5 mg/ml) I 22', 30 rnin 37O, 10 min SDS, 0.275 mg/ml, 22', 30 min ( a t 4.0 mg p r o t e i n / m l )

100 66 15 77

99.9 98.6 87.6 103.7

100

62.6

83

85.1

Cont ro 1 SDS, 0.35 mg/ml, 22', 30 min ( a t 5.0 mg p r o t e i n / m l )

100

60.8

92

59.8

Control Tween 80, 25', 5 min Tween 80 (10 mM), 25',

100 75.6 42.9

74.6 73.8 81.3

120 minb

103.0 90.8 90.2 95.6 73.1 93.5

a

A 1 1 experimen ts were performed on washed membrane prepar a t i o n s from r a t b r a i n a s t r o c y t e s . Membranes were p r e i n c u b a t e d under t h e c o n d i t i o n s l i s t e d , and t h e n d i l u t e d 25-fold i n t o t h e Strophanthidin-sensitive ATPase a s s a y r e a c t i o n m i x t u r e a t 37'C. a c t i v i t y was measured i n t h e p r e s e n c e and absence of Na'. b C l a r i f i c a t i o n of t h e membrane suspension was s e e n . CLysophosphatidylcholine.

strophanthidin-sensitive a c t i v i t y . The e x t r a c t i o n d o e s n o t a f f e c t t h e m o b i l i t y of t h e a - s u b u n i t s on SDS g e l s , o r any of t h e i r o t h e r p h y s i c a l c h a r a c t e r i s t i c s , so i t does n o t c o n v e r t a t o a ( + ) . S i n c e m i l d e r d e t e r g e n t s and o t h e r a g e n t s t h a t d i s r u p t t h e membrane do n o t b r i n g about t h i s change, and s i n c e t h e SDS i s known t o be removing contaminating p r o t e i n s from t h e membrane p r e p a r a -

TWO Na,K-ATPases OF THE BRAIN

777

t i o n , it i s a t t r a c t i v e t o p r o p o s e t h a t t h e SDS m i g h t e i t h e r d e n a t u r e o r s o l u b i l i z e a n endogenous p r o t e i n f a c t o r t h a t c o n f e r s i o n independence on t h e Na,K-ATPase. T h i s h y p o t h e t i c a l f a c t o r " u n c o u p l e s " ATPase a c t i v i t y from c o n t r o l by N a and K ; i t would be i n t e r e s t i n g t o see if t h e Na,K-independent A T P a s e a c t i v i t y i s a l s o unc o u p l e d from i o n t r a n s p o r t i n l i p i d v e s i c l e s . The c h i e f j u s t i f i c a t i o n f o r a s c r i b i n g t h i s N a , K i n d e p e n d e n t a c t i v i t y t o Na,K-ATPase i s i t s s e n s i t i v i t y to strophanthidin. S e n s i t i v i t y t o cardiac glycosides and t h e i r d e r i v a t i v e s h a s become g e n e r a l l y a c c e p t e d a s a c r i t e r i o n f o r t h e measurement o f Na,K-ATPase a c t i v i t y , b u t it i s a n assumption t h a t needs t o be t e s t e d more c a r e f u l l y b e f o r e p r o c e e d i n g w i t h a n i n v e s t i g a t i o n of t h e a p p a r e n t u n c o u p l i n g d e s c r i b e d h e r e . Because t h e e x p e r i ments were done w i t h r a t a s t r o c y t e membranes, t h e s t r o p h a n t h i d i n a f f i n i t i e s a r e low ( ' ~m1 ~ ) ,a c h a r a c t e r i s t i c of t h e s p e c i e s . Consequently t h e a c t i v i t i e s were measured w i t h c o n c e n t r a t i o n s o f s t r o p h a n t h i d i n which might c o n c e i v a b l y have a s p u r i o u s e f f e c t on a Mg-ATPase. I f t h i s w e r e t r u e , o n e would have t o e x p l a i n t h e absence o f Na,K-stimulated a c t i v i t y i n f r e s h membranes by s u p p o s i n g t h a t it i s l a r g e l y l a t e n t ; i f so, i t i s s u r p r i s i n g t h a t it was n o t exposed by l y s o p h o s p h a t i d y l c h o l i n e , a l a m e t h i c i n , o r Tween 8 0 . The a t t r a c t i v e f e a t u r e o f t h e s e r e s u l t s i s t h a t t h e a p p a r e n t uncoupling a f f e c t s o n l y one of t h e two N a , K ATPases, e v e n when t h e i r a c t i v i t i e s are measured i n b r a i n microsomes, which c o n t a i n b o t h forms i n t h e same p r e p a r a t i o n ( d a t a n o t shown). T h i s s u g g e s t s a f u n c t i o n a l d i f f e r e n c e : s u s c e p t i b i l i t y t o r e g u l a t i o n by coupling/unc o u p l i n g . The a ( + ) form might d i f f e r from t h e a form f o r t h e p u r p o s e o f e s c a p i n g from a k i n d o f r e g u l a t i o n t h a t would o t h e r w i s e a c t on it. The r e g u l a t i o n of i o n t r a n s p o r t would be b a s e d o n t h e c o n t r o l o f t h e c o n v e r s i o n o f t h e enzyme from an uncoupled t o a coupled s t a t e ; i n t h e uncoupled s t a t e , ATP h y d r o l y s i s would c o n t i n u e b u t i o n t r a n s p o r t would b e t u r n e d o f f . The C a - A T P a s e o f s a r c o p l a s m i c r e t i c u l u m h a s a l s o been proposed t o e x i s t i n c o u p l e d and uncoupled s t a t e s , based o n s i m i l a r o b s e r v a t i o n s ( I n e s i et a i , 1976) The s t i m u l a t i o n of N a and K t r a n s p o r t by i n s u l i n , n e r v e growth f a c t o r , e p i d e r m a l growth f a c t o r , and l e c t i n s such as PHA h a s been r e p o r t e d (Resh et a l . , 1980; V i n o r e s and G u r o f f , 1980; C a r p e n t e r and Cohen, 1979; Kaplan, 19781, b u t few i f any e f f e c t s have been found o n A T P a s e a c t i v i t y i n broken c e l l p r e p a r a t i o n s , l e a v i n g open t h e p o s s i b i l i t y t h a t t h e s t i m u l a t i o n i s by r e g u l a t i o n o f c o u p l i n g .

.

.

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KATHLEEN J. SWEADNER

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Macara, I. G . ,

779

K u s t i n , K . , and C a n t l e y , L. C. ( 1 9 8 0 ) . G l u t a t h i o n e r e d u c e s c y t o p l a s m i c v a n a d a t e . Mechanism and p h y s i o l o g i c a l B i o c h i m . B i o p h y s . A c t a 6 2 9 , 95-106. implications. Marks, M. J . , and S e e d s , N. W. ( 1 9 7 8 ) . Ouabain-ATPase i n t e r a c t i o n i n b r a i n c e l l s m a i n t a i n e d as r e a g g r e g a t e s o r s u r f a c e c u l L i f e S c i . 2 3 , 2745-2755. tures. N e v i l l e , M. C . , P a t e r s o n , C. A . , and Hamilton, P. M. ( 1 9 7 8 ) . Evid e n c e f o r t w o sodium pumps i n t h e c r y s t a l l i n e l e n s o f t h e rabbit eye. E x p . E y e R e s . 2 7 , 637-648. P e t e r s o n , G. L . , Ewing, R. D., Hootman, S. R . , and C o n t e , F. P. ( 1 9 7 8 ) . Large scale p a r t i a l p u r i f i c a t i o n and m o l e c u l a r a n d k i n e t i c properties o f t h e Na,K-ATPase from A r t e m i a s a l i n a n a u p l i i . J . B i o l . Chem. 2 5 3 , 4762-4770. P e t r a l i , E. H . , and S u l a k h e , P. V. ( 1 9 8 0 ) . I s o l a t i o n o f a plasma membrane f r a c t i o n h i g h l y e n r i c h e d i n o u a b a i n - s e n s i t i v e Int. J . B i o c h e m . Na,K-ATPase from r a t b r a i n w h i t e matter. 1 2 , 407-420. Resh, M. D . , Nemenoff, R. A., and G u i d o t t i , G. ( 1 9 8 0 ) . I n s u l i n s t i m u l a t i o n o f Na,K-ATPase-dependent 86-Rb u p t a k e i n r a t adipocytes. J. B i o l . Chem. 2 5 5 , 10938-10945. Rodriguez d e L o r e s A r n a i z , G . , and M i s t r o r i g o de Pachecho, M. ( 1 9 7 8 ) . R e g u l a t i o n o f Na,K-ATPase o f n e r v e e n d i n g membranes: A c t i o n of n o r e p i n e p h r i n e and a s o l u b l e f a c t o r . N e u r o c h e m . R e s . 3, 733-744. R o z h a n e t s , V. V . , P r o m i s l o v , M. S . , G a b r i e l y a n , N . I . , and S h e r b a n j o v a , 0. I . ( 1 9 7 9 ) . I n h i b i t o r y e f f e c t o f l i q u o r c e r e b r o s p i n a l i s on t h e a c t i v i t y of Na,K-ATPase f r o m mammalian b r a i n s y n a p t i c membranes. V o p r . Med. K h i m . 2 5 , 71-74. S c h a e f e r , A . , Unyi, G . , and P f e i f e r , A. K. ( 1 9 7 2 ) . The e f f e c t s o f a s o l u b l e f a c t o r and of c a t e c h o l a m i n e s o n t h e a c t i v i t y o f Biochem. ATPase i n s u b c e l l u l a r f r a c t i o n s o f r a t b r a i n . Pharmacol. 2 1 , 2289-2294. S c h a e f e r , A., S e r e g i , A . , a n d Komlos, M. ( 1 9 7 4 ) . Ascorbic a c i d l i k e e f f e c t s o f t h e s o l u b l e f r a c t i o n o f r a t b r a i n o n ATPases and i t s r e l a t i o n t o c a t e c h o l a m i n e s and c h e l a t i n g a g e n t s . B i o c h e m . P h a r m a c o l . 2 3 , 2257-2271. S c h w a r t z , M . , E r n s t , S. A., S i e g e l , G . J . , a n d A g r a n o f f , B. W. (1981). Immunocytochemical l o c a l i z a t i o n o f Na,K-ATPase i n J . N e u r o c h e m . 36, 107-115. t h e g o l d f i s h o p t i c nerve. Simons, T. J. B. ( 1 9 7 9 ) . Vanadate--A new t o o l for b i o l o g i s t s . N a t u r e ( L o n d o n ) 2 8 1 , 337-338. Sweadner, K. J. ( 1 9 7 8 ) . P u r i f i c a t i o n from b r a i n o f a n i n t r i n s i c Biochim. membrane p r o t e i n f r a c t i o n e n r i c h e d i n Na,K-ATPase. B i o p h y s . A c t a 4 0 8 , 486-499. Sweadner, K. J. ( 1 9 7 9 ) . Two m o l e c u l a r forms o f ( N a + K ) - s t i m u l a t e d ATPase i n b r a i n . S e p a r a t i o n , and d i f f e r e n c e i n a f f i n i t y f o r J . B i o l . Chem. 2 5 4 , 6060-6067. strophanthidin. Takemoto, L . J . , Hansen, J. S . , and Hokin, L. E. ( 1 9 8 1 ) . Phosp h o r y l a t i o n of l e n s membrane: I d e n t i f i c a t i o n o f t h e c a t a l y t i c s u b u n i t o f Na,K-ATPase. B i o c h e m . B i o p h y s . R e s . Commun. 100, 58-64.

KATHLEEN J. SWEADNER

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Taniguchi, K. , and I i d a , S. (1972). Two a p p a r e n t l y d i f f e r e n t Biochim. Biophys. ouabain b i n d i n g s i t e s o f Na,K-ATPase Acta 288, 98-102. Urayama, 0. , and Nakao, M. ( 1 9 7 9 ) . Organ s p e c i f i c i t y of r a t Na,K-ATPase. J. Biochem. ( T o k y o ) 86, 1371-1381. V i n o r e s , S. , and Guroff , G. ( 1 9 8 0 ) . Nerve growth f a c t o r : Mechanism of a c t i o n . Annu. Rev. Biophys. Bioeng. 9, 223257. Wood, J. G., J e a n , D. H., Whitaker, J. N . , McLaughlin, B. J . , and Immunocytochemical l o c a l i z a t i o n of A l b e r s , R . W. (1977) Na,K-ATPase i n k n i f e f i s h b r a i n . J. N e u r o c y t o l . 6 , 571-581.

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CURRENT TOPICS IN MEMBRANES AND TRANSPORT, VOLUME 19

Antigenic Properties of the CY-, 0-, and 7-Subunits of Na,K-ATPase W I U l A M BALL, JR., JOHN H . COLLINS,

LOISLAND,AND ARNOLD SCHWARTZ Department of Pharmacology and Cell Biophysics University of Cincinnati College of Medicine Qncinrrati. Ohio

I.

INTRODUCTION

Numerous i n v e s t i g a t o r s have r a i s e d a n t i b o d i e s a g a i n s t v a r i o u s p r e p a r a t i o n s of Na,K-ATPase and i t s c a t a l y t i c ( a - ) and g l y c o p r o t e i n ( 8 - 1 s u b u n i t s . These a n t i b o d i e s have been found t o have v a r y i n g and sometimes a p p a r e n t l y c o n t r a d i c t o r y e f f e c t s on m e a s u r a b l e f u n c t i o n s of t h e enzyme ( W a l l i c k e t a l . , 1 9 7 9 ) . These c o n f l i c t i n g r e s u l t s i n l a r g e p a r t are due t o a l a c k of i n f o r m a t i o n a b o u t t h e b a s i c a n t i g e n i c p r o p e r t i e s of t h e enzyme. P r e v i o u s l y we have d e m o n s t r a t e d t h a t holoenzyme-specific a n t i b o d i e s a r e d i r e c t e d toward b o t h t h e a - and $ - s u b u n i t s , b u t n o t toward t h e l i p i d components o f t h e enzyme. W e have now o b t a i n e d r a b b i t a n t i b o d i e s r a i s e d t o t h e i s o l a t e d a- and @ - s u b u n i t s of t h e enzyme and mouse monoclonal a n t i b o d i e s d i r e c t e d toward t h e a-subuni t.

781

Copyright 0 1983 by Academic Ress. Inc. All righrs of reproductionin any form reserved.

ISBN 012-1533190

782

WILLIAM BALL, JR eta/.

100

>,

80

.E

.->

3 60

* 20 01 0

I

I

10

20

Protein (mg/ml)

Fig. 1. Effects o f v a r y i n g c o n c e n t r a t i o n s o f immunoglobulins on Na ,K-ATPase a c t i v i t y : T h e e f f e c t s o f h o l o e n z y m e - d i r e c t e d imm u n o g l o b u l i n s on the Na,K-ATPase a c t i v i t y ( 0 ), the e f f e c t s o f cat a l y t i c - s u b u n i t - d i r e c t e d a n t i b o d i e s f r o m two different a n t i s e r a ( O ) , and the e f f e c t s o f glycoprotein-subunit-directed a n t i b o d i e s f r o m two different a n t i s e r a ( A ) (Reprinted from Ball et a l . (1983), w i t h p e r m i s s i o n . )

.

11.

METHODS

The Na,K-ATPase was p u r i f i e d from s h e e p kidney and and y- ( o r p r o t e o l i p i d ) components were s e p a r a t e d by chromatography on B i o G e l A-5m a c c o r d i n g t o t h e procedure of Reeves e t a l . , (1980). R a b b i t s were i m munized w i t h t h e p r o t e i n s (SDS f r e e ) e m u l s i f i e d i n comp l e t e Freunds a d j u v a n t . Rabbit a n t i b o d y - a n t i g e n b i n d i n g was d e t e c t e d u s i n g a s o l i d s u r f a c e a d s o r p t i o n a s s a y u t i l i z i n g 1 2 5 I - l a b e l e d p r o t e i n A. C e l l f u s i o n s were done u s i n g s p l e e n cells from immunized mice and Sp2/0Ag14 myeloma cells a c c o r d i n g t o t h e procedure of G a l f r e e t al. (1977). Hybridoma c e l l a n t i b o d i e s were d e t e c t e d by a n (ELISA) a s s a y u s i n g a 8 - g a l a c t o s i d a s e sheep a n t i mouse IgG F ( a b ) c o n j u g a t e .

i t s a-, 8-,

;

111.

RESULTS AND DISCUSSION

A n t i b o d i e s were r a i s e d t o t h e a- a n d , @ - s u b u n i t s of t h e sheep kidney enzyme. These s u b u n i t s were shown t o m i g r a t e a s s i n g l e p r o t e i n bands when analyzed by sodium

783

IMMUNOCHEMICAL STUDIES OF Na,K-ATPase

100

80

h '0 0

c U J

40

20

0

0.04

0.02

0.01

0

Antibody Dilution Ratio F i g . 2 . Determination o f a n t i b o d y b i n d i n g t o the p r o t e o l i p i d (y p r o t e i n ) as a f u n c t i o n of a n t i s e r u m c o n c e n t r a t i o n . Holoenzymes p e c i f i c a n t i b o d y b i n d i n g t o Na,K-ATPase ( a ) and b i n d i n g t o the p r o t e o l i p i d ( A ) . V a l u e s for both are g i v e n a s p e r c e n t a g e s o f a n t i b o d y bound r e l a t i v e t o the maximum bound t o Na,K-ATPase. ( R e p r i n t e d f r o m B a l l and S c h w a r t z (1982), w i t h p e r m i s s i o n . )

d o d e c y l s u l f a t e - p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s (SDS( 7 . 5 % ) . Agar g e l immunoe.1ectrophoresis and d o u b l e d i f f u s i o n i m m u n o p r e c i p i t a t i o n t e c h n i q u e s have demons t r a t e d s i n g l e p r e c i p i t a t i o n a r c s between t h e a n t i s e r a and t h e immunizing a n t i g e n s , w i t h no a n t i b o d y c r o s s r e a c t i v i t y toward t h e o t h e r enzyme s u b u n i t . The 1251l a b e l e d p r o t e i n A s u r f a c e a d s o r p t i o n a s s a y w a s t h e n used t o d e t e r m i n e a n t i s e r u m t i t e r s , o r t h e d i l u t i o n s needed f o r 5 0 % maximum d e t e c t a b l e b i n d i n g . A n t i b o d i e s r a i s e d t o t h e a - s u b u n i t were found t o have s i m i l a r a f f i n i t i e s f o r b o t h t h e s u b u n i t and t h e holoenzyme w i t h a b o u t a 20%, l o w - a f f i n i t y c r o s s - r e a c t i v i t y t o t h e B-subunit. The a - s u b u n i t s p e c i f i c a n t i b o d i e s a l s o e f f e c t i v e l y i n h i b i t t h e Na,K-ATPase a c t i v i t y (Fig. 1). I n c o n t r a s t , t h e B - s u b u n i t - d i r e c t e d a n t i b o d i e s were found t o have a h i g h a f f i n i t y f o r t h e a n t i g e n b u t l i t t l e c r o s s - r e a c t i v i t y ( < 8 % ) o r a f f i n i t y toward t h e holoenzyme. These a n t i b o d i e s had no e f f e c t on enzyme a c t i v i t y ( F i g . 1 ) . The B - s u b u n i t - d i r e c t e d a n t i b o d i e s a l s o have a lowa f f i n i t y c r o s s - r e a c t i v i t y with t h e a-subunit. CompetiPAGE)

WILLIAM BALL, JR eta/.

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TABLE I.

Anti-Sheep Kidney Na,K-ATPase Antibody Producing Hybridoma C e l l s a

Cell line

and c l o n e designation

Isotype

Binding c h a r a c t e r i s t i c s (binding t i t e r o r binding s p e c i f i c i t y ) : Holoenzyme GlycoHoloenzyme (rat (sheep Catalytic protein kidney) subunit subunit kidney)

1 M7-PB-E9

IyGl (k) L chain

5 x 10m9M

3

X

lO-’M

0

0

2

IyGl (k) L chain

7

3

X

lO-’M

0

0

M8-Pl-A3

X

lO-’M

a

Hybridoma-produced a n t i b o d i e s were p u r i f i e d b y chromatography on p r o t e i n A-Sepharose. A n t i b o d y b i n d i n g t o the v a r i o u s a n t i g e n s was d e t e c t e d u s i n g 0.5 mg/ml o f a n t i g e n a b s o r b e d t o m i crotiter p l a t e s and the ( E L I S A ) a n t i b o d y s c r e e n i n g r e a g e n t t o d e t e c t bound a n t i b o d y . T i t e r v a l u e s a r e c o n c e n t r a t i o n s of a n t i b o d y g i v i n g 50% maximal b i n d i n g .

t i o n binding s t u d i e s suggest t h a t t h e c r o s s - r e a c t i v i t y of b o t h a n t i b o d y p o p u l a t i o n s does n o t r e s u l t from cont a m i n a t i o n of t h e a n t i g e n s b u t t h a t t h e r e may be p a r t i a l homologies of some s u b u n i t a n t i g e n i c s i t e s . I n a d d i t i o n , a l t h o u g h o n l y l i m i t e d q u a n t i t i e s of t h e y ( o r p r o t e o l i p i d ) p r o t e i n as i s o l a t e d from Na,KATPase were a v a i l a b l e , a n t i b o d y b i n d i n g s t u d i e s have been done which demonstrate t h a t a s i g n i f i c a n t f r a c t i o n of holoenzyme-directed a n t i b o d i e s b i n d t o y . W e find a maximal b i n d i n g and an a p p a r e n t a v i d i t y of a b o u t 20% t h a t of t h e holoenzyme ( F i g . 2 ) . S i m i l a r l y , t h e a n t i - a and t h e a n t i - 8 a n t i b o d i e s have also been found t o b i n d a - S u b u n i t - d i r e c t e d a n t i b o d i e s have a 25-35% t o y. c r o s s - r e a c t i v i t y and t h e 8 - s u b u n i t - d i r e c t e d a n t i b o d i e s about a 1 5 % c r o s s - r e a c t i v i t y w i t h y . These s t u d i e s have demonstrated t h a t t h e p r o t e o l i p i d i s an a n t i g e n i c a l l y a c t i v e component of t h e enzyme p r e p a r a t i o n , b u t t h e y have n o t demonstrated t h e p r e s e n c e of a n t i g e n i c s i t e s which a r e unique t o t h i s p r o t e i n . The d e m o n s t r a t i o n of a unique r o l e f o r y o r i t s p r e s e n c e a s a s p e c i f i c compon e n t of Na,K-ATPase a w a i t s f u r t h e r s t u d i e s u s i n g ys p e c i f i c antibodies. W e a l s o r e p o r t f o r t h e f i r s t time t h e i s o l a t i o n of mouse s p l e e n mouse myeloma c e l l hybridomas which secrete

IMMUNOCHEMICALSTUDIES OF Na,K-ATPase

785

monoclonal a n t i b o d i e s which bind t o t h e holoenzyme and a r e a-subunit-specific. Two d i s t i n c t c e l l l i n e s have been c l o n e d and c u l t u r e d i n v i v o w i t h t h e i r a n t i b o d i e s p u r i f i e d by p r o t e i n A-Sepharose a f f i n i t y column chromatography. These a n t i b o d i e s have been i d e n t i f i e d as I g G ( y l ) , l i g h t c h a i n (K) s u b c l a s s a n t i b o d i e s and b o t h have similar t i t e r values o r a f f i n i t i e s f o r the a-subunit (approximately 3 x 10-9 M ) and t h e holoenzyme ( T a b l e I ) . Competition b i n d i n g s t u d i e s have shown t h a t t h e s e a n t i b o d i e s b i n d s e p a r a t e s i t e s and s t u d i e s of t h e i r e f f e c t s on enzyme f u n c t i o n s a r e c u r r e n t l y under way.

REFERENCES

J., Jr., C o l l i n s , J . H . , Lane, L. K . , and Schwartz, A. 11983). S t u d i e s of t h e a n t i g e n i c p r o p e r t i e s of t h e catal y t i c and g l y c o p r o t e i n subunits of Na+,K+-ATPase. Arch. Biochem. Biophys. 221, i n p r e s s . B a l l , W. J . , Jr., and Schwartz, A . ( 1 9 8 2 ) . S t u d i e s of t h e a n t i Arch. g e n i c p r o p e r t i e s of sheep kidney Na+,K--ATPase. Biochem. Biophys. 217, 110-119. G a l f r e , G . , Howe, S . C . , M i l s t e i n , D., Butcher, G. W . , and Howard, J. C. ( 1 9 7 7 ) . A n t i b o d i e s t o major h i s t o c o m p a t i b i l i t y a n t i gens produced by h y b r i d c e l l l i n e s . N a t u r e (London) 266, 550-552. Reeves, A. S., C o l l i n s , J . H . , and Schwartz, A. (1980). I s o l a t i o n and c h a r a c t e r i z a t i o n of (Na,K) -ATPase p r o t e o l i p i d . Biochem. Biophys. R e s . Commun. 94, 1591-1598. W a l l i c k , E . T., Lane, L. K., and Schwartz, A. (1979). Biochemical mechanism of t h e sodium pump. Annu. Rev. P h y s i o l . 41, 397419. B a l l , W.

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CURRENT TOPICS IN MEMBRANESAND TRANSPORT,VOLUME 19

Antibodies to Na,K-ATPase: Characterization and Use in Cell-Free Synthesis Studies ALICIA McDONOUGH,' ANDREW HIAlT, AND ISIDORE EDELMAN Depanment of Physiology and Biophysics University of California School of Medicine Los Angeles, California

I.

INTRODUCTION

To examine t h e r e g u l a t i o n of t h e r a t e s o f t r a n s c r i p t i o n and t r a n s l a t i o n o f Na,K-ATPase and t o s t u d y t h e p r o c e s s i n g and a s s e n b l y o f t h e s u b u n i t , development o f a c e l l - f r e e s y n t h e s i s s y s t e m f o r t h e enzyme would b e advantageous. D e t e c t i o n of t r a n s l a t i o n a l p r o d u c t s i s dependent upon t h e a v a i l a b i l i t y of a n t i b o d i e s s p e c i f i c f o r t h e a- and 8 - s u b u n i t s of t h e enzyme. These a n t i bodies a l s o provide u s e f u l probes f o r t h e a n a l y s i s of enzyme s t r u c t u r e - f u n c t i o n r e l a t i o n s h i p s .

11.

PRODUCTION AND CHARACTERIZATION OF A N T I B O D I E S

T h r e e r a b b i t s were immunized w i t h N a , K - A T P a s e pur i f i e d from g u i n e a p i g r e n a l o u t e r medulla (Jqjrgensen, 1 9 7 4 ) . A n t i s e r a were t e s t e d by t h e immunoblotting ' P r e s e n t a d d r e s s : Department of P h y s i o l o g y , L h i v e r s i t y o f S o u t h e r n C a l i f o r n i a School of Y e d i c i n e , 20 2 5 Zonal A v e n u e , LOS A n g e l e s , C a l i f o r n i a 90033 787

Copyright 0 1983 by Academic Press, Inc. MI rightsof reproduction in any form reserved. ISBN 0-12-1533194

ALICIA McDONOUGH eta/.

788

method o f R e n a r t e t al., 1 9 7 9 : Na,K-ATPase w a s r e s o l v e d by SDS-PAGE, coupled t o d i a z o t i z e d p a p e r , i n c u b a t e d w i t h t e s t sera, and t h e n 1 2 5 I - l a b e l e d p r o t e i n A. The 1251l a b e l e d p r o t e i n A a d s o r p t i o n s i t e s were r e v e a l e d by a u t o r a d i o g r a p h y . A l l t h r e e r a b b i t s produced a n t i b o d i e s a g a i n s t b o t h a and B , and t h e t i t e r s v a r i e d w i t h t i m e .

111.

CROSS-REACTIVITY

When pooled a n t i s e r a were t e s t e d a g a i n s t r e n a l m i crosomal f r a c t i o n s (by immunoblotting) t h e a n t i - a a n t i b o d i e s c r o s s - r e a c t e d w i t h a l l a - s u b u n i t s t e s t e d (human, b e e f , dog, r a b b i t , r a t , mouse, t u r t l e , and t o a d ) w i t h v a r y i n g i n t e n s i t i e s . The a n t i - 8 a n t i b o d i e s , however, r e a c t e d o n l y t o human, r a t , and mouse @ - s u b u n i t s . No c r o s s - r e a c t i v i t y was s e e n w i t h p a r t i a l l y p u r i f i e d C a ATPaSe from g u i n e a p i g s a r c o p l a s m i c r e t i c u l u m .

IV.

B I N D I N G OF ANTIBODIES TO PROTEOLYTIC FRAGMENTS

T o determine t h e d i s t r i b u t i o n of a n t i g e n i c s i t e s a l o n g t h e l e n g t h of t h e i n d i v i d u a l s u b u n i t s , N a , K A T P a s e w a s e n z y m a t i c a l l y c l e a v e d a s d e s c r i b e d by F a r l e y e t al. (1980) and t h e fragments were r e s o l v e d by SDSPAGE, b l o t t e d , and t e s t e d a s d e s c r i b e d above. Anti-a and a n t i - 8 a n t i b o d i e s w e r e p u r i f i e d by a f f i n i t y chromatography. Pooled a n t i - a a n t i b o d i e s r e a c t e d w i t h f r a g ments of 7 7 , 0 0 0 , 58,000, and 41,000 M r I b u t n o t w i t h fragments of 4 0 , 0 0 0 o r 35,000 M y . R a b b i t s 1 and 2 made a n t i b o d i e s a g a i n s t b o t h t h e M y 58,000 and 4 1 , 0 0 0 f r a g ments, whereas a n t i - a from R a b b i t 3 d i d n o t r e a c t w i t h t h e M y 4 1 , 0 0 0 fragment. The a n t i - 8 a n t i b o d i e s r e a c t e d w i t h fragments i n t h e r a n g e of M r 35,000 t o 4 0 , 0 0 0 seen when t h e enzyme was d i g e s t e d w i t h chymotrypsin.

V.

CELL-FREE

SYNTHESIS

RNA from g u i n e a p i g kidney was t r a n s l a t e d i n r a b b i t r e t i c u l o c y t e l y s a t e s and wheat germ l y s a t e s . Addition of a n t i - a Na,K-ATPase a n t i b o d i e s p r e c i p i t a t e d n a s c e n t ac h a i n s l a b e l e d w i t h [35S]methionine which were charac-

USE OF ANTIBODIES IN STUDIES OF Na,K-ATPase

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t e r i z e d by SDS-PAGE and f l u o r o g r a p h y . The m o l e c u l a r w e i g h t o f t h e n a s c e n t a - c h a i n w a s 96,000. The i d e n t i t y of t h i s p e p t i d e as t h e a - s u b u n i t w a s e s t a b l i s h e d w i t h a f f i n i t y - c h r o m a t o g r a p h y - p u r i f i e d a n t i - a a n t i b o d i e s and by immunocompetition s t u d i e s which d e m o n s t r a t e d t h a t p u r i f i e d Na,K-ATPase immunocompeted , w h e r e a s Ca-ATPase d i d n o t . A n t i s e r a from R a b b i t s 1 and 2 p r e c i p i t a t e d M~ 96,000 p e p t i d e , whereas p r e c i p i t a t i o n w i t h R a b b i t 3 a n t i s e r a was b a r e l y d e t e c t a b l e . This s u g g e s t s t h a t i m munoreactivity with t h e region t h a t y i e l d s t h e M r 41,000 f r a g m e n t s of t h e a - s u b u n i t i s c r u c i a l f o r immunoprecipit a t i o n o f t h e t r a n s l a t i o n p r o d u c t s i n c e o n l y antiserum 3 f a i l e d t o b i n d t o t h i s f r a g m e n t . By f r a c t i o n a t i o n on p r e p a r a t i v e a g a r o s e g e l s , mRNA c o d i n g f o r t h e M r 96,000 p e p t i d e w a s c o n c e n t r a t e d i n t h e 22-28s f r a c t i o n , a s i z e a p p r o p r i a t e f o r t h e t r a n s l a t i o n of a M r 96,000 p e p t i d e , i n d i c a t i n g t h a t a and B must b e encoded o n s e p a r a t e m e s sengers. T r a n s l a t i o n of 0 w a s n o t d e t e c t e d i n t h i s system.

REFERENCES

F a r l e y , R. A . , Goldman, D. W . , and Bayley, H. (1980). J. Biol. Chem. 2 5 5 , 860-864. J d r g e n s e n , P. L. (1974). Blochim. Biophys. A c t a 3 5 6 , 36-52. Renart, J., Reiser, J . , and S t a r k , G. R . (1979). P r o c . Natl. A c a d . S c i . USA 7 6 , 3116-3120.

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CURRENT TOPICS IN MEMBRANES AND TRANSPORT, VOLUME 19

lmmunoreactivityof the a-and CY( +)-Subunits of Na,K-ATPase in Different Organs and Species GERARD D.SCHEUENBERG, IRENE V. PECH, AND WIU'L.

STAHL

VeteransAdministrationMedical Center and the Departmeas of Medicine (Neurology)and Physiology and Biophysics University of Washington School of Medicine Seattle, Washington

I.

INTRODUCTION

Sweadner (1979) h a s r e p o r t e d t h a t b r a i n c o n t a i n s t w o forms of t h e l a r g e s u b u n i t o f t h e N a , K - A T P a s e [ d e s i g n a t e d a and a ( + ) ] , w h e r e a s k i d n e y and o t h e r t i s s u e s c o n t a i n o n l y a s i n g l e form ( a ) . W e are interested i n u s i n g a n t i s e r a t o t h e lamb k i d n e y Na,K-ATPase f o r enzyme l o c a l i z a t i o n s t u d i e s i n k i d n e y ( B a s k i n a n d S t a h l , 1982) and b r a i n . I n t h e l a t t e r case, i t becomes e s s e n t i a l t o e s t a b l i s h t h a t a n t i - l a m b k i d n e y Na,K-ATPase ant i b o d i e s r e a c t w i t h b o t h forms of t h e b r a i n N a , K - A T P a s e large subunit.

791

Copyright 0 1983 by Academic Press. Inc. All rights of reproductionin any form reserved ISBN 0.12-153319-0

GERARD D. SCHELLENBERG

792

F i g . 1 . R e s o l u t i o n o f the a - a n d 8 - s u b u n i t s of the p u r i f i e d l a m b k i d n e y Na,K-ATPase b y S D S - p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s . P o l y p e p t i d e s w e r e v i s u a l i z e d b y C o o m a s s i e b l u e s t a i n i n g or a u t o r a d i o g r a p h y a f t e r b i n d i n g of 1 2 5 1 - l a b e l e d p r o t e i n A a n d a n t i b o d i e s . S a m p l e s (5 p q / l a n e ) w e r e s e p a r a t e d b y SDS-pol y a c r y l a n r i d e s l a b g e l e l e c t r o p h o r e s i s u s i n g 7%acrylamide r e s o l v i n g g e l s . The sample i n l a n e A w a s s t a i n e d w i t h C o o m a s s i e blue. S a m p l e s i n l a n e s B and C were t r a n s f e r r e d t o n i t r o c e l l u l o s e paper, r e a c t e d w i t h primary a n t i s e r u m a g a i n s t t h e Na,K-ATPase ( l a n e B ) or p r e i m m u n e s e r u m ( l a n e C). T h e b o u n d a n t i b o d y was d e t e c t e d u s i n g 1 2 5 1 - l a b e l e d p r o t e i n A f o l l o w e d b y a u t o r a d i o g r a p h y ( 2 ) . "a" and " B " i n d i c a t e t h e p o s i t i o n of the l a r g e a n d s m a l l s u b u n i t s of the e n z y m e .

11.

RESULTS AND DISCUSSION

Rabbits were immunized (Schellenberg e t a ] . , 1 9 8 2 ) using a purified lamb kidney Na,K-ATPase (Fig. 1, lane A). The antiserum obtained inhibited Na,K-ATPase enzyme activity from several sources including lamb kidney, dog kidney, dog brain, and rat brain (Schellenberg e t a l . , 1982). In order to investigate the immunologic crossreactivity of the Na,K-ATPase subunits in crude membranes

IMMUNOREACTIVITY OFTHE (Y AND a(+)SUBUNITS

H

I I

4

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K

L

M

. I

F i g . 2 . I m m u n o l o g i c c r o s s - r e a c t i v i t y o f the a- and a ( + ) s u b u n i t s o f the Na ,K-ATPase. Sampl es w e r e s u b j e c t e d t o el ectrop h o r e s i s (5% a c r y l a m i d e r e s o l v i n g g e l s ) , t r a n s f e r r e d t o n i t r o c e l l u l o s e p a p e r , and r e a c t e d w i t h r a b b i t a n t i - l a m b k i d n e y Na,K-ATPase s e r u m . Bound a n t i b o d i e s w e r e d e t e c t e d w i t h 1 2 5 1 - l a b e l e d p r o t e i n A f o l l o w e d b y a u t o r a d i o g r a p h y . O n l y the a - s u b u n i t r e g i o n s of the g e l s are shown. S a m p l e s a r e as f o l l o w s : ( A ) r a t b r a i n rnicrosomes (5 p g ) ; ( B ) N a L - t r e a t e d r a t b r a i n m i c r o s o m e s ( 7 p g ) ; ( C ) r a t k i d n e y m i c r o s o m e s (10 pg); ( D ) dog k i d n e y microsomes ( 1 p g ) ; (E,J, and M ) p a r t i a l l y p u r i f i e d d o g b r a i n Na,K-ATPase (2 p g ) ; ( F ) l a m b k i d n e y m i c r o s o m e s (1 pg); ( G , H , and K ) p u r i f i e d l a m b k i d n e y Na,K-ATPase ( 0 . 4 pg); (I) human c e r e b r a l cortex m i c r o s o m e s (5 p g ) ; and ( L ) Manduca s e x t a b r a i n m i c r o s o m e s ( 5 pg).

and p u r i f i e d p r e p a r a t i o n s of enzyme, t h e p o l y p e p t i d e s w e r e f i r s t r e s o l v e d by SDS-polyacrylamide s l a b g e l electrophoresis. The s e p a r a t e d p o l y p e p t i d e s w e r e t r a n s f e r r e d t o unmodified n i t r o c e l l u l a r p a p e r (Towbin e t a l . , 1979; B u r n e t t e , 1981) and r e a c t e d w i t h r a b b i t a n t i - l a m b k i d n e y Na,K-ATPase serum. The a n t i g e n - a n t i b o d y complexes F i g u r e 1, were d e t e c t e d u s i n g 1251-labeled p r o t e i n A. l a n e s B and C , i l l u s t r a t e s t h e r e a c t i v i t y o f t h e p u r i f i e d lamb k i d n e y N a , K - A T P a s e w i t h immune and preimmune serum, r e s p e c t i v e l y . The predominant immunoreactive bands c o r respond t o t h e a - and @ - s u b u n i t s . No r e a c t i v i t y o r background b i n d i n g was o b s e r v e d w i t h preimmune serum. The a n t i b o d i e s p r e s e n t a p p e a r t o be h i g h l y s p e c i f i c f o r t h e a - and $ - s u b u n i t s . The e x i s t e n c e o f b o t h a - and a ( + ) - s u b u n i t s of t h e N a , K - A T P a s e i n b r a i n w a s v e r i f i e d by e l e c t r o p h o r e s i s of b r a i n and kidney samples p h o s p h o r y l a t e d i n t h e p r e s e n c e of [ Y - ~ ~ P I A T P , Mg2+, and N a + ( d a t a n o t shown). The i m munologic c r o s s - r e a c t i v i t y of t h e a - and a ( + ) - p o l y p e p I n lamb, dog, and r a t kidney t i d e s are shown i n F i g . 2 . p r e p a r a t i o n s o n l y a s i n g l e immunoreactive band ( a ) w a s

GERARD D. SCHELLENBERG

794

observed ( F i g . 2 , l a n e s C , DI F, G I H I and K ) . I n cont r a s t , t h e dog b r a i n p r e p a r a t i o n ( F i g . 2 , l a n e s E , J , and M) c o n t a i n e d a band i d e n t i c a l i n m o b i l i t y ( a p p a r e n t M y 9 6 , 6 0 0 & 6 1 0 ) t o t h e a band of k i d n e y , a s w e l l a s a second band w i t h a h i g h e r molecular weight ( a p p a r e n t M r 1 0 6 , 0 0 0 & 560) c o r r e s p o n a i n g t o t h e a ( + ) form. Rat b r a i n p r e p a r a t i o n s showed an a ( + ) immunoreactive band and a r a t h e r f a i n t a band ( F i g . 2 , l a n e s A and B). Both t y p e s of l a r g e s u b u n i t were a l s o observed i n r a t b r a i n membranes p r e p a r e d i n t h e p r e s e n c e of a combination of p r o t e a s e i n h i b i t o r s (phenylmethylsulfonylfluoride, d i i s o p r o p y l f l u o r o p h o s p h a t e , EDTA, p h e n a n t h r o l i n e , peps t a t i n A , and i o d o a c e t a m i d e ) . Human b r a i n ( F i g . 2 , l a n e I ) had both a and a (+) forms p r e s e n t , whereas t h e i n s e c t Manduca s e x t a ( l a n e L) had o n l y a s i n g l e band w i t h a m o b i l i t y i n t e r m e d i a t e t o t h e a and a ( + ) forms of dog b r a i n ( l a n e M ) . The 8-subunit of dog and r a t b r a i n a l s o c r o s s r e a c t e d with t h e lamb kidney Na,K-ATPase a n t i s e r u m . However, t h e b r a i n @ - s u b u n i t had a lower m o l e c u l a r weight than t h e kidney 8-subunit. The molecular w e i g h t of t h e r a t b r a i n s m a l l s u b u n i t was n o t changed when samples were p r e p a r e d i n t h e p r e s e n c e of t h e p r o t e a s e i n h i b i t o r s mentioned above.

ACKNOWLEEMENTS This work was supported i n p a r t by NIH g r a n t NS-05424 and by G. Schellenberg was supported by a t h e Veterans Administration. National Research Service Fellowship from t h e NIH (NS 06388). I r e n e Pech received a s t i p e n d f r m t h e Graduate School Research Fund o f t h e University of Washington.

REFERENCES

Baskin, D. G . , and S t a h l , W. L. (1982). Immunocytochemical l o c a l i z a t i o n of .Na+,K+-ATPase i n t h e r a t kidney. Histochemistry 73, 535-548. Burnette, W. N . (1981) "Western b l o t t i n g " : E l e c t r o p h o r e t i c t r a n s f e r of p r o t e i n s from sodium dodecyl s u l f a t e - p o l y a c r y l a mide g e l s t o unmodified n i t r o c e l l u l o s e and radiographic deAnal. t e c t i o n w i t h antibody and r a d i o i o d i n a t e d p r o t e i n A. Biochem. 112 , 195-203.

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S c h e l l e n b e r g , G. D . , Pech, I. V., and S t a h l , W. L. (1982). Immunoreactivity of s u b u n i t s of t h e Na+,K+-ATPase : Crossr e a c t i v i t y of t h e a, a+ and 6 forms i n d i f f e r e n t o r g a n s and s p e c i e s . B i o c h i m . B i o p h y s . A c t a 6 4 9 , 691-700+ + Sweadner, K. J. (1979). Two m o l e c u l a r forms of (Na + K )ATPase i n b r a i n . J . Biol. Chem. 2 5 4 , 6060-6067. Towbin, H . , S t a c h e l i n , T. , and Gordon, J. (1979). E l e c t r o p h o r e t i c t r a n s f e r of p r o t e i n s from polyacrylamide g e l s to n i t r o c e l l u l o s e s h e e t s : Procedure and some a p p l i c a t i o n s . Proc. N a t l . A c a d . Sci. USA 7 6 , 4350-4354.

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CURRENT TOPICS IN MEMBRANES ANDTRANSFORT. VOLUME 19

Role of Na+ and Ca2+ Fluxes in Terminal Differentiationof Murine ErythroleukemiaCells I. G.MACARA, R. D.SMITH, ANDL. C. CANTLEY Department of Biochemistry and Molecular Biulogv Harvard Universify Cambridge, Massachusens

I.

INTRODUCTION

Ion f l u x e s a c r o s s t h e plasma membrane are now r e c o g n i z e d a s p l a y i n g a c e n t r a l r o l e i n c o n t r o l of c e l l p r o l i f e r a t i o n and d i f f e r e n t i a t i o n (Harold, 1977; Rozeng u r t , 1 9 8 1 ) . However, t h e mechanisms by which t h e s e f l u x e s a r e r e g u l a t e d and c o u p l e d t o c h a n g e s i n gene exp r e s s i o n remain o b s c u r e . A u s e f u l model f o r i n v e s t i gating the regulation of terminal d i f f e r e n t i a t i o n is t h e F r i e n d murine e r y t h r o l e u k e m i a (MEL) c e l l l i n e . These c e l l s c a n m u l t i p l y i n d e f i n i t e l y i n c u l t u r e , b u t i f t r e a t e d w i t h d i m e t h y l s u l f o x i d e (DMSO) ( o r a v a r i e t y of o t h e r a g e n t s ) , t h e y become committed t o t e r m i n a l d i f f e r e n t i a t i o n . Committed c e l l s d i f f e r e n t i a t e i n t o “ r e t i c u l o c y t e s , ” and cease d i v i d i n g , e v e n i f t h e i n d u c i n g a g e n t h a s been removed ( H a r r i s o n , 1976; Housman et a l . , 1978). A d e c r e a s e i n Na,K-ATPase a c t i v i t y h a s been i m p l i c a t e d as an e a r l y s t e p i n commitment ( B e r n s t e i n et a l . , 1 9 7 6 ) . However, w e have found a l s o t h a t a n i n c r e a s e i n 797

Copynghr B 1983 by Academic Press, Inc. All rights of repduction in any form reserved. ISBN O-lZ-l533l94l

TIME ( m i d

TIME (min)

TIMEirnin)

( A ) E f f e c t o f a m i l o r i d e and e x t e r n a l Na on 45Ca i n f l u x i n t o MEL C e l l s . C e l l s were s u s Fig. 1. pended i n c h o l i n e - R i n g e r ' s ( 0 , @ ) or N a - R i n g e r ' s ( 0,m) [ 1 4 5 mM NaCl or choline c h l o r i d e , 5 mM KCI, 2 mM KH2PO4, 5 0 p M CaC12, 20 mM HEPES (pH 7 . 4 0 ) ] . S u s p e n s i o n s were c o o l e d t o O°C and 45CaC12 w a s a d d e d . A l i q u o t s were p e r i o d i c a l l y d i l u t e d i n t o c o l d c h o l i n e - R i n g e r ' s and c e n t r i f u g e d t h r o u g h o i l . d m i l o r i d e , where p r e s e n t ( @ , m ) , was i n c u b a t e d a t 40 p M w i t h the cells for 4 hr b e f o r e the s t a r t of the e x p e r i m e n t . ( B ) E f f e c t o f m i l o r i d e and external Na on 4% e f f l u x . C e l l s were l o a d e d w i t h 45Ca b y preincubation i n Na-Ringer's. T h e y were then c e n t r i f u g e d and r e s u s p e n d e d i n choline- ( m ) or NaR i n g e r ' s ( 0 ) a t O°C. Samples were removed and t r e a t e d as d e s c r i b e d above. S e p a r a t e cell suspensions were p r e i n c u b a t e d w i t h 4 0 pM a m i l o r i d e f o r 4 hr a n d r e s u s p e n d e d i n choline- ( 0 )or N a - R i n g e r ' s ( 0 ) c o n t a i n i n g 4 0 pM m i l o r i d e . ( C ) E f f e c t o f o u a b a i n on 4% i n f l u x . C e l l s were p r e i n c u b a t e d f o r 30 m i n (23OC) e i t h e r i n normal a(+) medium ( o ) or i n medium + 1 mM o u a b a i n ( @ ) T h e y w e r e then c e n t r i f u g e d and r e s u s p e n d e d i n Na-Ringer 's 45Ca i n f l u x was measured as d e s c r i b e d above .e R e p r i n t e d from S m i t h

.

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Na+AND Ca2+FLUXES IN TERMINAL DIFFERENTIATION

799

4 5 C a u p t a k e accompanies commitment, and t h a t commitment

can be b l o c k e d by c h e l a t i o n o f e x t e r n a l C a (Levenson et 1980). W e have s t u d i e d t h e N a dependence of 4 5 C a f l u x e s i n uninduced MEL c e l l s and found t h a t t h e i o n f l u x e s are c o u p l e d by a Na/Ca exchange system. The d i u r e t i c d r u g a m i l o r i d e , which b l o c k s t h e commitment o f MEL c e l l s (Levenson et al., 1 9 8 0 1 , p o t e n t l y i n h i b i t e d t h e N a / C a exchange. However, a m i l o r i d e d i d n o t r e v e r s e t h e f a l l i n 86Rb+ u p t a k e i n d u c e d by DMSO, s u g g e s t i n g t h a t a decrease i n N a , K - A T P a s e a c t i v i t y i s e s s e n t i a l t o commitment a n d p r e c e d e s t h e change i n C a i n f l u x . T h i s work i s d e s c r i b e d i n more d e t a i l i n Smith et a l . , ( 1 9 8 2 ) . al.,

11.

RESULTS

C a i n f l u x through a Na/Ca a n t i p o r t should be i n h i b i t e d by h i g h e x t e r n a l N a and s t i m u l a t e d by h i g h i n ternal Na. C a e f f l u x s h o u l d b e s t i m u l a t e d by h i g h external Na. These c r i t e r i a w e r e f u l f i l l e d f o r f l u x e s i n uninduced MEL c e l l s . Replacement of 150 mM c h o l i n e c h l o r i d e i n t h e medium w i t h N a C l d e c r e a s e d t h e i n i t i a l r a t e o f 45Ca i n f l u x by 4 0 k 1 5 % ( F i g . 1 A ) . A s i m i l a r d e c r e a s e w a s produced by 4 0 pM a m i l o r i d e . The p r e s e n c e of 150 mM e x t e r n a l N a s t i m u l a t e d t h e i n i t i a l r a t e o f n e t 45Ca e f f l u x from 45Ca-loaded MEL c e l l s by 5 0 % , and t h i s s t i m u l a t i o n w a s b l o c k e d by 4 0 P M a m i l o r i d e ( F i g . 1B). R a i s i n g i n t e r n a l N a by p r e i n c u b a t i o n of t h e c e l l s w i t h 1 m M o u a b a i n (25OC) d o u b l e d t h e r a t e of 45Ca i n f l u x (Fig. 1 C ) . The c o n c e n t r a t i o n o f a m i l o r i d e r e q u i r e d t o i n h i b i t N a / C a exchange ( 4 0 U M ) i s t h e same a s t h a t which b l o c k s commitment of MEL c e l l s i n 1 . 5 % DMSO (Levenson e t a l . , 1 9 8 0 ) . T h i s c o n c e n t r a t i o n had no d e t e c t a b l e e f f e c t on n e t N a + uptake. Thus, i n h i b i t i o n o f N a + i n f l u x i s n o t n e c e s s a r y t o b l o c k commitment, and N a / C a exchange must account f o r only a small f r a c t i o n of t h e t o t a l N a f l u x . T r e a t m e n t o f MEL c e l l s w i t h 1 . 5 % DMSO f o r 1 8 h r d e c r e a s e d o u a b a i n - i n h i b i t a b l e 86Rb+ u p t a k e by 3 0 % ( F i g . 2 ) . The d e c r e a s e w a s n o t overcome by t h e a d d i t i o n of 40 V M a m i l o r i d e . Thus, t h e s t e p a t which a m i l o r i d e b l o c k s commitment must be s u b s e q u e n t t o t h e c h a n g e s i n Na,K-ATPase a c t i v i t y .

800

I. G.MACARA

3000

X

3

-I LL

z

/

u

lOOC,

+a

a

(D

co

1

F i g . 2. E f f e c t of DMSO and a m i l o r i d e on 86Rb+ i n f l u x i n t o MEL c e l l s . C e l l s were grown i n a(+) medium w i t h 1.5% Me2SO ( 0 , W ), 1.5% Me2SO + 40 pM a m i l o r i d e ( A ,A ) , or w i t h o u t d r u g ( 0 , 0 ) f o r 40 hr before use. 86RbC1 was then a d d e d and I - m l a l i q u o t s were p e r i o d i c a l l y c e n t r i f u g e d t h r o u g h o i l . O u a b a i n (1 mM) was a d d e d t o three o f the f r a c t i o n s ( W ,A , 0 ) i m m e d i a t e l y b e f o r e the 86Rb+. Inset shows relative i n i t i a l r a t e s o f 86Rb+ u p t a k e . R e p r i n t e d from S m i t h e t a l . ( 1 9 8 2 ) w i t h p e r m i s s i o n o f the Journal o f Biological Chemistry.

111.

DISCUSSION

W e have p r e s e n t e d e v i d e n c e f o r an a m i l o r i d e - s e n s i t i v e Na/Ca a n t i p o r t system i n t h e MEL c e l l plasma memb r a n e , t h e o p e r a t i o n of which i s e s s e n t i a l t o a l l o w terminal d i f f e r e n t i a t i o n of t h e c e l l s . The r e s u l t s sugg e s t , moreover, t h a t t h e primary s i g n a l t h a t commits t h e c e l l s t o d i f f e r e n t i a t e i s a d e c r e a s e i n Na,K-ATPase ac-

Na+AND Ca2+FLUXES IN TERMINAL DIFFERENTIATION

801

t i v i t y . T h i s h y p o t h e s i s i s s u p p o r t e d by B e r n s t e i n ' s d e m o n s t r a t i o n t h a t ouabain a l o n e i n d u c e s d i f f e r e n t i a t i o n i n some MEL c e l l l i n e s ( B e r n s t e i n e t a l . , 1 9 7 6 ) . W e s u g g e s t t h a t t h e i n h i b i t i o n of Na,K-ATPase a c t i v i t y by i n d u c e r s c a u s e s a s m a l l i n c r e a s e i n c y t o s o l i c N a + concenIf t r a t i o n and d e p o l a r i z a t i o n of t h e plasma membrane. t h e MEL c e l l Na/Ca a n t i p o r t i s similar t o t h a t i n c a r d i a c t i s s u e s ( i . e . , i s e l e c t r o g e n i c and exchanges 3-4 Na atoms p e r Ca atom) , a l a r g e i n c r e a s e i n c y t o s o l i c C a 2 + w i l l res u l t and t r i g g e r t e r m i n a l d i f f e r e n t i a t i o n o f t h e c e l l s . The h y p o t h e s i s t h a t d e c r e a s e d Na,K-ATPase a c t i v i t y i s t h e key i n i t i a l e v e n t i n commitment of MEL c e l l s i s s u p p o r t e d by t h e o b s e r v a t i o n t h a t t h e t i m e r e q u i r e d f o r i n d u c e r s t o i n h i b i t Na,K-ATPase a c t i v i t y ( % 1 2 h r ) i s s i m i l a r t o t h e l a g p e r i o d t h a t o c c u r s b e f o r e commitment b e g i n s (Mager and B e r n s t e i n , 1 9 7 8 ) . Moreover, r a i s i n g t h e c y t o p l a s m i c Na+ l e v e l by a b r i e f i n c u b a t i o n w i t h o u a b a i n p l u s monens i n a c c e l e r a t e s commitment of MEL c e l l s (Smith e t a l . , 1 9 8 2 ) . The l e n g t h of t i m e r e q u i r e d b e f o r e maximum i n h i b i t i o n o r a c t i v a t i o n of Na,K-ATPase i s achieved (6-12 h r ) s u g g e s t s t h a t changes i n p r o t e i n s y n t h e s i s might be req u i r e d . The p o s s i b i l i t y t h a t t h e Na,K-ATPase copy number a l t e r s upon commitment of MEL c e l l s i s now b e i n g i n v e s t i gated.

ACKNOWLEDGMENTS

T h i s work w a s s u p p o r t e d by g r a n t GM 28538 ( t o L.C.) from t h e N a t i o n a l I n s t i t u t e s o f H e a l t h and g r a n t 791040 ( t o L.C.) from t h e I . G . M . was s u p p o r t e d by a C h a r l e s A. American H e a r t A s s o c i a t i o n . King F e l l o w s h i p (Medical Foundation, I n c . ) .

REFERENCES

B e r n s t e i n , A . , Hunt, D. M . , C r i c h l e y , V . , and Mak, T. W. ( 1 9 7 6 ) . C e l l 9 , 375-381. B r i d g e s , K . , Levenson, R . , Housman, D . , and C a n t l e y , L. C. ( 1 9 8 1 ) . 3. Cell Biol. 90, 542-544. Harold, F. M. (1977). Annu. Rev. M i c r o b i o l . 3 1 , 181-203. H a r r i s o n , P. R. (1976). Nature (London) 262, 353-356. Houman, D., G u s e l l a , J . , G e l l e r , R., Levenson, R . , and W e i l , S. (1978). In " D i f f e r e n t i a t i o n of Normal and N e o p l a s t i c Hemat o p o i e t i c C e l l s " (B. CLarkson, P. A Marks, and J. T i l l , e d s . ) , p p . 193-207. Cold S p r i n g Harbor L a b . , Cold S p r i n g Harbor, New York.

1. G.MACARA

802

Levenson, R., Housnan, D., and Cantley, L. C. (1980). Proc. N a t l . A c a d . Sci. USA 77, 5948-5952. Rozengurt, E. (1981). Adv. Enzyme R e g u l . 19, 61-85. Smith, R., Macara, I. G., Levenson, R., Housman, D . , and C a n t l e y , L. C. (1982). J. Biol. Chem. 257, 773-780. Yamasaki, H., Fibach, E . , Nudel, U . , Weinstein, B., R i f k i n d , A . , and Marks, P. A. (1977). Proc. N a t l . A c a d . Sci. USA 7 4 ,

3451.

CURRENT TOPICS IN MEMBRANES AND TRANSPORT, VOLUME 19

NalK Pumps and Passive K Transport in Large and Small Reticulocytes of Anemic Low- and High-Potassium Sheep P. K. LA UF AND G. VMET Department of Physiologv Duke University Medical Center Durham, North Carolina and Mar Planck Institute of Biochemistry 8033 Maninsried, Federal Republic of Germany

I.

INTRODUCTION

The h i g h p o t a s s i u m (HK)/low p o t a s s i u m (LK) dimorphism of mature e n u c l e a t e s h e e p r e d c e l l s i s c h a r a c t e r i z e d by major d i f f e r e n c e s i n t h e q u a n t i t a t i v e ( s i t e s ) and q u a l i t a t i v e ( k i n e t i c ) N a / K pump a c t i v i t i e s as w e l l as coupled t o s p e c i f i c o u a b a i n - i n s e n s i t i v e , p a s s i v e c a t i o n " l e a k s " ( T o s t e s o n and Hoffman, 1960; Hoffman and T o s t e s o n , 1 9 7 1 ; J o i n e r and L a u f , 1 9 7 8 a , b ) . G e n e t i c a l l y a s s o c i a t e d w i t h t h e above membrane t r a n s p o r t p r o p e r t i e s are t h e M and L s u r f a c e a n t i g e n s i n HK and LK s h e e p r e d c e l l s , r e s p e c t i v e l y (Rasmusen and H a l l , 1 9 6 6 ; Rasmusen, 1 9 6 9 ) . Because t h e a n t i b o d i e s a g a i n s t L a n t i g e n s b o t h a c t i v a t e N a / K pump f l u x ( E l l o r y and Tucker, 1969; Lauf e t a z . , 1970) and r e d u c e p a s s i v e K+ l e a k f l u x (Dunham, 1 9 7 6 ; Lauf e t al., 1 9 7 7 ) , and because of a v a r i e t y of a d d i t i o n a l r e a s o n s (Lauf, 1981) , a t l e a s t two L a n t i g e n s have been r e c o g n i z e d : t h e Lp a n t i g e n , a s s o c i a t e d w i t h t h e i n h i b i t e d s t a t e of t h e N a / K pump, and t h e Le a n t i g e n s i g n i f y i n g t h e a c t i v a t e d s t a t e of a K+ " l e a k " system. 803

Copynght 0 1983 by Aedemc Press. Inc. All nghu of reproduction UI any form wrved. ISBN 012-1533190

P. K. LAUF AND G.VALET

004

+

There i s new e v i d e n c e t h a t pump-independent p a s s i v e K movements may be mediated by a t l e a s t t w o components (Dunham and E l l o r y , 1981; Lauf and Theg, 1980; L a u f , 1981). The HK/LK polymorphism d e v e l o p s j u s t b e f o r e matur a t i o n of a sheep r e d c e l l . The immediate p r e c u r s o r of both HK and LK e r y t h r o c y t e s i s t h e high-K+ r e t i c u l o c y t e which, c e r t a i n l y i n t h e case of LK sheep, m u s t be g e n e t i c a l l y endowed w i t h membrane t r a n s p o r t mechanisms i n s t r u m e n t a l f o r t h e t r a n s f o r m a t i o n t o t h e f i n a l LK s t e a d y - s t a t e c e l l . The work p r e s e n t e d h e r e a t t e m p t e d t o shed f u r t h e r l i g h t on t h e membrane t r a n s p o r t e v e n t s accompnaying t h e c e l l u l a r m a t u r a t i o n of e r y t h r o c y t e p r e c u r s o r s i n t o both e n u c l e a t e HK as w e l l as LK r e d c e l l s . Such a s t u d y i s a l s o of i n t e r e s t because of i t s g e n e r a l a s p e c t of t h e r e l a t i o n s h i p between a c t i v e and p a s s i v e c a t i o n t r a n s p o r t systems d u r i n g c e l l u l a r d i f f e r e n t i a t i o n and m a t u r a t i o n .

11.

RESULTS AND D I S C U S S I O N

The b a s i c s t r a t e g y of t h i s work was f i r s t t o i s o l a t e by c e n t r i f u g a l e l u t r i a t i o n and t o c h a r a c t e r i z e by flow cytophotometry ( V a l e t e t al., 1 9 7 8 ; Lauf and V a l e t , 1 9 8 0 ) t h e r e t i c u l o c y t e p o p u l a t i o n newly produced i n response t o massive b l e e d i n g , avd t h e n t o measure i n t h e s e p a r a t e d c e l l s a c t i v e K+ pump and p a s s i v e K+ l e a k f l u x e s , a s w e l l a s t h e number of ouabain b i n d i n g and hence pump s i t e s p e r c e l l . I n t h e c o u r s e of t h e r e s p o n s e o f b o t h HK and LK sheep t o massive hemorrhage, w e d e t e c t e d c y t o p h o t o m e t r i c a l l y ( a c r i d i n e orange f l u o r e s c e n c e compared with conventional s t a i n i n g techniques) t h e simultaneous appearance i n t h e p e r i p h e r a l blood of two d i s t i n c t r e t i culocyte populations: a l a r g e , macrocytic population ( p o p u l a t i o n M c e l l s ) , and a s m a l l p o p u l a t i o n w i t h r a t h e r normocytic r e t i c u l o c y t e s whose c e l l volume was i n d i s t i n g u i s h a b l e from t h a t of t h e o l d e r LK r e d c e l l s . I n Table I t h e membrane t r a n s p o r t p a r a m e t e r s of m a c r o c y t i c r e t i c u l o c y t e s and of c e l l s from t h e volume p o p u l a t i o n c o n t a i n i n g t h e s m a l l r e t i c u l o c y t e s were compared w i t h t h e d a t a found i n r e d c e l l s from u n s e p a r a t e d blood of two LK and one HK sheep b e f o r e and 6 days a f t e r massive hemorrhage. On b a s i s of t h e d a t a of u n s e p a r a t e d r e d c e l l s i n t h e t h r e e sheep b e f o r e and on t h e s i x t h day a f t e r b l e e d i n g (Table IA and B ) , it i s e v i d e n t t h a t massive changes of c e l l u l a r c a t i o n s and of K+ pump and l e a k f l u x e s had

(=ME)

TABLE I.

Membrane Transport Parameters ~

~~~

~

Cell

mperimental step

Sheep (cations, antigens)

A. Unseparated cells; unbled a n i mals

LK 7(LM) 32.1 LK 10(LL) 35.4 HK 8(m) 29.9

Unseparat e d cells, 6 days a f t e r bleeding

volume Reticu(10-l~ l o c y t e s (%) liter)

1.2 1.7 4.0

C e l l K+

content M/cell)

K+ pump influx (W/liter cell/hr)

~~~~

Pump turnover (Kt i o n s / pump/sec)

Pump

density (ouabain sites/mu2)

$PP

(cm/sec x lo9)

2.31

0.17 0.26 0.58

23 26 27

0.7 1.1 2.1

0.59 2.81 3.16

0.28 0.28

B.

Separated large re t i c u l o cytes (population

37.7 39.5 36.1

12.5 17.0 15.0

1.04 0.78 2.70

0.30 1.07 1.27

36 121 37

0.9 1.0 3.6

1.93 6.01 1.40

LK 7(LM) LK lO(LL) LK 8(MM)

68.7 58.5 69.1

26.5 33.0 55.0

3.96 2.52 5.96

1.15 8.42 6.45

84 162 165

1.7 6.4 5.0

5.80 19.60 8.9

LK 7(LM) LK lO(LL) HK 8(MM)

37.2 38.2 37.9

18.0 18.0 27.0

0.55 0.25 2.48

0.14 0.19 1.53

35 25 48

0.4 0.8 3.6

1.20 6.50 1.40

LK 7(LM) LK lO(LL) HK

8(MM)

C.

M) D. Separat e d small re t i c u l o cytes (population 111)

P. K. LAUF AND G.VALET

806

o c c u r r e d . Note t h a t t h e r e d c e l l p a r a m e t e r s of t h e unb l e d animals a r e c h a r a c t e r i s t i c f o r LK (sheep 7 and 1 0 ) and HK (sheep 8) a n i m a l s : With t h e s m a l l c e l l volume d i f f e r e n t i a l s i n mind, c e l l u l a r K+ c o n t e n t of LK c e l l s i s about one-seventh of t h a t seen i n HK c e l l s , and cell u l a r Na+ c o n t e n t 5- t o 6 - f o l d h i g h e r . The h i g h e r K+ pump a c t i v i t y s e e n i n HK c e l l s i s p r i m a r i l y due t o a h i g h e r pump d e n s i t y , i . e . , 2 pumps/mu2 s u r f a c e area i n s t e a d of 1 i n LK c e l l s . Note t h a t t h e a p p a r e n t p a s s i v e K+ p e r m e a b i l i t y was found t o be much h i g h e r t h a n i n HK c e l l s , p a r t i c u l a r l y i n LK sheep 1 0 r e d c e l l s . T h i s f i n d . i n g i s c o n s i s t e n t w i t h e a r l i e r o b s e r v a t i o n s of Tosteson and Hoffman ( 1 9 6 0 ) . S i x days a f t e r b l e e d i n g , however, t h e mean c e l l volume was i n c r e a s e d due t o e n t r a n c e of Furthermore , t h e c e l l u l a r K+ r e t i c u l o c y t e s (Table 1 B ) c o n t e n t was h i g h e r i n b o t h LK and HK c e l l s and a remarka b l e r i s e i n ”&$(o c c u r r e d which i n p a r t may be e x p l a i n e d by a h i g h e r pump t u r n o v e r a s w e l l as by a h i g h e r pump d e n s i t y . I n t e r e s t i n g l y , t h e r e was a r a t h e r s i g n i f i c a n t upward change i n t h e a p p a r e n t p a s s i v e K+ p e r m e a b i l i t y of a l l t h r e e sheep r e d c e l l s , confirming some of o u r e a r l i e r work on anemic LK sheep ( K i m e t al., 1 9 8 0 ) . Analyzing t h e l a r g e r e t i c u l o c y t e s s e p a r a t e d by cent r i f u g a l e l u t r i a t i o n , it i s r e a d i l y a p p a r e n t t h a t t h e y are t h e s o u r c e of t h e massive t r a n s p o r t changes s e e n i n t h e whole blood (Table 1 C ) . Note t h a t t h e c e l l volumes a r e almost twice t h a t s e e n i n u n s e p a r a t e d c e l l s and t h a t t h e K+ c o n t e n t p e r c e l l is much h i g h e y , exceeding by f a r t h e volume increment. The i n c r e a s e d values apparentl y a r e due t o s i g n i f i c a n t changes i n pump t u r n o v e r and pump d e n s i t y . These e v e n t s were accompanied by an i n c r e a s e of t h e a p p a r e n t p a s s i v e K+ p e r m e a b i l i t y by a b o u t 10-fold a s compared t o t h a t s e e n i n r e d c e l l s of unbled animals. The t r a n s p o r t d a t a of s m a l l r e t i c u l o c y t e s o b t a i n e d from s m a l l e r c e l l p o p u l a t i o n s ( p o p u l a t i o n 111, V a l e t e t al., 1 9 7 8 ) by t h e u s e of a p e r c o l g r a d i e n t i n combinat i o n w i t h c e n t r i f u g a l e l u t r i a t i o n are q u i t e i n c o n t r a s t t o t h o s e of t h e l a r g e c e l l s . I t can be seen t h a t t h e s e c e l l s had t y p i c a l LK o r HK potassium l e v e l s , t h a t K+ pump and l e a k a c t i v i t i e s , a s w e l l as t h e pump d e n s i t y and pump t u r n o v e r number, p e r m i t t e d a c l e a r d i s t i n c t i o n between LK and HK r e d c e l l s . These d a t a s u g g e s t t h a t , p a r t i c u l a r l y i n LK a n i m a l s , 10-308 s m a l l r e t i c u l o c y t e s had most l i k e l y much lower t r a n s p o r t a c t i v i t i e s , p e r h a p s c l o s e t o t h o s e of mature a d u l t LK (or HK) r e d c e l l s . Aside from t h e t r a n s p o r t d a t a shown h e r e , t h e r e i s f u r t h e r e v i d e n c e f o r t h e p r e s e n c e of two r e t i c u l o c y t e p o p u l a t i o n s : I n hemoglobin A or AB t y p e sheep w e found t h a t t h e r e t i c u l o c y t e s of t h e p o p u l a t i o n M c o n t a i n

.

ME

NalK PUMPSAND PASSIVE K+ TRANSPORT

007

hemoglobin C , whereas t h e l a t t e r w a s a b s e n t i n t h e smaller r e t i c u l o c y t e s ( V a l e t and L a u f , 1 9 8 0 ) . The f i n d i n g s r e p o r t e d h e r e are r e l e v a n t f o r choosi n g t h e c o r r e c t e x p e r i m e n t a l model t o s t u d y t h e membrane t r a n s p o r t changes d u r i n g t h e t r a n s i t i o n from HK r e t i c u l o c y t e s t o m a t u r e LK red c e l l s . As t h e r e e x i s t s a l r e a d y a c o n s i d e r a b l e l e v e l of c o m p l e x i t y stemming from t h e k i n e t i c d i f f e r e n c e s i n c a t i o n pumps and l e a k s between t h e t w o t y p e s of s h e e p r e d c e l l s , d i s c o v e r y o f two d i s t i n c t r e t i c u l o c y t e p o p u l a t i o n s , of which o n e may be due t o s k i p p i n g a c e l l d i v i s i o n , adds t o t h e f a c t o r s t o be c o n s i d e r e d i n t h e a n a l y s i s of t h e HK/LK t r a n s i t i o n . U n f o r t u n a t e l y , t h e y i e l d s o f t h e normally produced r e t i c u l o c y t e s are s m a l l , whereas t h o s e o f t h e p e r h a p s p h y s i o l o g i c a l l y less r e p r e s e n t a t i v e l a r g e r e t i c u l o c y t e s are h i g h e r . F u t u r e s t u d i e s must d e c i d e whether and which o f t h e t e m p o r a l changes of t r a n s p o r t p a r a m e t e r s o b s e r v e d i n t h e l a r g e r e t i c u l o c y t e s warrant f u r t h e r study i n ord e r t o u n d e r s t a n d t h e n a t u r a l o r i g i n of t h e LK s h e e p r e d cell.

ACKNOWLEXMENT

T h i s work w a s s u p p o r t e d i n p a r t by N I H g r a n t AM 28236/HEM.

REFERENCES

Dunham, P. B. (1976). P a s s i v e potassium transport i n LK s h e e p r e d c e l l s . E f f e c t s o f a n t i - L a n t i b o d y and i n t r a c e l l u l a r potass i u m . J. G e n . P h y s i o l . 6 8 , 567-581. Dunham, P. B . , and E l l o r y , J. C. (1981). P a s s i v e potassium t r a n s port i n low potassium sheep red c e l l s : Dependence upon c e l l volume and c h l o r i d e . J. P h y s i o l . ( L o n d o n ) 3 1 8 , 511-530. E l l o r y , J. C . , and Tucker, E. M. (1969). S t i m u l a t i o n of t h e potassium transport system i n l o w potassium type r e d blood c e l l s by a s p e c i f i c a n t i g e n a n t i b o d y r e a c t i o n . N a t u r e (Lond o n ) 2 2 2 , 477-478. Hoffman, P. G . , and T o s t e s o n , D. C. ( 1 9 7 1 ) . A c t i v e sodium and potassium t r a n s p o r t i n h i g h p o t a s s i u m and low potassium sheep r e d c e l l s . J. Gen. P h y s i o l . 5 8 , 438-466. J o i n e r , C. H. , and L a u f , P. K. (1978a). The c o r r e l a t i o n between o u a b a i n b i n d i n g and K+ pump flux i n h i b i t i o n i n human and J . P h y s i o l . ( L o n d o n ) 283, 155-175. sheep erythrocytes.

P.K. LAUF AND G. VALET

808

J o i n e r , C. H . , and Lauf, P. K. (197833). Modulation o f ouabain binding and K+ pump f l u x by c e l l u l a r Na+ and K+ i n human and sheep e r y t h r o c y t e s . J. P h y s i o l . ( L o n d o n ) 2 8 3 , 177-196. K i m , H. D., Theg, B. E., and Lauf, P. K. (1980). LK sheep r e t i c u l o c y t o s i s . E f f e c t of anti-L on K f l u x and i n v i t r o maturat i o n . J. G e n . P h y s i o l . 76, 109-121. Lauf, P. K. (1981a). A chemically unmasked, c h l o r i d e dependent K+ t r a n s p o r t i n l o w K+ sheep red c e l l s : Genetic and evolutiona r y a s p e c t s . I n "Erythrocyte Membranes 2 : Recent C l i n i c a l and Experimental Advances: (W. C. Kruckeberg, J. W. Eaton, Alan R. L i s s , I n c . , New and G . J. B r e w e r , e d s . ) , pp. 13-30. York. Lauf, P. K. (1981b). Active and p a s s i v e c a t i o n t r a n s p o r t and i t s z. s o c i a t i o n with membrane a n t i g e n s i n sheep e r y t h r o c y t e s : Developments and t r e n d s . I n "Membranes and Transport" (A. N . Martonosi, e d . ) . Plenum, New York ( i n p r e s s ) . + Lauf, P. K., and Theg, B. E. (1980). A c h l o r i d e dependent K f l u x induced by El-ethylmaleimide i n g e n e t i c a l l y l o w K+ sheep and g o a t e r y t h r o c y t e s . B i o c h e m . B i o p h y s . R e s . C o m u n . 9 2 , 14221428. Lauf , P. K., and V a l e t , G. (1980). Cation t r a n s p o r t i n d i f f e r e n t volume populations of g e n e t i c a l l y low K+ lamb red c e l l s . J. C e l l . P h y s i o l . 1 0 4 , 283-293. Lauf , P. K. , Rasmusen , B. A. , Hoffman, P. G., Dunham, P. B. , Cook, P . , Pannelee, M. L., and Tosteson, D. C. (1970). Stimulation of a c t i v e potassium t r a n s p o r t i n LK sheep red c e l l s by blood group-L antiserum. J. Membr. B i o l . 3 , 1 - 1 3 . Lauf, P. K . , S t i e h l , B. J . , and J o i n e r , C. H. (1977). Active and passive c a t i o n t r a n s p o r t and L a n t i g e n h e t e r o g e n e i t y i n low potassium sheep r e d cells. Evidence a g a i n s t t h e concept of leak-pump i n t e r c o n v e r s i o n . J. G e n . P h y s i o l . 70, 221-242. Rasmusen, B. A. (1969). A blood group antibody which reacts exc l u s i v e l y w i t h LK sheep red blood cells. G e n e t i c s 61, 49s. Rasmusen, B. A., and H a l l , J. G. (1966). Association between potassium c o n c e n t r a t i o n and s e r o l o g i c a l type of sheep red blood c e l l s . S c i e n c e 1 5 1 , 1551-1552. F. (1960). Regulation of c e l l Tosteson, D. C. , and Hoffman, volume by a c t i v e t r a n s p o r t i n high and low potassium sheep red c e l l s . J. G e n . P h y s i o l . 4 4 , 169-194. Valet, G . , Franz, G., and Lauf, P. K. (1978). D i f f e r e n t red c e l l populations i n newborn, g e n e t i c a l l y low potassium sheep: Relation t o hematopoietic, immunologic and p h y s i o l o g i c d i f f e r e n t i a t i o n . J C e l l . P h y s i o l . 9 4 , 215-228.

J.

CURRENT TOPICS IN MEMBRANES AND TRANSPORT, VOLUME 19

Enhancement of Biosynthesis of Na,K-ATPase in the Toad Urinary Bladder by Aldosterone but Not T3 K. GEERING, M. GIRARDET, C. BRON, J.-P. KRAEHENBUHL, AND B. C. ROSSIER Institut de Pharmacologie Universite de Luusanne CH-1011 Lausanne, Switzerland and Institut de Biochimie Universite' de Lausanne CH-I066 Epalinges, Switzerland

I.

INTRODUCTION

I n t h e u r i n a r y b l a d d e r of t h e t o a d B u f o M a r i n u s , aldosterone stimulates t r a n s e p i t h e l i a l Na+ transport. A f t e r a l a t e n t p e r i o d of a b o u t 1 h r t h e r e i s a s h a r p i n c r e a s e i n t h e t r a n s e p i t h e l i a l Na+ t r a n s p o r t w i t h a c o n c o m i t a n t d r o p i n t r a n s e p i t h e l i a l e l e c t r i c a l res i s t a n c e ( R ) . T h i s e a r l y e f f e c t l a s t s a b o u t 3 h r and i s f o l l o w e d by a c o n t i n u o u s i n c r e a s e i n N a + t r a n s p o r t w i t h o u t f u r t h e r change i n R. A f t e r a b o u t 8 h r , t h e l a t e response of t h e t i s s u e reaches a steady s t a t e which i s m a i n t a i n e d up t o 2 0 h r o f i n c u b a t i o n . The e a r l y m i n e r a l o c o r t i c o i d e f f e c t i s l i k e l y t o be l i n k e d t o a change i n N a + conductance a t t h e a p i c a l membrane. L i t t l e i s known a b o u t t h e s u b c e l l u l a r e v e n t s which Interestingly, the late l e a d t o t h e l a t e response. r e s p o n s e can be i n h i b i t e d by t h y r o i d hormones which p e r s e have no e f f e c t on b a s e l i n e N a + t r a n s p o r t (Geering and R o s s i e r , 1 9 8 1 ) . I n t h e p r e s e n t s t u d y , w e asked t h e f o l l o w i n g q u e s t i o n s : (1) Does a l d o s t e r o n e 809

Copyright 0 1983 by Academic Press, Inc. All rights of reproductionin any form reserved. ISBN 0-12-153319-0

K. GEERING eta/.

810

i n c r e a s e t h e r a t e of s y n t h e s i s of t h e Na,K-ATPase?--a f a c t which c o u l d b e u l t i m a t e l y l i n k e d t o t h e l a t e m i n e r a l o c o r t i c o i d e f f e c t and ( 2 ) Is t h e a n t i m i n e r a l o c o r t i c o i d a c t i v i t y of T3 mediated by an i n h i b i t i o n of Na,K-ATPase s y n t h e s i s ?

11.

B.

RESULTS AND DISCUSSION

The b i o s y n t h e s i s of Na,K-ATPase was s t u d i e d i n m a r i n u s b l a d d e r by i n c o r p o r a t i o n of r a d i o a c t i v e p r e -

c u r s o r s i n t o p r o t e i n s followed by i n d i r e c t immunoprecip i t a t i o n (Maccecchini et a l . , 1979; G i r a r d e t e t a l . , 1981; Geering e t al., 1 9 8 1 ) . A c e l l homogenate w a s p r e p a r e d from b l a d d e r s l a b e l e d f o r 30 min w i t h [35S]methion i n e ( 2 0 0 p C i / m l ) and i n c u b a t e d w i t h m o n o s p e c i f i c a n t i s e r a p r e p a r e d a g a i n s t t h e a- (96K) and t h e 8- ( 6 0 K ) subu n i t s ( G i r a r d e t et a l . , 1 9 8 1 ) . Anti-a serum p r e c i p i t a t e d a s i n g l e band (96K) from c e l l homogenates as shown by SDS-PAGE and a u t o r a d i o g r a p h y (Geering et al., 1 9 8 1 ) . With a 30-min p u l s e t h e a n t i - 8 serum p r e c i p i t a t e d a 42K p r o t e i n , however, w i t h l o n g e r p u l s e s ( 2 0 h r ) t h e a n t i - 8 serum p r e f e r e n t i a l l y immunoprecipitated t h e 60K s u b u n i t . Some b l a d d e r s w e r e i n c u b a t e d f o r 1 8 h r b e f o r e l a b e l i n g e i t h e r w i t h T3 ( 6 0 nM), a l d o s t e r o n e ( 8 0 nM), o r T3 p l u s a l d o s t e r o n e . Changes i n t h e r a t e of b i o s y n t h e s i s o f Na,K-ATPase w e r e a s s e s s e d by comparing t h e peak h e i g h t s of t h e immunoprecipitated a - s u b u n i t on a u t o r a d i o g r a p h i c s c a n s from u n t r e a t e d and t r e a t e d t i s s u e . T3 t r e a t m e n t d i d n o t modify t h e r a t e of s y n t h e s i s of t h e a - s u b u n i t (mean 2 SE of p e r c e n t change i n peak h e i g h t of a - s u b u n i t compared t o c o n t r o l s : -11.5 +- 11, n = 4, p > 0.3). On t h e o t h e r hand, a l d o s t e r o n e o r ( a l d o s t e r o n e p l u s T ) produced, r e s p e c t i v e l y , a 1 2 4 29% ( n = 5 , p < 0.027 and 1 4 3 f 1 8 % ( n = 4 , p < 0 . 0 0 5 ) i n crease i n s y n t h e s i s r a t e of t h e a - s u b u n i t . Amiloride (50 p ~ ,a dose which t o t a l l y i n h i b i t s t r a n s e p i t h e l i a l Na+ t r a n s p o r t ) , given alone o r i n t h e p r e s e n c e of a l d o s t e r o n e , was used t o check whether t h e e f f e c t of a l d o s t e r o n e w a s secondary t o an i n c r e a s e d N a + s u p p l y t o t h e c e l l b r o u g h t a b o u t by a n e l e v a t e d N a + conductance a t t h e a p i c a l membrane. N e i t h e r b a s a l n o r a l d o s t e r o n e - i n d u c e d rates of enzyme s y n t h e s i s were modif i e d by a m i l o r i d e . Anti-B serum immunoprecipitated a 42K p r o t e i n from homogenates of c e l l s l a b e l e d f o r a 30-min p u l s e . T3 d i d n o t i n f l u e n c e t h e r a t e o f s y n t h e s i s of t h i s p r o t e i n , b u t a l d o s t e r o n e produced an i n d u c t i o n which p a r a l l e l e d t h a t

*

BIOSYNTHESISOFNa,K-ATPaseIN TOAD

81 1

of t h e a - s u b u n i t . W e conclude t h a t , i n t h e t o a d u r i n a r y b l a d d e r , a l d o s t e r o n e enhances t h e r a t e of s y n t h e s i s of I t h a s y e t t o b e shown t h e a - s u b u n i t and a 42K p r o t e i n . whether t h e i n c r e a s e i n s y n t h e s i s r a t e i s synchronous w i t h t h e t i m e c o u r s e of a l d o s t e r o n e - i n d u c e d Na+ t r a n s p o r t and i s thus one of t h e d e t e r m i n a n t s of t h e l a t e m i n e r a l o c o r t i c o i d r e s p o n s e . However, t h e r e s u l t s o f t h e experiment c a r r i e d o u t w i t h a m i l o r i d e i m p l i e s t h a t t h e e f f e c t o f a l d o s t e r o n e on b i o s y n t h e s i s o f t h e Na,KA T P a s e i s independent of t h e e n t r y of N a + a t t h e a p i c a l membrane. F u r t h e r i n v e s t i g a t i o n s a r e a l s o needed f o r t h e i d e n t i f i c a t i o n of t h e 42K p r o t e i n . D i f f e r e n c e s i n t h e immunoprecipitation p a t t e r n s of t h e a n t i - 6 serum w i t h d i f f e r e n t l a b e l i n g t i m e s s u g g e s t t h a t t h e 42K prot e i n i s e i t h e r a p r e c u r s o r of t h e mature g l y c o p r o t e i n o r a p r o t e i n i n v o l v e d t r a n s i e n t l y i n t h e e x p r e s s i o n of a membrane p r o t e i n (D. Meyer, p e r s o n a l communication). T 3 does n o t modify t h e r a t e of s y n t h e s i s of t h e a - s u b u n i t i n t h e t o a d b l a d d e r . These r e s u l t s c o n f i r m t h a t t h e r o l e of T3 i s d i f f e r e n t i n p o i k i l o t h e r m i c a n i mals, such a s amphibia ( R o s s i e r e t al., 1 9 7 9 1 , f r o m i t s r o l e i n mammals, i n which t h i s hormone h a s been shown t o s t i m u l a t e Na,K-ATPase s y n t h e s i s ( L o and Edelman, 1 9 7 6 ) . I n a d d i t i o n , T3 seems t o e x e r t i t s a n t i m i n e r a l o c o r t i c o i d a c t i v i t y by a p r o c e s s o t h e r t h a n an i n h i b i t i o n of t h e r a t e of s y n t h e s i s of t h e Na,K-ATPase.

ACKNOWLEDGMENT

This work was supported by t h e Swiss National Foundation f o r S c i e n t i f i c Research (Grant no. 3.646.80) and t h e F r i t z HoffmannLa Roche Foundation f o r S c i e n t i f i c C o l l a b o r a t i o n (Grant no. 1 4 6 ) .

REFERENCES

Geering, K . , and R o s s i e r , B. C . (1981). Thyroid hormone-aldos t e r o n e antagonism on N a + t r a n s p o r t i n toad b l a d d e r . J . Biol. C h e m . 256, 5504-5510. Geering, K . , G i r a r d e t , M . , Bron, C. , KraehenbAl, J.-P., and R o s s i e r , B. C . (1981). B i o s y n t h e s i s o f t h e c a t a l y t i c subu n i t of (Na+,K+)-ATPase i n toad kidney and t o a d b l a d d e r e p i t h e l i a l cells. I n "Membranes i n Growth and Development" (J. F. Hoffman, G. H . Giebisch, and L. Bolis, e d s . ) , A. R. L i s s , N e w York, pp. 537-542.

812

K. GEERING eta/.

G i r a r d e t , M., Geering, K. , F r a n t e s , J. M. , Geser, D. , R o s s i e r , B. C. , Kraehenblihl, J . - P . , and Bron, C. (1981). Immunochemical evidence f o r a transmembrane o r i e n t a t i o n o f b o t h t h e (Na+,K+)-ATPase s u b u n i t s . Biochemistry 20, 6684-6692. Lo, C. S . , and Edelman, I. S . (1976). E f f e c t of t r i i o d o t h y r o n i n e on t h e s y n t h e s i s and d e g r a d a t i o n of renal c o r t i c a l ( N a + + K+) adenosine t r i p h o s p h a t a s e . J. B i o l . Chem. 251, 7834-7840. Maccecchini, M. L . , Rudin, Y., B l o b e l , G., and S c h a t z , G. (1979). Import of p r o t e i n s i n t o mitochondria p r e c u r s o r forms o f t h e e x t r a r n i t o c h o n d r i a l l y made F1-ATPase s u b u n i t s i n y e a s t . Proc. N a t l . A c a d . Sci. USA 76, 343-347. R o s s i e r , B. C. , R o s s i e r , M., and Lo, C. S. (1979). Thyroxine and Na' t r a n s p o r t i n toad: R o l e i n t r a n s i t i o n from p o i k i l o - t o homeothermy. Am. J. P h y s i o l . 236, C117-Cl24.

-

CURRENT TOPICS IN MEMBRANES AND TRANSPORT. VOLUME 19

Na,K-ATPase Activity in Rat Nephron Segments: Effect of Low Potassium Diet and Thyroid Deficiency LAL C.GARG AND C. CRAIG TISHER Depunmenr of Pharmacology and Division of Nephrology Depunmenr of Medicine University of Florida College of Medicine Guinesville, Florida

I.

INTRODUCTION AND METHODS

Na,K-ATPase i s i n v o l v e d i n N a + r e a b s o r p t i o n i n a l most a l l segments o f t h e mammalian nephron, b u t t h e enzyme a c t i v i t y and N a + t r a n s p o r t i s i n c r e a s e d by minera l o c o r t i c o i d hormones i n a f e w s p e c i f i c segments o n l y (Garg e t a l . , 1 9 8 1 ) . Thyroid hormone h a s been shown t o i n c r e a s e t h e a c t i v i t y o f r e n a l Na,K-ATPase (IsmailB e i g i and Edelman, 1 9 7 1 1 , b u t i t s s i t e o f a c t i o n i n t h e nephron i s n o t known. I n a d d i t i o n , t h e r e i s c o n s i d e r a b l e d e b a t e r e g a r d i n g t h e r o l e of Na,K-ATPase i n K+ exc r e t i o n . I n t h i s a r t i c l e , w e summarize t h e r e s u l t s o f o u r r e c e n t s t u d i e s on ( a ) N a , K - A T P a s e a c t i v i t y i n nephron segments o f normal r a t and i t s comparison w i t h t h e a n i m a l s f e d a low K+ d i e t ; ( b ) Na,K-ATPase a c t i v i t y i n f o u r m o r p h o l o g i c a l l y d i s t i n c t t h i n limb segments o f t h e r a t nephron; and ( c ) t h e e f f e c t o f t h y r o i d d e f i c i e n cy on Na,K-ATPase a c t i v i t y i n d i f f e r e n t segments of t h e r a t nephron. The methods used for t r e a t m e n t of a n i m a l s 813

Copyright 0 1983 by Academic Press, Inc. All rights of reproduction in any form reserved.

ISBN 0-12-153319-0

814

LAL C. GARG et a/.

B

4

b-1

MED TAL

IS

q M E D TAL

0s

L

1

CONTROL

LOW K+-DIET

MED COLL, 0s

MED COLL, 200

100

IS

0 pmol/mm/min

0

100

200 300 400

F i g . 1. ( A ) Mg- and Na,K-ATPase a c t i v i t y i n i n d i v i d u a l n e p h r o n s e g m e n t s f r o m normal r a t s (mean 5 SEM). T h e number o f r a t s i s g i v e n i n the b a r s on the l e f t . ( B ) Na,K-ATPase a c t i v i t y i n c o r t i c a l c o l l e c t i n g d u c t of control r a t s and r a t s f e d a l o w Kf d i e t (mean & SEM o f f o u r a n i m a l s i n each group). S i g n i f i c a n t change a t p 0.01.

and microdissection of nephron segments and assay of Na,K-ATPase have been described previously (Garg and Tisher, 1983; Garg e t a l . , 1981; Garg e t a l . , 1982a,b).

11.

RESULTS AND CONCLUSIONS

The Na,K-ATPase activities in normal rat segments (Fig. 1A) demonstrated three peaks : the initial portion of the proximal convoluted tubule (Sl), the medullary thick ascending limb from the inner stripe, and the distal convoluted tubule. Qualitatively these results are similar to those reported for normal rabbits (Garg e t a l . , 1981). However, quantitatively the Na,K-ATPase activity in rat nephron segments was greater than in the

Na,K-ATPase ACTIVITY IN RAT NEPHRON SEGMENTS

TABLE I.

815

Na,K-ATPase A c t i v i t y i n Thin Limb Segments of t h e Rat Nephrona

Type 1

Type 11

5 2 3

30 f 5b

Type I11

4 f 3

Type IV 3 2 2

a

Type I represents the descending t h i n limb segment o f the short-looped nephrons. Types 11 and III comprise the outer and inner m e d u l l a r y portions, r e s p e c t i v e l y , o f the descending limb of long-looped nephrons. !Pype N represents the ascending t h i n limb segment of the long-looped nephrons. Enzyme values are expressed as mean f SEM o f f i v e animals i n pmole ADP min-1 nun-1. b s i g n i f i c a n t l y d i f f e r e n t from 0 a t p < 0.01.

TABLE 11.

E f f e c t of Aminotriazole (ATZ) and L-Thyroxine Na,K-ATPase A c t i v i t y i n Rat Nephron Segments

Segment Proximal conv. S1 Proximal str. S2 Proximal s t r . S3 Medullary TALI IS Medullary TALI 0s C o r t i c a l TAL

(T4) on

% Change from c o n t r o l ATZ + T4 AT2

-57.3a -27. 7 -11.6 6.5 2.8 -20.2

-

a S i g n i f i c a n t l y d i f f e r e n t from control at p

-

9.1

+

6.9 +13.9 6.5 +27.3 +36.6

-

0.01.

same segments o f r a b b i t nephron as determined p r e v i o u s l y u s i n g t h e same a s s a y method (Garg e t a l . , 1 9 8 1 ) . These f i n d i n g s are c o n s i s t e n t w i t h t h e q u a n t i t a t i v e d i f f e r e n c e s i n t h e g l o m e r u l a r f i l t r a t i o n r a t e and Na+ r e a b s o r p t i o n i n t h e two s p e c i e s (Windhager, 1 9 7 9 ) . F u r t h e r m o r e , w e found t h a t i n t h e normal r a t t h e Na,K-ATPase a c t i v i t y o f t h e m e d u l l a r y t h i c k a s c e n d i n g l i m b from t h e i n n e r s t r i p e w a s g r e a t e r t h a n t h a t from t h e o u t e r s t r i p e . The f i n d i n g s are c o n s i s t e n t w i t h c e r t a i n m o r p h o l o g i c a l d i f f e r e n c e s t h a t e x i s t i n t h e t w o p o r t i o n s of t h i s segment o f t h e r a t nephron ( A l l e n and T i s h e r , 1 9 7 6 ) . F i g u r e 1 B d e m o n s t r a t e s t h a t t h e low-K+ d i e t produced a s i g n i f i c a n t d e c r e a s e i n Na,K-ATPase a c t i v i t y i n t h e c o r t i c a l c o l l e c t i n g d u c t . There w a s no s i g n i f i c a n t change i n Na,K-ATPase a c t i v i t y i n any o t h e r segment i n a n i m a l s f e d a low-K+ d i e t , and t h e r e w a s no change i n

LAL C.GARG et 81.

818

Mg-ATPase a c t i v i t y i n any nephron segment i n t h e same animals. Table I demonstrates t h a t s i g n i f i c a n t l e v e l s of Na,K-ATPase a c t i v i t y was found o n l y i n t h e o u t e r medull a r y p o r t i o n of t h e t h i n limbs of long-looped nephrons (Type 11). These results a r e c o n s i s t e n t w i t h t h e morp h o l o g i c a l c h a r a c t e r i s t i c s of t h i s segment of t h e r a t nephron (Garg and T i s h e r , 1 9 8 3 ) . Table I1 d e m o n s t r a t e s t h a t t h e r e was a s i g n i f i c a n t d e c r e a s e i n Na,K-ATPase a c t i v i t y i n t h e S1 segment of t h e proximal t u b u l e i n hypothyroid ( a m i n o t r i a z o l e t r e a t e d ) r a t s t h a t were c o r r e c t e d by a d m i n i s t r a t i o n of t - t h y r o x i n e . N o s i g n i f i c a n t changes were observed i n o t h e r segments t h a t were examined. W e conclude t h a t ( a ) t h e g r e a t e r a c t i v i t y of Na,K-ATPase i n r a t t h a n r a b b i t nephrons i s r e l a t e d t o t h e g r e a t e r r a t e of g l o m e r u l a r f i l t r a t i o n and N a + r e a b s o r p t i o n p e r r a t nephron; ( b ) t h e p r e s e n c e of s i g n i f i c a n t Na,K-ATPase a c t i v i t y i n Type I1 t h i n limb segments ( o u t e r medullary p o r t i o n of t h e long-looped nephron) s u g g e s t s t h a t t h e r e may b e a c t i v e t r a n s p o r t of K+ o r Na+ o r b o t h i n t h i s segment; (c) d i e t a r y p o t a s sium modulates K+ t r a n s p o r t through N a , K - A T P a s e i n t h e c o r t i c a l c o l l e c t i n g d u c t : and ( d ) t h e S 1 segment of t h e proximal t u b u l e i s a major s i t e of a c t i o n of t h y r o i d hormone on Na,K-ATPase i n t h e r a t nephron.

REFERENCES

A l l e n , F . , and T i s h e r , C. C. (1976). Morphology of t h e ascending t h i c k l i m b o f Henle. Kidney I n t . 9 , 8-22. Garg, L. C . , and T i s h e r , C. C . (1983). Na,K-ATPase a c t i v i t y i n t h i n l i m b s o f r a t nephron. K i d n e y I n t . 23, 255. Garg, L. C . , Knepper, M., and Burg, M. B. (1981). M i n e r a l o c o r t i c o i d e f f e c t s on sodium and potassium adenosine t r i p h o s p h a t a s e i n i n d i v i d u a l nephron segments. Am. J. Physiol. 240, F536-F544. Garg, L. C. , Mackie, S., and T i s h e r , C . C. (1982a). E f f e c t of low potassium d i e t on Na-K-ATPase i n r a t nephron segments. P f l i i g e r s Arch. 394, 113-117. Garg, L. C . , Mackie, S., and T i s h e r , C. C. (1982b). S i t e of act i o n of t h y r o i d hormone on Na-K-ATPase i n r a t nephron segments. Kidney I n t . 21, 274. G i e b i s c h , G. (1979) Renal potassium t r a n s p o r t . I n "Membrane T r a n s p o r t i n Biology" (G. Giebisch, D. C . Tosteson, and H. H . Ussing, e d s . ) V o l . 4, pp. 215-298. Springer-Verlag, B e r l i n and New York.

.

Na,K-ATPase ACTIVITY IN RAT NEPHRON SEGMENTS

817

I s m a i l - B e i g i , F., and Edelman, I . S. ( 1 9 7 1 ) . The mechanism of t h e c a l o r i g e n i c a c t i o n of t h y r o i d hormone. S t i m u l a t i o n of Na-K-ATPase. J. Gen. P h y s i o l . 57, 710-722. In “Membrane Windhager, E . E . ( 1 9 7 9 ) . Sodium c h l o r i d e t r a n s p o r t . T r a n s p o r t i n B i o l o g y ” ( G . Giebisch, D. C . T o s t e s o n , and H. H . U s s i n g , eds.) V o l . 4 , pp. 146-213. Springer-Verlag, B e r l i n and New York.

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CURRENT TOPICS IN MEMBRANES AND TRANSPORT. VOLUME 19

Axonal Transport of Na,K-ATPase in Optic Nerve of Hamster SUSAN C.SPECHT Department of Pharmacology School of Medicine University of Puerto Rico San Juan, Puerto Rico

The Na,K-ATPase of n e u r a l t i s s u e i s c o n c e n t r a t e d i n t h e axons and n e r v e e n d i n g s . Because t h e s e c e l l u l a r domains cannot c a r r y o u t p r o t e i n s y n t h e s i s , a x o n a l prot e i n s a r e s y n t h e s i z e d i n t h e c e l l body and d e l i v e r e d t o t h e i r f i n a l d e s t i n a t i o n by an e n e r g y - r e q u i r i n g i n t r a c e l l u l a r t r a n s l o c a t i o n system, a x o n a l t r a n s p o r t ( f o r reviews, see G r a f s t e i n and Forman, 1 9 8 0 ; Schwartz, 1 9 7 9 ) . Two r a t e s of axonal t r a n s p o r t a r e g e n e r a l l y r e c o g n i z e d : f a s t t r a n s p o r t (200-400 mm/day) and slow t r a n s p o r t ( 1 - 4 mm/day). The e v i d e n c e s u g g e s t s t h a t f a s t t r a n s p o r t c o n t a i n s membrane-bound p r o t e i n s , wherea s t h e slow phase c o n t a i n s c y t o s k e l e t a l p r o t e i n s , such a s t u b u l i n (Hoffman and Lasek, 1 9 7 5 ) . The s t u d i e s rep o r t e d h e r e w e r e i n i t i a t e d t o i n v e s t i g a t e t h e axonal t r a n s p o r t of Na,K-ATPase and t o d e t e r m i n e i t s h a l f - l i f e i n t h e nerve ending. The Na,K-ATPase of a d u l t hamster o p t i c n e r v e was r a d i o a c t i v e l y l a b e l e d a t i t s s i t e of s y n t h e s i s by unil a t e r a l i n t r a o c u l a r i n j e c t i o n of [35S]methionine ( N e w England N u c l e a r ) followed by i n c o r p o r a t i o n w i t h i n t h e 819

Copyright 0 1983 by Acpdemic Rcss, loc. All rightsof npmaucIim in any fom resmed. ISBN 0-12-1533194

820

SUSAN C. SPECHT

F i g . 1 . ( A ) Na,K-ATPase p r e p a r e d b y the m e t h o d of B e r t o n i and S i e g e 1 (1978) was e l e c t r o p h o r e s e d i n a 5% S D S - p o l y a c r y l a m i d e g e l (Cohen e t a l . , 1977). T h e p o s i t i o n of the M r 96,000 c a t a l y t i c s u b u n i t i s i n d i c a t e d . ( B ) A u t o r a d i o g r a m of the Na,K-ATPase p h o s p h o r y l a t e d w i t h [32P]ATP i n the p r e s e n c e o f 140 mM N a C l ( l e f t G i d e ) or 20 mM K C l ( r i g h t ) . T h e r e i s e s s e n t i a l l y no l a b e l i n g of the M, 96,000 band i n the p r e s e n c e o f K'.

r e t i n a l g a n g l i o n c e l l s , which form t h e o p t i c n e r v e . Each hamster w a s i n j e c t e d w i t h 0 . 1 7 m C i . A t 1, 3 , 7 , o r 1 5 days a f t e r i n t r a o c u l a r l a b e l i n g , two a n i m a l s were k i l l e d and t h e l a t e r a l g e n i c u l a t e n u c l e u s and sup e r i o r c o l l i c u l u s ( c o n t a i n i n g o p t i c nerve e n d i n g s ) d i s s e c t e d o u t f o r p r e p a r a t i o n of Na,K-ATPase. The r i g h t and l e f t s i d e s of t h e b r a i n s were p r e p a r e d s e p a r a t e l y . Because 95-98% of t h e o p t i c nerve f i b e r s d e c u s s a t e a t t h e o p t i c chiasma ( S c h n e i d e r , 1963) , t h e s i d e of t h e b r a i n r e c e i v i n g f i b e r s predominately from t h e u n l a b e l e d ( l e f t ) eye s e r v e d a s a measure of t h e i n t r a o c u l a r l y app l i e d l a b e l . The amount of s t a r t i n g m a t e r i a l from t h e two pooled b r a i n s was 80-100 mg/si.de.

AXONAL TRANSPORT IN OPTIC NERVE OF HAMSTER

821

The Na,K-ATPase w a s p a r t i a l l y p u r i f i e d by a NaI method ( B e r t o n i and S i e g e l , 1 9 7 8 ) . The s p e c i f i c a c t i v i t y a v e r a g e d 1 . 7 umoles Pi/mg/min, d e t e r m i n e d by t h e l i b e r a t i o n of i n o r g a n i c p h o s p h a t e ( S p e c h t and Robinson, 1 9 7 3 ) . The c r u d e enzyme p r e p a r a t i o n w a s s e p a r a t e d by SDS-polyacrylamide s l a b g e l e l e c t r o p h o r e s i s o n a 5-15% p o l y a c r y l a m i d e g r a d i e n t (Cohen e t a l . , 1 9 7 7 ) . A b r o a d major band w a s found a t M y 9 6 , 0 0 0 w i t h minor PASWhen f r e s h l y p o s i t i v e bands a t M r 50,000 and 6 3 , 0 0 0 . p r e p a r e d enzyme w a s u s e d , t h e M y 9 6 , 0 0 0 band c o u l d b e r e s o l v e d i n t o two bands w i t h t h e h i g h e r m o l e c u l a r w e i g h t component d o m i n a t i n g ; t h e s e r e p r e s e n t e d t h e a ( + ) and a forms of t h e c a t a l y t i c s u b u n i t (Sweadner, 1 9 7 9 ) . To d e m o n s t r a t e t h a t t h e M~ 96,000 band was t h e c a t a l y t i c subunit, t h e preparation w a s phosphorylated w i t h [32P]ATP (2600 Ci/mmole; N e w England N u c l e a r ) i n t h e p r e s e n c e o f e i t h e r 1 4 0 mM N a C l o r 2 0 m~ K C 1 f o r 1 0 o r 2 0 sec a t room t e m p e r a t u r e . After precipitation w i t h 5% TCA, t h e p r o t e i n was washed w i t h TCA and water, t h e n d i s s o l v e d i n sample b u f f e r and e l e c t r o p h o r e s e d i n a 5% SDS-polyacrylamide s l a b g e l . F i g u r e 1 A shows a Coomassie B l u e - s t a i n e d g e l ; F i g . 1 B i s an a u t o r a d i o g r a m of t h e same g e l . The M~ 9 6 , 0 0 0 band w a s p h o s p h o r y l a t e d o n l y i n t h e p r e s e n c e of N a + , d e m o n s t r a t i n g t h a t t h e band r e p r e s e n t e d t h e c a t a l y t i c s u b u n i t o f t h e Na,K-ATPase. To d e t e r m i n e i f c a t a l y t i c s u b u n i t s s y n t h e s i z e d i n t h e r e t i n a l g a n g l i o n c e l l s were a x o n a l l y t r a n s p o r t e d i n t o t h e o p t i c nerve e n d i n g s , enzyme w a s p r e p a r e d from a n i m a l s l a b e l e d i n t r a o c u l a r l y w i t h [ 3 5 S ] m e t h i o n i n e and a n a l y z e d by b o t h s l a b g e l e l e c t r o p h o r e s i s and s c i n t i l l a t i o n c o u n t i n g . The g e l s were s l i c e d i n t o 2-mm s t r i p s i n t h e r e g i o n o f t h e M y 96,000 band, d i s s o l v e d , and c o u n t e d . F i g u r e 2 shows a t y p i c a l r e s u l t from a g e l o f enzymes p r e p a r e d a t 1 and 7 d a y s a f t e r i n t r a o c u l a r l a b e l i n g . The f o l l o w i n g s a l i e n t p o i n t s b e a r emphasis: 1. A t 1 day a f t e r i n t r a o c u l a r i n j e c t i o n l a b e l i n g o f t h e N a , K - A T P a s e band w a s c o n s i d e r a b l y h i g h e r on t h e l e f t s i d e t h a n on t h e r i g h t . This i n d i c a t e s t h a t t h e l a b e l e d enzyme o r i g i n a t e d p r i n c i p a l l y i n t h e r e t i n a l g a n g l i o n c e l l s o f t h e r i g h t e y e and w a s n o t s y n t h e s i z e d l o c a l l y from r a d i o a c t i v e p r e c u r s o r which had e n t e r e d t h e bloodstream. 2 . The presence of l a b e l e d enzyme i n t h e n e r v e e n d i n g s a t 1 day a f t e r i n t r a o c u l a r l a b e l i n g d e m o n s t r a t e s t r a n s l o c a t i o n a t t h e r a p i d r a t e of axonal t r a n s p o r t . 3. L a b e l i n g w a s e i t h e r e q u a l t o t h e 1-day v a l u e o r h i g h e r a f t e r 7 days. These and o t h e r d a t a n o t p r e s e n t e d i n d i c a t e t h a t t h e h a l f - l i f e of t h e enzyme i n a d u l t o p t i c n e r v e e n d i n g s i s a t l e a s t 1 week. N o n e t h e l e s s , t h e r e a l h a l f - l i f e may be c o n s i d e r a b l y s h o r t e r . S e v e r a l a l t e r n a t e

SUSAN C.SPECHT

822

110

loo

90

113

102

Molecular weight x

93

Id

F i g . 2. S c i n t i l l a t i o n c o u n t i n g o f 2-mm slices f r o m SDSp o l y a c r y l a m i d e g e l s of Na,K-ATPase p r e p a r e d f r o m the l a t e r a l g e n i c u l a t e n u c l e u s and s u p e r i o r c o l l i c u l u s o f a d u l t h a m s t e r s 1 (A) and 7 ( B ) d a y s a f t e r i n j e c t i o n o f [ 3 5 ~ ] m e t h i o n i n ei n t o the r i g h t e y e . T h e u p p e r part ( " l e f t " ) p o r t i o n o f e a c h g r a p h r e p r e sents l a b e l i n g i n the o p t i c nerve e n d i n g s f r o m the i n j e c t e d r i g h t e y e , w h e r e a s the lower ( " r i g h t " ) p a r t r e p r e s e n t s l a b e l i n g i n t i s s u e p r e d o m i n a t e l y r e c e i v i n g f i b e r s f r o m the u n l a b e l e d e y e . " R i g h t " l a b e l i n g is p r i n c i p a l l y d u e t o i n c o r p o r a t i o n o f [ 3 5 S ]methionine b y g l i a and n e u r o n e s . Molecular w e i g h t s w e r e e s t i m a t e d f r o m a c u r v e d e t e r m i n e d b y the r e l a t i v e m i g r a t i o n o f m o l e c u l a r w e i g h t s t a n d a r d s ( B - g a l a c t o s i d a s e , 116,000; p h o s p h o r y l a s e B , 94,000; bovine s e r u m a l b u m i n , 68,000; c a r b o n i c a n h y d r a s e , 43,000; s o y b e a n t r y p s i n inhibitor, 21,000; l y s o z y m e , 1 4 , 0 0 0 ) .

e x p l a n a t i o n s f o r t h e s u s t a i n e d l a b e l i n g of n e r v e ending Na,K-ATPase a r e t e n a b l e , i n c l u d i n g delayed release of t h e enzyme from a somata1 p o o l followed by f a s t t r a n s p o r t ( G r a f s t e i n e t a l . , 1975) and g l i a l r e u t i l i z a t i o n of r a d i o a c t i v e p r e c u r s o r . Slow axonal t r a n s p o r t i s t h e l e a s t l i k e l y cause. Experiments a r e i n p r o g r e s s t o resolve these issues.

ACKNOWLEDGMENT

Supported i n p a r t by NIH-PHS g r a n t EY 02334. Roberto OcasioRivera and Teresa Candelas provided t e c h n i c a l a s s i s t a n c e and Carmen Chico prepared t h e manuscript.

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REFERENCES

+

+

B e r t o n i , J. M., and S i e g e l , G. J. (1978). Development o f ( N a + K ) ATPase i n r a t cerebrum: C o r r e l a t i o n w i t h Na+-dependent phosp h o r y l a t i o n and K+ para-nitrophenylphosphatase. J. Neurochem. 31, 1501-1511. Cohen, R. S . , Blomberg, F . , B e r z i n s , K . , and S i e k e v i t z , P. ( 1 9 7 7 ) . The s t r u c t u r e o f p o s t s y n a p t i c d e n s i t i e s i s o l a t e d from dog c e r e b r a l c o r t e x . J. C e l l B i o l . 7 4 , 181-203. G r a f s t e i n , B . , and Forman, D. S. (1980). I n t r a c e l l u l a r t r a n s p o r t i n neurons. P h y s i o l . Rev. 60, 1167-1283. G r a f s t e i n , B . , Miller, J. A . , Ledeen, R . W., Haley, J . , and S p e c h t , S. C . ( 1 9 7 5 ) . Axonal t r a n s p o r t of p h o s p h o l i p i d i n g o l d f i s h o p t i c system. Exp. Neurol. 4 6 , 262-281. Hoffman, P. N . , and Lasek, R. J. (1975). The slow component o f axonal t r a n s p o r t . I d e n t i f i c a t i o n o f major s t r u c t u r a l polyp e p t i d e s of t h e axon and t h e i r g e n e r a l i t y among m a m m a l i a n neurons. J. C e l l Biol. 66, 351-366. S c h n e i d e r , G. E. (1963). Two v i s u a l systems. S c i e n c e 1 6 3 , 895902. Schwartz, J. H. ( 1 9 7 9 ) . Axonal t r a n s p o r t : Components, mechanisms and s p e c i f i c i t y . Annu. Rev. Neurosci. 2, 467-504. S p e c h t , S. C . , and Robinson, J. D. (1973). S t i m u l a t i o n o f t h e (Na+ + K+)-dependent ATPase by amino a c i d s and p h o s p h a t i d y l s e r i n e : C h e l a t i o n of heavy m e t a l i n h i b i t o r s . Arch. Biochem. Biophys. 154, 314-323. Sweadner, K. J. (1979). Two m o l e c u l a r forms o f (Na' +)'K s t i m u l a t e d ATPase i n b r a i n . S e p a r a t i o n and d i f f e r e n c e i n a f f i n i t y f o r s t r o p h a n t i d i n . J. B i o l . Chem. 2 5 4 , 6060-6067.

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Part IX

Na,K-ATPase and Positive lnotropy; Endogenous Glycosides

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CURRENT TOPICS IN MEMBRANES AND TRANSPORT, VOLUME 19

Positive lnotropic Action of Digitalis and Endogenous Factors: Na,K-ATPse and Positive Inotropy; ‘‘Endogenous Glycosides’’ A. SCHWAR’IZ Department of Phamcology and Cell Biophysics College of Medicine University of Cincinnati Cincinnati, Ohio

I.

HISTORICAL BACKGROUND

e x t r a c t e d c a r d i a c g l y c o s i d e s ( t h e words d i g i t a l i s , ouabain, and c a r d i a c g l y c o s i d e s w i l l be used i n t e r c h a n g e a b l y t h r o u g h o u t t h i s a r t i c l e , e x c e p t when i n d i c a t e d o t h e r w i s e ) from t h e Foxglove p l a n t a n d , a f t e r a d m i n i s t e r i n g i n f u s i o n s of t h e e x t r a c t s , o b t a i n e d d r a m a t i c improvement i n p a t i e n t s s u f f e r i n g from d r o p s y (edematous f l u i d c o l l e c t i o n ) a s s o c i a t e d w i t h c o n g e s t i v e heart failure. H i s b r i l l i a n t treatise (Withering, 1785) o n t h e t h e r a p e u t i c b e n e f i t s and t o x i c m a n i f e s t a t i o n s of t h e Foxglove r e m a i n s a s a t r i b u t e t o h i s enormous cont r i b u t i o n s and r e p r e s e n t s t h e f i r s t i n - d e p t h c l i n i c a l cardiological investigation. S i n c e t h a t t i m e no o t h e r d r u g h a s r e c e i v e d a s much a t t e n t i o n , n o t o n l y c l i n i c a l l y b u t i n t e r m s of fundament a l b a s i c s c i e n c e i n v e s t i g a t i o n , and y e t t h e mechanism of a c t i o n of t h i s i m p o r t a n t d r u g r e m a i n s an enigma. T h i s i s so, i n my o p i n i o n , b e c a u s e w e s t i l l do n o t have 825

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a complete u n d e r s t a n d i n g o f how t h e c a r d i a c c e l l i s a b l e t o l i n k t h e movements of sodium, p o t a s s i u m , and calcium a c r o s s membranes t o t h e p r o c e s s o f c o n t r a c t i o n . T h i s a s p e c t , v i z . , how i o n s c o n t r o l c e l l u l a r a c t i v i t y , of c o u r s e , i s fundamental t o a l m o s t e v e r y l i v i n g c e l l . F o r t u i t o u s l y , it t u r n e d o u t t h a t W i t h e r i n g ' s d r u g i s a h i g h l y s p e c i f i c i n h i b i t o r of t h e t r a n s p o r t o f sodium and p o t a s s i u m and t h e enzymatic machinery of t h e N a , K - A T P a s e o f t h i s pump (Schatzmann, 1953; Skou, 1 9 6 0 ) . I t i s log i c a l , t h e r e f o r e , t o e x p e c t a " r e c i p r o c a l r e a c t i o n , '' v i z . , t h a t o u a b a i n would be used t o i n v e s t i g a t e t h e mol e c u l a r n a t u r e o f t h i s pump and t h a t t h e pump i t s e l f would be i m p l i c a t e d i n t h e mechanism of i n o t r o p i c a c t i o n ( i n c r e a s e d f o r c e of c o n t r a c t i o n ) of d i g i t a l i s on t h e h e a r t ( L e e and Klaus, 1 9 7 1 ; Schwartz et a l . , 1975; Akera and Brody, 1 9 7 8 ) . A s i n t h e f i r s t two m e e t i n g s o n N a , K - A T P a s e ( A s k a r i , 1 9 7 4 ; Skou and Nbrby, 1 9 7 9 ) , d i g i t a l i s w a s a l s o cons i d e r e d i n t h i s t h i r d c o n f e r e n c e from t h e v i e w p o i n t of a s p e c i f i c i n h i b i t o r of t h e Na,K-ATPase and a s a p o w e r f u l cardiac stimulant. I n dedicating an e n t i r e session t o t h e l a t t e r , however, it i s hoped t h a t i n s i g h t s i n t o mechanism w i l l be r e v e a l e d t h a t w i l l n o t o n l y a i d u s i n u n d e r s t a n d i n g how an i n c r e a s e d c o n t r a c t i o n i s o b t a i n e d , b u t p e r h a p s p r o v i d e b a s i c i n f o r m a t i o n on t h e r o l e o f t h e N a / K pump i n c o n t r o l of c a r d i a c f u n c t i o n . To t h i s e n d , w e have a l s o i n c l u d e d a n e x c i t i n g new a r e a on endogenous It is l i k e l y s u b s t a n c e s t h a t resemble d i g i t a l i s a c t i o n . t h a t t h e r e are pharmacological r e c e p t o r s f o r every chemical ( d r u g ) d e r i v e d from n a t u r a l p r o d u c t s t h a t , when used i n v e r y s m a l l amounts, c a u s e s a t h e r a p e u t i c response. I t i s e q u a l l y l i k e l y t h a t t h e s e r e c e p t o r s e x i s t because t h e p l a n t p r i n c i p l e s resemble i n some way t h e s t r u c t u r e s of some endogenously s y n t h e s i z e d s u b s t a n c e s . I n r e v i e w i n g t h e t o p i c of d i g i t a l i s , i n o t r o p i c mechanisms, and t h e sodium pump, I s h o u l d l i k e t o emphas i z e t h a t t h e r e have been l i t e r a l l y t h o u s a n d s of a r t i c l e s p u b l i s h e d on t h e s e s u b j e c t s s i n c e 1953 ( L e e and Klaus, 1971; Schwartz et a l . , 1975; Akera and Brody, 1 9 7 8 ) . I n t h i s volume, Smith and B a r r y have reviewed t h e r e l a t i o n s h i p between i n h i b i t i o n of monovalent c a t i o n t r a n s p o r t and t h e p o s i t i v e i n o t r o p i c a c t i o n of d i g i t a l i s , and Haupert h a s p r e s e n t e d a r e v i e w o f endogenous d i g i t a l i s l i k e s u b s t a n c e s . Our i n t e n t , t h e r e f o r e , i s t o d i s c u s s a f e w c r i t i c a l a s p e c t s , and t o i d e n t i f y c o n t r o v e r s i a l i s s u e s , and p e r h a p s some problems t h a t need s o l v i n g . Repke p r e s e n t e d t h e f i r s t i n - d e p t h s t u d y i l l u s t r a t i n g t h e r e l a t i o n s h i p between Na,K-ATPase i n h i b i t i o n and t h e r a p e u t i c a c t i v i t i e s of c a r d i a c g l y c o s i d e s (Repke, 1964; W i l b r a n d t , 1 9 6 2 ) . H e , i n f a c t , s u g g e s t e d a

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mechanism by which t h e i n h i b i t i o n of t h e pump would supply a d d i t i o n a l calcium with each b e a t . T h e r e i s no q u e s t i o n t h a t a number o f i n v e s t i g a t o r s w e r e t h i n k i n g a l o n g t h e same l i n e s a t t h a t t i m e ( W i l b r a n d t , 1 9 6 2 ) . F r example, t h e r e m a r k a b l e s i m i l a r i t y between t h e i n h i b i t o r y e f f e c t of e i g h t d i f f e r e n t c a r d i a c g l y c o s i d e s on K+ i n f l u x i n t o human r e d c e l l s , and t h e i r l e t h a l e f f e c t s on c a t h e a r t s , l e d Solomon e t a l . (1956) t o sugg e s t t h a t " t h e mechanism of a c t i o n o f t h e s e d r u g s ( i . e . , c a r d i a c q l y c o s i d e s ) on t h e h e a r t may be s i m i l a r i n some r e s p e c t s t o t h e i r a c t i o n on r e d c e l l s . " What c r i t e r i a s h o u l d be f u l f i l l e d i n o r d e r t o acc e p t t h e c o n c e p t t h a t t h e Na,K-ATPase enzyme s y s t e m i s t h e pharmacological r e c e p t o r f o r d i g i t a l i s ? I n t h i s c a s e , l e t u s assume t h a t t h e Na,K-ATPase s y s t e m i s t h e r e c e p t o r , R , and t h a t d i g i t a l i s i s t h e d r u g , D , so t h a t R + D

kl, R D -

effect

-k-l

The c r i t e r i a f o r t h i s R-D i n t e r a c t i o n , t h e n , i n c l u d e t h e following: (1) a n a f f i n i t y which i s i n t h e same r a n g e i n which t h e d r u g i s a c t i v e p h a r m a c o l o g i c a l l y : (2) s a t u r a b i l i t y ; (3) r e v e r s i b i l i t y , consistent with t h e k i n e t i c s of p h a r m a c o l o g i c a l a c t i o n : ( 4 ) t i s s u e d i s t r i b u t i o n , c o n s i s t e n t w i t h t h e known p h a r m a c o l o g i c a l a c t i o n s ; ( 5 ) s p e c i f i c b i n d i n g removed by a q o n i s t o c c u p a t i o n of r e c e p t o r : ( 6 ) a binding isotherm r e l a t e d t o t h e doseresponse c u r v e : ( 7 ) t h e r e l a t i v e b i n d i n g a f f i n i t i e s o f a n a l o g s o f t h e d r u g s h o u l d be c o n s i s t e n t w i t h t h e i r b i o l o g i c a l e f f e c t s : (8) s p e c i e s s e n s i t i v i t y , i f it e x i s t s , s h o u l d r e f l e c t d i f f e r e n c e s ( e . q . , ease of d i s s o c i a t i o n o f t h e d r u g from t h e p u t a t i v e r e c e p t o r ) c o n s i s t e n t w i t h t h e s e n s i t i v i t y ( a f f i n i t y ) of t h e s p e c i e s t o t h e d r u g . These c r i t e r i a , f o r t h e most p a r t , w i t h r e s p e c t t o d i g i t a l i s i n t e r a c t i o n w i t h t h e pharmacological r e c e p t o r , Na,K-ATPase, h a v e b e e n f u l f i l l e d (Schwartz e t al., 1975: Akera and Brody, 1 9 7 8 ) . The p r i m a r y a r e a o f d i s a g r e e ment i s w i t h c r i t e r i o n 3 . O k i t a and h i s c o l l e a g u e s have p r e s e n t e d e v i d e n c e o n t h i s p o i n t , and t h e i r r e s u l t s w i l l be d i s c u s s e d l a t e r . L e t u s now t a k e t h i s argument t o t h e n e x t l e v e l , v i z . , i s i n h i b i t i o n of t h e enzyme a c t i v i t y s u b s e q u e n t t o t h e b i n d i n g o f d i g i t a l i s r e l a t e d t o t h e increase i n i n t r a c e l l u l a r c a l c i u m a c t i v i t y t h a t must o c c u r i f c o n t r a c t i o n i s t o b e augmented? Is i n t r a c e l l u l a r Na a c t i v i t y i n c r e a s e d p r i o r t o t h e development of i n o t o p y ? These a r e t h e areas of c o n s i d e r a b l e c o n t r o v e r s y , and t h e s e a r e c o v e r e d q u i t e w e l l by Smith and B a r r y i n t h i s volume.

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T o summarize, i n most o f t h e e x p e r i m e n t s i n v o l v i n g whole

a n i m a l s , i s o l a t e d t i s s u e s , and c e l l s i n which ATPase act i v i t y o r i n t r a c e l l u l a r N a + a r e measured, i n v e s t i g a t o r s have f o u n d t h a t t h e enzyme i s i n h i b i t e d by c o n c e n t r a t i o n s o f d i g i t a l o i d s t h a t produce an i n c r e a s e i n c o n t r a c t i o n S c h w a r t z e t al., 1975; Akera and Brody, 1978; L e e e t al., 1 9 8 0 ; L e e , 1 9 8 1 ) . I n some e x p e r i m e n t s , however, i n which t h e a c t i v i t y of t h e Na,K-ATPase i n v i v o i s measured by i o n c o n t e n t a n d / o r i o n f l u x , o r by m e a s u r i n g q u a n t i t a t i v e l y t h e e l e c t r o g e n i c pump of s p e c i a l i z e d c o n d u c t i n g t i s s u e , v e r y low c o n c e n t r a t i o n s of o u a b a i n o r s t r o p h a n t h i d i n (below M i n some c a s e s ) have been r e p o r t e d t o produce a s t i m u l a t i o n of t h e sodium pump and h e n c e , s u p p o s e d l y , a d e c r e a s e i n Na i o n a c t i v i t y (Noack e t a l . , 1 9 7 9 ; Noble, 1980; G o d f r a i n d , 1 9 8 1 ) . I n some e x p e r i m e n t s u s i n g Na e l e c t r o d e s , d a t a have been r e p o r t e d showing a s l i g h t d e c r e a s e o f Na, s u g g e s t i n g a t r a n s i e n t N a / K pump s t i m u l a t i o n produced by l o w c o n c e n t r a t i o n s ( < l O - 7 M ) of s t r o p h a n t h i d i n o r a c e t y l s t r o p h a n t h i d i n (Cohen e t a l , 1 9 7 6 ) . T h i s s t i m u l a t i n g e f f e c t i s accompanied i n some e x p e r i m e n t s by n e g a t i v e i n o t r o p y , i n o t h e r s by p o s i t i v e i n o t r o p y , a n d , i n s t i l l o t h e r s l by no change i n c o n t r a c tion. F u r t h e r m o r e , some i n v e s t i g a t o r s have f o u n d e v i dence f o r more t h a n one b i n d i n g s i t e € o r d i g i t a l i s , w i t h d i f f e r e n t apparent a f f i n i t i e s , a s s o c i a t e d with t h e sarcolemma ( G o d f r a i n d , 1 9 8 1 ) A "high-affinity" site, for example, a p p e a r s t o be i n c o n s i s t e n t w i t h a b i n d i n g t o t h e Na,K-ATPase ( s e e b e l o w ) . O t h e r p u b l i s h e d s t u d i e s have a l s o revealed disquieting data inconsistent with t h e Na, K-ATPase i n o t r o p i c h y p o t h e s i s . For example, O k i t a and c o l l e a g u e s ( 1 9 7 3 ) showed t h a t when t h e p o s i t i v e i n o t r o p i c e f f e c t of d i g i t a l i s w a s washed o u t of an i s o l a t e d r a b b i t h e a r t p r e p a r a t i o n , t h e i n h i b i t e d NalK-ATPase remained i n h i b i t e d , and t h a t t h i s i n h i b i t i o n d i d n o t i n c r e a s e when a n o t h e r " d o s e " o f o u a b a i n t h a t produced p o s i t i v e i n o t r o p y w a s a d m i n i s t e r e d (see c r i t e r i o n 3 ) . The same i n v e s t i g a t o r , a l o n g w i t h Kurobane and Nandi ( P a r t V I I I , t h i s v o l u m e ) , p r e s e n t e d a new s t u d y i n t h i s symposium i n which t h e y showed t h a t t h e i n o t r o p i c h a l f - l i f e of o u a b a i n (1.2 x M ) w a s 1 . 7 f 0 . 2 h r a t 3 O o C , i n a dog a t r i a l o r v e n t r i c u l a r t r a b e c u l a r p r e p a r a t i o n , whereas t h e h a l f l i f e d i s s o c i a t i o n r a t e of a p u r i f i e d kidney N a , K - A T P a s e o u a b a i n complex a t 3 O o C was 9 . 0 ? 0 . 2 h r . The d i s s o c i a t i o n h a l f - l i f e of a c a r d i a c sarcolemma-ouabain complex was 1 . 8 f 0 . 1 h r and 9 . 3 f 0 . 3 h r f o r t h e two a p p a r e n t o u a b a i n - b i n d i n g s i t e s which a number of i n v e s t i g a t o r s a r e r e p o r t i n g ( W e l l s m i t h and Lindenmayer , 1980; Erdmann, 1 9 8 1 ) . O k i t a c o n c l u d e d t h a t t h e Na,K-ATPase i s not t h e p h a r m a c o l o g i c a l r e c e p t o r f o r d i g i t a l i s , and t h a t t h e receptor o r binding s i t e t h a t i s involved i n posit.ive ino-

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tropy has d i f f e r e n t d i s s o c i a t i o n c h a r a c t e r i s t i c s than a d i g i t a l i s - p u r i f i e d Na,K-ATPase complex a n d may b e l o c a t e d n e a r t h e N a , K - A T P a s e i n o r on t h e sarcolemma. However, t h e s e e x p e r i m e n t s do n o t p r o v e t h a t a non-Na,KA T P a s e r e c e p t o r e x i s t s , b e c a u s e i t may b e t h a t m u l t i p l e forms o r i s o m e r s o f Na,K-ATPase a r e p r e s e n t , e a c h h a v i n g different affinities. I n f a c t , e v i d e n c e i n f a v o r of t h e l a t t e r h a s been p r e s e n t e d by Sweadner ( 1 9 7 9 ) and by W e l l s m i t h and Lindenmayer (1980) F u r t h e r m o r e , a compar i s o n o f k i n e t i c c h a r a c t e r i s t i c s of a p u r i f i e d , s o l u b i l i z e d k i d n e y N a , K - A T P a s e w i t h d a t a d e r i v e d from c a r d i a c p r e p a r a t i o n s may b e m i s l e a d i n g , s i n c e i m p o r t a n t d i f f e r e n c e s may e x i s t . Is t h e r e a s o l u t i o n t o a l l o f t h e s e d i v e r g e n t s t u d i e s and c o n c l u s i o n s ? Erdmann e t a l . (1980) r e p o r t e d an i n o t r o p i c mechanism i n r a t and g u i n e a - p i g v e n t r i c l e ( P o s t e r , t h i s s e s s i o n ) t h a t responded t o c o n c e n t r a t i o n s of o u a b a i n t h a t d i d n o t i n h i b i t t h e N a , K - A T P a s e . In cat h e a r t membranes, however, t h e K D f o r b i n d i n g of o u a b a i n t o t h e membrane p r e p a r a t i o n i s i n t h e s a m e r a n g e as i s t h e 150 f o r enzyme i n h i b i t i o n . T h e r e f o r e , Erdmann p r o poses t h e f o l l o w i n g s o l u t i o n t o t h e problems: Perhaps t h e r e a r e two i n o t r o p i c mechanisms, o n e t h a t i n v o l v e s t h e N a , K - A T P a s e i n h i b i t i o n and o n e t h a t d o e s n o t . This, i n a way, i s c o n s i s t e n t w i t h what w e and Lullmann, i n d e p e n d e n t l y , p u t f o r t h a few y e a r s a g o , v i z . , t h a t maybe d i g i t a l i s p r o d u c e s a n i n o t r o p i c a c t i o n by m o b i l i z i n g c a l cium a s s o c i a t e d w i t h l i p i d s o f t h e N a , K - A T P a s e ( f o r review, see Schwartz and Adams, 1 9 8 0 ) p r i o r t o any i n h i b i t i o n of enzyme a c t i v i t y . T h i s would be a n i c e comprom i s e , e x c e p t t h a t t h e c o n c e n t r a t i o n s of o u a b a i n w e u s e d were t o o h i g h . F u r t h e r m o r e , o u r r e c e n t e x p e r i m e n t s a t t e m p t i n g t o r e p r o d u c e t h e Erdmann r e s u l t s o n r a t vent r i c l e have l e d t o a n a l t e r n a t e c o n c l u s i o n . W e found t h a t t h e r e may i n d e e d b e "two mechanisms" i n t h e r a t v e n t r i c l e , b u t only 30% of t h e t o t a l i n o t r o p i c action of ouabain a p p e a r s t o be a s s o c i a t e d w i t h t h e h i g h - a f f i n i t y , l o w - c a p a c i t y s i t e . The r e m a i n d e r ( 7 0 % ) i s q u i t e c o n s i s t e n t w i t h an i n h i b i t i o n of t h e N a , K - A T P a s e . We feel that Erdmann d i d n o t go h i g h enough i n " t i t r a t i n g " o u a b a i n induced p o s i t i v e i n o t r o p y . L e t u s now c o n s i d e r t h e s i g n i f i c a n c e , if a n y , o f t h e s t i m u l a t i o n o f t h e pump by v e r y low c o n c e n t r a t i o n s of o u a b a i n t h a t h a s been r e p o r t e d by a number of i n v e s t i g a t o r s ( N o b l e , 1 9 8 0 ; G o d f r a i n d , 1 9 8 1 ) . Does t h i s phenomenon p l a y a d i r e c t r o l e i n t h e p o s i t i v e i n o t r o p i c a c t i o n of d i g i t a l o i d s o r i s i t a r e f l e c t i o n of i n d i r e c t e f f e c t s on c a t e c h o l a m i n e - d e p e n d e n t s y s t e m s ? I t c e r t a i n l y i s w e l l a p p r e c i a t e d t h a t o u a b a i n and o t h e r c a r d i a c g l y c o s i d e s c a n i n h i b i t t h e r e u p t a k e amine pump ( d e p e n d e n t on Na,K-ATPase

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830

A. SCHWARTZ

i n nerve endings) i n adrenergic nerve endings, thereby i n c r e a s i n g t h e c o n c e n t r a t i o n of f r e e norepinephrine i n h e a r t and o t h e r t i s s u e s (Shannar and B a n e r j e e , 1 9 8 0 ) . I n some e x p e r i m e n t s , o u a b a i n h a s been o b s e r v e d t o release s t o r e d c a t e c h o l a m i n e s ( S e i f e n , 1977; Duncan, 1 9 7 7 ) . I t h a s a l s o been known f o r some t i m e t h a t n o r e p i n e p h r i n e and o t h e r 8 - a d r e n e r g i c a g o n i s t s s t i m u l a t e monovalent cat i o n t r a n s p o r t i n s k e l e t a l m u sc l e and p e r h a p s o t h e r tiss u e s as w e l l (Togers e t a l . , 1 9 7 7 ) . I n f a c t , t h e s t i m u l a t i n g and i n h i b i t o r y a c t i o n s of c a r d i a c g l y c o s i d e s on s y m p a t h e t i c n e r v e a c t i v i t y have been i m p l i c a t e d i n t h e g e n e s i s of d i g i t a l i s - i n d u c e d c a r d i a c a r r h y t h m i a s ( G i l l i s , 1969). F i n a l l y , t h e r e c e n t r e p o r t s by Smith and h i s c o l l e a g u e s (see Smith and B a r r y , t h i s volume) and by Clausen e t a l . ( 1 9 8 2 ) s u g g e s t t h a t a c o n s i d e r a b l e p o r t i o n of t h e N a , K - A T P a s e p r e s e n t i n c a r d i a c p r e p a r a t i o n s i s d e r i v e d from n e r v e t e r m i n a l s , which would c o n t a i n n o r e p i n e p h r i n e and o t h e r a d r e n e r g i c a g o n i s t s . Perhaps t h e Na,K-ATPase a s s o c i a t e d w i t h n e r v e t e r m i n a l s reacts d i f f e r e n t l y t o o u a b a i n t h a n d o e s t h e Na,K-ATPase p r e s e n t i n t h e c e l l membranes of myocytic t i s s u e . The e x p e r i ments of Sweadner ( 1 9 7 9 ) showing d i f f e r e n c e s i n o u a b a i n s e n s i t i v i t y o f N a , K - A T P a s e i s o l a t e d from d i f f e r e n t subf r a c t i o n s of t h e b r a i n support t h i s contention. T h e i s s u e o f t h e N a / K pump s t i m u l a t i o n and t h e poss i b i l i t y of m u l t i p l e r e c e p t o r s f o r ouabain i n t h e h e a r t h a s been n i c e l y a r t i c u l a t e d by G o d f r a i n d and co-workers ( G o d f r a i n d , 1981; Godfraind and Ghysel-Burton, 1 9 7 7 ) . I n b r i e f , t h e s e i n v e s t i g a t o r s s u g g e s t t h a t t h e r e are two binding sites f o r ouabain, a " h i g h - a f f i n i t y " s i t e assoc i a t e d w i t h s t i m u l a t i o n of t h e N a / K pump, and a "lowa f f i n i t y " s i t e a s s o c i a t e d w i t h pump i n h i b i t i o n . The i n o t r o p i c a c t i o n of ouabain, according t o t h e i n v e s t i g a t o r s , may c o n s i s t o f more t h a n one p r o c e s s , one r e l a t e d t o i n h i b i t i o n of t h e N a / K pump, and t h e o t h e r e i t h e r unknown o r r e l a t e d t o pump s t i m u l a t i o n . I t w a s a l s o s t a t e d by Godfraind and h i s c o l l e a g u e s t h a t i n c o n t r a s t t o ouab a i n , d i h y d r o o u a b a i n produced o n l y pump i n h i b i t i o n (Godfraind, 1 9 8 1 ) . T o t e s t t h e s e i n t e r e s t i n g p r o p o s a l s , a number o f i n v e s t i g a t o r s have r e c e n t l y completed d i f f e r e n t t y p e s o f e x p e r i m e n t s . Smith and h i s c o l l e a g u e s ( t h i s volume) u s e d g u i n e a - p i g a t r i a , B-adrenergic b l o c k i n g a g e n t s , and chemical-induced d e p l e t i o n of endogenous c a t e c h o l a m i n e s , and showed t h a t low ( 3 nn) c o n c e n t r a t i o n s of o u a b a i n s t i m u l a t e d N a pump a c t i v i t y by r e l e a s i n g c a t e c h o l a m i n e s ( n o r e p i n e p h r i n e ) , which i n t u r n s t i m u l a t e d t h e pump. T h i s c o n c e n t r a t i o n of o u a b a i n , however, d i d n o t produce a p o s i t i v e i n o t r o p i c e f f e c t , p e r h a p s because a n i n s u f f i -

MECHANISMOF DIGITALIS ACTION; ENDOGENOUS FACTORS

831

d e n t amount of n o r e p i n e p h r i n e w a s r e l e a s e d . A concent r a t i o n of 1 0 mM o u a b a i n , i n some e x p e r i m e n t s , however, d i d i n c r e a s e developed t e n s i o n and t h i s a p p a r e n t l y w a s due t o t h e release o f enough endogenous c a t e c h o l a m i n e . Smith and h i s co-workers found t h a t t h e o n l y c l o s e c o r r e l a t i o n between a d i r e c t i n o t r o p i c a c t i o n of o u a b a i n r e l a t e d t o i n h i b i t i o n of N a / K pump a c t i v i t y w a s w i t h an i n h i b i t i o n of t h e l a t t e r a t c o n c e n t r a t i o n s h i g h e r M ouabain. The second t y p e o f e x p e r i m e n t than w a s c a r r i e d o u t by L e e and co-workers ( 1 9 8 0 ) , i n which it w a s shown t h a t d i h y d r o o u a b a i n and s t r o p h a n t h i d i n b o t h produced a n i n c r e a s e i n i n t r a c e l l u l a r N a a s measured w i t h a sodium e l e c t r o d e , and t h a t t h i s i n crease i n N a , which w a s undoubtedly due t o a n i n h i b i t i o n o f t h e pump, w a s d i r e c t l y r e l a t e d t o t h e p o s i t i v e i n o t r o p i c a c t i o n of t h e d r u g s . The most r e c e n t s t u d i e s of L e e and h i s c o l l e a g u e s ( L e e and V a s s a l l e , 1982; L e e and D a g o s t i n o , 1982, 1983) show t h a t no d e t e c t a b l e changes i n N a i were produced by s t r o p h a n t h i d i n a t 10-8 M . The t h r e s h o l d c o n c e n t r a t i o n f o r a n i n c r e a s e i n t e n s i o n , 5 x 10-8 M , produced a n i n c r e a s e i n N a i . This effect w a s dose-dependent and l i n e a r and t h e t i m e c o u r s e of changes i n N a i w a s s i m i l a r t o t h a t o f t h e change i n t e n s i o n and r e c o v e r y . No t r a n s i e n t s t i m u l a t i o n of t h e N a / K was n o t e d . E i s n e r e t a ] . ( P a r t V I I I , t h i s volume) reexamined t h e e f f e c t s o f Na-pump a c t i v i t y on c o n t r a c t i o n i n s h e e p P u r k i n j e f i b e r s by u s i n g a voltage-clamp p r o c e d u r e w i t h N a e l e c t r o d e s and a l t e r i n g t h e e x t e r n a l r u b i dium c o n c e n t r a t i o n . I t i s c l e a r from a l l of t h e s e new s t u d i e s t h a t a r i s e o f Nai a c t i v i t y produces an i n c r e a s e d t w i t c h and t o n i c t e n s i o n . The r i s e i n N a i t o 4 mM ( i f n o t g r e a t e r ) would c e r t a i n l y o c c u r i n t h e p r e s e n c e o f d i g i t a l i s . Upon r e a c t i v a t i o n o f t h e pump, b o t h i n t e r n a l N a and t h e e l e c t r o g e n i c pump decay r e t u r n e d t o normal. The r e l a t i o n s h i p between N a i and t e n s i o n may be complicated i n t h a t t h e t w i t c h t e n s i o n a c c o r d i n g t o E i s n e r i n t h e s h e e p P u r k i n j e system i s a n o n l i n e a r f u n c t i o n o f Nai (i.e. , "hysteresis") E i s n e r s u g g e s t s t h a t t h e r e may b e a p o o l of i n t r a c e l l u l a r N a (subsarcolemmal?) t h a t may exchange more r a p i d l y t h a n t h e b u l k c y t o p l a s m i c N a i . This would be c o n s i s t e n t w i t h t h e work t h a t Smith p r e s e n t e d i n t h i s c o n f e r e n c e and p u b l i s h e d r e c e n t l y ( B a r r y e t a l . , 1981) showing t h a t i n c u l t u r e d c h i c k h e a r t c e l l s b u l k c e l l N a may n o t r e p r e s e n t t h e t o t a l a c t i v i t y o f t h e Na pump. T h e r e i s n o t a s i m p l e r e l a t i o n s h i p between b u l k c e l l N a i and magnitude o f p o s i t i v e i n o t r o p i c e f f e c t produced by any i n t e r v e n t i o n i n c l u d i n g o u a b a i n . These v e r y i n t e r e s t i n g r e s u l t s might, i n f a c t , e x p l a i n t h e s t u d i e s p u b l i s h e d by a number o f i n v e s t i g a t o r s i n which e i t h e r no change i n a p p a r e n t pump a c t i v i t y o r s t i m u l a t i o n by

.

832

A. SCHWARTZ

o u a b a i n a r e r e p o r t e d , b a s e d o n l y upon measurements of c e l l c o n t e n t of i o n s . E i s n e r and h i s c o l l e a g u e s , howe v e r , d i d f i n d t h a t t h e e l e c t r o g e n i c Na pump c u r r e n t ( v o l t a g e - c l a m p measurement) h a s t h e same k i n e t i c s a s t h e N a i measured by t h e e l e c t r o d e , which s u g g e s t s t h a t t h e N a pump "sees" t h e same Nai a s t h e e l e c t r o d e . C l e a r l y more work n e e d s t o b e done i n t h i s a r e a . The t h i r d t y p e of e x p e r i m e n t b e a r s on t h e n a t u r e o f t h e "low d o s e " e f f e c t i n terms of r e p r o d u c i b l e changes i n contraction. I n c o l l a b o r a t i o n w i t h G o d f r a i n d and h i s c o l l e a g u e s , w e u s e d a t r i a and v e n t r i c l e s of g u i n e a p i g , c a t , and r a b b i t and measured i o n c o n t e n t , f l u x , and c o n t r a c t i l i t y i n t h e m u s c l e s and Na,K-ATPase a c t i v i t y i n enzyme i s o l a t e d from t h e m u s c l e s (Grupp e t a l . , 1 9 8 2 ) . Some i n t e r e s t i n g d a t a emerged from t h i s LouvainC i n c i n n a t i s t u d y . The "low-dose" e f f e c t i s o b s e r v e d o n l y when f i e l d s t i m u l a t i o n a t h i g h v o l t a g e was u s e d . I n some o f t h e e x p e r i m e n t s a n e g a t i v e i n o t r o p i c e f f e c t was o b s e r v e d , f o l l o w e d i n some c a s e s by a s l i g h t p o s i t i v e i n o t r o p i c a c t i o n w i t h c o n c e n t r a t i o n s of o u a b a i n around 3 0 - 8 JI. T h i s o c c u r r e d o n l y i n t h e l e f t a t r i a of g u i n e a p i g , and n e v e r o c c u r r e d i n v e n t r i c u l a r t i s s u e of any s p e c i e s . Higher d o s e s (from 1 0 - 7 M ) produced o n l y a pos i t i v e i n o t r o p i c e f f e c t . W e a l s o uncovered a n i m p o r t a n t t i s s u e e q u i l i b r a t i o n p r o b l e m , p a r t i c u l a r l y a t 37OC, which can l e a d t o a m i s i n t e r p r e t a t i o n o f a " n e g a t i v e i n o t r o p i c " a c t i o n o f a drug. Therefore, we f e e l t h a t the evidence t o d a t e i s t h a t any pump s t i m u l a t i o n t h a t i s found o c c u r s p r i m a r i l y i n g u i n e a - p i g l e f t a t r i a and p e r h a p s , a s i s t h e c a s e i n N o b l e ' s l a b o r a t o r y (Noble, 1 9 8 0 ) , i n s h e e p P u r k i n j e f i b e r s , and i s m o s t c l o s e l y a s s o c i a t e d w i t h a negative inotropic e f f e c t . This i s reasonable since a s t i m u l a t i o n o f t h e pump s h o u l d l e a d t o a d r o p i n Nai a c t i v i t y which, i n t u r n , o u g h t t o s t i m u l a t e a Na/Ca e x change t h e r e b y r e d u c i n g i n t r a c e l l u l a r Ca, n o t i n c r e a s i p g i t . The mechanism may i n v o l v e a s e l e c t i v e e f f e c t on Na,K-ATPase a s s o c i a t e d w i t h n e u r a l e l e m e n t s p r e s e n t i n t h e a t r i a of g u i n e a p i g s , b u t w e have no d a t a on t h i s point. The s i g n i f i c a n c e of such a phenomenon i n t h e mechanism of t h e t h e r a p e u t i c a c t i o n of d i g i t a l i s i s obs c u r e . There i s one o t h e r p o i n t . W e have t o be c a r e f u l i n e x t r a p o l a t i n g d a t a from a t r i a t o v e n t r i c l e s o r from s p e c i a l i z e d c o n d u c t i n g t i s s u e t o common m y o c a r d i a l c e l l s . There may be i m p o r t a n t d i f f e r e n c e s i n o r g a n e l l e d i s t r i b u t i o n and m e t a b o l i s m . Before l e a v i n g t h e t o p i c of s t i m u l a t i o n , I s h o u l d l i k e t o add o n e a d d i t i o n a l c o m p l e x i t y . Using v o l t a g e clamp t e c h n i q u e s , T s i e n and Marban ( 1 9 8 2 ; Marban, 1 9 8 1 , 1982; Marban and T s i e n , 1982) showed t h a t low c o n c e n t r a t i o n s o f o u a b a i n i n c r e a s e d s l o w inward c a l c i u m c u r r e n t ( I ~ ~and ) c o n t r a c t i l e fcrce

833

MECHANISM OF DIGITALISACTION; ENDOGENOUS FACTORS

i n f e r r e t p a p i l l a r y muscle M o u a b a i n ) and i n c a l f P u r k i n j e f i b e r s (10-8 M I , b u t t h a t t h i s e f f e c t w a s p r o b a b l y n o t due t o a d i r e c t e f f e c t on c a l c i u m c h a n n e l s . A scheme was p r e s e n t e d by T s i e n a t t h e E i g h t h I n t e r n a t i o n a l C o n g r e s s o f Pharmacology (Tokyo, J u l y , 1 9 8 1 ) which, i n t h e words of D r . T s i e n , g i v e s s o m e t h i n g f o r e v e r y o n e ‘I :

+

4 [NaIi

Ca-Na

1. [Cali

[Cali

diastole

S R systole

Twitch force

Scheme 1 The d a s h e d a r r o w acknowledges e a r l i e r s u g g e s t i o n s by o u r s e l v e s (Schwartz and Adams, 1980) t h a t p e r h a p s d i g i t a l i s c o u l d m o b i l i z e c a l c i u m from t h e N a , K - A T P a s e by c h a n g i n g t h e a f f i n i t y o f p h o s p h o l i p i d s s e c o n d a r y t o conf o r m a t i o n a l changes i n Na,K-ATPase. The f o u r t h type o f e x p e r i m e n t t h a t b e a r s on t h e problems o f p o s s i b l e pump s t i m u l a t i o n a n d h i g h - and lowa f f i n i t y b i n d i n g s i t e s f o r o u a b a i n i n v o l v e s t h e u s e of c e r t a i n s t e r o i d - l i k e d r u g s t h a t can a f f e c t t h e N a / K pump and y e t may n o t p r o d u c e a p o s i t i v e i n o t r o p i c e f f e c t a t a l l . W e c o l l a b o r a t e d w i t h D r s . F. L a b e l l a and I. B i h l e r , who p r e v i o u s l y have shown t h a t c h l o r m a d i n o n e a c e t a t e (CMA), a h y d r o x y p r o g e s t e r o n e d e r i v a t i v e , i n h i b i t s N a , K A T P a s e i s o l a t e d from g u i n e a - p i g b r a i n , c a u s e s c h a n g e s i n i o n c o n t e n t o f g u i n e a - p i g a t r i a , and y e t d o e s n o t p r o d u c e a p o s i t i v e i n o t r o p i c e f f e c t i n g u i n e a - p i g h e a r t , o r may even c a u s e n e g a t i v e i n o t r o p i s m . W e found t h a t t h i s d r u g a c t s a t a o u a b a i n s i t e i n v i t r o , b u t due t o l i m i t e d s o l u b i l i t y , s i m p l y c a n n o t r e a c h a c o n c e n t r a t i o n h i g h enough t o a f f e c t t h e Na,K-ATPase i n s i t u (Wehling e t a l . , 1 9 8 1 ) . I n o n e e x p e r i m e n t w e were a b l e t o push t h e c o n c e n t r a t i o n h i g h enough s o t h a t a modest p o s i t i v e i n o t r o p i c e f f e c t w a s o b t a i n e d ( d a t a n o t shown).

11.

RESULTS AND DISCUSSION

Now, what a b o u t t h e m u l t i p l e b i n d i n g - s i t e problem? W e l l s m i t h and Lindenmayer (1980) d e m o n s t r a t e d t h e p r e s e n c e o f low- and h i g h - a f f i n i t y b i n d i n g s i t e s f o r o u a b a i n , which t h e y i n t e r p r e t e d a s two forms o f t h e N a , K -

834

A. SCHWARTZ

ATPase. Only o n e "form" w a s i n h i b i t e d . Perhaps t h e s i t e t h a t d i d n o t l e a d t o i n h i b i t i o n o f t h e pump i s conn e c t e d t o t h e p o s i t i v e i n o t r o p i c e f f e c t of o u a b a i n , a l though no d a t a are a v a i l a b l e on t h i s p o i n t . O t h e r s (Akera e t a l . , 1 9 7 9 ) have r e p o r t e d s i m i l a r r e s u l t s , a l though w i t h d i f f e r e n t i n t e r p r e t a t i o n s i n v o l v i n g a N a , K A T P a s e " s i t e " and a n o t h e r " s i t e " n o t c o n n e c t e d w i t h t h e enzyme. The problem of m u l t i p l e b i n d i n g s i t e s f o r d i g i t a l i s i s a v e r y i n t r i g u i n g and i m p o r t a n t one t h a t r e p r e s e n t s , i n my view, a major i s s u e . W e r e c e n t l y (Adams e t ai., 1 9 8 2 ) examined t h e problem by u s i n g i n t a c t r a t v e n t r i c u l a r s t r i p s , myocytes p r e p a r e d from t h e same t i s s u e , sarcolemmal v e s i c l e s , and i s o l a t e d N a , K - A T P a s e preparations. O u r d a t a , d e r i v e d from c o n t r a c t i l i t y and [ 3H] ouabain binding s t u d i e s , c e r t a i n l y r e v e a l a "high a f f i n i t y " site i n r a t v e n t r i c l e t h a t i s not consistent w i t h t h e 150 f o r i n h i b i t i o n o f t h e N a , K - A T P a s e p r e p a r e d from r a t v e n t r i c l e . A "low a f f i n i t y " p o s i t i v e i n o t r o p i c s i t e i s a l s o p r e s e n t , which i s c o n s i s t e n t w i t h t h e I 5 p d e r i v e d from i n h i b i t i o n o f t h e enzyme. The " h i g h - a f f i n i t y " s i t e c a n be removed by p r o l o n g e d washing, implying a n i n t e r e s t i n g d e s e n s i t i z a t i o n phenomenon (Grupp . I 1 9 8 1 ) . W e conclude t h a t i n r a t v e n t r i c l e ( n o t a t r i a ) , but n o t i n c a t v e n t r i c l e o r a t r i a , t h e r e appear t o be two i n o t r o p i c mechanisms. I s h o u l d l i k e t o p r o p o s e t h a t t h e r e may e x i s t two mechanisms o r " s i t e s " i n a l l t i s s u e s , but only i n t h e glycoside-insensitive rat v e n t r i c l e a r e t h e a f f i n i t i e s f a r enough a p a r t t o be measured. F i g u r e 1 g r a p h i c a l l y d i s p l a y s my h y p o t h e s i s . In recent preliminary experiments, L e e (1983) have shown a r i s e i n N a produced i n r a t v e n t r i c l e by low c o n c e n t r a t i o n s o f ouabagenin.

.

A.

HYPOTHESIS

I n r a t v e n t r i c l e , h i g h - a f f i n i t y , low-capacity b i n d i n g s i t e s f o r o u a b a i n e x i s t which m e d i a t e a "lowconcentration," p o s i t i v e inotropic effect t h a t accounts f o r 2 0 - 4 0 % of t h e maximal i n o t r o p i c a c t i o n of o u a b a i n . The h i g h - a f f i n i t y s i t e ( A ) may be e n z y m a t i c a l l y i n a c t i v e o r may be t u r n i n g o v e r a t a v e r y slow r a t e . I n t h i s case, t h e binding of ouabain induces a conformational change i n a r e c e p t o r p r o t e i n which i n t u r n a l t e r s t h e sarcolemmal b i n d i n g of c o n t r a c t i o n - d e p e n d e n t calcium. A l t e r n a t i v e l y , t h e h i g h - a f f i n i t y s i t e ( A ) may be enzym a t i c a l l y a c t i v e and t h u s f u l l y i n h i b i t e d by low concent r a t i o n s o f o u a b a i n . However, i f t h e p r o p o r t i o n o f t h e s e s i t e s i s s m a l l r e l a t i v e t o a l a r g e p o p u l a t i o n o f t h e lowa f f i n i t y sites ( B ) , then the percent i n h i b i t i o n r e s u l t i n g from h i g h - a f f i n i t y b i n d i n g of o u a b a i n would b e s m a l l . Thus, t h e p o s i t i v e i n o t r o p i c e f f e c t mediated by h i g h -

MECHANISM OF DIGITALISACTION: ENDOGENOUS FACTORS

1 [OUABAIN ]

OUABAIN

K-I1

NA,K - ATPASE (?)

7

I

1

1 CONTRACTILE FORCE

15

i LOW AFFINITY

HIGH AFFINITY

1

835

NA,K - ATPASE I$ =30pM

4

CO:A::kTILE

F i g . 1 . Two i n o t r o p i c m e c h a n i s m s of d i g i t a l i s a c t i o n : Two-si t e h y p o t h e s i s .

a f f i n i t y - s i t e pump i n h i b i t i o n a n d e l e v a t e d i n t r a c e l l u l a r sodium would b e low. I f two s i t e s e x i s t i n t h e myocardium o f " s e n s i t i v e " species, t h e a f f i n i t i e s of A and B may b e v e r y s i m i l a r ; d i f f e r e n t i a l d e t e c t i o n would b e t e c h n i c a l l y d i f f i c u l t . T h i s h y p o t h e s i s may o f f e r a p o s r s i b l e e x p l a n a t i o n of t h e o b s e r v e d l a c k o f c o r r e l a t i o n between t h e low-ouabain-concentration p o s i t i v e i n o t r o p i c e f f e c t and Na+ pump i n h i b i t i o n .

111.

"ENDOGENOUS DIGITALIS" ( E N D O D I G I N )

T h i s t o p i c w a s c o v e r e d i n d e t a i l by H a u p e r t ( P a r t t h i s v o l u m e ) . The l i t e r a t u r e (Thorp and Cobbin, 1 9 6 7 ; Whitmer e t al., 1982) i s r e p l e t e w i t h r e f e r e n c e s t o a l l types o f endogenous s u b s t a n c e s t h a t d i s p l a y card i o t o n i c a c t i o n and f a c t o r s t h a t may be r e l e a s e d from areas i n t h e b r a i n and a p p e a r t o a c t s p e c i f i c a l l y o n t h e r e n a l t u b u l e s , and hence have been c a l l e d " n a t r i u r e t i c f a c t o r s " o r " t h i r d f a c t o r " o r " t h i r d hormone" (Brody and J o h n s o n , 1 9 8 0 ) . R e c e n t l y t h e r e h a s been a r e s u r g e n c e of

VIII,

836

A. SCHWARTZ

a c t i v i t y i n t h i s a r e a by H a u p e r t , F l i e r , Fishman, L i c h s t e i n , and o t h e r s , who r e p o r t t h e i s o l a t i o n from a c i d - a c e t o n e e x t r a c t s of b r a i n a s u b s t a n c e o r s u b s t a n c e s t h a t behave v e r y much l i k e d i g i t a l i s . Gruber, Buckalew, and t h e i r c o l l e a g u e s ( P a r t V I I I , t h i s volume) have demons t r a t e d t h e p r e s e n c e of a s u b s t a n c e i n plasma which cross-reacts with digoxin antibodies. I t i s not c l e a r why t h e r e s h o u l d be s u c h s p e c i f i c i t y of t h e s u b s t a n c e t o t h e d i g o x i n m o l e c u l e (and n o t t o , e . g . , o u a b a i n o r d i g i t o x i n ) , s i n c e t h e antibody t o digoxin i s s p e c i f i c f o r d i g o x i n and n o t f o r a l l c a r d i a c g l y c o s i d e s . T h i s s u b s t a n c e ( c a l l e d "Endoxin") was detected by t h e u s e of an a n t i d i g o x i n a n t i b o d y and i s o l a t e d by immunochemical p r e c i p i t a t i o n methods. Haddy and co-workers (Pamnani e t a l . , P a r t V I I I , t h i s volume) have r e p o r t e d a t t h i s meeti n g t h e p r e s e n c e o f a humoral s u b s t a n c e ( h e a t - s t a b l e f a c t o r ) t h a t i n h i b i t s t h e N a / K pump i n v a s c u l a r smooth m u s c l e and i n h e a r t microsomes These workers p r e v i o u s l y found t h a t o u a b a i n - s e n s i t i v e 8iRb u p t a k e i n r e d blood c e l l s and Na,K-ATPase a c t i v i t y of c a r d i a c microsomes a r e s u p p r e s s e d i n a n i m a l s w i t h low-renin, volume-expanded hypertension. T h i s , of c o u r s e , s u g g e s t s t h e i n t r i g u i n g p o s s i b i l i t y of a m o l e c u l a r l e s i o n i n c e r t a i n t y p e s of hyp e r t e n s i o n , a t o p i c t h a t was d i s c u s s e d i n a n o t h e r s e s s i o n and i s reviewed by T o s t e s o n and co-workers (Canessa e t a 1 , P a r t I X , t h i s volume) The main problem w i t h a l l o f t h e d a t a t h u s f a r p r e sented i s t h a t t h e r e i s very l i t t l e information a v a i l a b l e on the s t r u c t u r e o r n a t u r e of t h e s e p u t a t i v e f a c t o r s o r hormones. What i s u r g e n t l y needed i s a " c r a s h program" i n p r o t e i n c h e m i s t r y t o i s o l a t e , p u r i f y , and i d e n t i f y these substances. U n t i l s u c h t i m e as t h i s i s accomp l i s h e d , t h e s p e c t o r of a r t i f a c t c l o u d s t h e i s s u e . In f a c t , Kracke ( P a r t V I I I , t h i s volume) and Whitmer ( P a r t V I I I , t h i s volume) i n o u r group i n d e p e n d e n t l y p r e s e n t e d e v i d e n c e t h a t t h e e f f e c t s of t h e a c i d - a c e t o n e e x t r a c t s c o u l d be a t t r i b u t e d t o K+ o r t o o x i d i z e d l i p i d s u b s t a n ces. DePover ( P a r t , t h i s volume) p r e s e n t e d some i n t e r e s t i n g e x p e r i m e n t s which n o t o n l y b e a r o n endod i g i n , b u t , a c c o r d i n g t o t h e i n v e s t i g a t o r s , may e x p l a i n t h e i r p r e v i o u s r e p o r t s of two o u a b a i n - b i n d i n g sites. The r e a d e r w i l l r e c a l l my e a r l i e r d i s c u s s i o n c u s s i o n of t h e a p p a r e n t s t i m u l a t i o n by low c o n c e n t r a t i o n s of c a r d i a c g l y c o s i d e s on t h e Na/K pump i n g u i n e a - p i g l e f t a t r i u m . Digoxin, f o r example, a p p e a r s t o s t i m u l a t e 42Ks e n s i t i v e u p t a k e by a b o u t 5 0 % ( g u i n e a - p i g l e f t a t r i u m i n c u b a t e d a t 30" f o r 45 m i n ) . Godfraind (1980) h a s s u g g e s t e d t h a t t h i s s t i m u l a t i o n r e a l l y r e p r e s e n t s a "der e p r e s s i o n , " i . e . , a removal o f a s p e c i f i c endogenous

.

.

MECHANISM OF DIGITALISACTION: ENDOGENOUS FACTORS

837

d i g i t a l i s - l i k e r e p r e s s o r s u b s t a n c e . A w a t e r e x t r a c t of g u i n e a p i g h e a r t , f o l l o w e d by methanol e x t r a c t i o n , pet r o l e u m benzene e x t r a c t i o n , and chromatography on a n A m b e r l i t e MB3 column w i t h p y r i d i n e a c e t a t e and on Sephadex G-25 r e a c t e d s p e c i f i c a l l y w i t h a n t i d i g o x i n ant i b o d i e s . The e x t r a c t i n h i b i t e d N a , K-ATPase of g u i n e a pig h e a r t about 15%, i n d i c a t i n g t h e possible presence o f " d i g o x i n a c t i v i t y " of a b o u t 4 0 nM c o n c e n t r a t i o n . I n some r e s p e c t s , t h e n , t h e s e r e s u l t s a r e s i m i l a r t o t h o s e o f Gruber and h i s c o l l e a g u e s who may have i s o l a t e d t h e same f a c t o r from plasma. I n terms o f s u b s t a n c e s t h a t may r e g u l a t e t h e pump from t h e c y t o s o l i c s i d e o f t h e pump, v a n a d a t e i s s t i l l of some i n t e r e s t . Werdan e t a l . ( P a r t V I I I , t h i s volume) h a s d i s c u s s e d b o t h s t i m u l a t o r y and i n h i b i t o r y a c t i o n s of v a n a d a t e on t h e sodium pump of c u l t u r e d h e a r t c e l l s from r a t , g u i n e a p i g , c h i c k e n , and man. It was r e p o r t e d t h a t o n l y vanadium i n t h e (V) v a l e n c e s t a t e i n I n t h e i n t a c t c e l l , vanah i b i t s i s o l a t e d Na,K-ATPase. dium(V) can be reduced t o vanadium(1V) which can s t i m u l a t e 86Rb+ + K+ u p t a k e i n a n i n s u l i n - l i k e manner. In some h e a r t c e l l s , a p p a r e n t l y , t h i s r e d u c t i o n does n o t o c c u r , l e a v i n g vanadium i n t h e V v a l e n c e s t a t e , which i n h i b i t s t r a n s p o r t much l i k e o u a b a i n . These i n t e r e s t i n g r e s u l t s s u g g e s t t h a t p e r h a p s t h e redox s t a t e of t i s s u e s e x e r t s c o n t r o l o r r e g u l a t i o n of Na/K t r a n s p o r t through vanadium.

IV.

SUMMARY

W e have made some p r o g r e s s i n u n d e r s t a n d i n g how c a r d i a c g l y c o s i d e s produce t h e i r p o w e r f u l s t i m u l a t i n g a c t i o n on c a r d i a c c o n t r a c t i o n . The e v i d e n c e t o d a t e s u g g e s t s s t r o n g l y t h a t t h e Na,K-ATPase i s t h e p h a r m a c o l o g i c a l rec e p t o r o r , i f t h e r e a r e m u l t i p l e mechanisms, o n e of t h e It appears t h a t i n h i b i t i o n pharmacological r e c e p t o r s . of N a , K - A T P a s e enzyme a c t i v i t y and s u b s e q u e n t i n c r e a s e i n t h e a c t i v i t y of N a i o n a t t h e i n s i d e of t h e sarcolemmal membrane, which i n t u r n a c t i v a t e s a N a / C a exchange " c a r r i e r , " r e p r e s e n t s t h e sequence o f changes t h a t l e a d from a binding of d i g i t a l i s t o the Na,K-ATPase, t o t h e p o s i t i v e inotropic action. I n my o p i n i o n , t h e c r i t i c a l exp e r i m e n t s t h a t are now needed t o s o l i d i f y t h i s h y p o t h e s i s must i n v o l v e s i m u l t a n e o u s measurements ( p e r h a p s by microe l e c t r o d e s ) of a N a i and a C a i , c o n t r a c t i o n , and d i g i t a l i s b i n d i n g t o Na,K-ATPase and t o o t h e r s u g g e s t e d s i t e s . These e x p e r i m e n t s a r e c e r t a i n l y n o t e a s y . They r e q u i r e

A. SCHWARTZ

838

a r e l a t i v e l y s i m p l e t y p e of c a r d i a c p r e p a r a t i o n , s u c h as v i a b l e s i n g l e c e l l s d e v o i d of f i b r o b l a s t s , i n which c o n t r a c t i o n can be q u a n t i t a t i v e l y measured. The p o s s i b i l i t y t h a t endogenous s u b s t a n c e s e x i s t which r e g u l a t e t h e pump by an a c t i o n on t h e o u t s i d e of t h e sarcolemma is very e x c i t i n g .

ACKNOWLEWMENTS

The d a t a d i s c u s s e d i n t h i s review from t h e a u t h o r ' s l a b o r a t o r y were d e r i v e d from e x p e r i m e n t s s u p p o r t e d i n p a r t by PO1 HL 22619 ( I ) . I s h o u l d l i k e t o t h a n k D r s . E. T. W a l l i c k , L. K. Lane, K. Whitmer, and G . Grupp f o r t h e i r comments and s u g g e s t i o n s .

REFERENCES Adams, R. J., Schwartz, A., Grupp, G., Lee, S.-W., W a l l i c k , E. T . , Powell, D. T . , T w i s t , Y. W . , G i t h i r a m , P. ( 1 9 8 2 ) . High-aff i n i t y o u a b a i n b i n d i n g s i t e and low-dose p o s i t i v e i n o t r o p i c e f f e c t i n rat myocardium. N a t u r e 296, 167-169. Akera, T . , and Brody, T. M. ( 1 9 7 8 ) . The r o l e of Na,K-ATPase i n t h e P h a r m a c o l . R e v . 29, 187-218. i n o t r o p i c a c t i o n of d i g i t a l i s . Akera, T . , Brody, T. M . , and Wiest, S. A. ( 1 9 7 9 ) . S a t u r a b l e adenos i n e 5 ' -triphosphate-independent b i n d i n g o f [3H] o u a b a i n t o b r a i n and c a r d i a c t i s s u e i n v i t r o . B r . J. P h a r m a c o l . 65, 403-409. A s k a r i , A. , ed. ( 1 9 7 4 ) . P r o p e r t i e s and f u n c t i o n s of ( N a + + K+)a c t i v a t e d adenosinetriphosphatase. A n n . N . Y . A c a d . S c i . 2 4 2 , 1-174. B a r r y , W. H . , B i e d e r t , S . , Miura, D, S., and Smith, T. W. ( 1 9 8 1 ) . Changes i n c e l l u l a r N a + , K + and C a 2 + c o n t e n t s , monovalent cat i o n t r a n s p o r t s rates a n d c o n t r a c t i l e s t a t e d u r i n g washout of c a r d i a c g l y c o s i d e s from c u l t u r e d c h i c k h e a r t c e l l s . C i r c . R e s . 4 9 , 141-149. Brody, M. J. , and Johnson, A. K. ( 1 9 8 0 ) . Role of a n t e r o v e n t r a l t h i r d v e n t r i c l e r e g i o n i n f l u i d and e l e c t r o l y t e b a l a n c e , art e r i a l p r e s s u r e r e g u l a t i o n and h y p e r t e n s i o n . In F r o n t i e r s i n Neuroendocrinology, Vol. 6 (L. M a r t i n i and W. F. Ganong, e d s . ) , ~C h a p t e r 9, pp. 249-292. Raven P r e s s , N e w York. C l a u s e n , T. , Hansen, 0. , a n d Larsson, L . - I . ( 1 9 8 1 ) . Sympathetic n e r v e t e r m i n a l d e s t r u c t i o n h a s no e f f e c t on s p e c i f i c [3H]ouabain binding. E u r . J. P h a r m a c o l 7 2 , 331-335. Cohen, I. , Daut, J . , and Noble, D. ( 1 9 7 6 ) . An a n a l y s i s o f t h e act i o n s of low c o n c e n t r a t i o n s o f ouabain on membrane c u r r e n t s i n P u r k i n j e f i b e r s . J. P h y s i o l . ( L o n d o n ) 260, 75-103.

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MECHANISM OF DIGITALIS ACTION; ENDOGENOUS FACTORS

839

Duncan, C. J. ( 1 9 7 7 ) . The a c t i o n of ouabain i n promoting t h e rel e a s e of c a t e c h o l a m i n e s . E x p e r i e n t i a 33, 923. Erdmann, E. ( 1 9 8 1 ) . I n f l u e n c e o f c a r d i a c g l y c o s i d e s on t h e i r r e c e p t o r . Handb. E x p . P h a r m a k . 56 ( I ) , 337-380. Erdmann, E . , P h i l i p p , G . , and S c h o l z , H. ( 1 9 8 0 ) . C a r d i a c g l y c o s i d e r e c e p t o r , NKA a c t i v i t y and f o r c e of c o n t r a c t i o n i n r a t h e a r t . B i o c h e m . P h a r m a c o l . 2 9 , 3219-3229. G i l l i s , R. A. ( 1 9 6 9 ) . C a r d i a c s y m p a t h e t i c n e r v e a c t i v i t y : Changes induced by ouabain and p r o p r a n o l o l . S c i e n c e 1 6 6 , 508-510. Godfraind, T. ( 1 9 8 0 ) . S t i m u l a t i o n e t i n h i b i t i o n de l a pompe h sodium p a r l e s h g t 6 r o s i d e s c a r d i o t o n i q u e s . B u l l . Acad. R. Med. B e l g 1 3 5 , 174-192. Godfraind, T. ( 1 9 8 1 ) . S t i m u l a t i o n and i n h i b i t i o n o f t h e Na+,K+-pmp Handb. E x p . P h a r m a k o l . 56 ( I ) , 381-393. by c a r d i a c g l y c o s i d e s . Godfraind, T . , and Ghysel-Burton, J. ( 1 9 7 7 ) . Binding s i t e s r e l a t e d t o ouabain-induced s t i m u l a t i o n o r i n h i b i t i o n o f t h e sodium N a t u r e ( L o n d o n ) 2 6 5 , 165-166. pump. Grupp, G . , Grupp, I . L., Ghysel-Burton, J . , Godfraind, T . , and Schwartz, A. ( 1 9 8 2 ) . E f f e c t s of v e r y low c o n c e n t r a t i o n s o f ouabain on c o n t r a c t i l e f o r c e o f i s o l a t e d g u i n e a p i g , r a b b i t and c a t a t r i a and r i g h t v e n t r i c u l a r p a p i l l a r y muscles: An i n t e r i n s t i t u t i o n a l study. J . P h a r m a c o l . Exp. T h e r . 2 2 0 , 145-151. Grupp, I . L . , Grupp, G . , and Schwartz, A ( 1 9 8 1 ) . D i g i t a l i s r e c e p tor. D e s e n s i t i z a t i o n i n r a t v e n t r i c l e : Ouabain produces two L i f e S c i . 2 9 , 2789-2794. inotropic effects. Lee, C. 0. ( 1 9 8 1 ) . I o n i c a c t i v i t i e s i n c a r d i a c muscle c e l l s and a p p l i c a t i o n o f i o n s e l e c t i v e m i c r o e l e c t r o d e s . Am. J. P h y s i o l . 2 4 1 , H459-H478. Lee, C . O . , and Dagostino, M. ( 1 9 8 2 ) . E f f e c t of s t r o p h a n t h i d i n on i n t r a c e l l u l a r Na i o n a c t i v i t y and t w i t c h t e n s i o n of b e a t i n g dog c a r d i a c P u r k i n j e f i b e r s . B i o c h e m . J . 37, 342a. Lee, C. O . , and V a s s a l l e , M. ( 1 9 8 2 ) . Role o f i n t r a c e l l u l a r sodium i n s t r o p h a n t h i d i n and n o r e p i n e p h r i n e i n o t r o p y o f c a n i n e F e d . P r o c . , F e d . Am. SOC. E x p . B i o l . Purkinje f i b e r s . ( a b s t r . ) 41, 1504. L e e , C. O . , Kang, D. H . , Sokol, J. H . , and Lee, K. S . ( 1 9 8 0 ) . Rel a t i o n between i n t r a c e l l u l a r N a i n a c t i v i t y and t e n s i o n o f sheep c a r d i a c P u r k i n j e f i b e r s exposed t o dihydroouabain. B i o p h y s . J. 2 9 , 315-330. Lee, K. S . , and Klaus, W. (1971). The s u b c e l l u l a r b a s i s f o r t h e mechanism o f i n o t r o p i c a c t i o n of c a r d i a c g l y c o s i d e s . P h a r m a c o l . R e v . 2 3 , 193-261. Marban, E. ( 1 9 8 1 ) . AHA Meet., L o u i s K a t z Award S y m p . 1 9 8 1 , Paper

.

Marban, E . (1982). Ph.D. D i s s e r t a t i o n . Noack, E . , F e l g e n t r a g e r , J . , and Z e t t n e r , B. ( 1 9 7 9 ) . Changes i n myocardial Na+,K+ c o n t e n t d u r i n g t h e development of c a r d i a c J. Mol. C e l l . C a r d i o l . 1 1 , 1189-1194. glycoside inotropy.

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Noble , D. (1980). Mechanism o f a c t i o n of t h e r a p e u t i c l e v e l s of c a r d i a c g l y c o s i d e s . Cardiovasc. R e s . 1 4 , 495-914. O k i t a , G . , Richardson, F . , and Roth-Schechter, B. L. (1973). D i s s o c i a t i o n o f t h e p o s i t i v e i n o t r o p i c a c t i o n o f d i g i t a l i s from i n h i b i t i o n of Na,K-ATPase. J. Pharmacol. Exp. Ther. 1 8 5 ,

1-11. Repke, K. (1964). Ueber den Biochemische Wirkungsmode von Digitalis. K1 i n . Wochenschr. 42 , 157-165. Schatzmann, H. J. ( 1 9 5 3 ) . Herzglycoside a l s hemmstoffe f u r den a k t i v e n kalium und n a t r i u m t r a n s p o r t durch d i e e r y t h r o c y t e n membran. Helv. P h y s i o l . Acta 11, 346-354. Schwartz, A . , and Adams, R. J. ( 1 9 8 0 ) . S t u d i e s on t h e d i g i t a l i s C i r c . R e s . 4 6 , Suppl. I ) , 154-160. receptor. Schwartz, A., Lindenmayer, G. E . , and A l l e n , J. C. (1975). The sodium-potassium adenosine t r i p h o s p h a t a s e : Pharmacological , p h y s i o l o g i c a l and biochemical a s p e c t s . Pharmacol Rev. 2 7 , 3-134. S e i f e n , E. (1977). Evidence f o r p a r t i c i p a t i o n of c a t e c h o l a m i n e s i n Eur. J. Pharmacol 26 , 115-118. c a r d i a c a c t i o n o f ouabain. Sharmar, V. K., and B a n e r j e e , S. P . (1980). Ouabain s t i m u l a t i o n of n o r a d r e n a l i n e t r a n s p o r t i n guinea p i g h e a r t . N a t u r e (London) 286, 817-819. ++ + M a + , K +Skou, J. C. (1960). F u r t h e r i n v e s t i g a t i o n s on a Mg a c t i v a t e d ATPase p o s s i b l y r e l a t e d t o t h e a c t i v e l i n k e d t r a n s p o r t o f N a + and K+ a c r o s s t h e nerve membrane. Biochim. B i o p h y s . Acta 4 2 , 6-23. Skou, J. C . , and Ndrby, J. G . , e d s . (1979). "Na,K-ATPase: Struct u r e and K i n e t i c s . " Academic Press, N e w York. Solomon, A. K., G i l l , T. J . , 111, and Gold, G. L. (1956). The k i n e t i c s of c a r d i a c g l y c o s i d e i n h i b i t i o n o f potassium t r a n s p o r t i n human e r y t h r o c y t e s . J . Gen. P h y s i o l . 4 0 , 327-350. Sweadner, K. J. ( 1 9 7 9 ) . Two m o l e c u l a r forms of NKA i n b r a i n . J. B i o l . Chem. 254 , 6060-6067. Thorp, R . , and Cobbin, L. ( 1 9 6 7 ) . "Cardiac S t i m u l a n t S u b s t a n c e s , " pp. 223-272. Academic P r e s s , N e w York. Togers, E. M. , Cheng, L. C . , and Zierler, K. (1977). Betaa d r e n e r g i c e f f e c t on N ~ + , K + t r a n s p o r t i n r a t s k e l e t a l muscle. Biochim. Biophys. Acta 464 , 347-355. T s i e n , R. W . , and Marban, E. (1982). D i g i t a l i s and slow inward calcium c u r r e n t i n h e a r t muscle: Evidence f o r r e g u l a t i o n e f f e c t s of i n t r a c e l l u l a r calcium on calcium c h a n n e l s . Adv. Pharmacol. Ther., Proc. I n t . Congr. 8 t h , 1981 Abstract, pp. 150-151. Wehling, M . , Schwartz, A . , Whitmer, K., Grupp, G., Grupp, I. L . , and W a l l i c k , E . T. ( 1 9 8 1 ) . I n t e r a c t i o n of CMA w i t h t h e ouab a i n b i n d i n g s i t e o f Na,K-ATPase. Mol. Pharrnacol. 2 0 , 551557. Wellsmith, N . V. , and Lindenmayer, G . E. ( 1 9 8 0 ) . Two r e c e p t o r forms f o r o u a b a i n i n sarcolemma-enriched p r e p a r a t i o n s from C i r c . R e s . 4 7 , 710-720. canine v e n t r i c l e .

.

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MECHANISMOF DIGITALISACTION; ENDOGENOUS FACTORS

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Whitmer, K. , W a l l i c k , E. T., Epps, D. E . , Lane, L. K . , C o l l i n s , J . , and Schwartz, A. ( 9 8 2 ) . Minireview: I s t h e r e an endogenous d i g i t a l i s - l i k e f a c t o r ? L i f e S c i . 30, 2261-2275. Wilbrandt, W., ed. (1962). Proceedings of the F i r s t I n t e r n a t i o n a l Pharmacology Meeting, 1961, V o l . 3 . Pergamon, Oxford. Withering, W. (1785). A n account of Foxglove and some o f i t s medic a l uses. The C l a s s i c s of Medicine L i b r a r y , D i v i s i o n of Gryphon E d i t i o n s , Ltd., Birmingham, Alabama, 1979.

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CURRENT TOPICS IN MEMBRANES AND TRANSPORT,VOLUME 19

Endogenous Glycoside-Like Substances GARNER T. HAUPERT, JR. Renal Unit and Cellular and Molecular Research Laboratory Massachusetts General Hospital Boston, Massachusetts

I.

INTRODUCTION

S e v e r a l known endogenous s u b s t a n c e s have been i m p l i c a t e d i n t h e r e g u l a t i o n of Na,K-ATPase a c t i v i t y . C a t e c h o l a m i n e s ( P h i l l i s , 1978; L. C . C a n t l e y e t a l . , 1978; H o r w i t z , 19791, t h y r o i d hormone (Smith and Edelman, 1 9 7 9 ) , and vanadium ( L . C . C a n t l e y , J r . , et a l . , 1978) have a l l been l i n k e d t o e i t h e r d i r e c t o r i n d i r e c t e f f e c t s o n enzyme a c t i v i t y . I n r e c e n t y e a r s , however, a body of e x p e r i m e n t a l e v i d e n c e h a s been a c c u m u l a t i n g which f a v o r s t h e e x i s t e n c e of a presumed u n i q u e and a s y e t u n c h a r a c t e r i z e d r e g u l a t o r of N a , K-ATPase which f u n c t i o n s i n a n i n h i b i t o r y mode, and i n t h i s , a s w e l l a s o t h e r ways, f u n c t i o n a l l y r e s e m b l e s t h e c a r d i a c q l y c o s i d e s . T h i s rep o r t w i l l p r e s e n t a n o v e r v i e w of t h e s t a t u s o f work g e n e r a t e d from s e v e r a l l a b o r a t o r i e s p e r t i n e n t t o t h e b i o l o g i c a l e f f e c t s , c h e m i c a l n a t u r e , s o u r c e of product i o n , and p o t e n t i a l p h y s i o l o g i c i m p l i c a t i o n s a c c o r d e d t o t h i s p u t a t i v e endogenous g l y c o s i d e - l i k e compound. 043

Copyright 0 1983 by Academic Press, Inc. All rightsof reproduction in any form r e ~ e ~ e d . ISBN 0-12-153319-0

GARNER T. HAUPERT,JR.

044

Demonstration of endogenous g l y c o s i d e - l i k e a c t i v i t y h a s emanated from two a p p r o a c h e s . Even b e f o r e t h e d i s c o v e r y of n a t u r a l l y o c c u r r i n g l i g a n d s i n t h e mammalian b r a i n t o t h e o p i a t e r e c e p t o r l e d t o t h e h y p o t h e s i s of a p o t e n t i a l p a r a l l e l s i t u a t i o n f o r t h e Na,K-ATPase, workers i n t h e area of n a t r i u r e t i c hormone r e s e a r c h were r e p o r t i n g d a t a t o s u g g e s t t h a t t h e mechanism of a c t i o n o f t h e v a r i o u s a c t i v e e x t r a c t s might be m e d i a t e d t h r o u g h e f f e c t s on t h e sodium pump a l o n g t h e r e n a l t u b u l a r e p i t h e l i u m . Although C l a r k s o n and dewardener (1972) and Kaplan et a l . (1974) had d e m o n s t r a t e d i n t r a c e l l u l a r i o n c o n c e n t r a t i o n changes and a l t e r a t i o n s i n o x i d a t i v e metabolism of t r a n s p o r t i n g e p i t h e l i a r e f l e c t i n g sodium-pump i n h i b i t i o n , t h e r e c o g n i t i o n of an endogenous s u b s t a n c e w i t h o u a b a i n l i k e e f f e c t s w a s f i r s t a r t i c u l a t e d by Gonick and coworkers ( H i l l y a r d et al., 1 9 7 6 ) d u r i n g t h e i r a t t e m p t s t o c h a r a c t e r i z e a n a t r i u r e t i c p r i n c i p l e e x t r a c t e d from ren a l t i s s u e of volume-expanded r a t s .

11.

MATERIALS AND METHODS

Along t h e s e l i n e s , o u r own i n t e r e s t i n t h e p u t a t i v e n a t r i u r e t i c hormone l e d u s s e v e r a l y e a r s ago t o b e g i n t h e s t u d y of an endogenous compound w i t h sodium t r a n s p o r t i n h i b i t o r y p r o p e r t i e s (Haupert and Sancho, 1 9 7 9 ) . On t h e background of a s u b s t a n t i a l body o f i n d i r e c t e v i d e n c e f o r t h e e x i s t e n c e o f such a f a c t o r (dewardener, 19771, and w i t h t h e knowledge t h a t t h e hypothalamus h a s proved a r i c h s o u r c e o f r e g u l a t o r y hormonal s u b s t a n c e s , w e s c r e e n e d e x t r a c t s o f bovine hypothalamus f o r sodium t r a n s p o r t i n h i b i t o r y a c t i v i t y . S t a r t i n g w i t h kilogram q u a n t i t i e s , t i s s u e was e x t r a c t e d i n acetone-HC1 , and t h e l i p i d s removed by p e t r o l e u m e t h e r e x t r a c t i o n . The aqueous phase w a s t h e n chromatographed on Sephadex G-25. D e s a l t i n g was accomplished by ion-exchange chromatography, and f u r t h e r c o n c e n t r a t i o n of t h e a c t i v i t y by l i p o p h i l i c g e l chromatography and high-performance l i q u i d chromatography. The b a s i c b i o l o g i c a l a s s a y i n t h i s e a r l y work w a s t h e i s o l a t e d t o a d b l a d d e r p r e p a r a t i o n (Leaf e t a l . , 1 9 5 8 ) . I s o l a t e d hemibladders from B u f o m a r i n u s were s u s pended between two h a l v e s of a l u c i t e chamber c o n s t r u c t e d so a s t o p r o v i d e f o r s i m u l t a n e o u s e x p e r i m e n t a l and cont r o l o b s e r v a t i o n s on t h e same p i e c e of t i s s u e . T r a n s e p i t h e l i a l sodium t r a n s p o r t i s measured a s t h e s h o r t - c i r c u i t c u r r e n t (SCC), and r e s i s t a n c e measurements a r e made a t 10-sec i n t e r v a l s t h r o u g h o u t t h e s t u d y . A p p l i c a t i o n of

ENDOGENOUS GLYCOSIDE-LIKE SUBSTANCES

845

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'

'

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20 30 40 50 60 T/M€ (minuies)

70

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80

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F i g . 1 . I n h i b i t i o n of SCC ( a c t i v e Na t r a n s p o r t ) i n i s o l a t e d ) t o a d b l a d d e r . The 250-pl s a m p l e o f h y p o t h a l a m i c f a c t o r ( O--O was a p p l i e d t o 4 ml of a m p h i b i a n R i n g e r ' s s o l u t i o n b a t h i n g the s e r o s a l s u r f a c e a t To. T h e control ( 0---0 ) was 250 p l o f R i n g e r ' s s o l u t i o n . S e r o s a l s u r f a c e s were r i n s e d w i t h fresh R i n g e r ' s s o l u t i o n a t 48 m i n . H y p o t h a l a m i c f a c t o r p r o d u c e d a p r o m p t and s u s t a i n e d i n h i b i t i o n of a c t i v e s o d i u m t r a n s p o r t , reversible w i t h r i n s i n g . R e p r o d u c e d f r o m H a u p e r t and S a n c h o ( 1 9 7 9 ) , w i t h p e r m i s s i o n o f the publisher.

s m a l l amounts of a c t i v e d e s a l t e d e x t r a c t s , when a p p l i e d t o t h e s e r o s a l s u r f a c e o f t h e membrane, r e s u l t e d i n a prompt and s u b s t a n t i a l d r o p i n t h e SCC which w a s complet e l y r e v e r s i b l e by a g e n t l e r i n s e of t h e s e r o s a l s u r f a c e w i t h f r e s h R i n g e r ' s s o l u t i o n ( F i g . 1 ) . The d r o p i n c u r r e n t w a s accompanied by an increase i n r e s i s t a n c e a c r o s s t h e membrane, i n d i c a t i n g i n h i b i t i o n of sodium movement t h r o u g h t h e a c t i v e t r a n s p o r t pathway. A p p l i c a t i o n of a c t i v e e x t r a c t s t o t h e mucosal s i d e produced no c h a n g e s i n SCC, i n d i c a t i n g t h a t t h e o b s e r v e d e f f e c t s were n o t m e d i a t e d t h r o u g h a l t e r e d sodium e n t r y a t t h e a p i c a l membrane. These p h y s i o l o g i c a l e f f e c t s reminded u s of t h e a c t i o n of o u a b a i n i n s i m i l a r a n u r a n membrane p r e p a r a t i o n s . To e x p l o r e t h e p o s s i b i l i t y t h a t t h e unknown s u b s t a n c e had o t h e r o u a b a i n - l i k e f e a t u r e s , w e s t u d i e d i t s i n t e r a c t i o n w i t h o u a b a i n i n f r o g u r i n a r y b l a d d e r by t h e method of M i l l s and E r n s t ( 1 9 7 5 ) . I n t h e s e e x p e r i m e n t s f r o g u r i n a r y b l a d d e r w a s suspended i n t h e L u c i t e chamber and t h e e x p e r i m e n t a l h a l f p r e t r e a t e d w i t h a s t a n d a r d d o s e of f a c t o r and a l l o w e d t o r e a c t f o r 1 5 min, d u r i n g which t ' m e t h e u s u a l d r o p i n SCC o c c u r r e d . A t 15 min, [ Hlouabain w a s a p p l i e d t o b o t h e x p e r i m e n t a l and c o n t r o l

4

GARNER T. HAUPERT, JR.

a46

TABLE I .

Binding of O u a b a i n t o F r o g B l a d d e r B i n d i n g , prnol ouabain/mg

-__ Ouabain, PM

2 . 9 (n=5) 0 . 4 (n=4)

Ouabain

0.623 0.186

(wet w t ) t i s s u e

-

.-

Ouabain f2U

hypothalamic factor 0.442 0.125

A binding* 0.181 0.061

P

t

0.031k

<0.01

0.012t

cO.02

~

*

Mean d i f f e r e n c e ? SEM. +Probability t h a t A binding = 0.

h a l v e s of t h e membrane. A f t e r an a d d i t i o n a l 3 0 min t h e chamber w a s d r a i n e d , and t i s s u e from e a c h h a l f w a s p r e p a r e d f o r l i q u i d s c i n t i l l a t i o n c o u n t i n g . R e s u l t s showed t h a t a t two l e v e l s o f o u a b a i n t e s t e d t h e r e w a s s i g n i f i c a n t i n h i b i t i o n of o u a b a i n b i n d i n g t o b l a d d e r t i s s u e p r e t r e a t e d w i t h t h e sodium t r a n s p o r t i n h i b i t o r ( T a b l e I ) . I n a n a n a l o g o u s t y p e of e x p e r i m e n t , w e t e s t e d t h e e f f e c t s of t h e f a c t o r on o u a b a i n b i n d i n g t o g u i n e a - p i g b r a i n microsomal enzyme, a s a d a p t e d from t h e method publ i s h e d by Fishman ( 1 9 7 9 ) . F i g u r e 2 shows a d o s e - r e l a t e d i n h i b i t i o n of [3H]ouabain b i n d i n g t o t h e microsomal f r a c t i o n by i n c r e a s i n g amounts of h y p o t h a l a m i c f a c t o r . The p r e c i s e n a t u r e o f t h e i n t e r a c t i o n among o u a b a i n , f a c t o r , and r e c e p t o r r e m a i n s t o b e d e f i n e d . On t h e o n e hand, t h e p o s s i b i l i t y e x i s t s of a c o m p e t i t i v e - t y p e i n h i b i t i o n between f a c t o r and o u a b a i n . On t h e o t h e r hand, w e know t h a t i n s e v e r a l c e l l t y p e s s t u d i e d ( J o i n e r and L a u f , 1 9 7 8 ; M i l l s e t a l . , 1 9 7 9 ) [3H]ouabain b i n d i n g t o i t s r e c e p t o r i s a f u n c t i o n of t u r n o v e r of t h e pump. It i s p o s s i b l e , t h e r e f o r e , t h a t t h e d i s p l a c e m e n t of o u a b a i n may o c c u r a s a n e v e n t s e c o n d a r y t o d i m i n i s h e d pump act i v i t y c a u s e d by d i r e c t e f f e c t s of t h e f a c t o r on t h e enzyme. T o b e g i n t o s t u d y t h i s p r o b l e m , w e examined t h e d i r e c t e f f e c t s of t h e h y p o t h a l a m i c f a c t o r on N a , K - A T P a s e from r a b b i t r e n a l o u t e r m e d u l l a . Enzyme was p r e p a r e d , and had a Na,K-dependent A T P a s e a c t i v i t y of 3 0 pmoles i n o r g a n i c p h o s p h o r u s l i b e r a t e d p e r mg p r o t e i n p e r h o u r . Enzyme w a s i n c u b a t e d w i t h v a r y i n g d o s e s of o u a b a i n a n d hypothalamic f a c t o r . F i g u r e 3 shows a d o s e - r e l a t e d i n h i b i t i o n of enzyme a c t i v i t y c a u s e d by t h e f a c t o r . Of n o t e w a s t h a t when submaximal d o s e s of o u a b a i n and f a c t o r were c o - i n c u b a t e d w i t h t h e enzyme, i n h i b i t i o n of a c t i v i t y was a d d i t i v e .

847

ENDOGENOUS GLYCOSIDE-LIKESUBSTANCES

100

0

100

I

2

3

l-lypafhlamic factor, arbitrary units Fig. 2. Inhibition of [3H]ouabain binding t o guinea-pig brain microsomes by increasing concentrations of hypothalamic f a c t o r . ( I n s e r t ) Inhibition of binding by unlabeled ouabain.

I n summary, t h i s m a t e r i a l r e v e r s i b l y i n h i b i t s act i v e sodium t r a n s p o r t i n anuran membranes. It directly i n h i b i t s r e n a l Na,K-ATPase from a mammalian s o u r c e , and i t s o u a b a i n - l i k e a c t i v i t y i s f u r t h e r m a n i f e s t e d by i t s a b i l i t y t o i n h i b i t o u a b a i n b i n d i n g t o i t s c e l l u l a r rec e p t o r i n f r o g u r i n a r y b l a d d e r and g u i n e a - p i g b r a i n m i crosomes. Frog u r i n a r y b l a d d e r and r a b b i t r e n a l N a , K ATPase have been found t o be s i g n i f i c a n t l y more s e n s i t i v e t o t h e e f f e c t s of t h e compound t h a n t o a d u r i n a r y b l a d d e r and enzyme p r e p a r e d i n an i d e n t i c a l f a s h i o n from r a t r e n a l t i s s u e , r e s p e c t i v e l y . These i n t e r s p e c i e s d i f f e r e n c e s i n s e n s i t i v i t y a r e p a r a l l e l t o t h o s e of o u a b a i n i n t h e same p r e p a r a t i o n s . Several preliminary experiments i n o u r l a b o r a t o r y have shown t h e sodium t r a n s p o r t i n h i b i t o r t o be n a t r i u r e t i c when i n f u s e d i n t o an i s o l a t e d p e r f u s e d r a t k i d n e y . Most of t h e s e v e r a l g r o u p s s t u d y i n g n a t r i u r e t i c e x t r a c t s have now found them t o be i n h i b i t o r s I s h a l l r e t u r n t o a discussion of t h e of Na,K-ATPase. chemical n a t u r e of t h e hypothalamic f a c t o r .

GARNER T. HAUPERT, JR.

a48

f

Total lpM Na+-K+ Ou ATPase

2U HF

IU HF

.5U

HF

lpM

+

Ou

2 U HF

F i g . 3 . I n h i b i t i o n o f Na,K-ATPase f r o m r a b b i t r e n a l o u t e r m e d u l l a b y o u a b a i n (Ou) and v a r i o u s c o n c e n t r a t i o n s o f h y p o t h a l a m i c f a c t o r ( H F ) ; n = 6 , error b r a c k e t s i n d i c a t e f 1 SEM. T h e e f f e c t s o f h y p o t h a l a m i c f a c t o r were d o s e - r e l a t e d . Hypothalamic f a c t o r p l u s submaximal doses o f o u a b a i n p r o d u c e d a d d i t i v e i n h i b i t i o n . * p < 0.02-0.001 vs t o t a l Na,K-ATPase; t p < 0.001 vs 1 p M o u a b a i n . + p < 0.02 vs 2 U of h y p o t h a l a m i c f a c t o r . Reproduced from H a u p e r t and Sancho ( 1 9 7 9 ) w i t h p e r m i s s i o n o f the p u b l i s h e r .

111.

RESULTS AND D I S C U S S I O N

Other i n v e s t i g a t o r s have approached t h e q u e s t i o n of endogenous g l y c o s i d e s from a somewhat d i f f e r e n t p o i n t of view. The d i s c o v e r y t h a t n a t u r a l l i g a n d s t o t h e o p i a t e r e c e p t o r s e x i s t w i d e l y i n animals (Hughes e t a l . , 1975; Simantov and Snyder, 1976) l e a d s t o t h e h y p o t h e s i s t h a t p a r a l l e l s i t u a t i o n s might o b t a i n f o r o t h e r pharmacologic a l l y p o t e n t s u b s t a n c e s from t h e p l a n t kingdom which e x e r t t h e i r e f f e c t s through s p e c i f i c r e c e p t o r s i n animal t i s s u e s . Thus t h e Na,K-ATPase i n animal s p e c i e s might have an endogenous a n a l o g t o t h e c a r d i a c g l y c o s i d e s . F l i e r ( 1 9 7 8 ) and F l i e r e t a l . ( 1 9 7 9 ) f i r s t demonstrated endogenous g l y c o s i d e - l i k e a c t i v i t y i n t o a d s k i n and plasma. E x t r a c t s of s k i n and d i l u t i o n s of serum were shown t o d i s p l a c e [3H]ouabain b i n d i n g t o human e r y t h r o c y t e s , t o i n h i b i t Na,K-ATPase p r e p a r e d from e l e c t r i c e e l , and, i n t h e c a s e of s e r u m , t h e c r o s s - r e a c t i n a d i g i t a l i s immunoassay. These s t u d i e s i n t o a d s were extended t o a v a r i e t y of amphibian s p e c i e s , and e x t r a c t a b l e d i g i t a l i s l i k e a c t i v i t y was found t o e x i s t w i d e l y , i n c l u d i n g i n s p e c i e s which a p p a r e n t l y do n o t produce b u f o d i e n o l i d e t o x i n s which a r e known t o p o s s e s s g l y c o s i d e - l i k e a c t i v i -

ENDOGENOUS GLYCOSIDE-LIKESUBSTANCES

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ty. I t i s now c l e a r , f u r t h e r m o r e , t h a t endogenous g l y c o s i d e - l i k e a c t i v i t y can be r e c o v e r e d from b l o o d , b r a i n , and k i d n e y i n mammalian s p e c i e s . Thus Fishman (1979) p r e p a r e d e x t r a c t s o f whole g u i n e a - p i g b r a i n , which f o l l o w i n g p a r t i a l p u r i f i c a t i o n w a s found t o m i m i c d i g i t a l i s g l y c o s i d e s by competing w i t h o u a b a i n b i n d i n g t o N a , K - A T P a s e and by i n h i b i t i n g rubidium u p t a k e i n t o human e r y t h r o c y t e s . L i c h t s t e i n and Samuelov (1980) have l i k e w i s e p r e p a r e d an e x t r a c t o f whole r a t b r a i n which i n h i b i t s Na,K-ATPase a c t i v i t y and [3H]ouabain b i n d i n g i n r a t synaptosomal membranes. F i n a l l y , Gruber and co-workers (1980) have r e c e n t l y r e p o r t e d t h a t p a r t i a l l y p u r i f i e d d i a f i l t r a t e s of c a n i n e plasma c o r r e s p o n d i n g t o f r a c t i o n s which i n h i b i t SCC i n anuran membranes a l s o c r o s s - r e a c t w i t h a n t i b o d i e s r a i s e d a g a i n s t d i g o x i n . The c r o s s - r e a c t i n g a c t i v i t y w a s rec o v e r e d from plasma of h y d r o p e n i c a n i m a l s b u t w a s p r e s e n t i n g r e a t e r amounts i n a n i m a l s p r e v i o u s l y expanded w i t h s a l i n e . Chromatographic p r o f i l e s showed two s e p a r a t e peaks of a c t i v i t y , implying two m o l e c u l a r s p e c i e s w i t h differing structures. Chemical c h a r a c t e r i z a t i o n o f t h e g l y c o s i d e - l i k e s u b s t a n c e o r s u b s t a n c e s h a s proved c o n s i d e r a b l y more problematic than t h e demonstration of b i o l o g i c a l a c t i v i t y , and t h e l i t e r a t u r e i s q u i t e s p a r s e i n t h i s area. T a b l e I1 summarizes p u b l i s h e d f i n d i n g s . A l l i n v e s t i g a t o r s concur t h a t t h e m o l e c u l e o f i n t e r e s t i s o f l o w mol e c u l a r w e i g h t , w i t h t h e 500-1000 MW r a n g e b e i n g g e n e r a l l y c i t e d . There i s d i v i s i o n of o p i n i o n a s t o i t s g e n e r a l chemical n a t u r e . I n v e s t i g a t o r s i n t h e t o p p o r t i o n o f T a b l e I1 have r e p o r t e d p a r t i a l l y p u r i f i e d s u b s t a n c e s which a r e i n a c t i v a t e d by e i t h e r a c i d h y d r o l y s i s o r p r o t e o l y t i c d i g e s t i o n , o r both, suggesting t h e molecule t o be p e p t i d i c i n n a t u r e . Authors c i t e d i n t h e l o w e r p a r t of t h e t a b l e have found t h e i r r e s p e c t i v e a c t i v e f r a c t i o n s t o be r e s i s t a n t t o p r o t e o l y s i s and o r a c i d h y d r o l y s i s , s u g g e s t i n g a n o n p e p t i d i c m o l e c u l e . The h y p o t h a l a m i c f a c t o r h a s been found t o behave a s a weak b a s e , and t o b e r e s i s t a n t t o s t r i n g e n t acid h y d r o l y s i s . Its a c t i v i t y i s d e s t r o y e d by a s h i n g o r free r a d i c a l s u b s t i t u t i o n . R e s o l u t i o n of t h e d i v e r g e n t f i n d i n g s among t h e s e v e r a l groups awaits d e f i n i t i v e s t r u c t u r a l i d e n t i f i c a t i o n o f t h e compound o r compounds i n q u e s t i o n . The most sal i e n t d i s c r e p a n c y c e n t e r s on a p e p t i d i c v e r s u s nonpept i d i c m o l e c u l e . Based on r e s u l t s o f a c i d s t a b i l i t y t e s t i n g , o n e would have t o b e l i e v e t h a t a l l g r o u p s c a n n o t be s t u d y i n g t h e same s u b s t a n c e Although t h e s i t e o f o r i g i n o f endogenous g l y c o s i d e s u b s t a n c e remains t o be e s t a b l i s h e d , s e v e r a l l i n e s o f e v i d e n c e f a v o r t h e c e n t r a l nervous system. Organ a b l a -

.

TABLE 11.

Source, m r i f i c a t i o n Techniques and Chemical C h a r a c t e r i s t i c s of I s o l a t e s Reported t o Show Glycoside-Like A c t i v i t y a

Investigator

Source

H i l l y a r d (1976)

Kidney

Gruber (1978)

Canine plasma Urine

Bourgoinie (1976)

Purification Boiled homogenate Gel f i l t r a t i o n Diafiltration HPLC G e l filtration HPLC

Characteristics Trypsin-S Limited s t a b i l i t y Acid-S Acidic p e p t i d e Acid, base I S Pepsin, LAP

Amino a c i d a n a l y s i s F l i e r (1978, 1979)

Amphibian s k i n

Clarkson (1979)

Urine

Organic e x t r a c t i o n s Silica gel HPLC

Gel f i l t r a t i o n Organic e x t r a c t i o n

Fishman (1979) Haupert (1979)

a

Guinea p i g Brain Bovine Hypothalamus

S-sensitive; R - r e s i s t a n t ; l e u c i n e amino p e p t i d a s e .

()-partial;

G e l filtration

Heat I R Trypsin Var. Polarity Acid, Base - (R) Nitrous a c i d I S Prolidase Acid stable

Ion-exchange

Gel f i l t r a t i o n Ion-exchange HPLC

Acid s t a b l e Polar Weak base

HPLC-high performance l i q u i d chromatography;

LAP-

ENDOGENOUS GLYCOSIDE-LIKESUBSTANCES

851

t i o n e x p e r i m e n t s (Kaloyanides e t ai. , 1 9 7 7 ) have shown t h a t volume-expansion n a t r i u r e s i s o f a c r o s s - c i r c u l a t e d i s o l a t e d k i d n e y o c c u r s d e s p i t e t h e a b s e n c e of a d r e n a l , t h y r o i d , p a r a t h y r o i d , o r p i t u i t a r y g l a n d i n t h e donor a n i m a l . The n a t r i u r e t i c r e s p o n s e i n t h e i s o l a t e d o r g a n was a b o l i s h e d , however, f o l l o w i n g volume e x p a n s i o n o f a d e c a p i t a t e d donor (Kaloyanides e t a ] . , 1 9 7 8 ) . Evidence based on l e s s i n v a s i v e e x p e r i m e n t a l p r o c e d u r e s h a s a l s o p o i n t e d t o b r a i n and i n p a r t i c u l a r t o t h e hypothalamus. Thus t h e a c t i v i t y h a s been i s o l a t e d and p a r t i a l l y p u r i f i e d from hypothalamic t i s s u e (Haupert and Sancho, 1 9 7 9 ) , and w e have found t h e c o n c e n t r a t i o n of t h i s a c t i v i t y t o be a t l e a s t 1 0 - f o l d g r e a t e r t h a n i n e x t r a c t s of whole b o v i n e b r a i n . Two o t h e r l i n e s of e v i d e n c e f a v o r t h e hypothalamic h y p o t h e s i s . S i l v a - N e t t o and coworkers (1980) d e m o n s t r a t e d by r e c o l l e c t i o n micropunct u r e sampling i n h i b i t i o n o f t u b u l a r sodium and f l u i d t r a n s p o r t produced by s t i m u l a t i o n of t h e a n t e r i o r l a t e r a l hypothalamus. Such changes a p p e a r i n d e p e n d e n t of a l t e r a t i o n s i n r e n a l hemodynamics and hormonal e f f e c t s a t t r i b u t a b l e t o DOCA, v a s o p r e s s i n , and o x y t o c i n . C o n v e r s e l y , Bealer and c o l l e a g u e s ( 1 9 7 9 ) , i n c o l l a b o r a t i o n w i t h D r s . Gruber and Buckalew ( 1 9 7 8 ) , have rep o r t e d i m p a i r e d volume-expansion n a t r i u r e s i s i n r a t s w i t h l e s i o n s of t h e a n t e r o v e n t r a l t h i r d - v e n t r i c l e reg i o n , and a s s a y of plasma from t h e s e a n i m a l s showed no n a t r i u r e t i c a c t i v i t y . Sham-lesioned r a t s had a b r i s k n a t r i u r e s i s and a h i g h l e v e l o f n a t r i u r e t i c a c t i v i t y . I n a d d r e s s i n g t h e p o t e n t i a l r o l e f o r an endogenous sodium t r a n s p o r t i n h i b i t o r i n t h e g e n e s i s of volumee x p a n s i o n h y p e r t e n s i o n , Haddy and co-workers (Pamnani e t a l . , 1980) have found s u p e r n a t e s of b o i l e d plasma from volume-hypertensive dogs and r a t s t o c o n t a i n a subs t a n c e which i n h i b i t s o u a b a i n - s e n s i t i v e 86Rb u p t a k e i n t a i l a r t e r i e s of normal r a t s . When t h e h y p e r t e n s i o n w a s induced i n r a t s p r e v i o u s l y l e s i o n e d i n t h e a n t e r o v e n t r a l t h i r d - v e n t r i c l e r e g i o n , a s s a y o f t h e s u p e r n a t e s from t h e s e a n i m a l s f a i l e d t o produce pump s u p p r e s s i o n i n t h e t a i l a r t e r y of t h e a s s a y r a t . Taken t o g e t h e r t h e composite o b s e r v a t i o n s s u g g e s t t h e hypothalamus t o be i m p o r t a n t l y i n v o l v e d i n t h e release o r a c t i v a t i o n o f t h e sodium t r a n s p o r t i n h i b i t o r y a c t i v i t y , and p e r h a p s t o r e p r e s e n t a s i t e of i t s concent r a t i o n , i f n o t d e novo production. The p h y s i o l o g i c a l i m p l i c a t i o n s f o r a n endogenous s u b s t a n c e which r e g u l a t e s t h e N a , K - A T P a s e a r e p r o t e a n . I n a d d i t i o n t o p a r t i c i p a t i o n i n t h e m o d u l a t i o n of memb r a n e p o t e n t i a l s , c e l l volume, e l e c t r o l y t e e x c r e t i o n , and e x t r a c e l l u l a r f l u i d volume s t a t u s , such s u b s t a n c e c o u l d p r o v e t o e x e r c i s e i m p o r t a n t e f f e c t s on c a r d i a c

GARNER T. HAUPERT, JR.

852

c o n t r a c t i l i t y , c e l l u l a r t h e r m o g e n e s i s , and h o m e o s t a t i c adjustment of t u b u l a r f u n c t i o n i n p r o g r e s s i v e r e n a l i n s u f f i c i e n c y . Recent r e p o r t s of i o n - t r a n s p o r t abnormalit i e s i n b l o o d c e l l s of p a t i e n t s w i t h e s s e n t i a l hypert e n s i o n (Canessa e t al., 1980; Garay et a l . , 1980) have complemented t h e body of e x p e r i m e n t a l d a t a l i n k i n g t o n i c sodium-pump i n h i b i t i o n t o t h e g e n e s i s of volumee x p a n s i o n h y p e r t e n s i o n i n a n i m a l s (Haddy e t a l . , 1978) , t h e l a t t e r b e i n g mediated p e r h a p s t h r o u g h induced changes i n transmembrane sodium and c a l c i u m g r a d i e n t s i n a r t e r i o l a r smooth muscle c e l l s , l e a d i n g i n t u r n t o t o n i c i n c r e a s e s i n c o n t r a c t i l e t e n s i o n and e l e v a t e d v a s c u l a r r e s i s t a n c e ( B l a u s t e i n , 1 9 7 7 ) . Although t h e r o l e of N a , K A T P a s e remains i n q u e s t i o n v i s h v i s sodium/potassium c o t r a n s p o r t and s o d i u m / l i t h i u m c o u n t e r t r a n s p o r t abnormal i t i e s found i n e r y t h r o c y t e s of e s s e n t i a l h y p e r t e n s i v e s ( T o s t e s o n , 1981) , t h e f a l l i n t h e o u a b a i n - s e n s i t i v e comp o n e n t s o f t h e t o t a l sodium e f f l u x r a t e c o n s t a n t i n l e u k o c y t e s from e s s e n t i a l h y p e r t e n s i v e s and i n normal leukoc y t e s b a t h e d i n h y p e r t e n s i v e sera ( P o s t o n e t al., 1981) s u g g e s t s a r o l e f o r a c i r c u l a t i n g sodium t r a n s p o r t i n h i b i t o r i n t h e g e n e s i s of c e r t a i n o f t h e i o n - t r a n s p o r t abnormalities i n these patients. S i m i l a r e f f e c t s of pump s u p p r e s s i o n i n v a s c u l a r smooth muscle c e l l s have l i k e w i s e been c i t e d i n s u p p o r t of a c e n t r a l r o l e f o r a n endogenous sodium t r a n s p o r t i n h i b i t o r i n t h e g e n e s i s of volumee x p a n s i o n h y p e r t e n s i o n (Haddy e t a l . , 1 9 7 8 ) . The b r e a d t h of p o t e n t i a l p h y s i o l o g i c i m p o r t a n c e f o r a n endogenous r e g u l a t o r o f t h e Na,K-ATPase s e r v e s t o und e r s c o r e t h e major c h a l l e n g e i n t h e area of t h e endogenous g l y c o s i d e q u e s t i o n which c o n t i n u e s t o b e t h e d e f i n i t i v e s t r u c t u r a l i d e n t i f i c a t i o n of t h i s compound o r compounds which w i l l p e r m i t f u r t h e r s t u d i e s o f i t s mechanism of a c t i o n and t h e development of a s u i t a b l e a s s a y t o beg i n t o probe i t s i m p o r t a n c e i n p h y s i o l o g y .

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B e a l e r , S . , Haywood, J. R . , Johnson, A . K., Gruber, K. A . , Buckalew, V. M., and Brody, M. J. (1979). Impaired n a t r i u r e s i s and s e c r e t i o n of n a t r i u r e t i a hormone i n r a t s w i t h l e s i o n s of t h e a n t e r o v e n t r a l 3rd v e n t r i c l e . Fed. Am. SOC. Exp. B i o l . 3 8 , 1232. B l a u s t e i n , M. P. (1977). Sodium i o n s , aalcium i o n s , blood p r e s s u r e r e g u l a t i o n and h y p e r t e n s i o n : A reassessment and a h y p o t h e s i s . Am. J. P h y s i o l 232 (3), C165-Cl73.

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B o u r g o i g n i e , J. J . , F a v r e , H . , Kaplan, M. A . , Eun, C . , Hwang, K . H . , and B r i c k e r , N. S. ( 1 9 7 6 ) . On t h e n a t r i u r e t i c f a c t o r o f serum and u r i n e from p a t i e n t s w i t h c h r o n i c u r a e m i a . In " I n t e r n a t i o n a l Workshop a t Cologne on C e n t r a l Nervous C o n t r o l o f N a + B a l a n c e : R e l a t i o n t o t h e Renin-Angiotensin System" (w. Kaufman and D. K. K r a u s e , e d s . ) , pp. 133-140. Thieme, St u t t g a r t . Canessa, M., Adragna, N . , Solomon, H. S . , C o n n o l l y , T. M., a n d T o s t e s o n , D. C. ( 1 9 8 0 ) . I n c r e a s e d s o d i u m - l i t h i u m c o u n t e r t r a n s p o r t i n r e d c e l l s of p a t i e n t s w i t h e s s e n t i a l h y p e r t e n N. E n g l . J. Med. 302, 772-776. sion. C a n t l e y , L. C . , Jr., C a n t l e y , L. G . , a n d J o s e p h s o n , L. ( 1 9 7 8 ) . A c h a r a c t e r i z a t i o n o f v a n a d a t e i n t e r a c t i o n w i t h (Na,K)-ATPase: J. Biol. Chem. 253, M e c h a n i s t i c and r e g u l a t o r y i m p l i c a t i o n s . 7361-7368. C a n t l e y , L. C . , Ferguson, J. H . , and K u s t i n , K. ( 1 9 7 8 ) . Norepinephrine complexes and r e d u c e s vanadium (V) t o reverse v a n a d a t e J . Am. Chem. SOC. 1 0 0 , i n h i b i t i o n of t h e (Na,K)-ATPase. 5210-2. C l a r k s o n , E. M. , and d e l a r d e n e r , H. E. ( 1 9 7 2 ) . I n h i b i t i o n o f =odium and potassium t r a n s p o r t i n s e p a r a t e d r e n a l t u b u l e f r a g ments i n c u b a t e d i n e x t r a c t s of u r i n e o b t a i n e d from s a l t loaded individuals. C l i n S c i 42, 607-617. C l a r k s o n , E. M . , S h e e l a g h , M. R . , and dewardener, H. E. ( 1 9 7 9 ) . F u r t h e r o b s e r v a t i o n s on a low-molecular-weight n a t r i u r e t i c s u b s t a n c e i n t h e u r i n e o f normal man. Kidney I n t . 1 6 , 710721. dewardener, H. E. ( 1 9 7 7 ) . N a t r i u r e t i c hormone. Clin. S c i . M o l . Med. 5 3 , 1-8. Fishman, M. C. ( 1 9 7 9 ) . Endogenous d i g i t a l i s - l i k e a c t i v i t y i n mamm a l i a n b r a i n . Proc. N a t l . A c a d . S c i . USA 76, 4661-4663. F l i e r , J. S . ( 1 9 7 8 ) . O u a b a i n - l i k e a c t i v i t y i n t o a d s k i n and i t s i m p l i c a t i o n s f o r endogenous r e g u l a t i o n of i o n t r a n s p o r t . N a t u r e ( L o n d o n ) 274, 285-286. F l i e r , J. S . , M a r a t o s - F l i e r , E . , P a l l o t t a , J. A . , and McIsaac, D. Endogenous d i g i t a l i s - l i k e a c t i v i t y i n t h e plasma (1979) o f t h e t o a d B u f o m a r i n u s . N a t u r e ( L o n d o n ) 279, 341-343. Garay, R. P . , E l g h o z i , J. L., Dagher, G . , and Meyer, P. ( 1 9 8 0 ) . L a b o r a t o r y d i s t i n c t i o n between e s s e n t i a l and s e c o n d a r y hypert e n s i o n by measurement of e r y t h r o c y t e c a t i o n f l u x e s . N. Engl J . Med. 302, 769-771. G r u b e r , K. A . , a n d Buckalew, V. M . , Jr. ( 1 9 7 8 ) . F u r t h e r c h a r a c t e r i z a t i o n a n d e v i d e n c e f o r a p r e c u r s o r i n t h e f o r m a t i o n of plasma a n t i n a t r i f e r i c f a c t o r . P r o c . SOC. Exp. B i o l . Med. 1 5 9 , 463-467. Gruber, K. A . , W h i t a k e r , J. M . , and Buckalew, V. M . , Jr. ( 1 9 8 0 ) . Endogenous d i g i t a l i s - l i k e s u b s t a n c e i n p l a s m a o f volumeexpanded d o g s . N a t u r e (London) 287, 743-745. Haddy, F . , Pamnani, M . , a n d Clough, D. ( 1 9 7 8 ) . The sodiumpotassium pump i n volume expanded h y p e r t e n s i o n . C l i n . Exp. H y p e r t e n s i o n 1 , 295-336.

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Haupert, G. T., J r . , and Sancho, J. M. (1979). Sodium t r a n s p o r t i n h i b i t o r from bovine hypothalamus. Proc. N a t l . Acad. Sci. USA 76, 4658-4660. H i l l y a r d , S. D., Lu, E . , and Gonick, H. C. (1976). F u r t h e r charact e r i z a t i o n o f t h e n a t r i u r e t i c f a c t o r d e r i v e d from kidney t i s sue of volume expanded r a t s . E f f e c t s on short-circuit c u r r e n t Circ and sodium-potas sium-adenos i n e t r i p h o s p h a t a s e a c t i v i t y . R e s . 38, 250-255. Horwitz, B. A. (1979) C e l l u l a r e v e n t s u n d e r l y i n g catecholamineinduced thermogenesis: Cation t r a n s p o r t i n brown a d i p o c y t e s . Fed. Proc., Fed. Am. Soc. Exp. B i o l . 38, 2170-2176. Hughes, J . , Smith, T. W . , K o s t e r l i t z , H. W., F o t h e r g i l l , L. A . , Morgan, B. A. , and M o r r i s , H. R. (1975). I d e n t i f i c a t i o n o f two r e l a t e d p e n t a p e p t i d e s from t h e b r a i n w i t h p o t e n t o p i a t e a g o n i s t a c t i v i t y . Nature (London) 258, 577-579. J o i n e r , C. H. , and Lauf, P. K. (1978). Modulation of ouabain bindi n g and potassium pump f l u x e s by c e l l u l a r sodium and p o t a s s i u m i n human and sheep e r y t h r o c y t e s . J. P h y s i o l . (London) 283, 177-196. Kaloyanides, G . J . , Cohen, L . , and DiBona, G . F. (1977). F a i l u r e of s e l e c t e d e n d o c r i n e organ a b l a t i o n t o modify t h e n a t r i u r e sis o f blood volume expansion i n t h e dog. C l i n . Sci. Mol. Med. 52, 351-356. Kaloyanides, G. J . , Balabanian, M. B . , and Bowman, R. L. (1978). Evidence t h a t t h e b r a i n p a r t i c i p a t e s i n t h e humoral n a t r i u r e t i c mechanism o f blood volume expansion i n t h e dog. J. C l i n . I n v e s t . 62, 1288-1295. Kaplan, M. A , , Bourgoignie, J. J . , Rosecan, J . , and B r i c k e r , N. S. (1974). The e f f e c t s of t h e n a t r i u r e t i c f a c t o r from uremic u r i n e on sodium t r a n s p o r t , water and e l e c t r o l y t e c o n t e n t , and p y r u v a t e o x i d a t i o n by t h e i s o l a t e d toad b l a d d e r . J. C l i n . I n v e s t . 5 3 , 1568-1577. Leaf, A., Anderson, J . , and Page, L . B. (1958). Active sodium t r a n s p o r t by t h e i s o l a t e d t o a d b l a d d e r . J. Gen. P h y s i o l . 41, 657-667. L i c h t s t e i n , D., and Samuelov, S. (1980). Endogenous o u a b a i n - l i k e a c t i v i t y i n r a t brain. Biochem. Biophys R e s . Commun 96, 1518-1523. M i l l s , J. W . , and E r n s t , S. A. (1975). L o c a l i z a t i o n of sodium pump s i t e s i n f r o g u r i n a r y b l a d d e r . Biochim. Biophys. Acta 375, 268-273. M i l l s , J. W . , Mcknight, A . D. C . , J a r r e l l , J. A . , Dayer, J. M . , and A u s i e l l o , D. A. (1981). I n t e r a c t i o n o f ouabain w i t h t h e sodium. pump i n i n t a c t e p i t h e l i a l c e l l s . J. C e l l Biol. 88, 637-643. Pamnani, M., Bugge, J . , Huot, S . , Clough, D., and Haddy, F. (1980). Vascular sodium-potassium pump a c t i v i t y i n a c u t e l y s a l i n e loaded rats w i t h a n t e r o v e n t r a l t h i r d v e n t r i c l e l e s i o n s . Fed. Proc., Fed. Am. SOC. E x p . Biol. 4 0 , 390. P h i l l i s , J. W. (1978). P h y s i o l o g i c a l and pharmacological s t u d i e s on c e n t r a l s y n a p t i c t r a n s m i s s i o n . I n “ C e l l , T i s s u e and Organ

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C u l t u r e s i n Neurobiology" (S. F e d o r o f f and L. H e r t z , e d s . ) , pp. 93-97. Academic P r e s s , New York. P o s t o n , L . , S e w e l l , R. B . , W i l k i n s o n , S. P., R i c h a r d s o n , P. J . , W i l l i a m s , R . , C l a r k s o n , E. M . , MacGregor, G. A . , and dewardener, H. E. (1981). Evidence f o r a c i r c u l a t i n g sodium t r a n s p o r t i n h i b i t o r i n e s s e n t i a l hypertension. B r . Med. J. 282, 847-849. S i l v a - N e t t o , C. R., de Mello A i r e s , M . , and M a l n i c , G. ( 1 9 8 0 ) . A microHypothalamic s t i m u l a t i o n and e l e c t r o l y t e e x c r e t i o n : p u n c t u r e s t u d y . Am. J. P h y s i o l . 239, F206-F214. Simantov, R . , a n d Snyder, S. H. ( 1 9 7 6 ) . M o r p h i n e - l i k e p e p t i d e s i n mammalian b r a i n : I s o l a t i o n , s t r u c t u r e , e l u c i d a t i o n , and i n t e r a c t i o n s w i t h t h e o p i a t e receptor. Proc. N a t l . A c a d . S c i . USA 73, 2515-2519. S m i t h , T. J . , and Edelman, I. S. ( 1 9 7 9 ) . The r o l e of sodium F e d . Proc./ F e d . Am. t r a n s p o r t i n t h y r o i d thermogenesis. SOC. Exp. Biol. 38, 2150-2153. C a t i o n c o u n t e r t r a n s p o r t and c o t r a n s p o r t T o s t e s o n , D. C. (1981) i n human red c e l l s . F e d . P r o c . , Fed. Am. SOC. E x p . B i o l . 4 0 , 1429-1433.

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CURRENT TOPICS M MEMBRANES AND TRANSWRT. VOLUME 19

Monovalent Cation Transport and Mechanisms of Digitalis-Induced lnotropy THOMAS W. SMITH AND WILLIAM H. BARRY Cardiovascular Division, Brigham and Women 's Hospital and Department of Medicine Brigham and Women's Hospital and Harvard Medical School Boston, Massachusetts

I.

INTRODUCTION

V i r t u a l l y a l l known a s p e c t s o f m y o c a r d i a l c e l l u l a r a c t i v i t y have been s t u d i e d i n s e a r c h o f t h e b a s i c mechanism t h a t a c c o u n t s for t h e a b i l i t y of c a r d i a c g l y c o s i d e s t o e f f e c t an i n c r e a s e i n t h e c o n t r a c t i l e s t a t e of h e a r t m u s c l e (Smith and Braunwald, 1 9 8 0 ) . Lack o f e v i d e n t p r i m a r y e f f e c t s a t c l i n i c a l l y r e l e v a n t concent r a t i o n s on c o n t r a c t i l e o r r e g u l a t o r y p r o t e i n s , i n t e r mediary metabolism, o r e n e r g e t i c s h a s s e r v e d t o f o c u s r e c e n t i n v e s t i g a t i v e e f f o r t s i n t h e area of e x c i t a t i o n contraction coupling. I t i s w i d e l y t h o u g h t t h a t i n some way d i g i t a l i s g l y c o s i d e s a t " t h e r a p e u t i c " ( s u b t o x i c ) l e v e l s enhance t h e a v a i l a b i l i t y of C a 2 + t o m y o c a r d i a l c o n t r a c t i l e e l e m e n t s f o l l o w i n g e x c i t a t i o n . Direct e v i d e n c e f o r a n i n c r e a s e i n t h e magnitude of t h e i n t r a c e l l u l a r Ca2+ t r a n s i e n t i n r e s p o n s e t o a c e t y l s t r o p h a n t h i d i n i s now a v a i l a b l e t h a n k s t o t h e e l e g a n t s t u d i e s of A l l e n and B l i n k s (1978) u s i n g t h e p h o t o a c t i v e c a l c i u m - s e n s i t i v e The c e l l u l a r mechanism whereby t h i s p r o t e i n aequorin. 857

Copyright 0 1983 by Academic Press, Inc. Ail rights of reproducrion in any form resewed. ISBN 0-12-1533190

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augmentation of c a l c i u m a v a i l a b i l i t y o c c u r s w i l l be t h e p r i n c i p a l s u b j e c t of t h i s d i s c u s s i o n . Twenty y e a r s have p a s s e d s i n c e t h e p i o n e e r i n g works of Skou and of Repke l e d t o t h e s u g g e s t i o n t h a t i n h i b i t i o n o f t h e Mg2+- and ATP-dependent, N a + - and K + - a c t i v a t e d t r a n s p o r t enzyme complex i n t h e sarcolemma known as Na ,K-ATPase might m e d i a t e t h e i n o t r o p i c e f f e c t s of c a r d i a c g l y c o s i d e s (Repke, 1 9 6 2 ) . A s u b s t a n t i a l w e i g h t of c i r c u m s t a n t i a l e v i d e n c e i m p l i c a t i n g Na,K-ATPase as a r e c e p t o r f o r t h e i n o t r o p i c e f f e c t s of d i g i t a l i s now e x i s t s and h a s been summarized i n s e v e r a l r e v i e w s (Smith and Braunwald, 1980; Schwartz e t a l . , 1975; Akera and Brody, 1 9 7 8 ) . D e s p i t e t h e d e t a i l e d knowledge of t h e h i g h - a f f i n i t y , h i g h - s p e c i f i c i t y i n t e r a c t i o n between c a r d i a c g l y c o s i d e s and t h e N a , K - A T P a s e i n h i b i t o r y s i t e f a c ing t h e o u t e r c e l l surface discussed elsewhere i n t h i s symposium, however, d i r e c t evid.3nce o f a c a u s a l r e l a t i o n s h i p between sodium pump i n h i b i t i o n and t h e p o s i t i v e i n o t r o p i c r e s p o n s e h a s been d i f f i c u l t t o o b t a i n and remains c o n t r o v e r s i a l ( O k i t a , 1977; Rhee e t a l . , 1 9 8 1 ) . A s t u m b l i n g b l o c k i n t h e development o f c u r r e n t und e r s t a n d i n g of c a r d i a c g l y c o s i d e - i n d u c e d i n o t r o p y h a s been t h e l a c k o f b r o a d l y a c c e p t e d o p e r a t i o n a l d e f i n i t i o n s o f " t h e r a p e u t i c " ( n o n t o x i c ) and " t o x i c " d o s e s o r concent r a t i o n s of c a r d i o a c t i v e s t e r o i d s . T h i s problem stems, a t l e a s t i n p a r t , from t h e e x i s t e n c e o f i m p o r t a n t n e u r a l i n f l u e n c e s on t h e emergence o f d i g i t a l i s - t o x i c rhythm d i s t u r b a n c e s i n t h e i n t a c t animal ( G i l l i s and Q u e s t , 1980; Somberg e t a l . , 1978; Somberg and Smith, 1 9 7 9 ) . I n v i t r o p r e p a r a t i o n s , d e v o i d of n e u r a l i n p u t , g e n e r a l l y t o l e r a t e appreciably higher concentrations of cardiac g l y c o s i d e i n t h e b a t h i n g medium w i t h o u t o v e r t rhythm d i s t u r b a n c e s t h a n can be t o l e r a t e d i n t h e plasma of t h e i n t a c t animal. I t i s a l s o i m p o r t a n t t o remember, i n comp a r i n g t h e r e s u l t s of v a r i o u s s t u d i e s , t h a t s u b s t a n t i a l d i f f e r e n c e s e x i s t i n t h e s u s c e p t i b i l i t y of Na,K-ATPase from d i f f e r e n t s p e c i e s t o i n h i b i t i o n by c a r d i a c g l y c o s i d e s . T h r e s h o l d and IC50 v a l u e s f o r o u a b a i n i n h i b i t i o n of monovalent c a t i o n a c t i v e t r a n s p o r t s h o u l d t h e r e f o r e be c o n s i d e r e d i n comparing t h e r a p e u t i c and t o x i c d i g i t a l i s l e v e l s i n various experimental preparations. Finall y , f o r reasons not a l t o g e t h e r clear, g r e a t e r e f f e c t i v e r e c e p t o r o c c u p a n c i e s are t y p i c a l l y a c h i e v e d a t a g i v e n plasma c o n c e n t r a t i o n o f g l y c o s i d e g i v e n i n v i v o t h a n a t comparable c o n c e n t r a t i o n s i n b a t h i n g o r p e r f u s i n g media in vitro.

ION TRANSPORTAND DIGITALIS-INDUCEDINOTROPY

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859

CORRELATION O F CARDIAC GLYCOSIDE-INDUCED INOTROPY W I T H I N H I B I T I O N O F MONOVALENT CATION ACTIVE TRANSPORT I N AN INTACT ANIMAL MODEL

Longstanding c o n t r o v e r s y s u r r o u n d s t h e i s s u e of whether sodium pump i n h i b i t i o n o c c u r s a t p o s i t i v e l y i n o t r o p i c b u t s u b t o x i c cardiac g l y c o s i d e d o s e s , o r whether sodium pump i n h i b i t i o n i s i n s t e a d a phenomenon l e a d i n g t o mechanical and e l e c t r o p h y s i o l o g i c a l t o x i c i t y , b u t n o t t o t h e d e s i r e d t h e r a p e u t i c ( i n o t r o p i c ) r e s p o n s e . ACc o r d i n g l y , w e s t u d i e d an i n t a c t ( a l b e i t a n e s t h e t i z e d ) dog model i n which a s u s t a i n e d i n o t r o p i c e f f e c t w a s a c h i e v e d by p r o l o n g e d i n t r a v e n o u s i n f u s i o n of ouabain a t 0.036 mg/kg/min a f t e r a n i n i t i a l l o a d i n g d o s e o f 30 pg/kg (Hougen and Smith, 1 9 7 8 ) . T h i s produced no o v e r t rhythm d i s t u r b a n c e s , a l l a n i m a l s r e m a i n i n g i n norm a l s i n u s rhythm e v e n a f t e r 5 h r of c o n t i n u o u s i n f u s i o n . S e r i a l l e f t v e n t r i c u l a r f u l l - t h i c k n e s s b i o p s i e s were t a k e n p r i o r t o , and 1 and 2 h r a f t e r , o u a b a i n i n f u s i o n had been begun, w h i l e s i m u l t a n e o u s l y m o n i t o r i n g l e f t v e n t r i c u l a r c o n t r a c t i l e s t a t e by measurement o f t h e maximum r a t e of i n t r a c a v i t a r y p r e s s u r e r i s e a t o n s e t of s y s t o l e (LV m a x i m u m d P / d t ) Ouabain-inhibitable uptake of t h e K+ a n a l o g 86Rb' was d e t e r m i n e d i n v i t r o i n t h e s e b i o p s y samples a s a measure of monovalent c a t i o n a c t i v e t r a n s p o r t , and compared w i t h t r a n s p o r t a c t i v i t y i n biopsies t a k e n p r i o r t o o u a b a i n a d m i n i s t r a t i o n and v a l u e s i n b i o p s i e s from dogs r e c e i v i n g v e h i c l e a l o n e . LV maximum d P / d t i n c r e a s e d above b a s e l i n e by 29 f 3(SEM)%a t 1 h r , accompanied by a 2 1 t 6 % r e d u c t i o n i n a c t i v e t r a n s p o r t o f Rb+. A t 2 h r , LV maximum d P / d t w a s enhanced by 46 t 9 % w h i l e Rb+ t r a n s p o r t w a s s i m u l t a n e o u s l y reduced by 33 f 5 % . C o n t r o l dogs showed changes n e i t h e r i n cont r a c t i l e s t a t e n o r i n monovalent c a t i o n t r a n s p o r t . In a n o t h e r group of dogs g i v e n o u a b a i n a c c o r d i n g t o t h e same p r o t o c o l , s i g n i f i c a n t i n h i b i t i o n by Rb+ t r a n s p o r t w a s e v i d e n t a t t h e e a r l i e s t t i m e ( 3 0 min) a c l e a r - c u t p o s i t i v e i n o t r o p i c e f f e c t was m a n i f e s t . I n c o n t r a s t , norep i n e p h r i n e i n d o s e s s u f f i c i e n t t o produce a comparable i n c r e a s e i n LV maximum d P / d t o v e r a p e r i o d of 30-120 min d i d n o t a l t e r a c t i v e Rb+ t r a n s p o r t . These f i n d i n g s p r o v i d e d i r e c t e v i d e n c e t h a t d o s e s o f o u a b a i n a s s o c i a t e d w i t h a marked p o s i t i v e i n o t r o p i c e f f e c t ( b u t no t o x i c a r r h y t h m i a s ) a r e a s s o c i a t e d w i t h i n h i b i t i o n o f m y o c a r d i a l monovalent c a t i o n a c t i v e t r a n s port. I n r e l a t e d s t u d i e s , we established t h a t nontoxic d i g o x i n d o s e s p r o d u c i n g a mean i n c r e a s e of 2 0 % i n LV maxi m u m d P / d t r e d u c e d Rb+ a c t i v e t r a n s p o r t i n b i o p s y samples by 2 5 % below c o n t r o l , whereas t o x i c doses d e c r e a s e d

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THOMAS W. SMITH AND WILLIAM H. BARRY

t r a n s p o r t 59% below b a s e l i n e v a l u e s a t o n s e t of d i g o x i n t o x i c a r r h y t h m i a s (Hougen et a l . , 1 9 7 9 ) . T r a n s p o r t i n m y o c a r d i a l b i o p s y s a m p l e s was r e d u c e d 8 0 % below c o n t r o l a t o n s e t o f f a t a l v e n t r i c u l a r f i b r i l l a t i o n f o l l o w i n g adm i n i s t r a t i o n of 0 . 3 mg/kg of d i g o x i n . I n t e r e s t i n g l y , d i g o x i n - s p e c i f i c 1gG or Fab f r a g m e n t s of h i g h a f f i n i t y f o r the glycoside reversed established digoxin-toxic a r r h y t h m i a s w i t h s i n u s rhythm r e t u r n i n g a t a t i m e when monovalent c a t i o n a c t i v e t r a n s p o r t w a s r e s t o r e d t o 51% of c o n t r o l , comparable t o t h e l e v e l of t r a n s p o r t i n h i b i t i o n p r e s e n t a t o n s e t of o v e r t rhythm d i s t u r b a n c e s (Hougen e t a l . , 1 9 7 9 ) . S i n c e it i s g e n e r a l l y h e l d t h a t v e n t r i c u l a r a r r h y t h mias c h a r a c t e r i s t i c o f advanced d i g i t a l i s t o x i c i t y a'rise i n t h e P u r k i n j e network, we a l s o a s s e s s e d t h e r e l a t i v e s e n s i t i v i t i e s of c a n i n e P u r k i n j e f i b e r s and myocardium t o i n h i b i t i o n o f monovalent c a t i o n t r a n s p o r t by d i g o x i n a t a n a c u t e t o x i c end p o i n t and a f t e r c h r o n i c , s t e a d y s t a t e , n o n t o x i c d o s e s p r e v i o u s l y shown t o p r o d u c e a s i g n i f i c a n t p o s i t i v e i n o t r o p i c e f f e c t (Somber9 e t a l . , 1 9 8 1 ) . The r e s u l t s o f t h e s e e x p e r i m e n t s a r e summarized i n F i g . 1. Comparing c o n t r o l t r a n s p o r t v a l u e s ( s t i p p l e d b a r s ) t o t h o s e o b s e r v e d a t o n s e t of o v e r t t o x i c i t y ( h o r i z o n t a l l y h a t c h e d b a r s ) i n m y o c a r d i a l t i s s u e , a mean i n h i b i t i o n of t r a n s p o r t of 4 4 ? 1 0 % was found compared t o a 76 f 3 % i n h i b i t i o n of t r a n s p o r t i n P u r k i n j e f i b e r s a t t h e same end p o i n t of v e n t r i c u l a r t a c h y c a r d i a . Greater s e n s i t i v i t y of P u r k i n j e f i b e r s t h a n of myocardium was a l s o found d u r i n g c h r o n i c a d m i n i s t r a t i o n of n o n t o x i c d i g o x i n d o s e s s u f f i c i e n t t o m a i n t a i n s t e a d y - s t a t e serum d i g o x i n c o n c e n t r a t i o n s a v e r a g i n g 2 . 1 ng/ml ( F i g . 1, c r o s s - h a t c h e d bars). Taken t o g e t h e r , t h e s e s t u d i e s e s t a b l i s h t h a t a n e c e s s a r y c o n d i t i o n i s m e t i n s u p p o r t of t h e h y p o t h e s i s t h a t i n h i b i t i o n o f t h e sodium pump i s c a u s a l l y r e l a t e d t o t h e p o s i t i v e i n o t r o p i c e f f e c t of c a r d i a c g l y c o s i d e s a t subtoxic doses o r c o n c e n t r a t i o n s i n t h e i n t a c t animal. These f i n d i n g s were n o t s u r p r i s i n g i n view o f t h e e x t r a o r d i n a r i l y c l o s e c o r r e l a t i o n found by F l a s c h and Heinz between t h e a b i l i t y of 1 7 c a r d e n o l i d e s , c a r d e n o l i d e g l u c u r o n i d e s , and s u l f a t e s t o i n h i b i t N a , K - A T P a s e o r monov a l e n t c a t i o n a c t i v e t r a n s p o r t and t h e i r a b i l i t y t o i n c r e a s e t h e c o n t r a c t i l i t y of g u i n e a - p i g i s o l a t e d p a p i l l a r y m u s c l e s ( F l a s c h and Heinz, 1 9 7 8 ) . F u r t h e r e v i d e n c e cons i s t e n t w i t h t h i s view i s d i s c u s s e d a t l e n g t h by Schwartz e t a l . ( 1 9 7 5 ) and by A k e r a and Brody ( 1 9 7 8 ) .

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+

F i g . 1 . Comparison of Rb a c t i v e t r a n s p o r t i n P u r k i n j e f i bers and myocardium o f d o g s not e x p o s e d t o d i g o x i n (n = 8 ; s t i p p l e d b a r s ) , c h r o n i c a l l y d o s e d a n i m a l s r e c e i v i n g 0.02 mg/kg d i g o x i n d a i l y (n = 8 ; d i a g o n a l l y h a t c h e d b a r s ) , and a n i m a l s a t onset o f v e n t r i c u l a r t a c h y c a r d i a a f t e r a c u t e a d m i n i s t r a t i o n of 0 . 3 m g / k g d i g o x i n ( n = 8 ; h o r i z o n t a l l y h a t c h e d b a r s ) . V a l u e s shown a r e p e r c e n t c h a n g e s compared t o a c t i v e t r a n s p o r t i n a n i m a l s not r e c e i v i n g d i g o x i n . T h e p e r c e n t c h a n g e i s s i g n i f i c a n t l y ( p < 0.01) g r e a t e r for P u r k i n j e f i b e r s t h a n f o r myocardium u n d e r both a c u t e and chronic d o s a g e r e g i m e n s . R e p r o d u c e d , w i t h p e r m i s s i o n , from J . C1 i n . Invest. (Somberg e t a l . , 1 9 8 1 ) .

111.

SODIUM PUMP I N H I B I T I O N AND THE POSITIVE I N O T R O P I C RESPONSE

A p l a u s i b l e mechanism l i n k i n g sodium pump i n h i b i t i o n t o a p o s i t i v e i n o t r o p i c e f f e c t on h e a r t m u s c l e w a s p u t f o r w a r d by Baker and c o l l e a g u e s i n 1969, b a s e d on s t u d i e s o f a sodium/calcium exchange c a r r i e r found i n s q u i d axon and a l s o i n mammalian c a r d i a c muscle by R e u t e r and c o l l e a g u e s ( G l i t s c h e t a ~ . , 1 9 7 0 ) . The s e q u e n c e s u g g e s t e d t h e n and s i n c e by a number o f w o r k e r s i n t h a t c a r d i a c g l y c o s i d e b i n d i n g t o t h e i n h i b i t o r y s i t e on sarcolemmal Na,K-ATPase i n h i b i t s t h e outward t r a n s p o r t of Na+* l e a d i n g t o i n c r e a s e d “a+] i ; t h r o u g h t h e Na-Ca exchange mechanism t h i s l e a d s t o enhanced C a 2 + i n f l u x a n d / o r

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THOMAS W. SMITH AND WILLIAM H. BARRY

reduced Ca2+ e f f l u x . The consequent i n c r e a s e i n [Cali, i n t u r n , i n c r e a s e s t h e amount o f C a 2 + a v a i l a b l e t o t h e c o n t r a c t i l e e l e m e n t s and t h u s t o a p o s i t i v e i n o t r o p i c e f f e c t . T h i s sequence o f e v e n t s h a s been d i f f i c u l t t o p r o v e , however, and i s by no means t h e o n l y mechanism u n d e r r e c e n t c o n s i d e r a t i o n . Lullmann and c o l l e a g u e s , f o r example, i n t e r p r e t t h e i r e x p e r i m e n t a l r e s u l t s ( B e n t f e l d e t a l . , 1977) as s u g g e s t i n g a g l y c o s i d e - i n d u c e d l a b i l i z a t i o n o f C a 2 + b i n d i n g by sarcolemmal s i t e s t h a t i s dependent on g l y c o s i d e b i n d i n g t o N a , K - A T P a s e , b u t t h a t does n o t r e q u i r e measurable d i s t u r b a n c e of normal N a + or K+ g r a d i e n t s a t t h e r a p e u t i c d o s e s s i n c e under p h y s i o l o g i c a l c o n d i t i o n s t h e r e i s e x c e s s Na+ pump c a p a c i t y (Lullmann and P e t e r s , 1 9 7 9 ) . A s i m i l a r mechanism w a s s u g g e s t e d by G e r v a i s e t a l . ( 1 9 7 7 ) . Weingart e t a l . (1978) f i r s t s u g g e s t e d t h a t , c o n t r a r y t o e a r l i e r f i n d i n g s , enhancement of slow inward c a l cium c u r r e n t ( I s i ) d u r i n g t h e a c t i o n p o t e n t i a l o c c u r s i n cardiac Purkinje f i b e r s a t p o s i t i v e l y inotropic strophant h i d i n c o n c e n t r a t i o n s p r i o r t o t h e development o f e v i d e n t t o x i c i t y . These f i n d i n g s have been e x t e n d e d by Marban and T s i e n (1979, 1 9 8 2 ) , who s t u d i e d ouabain-induced enhancement of Isi i n c a l f P u r k i n j e f i b e r s and i n f e r r e t p a p i l l a r y muscles u s i n g voltage-clamp t e c h n i q u e s . L i k e o u a b a i n , t h e a l k a l o i d n e u r o t o x i n v e r a t r i d i n e , which i s known t o i n c r e a s e [ N a ] i , a l s o i n c r e a s e d b o t h f o r c e and I s i . Marban and T s i e n s u g g e s t t h a t a r i s e i n [ N a ] i c a n p r o bia b l,y t h r o u g h a s e c o n d a r y r i s e lead t o increased ~ ~ i n [ C a l i mediated by t h e N a / C a exchange mechanism n o t e d above. Thus, t h e enhancement of Isi o b s e r v e d may be dependent on a c r i t i c a l l e v e l of sodium pump i n h i b i t i o n acc o r d i n g t o t h e i r f o r m u l a t i o n . They warn, however, t h a t d i g i t a l i s enhancement of I s i may a l s o o c c u r a t t h r e s h o l d i n o t r o p i c d o s e s i n t h e a b s e n c e of a measurable rise i n i n t r a c e l l u l a r Na , and d i s c u s s t h e a l t e r n a t i v e p o s s i b i l i t y t h a t d i a s t o l i c [ C a l i may r i s e due t o some a c t i o n of d i g i t a l i s o t h e r t h a n i n h i b i t i o n of t h e sodium pump. Lederer and E i s n e r have o b s e r v e d an i n c r e a s e i n ~~iaccompanying t h e i n c r e a s e i n t w i t c h f o r c e produced by s u b t o x i c s t r o phanthidin c o n c e n t r a t i o n s i n sheep c a r d i a c Purkinje f i b e r s ( L e d e r e r and E i s n e r , 1 9 8 2 ) , and c o n c l u d e t h a t t h e e f f e c t o n Isi is p r o b a b l y m e d i a t e d by N a pump i n h i b i t i o n .

IV.

STUDIES ON CULTURED HEART CELLS

Our own s t u d i e s i n i n t a c t animal models, w h i l e necessary t o permit a c l i n i c a l l y relevant operational defin i t i o n o f t o x i c i t y , a r e l i m i t e d i n terms of d e f i n i n g u n i -

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d i r e c t i o n a l and n e t f l u x e s o f Na', ,'K and Ca2+ b e c a u s e of t h e series d i f f u s i o n b a r r i e r s and i n t e r s t i t i a l s p a c e u n c e r t a i n t i e s t h a t b e s e t e x p e r i m e n t s on i n t a c t t i s s u e . I n order t o test the hypothesis t h a t p o s i t i v e i n o t r o p i c e f f e c t s o f d i g i t a l i s g l y c o s i d e s are m e d i a t e d by N a + pump i n h i b i t i o n v i a i n c r e a s e d [Na] i and c o n s e q u e n t l y i n c r e a s e d [ C a l i t h r o u g h t h e N a / C a exchange c a r r i e r mechani s m , w e sought a cardiac preparation with t h e following (1) a s u b s t a n t i a l p o s i t i v e i n o t r o p i c reproperties: sponse t o c a r d i a c g l y c o s i d e s , and a means t o m o n i t o r and r e c o r d t h a t r e s p o n s e ; (2) r a p i d o n s e t o f a s t a b l e i n o t r o p i c response t o glycoside exposure, with r a p i d o f f s e t f o l l o w i n g washout: ( 3 ) t h e means t o assess, i n a s d i r e c t a way a s p o s s i b l e , t h e e f f e c t s o f c a r d e n o l i d e s o n u n i d i r e c t i o n a l and n e t f l u x e s of N a + , K+, and Ca2+ w i t h adeq u a t e t e m p o r a l r e s o l u t i o n under c o n d i t i o n s i d e n t i c a l t o t h o s e u n d e r which i n o t r o p i c s t a t e i s d e t e r m i n e d , and (4) t h e means of r e s o l v i n g components o f C a f l u x e s i n b e a t i n g p r e p a r a t i o n s under b a s a l and g l y c o s i d e - s t i m u l a t e d conditions. These c o n d i t i o n s are m e t by s p o n t a n e o u s l y b e a t i n g monolayers o f c u l t u r e d c h i c k embryo v e n t r i c u l a r c e l l s , which i n a d d i t i o n have t h e s i m p l i f y i n g a d v a n t a g e o f t h e absence o f o t h e r f a c t o r s t h a t c o u l d modulate i o n t r a n s p o r t and/or c o n t r a c t i l e s t a t e , s u c h as endogenous catecholamine c o n t e n t . The system u s e d t o q u a n t i f y c o n t r a c t i l e a c t i v i t y i n t h i s p r e p a r a t i o n h a s been d e s c r i b e d i n d e t a i l p r e v i o u s l y ( B i e d e r t e t a l . , 1 9 7 9 1 , and c o n s i s t s of an o p t i c a l - v i d e o system t h a t m o n i t o r s and r e c o r d s c o n t r a c t i l e s t a t e of i n d i v i d u a l c e l l s o v e r p e r i o d s o f h o u r s w i t h o u t i n any way d i s t u r b i n g t h e i r p h y s i o l o g i c s t a t e . Ten-day-old c h i c k embryo h e a r t s are removed und e r s t e r i l e c o n d i t i o n s and t h e v e n t r i c l e s c u t i n t o f r a g ments and g e n t l y a g i t a t e d i n C a - and Mg-free Hanks' s o l u t i o n c o n t a i n i n g t r y p s i n . D i s s o c i a t e d c e l l s a r e p l a t e d on c i r c u l a r g l a s s c o v e r s l i p s and i n c u b a t e d for 2-3 d a y s i n a h u m i d i f i e d 5 % C02-95% a i r atmosphere a t 37OC, a t which t i m e c o n f l u e n t monolayers have formed i n which a t l e a s t 80% o f t h e cells c o n t r a c t synchronously a t r a t e s of about 1 2 0 p e r minute. For c o n t r a c t i l i t y measurements, a g l a s s c o v e r s l i p w i t h a t t a c h e d monolayer of h e a r t c e l l s i s p l a c e d i n a Sykes-Moore chamber w i t h i n l e t and o u t l e t p o r t s f o r p e r f u s i o n . The chamber i s p l a c e d on t h e s t a g e of an i n v e r t e d phase c o n t r a s t microscope e n c l o s e d i n a L u c i t e box w i t h c o n t r o l l e d atmosphere and t e m p e r a t u r e . Flow c h a r a c t e r i s t i c s o f t h e chamber a l l o w medium t o exchange w i t h a t i m e c o n s t a n t of a b o u t 15 sec a t a f l o w r a t e of 0 . 9 6 ml/min. T h e microscope image i s m o n i t o r e d by a l o w l i g h t l e v e l TV camera, t h e o u t p u t from which i s c o n n e c t e d t o a

THOMAS W. SMITH AND WILLIAM H. BARRY

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F i g . 2. E f f e c t o f o u a b a i n on c o n t r a c t i l i t y o f c u l t u r e d h e a r t cells. (A) R e c o r d i n g o f a m p l i t u d e o f c o n t r a c t i o n i n micrometers (upper trace) P r e - o u a b a i n control a m p l i t u d e and v e l o c i t y ( l e f t ) w e r e o b t a i n e d d u r i n g p e r f u s i o n o f t h e c u l t u r e w i t h medium c o n t a i n i n g 0.6 mM C a a t 37OC. The m i d d l e p a n e l shows the c h a n g e i n a m p l i t u d e and v e l o c i t y o f cell w a l l motion 7 m i n a f t e r i n i t i a t i o n o f p e r f u s i o n w i t h medium c o n t a i n i n g 0.6 mM Ca and 5 X M ouabain. The post-ouabain r e c o r d i n g s ( r i g h t ) were o b t a i n e d a f t e r 7 m i n o f p e r f u s i o n of the c u l t u r e w i t h o u a b a i n - f r e e medium. T h e u p p e r v e r t i c a l b a r i n d i c a t e s 1 pm of m o t i o n a m p l i t u d e and the l o w e r v e r t i c a l b a r 24 p m / s e c o f m o t i o n v e l o c i t y . ( B ) Mean ( & SEM) v a l u e s M o b t a i n e d i n 1 2 c u l t u r e s d u r i n g e x p o s u r e t o and w a s h o u t o f ouabain. A m p l i t u d e o f cell w a l l motion i n micrometers i s p l o t t e d a g a i n s t t i m e i n m i n u t e s . A f t e r 10 m i n o f e q u i l i b r a t i o n , c u l t u r e s w e r e p e r f u s e d w i t h 10-6 M o u a b a i n f o r 7 m i n . T h e c o n t r a c t i o n amp l i t u d e b e g a n t o i n c r e a s e a f t e r 2 min o f e x p o s u r e t o o u a b a i n and

.

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F i g . 3 . C o n c e n t r a t i o n - e f f e c t c u r v e showing r e s p o n s e s t o ouab a i n of a m p l i t u d e of c e l l w a l l m o t i o n ( l e f t s c a l e ) a n d i n h i b i t i o n of Rb+ a c t i v e t r a n s p o r t ( r i g h t s c a l e ) . Po i n t s a r e m e a n ? SEM of 4 - 1 4 experiments. The i n c r e a s e i n c o n t r a c t i o n amplitude i n p e r c e n t shows the m a x i m a l e f f e c t a t e a c h o u a b a i n c o n c e n t r a t i o n . T h r e s h o l d s f o r s i g n i f i c a n t i n h i b i t i o n of Rb+ a c t i v e t r a n s p o r t a n d i n c r e a s e o f cont r a c t i l i t y w e r e b e t w e e n 10-7 a n d 2 . 5 x 1 0 - 7 M o u a b a i n . T h e s i m i l a r d e p e n d e n c e of p e r c e n t increase i n a m p l i t u d e of w a l l notion a n d p e r cent i n h i b i t i o n o f Rb+ t r a n s p o r t on o u a b a i n c o n c e n t r a t i o n i s e v i d e n t . R e p r o d u c e d , w i t h p e r m i s s i o n , f r o m J . Gen. P h y s i o l . ( B i e d e r t e t a l . , 1979)

.

( F i g . 2 cont'd) r e a c h e d a p l a t e a u (47%mean i n c r e a s e ) w i t h i n 6 m i n . D u r i n g w a s h u t o f o u a b a i n , the e f f e c t w a s r a p i d l y reversed a n d control level w a s r e s t o r e d a f t e r 6 m i n . R e p r o d u c e d , w i t h p e r m i s s i o n , 1979). f r o m J . Gen. P h y s i o l . ( B i e d e r t e t a1

.,

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THOMAS W. SMITH AND WILLIAM H. BARRY

v i d e o motion d e t e c t o r t h a t a n a l y z e s c e l l motion a l o n g a s e l e c t e d raster l i n e segment and p l o t s p o s i t i o n d a t a e v e r y 1 6 msec. The a n a l o g v o l t a g e o u t p u t from t h e mot i o n d e t e c t o r i s c a l i b r a t e d t o i n d i c a t e a c t u a l microns of motion; t h e f i r s t d e r i v a t i v e w i t h r e s p e c t t o t i m e i s determined by a n RC d i f f e r e n t i a t o r , and b o t h s i g n a l s are r e c o r d e d , as shown i n F i g . 2 . P o s i t i v e i n o t r o p i c e f f e c t s of o u a b a i n , r e f l e c t e d i n i n c r e a s e s i n t h e a m p l i t u d e and v e l o c i t y of c e l l w a l l mot i o n , were m a n i f e s t by 1.5-2 min, and r e a c h e d a s t a b l e p l a t e a u w i t h i n 5-6 min ( B i e d e r t e t a l . , 1 9 7 9 ) . The i n o t r o p i c e f f e c t w a s r e v e r s e d w i t h i n a b o u t 5 min o f washout o f o u a b a i n ( F i g . 2 ) . C o i n c i d e n t w i t h t h e o n s e t of p o s i t i v e i n o t r o p y w a s s i g n i f i c a n t i n h i b i t i o n of 42K+ u p t a k e ( o r 86Rb+ u p t a k e ) , accompanied by i n h i b i t i o n of 24Na+ eff l u x . A s summarized i n F i g . 3 , t h e d e g r e e o f i n h i b i t i o n o f t r a n s p o r t w a s c l o s e l y r e l a t e d t o t h e magnitude of the i n o t r o p i c e f f e c t , w i t h t h e t h r e s h o l d f o r b o t h phenomena j u s t above 1 0 - 7 M and maintenance o f p a r a l l e l i s m from 0 ' 6 M ouabain. 2.5 x 10-7 M t h r o u g h 1 . 5 x 1 Taking advantage of t h e l a c k o f d i f f u s i o n b a r r i e r s and i n t e r s t i t i a l s p a c e u n c e r t a i n t i e s i n t h e c u l t u r e d mon o l a y e r p r e p a r a t i o n , w e have been a b l e t o r e s o l v e t h e time c o u r s e o f C a exchange i n t o t w o components a s shown i n F i g . 4 . A r a p i d p h a s e of exchange w i t h a r a t e cons t a n t o f 3.91 min-1 a c c o u n t e d f o r a b o u t 37% of t h e t o t a l C a c o n t e n t and w a s e v i d e n t between 1 sec and 1 min. A s l o w e r e x p o n e n t i a l phase w i t h a r a t e c o n s t a n t of

F i g . 4 . 45Ca u p t a k e b y m o n o l a y e r s o f c u l t u r e d h e a r t c e l l s . (A) For e a c h d a t a p o i n t , c o v e r s l i p s were immersed i n c u l t u r e medium for the t i m e i n d i c a t e d on the h o r i z o n t a l a x i s . A f t e r w a s h i n g t o remove e x t r a c e l l u l a r f l u i d , Ca content was c a l c u l a t e d f r o m the 45Ca i n the u p t a k e medium. The d a t a w e r e n o r m a l i z e d r e l a t i v e t o my p r o t e i n p r e s e n t on e a c h c o v e r s l i p . T h e c a l c u l a t e d Ca content (nmoles/mg p r o t e i n , v e r t i c a l a x i s ) d o e s not e q u a l the a c t u a l t o t a l e x c h a n g e a b l e Ca content u n t i l l a b e l i n g h a s been a c h i e v e d . Each p o i n t p l o t t e d i s the mean 2 SEN for 10 c o v e r s l i p s . U p t a k e b y c o v e r s l i p s w i t h o u t a t t a c h e d c e l l s i s shown b y the d o t t e d l i n e ; i n t h i s p l o t , the mean p r o t e i n content from the c o v e r s l i p s w i t h a t t a c h e d c e l l s was u s e d t o e s t i m a t e nmoles Ca/mg p r o t e i n c o n t r i b u t e d b y the g l a s s a l o n e , ( B ) A p l o t on a l o g v e r t i c a l s c a l e o f c e l l u l a r u p t a k e f r o m t h e d a t a a b o v e for the f i r s t 30 min, e x p r e s s e d a s C a l z o - C a t , w h e r e Ca120 i s the content o f Ca a f t e r l a b e l i n g t o e q u i l i b r i u m (120 m i n ) . W i g h t e d nonlinear r e g r e s s i o n a n a l y s i s showed t h a t the d a t a c o u l d be best f i t b y t w o e x p o n e n t i a l c u r v e s ( p < 0.001). R e p r o d u c e d , w i t h p e r m i s s i o n , from J . P h y s i o l . (London) ( B a r r y and S m i t h , 1 9 8 2 ) .

ION TRANSPORTAND DIGITALIS-INDUCEDINOTROPY

Fig. 4A

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THOMAS W. SMITH AND WILLIAM H. BARRY

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F i g . 5 . E f f e c t s of o u a b a i n on Na a n d Ca content. ( A ) D e p e n d e n c e o f c e l l u l a r s o d i u m c o n t e n t on o u a b a i n c o n c e n t r a t i o n ( l o g s c a l e ) . M o n o l a y e r s w e r e e x p o s e d t o 24Na' for 30 m i n . A t t h i s t i m e e q u i l i b r i u m w i t h e x c h a n g e a b l e i n t r a c e l l u l a r Na+ w a s r e a c h e d . P o i n t s a r e m e a n s 2 SEM o f 25-38 e x p e r i m e n t s . A t 10-7 M o u a b a i n , t h e r e was no c h a n g e i n s o d i u m c o n t e n t , w h e r e a s t h e r e w a s a s i g n i f i c a n t i n crease a t 5 X M ( p < 0 . 0 1 ) a n d a t lod6 M o u a b a i n ( p < 0 . 0 1 ) . ( B ) D e p e n d e n c e of c e l l u l a r c a l c i u m content on o u a b a i n c o n c e n t r a t i o n . M o n o l a y e r s w e r e e x p o s e d t o 45Ca2+ for 10 m i n . P o i n t s a r e m e a n s ? M o u a b a i n , t h e c h a n g e i n Ca c o n SEM of 28-35 e x p e r i m e n t s . A t t e n t w a s n o t s t a t i s t i c a l l y s i g n i f i c a n t , b u t a n i n c r e a s e c o u l d be demonstrated a t 5 x ( p < 0 . 0 1 ) and a t M ouabain ( p < 0.001). Reproduced, w i t h p e r m i s s i o n , f r o m J . G e n . P h y s i o l . (Biedert et a l . , 1979).

-1

0 . 0 6 9 min , c o m p l e t e b y 1 2 0 m i n , a c c o u n t e d for t h e rem a i n i n g e x c h a n g e a b l e C a c o n t e n t . T h e e f f e c t s of t h e s l o w c h a n n e l b l o c k i n g a g e n t v e r a p a m i l and t h e t r i v a l e n t c a t i o n

ION TRANSPORTAND DIGITALIS-INDUCEDINOTROPY

869

lanthanum o n t h e s e C a f l u x e s have been examined and conf i r m t h a t t h e r a p i d l y e x c h a n g e a b l e Ca p o o l i s i n t i m a t e l y r e l a t e d t o c o n t r a c t i l e s t a t e ( B a r r y and S m i t h , 1 9 8 2 ) . Changes i n t h e t r a n s s a r c o l e m m a l N a g r a d i e n t had marked e f f e c t s on t h i s r a p i d component o f C a u p t a k e , c o n s i s t e n t w i t h t h e p r e s e n c e of an a c t i v e N a - C a exchange c a r r i e r mechanism i n t h e s e c e l l s ( B a r r y and S m i t h , 1 9 8 2 ) . R e t u r n i n g t o r e s p o n s e s o f c u l t u r e d c h i c k embryo vent r i c u l a r c e l l s t o o u a b a i n , F i g . 5 summarizes t h e c h a n g e s i n i n t r a c e l l u l a r N a + c o n t e n t and i n t h e c o n t e n t of t h e r a p i d l y e x c h a n g e a b l e Ca2+ p o o l a f t e r e x p o s u r e t o c o n t r o l c o n d i t i o n s o r t o graded c o n c e n t r a t i o n s of ouabain. J u s t below t h e t h r e s h o l d f o r a n i n o t r o p i c r e s p o n s e ( 1 0 - 7 M ) , no change i n N a o r r a p i d l y e x c h a n g e a b l e C a c o n t e n t w a s present. A t c o n c e n t r a t i o n s of 5 x 1 0 - 7 M and 1 0 - 6 M , corresponding t o s u s t a i n e d p o s i t i v e i n o t r o p i c e f f e c t s , i n c r e m e n t s i n c e l l u l a r Na c o n t e n t were o b s e r v e d and were accompanied by a n i n c r e a s e i n t h e s i z e o f t h e r a p i d l y exchangeable Ca pool ( B i e d e r t e t a l . , 1 9 7 9 ) . These f i n d i n g s s u p p o r t t h e h y p o t h e s i s t h a t a c a u s a l r e l a t i o n s h i p e x i s t s between sodium pump i n h i b i t i o n and t h e p o s i t i v e i n o t r o p i c e f f e c t of c a r d i a c g l y c o s i d e s , b u t l e a v e open t h e q u e s t i o n o f w h e t h e r i n s t a n t a n e o u s N a + pump r a t e , c e l l u l a r N a + c o n t e n t , o r b o t h a r e i m p o r t a n t i n modulating C a 2 + c o n t e n t i n a r a p i d l y exchangeable pool c l o s e l y l i n k e d t o c o n t r a c t i l e s t a t e . F u r t h e r m o r e , work from o t h e r l a b o r a t o r i e s , summarized by O k i t a ( 1 9 7 7 1 , h a s been i n t e r p r e t e d a s s u g g e s t i n g a t e m p o r a l d i s s o c i a t i o n o f d i g i t a l i s - i n d u c e d i n o t r o p y from i n h i b i t i o n of N a , K A T P a s e d u r i n g r e v e r s a l of t h e i n o t r o p i c e f f e c t w i t h g l y c o s i d e washout. To e x p l o r e t h e s e i s s u e s f u r t h e r , w e s t u d i e d t h e t i m e r e l a t i o n s of r e t u r n t o b a s e l i n e i n o t r o p i c s t a t e , r e c o v e r y o f monovalent c a t i o n a c t i v e t r a n s p o r t r a t e , and r e t u r n toward normal o f c e l l u l a r c o n t e n t s o f N a + , K+, and r a p i d l y e x c h a n g e a b l e Ca2+ a f t e r removal of o u a b a i n from t h e b a t h i n g medium ( B a r r y e t a l . , 1 9 8 1 ) . Monovalent c a t i o n t r a n s p o r t , judged by t h e r a t e o f 4 2 K u p t a k e , r e t u r n e d from 55% of c o n t r o l d u r i n g e x p o s u r e t o 1 0 - 6 M o u a b a i n t o a v a l u e s l i g h t l y b u t s i g n i f i c a n t l y above c o n t r o l by o n e min a f t e r removal o f o u a b a i n . As summarized i n F i g . 6 , by 7 min a f t e r removal of o u a b a i n from b a t h i n g media, a t a t i m e when c o n t r a c t i l e s t a t e had r e t u r n e d t o b a s e l i n e , i n s t a n t a n e o u s pump r a t e and a l s o t h e s i z e of t h e r a p i d l y e x c h a n g e a b l e C a p o o l had r e t u r n e d t o c o n t r o l . I n cont r a s t , e l e v a t e d l e v e l s o f [ N a ] i and r e d u c e d [ K ] i l e v e l s had n o t r e t u r n e d t o b a s e l i n e e v e n a f t e r a p e r i o d of 6 0 m i n had e l a p s e d f o l l o w i n g removal o f o u a b a i n . Thus, washout of o u a b a i n w a s accompanied by a r a p i d r e t u r n of monovalent c a t i o n t r a n s p o r t r a t e , r a p i d l y e x c h a n g e a b l e

870

THOMAS W. SMITH AND WILLIAM H. BARRY

B

C

MINUTES F i g . 6 . S e q u e n t i a l c h a n g e s i n mean K+ u p t a k e r a t e (A) mean a m p l i t u d e o f c o n t r a c t i o n ( B ) , and r a p i d l y e x c h a n g e a b l e Ca content ( C ) before o u a b a i n e x p o s u r e , 7 min a f t e r e x p o s u r e t o M ouabain, and 7 min a f t e r r e m o v a l of o u a b a i n f r o m m e d i a b a t h i n g the c u l t u r e d h e a r t cells. Data a r e f r o m B a r r y e t a l . ( 1 9 8 1 ) .

c e l l u l a r C a c o n t e n t , and c o n t r a c t i l e s t a t e t o b a s e l i n e l e v e l s w h i l e [ N a ] i and [ K ] i r e t u r n e d c o n s i d e r a b l y more s l o w l y toward normal. The i n o t r o p i c s t a t e i n t h i s exp e r i m e n t a l p r e p a r a t i o n , t h e r e f o r e , seems t o c o r r e l a t e more c l o s e l y w i t h i n s t a n t a n e o u s sodium pump r a t e (and w i t h r a p i d l y e x c h a n g e a b l e Ca c o n t e n t ) t h a n w i t h b u l k i n t r a c e l l u l a r N a c o n t e n t . Analogous f i n d i n g s , u s i n g i o n - s e n s i t i v e i n t r a c e l l u l a r m i c r o e l e c t r o d e s , have b e e n n o t e d by E l l i s (1977) and by Cliura and Rosen ( 1 9 7 8 ) . These f i n d i n g s are n o t e a s y t o i n t e r p r e t , b u t r a i s e t h e q u e s t i o n of a subsarcolemmal s p a c e i n which t h e e f f e c t i v e N a a c t i v i t y available t o influence Ca content t h r o u g h N a / C a exchange depends on t h e i n t e r a c t i o n s o f t r a n s s a r c o l e m m a l N a t r a n s p o r t r a t e , t h e r a t e of N a i n f l u x from t h e e x t r a c e l l u l a r s p a c e , and t h e r a t e of exchange w i t h o t h e r i n t r a c e l l u a r N a p o o l s .

ION TRANSPORT AND DIGITALIS-INDUCED INOTROPY

V.

871

MECHANISMS OTHER THAN SODIUM PUMP I N H I B I T I O N I N THE MEDIATION OF CARDIAC GLYCOSIDE EFFECTS: THE ROLE O F CATECHOLAMINES

These f i n d i n g s , t o g e t h e r w i t h s t u d i e s of L e e e t a i . (19801, E i s n e r and L e d e r e r (19791, and Marban and T s i e n (1982) u s i n g e l e c t r o p h y s i o l o g i c t e c h n i q u e s , s u p p o r t t h e v i e w t h a t sodium pump i n h i b i t i o n i s i n t i m a t e l y r e l a t e d t o t h e p o s i t i v e i n o t r o p i c e f f e c t s of c a r d i a c g l y c o s i d e s . R e s e r v a t i o n s have been e x p r e s s e d , however, by a number of w o r k e r s i n c l u d i n g several c i t e d above ( O k i t a , 1 9 7 7 ; Lullmann and Peters, 1 9 7 9 ) . The e v i d e n c e b e a r i n g on w h e t h e r sodium pump i n h i b i t i o n c a u s e s t h e t h e r a p e u t i c , i n o t r o p i c e f f e c t s o f d i g i t a l i s ( a s opposed t o t h e t o x i c e f f e c t s o f a r r h y t h m i a s and c o n t r a c t u r e ) h a s been c r i t i c a l l y r e v i e w e d by Noble, who c o n c l u d e s t h a t mechanisms o t h e r t h a n sodium pump i n h i b i t i o n may w e l l b e i n v o l v e d i n t h e i n o t r o p i c e f f e c t s o f c a r d i a c g l y c o s i d e s a t low d o s e s o r c o n c e n t r a t i o n s , and t h a t " e x p e r i m e n t a l r e s u l t s now a v a i l a b l e s h o u l d warn u s t h a t w e a r e d e a l i n g w i t h a m u l t i f a c t o r s i t u a t i o n t h a t may w e l l n o t e a s i l y succumb t o a s i n g l e u n i f y i n g h y p o t h e s i s " (Noble, 1 9 8 0 ) . The b a s i s f o r t h i s c a r e f u l l y c o n s i d e r e d c o n c l u d i n g s t a t e m e n t rests i n l a r g e p a r t o n e v i d e n c e from Liillmann and P e t e r s ( 1 9 7 9 ) and from G o d f r a i n d and c o l l e a g u e s (Ghysel-Burton and G o d f r a i n d , 1979; G o d f r a i n d and GhyselB u r t o n , 1 9 8 0 ) , a s w e l l as d a t a from N o b l e ' s own l a b o r a t o r y (Cohen e t a l . , 1976) , i n d i c a t i n g t h a t p o s i t i v e i n o t r o p i c e f f e c t s of c a r d i a c g l y c o s i d e s can be e l i c i t e d i n i n v i t r o t i s s u e p r e p a r a t i o n s a t low c a r d e n o l i d e concent r a t i o n s where no i n h i b i t i o n of sodium pump a c t i v i t y i s evident. I n d e e d , s t u d i e s of a t l e a s t two t i s s u e p r e p a r a t i o n s have shown a p p a r e n t s t i m u l a t i o n of monovalent c a t i o n a c t i v e t r a n s p o r t by low g l y c o s i d e c o n c e n t r a t i o n s i n t h e nanomolar r a n g e (Ghysel-Burton and G o d f r a i n d , l 9 7 9 ; Cohen e t a ] . , 1 9 7 6 ; Blood and Noble, 19781, w i t h i n h i b i t i o n only a t higher concentrations. S e v e r a l l i n e s o f e v i d e n c e l e d u s t o t e s t t h e hyp o t h e s i s t h a t t h e pump s t i m u l a t o r y component of t h i s b i p h a s i c r e s p o n s e i s m e d i a t e d by endogenous c a t e c h o l a m i n e s (Hougen e t a l . , 1 9 8 1 ) . F i r s t , i t h a s been shown t h a t 8 - a d r e n e r g i c a g o n i s t s can s t i m u l a t e monovalent c a t i o n a c t i v e t r a n s p o r t i n i n t a c t t i s s u e s (Rogus e t a l . , 1 9 7 7 ) . Second, mammalian m y o c a r d i a l t i s s u e c o n t a i n s a p p r e c i a b l e n o r e p i n e p h r i n e stores i n a d r e n e r g i c n e r v e t e r m i n a l s , and these terminals appear t o contain a s u b s t a n t i a l portion of t h e N a , K - A T P a s e c o n t e n t o f whole m y o c a r d i a l homog e n a t e s (Somberg e t a l . , 1 9 8 1 ) . F i n a l l y , o u a b a i n p r o motes endogenous n o r e p i n e p h r i n e release i n p e r f u s e d

THOMAS W. SMITH AND WILLIAM H. BARRY

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F i g . 7 . E f f e c t o f o u a b a i n on uptake into guinea-pig l e f t SEM f o r 5-18 t i s s u e s a t e a c h p i n t . a t r i a . V a l u e s a r e mean S a m p l e s w e r e i n c u b a t e d f o r 30 m i n i n the p r e s e n c e o f the i n d i c a t e d o u a b a i n c o n c e n t r a t i o n and then i n the p r e s e n c e o f the same o u a b a i n c o n c e n t r a t i o n and 86Rb+ f o r a n a d d i t i o n a l 30 m i n . S i g n i f i c a n t (p 0 . 0 5 ) s t i m u l a t i o n o f Rb+ t r a n s p o r t o c c u r r e d a t 3 X 10-9 M o u a b a i n , and i n h i b i t i o n was observed a t o u a b a i n c o n c e n t r a t i o n s o f 10-7 M or g r e a t e r . Reproduced, w i t h p e r m i s s i o n , f r o m J . C l i n . Invest. (Hougen e t a l . , 1 9 8 1 ) .

*

o r g a n p r e p a r a t i o n s ( S e i f e n , 1974; Garcia and K i r p e k a r , 1973) and a l s o i n h i b i t s n o r e p i n e p h r i n e r e u p t a k e i n g u i n e a - p i g myocardium a t o u a b a i n c o n c e n t r a t i o n s i n t h e nanomolar r a n g e (Sharma and B a n e r j e e , 1 9 8 0 ) . Our f i r s t o b j e c t i v e i n t h e s e s t u d i e s w a s t o confirm t h e o b s e r v a t i o n s o f Godfraind and c o l l e a g u e s t h a t low c o n c e n t r a t i o n s of o u a b a i n i n t h e nanomole r a n g e s t i m u l a t e t h e sodium pump i n i s o l a t e d g u i n e a - p i g l e f t a t r i a l t i s s u e . Accordingly, we used c o n d i t i o n s similar t o those d e s c r i b e d (Ghysel-Burton and G o d f r a i n d , 1979) e x c e p t t h a t Rubidium t h e a t r i a i n o u r e x p e r i m e n t s were q u i e s c e n t . was used a s a K+ a n a l o g a t a f i n a l c o n c e n t r a t i o n of 0 . 1 mM, u s i n g 86Rb a s t r a c e r , i n t h e p r e s e n c e of 4 . 0 mM K+, and a t r i a l samples w e r e s t u d i e d w i t h c o n d i t i o n s und e r which Rb+ u p t a k e w a s l i n e a r w i t h r e s p e c t t o b o t h t i m e and t i s s u e w e i g h t (Hougen e t a l . , 1 9 8 1 ) . When a t r i a l samples were i n c u b a t e d i n t h e p r e s e n c e of 3 nM ouabain--a c o n c e n t r a t i o n a t which Ghysel-Burton

ION TRANSPORT AND DIGITALIS-INDUCEDINOTROPY

0.400r

873

T

P!O. I so. 128’21

122’21

/6/

F i g . 8 . The e f f e c t s o f i s o p r o t e r e n o l and p r o p r a n o l o l on a c t i v e t r a n s p r t i n g u i n e a - p i g l e f t a t r i a . A f t e r a 60-min i n c u b a t i o n a t 3OoC i n t h e p r e s e n c e o f i s o p r o t e r e n o l M ) , Rb’ a c t i v e t r a n s p o r t was i n c r e a s e d 33 f 1 0 % a b o v e b a s e l i n e v a l u e s (p 0.01). When t i s s u e s were p r e i n c u b a t e d f o r 1 5 m i n i n p r o p r a n o lo1 M ) , then i n both p r o p r a n o l o l and i s o p r o t e r e n o l , no s t i m u l a t i o n of Rb+ t r a n s p o r t o c c u r r e d . R e p r o d u c e d , w i t h p e r m i s s i o n , from J . C l i n . Invest. (Houqen e t a l . , 1 9 8 1 ) . Rb

+

and G o d f r a i n d found sodium pump s t i m u l a t i o n - - w e o b s e r v e d a mean 2 0 f 8 (SEMI% s t i m u l a t i o n of Rb+ a c t i v e u p t a k e above b a s e l i n e v a l u e s . I n t h e e x p e r i m e n t summarized i n F i g . 7 , a r a n g e of o u a b a i n c o n c e n t r a t i o n s w a s p r e s e n t i n i n c u b a t i o n media. A s i g n i f i c a n t i n c r e a s e i n R b + a c t i v e u p t a k e above c o n t r o l v a l u e s was a g a i n o b s e r v e d a t 3 nM o u a b a i n . A n o n s i g n i f i c a n t i n c r e a s e o c c u r r e d a t 10-8 M , whereas c o n c e n t r a t i o n s o f 1 0 - 7 M and above r e s u l t e d i n p r o g r e s s i v e i n h i b i t i o n of t r a n s p o r t . W e t h e n examined t h e e f f e c t s of a- and B - a d r e n e r g i c a g o n i s t s and a n t a g o n i s t s on monovalent c a t i o n t r a n s p o r t . N o r e p i n e p h r i n e (10-8 M ) s i g n i f i c a n t l y i n c r e a s e d a c t i v e The t r a n s p o r t o f Rb+ by 29 k 1 0 % above c o n t r o l l e v e l s . pure 8-adrenergic a g o n i s t i s o p r o t e r e n o l ( l o m 8 M ) a l s o s t i m u l a t e d t r a n s p o r t by 33 f 1 0 % above b a s e l i n e l e v e l s , and t h i s e f f e c t w a s b l o c k e d by a 1 0 - 6 M c o n c e n t r a t i o n of t h e 8-adrenergic blocking agent propranolol (Fig. 8 ) .

874

THOMAS W. SMITH AND WILLIAM H. BARRY

A d d i t i o n a l e x p e r i m e n t s d e m o n s t r a t e d t h a t 8-, b u t n o t a - a d r e n e r g i c e f f e c t s mediated t h e s t i m u l a t i o n of monov a l e n t c a t i o n t r a n s p o r t i n t h e guinea-pig l e f t a t r i a l samples. I f t h e h y p o t h e s i s w e r e c o r r e c t t h a t sodium pump s t i m u l a t i o n by low o u a b a i n c o n c e n t r a t i o n s i s c a t e c h o l amine-mediated, t h e n pharmacologic blockade o f 8 - a d r e n e r g i c r e c e p t o r s w i t h p r o p r a n o l o l would be e x p e c t e d t o a b o l i s h t h e s t i m u l a t o r y e f f e c t of 3 nM ouabain. T h i s experiment w a s done and showed t h a t i n t h e p r e s e n c e of p r o p r a n o l o l , no s t i m u l a t o r y e f f e c t of 3 nM o u a b a i n occ u r r e d . A d d i t i o n a l e x p e r i m e n t s showed t h a t p r o p r a n o l o l a l o n e had no m e a s u r a b l e e f f e c t s on Rb+ t r a n s p o r t . Our working h y p o t h e s i s f u r t h e r p r e d i c t e d t h a t endogenous n o r e p i n e p h r i n e c o u l d be shown t o s t i m u l a t e monoUsing 10-7 M tyramine t o c a u s e valent cation transport. release of n o r e p i n e p h r i n e from n e r v e t e r m i n a l s , w e obs e r v e d a 26 f 1 2 % enhancement of R b + a c t i v e t r a n s p o r t . Sodium pump a c t i v i t y i n t h i s p r e p a r a t i o n , t h e n , i s s t i m u l a t e d by e i t h e r exogenous o r endogenous c a t e c h o l a m i n e s w i t h B-adrenergic a c t i v i t y . F i n a l l y , t o examine f u r t h e r t h e r o l e of endogenous c a t e c h o l a m i n e s i n t h e m e d i a t i o n of monovalent c a t i o n t r a n s p o r t s t i m u l a t i o n by low c o n c e n t r a t i o n s o f o u a b a i n , i n v i v o c a t e c h o l a m i n e d e p l e t i o n w a s a c h i e v e d by t h e u s e of 6-hydroxydopamine i n one group of a n i m a l s and by r e s e r p i n e i n a n o t h e r a c c o r d i n g t o s t a n d a r d methods (Hougen et al. , 1 9 8 1 ) . Responses o f l e f t a t r i a l Rb' upt a k e i n samples from t h e s e a n i m a l s i n t h e p r e s e n c e and a b s e n c e o f 3 nM o u a b a i n are summarized i n F i g . 9. Samples from r e s e r p i n e - t r e a t e d a n i m a l s showed no t r a n s p o r t s t i m u l a t i o n i n r e s p o n s e t o 3 nM o u a b a i n , b u t r a t h e r a s m a l l , b u t s i g n i f i c a n t i n h i b i t i o n o f Rb+ u p t a k e t o 80 -+ 8 % o f c o n t r o l o c c u r r e d . S i m i l a r l y , l e f t a t r i a from 6-hydroxydopamine-treated g u i n e a p i g s showed i n h i b i t i o n r a t h e r t h a n s t i m u l a t i o n of t r a n s p o r t i n r e s p o n s e t o 3 nM o u a b a i n ( F i g . 9 ) . The r e s u l t s from t h e s e e x p e r i m e n t s i n d i c a t e t h a t s t i m u l a t i o n of sodium pump a c t i v i t y by low c o n c e n t r a t i o n s o f o u a b a i n r e q u i r e s c a t e c h o l a m i n e s t o be p r e s e n t i n amounts g r e a t e r t h a n t h o s e remaining a f t e r exposure i n v i v o t o r e l a t i v e l y l a r g e doses of r e s e r p i n e o r 6-hydroxydopamine .

Thus, w e have confirmed e a r l i e r r e p o r t s t h a t low o u a b a i n c o n c e n t r a t i o n s i n t h e nanomolar r a n g e s t i m u l a t e monovalent c a t i o n a c t i v e t r a n s p o r t i n myocardium, and f i n d t h a t t h i s s t i m u l a t o r y e f f e c t i s m e d i a t e d by 6 - a d r e n e r g i c e f f e c t s of endogenous c a t e c h o l a m i n e s .

ION TRANSPORT AND DIGITALIS-INDUCEDINOTROPY

875

RESERPINE

*

T

* T

n

l

"

CONTROL

OUABAIN 3nM

I'

CONTRI

OUABAIN 3 nM

F i g . 9 . T h e e f f e c t o f i n vivo c a t e c h o l a m i n e d e p l e t i o n on a c t i v e t r a n s p o r t i n guinea-pig l e f t a t r i a . L e f t a t r i a from r e s e r p i n e - t r e a t e d g u i n e a p i g s w e r e i n c u b a t e d f o r 60 m i n a t 30°C i n the p r e s e n c e or a b s e n c e o f 3 n M o u a b a i n . No o u a b a i n - i n d u c e d e n h a n c e m e n t o f Rb+ a c t i v e t r a n s p o r t o c c u r r e d ; r a t h e r , a s m a l l i n h i b i t i o n (80 ? 8 % of b a s e l i n e v a l u e s , P < 0 . 0 5 ) was observed. A s i m i l a r small inhibition (83 ? 7% o f b a s e l i n e v a l u e s , P 0.05 + i n R b a c t i v e t r a n s p o r t was m e a s u r e d i n l e f t a t r i a l t i s s u e removed f r o m a n i m a l s t r e a t e d w i t h 6-OHDA. V a l u e s shown a r e m e a n s f SEM for the n u m b e r s of t i s s u e s a m p l e s i n d i c a t e d i n p a r e n t h e s e s . R e p r o d u c e d , w i t h p e r m i s s i o n , f r o m J. C l i n . I n v e s t . (Hougen e t a l . , 1 9 8 1 ) .

+

Rb

VI.

AN OVERVIEW: UNITARY VS PLURALISTIC VIEWS OF CARDIAC GLYCOSIDE ACTION

I t seems f a i r t o c o n c l u d e from a s u r v e y o f a v a i l a b l e evidence t h a t t h e only t r u l y unifying concept regarding c a r d i a c g l y c o s i d e a c t i o n i s t h a t t h i s f a m i l y of compounds, a t p h a r m a c o l o g i c a l l y r e l e v a n t d o s e s o r c o n c e n t r a t i o n s , a c t s by b i n d i n g w i t h h i g h a f f i n i t y and s p e c i f i c i t y t o a s i t e on t h e N a , K - A T P a s e complex t h a t f a c e s t h e o u t e r s u r f a c e of v i r t u a l l y a l l e u k a r y o t i c c e l l s . A l t e r n a t i v e r e c e p t o r s , i f t h e y e x i s t , have e l u d e d r e c o g n i t i o n (Smith e t a l . , 1 9 7 4 ) . An e x t r a o r d i n a r i l y b r o a d a r r a y of b i o l o g i c e f f e c t s - - f r o m p r o t e c t i o n o f monarch b u t t e r f l i e s from p r e d a t o r s (Brower, 1 9 6 9 ) t o i n f l u e n c i n g ( p e r h a p s ) t h e a r t i s t i c s t y l e o f V i n c e n t Van Gogh ( L e e , 1 9 8 1 ) - - a p p e a r s t o s t e m from t h i s b a s i c i n t e r a c t i o n . I n t h e n a r r o w e r c l i n i -

876

THOMAS W. SMITH AND WILLIAM H. BARRY

c a l s e t t i n g , well-documented e f f e c t s of c a r d i a c glycos i d e s range from enhancement of myocardial c o n t r a c t i l i t y t o changes i n t h e p r o p e r t i e s of t h e c a r d i a c conduction system, and t h e l a t t e r stem from b o t h d i r e c t and autonom i c a l l y mediated e f f e c t s (Smith and Braunwald, 1 9 8 0 ) . Autonomic e f f e c t s , i n t u r n , may be m a n i f e s t i n a l t e r a t i o n s i n b o t h p a r a s y m p a t h e t i c and s y m p a t h e t i c a c t i v i t y , and t h e s e autonomic i n f l u e n c e s s t e m from b o t h CNS and p e r i p h e r a l mechanisms. On r e f l e c t i o n , i t i s n o t s u r p r i s i n g t h a t i n t e r a c t i o n w i t h such a fundamental and h i g h l y conserved m o l e c u l a r s p e c i e s a s t h a t which maint a i n s transmembrane Na+ and K+ g r a d i e n t s r e s u l t s i n s u c h a m u l t i p l i c i t y of b i o l o g i c a l e f f e c t s . I n s h o r t , t h e r e a r e ample grounds t o heed N o b l e ' s warning t h a t g l y c o s i d e e f f e c t s may be m u l t i f a c t o r i a l (Noble, 1 9 8 0 ) . I n o u r view, t h e r e i s convincing e v i d e n c e t h a t one mechanism l e a d i n g t o s u s t a i n e d p o s i t i v e i n o t r o p i c e f f e c t s of c a r d i a c g l y c o s i d e s i n h e a r t muscle i s p a r t i a l i n h i b i t i o n of sodium t r a n s p o r t . E a r l i e r c i r c u m s t a n t i a l e v i d e n c e (Schwartz et a l . , 1975; Akera and Brody, 1 9 7 8 ) i s now s u p p o r t e d by s t u d i e s u s i n g e l e c t r o p h y s i o l o g i c approaches (Marban and T s i e n , 1 9 8 2 ; Lederer and E i s n e r , 1 9 8 2 ; E i s n e r and L e d e r e r , 1 9 7 9 , 19801, i n t r a c e l l u l a r i o n - s e n s i t i v e m i c r o e l e c t r o d e t e c h n i q u e s (Lee et a l . , 1980) , and i o n f l u x s t u d i e s u s i n g r a d i o i s o t o p i c t r a c e r methods (Langer and S a r e n a , 1 9 7 0 ; B i e d e r t et a l . , 1 9 7 9 ; Barry et a l . , 1 9 8 1 ) . Data reviewed above f u r t h e r i n d i c a t e t h a t myocardial monovalent c a t i o n t r a n s p o r t i n h i b i t i o n can be documented i n i n t a c t animal models u s i n g c a r d i a c g l y c o s i d e - s e n s i t i v e s p e c i e s a t d o s e s and plasma and myocardial l e v e l s c a u s i n g a p o s i t i v e i n o t r o p i c e f f e c t w i t h o u t e v i d e n t t o x i c i t y (Hougen and Smith, 1 9 7 8 ) . However, t h e s e f i n d i n g s by no means p r e c l u d e t h e e x i s t ence of o t h e r mechanisms t h a t may be o p e r a t i v e i n a d d i t i o n t o , o r i n some c i r c u m s t a n c e s i n s t e a d o f , myocardial sodium-pump i n h i b i t i o n . Indeed, a d d i t i o n a l mechanisms a r e r e q u i r e d t o r e s o l v e problems r a i s e d by seemingly conf l i c t i n g e v i d e n c e r e p o r t e d ( B e n t f e l d et al., 1 9 7 7 ; Ghysel-Burton and Godfraind, 1 9 7 9 ; Godfraind and GhyselBurton, 1 9 8 0 ; Cohen et a l . , 1 9 7 6 ; Blood and Noble, 1 9 7 8 ) . I n t h i s c o n t e x t , t h e h y p o t h e s i s advanced by Akera and Brody ( 1 9 7 8 ) i s of i n t e r e s t . They s u g g e s t t h a t i n t e r a c t i o n of low, s u b t o x i c g l y c o s i d e c o n c e n t r a t i o n s w i t h myocardial Na,X-ATPase r e d u c e s sodium pump c a p a c i t y , res u l t i n g i n an enhanced t r a n s i e n t i n c r e a s e i n [NaIi close t o t h e sarcolemma o c c u r s d u r i n g t h e e a r l y phase of t h e c a r d i a c c y c l e . The i n c r e a s e i n subsarcolemmal Na+ r e s u l t s i n an enhanced Ca2+ i n f l u x v i a Na/Ca exchange and t h u s i n a p o s i t i v e i n o t r o p i c e f f e c t , b u t t h e Na+ i n c r e a s e i s c y c l i c and n o t cumulative. T h i s scheme

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p r o v i d e s a mechanism whereby sodium pump i n h i b i t i o n could mediate a p o s i t i v e i n o t r o p i c e f f e c t without causi n g measurable a l t e r a t i o n s i n s t e a d y - s t a t e c e l l u l a r N a + o r K+ c o n t e n t , e s p e c i a l l y g i v e n t h e n o i s e l e v e l s i n h e r e n t i n c h e m i c a l estimates of i n t r a c e l l u l a r N a c o n t e n t of i n t a c t t i s s u e . A v a l i d t e s t of t h i s h y p o t h e s i s w i l l a w a i t t e c h n o l o g i c a l developments t o p e r m i t q u a n t i t a t i o n of t h e i n t r a c e l l u l a r sodium t r a n s i e n t , a measurement n o t c u r r e n t l y p o s s i b l e b e c a u s e of t h e r e l a t i v e l y s l o w r e s p o n s e t i m e o f t h e p r e s e n t g e n e r a t i o n of sodiums e n s i t i v e microelectrodes. Also of i n t e r e s t , b u t d i f f i c u l t t o prove d i r e c t l y , i s t h e c o n c e p t advanced by Lullmann and c o l l e a g u e s t h a t c a r d i a c g l y c o s i d e b i n d i n g t o N a , K - A T P a s e f a v o r s a conf o r m a t i o n a l s t a t e o f t h e enzyme t h a t i s c o n n e c t e d w i t h an i n c r e a s e d l a b i l i t y of a plasmalemmal C a p o o l t h a t m e d i a t e s e x c i t a t i o n - c o n t r a c t i o n c o u p l i n g (Lullmann and P e t e r s , 1 9 7 9 ) . Occupancy of a f r a c t i o n o f N a , K - A T P a s e s i t e s i s s u g g e s t e d t o p r o d u c e t h e i n c r e a s e d l a b i l i t y of C a 2 + b i n d i n g t o p lasmalemmal pho s p h a t i d y lser i n e m o l e c u l e s , while a t s u b t o x i c glycoside l e v e l s t h e remaining unoccupied t r a n s p o r t s i t e s i n c r e a s e t h e i r a c t i v i t y t o m a i n t a i n i n t r a c e l l u l a r N a + and K + h o m e o s t a s i s . The conc e p t o f e x c i t a t i o n - c o n t r a c t i o n c o u p l i n g embodied i n t h i s model d i f f e r s somewhat from t h a t advanced by o t h e r w o r k e r s (Winegrad, 1979; F a b i a t o and F a b i a t o , 1 9 7 9 ) . The work o f G e r v a i s and c o l l e a g u e s ( 1 9 7 7 1 , w h i l e supp o r t i v e o f t h e p o s s i b i l i t y of s u c h a mechanism, u s e d a s h e e p k i d n e y Na,K-ATPase p r e p a r a t i o n , and a l t e r a t i o n s i n C a b i n d i n g were n o t r e p o r t e d a t o u a b a i n c o n c e n t r a t i o n s below 1 0 - 5 M. C e n t r a l t o t h e scheme advanced by Lullmann and P e t e r s ( 1 9 7 9 ) and d i s c u s s e d i n d e t a i l by Noble (1980) i s t h e e x i s t e n c e o f a " s u p e r f i c i a l lanthanum-di s p l a c e a b l e C a p o o l a l s o emphasized i n t h e work of N a y l e r ( 1 9 7 3 ) and o f Langer and Frank ( 1 9 7 2 ) . I t i s i m p o r t a n t , i n o u r view, t o n o t e t h a t w e a l s o o b s e r v e d i s t i n c t enhancement of a r a p i d l y exchangeable C a pool i n c u l t u r e d h e a r t cells i n r e s p o n s e t o c a r d i a c g l y c o s i d e s ; t h a t t h e change i n t h i s C a p o o l o c c u r s a t e x a c t l y t h e t h r e s h o l d o u a b a i n conc e n t r a t i o n t h a t produces a p o s i t i v e i n o t r o p i c e f f e c t ( B i e d e r t e t a l . , 1 9 7 9 ) ; and t h a t t h e t i m e c o u r s e o f changes i n t h e s i z e of t h i s C a p o o l c l o s e l y p a r a l l e l s t h e o n s e t and o f f s e t o f t h e p o s i t i v e i n o t r o p i c e f f e c t d u r i n g b o t h i n i t i a l e x p o s u r e t o ( B i e d e r t e t a l . , 1 9 7 9 ) and washo u t of ouabain (Barry e t a l . , 1981) , r e s p e c t i v e l y . The c l o s e r e l a t i o n s h i p of p o s i t i v e i n o t r o p i c e f f e c t s t o t h e d e g r e e of monovalent c a t i o n t r a n s p o r t i n h i b i t i o n h a s been i l l u s t r a t e d i n F i g . 3 . W e would, however, add a word of c a u t i o n r e g a r d i n g t h e a c c e p t a n c e of p r e v i o u s e x p e r i m e n t a l

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o b s e r v a t i o n s a s p r o v i n g t h e e x i s t e n c e of a p h y s i o l o g i c a l l y i m p o r t a n t C a p o o l t h a t i s " s u p e r f i c i a l " and " l a n t h a n u m - d i s p l a c e a b l e ,I' s i n c e o u r s t u d i e s i n b e a t i n g c u l t u r e d h e a r t c e l l s o f f e r an a l t e r n a t i v e e x p l a n a t i o n of the experimental findings. Having d e v e l o p e d methods t o measure u n i d i r e c t i o n a l C a f l u x e s i n b e a t i n g monolayers of c u l t u r e d h e a r t c e l l s o v e r i n t e r v a l s a s s h o r t a s 5 sec ( B a r r y and S m i t h , 1 9 8 2 1 , w e a s s e s s e d t h e e f f e c t s of l a n thanum ( L a ) i n t h i s p r e p a r a t i o n . Ca u p t a k e by c u l t u r e d c e l l s c o u l d b e a l m o s t c o m p l e t e l y i n h i b i t e d by e x p o s u r e t o 1 mM Lac13 w i t h i n 5 sec. T h i s c o n c e n t r a t i o n o f La a l s o i n h i b i t e d c o n t r a c t i o n c o m p l e t e l y w i t h i n 5 sec. During e f f l u x o f 45Ca from c e l l s , however, e x p o s u r e t o 1 mM La produced no change i n t h e r a t e of C a e f f l u x o v e r t h e i n i t i a l 2 0 s e c , and o n l y m i n i m a l i n h i b i t i o n w a s e v i d e n t a t 30 sec. More marked i n h i b i t i o n o f C a e f f l u x d i d d e v e l o p a f t e r 3 min of L a e x p o s u r e . T h e r e w a s no e v i dence f o r a component o f s u p e r f i c i a l L a - d i s p l a c e a b l e C a i n t h e s e e x p e r i m e n t s . The i n i t i a l , much g r e a t e r i n h i b i t o r y e f f e c t of L a on C a i n f l u x t h a n on C a e f f l u x c o u l d ' e x p l a i n t h e f i n d i n g s o f Lan e r and Frank ( 1 9 7 2 ) of a n The e x p e r i m e n t a l a p p a r e n t " d i s p l a c e m e n t " o f 15Ca by L a . d e s i g n o f N a y l e r (1973) u s i n g an i n t a c t p a p i l l a r y m u s c l e p r e p a r a t i o n l e a v e s open t h e p o s s i b i l i t y t h a t an a p p a r e n t d i s p l a c e m e n t of 45Ca w i t h L a e x p o s u r e c o u l d b e due t o a r a p i d i n h i b i t i o n of 45Ca back f l u x i n t o t h e c e l l s from a r e l a t i v e l y slowly exchanging i n t e r s t i t i a l space ( A t t w e l l et a l . , 1 9 7 9 ) . O t h e r s have s u g g e s t e d r e c e n t l y t h a t C a bound t o t h e basement membrane-glycocalyx complex may n o t be n e c e s s a r y € o r e x c i t a t i o n - c o n t r a c t i o n coupling i n h e a r t muscle. I s e n b e r g and Klockner ( 1 9 8 0 ) found t h a t I p e r s i s t e d i n i s o l a t e d r a t h e a r t myocytes i n which tge g l y c o c a l y x was removed by t r e a t m e n t w i t h c o l l a g e n a s e hyaluronidase. Harding and H a l l i d a y ( 1 9 8 0 ) removed 7 9 % o f t h e s i a l i c a c i d r e s i d u e s by n e u r a m i n i d a s e t r e a t m e n t o f g u i n e a - p i g a t r i a and found no change i n t h e c a l c i u m s e n s i t i v i t y of c o n t r a c t i o n . Winegrad ( 1 9 7 9 ) f u r t h e r sugg e s t s t h a t t h e basement membrane complex i s u n l i k e l y t o be t h e source of a c t i v a t o r calcium i n f r o g h e a r t , s i n c e n o t all c a r d i a c c e l l s from t h a t s o u r c e a r e i n v e s t e d by this structure. None of t h e e x p e r i m e n t a l o b s e r v a t i o n s j u s t cons i d e r e d e x c l u d e s w i t h c e r t a i n t y t h e e x i s t e n c e of a mechanism f o r c a r d i a c g l y c o s i d e - i n d u c e d p o s i t i v e i n o t r o p y i n mammalian myocardium t h a t depends on g l y c o s i d e b i n d i n g t o , b u t n o t i n h i b i t i o n o f , Na,K-ATPase. Nevertheless, w e a r e drawn t o t h e s i m p l e s t model t h a t c a n accommodate the available experimental observations. The r a p i d l y growing l i t e r a t u r e on t h e Na/Ca exchange c a r r i e r i n myoc a r d i u m (Baker e t a l . , 1969; G l i t s c h e t a l . , 1 9 7 0 ;

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Reeves and S u t k o , 1 9 7 9 ; P i t t s , 1 9 7 9 ; Horackova and V a s s o r t , 1 9 7 9 ; L e e e t al., 1980; Coraboeuf e t a ] . , 1981) now p r o v i d e s t h e c r i t i c a l l i n k needed between Na-pump i n h i b i t i o n and an enhanced c o n t r a c t i l e C a p o o l . W e have a l r e a d y n o t e d t h a t t h e enhancement o f s l o w i n ward c u r r e n t w i t h g l y c o s i d e e x p o s u r e shown by W e i n g a r t e t a l . (19781, Marban and T s i e n ( 1 9 7 9 ) and L e d e r e r and E i s n e r (1982) h a s been s u g g e s t e d by t h o s e a u t h o r s t o b e s e c o n d a r y t o Na-pump i n h i b i t i o n and c o n s e q u e n t l y enhanced [ C a l i . How can w e e x p l a i n t h e a p p a r e n t l y d i s c r e p a n t f i n d i n g s o f G o d f r a i n d and Ghysel-Burton (1980) r e g a r d i n g an a b s e n c e of i n h i b i t i o n , o r e v e n s t i m u l a t i o n o f monovalent c a t i o n t r a n s p o r t i n g u i n e a - p i g l e f t a t r i a a t c o n c e n t r a t i o n s i n t h e nanomole r a n g e t h a t e l i c i t a s m a l l b u t s i g n i f i c a n t i n o t r o p i c e f f e c t ? Our f i n d i n g s summarized i n F i g s . 7 , 8 , and 9 i n d i c a t e t h a t s t i m u l a t i o n o f Na-pump a c t i v i t y i n g u i n e a - p i g l e f t a t r i a by low ( 3 nM) o u a b a i n c o n c e n t r a t i o n s i s c a t e c h o l a m i n e d e p e n d e n t (Hougen e t a l . , 1 9 8 1 ) . D r s . L e c h a t and Malloy i n o u r l a b o r a t o r y have r e c e n t l y o b t a i n e d e v i d e n c e sugg e s t i n g t h a t endogenous c a t e c h o l a m i n e s may a l s o m e d i a t e p o s i t i v e i n o t r o p i c e f f e c t s o f low c o n c e n t r a t i o n s of ouab a i n u n d e r c o n d i t i o n s i n which no monovalent c a t i o n t r a n s p o r t i n h i b i t i o n i s e v i d e n t . W e determined t h e inot r o p i c e f f e c t o f 1 0 nM o u a b a i n i n a t r o p i n e - t r e a t e d i s o l a t e d l e f t a t r i a of normal g u i n e a p i g s and from a n i m a l s p r e t r e a t e d w i t h d o s e s o f 6-hydroxydopamine t h a t d e p l e t e d more t h a n 9 0 % of endogenous c a t e c h o l a m i n e s . Developed t e n s i o n was s i g n i f i c a n t l y i n c r e a s e d by 1 0 % w i t h e x p o s u r e of a t r i a from normal a n i m a l s t o 1 0 nM o u a b a i n , a concent r a t i o n t h a t d i d n o t i n h i b i t a c t i v e u p t a k e o f 86Rb'. In c o n t r a s t , a t r i a from a n i m a l s t r e a t e d w i t h 6-hydroxydopamine d i d n o t show a p o s i t i v e i n o t r o p i c r e s p o n s e t o 1 0 nM o u a b a i n . A t h i g h e r o u a b a i n c o n c e n t r a t i o n s (above 1 0 - 7 M I , t h e r e w a s a c l o s e c o r r e l a t i o n between t h e e x t e n t o f N a pump i n h i b i t i o n and t h e magnitude of t h e p o s i t i v e i n o t r o p i c r e s p o n s e ; t h i s c o r r e l a t i o n w a s found i n a t r i a from b o t h normal and c a t e c h o l a m i n e - d e p l e t e d a n i m a l s . I n cont r a s t t o t h e r e p o r t of B e n t f e l d e t a l . ( 1 9 7 7 ) , w e f o u n d c l e a r - c u t i n h i b i t i o n o f monovalent c a t i o n a c t i v e t r a n s p o r t i n r e s p o n s e t o t h e p o s i t i v e l y i n o t r o p i c o u a b a i n conc e n t r a t i o n of 4 x 1 0 - 7 M . Our t e n t a t i v e c o n c l u s i o n i s t h a t i n h i b i t i o n of monov a l e n t c a t i o n a c t i v e t r a n s p o r t i s d i r e c t l y and c a u s a l l y r e l a t e d t o t h e p o s i t i v e i n o t r o p i c e f f e c t of c a r d i a c g l y c o s i d e s i n most p r e p a r a t i o n s s t u d i e d t o d a t e i n which r e a s o n a b l y d i r e c t measurements of sodium pump a c t i v i t y can b e made. I t seems c l e a r t h a t i n h i b i t i o n c a n b e demo n s t r a t e d under c o n d i t i o n s t h a t p e r m i t a s u s t a i n e d p o s i t i v e i n o t r o p i c e f f e c t w i t h o u t emergence of o v e r t t o x i c i -

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t y . N e v e r t h e l e s s , it i s l i k e l y t h a t a d d i t i o n a l mechani s m s can come i n t o p l a y under s e l e c t e d c i r c u m s t a n c e s , p a r t i c u l a r l y i n i n t a c t myocardium. W e b e l i e v e t h a t enhanced release o r reduced r e u p t a k e of endogenous norep i n e p h r i n e i s one s u c h mechanism t h a t may o b t a i n i n i s o l a t e d g u i n e a - p i g a t r i a l t i s s u e . More d e f i n i t i v e s t u d i e s are needed, e s p e c i a l l y unde*r c i r c u m s t a n c e s t h a t p e r m i t adequate o p e r a t i o n a l d e f i n i t i o n s of " t h e r a p e u t i c " (inot r o p i c ) and t o x i c end p o i n t s . Such s t u d i e s a w a i t more s a t i s f a c t o r y means t o a s s e s s monovalent c a t i o n a c t i v e t r a n s p o r t i n t h e myocardium of i n t a c t animal models.

ACKNOWLEDGMENT

p a r t s of t h e work d e s c r i b e d were s u p p o r t e d by N I H G r a n t s HL-18003 and HL-23893.

REFERENCES

+ +

Akera, T . , and Brody, T. M. ( 1 9 7 8 ) . The r o l e o f N a ,K -ATPase i n P h a r m a c o l . R e v . 29, t h e inotropic action of d i g i t a l i s . 187-220. A l l e n , D. G., and B l i n k s , J. R. ( 1 9 7 8 ) . C a l c i u m t r a n s i e n t s i n a e q u o r i n - i n j e c t e d f r o g c a r d i a c muscle. N a t u r e (London) 2 7 3 , 509-513. A t t w e l l , D., E i s n e r , D. , and Cohen, I. ( 1 9 7 9 ) . V o l t a g e clamp and tracer f l u x d a t a : E f f e c t s o f a r e s t r i c t e d e x t r a c e l l u l a r space. Q . R e v . B i o p h y s . 1 2 , 213-261. Baker, P. F . , B l a u s t e i n , M. P . , Hodgkin, A. L . , and S t e i n h a r d t , R . A. (1969). The i n f l u e n c e o f calcium on sodium e f f l u x i n J . P h y s i o l . ( L o n d o n ) 2 0 0 , 431-458. s q u i d axons. B a r r y , W. H . , and Smith, T. W. ( 1 9 8 2 ) . Mechanisms o f transmemb r a n e calcium movement i n c u l t u r e d c h i c k embryo v e n t r i c u l a r c e l l s . J . P h y s i o l . (London) (In p r e s s ) . Barry, W. H . , B i e d e r t , S., Miura, D. S . , and Smith, T. W. ( 1 9 8 1 ) . Changes i n c e l l u l a r N a , K , and C a c o n t e n t s , monovalent cat i o n t r a n s p o r t r a t e , and c o n t r a c t i l e s t a t e d u r i n g washout of c a r d i a c g l y c o s i d e s from c u l t u r e d c h i c k h e a r t c e l l s . Circ. R e s . 49, 141-149. B e n t f e l d , M . , Lihlmann, H . , Peters, T . , and Proppe, D. ( 1 9 7 7 ) . I n t e r d e p e n d e n c e of i o n t r a n s p o r t and t h e a c t i o n o f ouabain i n h e a r t muscle. Br. J . P h a r m a c o l . 6 1 , 19-27. B i e d e r t , S. , B a r r y , W. H . , and Smith, T. W. ( 1 9 7 9 ) . I n o t r o p i c e f f e c t s and changes i n sodium and calcium c o n t e n t s a s s o c i a t e d w i t h i n h i b i t i o n of monovalent c a t i o n a c t i v e t r a n s p o r t by ouab a i n i n c u l t u r e d myocardial c e l l s . J . G e n . P h y s i o l . 7 4 , 479-494.

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Blood, B. E . , and Noble, D. ( 1 9 7 8 ) . Two mechanisms f o r t h e i n o t r o p i c a c t i o n o f ouabain on s h e e p c a r d i a c P u r k i n j e f i b e r cont r a c t i l i t y . I n " B i o p h y s i c a l Aspects o f C a r d i a c Muscle" (M. Morad and M. Tabatabai, e d s . ) , pp. 369-379. Academic Press, N e w York. Sci. Am. 220(2) Brower, L. P. ( 1 9 6 9 ) . E c o l o g i c a l c h e m i s t r y . 22-29. Cohen, I . , Daut, J., and Noble, D. ( 1 9 7 6 ) . An a n a l y s i s o f t h e act i o n s o f l o w c o n c e n t r a t i o n s of o u a b a i n on membrane c u r r e n t s J. P h y s i o l . (London) 260, 75-103. i n Purkinje fibers. Coraboeuf, E . , G a u t i e r , P . , and Giuraudou, P. ( 1 9 8 1 ) . P o t e n t i a l and t e n s i o n changes induced by sodium removal i n dog P u r k i n j e f i b r e s : Role of an e l e c t r o g e n i c sodium-calcium exchange. J . P h y s i o l . (London) 311, 605-622. E i s n e r , D . A . , and L e d e r e r , W. J. ( 1 9 7 9 ) . Does sodium pump i n h i b i t i o n produce t h e p o s i t i v e i n o t r o p i c e f f e c t s o f s t r o p h a n t h i d i n i n mammalian c a r d i a c muscle? J. P h y s i o l . (London) 296, 75P-76P. E i s n e r , D. A., a n d L e d e r e r , W. J. ( 1 9 8 0 ) . The r e l a t i o n s h i p between sodium pump a c t i v i t y and t w i t c h t e n s i o n i n c a r d i a c P u r k i n j e fibers. J . P h y s i o l . (London) 303, 475-494. E l l i s , D. ( 1 9 7 7 ) . The e f f e c t s o f e x t e r n a l c a t i o n s and ouabain on t h e i n t r a c e l l u l a r sodium a c t i v i t y o f sheep h e a r t P u r k i n j e J . P h y s i o l . (London) 273, 211-240. fibers. F a b i a t o , A . , and F a b i a t o , F . ( 1 9 7 9 ) . C a l c i u m and c a r d i a c e x c i t a t i o n - c o n t r a c t i o n c o u p l i n g . Annu. Rev. P h y s i o l . 41, 473-484. F l a s c h , H . , and Heinz, N. ( 1 9 7 8 ) . C o r r e l a t i o n between i n h i b i t i o n o f (Na+,K+)-membrane-ATPase and p o s i t i v e i n o t r o p i c a c t i v i t y of c a r d e n o l i d e s i n i s o l a t e d p a p i l l a r y m u s c l e s of g u i n e a p i g . Naunyn-Schmiedeberq's Arch. Pharmacol. 304, 37-44. Garcia, A. G . , and K i r p e k a r , S. M. ( 1 9 7 3 ) . Release o f n o r a d r e n a l i n e from s l i c e s of c a t s p l e e n by p r e t r e a t m e n t w i t h calcium, s t r o n t i u m and barium. J . P h y s i o l . (London) 235, 693-713. G e r v a i s , A . , Lane, L. K. , Anner, B. M. , Lindenmayer, G . E . , and Schwartz, A. ( 1 9 7 7 ) . A p o s s i b l e m o l e c u l a r mechanism o f t h e C i r c . R e s . 4 0 , 8-14. action of d i g i t a l i s . Ghysel-Burton, J., and G o d f r a i n d , T. ( 1 9 7 9 ) . S t i m u l a t i o n and i n h i b i t i o n of t h e sodium pump by c a r d i o a c t i v e s t e r o i d s i n r e l a t i o n t o t h e i r b i n d i n g s i t e s and t h e i r i n o t r o p i c e f f e c t on guinea-pig i s o l a t e d a t r i a . B r . J . Pharmacol. 66, 175-184. G i l l i s , R. A. , and Q u e s t , J. A. ( 1 9 8 0 ) . The r o l e of t h e n e r v o u s system i n t h e c a r d i o v a s c u l a r e f f e c t s o f d i g i t a l i s . Pharmacol Rev. 131, 19-97. G l i t s c h , H. G . , R e u t e r , H. , and S c h o l z , H. ( 1 9 7 0 ) . The e f f e c t o f t h e i n t e r n a l sodium c o n c e n t r a t i o n o n calcium fluxes i n i s o l a t e d guinea-pig a u r i c l e s . J . P h y s i o l . (London) 209, 25-43. G o d f r a i n d , T . , and Ghysel-Burton, J . ( 1 9 8 0 ) . Independence of t h e p o s i t i v e i n o t r o p i c e f f e c t o f ouabain from t h e i n h i b i t i o n o f t h e h e a r t N a + , K + pump. P r o c . N a t l . Acad. S c i . USA 77, 3067-3069.

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Harding, W. E., a n d H a l l i d a y , J . ( 1 9 8 0 ) . Removal of s i a l i c a c i d f r a n c a r d i a c sarcolemma d o e s n o t a f f e c t c o n t r a c t i l e f u n c t i o n i n e l e c t r i c a l l y s t i m u l a t e d guinea p i g l e f t a t r i a . N a t u r e (London) 286, 819-821. Horackova, M. , and V a s s o r t , G. (1979) Sodium-calcium exchange i n r e g u l a t i o n of c a r d i a c c o n t r a c t i l i t y . J . Gen. P h y s i o l . 73, 403-424. Hougen, T . J., and Smith, T. W. ( 1 9 7 8 ) . I n h i b i t i o n o f myocardial monovalent c a t i o n a c t i v e t r a n s p o r t by s u b t o x i c d o s e s o f ouab a i n i n t h e dog. C i r c . R e s . 42, 856-863. Hougen, T. J., Lloyd, B. L . , a n d Smith, T. W. ( 1 9 7 9 ) . E f f e c t s of i n o t r o p i c and arrhythmogenic d i g o x i n d o s e s and o f d i g o x i n s p e c i f i c a n t i b o d y on m y o c a r d i a l monovalent c a t i o n t r a n s p o r t i n t h e dog. C i r c . Res. 44, 23-31. Hougen, T . J . , S p i c e r , N . , a n d Smith, T. W. ( 1 9 8 1 ) . S t i m u l a t i o n o f monovalent c a t i o n a c t i v e t r a n s p o r t by low c o n c e n t r a t i o n s of c a r d i a c g l y c o s i d e s : Role o f c a t e c h o l a m i n e s . J. C l i n . I n v e s t . 68, 1207-1214. I s e n b e r g , G . , and Klockner, V. ( 1 9 8 0 ) . Glycocalyx i s n o t r e q u i r e d f o r slow inward calcium c u r r e n t i n i s o l a t e d r a t h e a r t myoc y t e s . N a t u r e (London) 284, 358-360. Langer, G. A . , and F r a n k , J. S . ( 1 9 7 2 ) . Lanthanum i n h e a r t c e l l c u l t u r e . E f f e c t on calcium exchange c o r r e l a t e d w i t h i t s loc a t i o n . J. C e l l Biol. 54, 441-455. Langer, G . A., and S a r e n a , S. D. (1970). E f f e c t s o f s t r o p h a n t h i d i n upon c o n t r a c t i o n and i o n exchange i n r a b b i t v e n t r i c u l a r myocardium. J. M o l . C e l l . C a r d i o l . 1, 65-90. L e d e r e r , W. J . , and E i s n e r , D. A. ( 1 9 8 2 ) . The e f f e c t s of sodium pump a c t i v i t y on t h e slow inward c u r r e n t i n sheep c a r d i a c P u r k i n j e f i b e r s . Proc. R. Soc. London Ser. B ( I n p r e s s ) . Lee, C . O., Uhm, D. Y., and Dresdner, K. ( 1 9 8 0 a ) . Sodium-calcium exchange i n r a b b i t h e a r t muscle c e l l s : Direct measurement o f s a r c o p l a s m i c C a 2 + a c t i v i t y . S c i e n c e 209, 699-701. Lee, C. 0. , Kang, D. H. , Sokol, J. H. , and L e e , K. S. (1980b). R e l a t i o n between i n t r a c e l l u l a r N a i o n a c t i v i t y and t e n s i o n o f sheep c a r d i a c P u r k i n j e fibers exposed t o dihydro-ouabain. Biophys. J. 29, 315-330. Lee, T. C. ( 1 9 8 1 ) . Van Gogh's v i s i o n . D i g i t a l i s i n t o x i c a t i o n ? JAMA, J. Am. Med. Assoc. 245, 727-729. L h l m a n n , H., and P e t e r s , T. (1979). A c t i o n of c a r d i a c g l y c o s i d e s on t h e e x c i t a t i o n - c o n t r a c t i o n c o u p l i n g i n h e a r t muscle. Prog. P h a m a c o l . 2, 5-57. Marban, E . , and T s i e n , R . W. (1979). Ouabain increases t h e slow J. P h y s i o l . inward calcium c u r r e n t i n v e n t r i c u l a r muscle. (London) 292, 72P-73P. Marban, E., and T s i e n , R. W. (1982). Enhancement o f c a r d i a c calcium c u r r e n t d u r i n g d i g i t a l i s i n o t r o p y : P o s i t i v e f e e d b a c k (London) r e g u l a t i o n b y i n t r a c e l l u l a r calcium? J. P h y s i o l (In press). Miura, D. S . , and Rosen, M. R. ( 1 9 7 8 ) . The e f f e c t s o f ouabain o n t h e transmembrane p o t e n t i a l s and i n t r a c e l l u l a r p o t a s s i u m

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a c t i v i t y of canine c a r d i a c P u r k i n j e f i b e r s . Circ. R e s . 4 2 , 333-338. Nayler, W. G . (1973). An e f f e c t of ouabain on t h e s u p e r f i c i a l l y l o c a t e d s t o r e s of calcium i n c a r d i a c muscle c e l l s . J. Mol. C e l l . C a r d i o l . 5, 101-110. Noble, D. (1980). Mechanism of a c t i o n of t h e r a p e u t i c l e v e l s of c a r d i a c g l y c o s i d e s . C a r d i o v a s c . R e s . 1 4 , 495-514. Okita, G . T. (1977). D i s s o c i a t i o n of Na+,K+-ATPase i n h i b i t i o n from F e d . P r o c . , F e d . Am. SOC. E x p . Biol. 3 6 , d i g i t a l i s inotropy. 2225-2230. P i t t s , B. J. R. (1979). Stoichiometry of sodium-calcium exchange i n c a r d i a c sarcolemmal v e s i c l e s . J . Biol. Chem. 2 5 4 , 62326235. Reeves, J. P . , and Sutko, J . L. (1979). Sodium-calcium i o n exP r o c . N a t l . A c a d . Sci. change i n c a r d i a c membrane v e s i c l e s . USA 7 6 , 590-594. Repke, K. (1962). Metabolism of c a r d i a c g l y c o s i d e s . P r o c . Pharmac o l . Meet. l s t , 1 9 6 1 , Vol. 3 , p . 47. Rhee, H. M . , Huang, W.-H., and Askari, A. (1981). R e l a t i o n s h i p between t h e p o s i t i v e i n o t r o p i c a c t i o n of ouabain and i t s i n h i b i t o r y e f f e c t s on Na+,K+-ATPase and a c t i v e t r a n s p o r t of Rb+ i n t h e dog h e a r t . Eur. J . P h a r m a c o l . 7 0 , 273-278. Rogus, E. M., Cheng, L. C . , and Z i e r l e r , K . (1977). Beta-adrenergic Biochim. e f f e c t on Na+-K+ t r a n s p o r t i n r a t s k e l e t a l muscle. B i o p h y s . A c t a 4 6 4 , 347-355. Schwartz, A . , Lindenmayer, G . E . , and Allen, J . C. (1975). The sodium-p tass i u m ade nos i n e t r i p ho sph a t a s e : Pharmacological , p h y s i o l o g i c a l and biochemical a s p e c t s . Pharmacol R e v . 2 7 , 3-134. S e i f e n , E . (1974). Evidence f o r p a r t i c i p a t i o n o f catecholamines i n c a r d i a c a c t i o n o f ouabain. E u r . J . P h a r m a c o l . 2 6 , 115-118. Sharma, V. K . , and Banerjee, S. P. (1979). Regeneration of [3H]ouabain binding t o (Na++K+) -ATPase i n chemically sympathectomized c a t p e r i p h e r a l organs. Mol. P h a r m a c o l . 1 5 , 35-42. Sharma, V. K . , and Banerjee, S. P. (1980). Ouabain s t i m u l a t i o n of n o r a d r e n a l i n e t r a n s p o r t i n guinea p i g h e a r t . N a t u r e (London) 2 8 6 , 817-819. Smith, T. W . , and Braunwald, E . (1980). The management of h e a r t f a i l u r e . In "Cardiovascular Diseases" (E. Braunwald, ed. ) , pp. 509-570. Saunders, P h i l a d e l p h i a . Smith, T. W., Wagner, H . , J r . , and Young, M. (1974). Cardiac g l y c o s i d e i n t e r a c t i o n w i t h s o l u b i l i z e d myocardial sodium- and Mol Pharmacol potassium-dependent adenosine t r i p h o s p h a t a s e . 10, 626-633. Somberg, J. C . , and Smith, T. W. (1979). L o c a l i z a t i o n o f t h e n e u r a l l y mediated arrhythmogenic p r o p e r t i e s of d i g i t a l i s . S c i e n c e 2 0 4 , 321-323. Somberg, J. C . , R i s l e r , T . , and Smith, T. W. (1978). Neural f a c t o r s i n d i g i t a l i s t o x i c i t y : P r o t e c t i v e e f f e c t of C - 1 s p i n a l cord t r a n s e c t i o n . Am. J . p h y s i o l . 2 3 5 , H531-H536.

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Somberg, J. C., B a r r y , W. H., and Smith, T. W. (1981). D i f f e r i n g s e n s i t i v i t i e s of P u r k i n j e f i b e r s and myocardium t o i n h i b i t i o n of monovalent c a t i o n t r a n s p o r t by d i g i t a l i s . J. C l i n . I n v e s t . 67, 116-123. Weingart, R., Kass, R. S . , and T s i e n , R. W. (1978). Is d i g i t a l i s i n o t r o p y a s s o c i a t e d w i t h enhanced slow inward calcium curr e n t ? Nature (London) 273, 389-392. Electromechanical coupling i n h e a r t muscle. Winegrad , S . (1979) In "Handbook of Physiology" (R. M. Berne, N. S p e r e l a k i s , and S. R. Geiger , e d s . ) , 2nd e d . , S e c t . 2, Vol. I , p p . 393-428. Am. P h y s i o l . SOC., Washington, D.C.

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CURRENT TOPICS IN MEMBRANES AND TRANSPORT, VOLUME 19

Effects of Sodium Pump Inhibitionon Contraction in Sheep Cardiac Purkinje Fibers D. A. EZSNER Department of Physiology University College London London, United Kingdom

W. J . LEDERER Depanment of Physiology University of Maryland Baltimore, Maryland

R. D. VAUGHAN-JONES Department of Pharmacology Oxford, United Kingdom

I.

INTRODUCTION AND METHODS

I t i s w e l l e s t a b l i s h e d t h a t i n h i b i t i n g t h e sodium pump i n c r e a s e s t h e f o r c e of c o n t r a c t i o n o f c a r d i a c muscle. T h i s e f f e c t a p p e a r s t o be mediated v i a an i n crease o f i n t r a c e l l u l a r sodium a c t i v i t y , a i a . One t h e o r y f o r t h e mechanism of t h i s e f f e c t i n v o l v e s i a Na/Ca exchange ( R e u t e r and S e i t z , 1 9 6 8 ) . I n c r e a s i n g d N a would p r o d u c e an i n c r e a s e o f C a i n f l u x and d e c r e a s e C a e f f l u x t h r o u g h s u c h an e x c h a n g e , l e a d i n g t o i n c r e a s e d c o n t r a c tion. I n o r d e r t o i n v e s t i g a t e t h e mechanism of t h e pos i t i v e i n o t r o p i c e f f e c t s o f a r a i s e d d i a , one n e e d s t o measure a1 and t e n s i o n s i m u l t a n e o u s l y . As w e l l a s . i n Na v e s t i g a t i n g t h e s t e a d y - s t a t e r e l a t i o n s h i p between a i a and t e n s i o n , i t i s a l s o o f i n t e r e s t t o c h a r a c t e r i z e t h e e f f e c t s o f sydden c h a n g e s of a 1 The o n l y f e a s i b l e way t o measure a1 w i t h adequate N a t i m e r e s o l u t i o n i s w i t h a n ion-selec!?ve m i c r o e l e c t r o d e . W e used r e c e s s e d - t i p N a - s e n s i t i v e m i c r o e l e c t r o d e s (Thomas, 1 9 7 8 ) . The exp e r i m e n t s w e r e performed on v o l t a g e - c l a m p e d s h e e p c a r d i a c

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F i g . 1 . S i m u l t a n e o u s measurement of c u r r e n t , aid, tension, and vol tage-clamped membrane p o t e n t i a l . A 500-msec d e p o l a r i z i n g p u l s e was a p p l i e d f r o m the h o l d i n g p o t e n t i a l ( - 6 8 mV) t o -35 mV a t 0.1 Hz t o e l i c i t a t w i t c h . F r o m Eisner e t a l . ( 1 9 8 0 ) .

P u r k i n j e f i b e r s . F u r t h e r d e t a i l s of t h e methods u s e d have a l r e a d y a p p e a r e d ( E i s n e r and L e d e r e r , 1979a; E i s n e r e t a l . , 1 9 8 1 ) . The use of t h e v o l t a g e clamp makes i t p o s s i b l e t o i n v e s t i g a t e t h e e f f e c t s of changes of aBa w i t h o u t s e c o n d a r y e f f e c t s due t o changes of membrane p o t e n t i a l . Sodium pump a c t i v i t y w a s c o n t r o l l e d by changing t h e e x t e r n a l rubidium c o n c e n t r a t i o n (K-free sol u t i o n s were used t h r o u g h o u t ) .

SODIUM PUMP INHIBITION IN SHEEP CARDIAC PURKINJE FIBERS

11.

887

RESULTS AND D I S C U S S I O N

F i g u r e 1 shows s i m u l t a n e o u s measurements o f i n t r a c e l l u l a r N a a c t i v i t y , a h a , t e n s i o n and c u r r e n t i n a voltage-clamped s h e e p c a r d i a c P u r k i n j e f i b e r . The sodium pump w a s i n h i b i t e d by r e d u c i n g [ % l o from 1 0 t o 0 mM. T h i s produced a r i s e o f a 1 ( c f . D e i t m e r and E l l i s , 1978) and a l s o i n c r e a s e d Na t w i t c h and t o n i c t e n s i o n . Following t h i s e x p o s u r e t o Rb-free s o l u t i o n , [RbIo was i n c r e a s e d t o 4 mM r e s u l t i n g i n a decrease of a1 and both t w i t c h and t o n i c t e n s i o n . A t t h e same t i m e c u r r e n t t r a c e shows a peak of outward c u r r e n t which d e c a y s away. T h i s c u r r e n t t r a n s i e n t i s t h e e l e c t r o g e n i c Na-pump c u r r e n t t r a n s i e n t ( E i s n e r and L e d e r e r , 1979b; Gadsby and C r a n e f i e l d , 1 9 7 9 ) . A n a l y s i s of r e c o r d s s u c h a s F i g . 1 shows t h a t b o t h a i a and t h e c u r r e n t decay exp o n e n t i a l l y upon r e a c t i v a t i n g t h e N a pump w i t h rubidium. T h e h a l f - t i m e o f d e c a y , o f c u r r e n t i s always s i m i l a r t o t h a t o f t h e decay of a 1 ( E i s n e r et al., 1 9 8 1 ) . The r e l a t i o n s h i p @tween a N1 a and t e n s i o n i s s t u d i e d i n more d e t a i l i n F i g . 2 . Reducing [Rb], t o z e r o a g a i n produces a n i n c r e a s e o f p h a s i c t e n s i o n . F i g u r e 2 B shows p h a s i c t e n s i o n a s a f u n c t i o n of a 1 d u r i n g t h e e x p o s u r e It is clearN$hat t h e twitch tent o Rb-free s o l u t i o n . s i o n i s a n o n l i n e a r , f u n c t i o n of a i There i s v e r y l i t t l e t e n s i o n a t a i a v a l u e s o f 182s t h a n a b o u t 6 mM. I n c r e a s i n g a a above t h i s l e v e l i s a s s o c i a t e d w i t h a s t e e p rise o t e n s i o n . T h i s n o n l i n e a r r e l a t i o n s h i p between t e n s i o n and a 1 c o n t r a s t s w i t h t h e r e p o r t o f L e e et a i . ( 1 9 8 0 ) . The88 workers found t h a t t e n s i o n w a s a l i n e a r f u n c t i o n o f a k a d u r i n g N a pump i n h i b i t i o n by d i hydroouabain. W e found a n o n l i n e a r r e l a t i o n s h i p e v e n under comparable c o n d i t i o n s t o t h o s e u s e d by L e e e t a l . Although t h e r e a r e many p o s s i b l e e x p l a n a t i o n s f o r t h i s nonlinear r e l a t i o n s h i p , an a t t r a c t i v e p o s s i b i l i t y i s b a s e d upon t h e known p r o p e r t i e s of N a / C a exchange i n o t h e r t i s s u e s s u c h as t h e s q u i d axon. A r e c e n t model ( M u l l i n s , 1977) s u g g e s t s t h a t f o u r N a and one c a l c i u m i o n must complex w i t h t h e c a r r i e r f o r t r a n s p o r t t o OCc u r . I n t h i s . c a s e t h e c a l c i u m i n f l u x w i l l be a s t e e p T h i s might u p d e r l i e t h e s t e e p r e l a f u n c t i o n of a k a . I t i s , however, t i o n s h i p between t e n s i o n and a',. e q u a l l y p o s s i b l e t h a t i n c r e a s e i loading of i n t r a c e l l u l a r c a l c i u m s t o r e s o r a n i n c r e a s e of t h e calcium c u r r e n t c a n a l s o c o n t r i b u t e t o t h e s t r o n g dependence of t e n s i o n o n

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a1

aia

Na' W e have a l s o examined t h e r e l a t i o n s h i p between and t e n s i o n d u r i n g t h e r e a c t i v a t i o n o f t h e N a pump. The c o m p l i c a t e d r e s u l t o b t a i n e d can be s e e n i n F i g . 1. When t h e N a pump i s i n h i b i t e d , b o t h a k a and t e n s i o n rise

888

D. A. EISNER eta/.

5 min

0

4

8

12

i and twitch tension. The relationship between a ( A ) E f f e c t o f changing [Rb], from 10 t o o m~ on a i a and tension. Throughout the experiment a depolarizing pulse was applied a t 0 . 1 Hz from the holding p t e n t i h l (-70 mV) t o - 3 3 mV. The arrow denotes an a r t i f a c t due t o change o f solution l e v e l . ( B ) Twitch tension a s a function o f a i a during the exposure t o 0 [RbIo. Data from ( A ) . From Eisner e t al. ( 1 9 8 1 ) . F i g . 2.

SODIUM PUMP INHIBITION IN SHEEP CARDIAC PURKINJE FIBERS

889

a t comparable r a t e s . However, on r e a c t i v a t i n g t h e pump w i t h e x t e r n a l R b , t o n i c t e n s i o n f a l l s much f a s t e r t h a n does a k a . I n o t h e r words t h e r e i s a h y s t e r e s i s i n t h e r e l a t i o n s h l p between a’ and t o n i c t e n s i o n . A g i v e n l e v e l of a i a i s a s s o c i g t e d w i t h a h i g h e r l e v e l of t e n s i o n d u r i n g Na-pump i n h i b i t i o n ( R b - f r e e ) t h a n d u r i n g N a pump r e a c t i v a t i o n . Although n o t a p p a r e n t i n F i g . 1 t h e r e . i s sometimes a h y s t e r e s i s between t w i t c h t e n s i o n although t h e h y s t e r e s i s f o r t o n i c tension is more and ramatic. T h i s h y s t e r e s i s i s n o t e a s i l y e x p l a i n e d on a N a / C a exchange model. On s u c h a scheme t e n s i o n s h o u l d f a l l e i t h e r a t t h e same r a t e o r more s l o w l y t h a n a i a . There a r e s e v e r a l p o s s i b l e e x p l a n a t i o n s f o r t h e o b s e r v e d hysteresis. A t r i v i a l p o s s i b i l i t y i s t h a t t h e N a a c t i v i t y s e e n by t h e N a / C a exchange changes more q u i c k l y t h a n t h a t r e c o r d e d by t h e N a e l e c t r o d e . It is possible that N a i n a subsarcolemmal s p a c e exchanges much more r a p i d l y than t h e bulk, cytoplasmic afi This explanation is, however, made less c r e d i b l e by t8e e x p e r i m e n t o f F i g . 1 which d e m o n s t r a t e s t h a t t h e e l e c t r o g e n i c N a pump c u r r e n t h a s t h e same k i n e t i c s as aka measured by t h e e l e c t r o d e . T h i s s u g g e s t s t h a t t h e N a pump sees t h e same a h a a s t h e e l e c t r o d e and t h e r e f o r e t h a t Na n e a r t h e membrane changes i n p h a s e w i t h t h a t r e c o r d e d by t h e e l e c t r o d e . I t i s t h e r e f o r e u n l i k e l y t h a t t h e Na/Ca exchange i s cont r o l l e d by a d i f f e r e n t N a a c t i v i t y from t h a t r e c o r d e d . An a l t e r n a t i v e p o s s i b i l i t y i s b a s e d on t h e o b s e r v a t i o n t h a t i n c r e a s e s of a1 produced by N a pump i n h i b i t i o n l e a d t o a n i n t r a c e l y g l a r a c i d i f i c a t i o n ( D e i t m e r and E l l i s , 1 9 8 0 ) . T h i s a c i d i f i c a t i o n . w i . 1 1 r e d u c e t h e amount ( F a b i a t o and Fabiato, of t e n s i o n developed a t a g i v e n a1 1 9 7 8 ) . I t i s t h e r e f o r e p Q s s i b l e % a t t h e r e i s no hyst e r e s i s between and a i a . In t h i s case the hysteresis between a 1 and t e n s i o n would r e f l e c t changes of t h e s e n s i t i v i t y 8? t h e c o n t r a c t i l e p r o t e i n s t o calcium. Direct measurements of a C1 a u s i n g e i t h e r m i c r o e l e c t r o d e s o r a e g u o r i n w i l l be r e q u i r e d t o s e t t l e t h i s problem.

.

ata

REFERENCES

Deitmer, J. W . , a n d E l l i s , D (1978). The i n t r a c e l l u l a r sodium act i v i t y o f c a r d i a c P u r k i n j e f i b r e s d u r i n g i n h i b i t i o n and rea c t i v a t i o n of t h e Na-K pump. J. P h y s i o l . (London) 284, 241259. Deitmer, J . W . , and E l l i s , D. (1980). I n t e r a c t i o n s between t h e r e g u l a t i o n of t h e i n t r a c e l l u l a r pH a n d sodium a c t i v i t y of sheep c a r d i a c Purkinje f i b r e s . J. P h y s i o l , (London) 204, 471-488.

D. A. EISNER eta/.

890

E i s n e r , D. A . , and L e d e r e r , W. J. (1979a). I n o t r o p i c and arrhythmogenic e f f e c t s o f potassium d e p l e t e d s o l u t i o n s on mammalian c a r d i a c muscle. J. P h y s i o l . (London) 284, 255277. E i s n e r , D. A . , and L e d e r e r , W. J. (1979b). The r o l e o f t h e sodium pump i n t h e e f f e c t s o f p o t a s s i u m d e p l e t e d s o l u t i o n s on J. P h y s i o l . (London) 294, 279mammalian c a r d i a c muscle. 301. E i s n e r , D. A . , L e d e r e r , W. J . , and Vaughan-Jones, R. D. ( 1 9 8 0 ) . E l e c t r o g e n i c sodium pumping i n c a r d i a c muscle: Shultaneo u s measurement of i n t r a c e l l u l a r sodium a c t i v i t y , membrane J. P h y s i o l . (London) 3 0 0 , 42P-43P. c u r r e n t and t e n s i o n . E i s n e r , D. A., L e d e r e r , W. J., and Vaughan-Jones, R. D. (1981). The dependence of sodium pumping and t e n s i o n on i n t r a c e l l u l a r sodium a c t i v i t y i n voltage-clamped s h e e p P u r k i n j e J. P h y s i o l . (London) 3 1 7 , 163-187. fibres. F a b i a t o , A., and F a b i a t o , F. (1978). E f f e c t s of p H o n t h e myof i l a m e n t s and t h e s a r c o p l a s m i c r e t i c u l u m o f s k i n n e d cells from c a r d i a c and s k e l e t a l muscles. J. P h y s i o l . (London) 276, 233-255. Gadsby, D. C ., and C r a n e f i e l d , P. F. ( 1 9 7 9 ) . D i r e c t measurement o f changes i n sodium pump c u r r e n t i n c a n i n e cardiac P u r k i n j e f i b r e s . Proc. N a t l . Acad. S c i . USA 76, 1783-1787. Lee, C. O . , Kang, D. H . , Sokol, J. H . , and L e e , K. S . ( 1 9 8 0 ) . Rel a t i o n between i n t r a c e l l u l a r N a i o n a c t i v i t y and t e n s i o n of s h e e p c a r d i a c P u r k i n j e f i b r e s exposed t o dihydro-ouabain. B i o p h y s . J. 29, 315-330. J . Gen. M u l l i n s , L . J. ( 1 9 7 7 ) . A mechanism f o r Na/Ca t r a n s p o r t . Physiol 70, 681-695. R e u t e r , H . , and S e i t z , H. ( 1 9 6 8 ) . The dependence of calcium e f flux from c a r d i a c muscle on t e m p e r a t u r e and i o n i c composiJ. P h y s i o l . (London) 1 9 5 , 451-470. tion. Thomas, R. C. (1978). " I o n - S e n s i t i v e I n t r a c e l l u l a r M i c r o e l e c t r o d e s : How t o Make and U s e Them." Academic Press, New York.

.

CURRENT TOPICS IN MEMBRANESAND TRANSPORT,VOLUME 19

Quantitative Evaluation of rH] Ouabain Binding to Contracting Heart Muscle, Positive Inotropy, Na,K-ATPase Inhibition, and @Rb+ Uptake in Several Species ERLAND ERDMANN, LDVDSAY BROWN, KARL WERDAN, AND WOLFGANG KRA WIElZ Medizinische KIinik I Universitat Miinchen Klinikum Grosshadern Miinchen, Federal Republic of Germany

I.

INTRODUCTION

Na,K-ATPase, s u p p o s e d l y t h e r e c e p t o r enzyme f o r t h e p h a r m a c o l o g i c a l e f f e c t s of c a r d i a c g l y c o s i d e s , i s s p e c i f i c a l l y i n h i b i t e d by c a r d i a c g l y c o s i d e s (Akera and Brody, 1 9 7 8 ) . E x p e r i m e n t a l e v i d e n c e f o r t h e i m p o r t a n c e of a c a r d i a c g l y c o s i d e - i n d u c e d i n h i b i t i o n of t h e Na/K pump i n t h e i n t a c t c a r d i a c p r e p a r a t i o n i s c o n t r o v e r s i a l (Noble, 1 9 8 0 ) . C a r d i a c g l y c o s i d e s i n c o n c e n t r a t i o n s causing p o s i t i v e inotropy are s a i d t o i n h i b i t Na/K t r a n s p o r t a n d Na,K-ATPase i n v i v o (Akera and Brody, 1 9 7 8 ) , s t i m u l a t e t h e enzyme (Ghysel-Burton a n d G o d f r a i n d , 1 9 7 9 ) , o r n o t t o a f f e c t t h e N a pump (Rhee e t al., 1 9 8 1 ) . I n r a t h e a r t t h e r e i s a s e r i o u s d i s c r e p a n c y between pos i t i v e i n o t r o p y (which c o i n c i d e d w i t h a s p e c i f i c b i n d i n g of [3H]ouabain) and i n h i b i t i o n of Na,K-ATPase o r 86Rb u p t a k e (which w a s d e t e c t a b l e o n l y i n 50- t o 1 0 0 - f o l d h i g h e r c o n c e n t r a t i o n s , when a r r h y t h m i a o r c o n t r a c t u r e o c c u r r e d ) (Erdmann et al., 1 9 8 0 ) . A s t h e r a t i s known t o be r a t h e r i n s e n s i t i v e t o t h e e f f e c t s of c a r d i a c g l y c o 891

Copyright Q 1983 by Academic Press, Inc. All rights of repmductionin any form reserved. ISBN 0-12-153319-0

ERLAND ERDMANNeta/.

892

sides ( D e t w e i l e r , 1967), we investigated the f i r s t steps of c a r d i a c g l y c o s i d e a c t i o n s i n more s e n s i t i v e s p e c i e s ( g u i n e a p i g and c a t ) , i n o r d e r t o f i n d o u t w h e t h e r N a , K ATPase i s i n h i b i t e d by p o s i t i v e i n o t r o p i c c o n c e n t r a t i o n s of o u a b a i n .

11.

METHODS AND MATERIALS

D e t a i l s have been d e s c r i b e d i n a p r e v i o u s p a p e r (Erdmann et a l . , 1 9 8 0 ) . I n b r i e f , t h e f o l l o w i n g method w a s a d o p t e d : g u i n e a - p i g l e f t a t r i a and c a t r i g h t p a p i l l a r y m u s c l e s were s t i m u l a t e d a t 1 Hz a t 35OC u n d e r a b o u t 1 g t e n s i o n (maximal p r e l o a d ) . A f t e r 60-min e q u i l i b r a t i o n , d i f f e r e n t c o n c e n t r a t i o n s of [3H]ouabain were added. U n l a b e l e d o u a b a i n was u s e d f o r t h e 86Rb+-uptake e x p e r i ments. A f t e r e q u i l i b r a t i o n of o u a b a i n e f f e c t s , 86RbC1 ( 1 . 5 V C i ) w a s added t o 1 0 min. The f o r c e of c o n t r a c t i o n , [3H]ouabain bound, o r 86Rb+ u p t a k e were t h e n measured. Each t i s s u e p r e p a r a t i o n was u s e d f o r o n l y one d r u g conc e n t r a ti o n .

111.

RESULTS

3 [ HIOuabain augments f o r c e of c o n t r a c t i o n i n u i n e a p i g a t r i a i n a c o n c e n t r a t i o n r a n q e of 1 0 - 7 - 2 x 1 0 - 2 M up t o about 5-fold. The s p e c i f i c [ d H ] o u a b a i n b i n d i n g t o t h e c o n t r a c t i n g a t r i a p a r a l l e l s t h e dose-response curve ( F i g . 1 ) . A t maximum p o s i t i v e i n o t r o p y t h e r e i s a b o u t 25% o f t o t a l s p e c i f i c [3H]ouabain bound, i f t h e bound ouabain a t 1 0 - 4 M i s defined a s n o n s p e c i f i c binding. A S c a t c h a r d p l o t ( n o t shown) o f [3H]ouabain b i n d i n g t o cont r a c t i n g guinea-pig a t r i a a t equilibrium c o n d i t i o n s ( i . e . , a f t e r f o r c e of c o n t r a c t i o n r e m a i n s s t a b l e f o r t h a t r e s p e c t i v e [3H]ouabain c o n c e n t r a t i o n ) shows a t l e a s t two d i f f e r e n t b i n d i n g s i t e s f o r t h e c a r d i a c g l y c o s i d e . The h i g h - a f f i n i t y s i t e ( K D = 2 x 1 0 - 6 M I Bmax 2, 0 . 8 pmole [3H]ouabain/mg w e t w t . ) i s s a t u r a t e d t o a b o u t 4 0 % a t maximal i n o t r o p y . The 86Rb+ u p t a k e , d e t e r m i n e d a t e q u i l i b r i u m c o n d i t i o n s , i s n o t i n h i b i t e d by low, i n o t r o p i c conc e n t r a t i o n s of ouabain, b u t i n c o n c e n t r a t i o n s h i g h e r t h a n 1 0 - 6 M (when a r r h y t h m i a o r c o n t r a c t u r e i s s e e n r e g u l a r l y ) . Thus, t h e r e i s [3H]ouabain b i n d i n g t o a h i g h - a f f i n i t y b i n d i n g s i t e i n g u i n e a - p i g a t r i a and i n c r e a s e i n f o r c e of c o n t r a c t i o n a t t h e same o u a b a i n con-

893

SPECIFIC OUABAIN BINDING TO CONTRACTING HEART

Z

0 IV

U

P Z

x

n=6-7 0 10-8

10-7

lo*

10-5

104

OUABAIN CONCENTRATION (M 1

Fig. 1. S p e c i f i c force o f c o n t r a c t i o n a n d l e f t a t r i a . For methods maximal e f f e c t s o c c u r a t 5 x 10-6 M (g6Rb+ u p t a k e

o u a b a i n b i n d i n g and i t s e f f e c t s on 86Rb+ u p t a k e i n c o n t r a c t i n g g u i n e a - p i g see ( E r d m a n n e t a l . , 1 9 8 0 ) . H a l f 4 X 1 0 - 7 M (positive i n o t r o p y ) a n d inhibition). 3H

M ) b u t no i n h i b i t i o n o f c e n t r a t i o n range (10-7-2 x 86Rb+ u p t a k e , which e n s u e s o n l y a t o u a b a i n c o n c e n t r a t i o n s above 2 x 1 0 - 6 M and p o s s i b l y c o n c o m i t a n t w i t h [3H]ouabain b i n d i n g t o a l o w - a f f i n i t y b i n d i n g s i t e . In c a t p a p i l l a r y muscles, t h e t h r e e experimental curves f o r [3H]ouabain b i n d i n g , i n c r e a s e i n f o r c e of c o n t r a c t i o n , and i n h i b i t i o n o f 86Rb+ u p t a k e a r e c o i n c i d e n t a l ( r e s u l t s n o t s h o w n ) . These e x p e r i m e n t s conform t o p r e v i o u s work o f Michael e t a i . ( 1 9 7 9 ) i n c a t h e a r t .

894

IV.

ERLAND ERDMANN eta/.

DISCUSSION

P r e v i o u s e x p e r i m e n t s have shown t h a t t h e r e i s no i n h i b i t i o n of 86Rb' u p t a k e i n e l e c t r i c a l l y s t i m u l a t e d v e n t r i c u l a r s t r i p s o f r a t h e a r t s by p o s i t i v e i n o t r o p i c c o n c e n t r a t i o n s o f 3H o u a b a i n , a l t h o u g h a h i g h - a f f i n i t y b i n d i n g s i t e was p a r t i a l l y s a t u r a t e d by t h e d r u g (Erdmann e t al., 1 9 8 0 ) . O t h e r i n v e s t i g a t o r s r e p o r t e d a c o i n c i d e n c e of p o s i t i v e i n o t r o p y and i n h i b i t i o n o f 86Rb+ u p t a k e i n dog h e a r t (Houghen e t al., 1 9 7 9 ) and c a t h e a r t (Michael et a l . , 1 9 7 9 ) . R e c e n t l y , Rhee e t a l . (1981) r e p o r t e d t h a t Na,K-ATPase a c t i v i t y may n o t be i n h i b i t e d by v e r y low, p o s i t i v e i n o t r o p i c c o n c e n t r a t i o n s of ouabain. I n t h e s e e x p e r i m e n t s , a f t e r i n t r a v e n o u s i n f u s i o n of o u a b a i n i n t h e i n s t r u m e n t e d d o g , sarcolemma was i s o l a t e d from t h e h e a r t , and K+-MFPase a c t i v i t y a s w e l l a s 86Rbf uptake i n h e a r t s l i c e s w a s determined. C r i t i c a l l y , one c o u l d a r g u e t h a t o u a b a i n m i g h t have d i s s o c i a t e d from t h e s p e c i f i c r e c e p t o r s d u r i n g t h e c o u r s e of p r e p a r a t i o n , although t h e authors t r i e d t o avoid t h a t . Under o u r exp e r i m e n t a l c o n d i t i o n s , f o r c e of c o n t r a c t i o n , [3H]ouabain b i n d i n g , and 86Rb+ u p t a k e w e r e d e t e r m i n e d i n t h e same preparations. Thus, a d e c r e a s e i n p e r c e n t a g e o f a c t i v e , o c c u p i e d c a r d i a c g l y c o s i d e r e c e p t o r s may be r u l e d o u t . B e s i d e s t h i s , t h e d i s c r e p a n c y f o u n d between t h e c u r v e s f o r i n c r e a s e i n f o r c e o f c o n t r a c t i o n and i n h i b i t i o n o f 86Rb+ u p t a k e ( a b o u t 1 2 - f o l d a t 5 0 % e f f e c t ) is t o o l a r g e t o be due t o e x p e r i m e n t a l e r r o r s . F u r t h e r m o r e , t h e r e w a s no s u c h d i s c r e p a n c y i n c a t p a p i l l a r y m u s c l e s i n a g r e e m e n t w i t h t h e work of M i c h a e l e t al. ( 1 9 7 9 ) . Thus w e c o n c l u d e t h a t , a l t h o u g h i n h i b i t i o n o f a c t i v e 86Rb' u p t a k e ( o r Na,K-ATPase) (Erdmann et al., 1 9 8 0 ) i s c o n c o m i t a n t w i t h p o s i t i v e i n o t r o p i c e f f e c t s of o u a b a i n i n t h e c a t h e a r t , t h e r e i s a d i s s o c i a t i o n between t h e s e e f f e c t s i n r a t and g u i n e a p i g h e a r t s . The S c a t c h a r d p l o t a n a l y s e s o f s p e c i f i c [3H]ouabain b i n d i n g t o cont r a c t i n g g u i n e a p i g a t r i a are s u g g e s t i v e of two d i f f e r The h i g h - a f f i n i t y r e c e p t o r c e r e n t t y p e s of r e c e p t o r s . t a i n l y i s connected with p o s i t i v e inotropy. The lowa f f i n i t y o u a b a i n - b i n d i n g s i t e i s o c c u p i e d a t o u a b a i n conc e n t r a t i o n s c a u s i n g i n h i b i t i o n of 86Rb+ u p t a k e and arrhythmia.

ACKNOWLEDGMENT

Supported by t h e Deutsche Forschungsgemeinschaft ( E r 6 5 / 4 - 3 ) .

895

SPECIFIC OUABAIN BINDING TO CONTRACTING HEART

REFERENCES

+ +

Akera, T . , and Brcdy, T. M. ( 1 9 7 8 ) . The r o l e o f N a , K -ATPase i n t h e i n o t r o p i c a c t i o n of d i g i t a l i s . P h a r m a c o l . Rev. 2 9 , 187-220. D e t w e i l e r , D. K. ( 1 9 6 7 ) . Comparative pharmacology of c a r d i a c g l y c o s i d e s . Fed. P r o c . Fed. Am. SOC. Exp. B i o l . 2 6 , 1119-1124. Erdmann, E. , P h i l i p p , G . , and Scholz, H . (1980) C a r d i a c glycos i d e r e c e p t o r , (Na+ + K+)-ATPase a c t i v i t y and f o r c e o f cont r a c t i o n i n r a t h e a r t . B i o c h e m . P h a r r n a c o l . 2 9 , 3219-3229. Ghysel-Burton, J . , and Godfraind, T. ( 1 9 7 9 ) . S t i m u l a t i o n and i n h i b i t i o n of sodium pump by c a r d i o a c t i v e s t e r o i d s i n r e l a t i o n t o t h e i r b i n d i n g s i t e s and t h e i r i n o t r o p i c e f f e c t on g u i n e a p i g i s o l a t e d a t r i a . B r . J . P h a r m a c o l . 6 6 , 175-184. Hougen, T. J . , Lloyd, B. L . , and Smith, T. W. (1979). E f f e c t s of i n o t r o p i c and arrhythmogenic d i g o x i n d o s e s and of digoxins p e c i f i c a n t i b o d y on myocardial monovalent c a t i o n t r a n s p o r t C i r c . Res. 4 4 , 23-31. i n t h e dog. Michael, L. H . , Schwartz, A . , and W a l l i c k , E . T. (1979). Nature of t h e t r a n s p o r t a d e n o s i n e triphosphatase-digitalis complex. XIV. Mol. P h a r m a c o l . 1 6 , 155-146. Rhee, H. M., Huang, W . , and A s k a r i , A. (1981). R e l a t i o n s h i p between t h e p o s i t i v e i n o t r o p i c e f f e c t of ouabain and i t s i n h i b i t o r y e f f e c t s on N a + , K + - A T P a s e and a c t i v e t r a n s p o r t of Rb+ i n t h e dog h e a r t . Europ. J . P h a r m a c o l . 7 0 , 273-278.

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CURRENT TOPICS IN MEMBRANES AND TRANSPORT, VOLUME 19

Contractile Force Effects of Low Concentrations of Ouabain in Isolated Guinea Pig, Rabbit, Cat, and Rat Atria and Ventricles G. GRUPP, I. L. GRUPP, J. GHYSEL-BURTON, T. GODFRAIND, A. DE POVER, AND A. SCHWAR7Z Departments of Pharmacology and Cell Biophysics Physiology, and Medicine University of Cincinnati College of Medicine Cincinnati, Ohio and Universiti Catholique de Louvain Bruxelles, Belgium

I.

INTRODUCTION

I t i s w e l l e s t a b l i s h e d t h a t a c t i v e t r a n s p o r t of Na,K-ATPase a c r o s s c e l l membranes i s c a t a l y z e d by N a , K - A T P a s e (Schwartz e t a l . , 1975) and t h a t t h e b i n d i n g of o u a b a i n t o i s o l a t e d N a , K - A T P a s e and t h e s u b s e q u e n t i n h i b i t i o n of enzyme a c t i v i t y a r e d i r e c t l y rel a t e d (Wallick et a l . , 1 9 7 9 ) The r e l a t i o n s h i p between t h e s e e v e n t s and a p o s i t i v e i n o t r o p i c a c t i o n o n h e a r t m u s c l e i s s t i l l t h e s u b j e c t of c o n s i d e r a b l e and i n c r e a s i n g c o n t r o v e r s y . The p o s s i b i l i t y t h a t two o r more s i t e s f o r d i g i t a l i s b i n d i n g e x i s t i n c a r d i a c sarcolemma e i t h e r a s s o c i a t e d , or n o t , w i t h t h e Na,K-ATPase, h a s a l s o been p r o m u l g a t e d for y e a r s ( G o d f r a i n d and GhyselB u r t o n , 1 9 8 0 ) . S t i m u l a t i o n of N a , K - A T P a s e , t r a n s i e n t d e c r e a s e i n i n t r a c e l l u l a r sodium, s t i m u l a t i o n of 42K+ u p t a k e ( G o d f r a i n d and Ghysel-Burton, 1980) and more n e g a t i v e r e v e r s a l p o t e n t i a l s have been r e p o r t e d w i t h v e r y low c o n c e n t r a t i o n s of g l y c o s i d e s ( 1 0 - 1 0 t o 10-8 M ouabain)

.

.

897

Copyright 0 1983 by Academic Press, Inc. A I Irights of reproduction in any form reserved. ISBN 0-12-153319-0

E f f e c t s of Low Doses of Ouabain a f t e r 1 5 m i n Exposure on C o n t r a c t i l e F o r c e of A t r i a l and V e n t r i c u l a r Myocardium and on Na, K-ATPase

TABLE I .

Ouabain Species

Control

N

C o n t r a c t i l e f o r c e (gm) Guinea I a t e pig IIa IIIa

IF Rabbit I a Cat Ia Guinea I b pig IIb IIIb Rabbit I b Cat ~b

7 5 4 12 7 5

0.81 0.53 0.81 0.77 0.76 0.87

? 0.04

7 5

0.51 0.51 0.52 0.42 0.81

f 0.03 f 0.06 f 0.03 f 0.04 2 0.04

4 7 5

2 0.04 2 0.06 2 0.04 2 0.07 f 0.05

0.77 0.51

0.04 0.08

0.74 ? 0.07 0.86 2 0.05 0.50 f 0.03 0.50 2 0.06 0 . 4 1 2 0.04 0.80 ? 0.03

Na,K-ATPase a c t i v i t y ( % of c o n t r o l ) 1 6 98.5 f 3 . 3 % 97.7 2 3 . 0 % Purified sheep kidney 6 100% Guinea p i g

0 . 6 8 f 0.06" 0 . 5 1 ? 0.04

0.84 2 0.06 0.69 2 0 . 0 5 * 0.88 o.o7*

*

1 . 2 9 2 0.05* 1 . 3 8 2 0.15* 1.44 2 0.12"

0 . 7 1 ? 0.06 0.84 f 0 . 0 4

0.72 f 0.06 1 . 0 2 0.07*

1.16 ? O . l l * 1 . 2 8 2 0.07"

0.50 5 0.03 0.51 0.06 0.42 2 0.04 0.79 f 0.04

0.58 0.61 0.59 0.44 0.94

9 5 . 8 25.3%

9 7 . 5 26.9%

70.2 +0.57%

13.7 io.49%

100%

96.7 21.6%

91.6 f 2 . 1 %

49.2 f 1 . 3 %

2 0.04*

0.71 0.51 0.78 0.65 0.73 0.84

2 0.09 f 0.05 f 0.3* f 0.06 2 0.05

0.50 0.51 0.52 0.41 0.79

f 0.03 f 0.06 2 0.04 f 0.05 2 0.03

*

f 0.03"

t 0.05" 2 0.04*

2 0.04 2 0.05

0.88 1.08 0.91 0.84 1.13

? 0.03*

f 0.13* 2 0.07" 2 0.13* 2 0.13*

heart

a L e f t a t r i a l s t r i p s i n 5 . 9 mM K+, 2 mM Ca2+, 1.2 mM M g 2 + . b p a p i l l a r y m u s c l e s i n 5 . 9 m~ K+, 2 m~ ~ a 2 + ,1 . 2 m~ ~ g 2 + . CWhole l e f t a t r i a i n 6 mM K', 1 . 8 mM Ca2+, 0 . 1 0 5 mM M g 2 + . dNumber of e x p e r i m e n t s ; v a l u e s a r e m e a n s f SEM; *, s i g n i f i c a n t l y d i f f e r e n c e f r o m control ( p < . O 5 ) . e l , 37OC, 9 0 - m i n c o n t r o l ; 11, 37'C, ZOO-min control; 111, 3OoC, 1ZO-min control; I V , 3OoC, 3 0 - m i n

control

.

ERLAND ERDMANN eta/.

899

11.

RESULTS AND DISCUSSION

W e f i n d t h a t a t 30' and 37OC, a t c o n c e n t r a t i o n s of 5 . 9 m~ ,'K 0 . 9 , 1 . 8 2 , and 2.5 m~ C a 2 + , and 1 . 2 and 0.105 m~ Mg2+, low c o n c e n t r a t i o n s of o u a b a i n (10-10, 1 0 - 9 , and 10-8 M ) have no e f f e c t on c o n t r a c t i l e f o r c e o r d F / d t of p a p i l l a r y m u s c l e s o f g u i n e a p i g , r a b b i t , and c a t ( T a b l e I ) . Higher c o n c e n t r a t i o n s of o u a b a i n ( 1 0 - 7 M and h i g h e r ) p r o d u c e i n t h e s e v e n t r i c u l a r p r e p a r a t i o n s t h e e x p e c t e d i n c r e a s e ( w i t h o u t a t r a n s i e n t decrease) i n c o n t r a c t i l e f o r c e , w i t h subsequent development of t o x i c i t y a t c o n c e n t r a t i o n s of 10-6 M and h i g h e r . I n a t r i a l myocardium of t h e t h r e e s p e c i e s , t h e r e s u l t s a r e more complex. I n s t r i p s p r e p a r e d from l e f t a t r i a , low d o s e s ( 1 0 - 1 0 , 1 0 - 9 , and 10-8 M ) o f o u a b a i n produced a p p a r e n t n e g a t i v e i n o t r o p i c e f f e c t s which w e r e shown t o be a n e x t e n s i o n o f a c o n t i n u o u s n a t u r a l d e c l i n e o f c o n t r a c t i l e force (Fig. 1 ) . This decline w a s particul a r l y s i g n i f i c a n t a t 37OC and w a s t h e same i n t h e p r e s e n c e o r a b s e n c e of 10-10 t o 10-8 M o u a b a i n . A t 3OoC no a p p a r e n t d e c l i n e was s e e n ( F i g . 2 ) . I n whole l e f t a t r i a from t h e g u i n e a p i g , however, M ouabain produced a t r a n s i e n t n e g a t i v e i n o t r o p i c e f f e c t when t h e t i s s u e w a s f i e l d s t i m u l a t e d , b u t n o t when s t i m u l a t e d by n e e d l e e l e c t r o d e s . W e o b s e r v e d t h a t when t h e Na+ and C a 2 + c o n t e n t o f t h e t i s s u e w a s h i g h , whole g u i n e a - p i g l e f t a t r i a w e r e less r e s p o n s i v e t o low d o s e s of o u a b a i n . I t i s p o s s i b l e t h a t t h e p r e s e n c e of n e u r a l t i s s u e , cont a i n i n g an isozyme of Na,K-ATPase i n whole a t r i a , i s r e s p o n s i b l e f o r t h e o c c a s i o n a l l y observed negative inot r o p y i n whole a t r i a s t i m u l a t e d w i t h f i e l d s t i m u l a t i o n . P u r i f i e d s h e e k i d n e y Na,K-ATPase w a s i n h i b i t e d by ouabain a t 5 x M and h i g h e r , w h e r e a s lower concent r a t i o n s o f o u a b a i n had no d e t e c t a b l e e f f e c t - - n e i t h e r i n h i b i t i o n n o r s t i m u l a t i o n - - d u r i n g t h e 30-min a s s a y period. The same o b s e r v a t i o n s w e r e made w i t h e n r i c h e d g u i n e a - p i g h e a r t N a , K - A T P a s e ; w e found o n l y i n h i b i t i o n by c o n c e n t r a t i o n s o f o u a b a i n 5 x M and h i g h e r (Table I ) . I n r a t v e n t r i c u l a r s t r i p s , b u t not i n r a t a t r i a , we d i d o b s e r v e p o s i t i v e i n o t r o p i c e f f e c t s o f low c o n c e n t r a t i o n s o f o u a b a i n which were d i f f e r e n t i n t h e i r ED50 from t h e p o s i t i v e i n o t r o p i c effects of high c o n c e n t r a t i o n s . W e found i n r a t v e n t r i c u l a r s t r i p s a t 35' and 37'12, a t 1 and 3 Hz s t i m u l a t i o n r a t e , and i n K r e b s - H e n s e l e i t and Tyrode s o l u t i o n , c u m u l a t i v e d o s e - r e s p o n s e c u r v e s o f o u a b a i n which showed a "low-dose" e f f e c t w i t h a h a l f maximal i n c r e a s e i n c o n t r a c t i l e f o r c e (ED501 of 0 . 5 U M o u a b a i n r e p r e s e n t i n g a b o u t 3 0 % of t h e t o t a l i n o t r o p y , and a " h i g h - d o s e " e f f e c t of 1 9 - t o 35 U M o u a b a i n r e p r e -

900

G.GRUPPetal. -log [Ouabain] 109 8 7 6

-log [Ouoboin] 1 0 9 8 7 6

g 0.8

f

0.6

0.4

_ _ ,,,I o.2 0

0

30

60 90 120 150 180 210 240 270 Minutes

F i g . 1. "Apparent" n e g a t i v e i n o t r o p i c e f f e c t s (upper graph) o f v e r y l o w d o s e s of o u a b a i n i n g u i n e a - p i g a t r i a t h a t d i s a p p e a r when t h e a t r i a a r e e q u i l i b r a t e d f o r 200 m i n ( l o w e r g r a p h ) . T i m e c o u r s e o f the e f f e c t s o f i n c r e a s i n g o u a b a i n c o n c e n t r a t i o n s to M ) on the l e f t a t r i a l a n d r i g h t v e n t r i c u l a r p a p i l l a r y m u s c l e s o f t h e g u i n e a p i g a t 37OC i n Krebs-Henseleit s o l u t i o n . ( 5 . 9 mM K f , 2.0 mM Ca2+, 1 . 2 mM Mg2+, pH 7 . 4 , ) . U p p e r g r a p h : E f f e c t s on l e f t a t r i a l s t r i p s a f t e r 90-min e q u i l i b r a t i o n . Lower g r a p h : E f f e c t s on a t r i a l s t r i p s ( 0 ) and v e n t r i c u l a r p a p i l l a r y m u s c l e s ( 0 ) a f t e r 200-min e q u i l i b r a t i o n .

s e n t i n g a b o u t 7 0 % of t o t a l i n o t r o p y . The o v e r a l l o r combined ED50 w a s 1 6 U M o u a b a i n ( F i g . 3 ) . The main component of t h e p o s i t i v e i n o t r o p i c e f f e c t of o u a b a i n i n r a t v e n t r i c l e , t h e " h i g h - d o s e " e f f e c t , seemed t o be a s s o c i a t e d w i t h i n h i b i t i o n of Na,K-ATPase; t h e ED50 f o r i n o t r o p y w a s v e r y c l o s e t o t h e 150 € o r i n h i b i t i o n of Na,K-ATPase. The low-dose e f f e c t o c c u r r e d a t a concent r a t i o n of o u a b a i n t h a t d o e s n o t i n h i b i t t h e Na,K-ATPase o f t h e r a t h e a r t ; however, t h e b i n d i n g of [ 3 H ] o u a b a i n t o a r a t h e a r t N a , K - A T P a s e membrane p r e p a r a t i o n o c c u r s a t a h i g h - a f f i n i t y s i t e (see S c h w a r t z e t a l . , 1 9 7 5 , i n t h e s e p r o c e e d i n g s ) , which s u g g e s t s t h a t t h e r e c e p t o r f o r t h e "low-dose" e f f e c t i s Na,K-ATPase b u t t h a t t h e enzyme i s n o t i n h i b i t e d . W e f o u n d t h a t t h e "low-dose" e f f e c t c o u l d n o t be removed by a - o r 8 - a d r e n e r g i c b l o c k a d e , by Howr e s e r p i n i z a t i o n , o r by a h i s t a m i n e H 2 a n t a g o n i s t . e v e r , when t h e t i s s u e p r e p a r a t i o n s w e r e washed f o r

901

CONTRACTILE FORCE EFFECTS OF LOW CONCENTRATIONS OF OUABAIN

IOuoboinl M

10-810-~ ~ X I O - ~ [Ouoboinl M

10-9~

Ouoboin I

0

I

60

I

I

120

I

I

180

I

I

24 0

Minutes

F i g . 2 . Absence o f p o s i t i v e or n e g a t i v e i n o t r o p i c e f f e c t s o f v e r y l o w concentrations o f guinea-pig l e f t a t r i a s t r i p s : M (N = 1 2 ) and M o u a b a i n (N = 1 2 ) a t 3OoC effects of i n T y r o d e s o l u t i o n ( 0 . 9 mM Ca2+, 0.105 mM M q 2 + , 6 mM K+). ( ) Low d o s e e x p o s u r e t o 1 0 - 9 and lo-* M o u a b a i n f o r 1 0 0 m i n followed b y increasing doses o f ouabain. ( 0 ) Control o b s e r v a t i o n s f o r 240 m i n w i t h o u t the a d d i t i o n of o u a b a i n (N = 4 ) .

6 0 - 1 2 0 min a f t e r c o m p l e t i o n o f t h e f i r s t d o s e - r e s p o n s e experiment, a second dose-response curve w i t h ouabain r e v e a l e d a b s e n c e o r d i s a p p e a r a n c e o f t h e "low-dose" e f f e c t and a normal " h i g h - d o s e " e f f e c t . The "low-dose" e f f e c t i s apparently r e l a t e d t o pretreatment with o u a b a i n . While i t i s p o s s i b l e t h a t t h e d i s a p p e a r a n c e o f "low-dose" i n o t r o p y i s due s i m p l y t o o c c u p a t i o n of r e c e p t o r s w i t h o u a b a i n , w e d e l i b e r a t e l y washed t h e t i s s u e r e p e a t e d l y f o r up t o 2 h r , which i s c o n s i d e r a b l y beyond t h e c a l c u l a t e d t i m e f o r c o m p l e t e removal o f o u a b a i n from t h i s h i g h - a f f i n i t y s i t e . I t i s also poss i b l e t h a t t h e ''two i n o t r o p i c e f f e c t s " a r e due t o i s o zymes o f N a , K - A T P a s e p r e s e n t i n t h e r a t v e n t r i c l e , as they a r e i n t h e r a t brain.

902

G . GRUPPetal.

RAT RV STRIP (Receptor Memory)

.9 al .8U

.-m c

; .7-

+.

0" .6.5 ,

1~ I -6

10-5 I

, lo-' I

1

Ouabain

F i g . 3. Two c u m u l a t i v e d o s e - r e s p o n s e c u r v e s of o u a b a i n i n the s a m e r a t v e n t r i c u l a r s t r i p s e p a r a t e d b y a 120-min w a s h o u t p e r i o d ( 3 5 ' C , 1 Hz, 5 . 4 mM K+, 1.8 mM C a 2 + , 1 . 0 5 mM Mg2+, pH 7 . 0 ) . ( 0 ) I n i t i a l dose-response c u r v e ( " f i r s t exposure") showing t w o p o s i t i v e i n o t r o p i c e f f e c t s , a l o w - d o s e e f f e c t o f +26% i n o t r o p y w i t h a h a l f - m a x i m a l (ED50) i n c r e a s e o f c o n t r a c t i l e f o r c e ( 4 ) p r o d u c e d b y 0.8 yM o u a b a i n a n d a " h i g h - d o s e " e f f e c t o f +88% i n o t r o p y ( 0 ) Dose-response c u r v e r e p e a t e d a f t e r w i t h a n ED50 o f 20 yM. 1 2 0 m i n washout ("second e x p s u r e " ) showing a b s e n c e o f "low-dose'' e f f e c t a n d s i n g l e " h i g h - d o s e " e f f e c t o f +86% i n o t r o p y a n d ED50 o f 22 yM.

REFERENCES

Godfraind, T . , and Ghysel-Burton, J . ( 1 9 8 0 ) . Independence o f t h e p o s i t i v e i n o t r o p i c e f f e c t of ouabain from t h e i n h i b i t i o n o f t h e h e a r t Na+-K+ pump. P r o c . N a t l . A c a d . S c i . USA 7 7 , 30673069. Schwartz, A . , Lindenmayer, G . E . , and A l l e n , J . C. ( 1 9 7 5 ) . The s o d i u m - p t a s s i u m adenosine t r ipho s p h a t a s e : Pharmacolog i c a 1 , p h y s i o l o g i c a l and b i o c h e m i c a l a s p e c t s . P h a r m a c o l . R e v . 2 7 , 3-134. W a l l i c k , E. T . , Lane, L . K . , and Schwartz, A. ( 1 9 7 9 ) . Biochemical mechanism of t h e sodium pump. A n n u . R e v . P h y s i o l . 4 1 , 397411.

CURRENT TOPICS IN MEMBRANES AND TRANSPORT, VOLUME 19

Difference of Digitalis Binding to Na,K-ATPase and Sarcolemma Membranes I. KUROBANE, D. L. NANDI, ANDG. T. OKITA Department of Pharmacology Northwestern University Medical School Chicago, Illinois

To c o n f i r m o u r p r e v i o u s o b s e r v a t i o n s t h a t i n h i b i t i o n of N a , K - A T P a s e i s n o t a s s o c i a t e d w i t h t h e i n o t r o p i c a c t i o n o f t h e r a p e u t i c c o n c e n t r a t i o n s of d i g i t a l i s ( O k i t a e t a l . , 1973) , we have compared t h e k i n e t i c s of [3H]ouab a i n d i s s o c i a t i o n from h i g h l y p u r i f i e d , s o l u b i l i z e d N a , K - A T P a s e and h i g h l y e n r i c h e d sarcolemma a g a i n s t t h e k i n e t i c s o f washout o f t h e i n o t r o p i c a c t i o n o f o u a b a i n i n canine h e a r t preparations.

I.

DISSOCIATION HALF-LIVES FROM SOLUBILIZED N a , K - A T P a s e AND SARCOLEMMA

Highly p u r i f i e d , s o l u b i l i z e d N a , K - A T P a s e p r e p a r e d from t h e o u t e r m e d u l l a o f c a n i n e k i d n e y s w a s u s e d i n s t e a d of c a r d i a c Na,K-ATPase b e c a u s e i t i s n o t p o s s i b l e a t p r e s e n t t o i s o l a t e h i g h l y p u r i f i e d Na,K-ATPase from cardiac cells. S o l u b i l i z e d Na,K-ATPase w a s incubated w i t h 1 . 2 x 1 0 - 7 M [3H]ouabain ( 0 . 5 u C i / m l ) u n d e r Type I b i n d i n g c o n d i t i o n s ( 1 3 0 mM NaC1, 3 mM MgC12, 2 mM ATP, 903

Copyright 0 1983 hy Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-153319-0

904

I. KUROBANE eta/.

3 0 mM T r i s - H C 1 , pH 7 . 4 ) . A f t e r 30-min i n c u b a t i o n s a t 3OoC, a n e x c e s s amount o f c o l d o u a b a i n (10-3 M ) w a s added. I m m e d i a t e l y a f t e r a n d a t 15-60 min i n t e r v a l s , e a c h sample w a s c o o l e d q u i c k l y and c e n t r i f u g e d a t 4 O C f o r 6 0 min a t 1 4 5 , 0 0 0 g t o remove f r e e and d i s s o c i a t e d o u a b a i n . The p e l l e t was t h e n r e s u s p e n d e d i n t h e medium and t h e r a d i o a c t i v i t y w a s c o u n t e d . P l o t t i n g r a d i o a c t i v i t y o f Na,K-ATPase v e r s u s i n c u b a t i o n t i m e d e m o n s t r a t e d o n l y one e x p o n e n t i a l component w i t h a In order t o d i s s o c i a t i o n h a l f - l i f e of 9 . 0 i 0 . 2 h r . d e t e r m i n e w h e t h e r a m a c r o m o l e c u l a r c o n s t i t u e n t may have been l o s t d u r i n g t h e p u r i f i c a t i o n p r o c e s s of Na,K-ATPase, s i m i l a r d i s s o c i a t i o n e x p e r i m e n t s w e r e c o n d u c t e d on c r u d e microsomes and p a r t i a l l y p u r i f i e d microsomes. A l l p r e p a r a t i o n s g a v e o n l y a s i n g l e e x p o n e n t i a l component which was n o t s i g n i f i c a n t l y d i f f e r e n t from s o l u b i l i z e d Na,KATPase (see T a b l e I ) . The same d i s s o c i a t i o n e x p e r i m e n t a l s o was c o n d u c t e d on h i g h l y e n r i c h e d sarcolemma p r e p a r e d from c a n i n e h e a r t s . P l o t t i n g r a d i o a c t i v i t y of sarcolemma v e r s u s i n c u b a t i o n t i m e d e m o n s t r a t e d two e x p o n e n t i a l components. The h a l f - l i f e of t h e f i r s t component was 1 . 8 i 0 . 1 h r and of t h e s e c o n d component w a s 9 . 3 2 0 . 3 h r . A h e a v i e r d e n s i t y sarcolemma f r a c t i o n a s w e l l a s a 0 . 2 % deoxyc h o l a t e ( D O C ) - t r e a t e d f r a c t i o n a l s o d e m o n s t r a t e d two comp o n e n t s w i t h t h e same h a l f - l i v e s (see T a b l e I ) . F o r t h e D O C - t r e a t e d f r a c t i o n t h e b i n d i n g r a t i o ( s l o w / f a s t compon e n t ) of [3H]ouabain was d e c r e a s e d a f t e r DOC t r e a t m e n t , i n d i c a t i n g s i g n i f i c a n t r e t e n t i o n of t h e f a s t component r e c e p t o r w i t h p a r t i a l loss o f Na,K-ATPase.

11.

I N O T R O P I C ACTION HALF-LIFE

I s o l a t e d c a n i n e v e n t r i c u l a r t r a b e c u l a were i n c u b a t e d i n a m u s c l e b a t h c o n t a i n i n g m o d i f i e d Krebs sol u t i o n (118 mM NaC1, 4 . 7 mM K C 1 , 2 . 1 mM MgC12, 1 . 8 mM C a C 1 2 , 1 . 0 mM NaH2PG4, 11 mM g l u c o s e , 2 5 m N a H C 0 3 ) w i t h c o n t i n u o u s o x y g e n a t i o n ( 9 5 % 02-5% C 0 2 ) . The p r e p a r a t i o n s w e r e s t i m u l a t e d a t 0 . 5 Hz, 15-20% above threshold voltage with f i e l d electric stimulation and 0 . 5 gm r e s t i n g t e n s i o n . A f t e r a 90-120 min e q u i l i b r a t i o n period a t 3OoC, ouabain ( 1 . 2 x M) w a s added t o t h e b a t h . A f t e r 40-60 min of d r u g t r e a t m e n t , washout of t h e i n o t r o p i c e f f e c t w a s i n i t i a t e d w i t h o u a b a i n - f r e e medium, a n d t h e h a l f - l i f e o f t h e i n o t r o p i c e f f e c t was c a l c u l a t e d t o b e 1 . 8 f 0 . 3 h r .

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DIGITALIS BINDING IN Na,K-ATPaseAND SARCOLEMMA MEMBRANES

3 Comparison of [ HIOuabain D i s s o c i a t i o n Half-Lives from Na,K-ATPase P r e p a r a t i o n s and Sarcolemma Membrane and t h e I n o t r o p i c Half-Life of Ouabain

TABLE I .

~~

Preparations

Half - l i f e a ( h r ) F a s t component Slow component

[3H] Ouabain d i s s o c i a t i o n :

Canine kidney p r e p a r a t i o n s Crude microsomes P a r t i a l l y p u r i f i e d microsomes S o l u b i l i z e d Na,K-ATPase Canine h e a r t sarcolemma Highly e n r i c h e d membrane Heavier d e n s i t y f r a c t i o n s DOC-treated h e a v i e r d e n s i t y fractions Inotropic effect: I s o l a t e d canine v e n t r i c u l a r trabecula

10.8 f 0.5 S . E . 9.4 f 0.3 9.0 f 0 . 2 1.8 f 0.1 1 . 7 f 0.2

9.3 f 0.3 9 . 2 f 0.5

1 . 8 f 0.1

8 . 9 f 0.4

1 . 7 i 0.2

aData r e p r e s e n t mean f S E o b t a i n e d f r o m 5 e x p e r i m e n t s f o r each 3H-ouabain d i s s o c i a t i o n d a t a and 1 0 e x p e r i m e n t s f o r t h e i n o t r o p i c d a t a . See t e x t f o r methodology.

111.

SCATCHARD ANALYSES O F SOLUBILIZED Na,K-ATPase

AND

SARCOLEMMA Highly p u r i f i e d , s o l u b i l i z e d c a n i n e k i d n e y N a , K A T P a s e w a s i n c u b a t e d f o r 30 min a t 30°C w i t h v a r y i n g conc e n t r a t i o n s of o u a b a i n ( 1 0 - 8 t o 10-5 M ) c o n t a i n i n g [3H]o u a b a i n ( 2 . 0 u C i / m l ) , u s i n g t h e same p r o t o c o l as f o r t h e previous d i s s o c i a t i o n experiments. Plotting the ratios o f bound t o f r e e [ R A ] / [ A ] v e r s u s bound [RA] d r u g a t d i f f e r e n t ouabain c o n c e n t r a t i o n s demonstrated only one l i n e a r component, i n d i c a t i n g o n e c l a s s of b i n d i n g s i t e s ( K = ~ 1.6 f 0 . 2 ~ n = 4 ) on t h e Na,K-ATPase molecule. P l o t t i n g [RA] v e r s u s o u a b a i n c o n c e n t r a t i o n showed half-maximal b i n d i n g a t 1 . 6 x M. Enzyme a s s a y i n a p a r a l l e l s e r i e s of e x p e r i m e n t s u s i n g t h e same s a m p l e s showed ID50 t o b e 1 . 8 10-7 M . The same S c a t c h a r d a n a l y s i s u n d e r t h e same c o n d i t i o n s b u t w i t h t h e a d d i t i o n of 1 0 m M K C 1 a l s o d e m o n s t r a t e d o n l y one l i n e a r component (KD = 1 . 3 x M ) w i t h half-maximal b i n d i n g a t 2 . 0 x 10-6 M . P a r a l l e l enzyme a s s a y e x p e r i m e n t s demon s t r a t e d a n ID50 a t 2 . 9 x 10-6 M . Scatchard a n a l y s i s a l s o was c o n d u c t e d on h i g h l y e n r i c h e d sarcolemma p r e p a r e d from canine hearts. I n agreement w i t h t h e [3H]ouabain d i s s o -

906

I. KUROBANE eta/.

c i a t i o n h a l f - l i f e d a t a , two l i n e a r components w e r e obt a i n e d ( K D = 0 . 6 - 1.3) x 10-8 M a n d ( 0 . 4 - 1 . 2 ) x 10-6 M ) , i n d i c a t i n g two d i f f e r e n t c l a s s e s (of o u a b a i n - b i n d i n g s i t e s on t h e sarcolemma membrane.

IV.

DISCUSS I O N

A s shown i n T a b l e I , t h e d i s s o c i a t i o n h a l f - l i f e of [ 3 H ] o u a b a i n from h i g h l y p u r i f i e d , s o l u b i l i z e d N a , K - A T P a s e i s v e r y d i f f e r e n t from t h e i n o t r o p i c h a l f - l i f e o f o u a b a i n (9 hr v s . 1 . 8 h r , r e s p e c t i v e l y ) . This very long, singlecomponent h a l f - l i f e w a s a l s o o b s e r v e d i n o t h e r c a n i n e k i d n e y p r e p a r a t i o n s c o n t a i n i n g p a r t i a l l y p u r i f i e d and On t h e o t h e r h a n d , h i g h l y c r u d e microsomal N a , K - A T P a s e . e n r i c h e d sarcolemma p r e p a r e d f r o m c a n i n e h e a r t s g a v e two d i f f e r e n t h a l f - l i f e values. The s h o r t e r h a l f - l i f e i s t h e same a s t h a t f o r t h e i n o t r o p i c a c t i o n , a n d t h e l o n g e r o n e i s s i m i l a r t o t h a t f o r highly purifield s o l u b i l i z e d N a , K ATPase. Scatchard analyses of highly p u r i f i e d , s o l u b i l i z e d N a , K - A T P a s e bound u n d e r Type I b i n d i n g c o n d i t i o n s w i t h o r w i t h o u t K C 1 p l u s t h e enzyme i n h i b i t i o n d a t a i n d i c a t e t h a t ouabain binds t o t h e a c t i v e i n h i b i t o r y s i t e of t h e Na,K-ATPase molecule. On t h e o t h e r h a n d , S c a t c h a r d a n a l y s e s o f sarcolemma membrane p r e p a r a t i o n s h a v e d e m o n s t r a t e d two l i n e a r c o m p o n e n t s w i t h o n e component h a v i n g a K,, v a l u e s i m i l a r t o t h a t o f h i g h l y p u r i f i e d , Therefore, present findings s o l u b i l i z e d Na,K-ATPase. s u g g e s t t h a t t h e p h a r m a c o l o g i c a l r e c e p t o r f o r t h e pos i t i v e i n o t r o p i c a c t i o n of o u a b a i n i s d i f f e r e n t from t h e i n h i b i t o r y s i t e of t h e Na,K-ATPase molecule therapeutic concentrations.

ACKNOWLEDGMENT

S u p p o r t e d i n p a r t b y N H L B I G r a n t HL-18598 and t h e Chicaqo Heart Association A81-18.

REFERENCE

O k i t a , G . , R i c h a r d s o n , F . , and R o t h - S c h e c h t e r , B . L. ( 1 9 7 3 ) . Dis s o c i a t i o n of the p o s i t i v e i n o t r o p ic a c t i o n of d i g i t a l i s f r o m i n h i b i t i o n o f sodium and p o t a s s ium-ac t i v a t e d a d e n o s i n e t r iphosphatase. J . P h a r m a c o l . Exp. T h e r . 185, 1-11.

CURRENT TOPICS IN MEMBRANES AND TRANSPORT, VOLUME 19

Pharmacological and Biochemical Studies on the Digitalis Receptor: A Two-Site Hypothesis for Positive lnotropic Action M O L D SCHWARlZ, INGRID GRUPP, ROBERTJ. ADAMS, TREVOR POWELL,' GUNTER GRUPP, AND E. T. WALLICK Departments of Pharmacology and Cell Biophysics University of Cincinnati College of Medicine Cincinnati. Ohio

I.

INTRODUCTION

The mechanism of t h e p o s i t i v e i n o t r o p i c e f f e c t of d i g i t a l i s g l y c o s i d e s has n o t been f u l l y e l u c i d a t e d . A c a s u a l r e l a t i o n s h i p may e x i s t between t h e b i n d i n g t o and consequent i n h i b i t i o n of Na,K-ATPase ( t h e sodium pump) by d i g i t a l i s and an i n c r e a s e d myocardial c o n t r a c t i l e f o r c e , and/or t h e mechanism may have something t o do w i t h an i n c r e a s e i n a sarcolemmal calcium pool w i t h o u t i n h i b i t i o n of t h e enzyme (Repke, 1 9 6 2 ; Schwartz and Adams, 1 9 8 0 ; Schwartz e t a l . , 1 9 7 5 ) .

' D e p a r t m e n t of P h y s i c s a s A p p l i e d t o M e d i c i n e , M i d d l e s e x H o s p i t a l M e d i c a l S c h o o l , London, England

907

Copyright 0 1983 by Academic Reas, Inc. All rights of reproduction in any form reserved. ISBN 0-12-1533194

908

ARNOLD SCHWARTZetal.

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0.9

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2

0.8

0.5 I

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1 0 - ~1 0 - ~ 1 0 - ~

Ouabain Concentration (MI F i g . 1 . Isometric c o n t r a c t i l e f o r c e , measured i n i s o l a t e d a d u l t r a t r i g h t v e n t r i c u l a r s t r i p s , which were e q u i l i b r a t e d a t 1 g p r e l o a d f o r 1 2 0 m i n i n t y r o d e s o l u t i o n or i n Krebs-Henseleit s o l u t i o n . S t r i p s w e r e e l e c t r i c a l l y p a c e d a t either 1 Hz or 3 Hz v i a p u n c t a t e e l e c t r o d e s a t 20% s u p r a t h r e s h o l d ( 3 - 5 V ) a t 2-msec d u r a tion.

11.

RESULTS AND D I S C U S S I O N

W e now r e p o r t t h a t two d i s t i n c t p o s i t i v e i n o t r o p i c sites f o r ouabain e x i s b i n r a t v e n t r i c u l a r s t r i p s b u t n o t i n a t r i a . The h i g h e r - a f f i n i t y s i t e ( ~ ~ =5 00. 5 P M ) c o r r e l a t e s w i t h [3H]ouabain b i n d i n g t o i n t a c t r a t vent r i c u l a r myocytes and c a r d i a c sarcolemma, b u t n o t w i t h i n h i b i t i o n of Na,K-ATPase a c t i v i t y ( F i g . 1 ) . T h i s "low concentration positive inotropic e f f e c t " represents 3 4 2 5 % ( N = 2 5 ) of t o t a l p o s i t i v e i n o t r o p y , w h e r e a s 0 4 2 9 % ( N = 2 5 ) o c c u r r e d a t o u a b a i n c o n c e n t r a t i o n s between 8 0 pM and 1 6 0 V M o u a b a i n . T h i s " h i g h c o n c e n t r a t i o n e f f e c t " i s c o n s i s t e n t w i t h a n i n h i b i t i o n of t h e Na,K-ATPase. The b i p h a s i c i n o t r o p i c r e s p o n s e i s n o t a f f e c t e d by 6 - a d r e n o c e p t o r b l o c k a d e ( 2 0 pM s o t a l o l ) o r by p r e t r e a t m e n t w i t h r e s e r p i n e . Only a monophasic conc e n t r a t i o n r e s p o n s e c u r v e i s c h a r a c t e r i s t i c of r a t l e f t a t r i a ( E D 5 0 = 4 5 2 9 V M ) and of c a t , r a b b i t , and g u i n e a pig v e n t r i c u l a r p a p i l l a r y muscles o r l e f t a t r i a (Table I ) , which i n d i c a t e s a s i n g l e c l a s s of p o s i t i v e i n o t r o p i c r e c e p t o r s i t e s f o r o u a b a i n . Although a m a j o r i t y of s t u d i e s o f [3H]ouabain b i n d i n g t o v a r i o u s membrane p r e p a r a t i o n s and p u r i f i e d N a , K - A T P a s e r e v e a l a s i n g l e C l a s s of o u a b a i n b i n d i n g s i t e s , s e v e r a l w o r k e r s h a v e r e c e n t l y

909

STUDIES ON THE DIGITALIS RECEPTOR

TABLE I.

P o s i t i v e I n o t r o p i c Action of Ouabain: "Sensitivity"

Species Cat Guinea p i g Rabbit Rat

Species

Atrial myocardiuma

Ventricular myocardiumb

ED50

ED50

0.16 0.16 0.52 55

0.17 0.19 0.55 A = 0.5 B = 20

( W)

(W)

aWhole l e f t a t r i a o r l e f t a t r i a l s t r i p s . bRight v e n t r i c u l a r p a p i l l a r y m u s c l e s o f c a t , guinea p i g , and r a b b i t , r i g h t v e n t r i c u l a r f r e e w a l l s t r i p s of r a t .

o b t a i n e d r e s u l t s which are c o n s i s t e n t w i t h t h e e x i s t e n c e o f two p o p u l a t i o n s o f o u a b a i n b i n d i n g s i t e s . Using c o n c e n t r a t i o n s of l a b e l e d o u a b a i n o f 0 . 1 P M and 0.2 p M , p r o b i t p l o t Co.5 v a l u e s (0.225 t o 0.325 p M ) y i e l d e d an a v e r a g e KD i S D o f 0.125 k 0.090 y M f o r i s o l a t e d m y o c i t e s . F o r c a r d i a c sarcolemma, a K D v a l u e of 0.352 ? 0.09 W M w a s o b t a i n e d . The e s t i m a t e d c a p a c i t y o f t h i s h i g h - a f f i n i t y s i t e (Bmax) i n sarcolemma w a s 8 . 7 k 0.93 pmoles/mg (see F i g . 2). A l o w e r - a f f i n i t y s i t e f o r o u a b a i n b i n d i n g must e x i s t , s i n c e t h e 150 f o r i n h i b i t i o n o f o u r r a t c a r d i a c sarcolemma p r e p a r a t i o n i s a b o u t 50 V M , s i m i l a r t o what w e and o t h e r s have rep o r t e d f o r r a t h e a r t N a , K - A T P a s e (see T a b l e 11). The l o w - a f f i n i t y s i t e ( 5 0 L I M ) f o r enzyme i n h i b i t i o n a g r e e s w e l l with t h e low-affinity site €or p o s i t i v e inotropy ( F i g . 1 ) . W e f e e l t h a t b o t h h i g h - and l o w - a f f i n i t y s i t e s a r e a s s o c i a t e d w i t h t h e N a , K - A T P a s e , p e r h a p s two d i f f e r e n t c o n f o r m e r s , one t h a t i s i n h i b i t e d by o u a b a i n and one t h a t i s n o t i n h i b i t e d .

910

ARNOLD SCHWARTZ eta/.

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C

2 m t

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5

60 40

20

1

t

-_

L--4/

I

a 10-8

(

10-7

I

(

10-6

I

10-5

I

10-4

I

10-3

Unlabelled Ouabain Concentration (MI F i g . 2 . D i s p l a c e m e n t ( b y u n l a b e l e d o u a b a i n ) of [ 3 H-J o u a b a i n b o u n d t o i n t a c t , c a l c i u m - t o l e r a n t v e n t r i c u l a r m y o c y t e s ( 0 ) and v e n t r i c u l a r sarcolemma ( 0) i s o l a t e d f r o m a d u l t r a t myocardium. The c u r v e shown was c a l c u l a t e d f r o m the d i s p l a c e m e n t e q u a t i o n f o r a s i n g l e c l a s s of s i t e s ( A k e r a and Chenq, 1 9 7 7 ) .

TABLE 11.

Na,K-ATPase I n h i b i t i o n by Ouabain: "Sensitivity"

Species Cat

Guinea p i g Rabbit Rat

C a r d i a c Na,K-ATPasea:

Specific

Is0 (UM)

0.03 0.8 1.3 23

aNa,K-ATPase p a r t i a l l y p u r i f i e d f r o m v e n t r i c u l a r myocardium. S p e c i f i c o u a b a i n - i n h i b i t a b l e a c t i v i t y o f enzyme p r e p a r a t i o n s r a n g e d b e t w e e n 20-30 u m o l e s P i / m g / h r , w h i c h a c c o u n t e d f o r m o r e t h a n 80% o f the t o t a l ATPase a c t i v i t y .

REFERENCES

Akera, T . , a n d Cheng, V. J . K . ( 1 9 7 7 ) . A simple method f o r t h e d e t e r m i n a t i o n o f a f f i n i t y and b i n d i n g s i t e c o n c e n t r a t i o n i n receptor binding s t u d i e s . Biochim. B i o p h y s . A c t a 470, 412-42 3.

STUDIES ON THE DIGITALIS RECEPTOR

91 1

Repke, K. H . R. (1962). P r o c . Int. P h a r m a c o l . Meet., lst, 1 9 6 1 VOl. 3 , pp. 47-73. Schwartz, A . , and Adams, R . J . ( 1 9 8 0 ) . S t u d i e s on t h e d i g i t a l i s r e c e p t o r . C i r c . Res. 4 6 , 1154-1160. Schwartz, A . , Lindenmayer, G. E . , and Allen, J. C. (1975). The sodium-potassium adenosine t r i p h o s p h a t a s e : Pharmacological, p h y s i o l o g i c a l and biochemical a s p e c t s . P h a r m a c o l . Rev. 2 7 , 3-134.

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CURRENT TOPICS IN MEMBRANES AND TRANSPORT, VOLUME 19

Hypothesis for the Mechanism of Stimulation of the NdK Pump by Cardiac Glycosides-Role of Endogenous Digitalis-Like Factor T. WDFRAZND, G. CASTMEDA-HERNANDEZ, J. GHYSEL-BURTON, AND A. DE POVER Laboratoire de Pharmacodynamie Gdnirale et de Pharmacologie Universiti Catholique de Louvain Brussels, Belgium

I.

INTRODUCTION

C a r d i a c g l y c o s i d e s e x e r t a b i p h a s i c a c t i o n on t h e a c t i v i t y of t h e Na/K pump i n i s o l a t e d h e a r t p r e p a r a t i o n s . A t h i g h c o n c e n t r a t i o n s t h e y i n h i b i t t h e pump, whereas a t low c o n c e n t r a t i o n s t h e y s t i m u l a t e it. I n g u i n e a p i g l e f t a t r i a i n c u b a t e d a t 3OoC f o r 45 min, d i g o x i n s t i m u l a t e s t h e o u a b a i n - s e n s i t i v e 42K uptake by a b o u t 5 0 % . T h i s e f f e c t wanes a f t e r a prolonged incub a t i o n (Godfraind and Ghysel-Burton, 1 9 7 9 ) . The stimul a t i o n of t h e pump h a s been observed i n s e v e r a l l a b o r a t o r i e s u s i n g d i f f e r e n t methodologies and p r e p a r a t i o n s (see Godfraind, 1 9 8 1 ) . T h i s a c t i o n i s a n t a g o n i z e d by K C 1 and r e q u i r e s an u n s a t u r a t e d l a c t o n e r i n g . I t i s d i f f i c u l t t o r e c o n c i l e t h e o b s e r v a t i o n s on i n t a c t t i s s u e w i t h t h o s e on p u r i f i e d Na,K-ATPase. Indeed, c o n c e n t r a t i o n s of g l y c o s i d e which s t i m u l a t e t h e pump a r e w i t h o u t e f f e c t o r a r e s l i g h t l y i n h i b i t o r y on g u i n e a p i g h e a r t N a , K - A T P a s e p r e p a r a t i o n s ( D e Pover and Godfraind, 1 9 7 9 ) . One of us h a s proposed t h a t t h e 913

Copynght 0 1983 by Academic h s , Inc. All rights of reproduction in any form reserved. ISBN 0-12-153319-0

T. GODFRAIND eta/

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a c t i o n o f low d o s e s o f g l y c o s i d e s s h o u l d b e c o n s i d e r e d a s a derepression r a t h e r than a t r u e stimulation, p o s t u l a t i n g t h e e x i s t e n c e of a s p e c i f i c endogenous rep r e s s o r s u b s t a n c e ( G o d f r a i n d , 1 9 8 0 ) . I n view of t h e d a t a mentioned a b o v e , t h e pump c o u l d be r e p r e s s e d down t o 7 0 % i n g u i n e a p i g a t r i a . R e c e n t s t u d i e s show t h a t t h e endogenous d i g i t a l i s - l i k e f a c t o r c a n be i d e n t i f i e d i n s e v e r a l t i s s u e s (Fishman, 1979; H a u p e r t and Sancho, 1 9 7 9 ; Gruber e t a l . , 1 9 8 0 ) . T h e r e f o r e , w e have examined t h e p o s s i b l e e x i s t e n c e of t h i s f a c t o r i n g u i n e a p i g heart.

11.

METHODS AND RESULTS

T h i r t y grams of g u i n e a p i g h e a r t was homogenized i n 3 0 0 m l of d e i o n i z e d water and c e n t r i f u g e d a t 1 0 0 , 0 0 0 g for 1 hr. The s u p e r n a t e was l y o p h i l i z e d , and t h e d r y m a t t e r w a s suspended i n 5 0 m l of m e t h a n o l . A f t e r cent r i f u g a t i o n , t h e p e l l e t w a s d i s c a r d e d and m e t h a n o l e v a p o r a t e d u n d e r low p r e s s u r e . The r e s i d u e w a s s u s p e n d e d i n 5 m l o f w a t e r and t r e a t e d w i t h 50 ml of p e t r o l e u m benzene. A f t e r removal o f t h e o r g a n i c p h a s e , t h e aqueous p h a s e w a s a p p l i e d t o a 4 0 0 - m l column c o n t a i n i n g A m b e r l i t e MB3 ( F l u k a ) e q u i l i b r a t e d w i t h 4 M p y r i d i n e a c e t a t e . The e l u a t e w a s e v a p o r a t e d u n d e r low p r e s s u r e . The r e s i d u e was suspended i n 5 m l of 0 . 1 M a c e t i c a c i d and a p p l i e d t o a 6 0 0 - m l column c o n t a i n i n g Sephadex G-25 (Pharmacia) e q u i l i b r a t e d w i t h 0 . 1 M acetic a c i d (20 ml/hr). T w o - m i l l i l i t e r s a m p l e s w e r e c o l l e c t e d and assayed f o r r e a c t i v i t y with antidigoxin antibodies ( D i a g n o s t i c P r o d u c t Corp. k i t ) . Peak f r a c t i o n s w e r e p o o l e d , l y o p h i l i z e d , and t a k e n up i n 0 . 5 m l o f w a t e r (water e x t r a c t ) . Water e x t r a c t s i n t e r a c t e d w i t h s p e c i f i c a n t i d i g o x i n a n t i b o d i e s . When d a t a of p e r c e n t a n t i g e n b i n d i n g v e r s u s e x t r a c t c o n c e n t r a t i o n were p l o t t e d o n a s e n i i l o g s c a l e , sigmoidal curves p a r a l l e l t o digoxin standard curves were o b t a i n e d . Two p i c o m o l e s o f e q u i v a l e n t d i g o x i n p e r gram o f w e t t i s s u e were found i n e x t r a c t s . N a , K - A T P a s e from g u i n e a p i g h e a r t was a s s a y e d a c c o r d i n g t o B a i s (1975) i n 0 . 1 m l medium c o n t a i n i n g 3 mM [ Y - ~ ~ P ] A T P (Amersham), 3 mM MgCl2, 3 mM K C 1 , 1 0 0 mM N a C 1 , 1 mM EGTA, and 2 0 mM Tris-maleate (pH 7 . 4 ) . F i f t e e n percent i n h i b i t i o n w a s o b t a i n e d i n t h e p r e s e n c e of 4 nM d i g o x i n e q u i v a l e n t of e x t r a c t . The s a m e e f f e c t was o b t a i n e d w i t h 4 0 nM d i g o x i n . Assuming a mole t o mole c o m p e t i t i o n r a t i o i n radioimmunoassay, it c a n b e a n t i c i p a t e d t h a t t h e f a c t o r i s 1 0 - f o l d more p o t e n t t h a n d i g o x i n .

HYPOTHESIS FOR STIMULATION OF THE NalK PUMP BY CARDIAC GLYCOSIDES

111.

915

DISCUSSION

These r e s u l t s s u g g e s t t h e e x i s t e n c e i n g u i n e a p i g h e a r t o f a d i g i t a l i s - l i k e f a c t o r , which c o u l d b e a An i n t e r a c t i o n p h y s i o l o g i c a l r e p r e s s o r of N a , K - A T P a s e . between t h i s f a c t o r and c a r d i a c g l y c o s i d e s c o u l d a c c o u n t f o r N a / K pump d e r e p r e s s i o n i n s i t u .

REFEReNCES

( 1 9 7 5 ) . A r a p i d and s e n s i t i v e r a d i o m e t r i c a s s a y f o r a d e n o s i n e t r i p h o s p h a t a s e a c t i v i t y u s i n g Cerenkov r a d i a t i o n . A n a l . B i o c h e m . 6 3 , 271-273. D e P o v e r , A . , and G o d f r a i n d , T. ( 1 9 7 9 ) . I n t e r a c t i o n o f o u a b a i n w i t h Na,K-ATPase from human h e a r t and g u i n e a - p i g h e a r t . B i o c h e m . P h a r m a c o l . 28, 3051-0000. Fishman, M. C . ( 1 9 7 9 ) . Endogenous d i g i t a l i s - l i k e a c t i v i t y i n mammalian b r a i n . P r o c . N a t l . A c a d . Sci. USA 76, 4661-4663. G o d f r a i n d , T. ( 1 9 8 0 ) . S t i m u l a t i o n e t i n h i b i t i o n d e l a pompe h sodium par l e s h 6 t 6 r o s i d e s c a r d i o t o n i q u e s . B u l l . A c a d . R. M e d . B e l g . 1 3 5 , 174-192. G o d f r a i n d , T. ( 1 9 8 1 ) . S t i m u l a t i o n and i n h i b i t i o n o f t h e N a , K-pump by c a r d i a c g l y c o s i d e s . H a n d b . E x p . P h a r m a k o l . 5 6 , 381-393. G o d f r a i n d , T . , and Ghysel-Burton, J. ( 1 9 7 9 ) . The c a r d i o a c t i v e p r o p e r t i e s of SC 4453, a d i g o x i n a n a l o g u e w i t h a C17p y r i d a z i n e r i n g . E u r . J. P h a r m a c o l . 6 0 , 337-344. G r u b e r , K. A . , W h i t a k e r , J. M . , and Buckalew, V. M . , Jr. ( 1 9 8 0 ) . Endogenous d i g i t a l i s - l i k e s u b s t a n c e i n plasma o f volume expanded dog. N a t u r e ( L o n d o n ) 287, 743-745. H a u p e r t , G . T . , and Sancho, J . M. ( 1 9 7 9 ) . Sodium t r a n s p o r t i n Proc. N a t l . Acad. S c i . h i b i t o r f r o m b o v i n e hypothalamus. (USA 76, 4658-4660. B a i s , R.

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CURRENT TOPICS IN MEMBRANESAND TRANSPORT, VOLUME 19

lmmunochemical Approaches to the Isolation of an Endogenous Digoxin-Like Factor KENNETH A. GRUBER, JANICE M. WHITAKER AND VARDAMANM . BUCKALEW, JR. Departmentsof Medicine and Physiology and Pharmacology Bowman Gray School of Medicine Winston-Salem, North Carolina

I.

INTRODUCTION

W e have p r e v i o u s l y r e p o r t e d t h e p r e l i m i n a r y i s o l a t i o n of a s u b s t a n c e i n plasma which i n h i b i t s N a , K A T P a s e and c r o s s - r e a c t s w i t h a n t i d i g o x i n a n t i b o d i e s (Gruber et a l . , 1 9 8 0 ) . The i s o l a t i o n p r o c e d u r e f o r t h i s endogenous d i g o x i n - l i k e s u b s t a n c e (endoxin) d i d n o t a l l o w t h e r a p i d p r o c e s s i n g of l a r g e volumes o f plasma, due t o t h e d i f f i c u l t y i n r e p r o d u c i b l y s e p a r a t i n g c r u d e plasma e x t r a c t s on high-performance l i q u i d chromatography ( H P L C ) . The immunological p r o p e r t i e s of endoxin s u g g e s t e d t h e use of immunochemical proc e d u r e s t o r a p i d l y and s p e c i f i c a l l y p u r i f y t h i s f a c t o r from plasma. W e h e r e i n r e p o r t t h e u s e of immunoprec i p i t a t i o n and a f f i n i t y chromatography t o i s o l a t e t h i s p u t a t i v e hormone.

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Copyright @ 1983 by Academic Press,Inc. All rights of reprcduction in any form reserved. ISBN 0-12-1533190

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11.

KENNETH A. GRUBER eta/.

MATERIALS AND METHODS

Plasma from e x t r a c e l l u l a r f l u i d volume-expanded dogs w a s p r e p a r e d a c c o r d i n g t o p r e v i o u s l y d e s c r i b e d p r o c e d u r e s ( G r u b e r and Buckalew, 1 9 7 8 ) , and a l i q u o t s e q u i v a l e n t t o 10 m l o f o r i g i n a l plasma w e r e s u b j e c t e d to Diafiltration t h r o u g h Amicon U M - 1 0 m o l e c u l a r s i e v e membranes w i t h 0 . 0 5 M a c e t i c a c i d . F o u r volumes p e r sample w e r e c o l l e c t e d . The f i l t r a t e was l y o p h i l i z e d Immunochemical i s o l a t i o n w a s by and s t o r e d a t - 4 O C . one of two p r o c e d u r e s .

(1) I m m u n o p r e c i p i t a t i o n was p e r f o r m e d on d i a f i l t r a t e s d i s s o l v e d i n 5 0 0 u 1 of 0 . 0 5 M phosphate b u f f e r A l a r g e excess of goat antidigoxin antibody(pH 7 . 4 ) . c o n t a i n i n g s e r u m w a s added t o e a c h s a m p l e , t h e m i x t u r e a l l o w e d t o i n c u b a t e f o r 2 h r , and t h e a n t i b o d y - a n t i g e n s a m p l e s p r e c i p i t a t e d by t h e a d d i t i o n o f r a b b i t a n t i g o a t IgG f o l l o w e d by a 30-min i n c u b a t i o n and c e n t r i f u g a t i o n a t 2 5 0 0 g . The r e s u l t i n g p e l l e t w a s r e d i s s o l v e d i n 0 . 0 5 M a c e t i c a c i d and r e d i a f i l t e r e d t h r o u g h U M - 1 0 membranes. The r e d i a f i l t e r e d s a m p l e s w e r e l y o p h i l i z e d and t e s t e d i n o u r d i g o x i n radioimmunoassay ( G r u b e r e t a l . , 1980). ( 2 ) A f f i n i t y chromatography w a s p e r f o r m e d on columns of a n t i d i g o x i n a n t i b o d i e s i m m o b i l i z e d on cyanogen b r o m i d e - a c t i v a t e d S e p h a r o s e . To p r e p a r e t h e s e columns, a n t i b o d y - c o n t a i n i n g serum w a s f i r s t s e p a r a t e d A 2 x 4 - c m column c o n t a i n i n g on p r o t e i n A-Sepharose. t h i s r e s i n w a s pumped by a B u c h l e r p e r i s t a l t i c pump a t a f l o w r a t e of 0 . 5 m l / m i n . The i n i t i a l column e l u e n t was 0 . 0 1 M p h o s p h a t e b u f f e r (pH 7 . 4 ) . One-milliliter a l i q u o t s o f serum were a p p l i e d t o t h e column and washed t h r o u g h w i t h t h e i n i t i a l column e l u e n t . The column e f f l u e n t w a s m o n i t o r e d by a UV d e t e c t o r a t 2 8 0 nm. A f t e r t h e e l u t i o n of a l a r g e UV p o s i t i v e p e a k , t h e column e l u e n t w a s changed t o 1 M a c e t i c a c i d t o r e v e r s e t h e s p e c i f i c b i n d i n g of IgG t o p r o t e i n A. IgG a l s o e l u t e d i n a UV p o s i t i v e peak which was c o l l e c t e d and l y o p h i l i z e d . A t o t a l of 4 0 m l of a n t i b o d y - c o n t a i n i n g serum were s e p a r a t e d on t h e p r o t e i n A columns t o a c q u i r e enough p u r i f i e d a n t i b o d y t o complex t o cyanogen bromidea c t i v a t e d S e p h a r o s e . The IgG was i m m o b i l i z e d a c c o r d i n g t o s t a n d a r d p r o c e d u r e s , and t h e r e s u l t i n g r e s i n packed i n t o 1 6 x 2-cm g l a s s columns w i t h a bed h e i g h t of 1 0 c m . The i n i t i a l column e l u e n t w a s 0 . 4 5 M N a C l t i t r a t e d t o pH 1 . 6 w i t h NaHC03. L y o p h i l i z e d d i a f i l t r a t e s e q u i v a l e n t t o 1 0 0 - 2 0 0 m l of o r i g i n a l plasma w e r e d i s s o l v e d i n t h e i n i t i a l e l u t i n g s o l v e n t i n a volume of 5 m l . These columns were a l s o pumped by a p e r i s t a l t i c Pump a t a f l o w

ISOLATION OF AN ENDOGENOUS DIGOXIN

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r a t e of 0.5 ml/min. The column e f f l u e n t was monitored a t 280 nm. A f t e r t h e a p p l i c a t i o n of plasma d i a f i l t r a t e s , a l a r g e UV p o s i t i v e peak e l u t e d . T h i s w a s considered t o be t h e nonadherent p o p u l a t i o n . A f t e r the peak approached b a s e l i n e , t h e column e f f l u e n t was changed t o 0.45 111 N a C 1 , t i t r a t e d t o pH 2 w i t h HC1, t o b r e a k t h e a n t i b o d y - a n t i g e n b i n d i n g and e l u t e t h e s p e c i f i c a l l y bound s u b s t a n c e s . These co.lumn e l u e n t s were chosen by d e t e r m i n i n g t h e c o n d i t i o n s which would a l l o w t h e s p e c i f i c b i n d i n g of 1 2 5 I - l a b e l e d d i g o x i n t o t h e column and e f f e c t i t s e l u t i o n i n a q u a n t i t a t i v e mann e r . The s p e c i f i c a l l y bound s u b s t a n c e s i n plasma a l s o e l u t e d i n a W p o s i t i v e peak and w e r e c o l l e c t e d and l y ophilized for further studies. The a c i d s t a b i l i t y of t h e d i g o x i n - l i k e a c t i v i t y i n plasma d i a f i l t r a t e s and immunochemically s e p a r a t e d samples was t e s t e d t o a s c e r t a i n i f t h e s u b s t a n c e ( s 1 res p o n s i b l e had p r o p e r t i e s s i m i l a r t o p e p t i d e s o r s t e r o i d s . Samples e q u i v a l e n t t o 10-25 m l of o r i g i n a l plasma w e r e d i s s o l v e d i n 0.5-2 m l of c o n s t a n t b o i l i n g 6 N HC1. The samples were t r a n s f e r r e d t o p r o t e i n hyd r o l y s i s t u b e s and c l o s e d under vacuum w i t h a propane t o r c h . The s e a l e d , e v a c u a t e d t u b e s were p l a c e d i n a l l O ° C oven f o r 2 4 h r . The t u b e s w e r e t h e n removed from t h e oven, t h e seal broken, and t h e a c i d removed under vacuum.

111.

RESULTS

Both immunoprecipitation and a f f i n i t y chromatography of plasma d i a f i l t r a t e s r e s u l t i n t h e i s o l a t i o n of d i g o x i n immunoreactivity. The amount of immunoreact i v i t y r e c o v e r e d a f t e r t h e immunochemical i s o l a t i o n s t e p s was 50-70 pg p e r m i l l i l i t e r of o r i g i n a l plasma. Compared t o t h e amount of immunoreactivity i n d i a f i l t r a t e s , t h i s i n d i c a t e d a r e c o v e r y r a t e of a t l e a s t 7 0 % . Table I shows t h e r e l a t i v e s u s c e p t i b i l i t y t o a c i d hyd r o l y s i s of plasma e x t r a c t s a t v a r i o u s s t a g e s of p u r i fication. These d a t a i n d i c a t e t h a t t h e a b i l i t y t o demonstrate t h e a c i d l a b i l i t y of endoxin i s dependent on t h e amount of H C 1 used t o h y d r o l y z e t h e sample and t h e r e l a t i v e p u r i t y of t h e sample. For example, d o u b l i n g t h e amount of a c i d used t o h y d r o l y z e d i a f i l t r a t e s i n c r e a s e s t h e i r a c i d l a b i l i t y by 7 - f o l d . I n a similar sense, 1 m l of HC1 w i l l d e s t r o y o n l y 15% of t h e immunoactivity

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KENNETH A. GRUBER eta/.

TABLE I .

S u s c e p t i b i l i t y of Plasma Samples t o Acid H y d r o l y s i s

Plasma samples

Acid d e s t r u c t i o n o f immunoactivity compared t o paired controls (%)

D i d f i l t r a t e s (10 m l plasma p e r sample) 1 ml H C 1 (1) 1 m l HC1 (2) 2 m l HC1 ( 3 ) 2 m l HC1 (4) 2 m l HC1 (5)

100

Immunoprecipitated (10 ml plasma p e r sample) 1 m l HC1

100

A f f i n i t y chromatography s p e c i f i c b i n d i n g peak (25 m l plasma p e r sample) 1 m l H C 1 (1) 1 m l HC1 (2) 1 m l HC1 (3)

51 54 100

15 18 100 100

i n a d i a f i l t r a t e of 1 0 m l o f o r i g i n a l p l a s m a , w h e r e a s t h e s a m e amount of a c i d w i l l d e s t r o y 5 0 - 1 0 0 % of t h e i m munoactivity i n a f f i n i t y chromatography-purified samples e q u i v a l e n t t o 2 5 m l of o r i g i n a l p l a s m a .

IV.

DISCUSSION

The a b i l i t y t o i s o l a t e an endogenous d i g o x i n - l i k e f a c t o r by immunochemical means, as w e l l a s d e t e c t i n a immunoassay, i n d i c a t e s t h a t t h i s f a c t o r t r u l y r e a c t s w i t h t h e b i n d i n g s i t e of t h e a n t i d i g o x i n a n t i b o d y . The d e m o n s t r a t i o n t h a t endogenous d i g o x i n i m m u n o r e a c t i v i t y i s s e n s i t i v e t o a s t a n d a r d p e p t i d e a c i d h y d r o l y s i s procedure provides f u r t h e r evidence t h a t t h e s u b s t a n c e ( s ) r e s p o n s i b l e i s n o t s t e r o i d and may i n f a c t be p e p t i d e i n n a t u r e . F i n a l p r o o f of t h i s h y p o t h e s i s a w a i t s t h e s t r u c t u r a l e l u c i d a t i o n of e n d o x i n . However, a f f i n i t y chromatography now p r o v i d e s u s w i t h a r a p i d and s p e c i f i c means of i s o l a t i n g a s u b s t a n c e which may p l a y a n impor-

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t a n t r o l e i n t h e r e n a l r e s p o n s e t o volume e x p a n s i o n ( G r u b e r et a 1 . I 1 9 8 0 ) and t h e e t i o l o g y o f h y p e r t e n s i o n (Gruber et a l . I 1 9 8 2 ) .

REFERENCES

G r u b e r , K. A . , and Buckalew, V. M . , Jr. ( 1 9 7 8 ) . F u r t h e r c h a r a c t e r i z a t i o n and e v i d e n c e f o r a p r e c u r s o r i n t h e f o r m a t i o n of p l a s m a a n t i n a t r i f e r i c f a c t o r . Proc. SOC. Exp. Biol. Med. 1 5 7 , 463-467. G r u b e r , K. A . , W h i t a k e r , J. M . , a n d Buckalew, V. M . , Jr. ( 1 9 8 0 ) . Endogenous d i g i t a l i s - l i k e s u b s t a n c e i n p l a s m a o f volumeexpanded d o g s . N a t u r e (London) 287, 743-745. G r u b e r , K. A . , R u d e l , L. L . , and B u l l o c k , B . C . ( 1 9 8 2 ) . I n c r e a s e d c i r c u l a t i n g l e v e l s of an endogenous d i g o x i n - l i k e f a c t o r i n h y p e r t e n s i v e non-human primates. Hypertension 4 , 348-354.

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CURRENT TOPICS IN MEMBRANES AND TRANSPORT, VOLUME 19

Demonstration of a Humoral NalK Pump Inhibitor in Experimental Low-Renin Hypertension M O T I U PAMNANI, STEPHEN HUOT, DAVID CLOUGH, JAMES BUGGY, AND FRANCIS J. HADDY Department of Physiology Uniformed Services University Bethesdu, Maryland

I.

INTRODUCTION

W e have p r e v i o u s l y shown t h a t o u a b a i n - s e n s i t i v e 86Rb u p t a k e and N a , K - A T P a s e a c t i v i t y are s u p p r e s s e d i n b l o o d v e s s e l s and c a r d i a c microsomes, r e s p e c t i v e l y , of a n i m a l s w i t h l o w - r e n i n , presumably volume-expanded hyp e r t e n s i o n (Clough e t a l . , 1 9 7 7 , 1978, 1980; Huot et a l . , 1980; Overbeck e t a 1 ., 1 9 7 6 ; Pamnani e t a 1 ., 1 9 7 8 a ) . These i n c l u d e dogs w i t h one-kidney, one-wrapped h y p e r t e n s i o n and r a t s w i t h (1) one-kidney, o n e - c l i p , ( 2 ) onek i d n e y , DOCA-saline, and ( 3 ) r e d u c e d r e n a l mass h y p e r tension. W e h a v e a l s o shown t h a t o u a b a i n - s e n s i t i v e 86% u p t a k e is a l s o s u p p r e s s e d i n r a t s and dogs f o l l o w i n g a c u t e volume e x p a n s i o n w i t h s a l i n e o r m a n n i t o l and t h a t s u p e r n a t e s of b o i l e d plasma from t h e s e r a t s and dogs r e d u c e 86Rb u p t a k e when a p p l i e d t o t a i l a r t e r i e s from normal r a t s (Pamnani e t a l . , 1978b, 1 9 8 1 ) .

923

Copyright 0 1983 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-153319-0

924

11.

MOTILAL PAMNANI eta/.

RESULTS AND DISCUSSION

This l a t t e r observation i n v i t e d t h e search €or s i m i l a r a c t i v i t y i n t h e plasma of t h e h y p e r t e n s i v e a n i mals. A new series o f p a i r e d dogs w i t h o n e - k i d n e y , one-wrapped h y p e r t e n s i o n and a sham o p e r a t i o n was t h e r e f o r e prepared. A f t e r a t l e a s t 4 w e e k s of s u s tained s i g n i f i c a n t hypertension i n t h e experimental anim a l and a s i m i l a r t i m e p e r i o d i n i t s p a i r e d shamoperated normotensive c o n t r o l animal, s u p e r n a t e s of b o i l e d plasma w e r e p r e p a r e d and a p p l i e d t o t a i l a r t e r i e s from normal r a t s . O u a b a i n - s e n s i t i v e 86Rb u p t a k e by t h e t a i l a r t e r y was s i g n i f i c a n t l y s u p p r e s s e d by t h e h y p e r t e n s i v e plasma s u p e r n a t e r e l a t i v e t o t h e n o r m o t e n s i v e plasma s u p e r n a t e , w h e r e a s o u a b a i n - i n s e n s i t i v e 8 6 ~ bupt a k e was u n a f f e c t e d , j u s t a s i s t h e c a s e f o r t h e h y p e r t e n s i v e a n i m a l s ' own b l o o d v e s s e l s . S u p e r n a t e s ofb o i l e d plasma w e r e a l s o p r e p a r e d from r a t s w i t h onek i d n e y , o n e - c l i p and r e d u c e d r e n a l mass h y p e r t e n s i o n and from a p p r o p r i a t e n o r m o t e n s i v e c o n t r o l a n i m a l s , and w e r e a p p l i e d t o t a i l a r t e r i e s from normal r a t s . I n both cases, t o t a l 86Rb u p t a k e by t h e t a i l a r t e r y was s u p p r e s s e d by t h e h y p e r t e n s i v e plasma s u p e r n a t e r e l a t i v e t o t h e n o r m o t e n s i v e plasma s u p e r n a t e . F i n a l l y , when r a t s w i t h a n AV3V l e s i o n o r sham l e s i o n w e r e a c u t e l y volumeexpanded w i t h s a l i n e , o u a b a i n - s e n s i t i v e 86Rb u p t a k e by t h e t a i l a r t e r y was h i g h e r i n t h e l e s i o n e d t h a n i n t h e s h a m - l e s i o n e d a n i m a l s . F u r t h e r m o r e , t o t a l 86Rb u p t a k e by a r t e r i e s from normal r a t s was g r e a t e r when i n c u b a t e d i n s u p e r n a t e s p r e p a r e d from l e s i o n e d r a t s t h a n when i n c u b a t e d i n s u p e r n a t e s from s h a m - l e s i o n e d r a t s . W e conc l u d e t h a t plasma from dogs w i t h o n e - k i d n e y , one-wrapped h y p e r t e n s i o n and r a t s w i t h o n e - k i d n e y , o n e - c l i p and reduced r e n a l mass h y p e r t e n s i o n c o n t a i n s a h e a t - s t a b l e f a c t o r which s u p p r e s s e s v a s c u l a r N a / K pump a c t i v i t y . It a p p e a r s i n r e s p o n s e t o volume e x p a n s i o n p e r se and app a r e n t l y t h e AV3V a r e a o f t h e b r a i n i n f l u e n c e s t h e p l a s ma c o n c e n t r a t i o n . I t i s known t h a t s u p p r e s s i o n of t h e v a s c u l a r N a / K pump w i t h o u a b a i n , f o r example, i n c r e a s e s c o n t r a c t i l e a c t i v i t y and t h e c o n t r a c t i l e r e s p o n s e s t o v a s o c o n s t r i c t o r a g e n t s . T h u s , t h e humoral pump i n h i b i t o r found i n t h i s s t u d y may b e i n v o l v e d i n t h e g e n e s i s and m a i n t e n a n c e of t h e h y p e r t e n s i o n .

HUMORAL Na/K PUMP INHIBITOR

925

REFERENCES

Clough, D. L . , Pamnani, M. B . , Overbeck, H . W . , and Haddy, F. J . ( 1 9 7 7 ) . Decreased myocardial N a , K - A T P a s e i n rats w i t h onekidney G l o d b l a t t h y p e r t e n s i o n . Fed. P r o c . , Fed. Am. S O C . Exp. B i o l . 36, 491. Clough, D. L . , Pamnani, M. B . , and Haddy, F. J. ( 1 9 7 8 ) . Decreased Na,K-ATPase a c t i v i t y i n l e f t v e n t r i c u l a r myocardium of rats w i t h one-kidney DCCA-saline h y p e r t e n s i o n . C l i n . Res. 26, 361. Clough, D . , Pamnani, M . , Huot, S., and Haddy, F. J . (1980). L e f t v e n t r i c u l a r Na,K-ATPase a c t i v i t y i n r a t s w i t h reduced r e n a l mass h y p e r t e n s i o n and spontaneous h y p e r t e n s i o n . P h y s i o l o g i s t 23, 91. Huot, S . , Pamnani, M. , Clough, D . , and Haddy, F. ( 1 9 8 0 ) . Depressed Na+-K+ pump a c t i v i t y i n t a i l a r t e r i e s o f reduced Fed. P r o c . , Fed. Am. SOC. r e n a l mass h y p e r t e n s i v e r a t s . Exp. B i o l . 39, 1188. Overbeck, H . W . , Pamnani, M. B . , Akera, T . , Brody, T. M . , and Haddy, F. J . ( 1 9 7 6 ) . Depressed f u n c t i o n of a ouabains e n s i t i v e sodium-potassium pump i n blood v e s s e l s from r e n a l h y p e r t e n s i v e dogs. C i r c . Res. 38, Suppl. 2 , 48-52. Pamnani, M. B . , Clough, D. L . , and Haddy, F. J. ( 1 9 7 8 a ) . A l t e r e d a c t i v i t y of t h e sodium-potassium pump i n a r t e r i e s of r a t s w i t h s t e r o i d h y p e r t e n s i o n . C l i n . S c i . Mol. Med. 55, 41s-43s. Pamnani, M. B . , Clouqh, D. L . , S t e f f e n , R. P . , and Haddy, F. J. (197833). Depressed Na+-K+ pump a c t i v i t y i n t a i l a r t e r i e s from a c u t e l y volume expanded rats. P h y s i o l o g i s t 2 1 , 88. Pamnani, M. B . , Clouqh, D. L . , Huot, S . J . , and Haddy, F. J. ( 1 9 8 1 ) . Sodium-potassium pump a c t i v i t y i n e x p e r i m e n t a l hyp e r t e n s i o n . In " V a s o d i l a t a t i o n " ( P . M. Vanhoutte and I. Leusen, e d s . ) , pp. 391-403. Raven Press, N e w York.

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CURRENT TOPICS IN MEMBRANES AND TRANSPORT, VOLUME 19

Absence of Ouabain-Like Activity of the Na,K-ATPase Inhibitor in Guinea Pig Brain Extract GEORGE R. KRACKE Depanment of Physiology and Biophysics Washington University School of Medicine St. Louis, Missouri

I.

MATERIALS AND METHOD

An acetone-HC1 e x t r a c t o f g u i n e a p i g b r a i n rep o r t e d t o have ouabain-like a c t i v i t y w a s prepared f o l lowing t h e p r o c e d u r e of Fishman ( 1 9 7 9 ) . The e x t r a c t w a s column-chromatographed on Sephadex G - 1 0 and f r a c t i o n s w e r e t e s t e d f o r i o n c o n t e n t and " a p p a r e n t o u a b a i n l i k e " a c t i v i t y , i . e . , a b i l i t y t o d i s p l a c e o u a b a i n from i t s b i n d i n g s i t e and t o i n h i b i t N a , K - A T P a s e under c o n d i t i o n s which o p t i m i z e one o r t h e o t h e r a s s a y ( F i g . 1 ) . F r a c t i o n s 33-37 had Na,K-ATPase i n h i b i t o r y a c t i v i t y i n b o t h a T r i s - and a h i s t i d i n e - b u f f e r e d a s s a y medium b u t no [3H]ouabain d i s p l a c i n g a c t i v i t y and were n o t t e s t e d f u r t h e r f o r " a p p a r e n t o u a b a i n - l i k e " a c t i v i t y ( F i g . 1B) F r a c t i o n s 46-48, i n c o n t r a s t , n o t only i n h i b i t e d t h e enzyme i n t h e T r i s - b u f f e r e d a s s a y medium b u t a l s o p r e v e n t e d b i n d i n g o f [3H]ouabain t o t h e N a , K - A T P a s e . S i n c e t h e " a p p a r e n t o u a b a i n - l i k e " a c t i v i t y of t h e s e f r a c t i o n s e l u t e d w i t h t h e s a l t peak ( F i g . l A ) , t h e rel a t i o n s h i p between t h i s a c t i v i t y and t h e i o n c o n t e n t o f

.

927

Copyright 0 1983 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-153319-0

928

GEORGE R. KRACKE

I

FRACTION

NUMBER

G e l f i l t r a t i o n chromatography o f guinea p i g b r a i n The column d i m e n s i o n s w e r e 1 . 5 X 100 cm; e l u a t e , 0 . 1 M a c e t i c a c i d ; f l o w r a t e , 1 1 . 3 m l / h r ; f r a c t i o n s i z e , 2 . 3 m l ; 23OC. The v o i d volume marker, b l u e d e x t r a n , e l u t e d i n f r a c t i o n s 2 5 - 2 8 . (A) Characteri s t i c 2 8 0 nm a b s o r p t i o n a n d s a l t e l u t i o n p r o f i l e s . ( B ) F r a c t i o n s w e r e t e s t e d f o r t h e i r a b i l i t y t o d i s p l a c e 1 nM [ 3 H ] o u a b a i n f r o m 1.1 p q o f p u r i f i e d d o g k i d n e y Na,K-ATPase and t o i n h i b i t Na,KA T P a s e a c t i v i t y i n e i t h e r a 100 mM T r i s - H C 1 - b u f f e r e d (pH 8 . 0 ) a s s a y m e d i u m (--) or a 30 mM h i s t i d i n e - H C 1 - b u f f e r e d (pH 7 . 5 ) a s s a y medium (---0---). ( C ) T h e Na, K , and P i c o n c e n t r a t i o n s and t h e pH o f f r a c t i o n s a r e s h o w n .

Fig. 1.

e x t r a c t ( 6 0 gm t i s s u e w e t w e i g h t ) on S e p h a d e x G - 1 0 .

3 t h e s a l t peak w a s i n v e s t i g a t e d . The [ H l o u a b a i n b i n d i n g i n h i b i t i o n of t h e f r a c t i o n s seemed t o c o r r e l a t e w i t h t h e p o t a s s i u m c o n t e n t ( F i g . 1 C ) . The e f f e c t of p o t a s s i u m c h l o r i d e on [3H]ouabain i s shown i n F i g . 2 . When t h e [ 3H] o u a b a i n d i s p l a c i n g a c t i v i t y o f f r a c t i o n s 43-51 i s p l o t t e d as a f u n c t i o n o f t h e i r i n d i v i d u a l potassium l e v e l s i n t h e binding assay, t h e displacement c u r v e i s c o m p l e t e l y s u p e r i m p o s a b l e on t h a t o f p u r e pot a s s i u m c h l o r i d e . Thus, t h e [3H]ouabain d i s p l a c i n g

929

Na,K-ATPase INHIBITOR IN GUINEA PIG BRAIN

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10 100 300 DESALTED EXTRACT VOL., yI

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Fig. 2. [3H]Ouabain d i s p l a c e m e n t a c t i v i t y o f p o t a s s i u m c h l o r i d e , u n l a b e l e d o u a b a i n , c o l u m n f r a c t i o n s , and d e s a l t e d b r a i n extract. The a b i l i t y o f u n l a b e l e d o u a b a i n (+-) , potassium column f r a c t i o n s o f F i g . 1 p l o t t e d a s a f u n c c h l o r i d e (*-), t i o n o f t h e i r p o t a s s i u m c o n c e n t r a t i o n s i n the b i n d i n g a s s a y (---@---), and d e s a l t e d b r a i n e x t r a c t p l o t t e d a s a f u n c t i o n o f v o l u m e t e s t e d i n the b i n d i n g a s s a y (---A---) t o d i s p l a c e 1 nM A 300 ~1 [ 3 H ] o u a b a i n f r o m p u r i f i e d d o g k i d n e y Na,K-ATPase. v o l u m e o f b r a i n e x t r a c t r e p r e s e n t s 0 . 4 5 b r a i n . Maximum [ 3 H ] o u a b a i n bound w a s l e s s t h a n 1 0 % . N o n s p e c i f i c [ 3 H ] o u a b a i n bound to the enzyme w a s l e s s t h a n 1% o f the t o t a l [ 3 H ] o u a b a i n b o u n d .

a c t i v i t y o f t h e s e f r a c t i o n s showing " a p p a r e n t o u a b a i n l i k e " a c t i v i t y c a n be e n t i r e l y a c c o u n t e d € o r by t h e potassium content.

11.

RESULTS AND DISCUSSI O N

The N a , K - A T P a s e i n h i b i t o r y a c t i v i t y of t h e s e s a l t peak f r a c t i o n s was d e m o n s t r a b l e i n t h e T r i s a s s a y medium b u t n o t i n a h i s t i d i n e ( m e t a l c h e l a t o r ) a s s a y medium ( F i g . 1B). T h i s a c t i v i t y w a s n o t d e s t r o y e d by a s h i n g ( 4 0 O o C f o r 30 m i n ) . Z i n c , b u t n o t vanadium, w a s det e c t e d i n f r a c t i o n s c o m p r i s i n g t h e s a l t peak. Thorough d e s a l t i n g of t h e p o o l e d f r a c t i o n s w i t h " a p p a r e n t o u a b a i n - l i k e " a c t i v i t y a b o l i s h e d t h i s a c t i v i t y . When a s s a y e d i n t h e h i s t i d i n e medium, a d d i t i o n a l Na,K-ATPase

930

GEORGE R. KRACKE

i n h i b i t o r y a c t i v i t y a p p e a r e d i n f r a c t i o n s d i s t i n c t from t h e s a l t peak ( f r a c t i o n s 49-50 and 5 2 - 5 5 ) . However, t h e s e f r a c t i o n s d i d n o t d i s p l a y o u a b a i n d i s p l a c i n g activity. The Na,K-ATPase i n h i b i t o r y a c t i v i t y c o u l d b e a c c o u n t e d f o r by t h e a c i d i t y of t h e s e f r a c t i o n s ( F i g . 1 C ) which w a s n o t e f f e c t i v e l y b u f f e r e d by t h e h i s t i d i n e medium. V a s o p r e s s i n , which d o e s n o t h a v e o u a b a i n - l i k e a c t i v i t y , w a s found i n t h e s e f r a c t i o n s e l u t i n g j u s t a f t e r t h e s a l t peak (James-Kracke e t a l . , 1 9 8 1 ) . The N a , K - A T P a s e i n h i b i t o r y a c t i v i t y o f f r a c t i o n s 33-37, demonstrable i n b o t h a s s a y systems, w a s n o t due t o t h e i n o r g a n i c p h o s p h a t e i n t h o s e f r a c t i o n s n o r t o vanadium (not present) I t i s concluded t h a t t h e " a p p a r e n t ouabain-like" a c t i v i t y of t h e g u i n e a p i g b r a i n e x t r a c t i s n o t t r u l y o u a b a i n - l i k e and c a n be e x p l a i n e d by i n t e r f e r i n g i o n s .

.

ACKNOWLEDGMENTS

I would l i k e t o t h a n k P a u l D e Weer f o r i n f o r m a t i v e d i s c u s s i o n s and a d v i c e . T h i s work w a s s u p p o r t e d by N I H G r a n t s N S 1 1 2 2 3 and HL 07275.

REFERENCES

Fishman, M . C . ( 1 9 7 9 ) . Endogenous d i g i t a l i s - l i k e a c t i v i t y i n mammalian b r a i n . P r o c . Natl. A c a d . S c i . USA 76, 4661-4663. James-Kracke, M. R . , K r a c k e , G. R . , and Lang, S . ( 1 9 8 1 ) . I d e n t i f i c a t i o n of a vasoactive substance (vasopressin) i n a b r a i n e x t r a c t c o n t a i n i n g a n unknown i n h i b i t o r of Na,E:ATPase. Clin. Exp. Hypertens. 3, 523-528.

CURRENT TOPICS IN MEMBRANES AND TRANSPORT, VOLUME 19

Brain Na,K-ATPase: Regulation by Norepinephrine and an Endogenous Inhibitor ALANC. S W A " Department of Psychiairy University of Texas Medical School Houston, Texas

I.

INTRODUCTION

One i m p o r t a n t q u e s t i o n a b o u t N a , K - A T P a s e i s w h e t h e r t h e r e a r e s p e c i f i c mechanisms whereby p o p u l a t i o n s o f enzyme m o l e c u l e s h a v i n g s p e c i a l i z e d p h y s i o l o g i c f u n c t i o n s are r e g u l a t e d . Sweadner h a s r e p o r t e d two forms o f Na,K-ATPase, d i f f e r i n g i n a f f i n i t y f o r c a r d i a c glycos i d e s ; t h e h i g h - a f f i n i t y form a p p e a r s t o b e l i m i t e d t o n e r v e c e l l s (Sweadner, 1 9 7 9 ) . T h i s n e r v e - s p e c i f i c form may a l s o b e s e l e c t i v e l y i n h i b i t e d by e r y t h r o s i n B ( S i l b e r g e l d , 1 9 8 1 ) . The p o s s i b l e p r e s e n c e of two enzyme forms raises t h e q u e s t i o n of w h e t h e r t h e y a r e d i f f e r e n t i a l l y r e g u l a t e d by p h y s i o l o g i c enzyme e f f e c t o r s . A compound i n b r a i n e x t r a c t s and plasma t h a t r e d u c e s c a r d i a c g l y c o s i d e b i n d i n g , c a t i o n t r a n s p o r t , and enzyme a c t i v i t y ( H a u p e r t and Sancho, 1 9 7 9 ; L i c h t s t e i n and Samuelov, 1980) i s one c a n d i d a t e f o r s u c h an e f f e c t o r , e s p e c i a l l y b e c a u s e t h e endogenous i n h i b i t o r a p p e a r s t o i n t e r a c t w i t h t h e c a r d i a c g l y c o s i d e b i n d i n g s i t e and a f f i n i t y f o r c a r d i a c g l y c o s i d e s may d i s t i n g u i s h t h e two 931

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932

ALAN C . SWANN

enzyme f o r m s . A second c a n d i d a t e i s n o r e p i n e p h r i n e , which i n c r e a s e s Na,K-ATPase a c t i v i t y in v i t r o (Wu and P h i l l i s , 1980) and i n v i v o (Swann e t a l . , 1 9 8 1 a ) . Whereas c h e m i c a l p r o p e r t i e s o f n o r e p i n e p h r i n e and t h e c o n d i t i o n s r e q u i r e d f o r s t i m u l a t i o n of enzyme in v i tro s u g g e s t t h a t s t i m u l a t i o n may r e s u l t from c h e l a t i o n o r p r o t e c t i o n a g a i n s t l i p i d peroxidation (Schaefer e t a ] . , 1974), stimulation i n vivo indicates that t h i s action of n o r e p i n e p h r i n e c a n o c c u r i n s i t u . The e x p e r i m e n t s r e p o r t e d h e r e examined (1) t h e i n t e r a c t i o n s o f endoqenous i n h i b i t o r and n o r e p i n e p h r i n e w i t h h i g h - a f f i n i t y o u a b a i n b i n d i n g , ( 2 ) i n t e r a c t i o n s between n o r e p i n e p h r i n e and endogenous i n h i b i t o r in v i t r o , and ( 3 ) t h e e f f e c t s of p e r s i s t e n t changes i n e x p o s u r e t o n o r e p i n e p h r i n e in vivo on h i g h - a f f i n i t y o u a b a i n b i n d i n g .

11.

A.

METHODS

ANIMALS,

DRUGS,

AND

T I S S U E PREPARATIONS

Male Sprague-Dawley r a t s w e i g h i n g 2 0 0 - 3 0 0 gm w e r e used. Drugs were g i v e n i n t r a p e r i t o n e a l l y . R a t s were k i l l e d 2 0 h r a f t e r t h e l a s t d r u g d o s e by d e c a p i t a t i o n a f t e r c h l o r a l h y d r a t e a n e s t h e s i a and b r a i n microsomes w e r e p r e p a r e d a s d e s c r i b e d by Swann e t a l . ( 1 9 8 1 a ) . F o r n o r a d r e n e r g i c l e s i o n s , t h e r i g h t d o r s a l noradr e n e r g i c b u n d l e was l o c a t e d s t e r e o t a c t i c a l l y under l i g h t c h l o r a l h y d r a t e a n e s t h e s i s and 4 ug 6-hydroxydopamine w a s i n j e c t e d a s w e have d e s c r i b e d p r e v i o u s l y ; l e s i o n s w e r e e v a l u a t e d by d e p l e t i o n o f n o r e p i n e p h r i n e i n cereb r a l c o r t e x compared t o t h e n o n l e s i o n e d s i d e (Swann e t a l . , 1981a).

B.

ASSAYS

+

K -p-nitrophenylphosphatase was a s s a y e d a s w e have p r e v i o u s l y d e s c r i b e d (Swann e t a]., 1 9 8 1 b ) . Ouabain b i n d i n g w a s measured i n t h e p r e s e n c e o f Mg2+, N a + , and

ATP a s w e have d e s c r i b e d ; n o n s p e c i f i c b i n d i n g w a s t h a t measured w i t h o u t Mg2+ and ATP (Swann e t a l . , 1 9 8 1 b ) . Ouabain i n h i b i t i o n c u r v e s f o r K+-p-nitrophenylphosphatase a c t i v i t y w e r e c a r r i e d o u t by p r e i n c u b a t i o n w i t h o u a b a i n , MgZ+, and i n o r g a n i c p h o s p h a t e a s i n o u r p r e v i ous work (Swann e t al., 1 9 8 0 ) .

933

BRAIN Na,K-ATPase AND NOREPINEPHRINE

.5

.4

1

2x104

4xW6

[Norepinephrine], M

+

F i g . 1 . Inhibition of n o r e p i n e p h r i n e s t i m u l a t i o n of K -NPPase b y b r a i n e x t r a c t . S o l i d l i n e w i t h o u t e x t r a c t . Dashed l i n e w i t h extract.

C.

PREPARATION

OF B R A I N

EXTRACT

Acid-acetone e x t r a c t i o n of c a t b r a i n t i s s u e w a s c a r r i e d o u t a s d e s c r i b e d by L i c h s t e i n and Samuelov ( 1 9 8 0 ) . The r e s u l t i n g m a t e r i a l p r o b a b l y c o n t a i n e d subs t a n t i a l r e s i d u a l K+, a s it s t i m u l a t e d -nitrophenylp h o s p h a t a s e a c t i v i t y i n t h e a b s e n c e of K+. Therefore, e x t r a c t i o n i n t o m e t h a n o l w a s r e p e a t e d and t h e r e s u l t i n g m a t e r i a l w a s d r i e d , d i s s o l v e d i n w a t e r , and a p p l i e d t o an A m b e r l i t e I R - 1 2 0 cation-exchange olumn i n t h e H+ form ( H a u p e r t and Sancho, 1 9 7 9 ) . The m a t e r i a l w i t h i n h i b i t o r y a c t i v i t y w a s e l u t e d with d i l u t e HC1. T h i s mat e r i a l d i d n o t s u b s t a n t i a l l y s t i m u l a t e p-nitrophenylp h o s p h a t a s e i n t h e a b s e n c e o f K+.

934

ALAN C. SWANN

.so0

-

,400-

:::j

,300-

.zoo-

, , ,

,

,

,

,

,

10"

10-7

10-6

10-5

10-4

10-3

100.

\ \ \ 0

0

[Ouabain], M

Fig. 2. b y ouabain.

I n h i b i t i o n of K'-P-nitrophenylphosphatase

enough b r a i n e x t r a c t t o d e c r e a s e enzyme a c t i v i t y b y h a l f : o u t norepinephrine; (B) w i t h 10-5 M norepinephrine.

111.

activity

T h e dashed l i n e shows i n h i b i t i o n i n t h e p r e s e n c e of (A)' w i t h -

RESULTS AND D ISC U S S I O N

The m a t e r i a l i n b r a i n e x t r a c t r e d u c e d s t i m u l a t i o n o f enzyme a c t i v i t y by n o r e p i n e p h r i n e , d e c r e a s i n g maximal s t i m u l a t i o n 3 - f o l d and a p p a r e n t a f f i n i t y 1 0 - f o l d ( F i g . 1) N o r e p i n e p h r i n e a l s o r e d u c e d i n h i b i t i o n by the1 b r a i n e x t r a c t . F i g u r e 2 shows t h a t t h e b r a i n e x t r a c t select i v e l y d e c r e a s e d h i g h - a f f i n i t y o u a b a i n b i n d i n g , which may c h a r a c t e r i z e a n e r v e - s p e c i f i c form of N a , K - A T P a s e (Sweadner, 1 9 7 9 ) . N o r e p i n e p h r i n e i n c r e a s e d t h e p r o p o r t i o n o f h i g h - a f f i n i t y enzyme; t h i s w a s p r e v e n t e d by brain extract. R e p e a t e d p h a r m a c o l o g i c n o r a d r e n e r g i c s t i m u l a t i o n by p i p e r o x a n e (Swann e t a l . , 1981b) i n v i v o i n c r e a s e d t h e h i g h - a f f i n i t y o u a b a i n b i n d i n g s e l e c t i v e l y , a s shown i n T a b l e I . Blockade of p o s t s y n a p t i c a - a d r e n e r g i c r e c e p t o r s w i t h p r a z o s i n , which i n c r e a s e d p r e s y n a p t i c n o r e p i n e p h r i n e r e l e a s e s i g n i f i c a n t l y as r e f l e c t e d by metab o l i t e l e v e l s , decreased ouabain binding. This implies t h a t b i n d i n g of n o r e p i n e p h r i n e t o i t s p o s t s y n a p t i c rec e p t o r s w a s necessary f o r s t i m u l a t i o n of h i g h - a f f i n i t y ouabain binding. The d a t a i n T a b l e I1 summarize t h e e f f e c t of l e s i o n s of t h e d o r s a l n o r a d r e n e r g i c b u n d l e on b r a i n Na,K-ATPase. These l e s i o n s d e p l e t e n o r e p i n e p h r i n e i n c e r e b r a l c o r t e x while i n c r e a s i n g norepinephrine release i n cerebellum. High-affinity ouabain binding w a s

.

935

BRAIN Na,K-ATPaseAND NOREPINEPHRINE

TABLE I .

E f f e c t of Repeated P i p e r o x a n e or P r a z o s i n on Ouabain B i n d i n g i n Cerebral C o r t e x a

High a f f i n i t y Low a f f i n i t y

C o n t r o l (13)

Piperoxane (8)

Prazosin (6)

1.000 f 0.079

1 . 3 2 5 f 0.212b 0.595 ? 0.211

0.725 5 O.17Oc 0.480 t 0.085c

0.618 f 0.388

a R a t s r e c e i v e d 1 mg/kg p i p e r o x a n e o r p r a z o s i n t w i c e d a i l y f o r 3 weeks. Ouabain b i n d i n g is e x p r e s s e d i n p r o p o r t i o n t o cont r o l h i g h - a f f i n i t y o u a b a i n b i n d i n g o f 26 p m o l e s / m g p r o t e i n f o r microsomes bPiperoxane h i g h - a f f i n i t y b i n d i n g d i f f e r e n t f r o m c o n t r o l , p < 0.05. CPrazosin high- o r low-affinity binding d i f f e r e n t f r o m c o n t r o l , p < 0.05.

.

TABLE 11.

E f f e c t o f 6-Hydroxydopamine L e s i o n o f t h e Dorsal N o r a d r e n e r g i c Bundle on Ouabain B i n d i n g t o B r a i n Microsomesa

Region

Side

Cortex

Ipsilateral Contralateral Ipsilateral Contralateral

Cerebellum

High a f f i n i t y 1.5 10.9 17.0 7.4

b f 1.2 f 2.0 f 5.4c t 2.4

Low a f f i n i t y 9.0 6.7 24.9 20.1

f f f f

2.4 1.3 1.2 6.5

aThe r i g h t d o r s a l n o r a d r e n e r g i c b u n d l e was l e s i o n e d w i t h Ouabain b i n d i n g 6-hydroxydopamine a s d e s c r i b e d i n t h e t e x t . is i n picomoles per m i l l i g r a m p r o t e i n f s t a n d a r d d e v i a t i o n ; h i g h - a f f i n i t y b i n d i n g is specific b i n d i n g t h a t was i n h i b i t a b l e M erythrosin B. with 'Ipsi 1a t e r a l d i f f e r e n t f r o m con t r a l a t e r a l , p a i r e d t = 7 . 1 4 , p < 0.005. C I p s i l a t e r a l d i f f e r e n t f r o m contralateral, paired t = 5.46, p < 0.02. dFrom Swann e t a l . (1982). Used w i t h p e r m i s s i o n f r o m Raven Press, I n c

.

markedly d e c r e a s e d i n c o r t e x and i n c r e a s e d i n c e r e b e l lum i n l e s i o n e d r a t s (Swann e t a l . ( 1 9 8 2 ) . T h e r e w e r e no c h a n g e s i n sham-operated r a t s . The d a t a i n t h i s a r t i c l e s u g g e s t t h a t a p o p u l a t i o n of N a , K - A T P a s e m o l e c u l e s , p o s s i b l y on p o s t s y n a p t i c t e r -

936

ALAN C . LSWANN

m i n a l s , i s r e g u l a t e d by n o r e p i n e p h r i n e , p o s s i b l y i n tandem w i t h an endogenous i n h i b i t o r of enzyme. This may b e one of t h e m e t a b o l i c f u n c t i o n s of t h e d i f f u s e n o r a d r e n e r g i c i n n e r v a t i o n of t h e c e r e b r a l c o r t e x . T h r e e i m p o r t a n t q u e s t i o n s t h a t r e m a i n , however, a r e (1) t h e i d e n t i t y of t h e enzyme i n h i b i t o r i n t i s s u e ext r a c t s , ( 2 ) t h e s p e c i f i c i t y of e r y t h r o s i n B s e n s i t i v i t y and h i g h o u a b a i n a f f i n i t y f o r t h e " n e r v e - s p e c i f i c " form of Na,K-ATPase w i t h h i g h m o l e c u l a r w e i g h t (Sweadner, 1 9 7 9 ) , and ( 3 ) t h e s p e c i f i c i t y o f t h e e f f e c t s of n o r e p i n e p h r i n e e x p o s u r e as opposed t o changes i n n e r v e a c t i v i t y p e r se. These q u e s t i o n s a r e now b e i n g i n v e s t i gated.

ACKNOWLEDGMENT

T h i s work was s u p p o r t e d i n p a r t by MH-07740 and by a Biom e d i c a l S c i e n c e s R e s e a r c h S u p p o r t G r a n t o f t h e U n i v e r s i t y o f Texas M e d i c a l S c h o o l a t Houston.

REFERENCES

H a u p e r t , G. T . , and Sancho, J . M. ( 1 9 7 9 ) . Sodium t r a n s p o r t i n P r o c . N a t l . Acad. Sci. h i b i t o r f r o m b o v i n e hypothalamus. USA 76, 4658-4660. L i c h t s t e i n , D . , and Samuelov, S. ( 1 9 8 0 ) . Endogenous "ouabainBiochern. Biophys. R e s . Commun. like" activity i n rat brain. 96, 1518-1523. S c h a e f e r , A . , S e r e g i , A . , and Komlos, M. ( 1 9 7 4 ) . A s c o r b i c a c i d l i k e e f f e c t o f t h e s o l u b l e f r a c t i o n o f r a t b r a i n on a d e n o s i n e t r i p h o s p h a t a s e s a n d i t s r e l a t i o n s h i p t o c a t e c h o l a m i n e s and chelating agents. Biochern. Pharmacol 2 3 , 2257-227L S i l b e r g e l d , E . K. ( 1 9 8 1 ) . E r y t h r o s i n B i s a s p e c i f i c i n h i b i t o r of h i g h a f f i n i t y ouabain b i n d i n g and i o n t r a n s p o r t i n r a t b r a i n . Neuropharmacology 20, 87-90. Swann, A. C . , M a r i n i , J . L . , S h e a r d , M. H . , and Maas, J . W . ( 1 9 8 0 ) . E f f e c t s of c h r o n i c d i e t a r y l i t h i u m on a c t i v i t y and r e g u l a t i o n o f (Na+,K+)-ATPase i n r a t b r a i n . Biochern. Pharrnacol. 2 9 , 2819-2823. Swann, A . C . , Crawley, J . N . , G r a n t , S . J . , a n d Maas, J . W. (1981a) N o r a d r e n e r g i c s t i m u l a t i o n i n vivo i n c r e a s e s ( N a + , K + ) - A T P a s e activity. L i f e S c i . 2 8 , 251-256. Swann, A. C . , G r a n t , S . J . , J a b l o n s , D. M . , and Maas, J. W'. I n c r e a s e d ouabain b i n d i n g a f t e r r e p e a t e d noradren(1981b) e r g i c s t i m u l a t i o n . B r a i n R e s . 213, 481-485.

.

.

BRAIN Na,K-ATPase AND NOREPINEPHRINE

937

Swann, A. C . , Grant, S . J . , and Maas, J . W. ( 1 9 8 2 ) . B r a i n (Na+,K+)-ATPase and n o r a d r e n e r q i c a c t i v i t y : E f f e c t s of h y p e r i n n e r v a t i o n and d e n e r v a t i o n on h i g h a f f i n i t y o u a b a i n b i n d i n g . J . N e u r o c h e m . 38, 836-839. Sweadner, K. J. ( 1 9 7 9 ) . Two m o l e c u l a r forms of (Na+,K+)-stimulated ATPase i n b r a i n . S e p a r a t i o n and d i f f e r e n c e i n a f f i n i t y f o r s t r o p h a n t h i d i n . J. Biol. C h e m . 2 5 4 , 6060-6067. Wu, P. H . , and P h i l l i s , J. W. ( 1 9 8 0 ) . C h a s a c t e r i z a t i o n of r e c e p t o r mediated catecholamine a c t i v a t i o n o f r a t b r a i n c o r t i c a l (Na+,K+)-ATPase. Int. J. B i o c h e m . 1 2 , 353-359.

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CURRENT TOPICS IN MEMBRANES AND TRANSPORT, VOLUME 19

Inhibitory and Stimulatory Effects of Vanadate on Sodium Pump of Cultured Heart Cells from Different Species KARL WERDAN, GERHARLI BAUMEDEL, WOLFGANG KRA WIEIZ, AND ERLAND ERDMANN Medizinische Klinik I der Universitat Miinchen Klinikum Grosshadern Munich, Federal Republic of Germany

I.

INTRODUCTION

Vanadate (Na3V04) i s a p o t e n t i n h i b i t o r of i s o l a t e d Na,K-ATPase. I n a g r e e m e n t w i t h t h i s f i n d i n g , act i v e K+ u p t a k e i s i n h i b i t e d i n human e r y t h r o c y t e s ( C a n t l e y e t a l . , 1 9 7 8 ) . I n r a t a d i p o c y t e s , however, u p t a k e i s u n a l t e r e d by Na3V04 (Dubyak and K l e i n z e l l e r , 19801, w h e r e a s i n c u l t u r e d r a t h e a r t c e l l s , even a s t i m u l a t i o n h a s been o b s e r v e d (Werdan e t a l . , 1 9 8 0 ) . T o d e t e r m i n e w h e t h e r t h e t r a c e e l e m e n t vanadium may a c t a s p h y s i o l o g i c a l r e g u l a t o r of N a , K - A T P a s e i n i n t a c t c e l l s , w e h a v e s t u d i e d t h e e f f e c t of Na3V04 on K+ upt a k e and on c e l l u l a r K+ i n c u l t u r e d h e a r t c e l l s o b t a i n e d from v a r i o u s s p e c i e s .

939

Copyright 0 1983 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-153319-0

940

11.

KARL WERDAN el al.

MATERIAL AND METHODS

E x p e r i m e n t s have been c a r r i e d o u t a t 37OC w i t h p r i mary c u l t u r e s of b e a t i n g h e a r t muscle c e l l s from neonat a l r a t s , w i t h G i r a r d i c e l l s - - a n e p i t h e l o i d - l i k e yrowing t r a n s f o r m e d c e l l l i n e from human h e a r t - - a n d w i t h lionmuscle c e l l s o b t a i n e d from h e a r t s of n e o n a t a l r a t s and g u i n e a p i g s (1- t o 5-day-old) and c h i c k e n embryos ( 9 t o 11-day-old). C e l l c u l t u r e t e c h n i q u e s , measurement o f K+ u p t a k e by 86Rb+ and o f 2-deoxy-D-[l-3H]glucose, and d e t e r m i n a t i o n of c e l l u l a r K+ by f l a m e p h o t o m e t r y have b e e n d e s c r i b e d p r e v i o u s l y (Werdan e t a l . , 1 9 8 0 , 1 9 8 2 ) .

111.

RESULTS

Na3V04 i n h i b i t s enzyme a c t i v i t y of Na,K-ATPasee n r i c h e d c e l l membrane f r a c t i o n s o r i g i n a t e d from h e a r t s of n e o n a t a l r a t s (Werdan e t a l . , 1 9 8 2 ) . I n c o n t r a s t t o t h i s f i n d i n g , a c o n c e n t r a t i o n - d e p e n d e n t s t i m u l a t i o n of 86Rb+ + K+ u p t a k e h a s been o b s e r v e d i n c u l t u r e d r a t h e a r t m u s c l e c e l l s d e r i v e d from t h e same t i s s u e ( F i g . l a ) . A s i m i l a r s t i m u l a t i o n h a s been a l s o found i.n h e a r t nonmuscle c e l l s from n e o n a t a l r a t s ( c o n t r o l . 5 . 3 nmoles/my p r o t e i n / m i n ; 6 0 % s t i m u l a t i o n by M Na3V04; EC50 = 1 . 4 x 1 0 - 4 M ) a s w e l l as i n G i r a r c l i c e l l s ( c o n t r o l 1 1 . 0 nmoles/mg p r o t e i n / m i n ; 65% s t i m u l a t i o n by 10-3 M Na3V04; ~ ~ = 53.50 x 10-5 M ) . In contrast, 86Rb+ + K+ u p t a k e i s i n h i b i t e d i n h e a r t nonmuscle c e l l s from g u i n e a p i g s ( F i g . l c ) and from c h i c k e n embryos ( c o n t r o l 1 1 . 7 nmoles/mg p r o t e i n / m i n ; 3 4 % i n h i b i t i o n by S t i m u l a t i o n of 1 0 - 3 M N a V04; EC50 = 1 . 0 x 1 0 - 4 M ) . 8 6 ~ b ++ K? u p t a k e by Na3V04 ( 3 x 1 0 - 4 - 10-3 M ) i n creases c e l l u l a r K+ (nmoles/my p r o t e i n , mean k SD, F i g . 1 . E f f e c t s o f Na3V04 and bovine i n s u l i n on u p t a k e o f and 2 - [ 3 H ] d e o x y - D - g l u c o s e i n r a t h e a r t m u s c l e c e l l s 8 6 R b f + K' ( a , b) and g u i n e a p i g h e a r t n o n r n u s c l e c e l l s ( c , d ) . C e l l s have been p r e i n c u b a t e d i n H E P E S - b u f f e r e d s a l t s o l u t i o n ( W e r d a n e t a l . , 1982) p l u s Na3VOg a n d bovine i n s u l i n r e s p e c t i v e l y f o r 2 4 0 rnin 0.52-0.71 rng p r o t e i n / f l a s k ; r a d i o a c t i v i t y : 86Rb+, ( 1 .O-2 . O ) x 106 c p m / f l a s k , 2 - [ 3 H ] d e o x y - D - g l u c o s e , 0 . 9 x lo6 c p r n / f l a s k ; f o r m a t e r i a l s and f u r t h e r e x p e r i m e n t a l d e t a i l s see W e r d a n e t a l . ( 1 9 8 0 , 1 9 8 2 ) . C o n t r o l v a l u e s o f 86Rb+ + K+ u p t a k e ( n m o l e s / m g p r o t e i n / rnin): ( a ) i n s u l i n e x p e r i m e n t , 1 5 . 8 ? 0 . 4 ; NajVOq e x p e r i m e n t , 10.0 ? 0.7; ( c ) i n s u l i n e x p e r i m e n t , 1 4 . 3 5 0 . 2 ; ( d ) Na3VO4 e x p e r i ment, 8.0 2 0.3. V a l u e s a r e g i v e n a s mean 2 SD ( n = 3 ) .

3

P .

2

Fb

Insulin

, (lOmU/ml)

I

r

0

u

Y

0

L

5

6

3 t (min)

I

u 0

3

6 t (min)

Fig. 1

942

KARL WERD.AN eta/.

n = 4-10)

i n r a t h e a r t muscle c e l l s ( c o n t r o l ( C ) 547 _+ 3 0 ; Na3V04 ( V ) 639 261, i n r a t h e a r t nonmuscle c e l l s (C 4 9 1 f 39; V 586 _+ 3 1 ) , and i n G i r a r d i c e l l s ( C 599 35; V 673 f 5 1 ) . Na3VOq-induced i n h i b i t i o n o f 86Rb+ + K+ u p t a k e decreases c e l l u l a r K+ i n h e a r t nonm u s c l e c e l l s from g u i n e a p i g s ( C 6 6 0 f 53; V 556 f 6 7 ) and from c h i c k e n embryos ( C 6 1 7 f 2 8 ; V 500 ? 3 9 ) . I n r a t h e a r t muscle c e l l s , a s t i m u l a t i o n of 86Rb+ + P:+ upt a k e , s i m i l a r t o t h a t w i t h v a n a d a t e , h a s been f o u n d , too, i n the presence of i n s u l i n (Fig. l a ) . Additionall y , v a n a d a t e m i m i c s i n s u l i n ' s enhancement of 2 - [ 3 € I ] deoxy-D-glucose u p t a k e i n t o t h e s e c e l l s ( F i g . lb).. S i m i l a r r e s u l t s t o t h o s e g i v e n i n F i g . l a and b h a v e been o b t a i n e d w i t h r a t h e a r t nonmuscle c e l l s and Girardi cells. However, i n h e a r t nonmuscle c e l l s from g u i n e a p i g s , Na3V04 d o e s n o t m i m i c i n s u l i n ' s s t i m u l a t i o n o f hexose t r a n s p o r t (Fig. I d ) ; uptake of 86Rb+ + K+ i s a l t e r e d i n a n o p p o s i t e manner by i n s u l i n and Na3V04 ( F i g . l c ) . No s t i m u l a t i o n o f 86Rb+ + K+ upt a k e by i n s u l i n h a s been o b s e r v e d i n h e a r t nonmuscle c e l l s from c h i c k e n embryos. _+

*

IV.

DISCUSSION

Our r e s u l t s c l e a r l y i n d i c a t e t h a t Na3V04 c a n e i t h e r s t i m u l a t e o r i n h i b i t 86Rb+ + K+ u p t a k e i n c u l t u r e d h e a r t c e l l s , t h e mode of a c t i o n d e p e n d i n g on t h e c e l l t y p e t e s t e d . C e l l u l a r K+ i s t h e r e b y e i t h e r i n c r e a s e d o r d e c r e a s e d . Only vanadium i n t h e V v a l e n c e s t a t e r e p r e s e n t s a p o t e n t i n h i b i t o r of N a , K - A T P a s e ( C a n t l e y and A i s e n , 1 9 7 9 ) , w h e r e a s o n l y vanadium i n t h e I V v a l e n c e s t a t e m i m i c s i n s u l i n ' s a c t i o n on h e x o s e m e t a b o l i s m ( S h e c h t e r and K a r l i s h , 1 9 8 0 ) . I n t h e i n t a c t c e l l , vanadium(V) c a n b e r e d u c e d t o vanadium(1V) ( C a n t l e y and A i s e n , 1 9 7 9 ) . T h e r e f o r e , t h e f o l l o w i n g h y p o t h e s i s may e x p l a i n t h e a n t a g o n i s t i c a c t i o n Gf Na3VG4 i n h e a r t c e l l s : v a n a d a t e i s t a k e n up i n t o h e a r t c e l l s (Werdan et a l . , 1 9 8 0 ) . I f i t i s r e d u c e d i n s i d e t h e c e l l s t o v a n a d i u m ( I V ) , it s t i m u l a t e s 8 6 ~ b ++ K+ u p t a k e i n a n i n s u l i n - l i k e manner. I n h e a r t c e l l types without s u f f i c i e n t c a p a b i l i t y f o r vanadate r e d u c t i o n , vanadium(V) may a c t as o u a b a i n - l i k e i n h i b i t o r of a c t i v e p o t a s s i u m t r a n s p o r t .

VANADATE ACTION ON SODIUM PUMP IN HEART CELLS

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ACKNOWLEDGMENTS

This work has been supported by Wilhelm Sander-Stiftung (14/1978-Wel) and by Deutsche Forschungsgemeinschaft (E 65/2).

REFERENCES

Cantley, L. C . , and Aisen, P. (1979). The f a t e o f cytoplasmic vanadium. J. Biol. Chem. 2 5 4 , 1781-1784. Cantley, L. C . , Resh, M. D . , and G u i d o t t i , G . (1978). Vanadate i n h i b i t s t h e r e d c e l l (Na+,K+)ATPase from t h e cytoplasmic s i d e . N a t u r e ( L o n d o n ) 2 7 2 , 552-554. Dubyak, G . R . , and K l e i n z e l l e r , A. (1980). The insulin-mimetic J. Biol. e f f e c t s o f vanadate i n i s o l a t e d rat adipocytes. Chem. 2 5 5 , 5306-5312. Shechter, Y . , and K a r l i s h , S . J . D. (1980). I n s u l i n - l i k e stimul a t i o n of glucose o x i d a t i o n i n r a t a d i p o c y t e s by vanadyl(1V) i o n s . N a t u r e ( L o n d o n ) 2 8 4 , 556-558. Werdan, K . , B a u r i e d e l , G . , Bozsik, M . , Krawietz, W . , and Erdmann, E. (1980). E f f e c t s of vanadate i n c u l t u r e d r a t h e a r t muscle c e l l s . B i o c h i m . Biophys. A c t a 597, 364-383. Werdan, K . , Bauriedel, G . , F i s c h e r , B . , Krawietz, W . , Erdmann, E . , Schmitz, W . , and Scholz, H . (1982). Stimulatory (insulin-mimetic) and i n h i b i t o r y (ouabain-like) a c t i o n o f vanadate on potassium uptake and c e l l u l a r sodium and potassium i n h e a r t c e l l s i n c u l t u r e . Biochim. Biophys. A c t a 6 8 7 , 79-93.

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CURRENT TOPICS IN MEMBRANES AND TRANSPORT,VOLUME 19

Endogenous Inhibitor of Na,K-ATPse: “Endodigin” K. R. HWITMER, D. EPPS, AND A. SCHWARZ Department of Phormacology and Cell Biophysics University of Cincinnati College of Medicine Cincinnati, Ohio

I.

INTRODUCTION

R e c e n t l y t h e r e have been r e p o r t s of a c i d - a c e t o n e e x t r a c t s of mammalian b r a i n which have been shown t o i n h i b i t v a r i o u s p a r a m e t e r s of Na,K-ATPase f u n c t i o n . I n t e r p r e t a t i o n of t h e s e r e p o r t s i s l i m i t e d by t h e i m p u r i t i e s of t h e enzyme p r e p a r a t i o n employed and t h e t y p e s of measurements made. To i d e n t i f y an endogenous d i g i t a l i s - l i k e substance, we suggest the following c r i t e r i a . The endogenous s u b s t a n c e m u s t , i n a dosedependent and d i g i t a l i s - l i k e manner, (1) b i n d t o t h e ouabain r e c e p t o r , ( 2 ) i n h i b i t Na,K-ATPase a c t i v i t y s p e c i f i c a l l y , and ( 3 ) c a u s e p o s i t i v e i n o t r o p y .

945

Copyright B 1983 by Academic F’ress, Inc. All righu of repro&ction in my form reserved. ISBN O-lZ-lS33l94

K. R. WHITMER eta/,

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11.

METHODS

R a t b r a i n s were q u i c k l y e x c i s e d , f r o z e n on d r y i c e , and e x t r a c t e d by t h e method of Carraway and Leeman (19731, Method A , o r by two m o d i f i c a t i o n s of t h i s method I n Methods B and C , an atmosphere of n i t r o g e n was subs t i t u t e d f o r a i r and i n Method C , water was s u b s t i t u t e d f o r a c i d i n t h e e x t r a c t i o n procedure. The a b i l i t y of t h e e x t r a c t s t o i n h i b i t Na,K-ATPase a c t i v i t y , [3H]ouabain a s s o c i a t i o n and d i s s o c i a t i o n w i t h t h e p u r i f i e d lamb kidney Na,K-ATPase, as w e l l a s C a 2 + A T P a s e a c t i v i t y of r a b b i t s k e l e t a l muscle s a r c o p l a s m i c r e t i c u l u m , w a s determined. Guinea p i g and r a b b i t a t r i a l m u s c l e s t r i p s were used t o a s s e s s t h e e f f e c t of e x t r a c t s on c o n t r a c t i l i t y . T o t a l p h o s p h o l i p i d was determined from a l i q u o t s of t h e l i p i d phases and p h o s p h o l i p i d comp o s i t i o n a s s e s s e d by two-dimensional t h i n - l a y e r chromatography (TLC)

.

111.

RESULTS AND DISCUSSION

S i n c e t h e method most widely used t o o b t a i n t h e put a t i v e endogenous d i g i t a l i s - l i k e s u b s t a n c e i n v o l v e s a i r - a c i d - a c e t o n e e x t r a c t i o n s of mammalian b r a i n (Method A ) , e x t r a c t s o b t a i n e d by t h i s method w e r e a n a l y z e d i n d e t a i l . Rat b r a i n e x t r a c t s o b t a i n e d from Method A i n h i b i t e d Na,K-ATPase a c t i v i t y i n a dose-dependent manner and t h e i n h i b i t i o n was n o t reduced by 5 mM EGTA, sugg e s t i n g t h a t t h e i n h i b i t i o n was n o t due t o calcium. T h i s e x t r a c t , however, a l s o i n h i b i t e d Ca2+-ATPase act i v i t y , i n d i c a t i n g t h a t t h e i n h i b i t i o n of Na,K-ATPase i s n e i t h e r s p e c i f i c nor d i g i t a l i s - l i k e . Air-acida c e t o n e e x t r a c t s i n h i b i t e d ouabain b i n d i n g i n a dosedependent manner. Experiments were designed t o a l l o w f o r t h e p r e s e n c e of potassium by i n c l u d i n g 1 0 mM K C 1 i n t h e r e a c t i o n medium. The i n h i b i t i o n of ouabain b i n d i n g i n c r e a s e d a t l o n g e r p e r i o d s of t i m e , s u g g e s t i n g nons p e c i f i c i n h i b i t i o n . The a d d i t i o n of t h e e x t r a c t t o preformed enzyme-ouabain complex caused a t i m e - and a dose-dependent l o s s of r a d i o a c t i v i t y which w a s f a s t e r t h a n t h e c h a s e by u n l a b e l e d ouabain, s u g g e s t i n g t h a t t h e e x t r a c t i s n o t simply competing w i t h ouabain f o r t h e ouabain b i n d i n g s i t e . C o n c e n t r a t i o n s of a i r - a c i d a c e t o n e e x t r a c t which had been demonstrated t o be a t t h e 150 f o r i n h i b i t i o n of Na,K-ATPase a c t i v i t y w e r e added t o a muscle b a t h c o n t a i n i n g guinea p i g and v e n t r i c u l a r

ENDOGENOUS INHIBITOR OF Na,K-ATPase: “ENDODIGIN”

947

s t r i p s . A t r a n s i e n t d e c r e a s e i n c o n t r a c t i l i t y was obt a i n e d and no p o s i t i v e i n o t r o p i c e f f e c t was o b s e r v e d d u r i n g a 20-min exposure. P o s s i b l e a r t i f a c t s which c o u l d account f o r t h e dat a o b t a i n e d w i t h t h e a i r - a c i d - a c e t o n e e x t r a c t i o n s of b r a i n i n c l u d e t h e p r e s e n c e of l y s o p h o s p h o l i p i d and p e r o x i d i z e d l i p i d , b o t h of which a r e known i n h i b i t o r s of Na,K-ATPase ( K a r l i et a l . , 1 9 7 9 ; Sun, 1 9 7 2 ) . Whole r a t b r a i n , and m a t e r i a l s o b t a i n e d from Methods A , B , and C were p a r t i t i o n e d by t h e method of Folch e t a l . (1957) and s u b j e c t e d t o TLC. The i d e n t i f i c a t i o n and q u a n t i f i c a t i o n of i n d i v i d u a l p h o s p h o l i p i d s p e c i e s a r e g i v e n i n Table I . The i n c r e a s e i n t h e number of s p e c i e s of p h o s p h o l i p i d s i n r a t b r a i n e x t r a c t s o b t a i n e d by Method A s u g g e s t s t h a t c o n s i d e r a b l e breakdown of phosp h o l i p i d o c c u r r e d a s a r e s u l t of t h e a i r - a c i d - a c e t o n e e x t r a c t i o n procedure. The e f f e c t of a c i d remains e v i d e n t i n t h e r e s u l t s o b t a i n e d from Method B. The phosp h o l i p i d composition from Method C s u g g e s t s l i t t l e o r no h y d r o l y s i s of p h o s p h o l i p i d . L i p i d p e r o x i d e s w e r e determined a f t e r Folch p a r t i t i o n i n g of e x t r a c t s p r e p a r e d by t h e t h r e e d i f f e r e n t methods. An a v e r a g e of 5 . 9 9 nmoles malonaldehyde/umole phosphorus was o b t a i n e d i n t h e l i p i d phase of r a t b r a i n s p r e p a r e d by a i r - a c i d a c e t o n e e x t r a c t i o n . D e t e c t a b l e amounts of p e r o x i d i z e d l i p i d w e r e not obtained i n m a t e r i a l prepared according t o Methods B and C . Both t h e aqueous and l i p i d phases o f Folch p a r t i t i o n s were t e s t e d f o r i n h i b i t i o n of Na,K-ATPase and Ca2+-ATPase a c t i v i t y (Table 11). The d e g r e e of enzyme i n h i b i t i o n v a r i e d w i t h t h e phase of t h e Folch p a r t i t i o n and t h e i n i t i a l method of e x t r a c t i o n , b u t i n h i b i t i o n of Na,K-ATPase was dose-dependent i n a l l c a s e s . Marked enzyme i n h i b i t i o n w a s o b t a i n e d i n t h e l i p i d phase of t h e Folch p a r t i t i o n i n a l l c a s e s , which s u g g e s t s a hydrophobic n a t u r e f o r t h e i n h i b i t i n g m a t e r i a l ( s 1 . Rat b r a i n s e x t r a c t e d by Methods A and B y i e l d e d m a t e r i a l which e x h i b i t e d t h e g r e a t e s t i n h i b i t i o n of enzyme act i v i t y p e r b r a i n f o r b o t h Na,K-ATPase and CaZ+-ATPase. However, Method C y i e l d e d m a t e r i a l which o v e r t h e same dose range i n h i b i t e d Na,K-ATPase a c t i v i t y i n a dosedependent manner , whereas Ca*+-ATPase a c t i v i t y appeared t o be s l i g h t l y and c o n s i s t e n t l y s t i m u l a t e d . S i n c e Methods A and B y i e l d e d m a t e r i a l ( s ) which were nons p e c i f i c i n h i b i t o r s , a s i n d i c a t e d by i n h i b i t i o n of b o t h Na,K-ATPase and Ca2+-ATPase a c t i v i t y , i t i s concluded t h a t e x t r a c t i o n methods, i n v o l v i n g t h e u s e of a i r o r a c i d o r b o t h , produce n o n s p e c i f i c i n h i b i t o r s . The o n l y method examined which y i e l d e d an e x t r a c t t h a t appeared

TABLE I.

Quantitative Analysis of Phosphorus Containing Species from TLCa

Abbreviation APG

BMP CL LPC LPE MLCL P PA Fc

PE PI PG PS Sm 0

a

Nomenclature Acyl phosphatidylglycerol Bis (monoacyl)glycerophosphate Cardiolipin Lysophosphatidylcholine Lysophosphatidylethanolamine Monolysocardiolipin Phosphorus-containing unknown lipids Phosphatidic acid Phosphatidylcholine Phosphatidylethanolamine Phosphatidylinositol Phosphatidylglycerol Phosphatidylserine Sphingomyelin Origin

rat brain

0.91

1.75 39.58 34.23

Air-acidacetone 1.4 1.24 1.81 9.74 31.06 1.14 14.5 2.2 18.27 3.49

2.79

2.2 1.17

13.1 6.54 1.07

1.24 6.98 3.8

N -acid2 acetone

N2/H20

acetone

6.2 2.14

60.58

23.73

33.77

41.17 25.5

3.4

3.2

Phosphorus composition is given as a percentage of the total phosphorus content.

TABLE 11.

R e l a t i v e I n h i b i t i o n of Na,K-ATPase and Ca

2+

-ATPase by Folch P a r t i t i o n i n g a

Aqueous phase

(D

.

L i p i d phase

% Inhibition:

Concn.

% Inhibition:

Rat b r a i n e x t r a c t i o n procedure

Concn (brain/ml)

A . Air-acid-acetone

0.32

6

9

0.008

53

63

B. N2-acid-acetone

0.16

11

0

0.16

41

54

C. H -H 0-acetone 2 2

0.32

10

0

0.32

11

2Ob

Na,K-ATPase

Ca

2+

-ATPase

(brain/ml)

Na,K-ATPase Ca

2+

-ATPase

P (0

aCalcium-ATPase a c t i v i t y (7 p g / m l ) was d e t e r m i n e d u n d e r the same c o n d i t i o n s as Na ,K-ATPase a c t i v i t y w i t h the e x c e p t i o n t h a t 0.1 mM EGTA was added t o the b u f f e r medium. Basal a c t i v i t y t S E The a d d i t i o n of r a b b i t s k e l e t a l m u s c l e s a r c o p l a s m i c r e t i c u l u m was 26 f 1 p m l e P i / h r / m g p r o t e i n . of 0 . 1 3 mM C a C l 2 (10 pM Ca2+ f r e e ) r e s u l t e d in a Ca2+-ATPase f S E of 419 f 21 p m o l e P i / h r / m g p r o -

tein. bAn a p p a r e n t s t i m u l a t i o n , not d o s e - d e p e n d e n t

.

K. R. WHlTMERetel.

950

to have a specific effect on Na,K-ATPase activity is Method C (nitrogen-water-acetone).

ACKNOWLEDGMENT

Supported by 5-T2 HL 07382 and PO1 HL 22619-04.

REFERENCES

Carraway, R . , and Leeman, S. (1973). The i s o l a t i o n o f a new hypotensive p e p t i d e , neurotensin, from bovine hypothalamus. J. B i o l . C h e m . 248, 6854-6861. Folch, J . , Lees, M . , and S t a n l e y , G. H . S. (1957). A simple method f o r t h e i s o l a t i o n and p u r i f i c a t i o n o f t o t a l l i p i d s from animal t i s s u e s . J. B i o l . Chem. 2 6 6 , 497-509. K a r l i , J. N . , Karikas, G. A . , Hatzipavlou, P. K . , L e w i s , G. M., and Moulopoulos, S. N. (1979). The i n h i b i t i o n o f Na+ and K+ s t i m u l a t e d ATPase a c t i v i t y i n r a b b i t and dog h e a r t sarcolemma by lysophosphatidyl c h o l i n e . L i f e S c i . 24, 1869-1876. S u n , A. Y. (1972). The e f f e c t o f l i p o x i d a t i o n on synaptosomal (Na+ + K+)-ATPase i s o l a t e d from t h e c e r e b r a l c o r t e x of s q u i r r e l monkey. B i o c h i r n . B i o p h y s . A c t a 2 6 6 , 350-360.

Part X

Physiology and Pathophysiologyof the NalK Pump

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CURRENT TOPICS IN MEMBRANES AND TRANSPORT,VOLUME 19

Disorders in Molecular Assemblies for Na Transport in Essential Hypertension MIlZY L. CANESSA, NORMA C. ADRAGNA, ISABEL BIZE, HAROLD SOLOMON,' AND DANIEL C. TOSTESON Department of Physiology Harvard Medical School Boston, Massachusetts

I.

INTRODUCTION

Three different transport systems translocate Na in human red cells: the phloretin-sensitive Na-Na exchange, the furosemide-sensitive Na-K cotransport, and the ouabain-sensitive Na pump. The three systems may have increasing levels of complexity in their molecular assemblies in order to function as Na translocators. The simplest unit might be the Na-Na exchange which couples the Na gradient to the counterflow of Na or Li. In the Na-K cotransport unit, the coupling of Na movement to the K gradient requires a higher degree of complexity in molecular assembly possibly afforded by a K-selective subunit. In the Na/K ATP-dependent pump, the coupling of Na transport to ATP hydrolysis may require the assembly of another subunit(s) to catalyze the chemical reactions. 'Affiliated with Brigham and Women's Hospital, Boston, Massachusetts. 951

Copyright 0 1983 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-153319.0

952

11.

MITZY L. CANESSA eta/.

RESULTS AND DISCUS S I O N

S e v e r a l p r o p e r t i e s of N a c o u n t e r t r a n s p o r t and cot r a n s p o r t p e r m i t t h e i r o p e r a t i o n a l c h a r a c t e r i z a t i o n as two d i f f e r e n t Na t r a n s p o r t p r o t e i n s . The systems d i f f e r i n t h e i r a f f i n i t y f o r N a and L i as w e l l as i n t h e i r sens i t i v i t y t o s e v e r a l i n h i b i t o r s , t o changes i n c e l l v o l ume, and t o replacement of c h l o r i d e by n i t r a t e . On t h e b a s i s o f t h e s e e x p e r i m e n t a l f i n d i n g s , w e have proposed t h a t c o u n t e r t r a n s p o r t and c o t r a n s p o r t a r e two d i f f e r e n t Na t r a n s p o r t p r o t e i n s . The two systems can a p p a r e n t l y f u n c t i o n a t maximal r a t e s i m u l t a n e o u s l y . The maximal r a t e o f Lii-Na, exchange can be s w i t c h e d on i n c e l l s loaded t o o b t a i n maximal a c t i v a t i o n of outward Li-K cotransport. N a c o u n t e r t r a n s p o r t and Na-K c o t r a n s p o r t can be simultaneously o r i n d e p e n d e n t l y a l t e r e d i n p a t i e n t s w i t h e s s e n t i a l h y p e r t e n s i o n and i n members of t h e i r f a m i l i e s . I n a Caucasian Bostonian p o p u l a t i o n , w e found t h a t elevated countertransport c o r r e l a t e d s i g n i f i c a n t l y with e l e v a t e d Na-K c o t r a n s p o r t i n p a t i e n t s w i t h e s s e n t i a l hyp e r t e n s i o n . I n P a r i s , however, i n t h o s e h y p e r t e n s i v e s u b j e c t s having a marked r e d u c t i o n i n t h e maximal a c t i v a t i o n of outward Na-K c o t r a n s p o r t , t h e Na c o u n t e r t r a n s p o r t was found o n l y s l i g h t l y e l e v a t e d . T h e r e f o r e , two t y p e s of a l t e r a t i o n s i n t h e o u a b a i n - i n s e n s i t i v e Na (1) e l e v a t e d t r a n s p o r t c a n be found i n h y p e r t e n s i o n : c o u n t e r t r a n s p o r t w i t h normal o r e l e v a t e d c o t r a n s p o r t , and ( 2 ) reduced c o t r a n s p o r t w i t h normal o r s l i g h t l y elevated countertransport. I t i s n o t c l e a r a t t h i s p o i n t whether t h e s e two t y p e s of Na t r a n s p o r t d i s o r d e r s i n h y p e r t e n s i o n r e f l e c t g e n e t i c d i f f e r e n c e s i n t h e p o p u l a t i o n s and/or environmental i n t e r a c t i o n s . Indeed, t h e d i s c o v e r y of t h e s e demes of r e d c e l l N a t r a n s p o r t may be a new i m p o r t a n t t o o l f o r r e s e a r c h on t h e g e n e t i c epidemiology of human hypertension. The s t r i k i n g r e l a t i o n s h i p found between a l t e r a t i o n s i n Na-K c o t r a n s p o r t and N a c o u n t e r t r a n s p o r t i n p a t i e n t s w i t h e s s e n t i a l h y p e r t e n s i o n seems t o i n d i c a t e t h a t , desp i t e d i f f e r e n c e s i n t h e i r f u n c t i o n a l p r o p e r t i e s , they might be somehow s i m i l a r l y r e g u l a t e d . A h y p o t h e s i s c o m p a t i b l e w i t h t h e s e f i n d i n g s i s t h a t t h e Na c o u n t e r t r a n s p o r t might r e f l e c t t h e g e n e t i c c o n t r o l of s y n t h e s i s and d e g r a d a t i o n p r o c e s s e s of Na-K c o t r a n s p o r t o r o t h e r N a t r a n s p o r t systems such a s t h e Na pump, t h e Na-Ca exchange, and t h e Na-coupled system.

CURRENT TOPICS IN MEMBRANES A N D TRANSPORT, VOLUME 19

The Na-K Cotransport System in Essential Hypertension R. P. GAMY, C.NAZARET, AND P.HANNAERT INSERM U 7c"SLA 318, Hepita1 Necker Paris. France

I.

INTRODUCTION

I n human e r y t h r o c y t e s , t h e p h y s i o l o g i c a l N a + conc e n t r a t i o n depends upon t h e b a l a n c e between t h e N a + ext r u s i o n by t h e N a / K pump and t h e N a + g a i n by p a s s i v e Na+ permeability. However, a n e t N a + e x t r u s i o n i n a n u p h i l l d i r e c t i o n a g a i n s t t h e e l e c t r o c h e m i c a l Na+ g r a d i e n t c o u l d s t i l l be o b s e r v e d i n t h e p r e s e n c e o f s a t u r a t i n g c o n c e n t r a t i o n s of o u a b a i n (Hoffman and Kregenow, 1966; S a c h s , 1 9 7 1 ) . I n a d d i t i o n , o u a b a i n - r e s i s t a n t , f u r o s e m i d e - s e n s i t i v e c o u p l e d movement o f N a + and K+ i n t h e inward (Wiley and Cooper, 1 9 7 4 ) and outward (Garay e t a l . , 1981) d i r e c t i o n s a c r o s s t h e membrane h a s been r e p o r t e d . T h i s Na-K c o t r a n s o r t system i s r e s p o n s i b l e f o r the ouabain-resistant Nap extrusion against t h e e l e c t r o c h e m i c a l N a + g r a d i e n t (Garay e t a l . , 1 9 8 0 ) . Under p h y s i o l o g i c a l c o n d i t i o n s , t h e outward cot r a n s p o r t i s s t i m u l a t e d by i n t e r n a l N a + and t h e inward c o t r a n s p o r t is s t i m u l a t e d by e x t e r n a l K+. Under basal c o n d i t i o n s t h e N a - K c o t r a n s p o r t system i s n e a r e q u i l i b 953

Copyright 0 1983 by Academic Press. Inc. All rights of reproduction in any form r e . ~ ~ e d . ISBN 0-12-153319-0

R. P.GARAY eta/.

954

r i u m , i . e . , inward and outward c o t r a n s p o r t f l u x e s show s i m i l a r magnitudes and t h u s no n e t c a t i o n f l u x e s a r e obs e r v e d . Any change i n i n t e r n a l N a + o r e x t e r n a l K+ conc e n t r a t i o n i s c o r r e c t e d by a n e t c o t r a n s p o r t f l u x i n t h e o p p o s i t e d i r e c t i o n of t h e i o n i c p e r t u r b a t i o n . Thus, t h e Na-K c o t r a n s p o r t system seems t o be a s e l f - r e g u l a t o r o f t h e N a + and K+ e l e c t r o c h e m i c a l g r a d i e n t s a c r o s s c e l l membranes. The N a - K c o t r a n s p o r t f l u x e s i n human e r y t h r o c y t e s are t e n t i m e s lower t h a n t h e pump f l u x e s . T h i s sugg e s t s t h a t i n human r e d c e l l s any r e g u l a t i o n o f t h e Na+ g r a d i e n t depends more on t h e pump (Garay and Garrahan, 1973) t h a n on t h e c o t r a n s p o r t system. However, t h i s seems n o t t o be t h e c a s e i n v a s c u l a r smooth muscle c e l l s i n which c o t r a n s p o r t and pump f l u x e s show a similar o r d e r of magnitude (Tuck e t a l . , 1 9 8 1 ) . T h i s s u g g e s t s t h a t i n t h e s e and p e r h a p s o t h e r c e l l s , t h e Na-K c o t r a f i s p o r t system may s e l f - r e g u l a t e any v a r i a t i o n i n i n t r a c e l l u l a r N a + c o n c e n t r a t i o n . On t h e o t h e r hand, t h e human red c e l l i s v e r y p a r t i c u l a r because i t s memb r a n e p o t e n t i a l i s v e r y c l o s e t o t h e C1- e q u i l i b r i u m poI n c e l l s l i k e v a s c u l a r smooth m u s c l e c e l l s , i n tential. which t h e membrane p o t e n t i a l i s v e r y c l o s e t o t h e e q u i l i b r i u m K+ p o t e n t i a l , t h e energy f o r n e t N a + movements may be s u p p l i e d by a c o u p l e d movement of C 1 - t h r o u g h a Na-K-C1 c o t r a n s p o r t (Haas e t a l . , 1982) W e have p r e v i o u s l y r e p o r t e d t h a t t h e outward f l u x e s c a t a l y z e d by t h e N a - K c o t r a n s p o r t system were abnormally l o w i n e r y t h r o c y t e s from most e s s e n t i a l h y p e r t e n s i v e pat i e n t s (Garay e t a l . t 1 9 8 0 ; Dagher and Garay, 1 9 8 0 ) . P r e l i m i n a r y r e s u l t s from o u r l a b o r a t o r y show t h a t t h e h y p e r t e n s i v e s w i t h low outward N a - K c o t r a n s p o r t f l u x e s (Co - h y p e r t e n s i v e s ) a r e d i f f e r e n t from t h o s e p r e v i o u s l y found by Canessa e t a l . (Canessa e t a l . , 1980) e x h i b i t i n g h i g h Na-Li c o u n t e r t r a n s p o r t f l u x e s (Counter + h y p e r t e n s i v e s ) . This e x p l a i n s t h e extremely v a r i a b l e proportion of Co - and/or Counter + h y p e r t e n s i v e s found i n s e v e r a l l a b o r a t o r i e s (see t h e t a b l e ) .

.

11.

A K I N E T I C ANALYSIS O F THE OUTWARD N a - K I N ESSENTIAL HYPERTENSION

COTRANSPORT

The r e g u l a t i o n of t h e i n t r a c e l l u l a r N a + c o n c e n t r a t i o n a p p e a r s t o be m e d i a t e d by t h e sigmoid s t i m u l a t i o n of t h e outward N a - K c o t r a n s p o r t by i n t e r n a l ' N a + ( F i g . 1 ) . The t h r e s h o l d p o i n t o f t h i s c u r v e i s v e r y c l o s e t o t h e p h y s i o l o g i c a l N a + c o n c e n t r a t i o n i n e r y t h r o c y t e s from

Na+,K+ COTRANSPORT IN HYPERTENSION

Frequency o f Co

-

and Counter

+

955

H y p e r t e n s i v e s i n D i f f e r e n t Samples ~

Lab0 ra t o r y Dagher and Garay Canessa e t a l . Cusi e t a l . G l e s s et al. Brunois e t a l . Ghione e t al. Herubel e t a l . Davidson e t a l . Woods e t a l . ~ Canessa e t a Adragna et a l . H a l p e r i n e t al. Garay e t a l . Tuck e t a l . La Rosa e t a l .

~~

P e r c e n t of t o t a l hypertensives co Counter

( P a r i s , 1980) (Boston, 1980) (Milan , 1981) (Heidelberg , 1981) (Reims , 1981) ( P i s a , 1981) (Rouen, 1981) (Capetown, 1982) (Chapel H i l l , 1982) . ( P~h i la d e l p h ia , 198 2 ) (Boston, 1982) (Caracas, 1982) ( P a r i s , 1982) (Los Angeles, 1982) (Peru 4500 m, 1982)

88.6

(44)

44.5 84.6 100 74

(45) (13) (33) (8)

100

(7)

34.7

(72)

58.8 4.6 83.3 70.8 68.2 76.2

(17) (22) (18) (65) (22) (21)

100 22.2

+

(36) (45)

100 5.9 86.4 28.6

(28)

aThese r e s u l t s have been o b t a i n e d u s i n g methods e s s e n t i a l 1 y s i m i l a r t o t h o s e p u b l i s h e d (Dagher and Garay, 1980; Canessa e t a l . , 1 9 8 0 ) . F o r e a c h l a b o r a t o r y , a normal r a n g e i n c l u d i n g 90% o f normoCo and Counter + h y p e r t e n s i v e s t e n s i v e c o n t r o l s was c a l c u l a t e d . i n d i c a t e s u b j e c t s with outward Na-K c o t r a n s p o r t or Na-Li c o u n t e r t r a n s p o r t below or above t h e normal range, r e s p e c t i v e l y . The numb e r o f h y p e r t e n s i v e s s t u d i e d is i n d i c a t e d i n b r a c k e t s . bNegro h y p e r t e n s i v e s ( f u r o s e m i d e - s e n s i t i v e K+ e f f l u x ) .

-

normotensive c o n t r o l s . In fact, the intracellular Na+ c o n c e n t r a t i o n f o r half-maximal s t i m u l a t i o n (K50%) of t h e outward c o t r a n s p o r t was 1 2 . 9 f 2 . 4 m m o l e s / l i t e r c e l l s (mean ? SD) i n 5 0 normotensive c o n t r o l s . I n 46 o u t o f 65 e s s e n t i a l h y p e r t e n s i v e s K50 % w a s h i g h e r t h a n 1 6 mmoles/liter c e l l s (Co - h y p e r t e n s i v e s ) . Thus, due t o a diminished apparent a f f i n i t y f o r i n t r a c e l l u l a r N a + (and t o a decreased maximal r a t e ) C o hypertensive p a t i e n t s do n o t show s u c h a s h a r p c o t r a n s p o r t s t i m u l a t i o n i n t h e c l o s e v i c i n i t y of t h e p h y s i o l o g i c a l N a + conc e n t r a t i o n ( F i g . 1 ) . Thus, it may be e x p e c t e d t h a t i n Co hypertensives the cells cannot w e l l regulate t h e d i s t u r b a n c e i n i n t r a c e l l u l a r Na+ c o n t e n t i n d u c e d by an e x c e s s N a + i n t a k e . T h i s may be p a r t i c u l a r l y t r u e i n e x c i t a b l e c e l l s o f h i g h surface/volume r a t i o s u c h a s v a s c u l a r smooth muscle c e l l s o r c a t e c h o l a m i n e r g i c neurons. B e f o r e t h e development of e s t a b l i s h e d hyper-

-

-

956

R. P. GARAY eta/.

0 = tSStWlIA1 H Y P E R l t W f l Y t P A l l t W l

z

h

.-

60-

I

5 r 0

W

0

x

I

i

_/

I

4

I

Fi5. 1. Percent response of the outward N a - K cotransport t o an increase i n i n t r a c e l l u l a r Na+ concentration i n a normotenNear s i v e subject ( 0 ) compared t o an hypertensive patient ( ) the physiological Na+ concentration, the essential hypertensive patient i s l e s s able t o respond t o a change i n internal Na+ concen t r a ti on.

.

t e n s i o n , a s l i g h t i n c r e a s e i n pump a c t i v i t y may compens a t e f o r t h e c o t r a n s p o r t a b n o r m a l i t y (Garay e t a l . , 1 9 8 0 ) . On t h e o t h e r hand, any temporary o r permanent i n c r e a s e i n i n t r a c e l l u l a r N a + c o n t e n t may l e a d t o a n i n crease i n v a s c u l a r r e s i s t a n c e t h r o u g h n o r a d r e n a l i n e release from c a t e c h o l a m i n e r g i c n e u r o n s (Nakazato e t a l . , 1978).

REFERENCES

Canessa, M . , Adragna, N . , Solomon, H., Connolly, T . , and T o s t e s o n , D. C. (1980) I n c r e a s e d sodium-lithium c o u n t e r t r a n s p o r t i n r e d c e l l s of p a t i e n t s w i t h e s s e n t i a l hypertension. New Eng. J . Med. 382, 7 7 2 .

.

Na+,K+ COTRANSPORT IN HYPERTENSION

957

+

+

Dagher, G. , and Garay, R. P. (1980). A Na , K c o - t r a n s p o r t a s s a y f o r e s s e n t i a l hypertension. Can. J . B i o c h e m . 5 8 , 1069. Garay, R., Adragna, N . , Canessa, M . , and Tosteson, D. C. (1981). Outward Na+, K+ c o - t r a n s p o r t i n human e r y t h r o c y t e s . J . Membrane B i o l . 6 2 , 169. Garay R. P . , Dagher, G., P e r n o l l e t , M. G . , Devynck, M. A., and Meyer, P. (1980). I n h e r i t e d d e f e c t s i n a Na+, K+ c o - t r a n s p o r t system i n e r y t h r o c y t e s from e s s e n t i a l hypertensive p a t i e n t s . Nature ( L o n d o n ) 2 8 4 , 281. Garay R., and Garrahan, P. (1973). The i n t e r a c t i o n of sodium and potassium w i t h t h e sodium pump i n r e d c e l l s . J. P h y s i o l . 2 3 1 , 297. Haas, M . , Schmidt, W. F . , and McManus, T . J. (1982). Cathecolamine-stimulated ion t r a n s p o r t i n duck r e d c e l l s . J. G e n . P h y s i o l . 8 0 , 125. Hoffman, J. F . , and Kregenow, F. M. (1966). The c h a r a c t e r i z a t i o n of new energy dependent c a t i o n t r a n s p o r t p r o c e s s e s i n red blood c e l l s . Ann. N.Y. A c a d . S c i . 1 3 7 , 566. Nakazato, Y., Ohga, A . , and Onoda, Y. (1978). The e f f e c t of ouabain o r noradrenaline o u t p u t from p e r i p h e r a l adrenergic neurons o f i s o l a t e d Guinea p i g Va d e f e r e n s . J . P h y s i o l . 2 7 8 , 45. Sachs, J. R. (1971). Ouabain i n s e n s i t i v e sodium movements i n t h e human red blood cells. J . Gen. P h y s i o l . 5 7 , 259. Tuck, M . , Garay, R., Russo-Marie, F., and Meyer, P. (1982). Na', K+ c o - t r a n s p o r t system i n v a s c u l a r smooth muscle c e l l s . S c i . Meet. I n t . SOC. H y p e r t e n s . 9 t h , 1 9 8 2 , p. Wiley, J. S., and Cooper, R. A . (1974). A furosemide-sensitive cot r a n s p o r t of sodium p l u s potassium i n t h e human red c e l l . J. C l i n . Invest. 5 3 , 745.

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CURRENT TOPICS M MEMBRANES AND TRANSPORT, VOLUME 19

Loss of Na,K-ATPase Activtty during Cataract Formation in Lens PARlMAL C. SEN

AND DOUGLAS R. PFEIFFER The Home1 Institute Universiry of Minnesota Austin, Minnesota

I.

INTRODUCTION

The maintenance o f t r a n s p a r e n c y i n l e n s i s c r i t i c a l l y dependent on t h e normal d i s t r i b u t i o n o f N a + and K+ i o n s a c r o s s f i b e r c e l l membranes, and t h u s on t h e a c t i v i t y o f l e n s N a , K - A T P a s e (see K i n o s h i t a , 1974, f o r r e v i e w ) . The plasma membrane o f f i b e r c e l l s i s u n u s u a l i n t h a t h i g h l e v e l s of c h o l e s t e r o l and s a t u r a t e d f a t t y acids i n the phospholipids r e s u l t i n a g e l t o l i q u i d p h a s e t r a n s i t i o n t e m p e r a t u r e of a p p r o x i m a t e l y 35OC (Sen and P f e i f f e r , 1 9 8 2 ) . Thus, l e n s Na,K-ATPase act i v i t y s h o u l d be h i g h l y s e n s i t i v e t o minor changes i n membrane l i p i d c o m p o s i t i o n . T r i p a r a n o l and re l a t e d amp h i p h i l i c p h a r m a c o l o g i c a l a g e n t s are c a t a r a c t o g e n i c and known t o a l t e r l i p i d metab o l i s m i n o t h e r t i s s u e s (see Lullmann-Rauch, 1 9 7 9 , f o r r e v i e w ) . I n t h i s r e p o r t , w e examine t h e t i m e c o u r s e of changes i n Na,K-ATPase a c t i v i t y , i o n c o n t e n t s , l i p i d comp o s i t i o n , and s u l f h y d r y l group c o n t e n t i n l e n s membranes d u r i n g c a t a r a c t f o r m a t i o n induced by t r i p a r a n o l f e e d i n g . 959

Copyright 0 1983 by Academic h s , Inc. All rights of reproduction in any form reserved. ISBN 0-12-153319-0

PARIMAL C.SEN AND DOUGLAS R. PFEIFFER

960

11.

EXPERIMENTAL

Weanling W i s t a r r a t s weighing approximately 50 gm

were maintained on r a t chow w i t h and w i t h o u t 0.075% (w/w) t r i p a r a n o l . P e r i o d i c a l l y l e n s e s were c o l l e c t e d and t h e p r e s e n c e of c a t a r a c t s w a s n o t e d . Measurements of l i p i d c o n t e n t and composition and Na,K-ATPase a c t i v i t y were made a s d e s c r i b e d p r e v i o u s l y (Mizuno et al., 1 9 8 1 ) . The i o n c o n t e n t s o f l e n s homogenates were measured by atomic a b s o r p t i o n s p e c t r o s c o p y . S u l f h y d r y l c o n t e n t s of t h e p a r t i a l l y p u r i f i e d Na,K-ATPase p r e p a r a t i o n s w e r e determined by r e a c t i o n w i t h DTNB (Murphy, 1 9 7 6 ) o r r e a c t i o n w i t h [1-14C]NEM (Shoot e t a l . , 1 9 7 8 ) .

111.

RESULTS AND DISCUSSION

F i g u r e 1 shows t h a t a r e c i p r o c a l r e l a t i o n s h i p e x i s t s between t h e i n c i d e n c e of c a t a r a c t f o r m a t i o n and l o s s of Na,K-ATPase a c t i v i t y i n l e n s e s from t r i p a r a n o l f e d r a t s . These r e s u l t s are c o n s i s t e n t w i t h t h e view t h a t t h e c a t a r a c t s a r e caused by i n h i b i t i o n of t h e enzyme. Na,K-ATPase a c t i v i t y i n kidney, b r a i n , l i v e r , and e r y t h r o c y t e s was n o t s i g n i f i c a n t l y diminished i n animals p o s s e s s i n g b i l a t e r a l c a t a r a c t s . E l e v a t e d Na+ and d e c r e a s e d K+ p r e s e n t i n t h e c a t a r a c t o u s l e n s e s (Table I ) confirms t h a t loss of t h e enzyme a c t i v i t y l e a d s t o abnormal c a t i o n d i s t r i b u t i o n i n t h i s unique t i s s u e . The c o n t e n t of Ca2+, a known i n h i b i t o r of l e n s Na,K-ATPase (Hamilton e t a l . , 1 9 7 9 ) , i s i n c r e a s e d i n t h e c a t a r a c t o u s samples ( T a b l e I ) . T r i p a r a n o l does n o t d i r e c t l y i n h i b i t Na,K-ATPase when added a t l e v e l s s u b s t a n t i a l l y i n e x c e s s of t h o s e expected t o accumulate i n l e n s d u r i n g t h e f e e d i n g p e r i o d (Mizuno et a l . , 1 9 8 1 ) . Lens Na,K-ATPase can be p a r t i a l l y p u r i f i e d by deoxycholate e x t r a c t i o n and dens i t y g r a d i e n t c e n t r i f u g a t i o n of t h e f i b e r c e l l plasma membrane f r a c t i o n (Sen and P f e i f f e r , 1 9 8 2 ) . Table I1 shows t h a t t h e d e o x y c h o l a t e t r e a t m e n t i n c r e a s e s t h e s p e c i f i c a c t i v i t y of t h e enzyme from normal b u t n o t c a t a r a c t o u s t i s s u e . The p a r t i a l l y p u r i f i e d enzyme i s n o t r e a c t i v a t e d by EGTA and/or bovine serum albumin (BSA). Thus, accumulation o f Ca2+, long-chain acyl-CoA, o r o t h e r p o t e n t i a l i n h i b i t o r s removable by p a r t i a l pur i f i c a t i o n does n o t a c c o u n t f o r enzyme i n h i b i t i o n . The c a t a r a c t o u s p r e p a r a t i o n c o n t a i n s fewer s u l f h y d r y l groups (Table 111), which could e x p l a i n t h e l o s s of ac-

Na,K-ATPase IN TRIPARANOL INDUCED CATARACT

961

Weeks of feedinq

Fig. 1 . T h e e f f e c t o f t r i p a r a n o l f e e d i n g on c a t a r a c t d e v e l o p m e n t and Na,K-ATPase i n r a t l e n s . Animals w e r e f e d a t r i p a r a n o l - c o n t a i n i n g or a n o r m a l d i e t f o r 6 8 d a y s , a f t e r w h i c h a l l a n i m a l s w e r e f e d n o r m a l c h o w . A t e a c h t i m e p o i n t lenses f r o m 7 or 8 a n i m a l s w e r e c o l l e c t e d , the p r e s e n c e o f c a t a r a c t s w a s n o t e d , and the a m o u n t o f Na,K-ATPase w a s d e t e r m i n e d on the p o o l e d h o m o g e n a t e a s d e s c r i b e d p r e v i o u s l y (Mizuno e t a l . , 1 9 8 1 ) : (.,El) The i n c i d e n c e o f c a t a r a c t s i n t r i p a r a n o l - f e d and normal r a t s , r e s p e c t i v e l y ; ( @ ,0 ) Na,K-ATPase i n lenses f r o m t r i p a r a n o l - f e d and n o r m a l r a t s , r e s p e c t i v e l y .

tivity if further studies show that they were lost from the Na,K-ATPase. Lipid analysis (Table IV) shows that lens phospholipid and sterol contents are not markedly altered in triparanol-fed animals. However, several differences in lipid composition are apparent. The percentage con-

962

PARIMALC. SEN AND DOUGLAS R. PFEIFFER

TABLE I.

C a t i o n Contents o f Normal and C a t a r a c t o u s Lensesa C a t a r a c t o u sb

Normalb Calcium Sodium Potassium Magnes ium

5.40 16.5 111.2 5.2

8.40 111.7 43.8 4.1

a

Lenses were homogenized and i o n c o n t e n t s w e r e determined a s d e s c r i b e d i n S e c t i o n II. b I n u n i t s of nmoles/mg p r o t e i n .

TABLE 11.

E f f e c t o f S e l e c t e d Agents on Na,K-ATPase A c t i v i t y from Normal and C a t a r a c t o u s Lenses A c t i v i t y ( nmo les/mg/hr) Normal Cataractous

Agenta

-

Deoxycholate EGTA BSA EGTA + BSA

34.9 f 4.9 80.0 f 0.5 110 110

-

+

102 f 1 2 . 3 81 f 1 . 5 50

--

7.6 f 2.0 11.4 f 3.2 16.5 16.5

+

9.1 k 2 . 1 10.2 f 2.8 11.2 12.2

a

When p r e s e n t , deoxycholate, EGTA, and BSA were u t i l i z e d a t 1 mg/mg p r o t e i n , 1 mM, and 1 mg/mg p r o t e i n , r e s p e c t i v e l y . The effects of E T A and BSA w e r e determined i n t h e presence of deoxycholate.

TABLE 111.

Comparison o f S u l f h y d r y l Group Contents o f Normal and C a t a r a c t o u s Lens Membrane ~~~

Normala 66.2 f 8.5 66.0 f 10.7

Cataractousa 53.5 f 7.1 47.0 f 6.8

a

~~

~

D i f f erencea

-12.7b -19. o c

I n u n i t s of nmoles SH/mg p r o t e i n . bDetermined s p e c t r o p h o t o m e t r i c a l l y by r e a c t i o n w i t h 5,s '-di t h i o b i s (2-ni t r o b e n z o i c a c i d ) CDetermined radiochemical 1y b y r e a c t i o n with [l-14C]NEM.

.

Na,K-ATPaseIN TRIPARANOL INDUCED CATARACT

TABLE I V .

963

C o n t e n t and Composition of L i p i d s from C o n t r o l and Triparanol-Fed R a t Lensesa

Lipid Phospholipids Control ( t o t a l ) PE PC PS SPh Triparanol ( t o t a l ) PE PC

4

73.0

72.0 35.8 40.0 12.8 8.6 72.0 35.2 38.0 13.0 10.4

66.0 35.0 39.0 13.4 8.4 66.0 34.0 31.8 12.0 15.6

6.2 97.5 2.5 5.2 80.0 12.5

6.0 97.5 2.5 5.4 70.0 20.8

-

73.0

-

PS SPh Sterol Control (total) Cholesterol Desm s t e r o l Triparanol ( t o t a l ) Cholesterol Desmsterol

Weeks of f e e d i n g 8 12

0

6.4 97.5 2.5 5.2 97.5 2.5

16

20

69.0 34.6 37.0 13.8 11.0 60.0 35.0 31.0 11.0 18.0

69.0 34.0 35.0 14.4 12.2 63.0 35.0 30.8 9.6 20.0

68.0 31.8 34.0 15.2 13.4 68.0 35 .O 30.0 9.8 21.0

6.1 97.5 2.5 4.8 70.0 20.0

6.3 97.5 2.5 6.8 75.0 18.0

6.4 97.5 2.5 7.0 79.0 15.0

a

Total values are given as pmoles/gm wet weight of lens. Values f o r individual components are given as percent of the total. PE, phosphatidylethanolamine; FC, phosphatidylcholine; PS, phosphatidylserine; SPh, sphingomyelin.

tent of phosphatidylserine in lens normally increases with age, whereas in triparanol-fed animals it decreases. An increase in sphingomyelin and decrease in phosphatidylcholine contents are seen in both groups of animals, although these alterations are accelerated markedly by triparanol. These findings suggest that the drug promotes activity of the base exchange pathway for sphingomyelin synthesis from phosphatidylcholine. Desmosterol also accumulates (Table IV) and changes in phospholipid fatty acid composition are observed (Mizuno e t al., 1981). Statistical analysis, employing cataracts produced by several agents, revealed that a loss of phosphatidylserine is the only change in lipid composition which always correlates with loss of lens Na,K-ATPase activity (Mizuno et al., 1981). Since this enzyme requires acidic phospholipid for maximal activity (Roelofsen and Van Deenen, 1973; Maura e t al., 1981), loss of phosphatidylserine, together with the membrane

PARIMALC. SEN AND DOUGLAS R. PFEIFFER

964

f l u i d i t y e f f e c t s of t h e o t h e r l i p i d a l t e r a t i o n s , may e x p l a i n t h e l o s s of Na,K-ATPase a c t i v i t y and t h u s t h e development of t h e s e t y p e s o f c a t a r a c t s .

ACKNOWLEDGMENTS

T h i s work w a s supported by g r a n t s EY 02307, EY 02613, and HL 08214 from t h e N a t i o n a l I n s t i t u t e s o f H e a l t h and t h e Hormel

e thank W i l l i a m L. A l b r e c h t o f Merrell-National Foundation. W L a b o r a t o r i e s for supplying t r i p a r a n o l .

REFERENCES Hamilton, P. M . , Delamere, N. A., and P a t e r s o n , C. A. (1979). The i n f l u e n c e of calcium i o n on l e n s ATPase a c t i v i t y . Invest. O p h t h a l m l . V i s u a l S c i . 1 8 , 434-436. K i n o s h i t a , J. H . (1974). Mechanisms i n i t i a t i n g cataract fonnat i o n . Invest. O p h t h a l m o l . 13, 713-724. Drug-induced lysosomal s t o r a g e d i s LUlmann-Rauch, R. (1979) In "Lysosomes i n Applied Biology and T h e r a p e u t i c s " orders. (J. T. Dingle e t al., e d s . ) , Vol. 6 , pp. 49-130. NorthHolland Publ., Amsterdam. Maura, F . , B o n e t t i , A. C . , and Carpends, F. (1981). I n c r e a s e o f Na+,K+-ATPase a c t i v i t y i n i n t a c t r a t b r a i n synaptosomes a f t e r t h e i r i n t e r a c t i o n with phosphatidylserine vesi cl es. B i o c h e m . B i o p h y s . Res. Commun. 1 0 1 , 1337-1344. Mizuno, G. R . , Chapman, C. J . , C h i p a u l t , J. R . , and P f e i f f e r , D. R. (1981). L i p i d composition and (Na+,K+) -ATPase act i v i t y of r a t lens d u r i n g t r i p a r a n o l - i n d u c e d cataract formation. B i o c h i m . B i o p h y s . A c t a 644, 1-12. Murphy, A. (1976). S u l f h y d r y l group m o d i f i c a t i o n of s a r c o p l a s m i c reticulum membranes. B i o c h e m i s t r y 25, 4492-4496. Roelofsen, B. , and Van Deenen, L. L. M. (1973). L i p i d requirements of membrane-bound ATPase. E u r . J. B i o c h e m . 4 0 , 245-257. Sen, P . C . , and P f e i f f e r , D. R. (1982). C h a r a c t e r i z a t i o n o f p a r t i a l l y p u r i f i e d (Na+,K+) -ATPase from p o r c i n e l e n s . B i o c h i m . B i o p h y s . A c t a 693, 34-44. Shoot, B. M . , Depont, J. J. H. H. M., and Bonting, S. L. (1978). S t u d i e s on (Na+,K+)-ATPase: Evidence f o r two classes of ess e n t i a l s u l f h y d r y l groups. B i o c h i m . B i o p h y s . A c t a 522, 602-613.

.

CURRENT TOPICS IN MEMBRANES AND TRANSPORT,VOLUME 19

NalK Pump: Effect of Obesity and Nutritional State M.DELUISE, P. USHER, AND J. FLIER Beth Israel Hospital Harvard Medical School Boston Massachusetts I

I.

INTRODUCTION

Na,K-ATPase, in its role as the major Na/K pump of cells, utilizes significant amounts of energy and thus could be responsible for a considerable proportion of overall cellular thermogenesis. Obesity may in part result from disordered cellular thermogenesis and some animal models of obesity have been shown to have both reduced cellular thermogenesis and reduced levels of Na,K-ATPase in tissues. The studies reported here have examined the status of the Na/K pump in some types of human obesity; in addition, an animal model has been used to determine the influence of dietary factors on tissue Na/K pump activity.

965

Copyright 0 1983 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-153319-0

966

11.

M. OeLUlSE eta/.

HUMAN OBESITY

The e r y t h r o c y t e h a s been used as t h e model i n t h e human s t u d i e s and t h e f o l l o w i n g measurements have been made i n f o u r s e p a r a t e groups of i n d i v i d u a l s : number of pump u n i t s / c e l l (from s a t u r a t i o n b i n d i n g of [3H]ouabain) i c a t i o n t r a n s p o r t a c t i v i t y bx t h e Na/K pump ( a s t h e o u a b a i n - s e n s i t i v e r a t e of 8 Rb u p t a k e ) ; i n t r a c e l l u l a r l e v e l s of Na+ and K + . The r e s u l t s are summarized i n Table I . I n markedly obese a d u l t s u b j e c t s t h e r e was a h i g h l y s i g n i f i c a n t ( p < 0 . 0 0 1 ) mean 22% d e c r e a s e i n t h e number of ouabain b i n d i n g s i t e s . T h i s was r e f l e c t e d i n equally s i g n i f i c a n t 16% decrease i n the c a t i o n t r a n s p o r t a c t i v i t y of t h e pump i n t h e same c e l l s . A d d i t i o n a l l y , t h e i n t r a c e l l u l a r l e v e l of sodium w a s a mean of 35% h i g h e r i n e r y t h r o c y t e s of obese s u b j e c t s . The magnitude of t h e r e d u c t i o n i n t h e number of pump u n i t s w a s d i r e c t l y c o r r e l a t e d w i t h t h e d e g r e e o f o b e s i t y of t h e s u b j e c t s , measured a s t h e p e r c e n t a g e of increase above i d e a l body I t i s probable t h a t t h e weight ( r = 0.56, p < 0 . 0 1 ) . d e f e c t i n t h e r e d c e l l Na/K pump i n o b e s i t y i s n o t secondary t o t h e obese s t a t e i t s e l f , s i n c e weight reduct i o n toward normal ( p a r t i a l i n 15 s u b j e c t s , complete i n 2 ) d i d n o t r e s u l t i n any change i n t h e s t a t u s of t h e Pump The f i n d i n g s i n a d u l t o b e s i t y were confirmed i n a group of a d o l e s c e n t (mean age 13.7 y r ) obese subjects whose o b e s i t y had no c l e a r e t i o l o g i c a l b a s i s a p a r t from a p o s s i b l e f a m i l i a l tendency t o t h e c o n d i t i o n . I n cont r a s t , w e found no a b n o r m a l i t y i n t h e mean number of pump u n i t s i n e r y t h r o c y t e s from a group of s i m i l a r l y obese a d o l e s c e n t s u b j e c t s (mean 187% of i d e a l body w e i g h t ) whose o b e s i t y was a s s o c i a t e d w i t h hypothalamic l e s i o n s ( u s u a l l y tumors). This finding i s a l s o consist e n t w i t h t h e h y p o t h e s i s t h a t t h e r e d c e l l N a / K pump def e c t seen i n i d i o p a t h i c o b e s i t y is n o t secondary t o t h e o b e s i t y p e r se.

111.

DIETARY INFLUENCE ON THE Na/K PUMP

T o i n v e s t i g a t e a p o s s i b l e r o l e f o r t h e N a / K pump i n t h e i n c r e a s e i n m e t a b o l i c r a t e s e e n a f t e r i n c r e a s e d cal o r i c i n t a k e ( s o - c a l l e d d i e t a r y - i n d u c e d thermogenesis) , we s t u d i e d pump-mediated c a t i o n t r a n s p o r t a c t i v i t y i n both l i v e r s l i c e s and i s o l a t e d s o l e u s muscles from mice which were induced t o i n c r e a s e c a l o r i c i n t a k e by having

NalK PUMP EFFECTOF OBESITY AND NUTRITIONAL STATE

TABLE I.

967

E r y t h r o c y t e Na/K Pump S t a t u s i n Human O b e s i t y

Groupa Adult c o n t r o l s (41) Adult obese (21) Adolescent c o n t r o l s (13) Adolescent i d i o p a t h i c o b e s e (10) Adolescent hypothalam i c obese (9)

Maximal ouabain binding capacity (pmoles/l09 c e l l s )

pump-mediated Rb uptake (nmoles/109 c e l l s / h r ) Na/K

0.596 2 0.017 0.474 t 0.018

83.5 t 1 . 6 70.2 1: 1 . 9

0.592 t 0.031

8 8 . 4 t 3.3

0.474 t 0.027

80.6 k 4 . 1

0.596 t 0.046

97.9 t 5.3

a

Numbers i n p a r e n t h e s e s refer t o the number of s u b j e c t s s t u d i e d . Results a r e e x p r e s s e d a s mean k SEM.

access t o a 1 0 % s u c r o s e s o l u t i o n i n a d d i t i o n t o chow. W e found t h a t 5 d a y s of access t o s u c r o s e r e s u l t e d i n a 3 0 % i n c r e a s e i n t o t a l c a l o r i c i n t a k e and i n concomit a n t 88% ( p < 0 . 0 0 1 ) and 2 2 % ( p < 0.02) i n c r e a s e s i n l i v e r and s o l e u s muscle N a / K pump a c t i v i t y , r e s p e c t i v e ly. I n c o n t r a s t t o t h e s e f i n d i n g s i n normal m i c e , o b / o b m i c e s i m i l a r l y exposed t o s u c r o s e showed no s i g n i f i c a n t i n c r e a s e i n t h e r a t e o f pump-mediated h e p a t i c 86Rb u p t a k e , e v e n though these a n i m a l s i n c r e a s e d t h e i r c a l o r i c i n t a k e by a n even g r e a t e r amount t h a n d i d t h e c o n t r o l animals. I n c o n c l u s i o n , w e have d e m o n s t r a t e d a r e d u c t i o n i n b o t h t h e number and t h e a c t i v i t y of r e d b l o o d c e l l N a / K pump u n i t s i n s o m e o b e s e humans; two l i n e s of e v i dence s u g g e s t t h a t t h e defect does n o t r e s u l t from o b e s i t y o r o v e r e a t i n g p e r se. I n d e e d , t h e i n c r e a s e d c a l o r i c i n t a k e t o which o b e s i t y i s u s u a l l y a t t r i b u t e d r e s u l t s i n i n c r e a s e d a c t i v i t y o f t h e pump i n a t l e a s t two t i s s u e s . T h i s d i e t a r y a c t i v a t i o n i s n o t s e e n i n g e n e t i c a l l y o b e s e mice which have been shown t o have a reduced t i s s u e complement of Na,K-ATPase u n i t s as w e l l as reduced rates of c e l l u l a r t h e r m o g e n e s i s .

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CURRENT TOPICS IN MEMBRANES AND TRANSPORT. VOLUME 19

Decreased Na,K-ATPase Activity in Erythrocyte Membranes and Intact Erythrocytes from Obese Man DA VIDM.MO7T, W A R W M E S ,' AND W D I L L. CLARK Phoenix Clinical Research Section National Institute of Anhritis, Diabetes, and Kidney Disease National Institutes of Health Phoenix, Arizona

I.

INTRODUCTION

Obesity i n rodents i s associated with increased m e t a b o l i c e f f i c i e n c y b a s e d a t l e a s t i n p a r t on r e d u c e d sodium pump a c t i v i t y ( L i n e t a l . , 1981; York e t a l . , 1 9 7 8 ) . S e v e r a l r e c e n t s t u d i e s i n man a l s o s u g g e s t a l t e r e d m e t a b o l i c r a t e s a s s o c i a t e d w i t h human o b e s i t y (Miller and P a r s o n a g e , 1975; S i m s e t a l . , 1 9 7 3 ) . Our s t u d i e s were u n d e r t a k e n t o e v a l u a t e t h e r e l a t i o n s h i p between Na,K-ATPase and o b e s i t y i n t h e P i m a I n d i a n s , a d e m o g r a p h i c a l l y w e l l - c h a r a c t e r i z e d and r e l a t i v e l y homogeneous p o p u l a t i o n w i t h an e x t r a o r d i n a r i l y h i g h p r e v a l e n c e of o b e s i t y (Knowler e t a l . , 1 9 8 1 ) . The r e s u l t s d e m o n s t r a t e a n i n v e r s e r e l a t i o n s h i p between sodium pump a c t i v i t y and t h e magnitude of o b e s i t y i n i n t a c t r e d c e l l s as w e l l a s i n i s o l a t e d e r y t h r o c y t e membranes. 'On leave of absence from the I n s t i t u t e of Experimental Endocrinology, Slovak Academy of Sciences, Bratislava, Czechoslovakia. 969

Copyright 0 1983 by Academic Press, Inc. All rights of reproduction inany form reserved. ISBN 0-12-1533190

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11.

DAVID M. MOTTet a/.

MATERIALS AND METHODS

Male Pima I n d i a n s u b j e c t s were i n good h e a l t h exc e p t f o r o b e s i t y a s judged by h i s t o r y , p h y s i c a l examinat i o n , and l a b o r a t o r y measurements. A f t e r an o v e r n i g h t f a s t and 30 min a t rest, blood was c o l l e c t e d i n Na2EDTA t u b e s and t h e plasma and b u f f y c o a t were removed. Red c e l l membranes were p r e p a r e d by a m o d i f i c a t i o n ( K l i m e s et ai. , 1982) of t h e osmotic l y s i s procedure rep o r t e d by B l o s t e i n ( 1 9 6 8 ) . Membrane Na,K-ATPase a c t i v i t y was measured a f t e r 3-hr i n c u b a t i o n a t 37OC u s i n g 0 . 4 mg/ml membrane p r o t e i n i n a 30 mM i m i d a z o l , 30 mM h i s t i d i n e b u f f e r (pH 8 . 0 ) , c o n t a i n i n g 88 mM N a C 1 , 0.9 mM MgC12, 0.5 mM Na2EDTA, and 1 . 0 mM T r i s - A T P p l u s o r minus 0 . 7 m~ ouabain. The change i n Na,K-ATPase a c t i v i t y was c a l c u l a t e d from t h e r e d u c t i o n i n r a t e of i n o r g a n i c phosp h a t e r e l e a s e w i t h and w i t h o u t ouabain. The r a t e of 86Rb uptake i n t o r e d blood c e l l s w a s determined a s p r e v i o u s l y d e s c r i b e d ( K l i m e s e t a l . , 1 9 8 2 ) . Na,K-ATPase a c t i v i t y was c a l c u l a t e d from t h e d i f f e r e n c e i n r a t e s of 86Rb uptake by c e l l s w i t h and w i t h o u t ouabain.

111.

RESULTS AND DISCUSSION

A s i g n i f i c a n t n e g a t i v e c o r r e l a t i o n was found between t h e r a t e of o u a b a i n - i n h i b i t e d 86Rb u p t a k e by r e d blood c e l l s and t h e o b e s i t y of a group o f 1 9 male Pima I n d i a n s ( F i g . 1 ) . These r e s u l t s confirm t h e n e g a t i v e c o r r e l a t i o n between 86Rb uptake and o b e s i t y r e p o r t e d by D e L u i s e et a l . ( 1 9 8 0 ) . I n o r d e r t o d e t e r m i n e t h e r e l a t i o n s h i p between o b e s i t y and sodium pump a c t i v i t y i n c e l l membranes, t h e r a t e of o u a b a i n - i n h i b i t e d r e l e a s e o f i n o r g a n i c phosphate was measured u s i n g p u r i f i e d p r e p a r a t i o n s of u n s e a l e d r e d c e l l membranes. Enzyme a c t i v i t y was i n v e r s e l y corr e l a t e d w i t h o b e s i t y ( F i g . 1) and was s i g n i f i c a n t l y c o r r e l a t e d w i t h t h e r a t e of 86Rb uptake by i n t a c t e r y t h r o c y t e s ( r = 0 . 5 0 ; p < 0.05, d a t a n o t shown). T h i s dec r e a s e d Na,K-ATPase a c t i v i t y i n i n t a c t c e l l s , t h e r e f o r e , i s n o t a d i r e c t f u n c t i o n of e l e c t r o c h e m i c a l g r a d i e n t o r substrate concentration differences i n erythrocytes f r o m obese s u b j e c t s . The r o l e of t h i s d e c r e a s e d e r y t h r o c y t e Na,K-ATPase i n t h e p a t h o g e n e s i s of human o b e s i t y i s s t i l l o b s c u r e . A p l a u s i b l e b u t unproved h y p o t h e s i s s u g g e s t s t h a t i n -

DECREASED Na,K-ATPase ACTIVITY IN ERYTHROCYTE

971

a'

r'"4 2 ITOt

*\

1 I

\

c 0

h

f z 4

140-

'1

110-

Body Mass Index ( k g / r n 2 )

F i g . 1 . R e l a t i o n s h i p b e t w e e n o b e s i t y and e r y t h r o c y t e Na,K-ATPase a c t i v i t y a s d e t e r m i n e d b y the r a t e of r u b i d i u m u p t a k e i n t o i n t a c t c e l l s ( A ) and the r e l e a s e of i n o r g a n i c p h o s p h a t e b y i s o l a t e d membranes (B). O b e s i t y i s e x p r e s s e d as b o d y m a s s i n d e x , which i s e q u a l t o the s u b j e c t ' s w e i g h t i n k i l o g r a m s d i v i d e d b y (A) r = -0.71, p < 0.001, n = 19; height in meters squared. ( B ) r = -0.70, p < 0 . 0 0 0 5 , n = 2 0 .

creased m e t a b o l i c e f f i c i e n c y , which r e s u l t s f r o m a reduced e x p e n d i t u r e o f e n e r g y by t h e sodium pump, c o u l d r e s u l t i n a n a c c u m u l a t i o n o f unused c a l o r i e s i n t h e

972

DAVID M. M O T et a/.

form of f a t . I f t h e abnormality i n e r y t h r o c y t e sodium pump r e f l e c t s a s i g n i f i c a n t l o s s of enzyme a c t i v i t y i n o t h e r t i s s u e s , t h e l e v e l of sodium pump a c t i v i t y could have a c a u s a l r e l a t i o n s h i p w i t h o b e s i t y . I n f o r m a t i o n on t h e r e g u l a t i o n of t h i s enzyme i n o t h e r t i s s u e s from obese subjects i s needed t o c l a r i f y t h e p o t e n t i a l cont r i b u t i o n of t h e sodium pump t o human o b e s i t y .

REFERENCES

B l o s t e i n , R. (1968). R e l a t i o n s h i p between e r y t h r o c y t e membrane p h o s p h o r y l a t i o n and adenosine t r i p h o s p h a t e h y d r o l y s i s . J. B i o l . Chem. 243, 1957-1965. DeLuise, M . , Blackburn, G . L . , and F l i e r , J. S . (1980). Reduced a c t i v i t y o f t h e r e d c e l l sodium-potassium pump i n human o b e s i t y . N. Engl. J. Med. 3 0 3 , 1017-1022. K l i m e s , I . , Nagulesparan, M . , Unger, R. H . , Aronoff, S. L., and Mott, D. M. (1982). Reduced Na+,K+-ATPase a c t i v i t y i n i n t a c t r e d c e l l s and i s o l a t e d membranes from obese man. J . C l i n . Endocrinol. Metab. 5 4 , 721-724. Knowler, W. C . , P e t t i t t , D. J., Savage, P. J . , and B e n n e t t , P. H. (1981). Diabetes i n c i d e n c e i n Pima I n d i a n s : C o n t r i b u t i o n o f o b e s i t y and p a r e n t a l d i a b e t e s . Am. J. Epidemiol. 113, 144-156. L i n t M. H . , Romosos, D. R . , Akera, T . , and L e v e i l l e , G. A . (1981). F u n c t i o n a l c o r r e l a t e s of N a + ,K+-ATPase i n l e a n and obese (ob/ob) mice. Metabolism 30, 431-432. Miller, D. S., and Parsonage, S. (1975). R e s i s t a n c e t o slimming: Adaption o r i l l u s i o n . Lancet 1, 713-175. Sims, E. A. H . , Danforth, E . , J r . , Horton, E. S . , Bray, G. A . , Glennon, J. S., and S a l a n s , L. B. (1973). Endocrine and m e t a b o l i c e f f e c t s o f experimental o b e s i t y i n man. Recent Prog. Horm. R e s . 29, 457-496. York, D. A . , Bray, G. A . , and Yuikimura, Y . (1978). An enzymatic d e f e c t i n t h e obese (ob/ob) mouse: Loss of thyroid-induced sodium and potassium-dependent adenosinetriphosphatase. Proc. N a t l . Acad. Sci. USA 75, 477-481.

CURRENT TOPICS IN MEMBRANES AND TRANSPORT. VOLUME 19

Functionally Abnormal Na/K Pump in Erythrocytes from a Morbidly Obese Subject J. FLIER, P. USHER,AND M.DELUISE Beth Israel Hospital Haward Medical School Boston, Massachusetts

I.

INTRODUCTION

A l t e r a t i o n s i n t h e number of N a / K pump u n i t s and a c t i v i t y i n e r y t h r o c y t e s have been d e s c r i b e d i n a v a r i e t y o f abnormal s t a t e s . Commonly, t h e s e changes a p p e a r t o be compensatory f o r an unknown p r i m a r y def e c t causing increased c e l l u l a r permeability t o t h e The b e s t - c h a r a c t e r monovalent c a t i o n s N a + and/or K + . i z e d examples of t h e s e c o n d i t i o n s i n c l u d e v a r i o u s hemol y t i c syndromes such a s h e r e d i t a r y s t o m a t o c y t o s i s and spherocytosis.

11.

DISCUSSION

W e now r e p o r t a p a t i e n t s u f f e r i n g from morbid o b e s i t y ( 2 5 2 % of i d e a l body w e i g h t ) who w a s found t o show a number of abnormal f e a t u r e s o f t h e red c e l l N a / K 973

Copyright 0 1983 by Academic Press. Inc. All rights of reproduction in any form reserved. ISBN 0-12-153319-0

J. FLlERetal.

974

TABLE I.

Erythrocyte Abnormalitiesa Measurement

Patient M?iJ

Controls ~~~

Maximal ouabain binding capacity (pmoles/l09 cells) Na,K-ATPase activity (pmoles Pi/hr/mg membrane protein) Erythrocyte Na+ (moles/liter cells) Erythrocyte K+ (moles/liter cells) Na/K pump-mediated R h uptake (nmoles/109 cells/hr) Ouabain-sensitive fractional K+ efflux Ouabain-sensitive Na+ efflux Ouabain-sensitive K+ influx Net ouabain-sensitive K+ influx Ouabain-sensitive lactate production

0.596 f 0.109 6.7

0.470 f 0.217

7.79 f 0.34 87.9 f 0.4 451 _+ 55

10.83 0.80 77.3 f 3.0 83.5 f 10.2

0.057 hr-l

0

0.76

2.04 f 0.30

0.45

1.07 f 0.37

a Results are expressed as mean f SD of at least f o u r separate determinations.

pump i n t h e absence of any o t h e r d e t e c t a b l e abnormalit i e s of h e r e r y t h r o c y t e s . Some of t h e s e a b n o r m a l i t i e s a r e g i v e n i n Table I. The i n i t i a l a b n o r m a l i t y d e t e c t e d i n t h i s p a t i e n t ' s c e l l s was an 18-fold i n c r e a s e i n t h e number o f pump u n i t s a s measured by [3H]ouabain s a t u r a t i o n b i n d i n g . A s i m i l a r i n c r e a s e was s e e n i n t h e Na,K-ATPase a c t i v i t y of e r y t h r o c y t e membranes. These i n c r e a s e d l e v e l s of Na,K-ATPase enzyme a r e n o t l i k e l y t o be due t o i n c r e a s e d p e r m e a b i l i t y of t h e r e d c e l l t o sodium s i n c e i n t r a c e l l u l a r l e v e l s of t h i s i o n were s i g n i f i c a n t l y lower t h a n norm a l i n t h e p a t i e n t ' s c e l l . Moreover, i n h i b i t i o n of t h e Na/K pump by ouabain r e s u l t e d i n a normal, moderate i n crease i n intracellular Na+. The i n c r e a s e d number of pump u n i t s was r e f l e c t e d i n a 5 - f o l d i n c r e a s e i n t h e o u a b a i n - s e n s i t i v e r a t e of 86F& uptake by t h e c e l l s . The s m a l l e r i n c r e a s e i n pump act i v i t y compared t o pump numbers would be e x p e c t e d from t h e lower t h a n normal l e v e l s of i n t r a c e l l u l a r Na+. The i n c r e a s e d r a t e o f rubidium uptake by t h e c e l l s was balanced by an e q u i v a l e n t i n c r e a s e i n t h e r a t e of e f f l u x of K from c e l l s , a s measured by 86Rb e f f l u x from c e l l s preloaded w i t h t h e i s o t o p e . I n s t r i k i n g c o n t r a s t t o normal c e l l s , however, a major p o r t i o n of t h i s e f f l u x ( 8 0 % ) was i n h i b i t e d by ouabain and t h u s appeared t o be mediated by t h e Na/K pump. Thus, i t was concluded t h a t t h e pump was m e d i a t i n g K+-K+ exchange, a p r o c e s s n o t

FUNCTIONALLY ABNORMAL NdK PUMP IN ERYTHROCYTES

975

s e e n i n c o n t r o l c e l l s under t h e s e e x p e r i m e n t a l c o n d i tions. + Simultaneous measurement of N a / K pump-mediated N a e f f l u x , K+ i n f l u x and o u a b a i n - s e n s i t i v e l a c t a t e product i o n a l l o w e d u s t o d e t e r m i n e t h e s t o i c h i o m e t r y and " e f f i c i e n c y " of t h e pump i n e r y t h r o c y t e s from o u r p a t i e n t . The v a l u e s o b t a i n e d (shown i n T a b l e I ) are o b v i o u s l y q u i t e d i f f e r e n t from c o n t r o l s . A f u r t h e r abnormality detected w a s a decrease i n t h e a f f i n i t y o f t h e pump f o r o u a b a i n . There w a s a 3-fold i n c r e a s e i n t h e c o n c e n t r a t i o n of l i g a n d r e q u i r e d t o h a l f - s a t u r a t e t h e binding c a p a c i t y of t h e cells o r of i s o l a t e d e r y t h r o c y t e membranes. A d d i t i o n a l l y , t h e c o n c e n t r a t i o n of o u a b a i n r e q u i r e d t o i n h i b i t 50% of Rb u p t a k e i n whole c e l l s o r Na,K-AT ase a c t i v i t y i n memb r a n e s w a s found t o be 4- and 3 - f o l d h i g h e r t h a n cont r o l s , respectively. I n c o n t r a s t t o t h e apparent reduction i n a f f i n i t y f o r o u a b a i n , t h e a f f i n i t y of t h e enzyme f o r K+ w a s found t o b e i n c r e a s e d , as measured i n d i r e c t l y by t h e concent r a t i o n of c a t i o n r e q u i r e d t o c a u s e 50% a c t i v a t i o n of Rb u p t a k e i n whole c e l l s o r Na,K-ATPase a c t i v i t y i n e r y t h r o c y t e membranes. I n c o n c l u s i o n , w e have d e s c r i b e d a unique c o n s t e l l a t i o n o f f u n c t i o n a l a b n o r m a l i t i e s of t h e Na/K pump i n o t h e r w i s e normal e r y t h r o c y t e s from an o b e s e p a t i e n t . I t i s p o s s i b l e t h a t t h e s e a b n o r m a l i t i e s may r e f l e c t a s t r u c t u r a l a l t e r a t i o n o f t h e enzyme, a l t h o u g h f u r t h e r s t u d i e s are r e q u i r e d t o f u l l y c h a r a c t e r i z e t h e N a , K ATPase i n t h i s p a t i e n t .

This Page Intentionally Left Blank

CURRENT TOPICS IN MEMBRANES AND TRANSPORT. VOLUME 19

Specific Insulin Binding to Purified Na,K-ATPase Associated with Rapid Activation of the Enzyme JULIE E. M.McGEOCH Harvard Medical School Boston, Massachusetts

I.

INTRODUCTION

W e r e p o r t t h e f i r s t o b s e r v a t i o n s of i n s u l i n i n t e r a c t i o n w i t h highly p u r i f i e d Na,K-ATPase. The enzyme was p r e p a r e d by t h e method o f J d r g e n s e n ( 1 9 7 4 ) from dog r e n a l o u t e r m e d u l l a . F i v e p r e p a r a t i o n s were u s e d , o f s p e c i f i c a c t i v i t y 3 3 umole Pi/mg p r o t e i n / m i n ( p r o t e i n d e t e r m i n e d by t h e method of Lowry). The a c t i v i t y was 9 7 % o u a b a i n i n h i b i t a b l e and t h e p r e p a r a t i o n s were 9 0 % p u r e f o r a- and 8 - s u b u n i t s on SDS g e l e l e c t r o p h o r e s i s . The c o u p l e d a s s a y o f B a r n e t t ( 1 9 7 0 ) w a s used t o measure t h e change i n N a , K - A T P a s e a c t i v i t y w i t h t i m e . The a s s a y w a s s t a r t e d by a d d i n g N a , K - A T P a s e i n 4 p 1 ( G i l s o n 0-20 p 1 p i p e t t e ) t o a c u v e t t e c o n t a i n i n g 1 m l of a mixt u r e of b u f f e r and s u b s t r a t e s , t o g i v e a f i n a l enzyme c o n c e n t r a t i o n o f 2.8 nM. The a s s a y m i x t u r e c o n t a i n e d 25 m~ i m i d a z o l e (pH 7 . 4 ) ; 1 0 pg/ml e a c h of l a c t i c dehydrogenase and p y r u v a t e k i n a s e ; 1 . 4 m~ PEP: 0 . 2 6 m~ NADH: 2 . 5 mM ATP; 1 0 mM N a C 1 ; 4 mM K C 1 ; 5 mM MgC12; 5 nM BSA k p o r c i n e i n s u l i n ( L i l l y ) . 977

Copyright 0 1983 by Academic &ss, Inc. All rights of reproduction in any form reserved. ISBN 0-12-1.533190

978

JULIE E. M. McGEOCH I

-

I

I

*I* Stimulation

40-

30-

P10

-

-

0- ' lOOpM

I

.

. - . I

.

. ..I

1nM lOnM lOOnM Insulin concentrntion

F i g . 1 . Dose r e s p o n s e c u r v e s f o r the i n i t i a l s t i m u l a t i o n of 2.8 nM (NaK)ATPase. A s s a y conditions a s i n t e x t w i t h Na+ ( a ) 37OC, i n c l u d i n g d a t a f r o m a t o t a l o f 6 4 10 mM; Kf 4 mM. t e s t and 100 control a s s a y s , for e a c h d a t a p o i n t n = 9 . (b) 22.SoC, i n c l u d i n g d a t a f r o m 22 t e s t and 11 control a s s a y s , f o r each d a t a p o i n t n = 3 .

11.

RESULTS AND DISCUSSION

F i g u r e 1 shows t h e i n i t i a l s t i m u l a t i o n o f a funct i o n of i n s u l i n c o n c e n t r a t i o n . A n a l y s i s of 66 t e s t exp e r i m e n t s w i t h 1 0 0 c o n t r o l s showed a h i g h l y s i g n i f i c a n t ( 2 0 . 5 % 5 2.5%) s t i m u l a t i o n of t h e s p e c i f i c a c t i v i t y , a l t h o u g h t h e s e n s i t i v i t y t o i n s u l i n was r a t h e r v a r i a b l e ( F i g . 2 b ) . The s t i m u l a t i o n was maximal w i t h i n 30 sec of i n s u l i n a d d i t i o n , and decayed t o h a l f - l e v e l i n 4 min ( F i g . 2 a ) . The s t i m u l a t i o n w a s reduced t o z e r o a t 100 mM N a o r 2 0 m K. A s i m i l a r e l e c t r o l y t e dependence w a s o b s e r v e d i n less p u r e Na,K-ATPase p r e p a r a t i o n s by Gavryck e t a l . ( 1 9 7 5 ) . The s t i m u l a t i o n was s p e c i f i c t o i n s u l i n as b o i l i n g t h e i n s u l i n , t r y p s i n d i g e s t i o n of i t , and p r o i n s u l i n s u b s t i t u t i o n gave no s t i m u l a t i o n . Also m i l d t r y p s i n d i g e s t i o n of t h e enzyme

INSULIN BINDING TO PURIFIED Na,K-ATPase

'I

979

Frequency 7

5

I

-

o n :

-30

0 */a

30

nn:

60

stimulation

90

Fig. 2. ( a ) T i m e d e c a y of i n s u l i n s t i m u l a t i o n of (NaK) ATPase i n c l u d i n g d a t a f r o m 14 t e s t and 1 4 control a s s a y s p e r p o i n t (NaK)a t each temperature. Assay c o n d i t i o n s a s i n text w i t h : ATPase 2.8 nM; Na+ 10 mM; K+ 4 mM; i n s u l i n 500 pM t o 1 5 nM. ( b ) F r e q u e n c y d i s t r i b u t i o n of i n i t i a l s t i m u l a t i o n of (NaK)ATPase b y i n s u l i n , i n c l u d i n g 66 t e s t and 100 control a s s a y s a t 37'C. Conditions: (NaK)ATPase 2 . 8 nM; Na+ 10 mM; K+ 4 mM; i n s u l i n 500 pM t o 20 nM. T h e mean s t i m u l a t i o n i s 20.5 f 2 . 5 % .

t o a d e g r e e t h a t d i d n o t change t h e s p e c i f i c a c t i v i t y removed i t s s e n s i t i v i t y t o i n s u l i n c o m p l e t e l y . S p e c i f i c i n s u l i n b i n d i n g t o t h e Na,K-ATPase p r e p a r a t i o n s w a s measured by t h e t e c h n i q u e s o f f i l t r a t i o n and c e n t r i f u g a t i o n . Both methods r e l i e d upon t h e f a c t t h a t t h e enzyme w a s p r e s e n t i n s h e e t s (Jfdrgensen, 1980) o f a b o u t 3 0 , 0 0 0 u n i t s , which allowed s e p a r a t i o n by 0.45 p m M i l l i p o r e f i l t r a t i o n , o r removal from t h e s u p e r n a t a n t by 2 min of c e n t r i f u g a t i o n a t 15,000 g on a Microfuge c e n t r i f u g e . The d e g r e e of b i n d i n g w a s determined from t h e c o u n t of 1 2 5 I - l a b e l e d i n s u l i n which e i t h e r remained i n t h e f i l t e r o r w a s removed from t h e s u p e r n a t a n t . A l l b i n d i n g e x p e r i m e n t s w e r e performed i n t h e p r e s e n c e of 0.25% BSA, which g r e a t l y r e d u c e d t h e n o n s p e c i f i c b i n d i n g t o l i p i d s h e e t s , tube w a l l s , and

980

JULIE E. M. McGEOCH

-

't

0.1

1 2 4610 Incubation time (min.)

100

F i s . 3 . T i m e c o u r s e of i n s u l i n b i n d i n g t o (NaK)ATPase. 100 pM 5I i n s u l i n was i n c u b a t e d a t 4OC w i t h 2 nM (NaK)ATPase f o r the t i m e s i n d i c a t e d i n a v o l u m e of 1 4 0 p 1 w h i c h c o n t a i n e d : Na+ 10 mM; K+ 4 mM; Mg2+ 5 mM; ATP 4 mV; 25 mM i m i d a z o l e and 0.25% BSA a t pH 7.4. B i n d i n g t o the e n z y m e was a s s a y e d b y the M i c r o f u g e c e n t r i f u g a t i o n method. T i m e d e c a y o f s t i m u l a t i o n shown a s dashed c u r v e . For e a c h p o i n t n = 6 .

f i l t e r . Where a p a r t i c u l a r i n c u b a t i o n t i m e i s i n d i cated i n the data, the e f f e c t i v e incubation t i m e is a b o u t 30 sec g r e a t e r owing t o t h e t i m e d e l a y i n f i l t r a t i o n o r ( t h e i n i t i a l phase o f ) c e n t r i f u g a t i o n . A l l t h e s h o r t i n c u b a t i o n t i m e d a t a were o b t a i n e d a t 4OC. The b i n d i n g was s t r o n g l y time-dependent ( F i g . 3 ) , showing c l e a r e v i d e n c e o f a n i n s u l i n i n t e r a c t i o n a t t i m e s less t h a n 30 sec. The b i n d i n g d a t a for a nominal 10-sec i n c u b a t i o n are shown i n F i g . 4 . By e a c h t e c h n i q u e a s p e c i f i c b i n d i n g l e v e l of 1 0 % w a s observed (130 ng i n s u l i n / m g p r o t e i n ) . On t h e assumption of e q u i l i b r i u m b i n d i n g k i n e t i c s , ( [ I ] - [ c ] ) - ( [ E ] - [ C ] ) = K D [ C ] , where [ I ] i s t h e t o t a l i n s u l i n concentration, [ E l i s t h e concentrat i o n of monovalent b i n d i n g s i t e s , and [ C ] i s t h e conc e n t r a t i o n of bound i n s u l i n , a computer l e a s t s q u a r e s r e g r e s s i o n a n a l y s i s w a s used t o f i n d t h e a v e r a g e K D = 1 . 9 5 f 1 . 2 n~ and t h e a v e r a g e [ E l = 0 . 2 0 2 0 . 1 2 nM. The c l a s s of b i n d i n g s i t e s [El w a s t h e r e f o r e e q u i v a l e n t i n number t o 1 0 % of t h e 2 nM Na,K-ATPase c o n c e n t r a t i o n .

INSULIN BINDING TO PURIFIED Na,K-ATPase

981

F i g . 4 . ( a ) P e r c e n t a g e o f i n s u l i n bound vs. i n s u l i n conc e n t r a t i o n by the f i l t r a t i o n m e t h o d . 100 pM 1 2 5 I - l a b e l e d i n s u l i n + c o l d i n s u l i n was i n c u b a t e d 10 sec a t 22.5OC w i t h 2 nM (NaK)ATPase i n a v o l u m e o f 140 p 1 c o n t a i n i n g : Na+ 10 mM; K+ 4 mM; Mg2+ 5 mM; ATP 4 mM; 25 mM i m i d a z o l e and 0.25% BSA a t pH 7 . 4 . B i n d i n g a s s a y e d b y f i l t r a t i o n m e t h o d . Each p o i n t n = 4 . ( b ) P e r c e n t a g e o f i n s u l i n bound v s . i n s u l i n c o n c e n t r a t i o n b y the M i c r o f u g e c e n t r i f u g a t i o n m e t h o d . R e a c t a n t s a s i n ( a ) , w i t h the 10 sec i n c u b a t i o n a t 4OC. Each p o i n t n = 6 . I n ( a ) and ( b ) the c u r v e s a r e the computed l e a s t s q u a r e s f i t t o the d a t a b a s e d on the e q u i l i b r i u m b i n d i n g a n a l y s i s .

The b i n d i n g o f i n s u l i n t o t h e enzyme w a s u n a f f e c t e d by o u a b a i n ; however, v a n a d a t e r e d u c e d t h e b i n d i n g as shown i n F i g . 5 . The h i g h c o n c e n t r a t i o n of b i n d i n g s i t e s and t h e dependence on v a n a d a t e s u g g e s t t h a t t h e b i n d i n g i s d i r e c t l y t o t h e Na,K-ATPase n e a r t h e phosphorylation s i t e . The o b s e r v e d K D f o r i n s u l i n b i n d i n g i s t h e same w i t h i n e r r o r a s t h a t €or t h e c l a s s i c a l i n s u l i n r e c e p t o r (Czech, 1 9 7 7 ) . The t i m e c o u r s e of b i n d i n g s u g g e s t s t h a t p o s s i b l y i n s u l i n r a p i d l y removes a " t h i r d f a c t o r " which a l l o w s i n s u l i n t o b i n d when t h e f a c t o r i s p r e s e n t on ( a f r a c t i o n o f ) t h e Na,K-ATPase. The s u b s e q u e n t increase i n binding could p o s s i b l y be a n i n v i t r o a r t i f a c t caused

JULIE E. M. McGEOCH

*/a

Insulin

I

V

A

I

n

t

a

I

0 500pM 50nM 5pM Vanadate concentration F i g . 5. E f f e c t o f vanadate (Na3V04). (a) 100 pM i n s u l i n incubated 10 sec a t 4OC with 2 nM (NaK)ATPase. Nai 10 mM; Kf 4 mM; Mg++ 5 mM; ATP 4 mM; imidazole 25 mM pH 7.4, and 0.25% BSA (b) I d e n t i c a l t o (a) except s u b s t r a t e s Na+, I@, Mgii and ATP abs e n t . Binding assayed by t h e c e n t r i f u g e method. Separate cont r o l s f o r each p o i n t , n = 2 .

by t h e l a c k o f a n o t h e r r e c e p t o r f o r t h e t h i r d f a c t o r i n t h e h i g h l y p u r i f i e d system. The s t i m u l a t i o n i s e x p l a i n e d i f t h e t h i r d f a c t o r i s an i n h i b i t o r of N a , K - A T P a s e . I n s u l i n i s known t o release a "secondary messenger" peptide following i t s i n t e r a c t i o n with t h e cell membrane ( J a r e t t and S e a l s , 1979; Larner et a l . , 1 9 7 9 ) . To t h i s e x t e n t , t h e h y p o t h e s i s of a " t h i r d f a c t o r " i s c o n s i s t e n t w i t h t h e known mechanism of i n s u l i n a c t i o n . The p r e s e n t r e s u l t s are c o m p a t i b l e w i t h t h e c o n c l u s i o n t h a t i n s u l i n i n t e r a c t s d i r e c t l y w i t h t h e Na,K-ATPase.

ACKNOWLEDGMENT

This work w a s supported by t h e Department of Medicine, Harvard Medical School.

INSULIN BINDING TO PURIFIED Na,K-ATPase

983

REFERENCES

B a r n e t t , R. E . ( 1 9 7 0 ) . E f f e c t o f monovalent c a t i o n s on t h e o u a b a i n i n h i b i t i o n o f t h e sodium and p o t a s s i u m i o n a c t i v a t e d adenosine t r i p h o s p h a t a s e . B i o c h e m i s t r y 9 , 4644-4648. Czech, M. P. ( 1 9 7 7 ) . Molecular b a s i s o f i n s u l i n a c t i o n . A n n u . Rev. B i o c h e m . 46, 359-384. Gavryck, W. A . , Moore, R. D . , and Thompson, R. C. ( 1 9 7 5 ) . E f f e c t of i n s u l i n upon membrane-bound ( N a K ) ATPase e x t r a c t e d from J. P h y s i o l . 252, 43-48. f r o g s k e l e t a l muscle. J a r e t t , L . , and S e a l s , J . R. ( 1 9 7 9 ) . P y r u v a t e dehydrogenase act i v a t i o n i n a d i p o c y t e m i t o c h o n d r i a by a n i n s u l i n - g e n e r a t e d S c i e n c e 206, 1407-1408. m e d i a t o r from muscle. Jgkgensen, P. L. ( 1 9 7 4 ) . P u r i f i c a t i o n and c h a r a c t e r i z a t i o n o f (NaK)ATPase 111. B i o c h i m . B i o p h y s . A c t a 356, 36-52. J#rgensen, P. L. (1980). Sodium and potassium i o n pump i n k i d n e y t u b u l e s . P h y s i o l . Rev. 60, 868-869. L a r n e r , J . , Galasko, G., Cheng, K . , De Paoli-Roach, A . A . , Huang, L. , Daddy, P., and K e l l o g , J . ( 1 9 7 9 ) . G e n e r a t i o n by i n s u l i n o f a chemical m e d i a t o r t h a t c o n t r o l s p r o t e i n p h o s p h o r y l a t i o n and d e p h o s p h o r y l a t i o n . Science 206, 14081410.

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CURRENT TOPICS IN MEMBRANES AND TRANSPORT, VOLUME 19

A Mechanism for Cholinergic Stimulationof Sodium Pump in Rat Submandibular Gland DA VIDJ . STEWART AND A WAR K. SEN Department of Pharmacology Faculty of Medicine University of Toronto Toronto. Ontario. Canada

W e r e c e n t l y d e m o n s t r a t e d t h a t N a / K pump a c t i v a t i o n by c a r b a c h o l i n r a t s u b m a n d i b u l a r g l a n d s l i c e s d o e s n o t depend on an i n f l u x o f sodium i n t o t h e c e l l s ( S h i e t a l . , 1980). I n t h a t study, evidence w a s a l s o presented t h a t pump a c t i v a t i o n may depend on a c y c l i c GMP-mediated mechanism. To f u r t h e r i n v e s t i g a t e t h e mechanism, N a / K pump a c t i v i t y w a s measured as o u a b a i n - s e n s i t i v e Rb uptake i n t i s s u e slices.

I.

METHODS

T i s s u e s l i c e s w e r e i n c u b a t e d a t 37' i n a Krebs b i c a r b o n a t e b u f f e r e d R i n g e r ' s medium e q u i l i b r a t e d w i t h 95% 02/5% C 0 2 . The medium w a s s l i g h t l y m o d i f i e d by rep l a c i n g a l l K i o n s w i t h an e q u i v a l e n t amount of R b i o n s . The s l i c e s were i n c u b a t e d w i t h t h e i n d i c a t e d a d d i t i o n s f o r 2 0 min and t h e n 86Rb (10 v C i ) w a s added and t h e i n c u b a t i o n c o n t i n u e d f o r a n a d d i t i o n a l 1 0 min. Slices 985

Copyright 0 1983 by Academic Ress, Inc. All rights of reproduction in any form reserved ISBN 0-12-1533194

DAVID J. STEWART AND AMAR K. SEN

986

TABLE I .

E f f e c t o f Carbachol on Ouabain-Sensitive Submandibular Gland S l i c e s

Control Carbachol

Rb Uptake i n

Calcium p r e s e n t

Calcium a b s e n t

1 . 2 8 k 0.22 2.27 k 0.31a

1.42 k 0.14 1.06 k 0.10

a

p > 0.05 with respect t o corresponding control; paired t-test w i t h n = 6 rats.

w e r e removed, weighed, and t h e r a d i o a c t i v i t y measured by Chernov r a d i a t i o n i n a l i q u i d s c i n t i l l a t i o n s p e c t r o meter. Uptake v a l u e s are e x p r e s s e d as Rb s p a c e s , i . e . , counts/gram t i s s u e d i v i d e d by c o u n t s / m i l l i l i t e r medium.

11.

RESULTS AND DISCUSSION

Sodium pump a c t i v a t i o n by c a r b a c h o l i s dependent on e x t r a c e l l u l a r calcium as shown i n T a b l e I. I n s l i c e s of p a r o t i d g l a n d , an i n c r e a s e d calcium i n f l u x o c c u r s d u r i n g c h o l i n e r g i c s t i m u l a t i o n (Putney, 1 9 7 6 ) . T o determine whether calcium e n t r y i n t o t h e c e l l i s , by its e l f , s u f f i c i e n t t o a c t i v a t e t h e sodium pump, t h e c a l c i u m ionophore A23187 was t e s t e d . A s shown i n Table 11, t h e ionophore s t i m u l a t e s Rb uptake only i n t h e p r e s e n c e of calcium. I t a p p e a r s u n l i k e l y t h a t calcium i t s e l f a c t i v a t e s t h e pump mechanism s i n c e c a l c i u m i s an i n h i b i t o r of Na,K-ATPase (Schwartz e t a l . , 1 9 6 3 ) . The e f f e c t s o f calcium on t h e pump a r e probably mediated by c y c l i c GMP. Both c a r b a c h o l and A23187 s t i m u l a t e a calcium-dependent r i s e i n c y c l i c GMP c o n c e n t r a t i o n i n submandibular g l a n d t i s s u e (Spearman and P r i t c h a r d , 1 9 7 9 ) . A s shown i n Table 111, d b - c y c l i c GMP a c t i v a t e s o u a b a i n - s e n s i t i v e Rb uptake. Note t h a t t h e u s e of c y c l i c GMP bypasses t h e requirement f o r calcium. The e f f e c t i s s p e c i f i c f o r c y c l i c GMP s i n c e d b - c y c l i c AMP has no e f f e c t on ouabains e n s i t i v e Rb uptake ( d a t a n o t shown). A scheme of how Na f l u x e s might be c o n t r o l l e d i n submandibular g l a n d c e l l s i s shown i n F i g . 1. Occupat i o n of t h e m u s c a r i n i c r e c e p t o r by a c e t y l c h o l i n e a c t i v a t e s calcium and sodium i n f l u x i n t o t h e c e l l . The c a l cium by some mechanism a c t i v a t e s g u a n y l a t e c y c l a s e and e l e v a t e s c e l l c y c l i c GMP l e v e l s . The c y c l i c GMP i n t u r n c o n v e r t s t h e Na,K-ATPase from a l a t e n t form t o an a c t i v e

MECHANISM FOR CHOLINERGIC STIMULATION OF SODIUM PUMP

TABLE 11.

E f f e c t of A23187 o n O u a b a i n - S e n s i t i v e Rb Uptake i n Submandibular Gland S l i c e s

Control A23187

a

987

Calcium present

Calcium absent

1 . 3 1 ? 0.08 2.49 ? 0.22a

1.36 ?r 0.09 1.37 f 0 . 2 4

p > 0.02 w i t h r e s p e c t t o c o r r e s p o n d i n g control; p a i r e d = 5.

t-test w i t h n

TABLE 111.

E f f e c t of & - C y c l i c GMP o n O u a b a i n - S e n s i t i v e Rb Uptake i n S u b m a n d i b u l a r G l a n d S l i c e s Calcium p r e s e n t

Control db-CGMP

1 . 1 7 f 0.10, 2.04 f 0.20

Calcium a b s e n t 1 . 1 2 f 0.22 2.42 ? 0.18,

' p > 0.05 w i t h r e s p e c t t o c o r r e s p o n d i n g c o n t r o l ; p a i r e d

t-test w i t h n = 7 .

form. The calcium a l s o activates a potassium efflux from the cell (Putney, 19791, to provide potassium for exchange with the Na/K pump mechanism. We have recently described a similar mechanism in the avian salt gland (Stewart e t ai., 1979; Stewart and Sen, 1981).

ACKNOWLEDGMENT

Supported i n p a r t by g r a n t s f r o m N a t i o n a l I n s t i t u t e o f H e a l t h (RO1-AM 2 0 4 2 5 ) , M e d i c a l R e s e a r c h C o u n c i l o f Canada (MT-2485) , a n d t h e C a n a d i a n C y s t i c F i b r o s i s F o u n d a t i o n .

DAVID J. STEWART AND AMAR K. SEN

988

Na'

STIMULATIONFig. 1. stimulation.

--+

M o d e l of a c t i v a t i o n of N a / K pump d u r i n g c h o l i n e r g i c

REFERENCES P u t n e y , J. W. (1976). J . P h a r m a c o l . E x p . T h e r . 1 9 9 , 526-537. P u t n e y , J. W. (1979). P h a r m a c o l . Rev. 30, 209-245. S c h w a r t z , A., L a s e t e r , H . , a n d K r a i n t z , L. (1963). J. C e l l . P h y s i o l . 6 2 , 193-205. Shi, M. , S t e w a r t , D. J. , and S e n , A. K. (1980). C a n . J. Biochem. 58, 1223-1229. Spearman, T. N . , and P r i t c h a r d , E . T. (1979). B i o c h i m . B i o p h y s . A c t a 588 , 55-62. S t e w a r t , D. J . , a n d Sen, A. K. (1981). Am. J . P h y s i o l . 2 4 0 ,

C207-C214.

S t e w a r t , D. J . , Sax, J . , Funk, R . , J . p h y s i o l . 237 , C200-C204.

a n d S e n , A. K.

(1979). Am.

CURRENT TOPICS IN MEMBRANES AND TRANSPORT, VOLUME 19

Evidence for an Aldosterone-Mediated, Na-Dependent Activation of Na,K-ATPase in the Cortical Collecting Tubule KEVIN J. PEZ", JUHA P. KOKKO, AND DIANA MARVER Departments of Internal Medicine and Biochemistry University of Texas Health Services Center Dallas, Texas

I.

INTRODUCTION

Numerous e f f o r t s have been made t o d e l i n e a t e t h e mechanisms by which a l d o s t e r o n e s t i m u l a t e s N a t r a n s p o r t i n v a r i o u s t a r g e t e p i t h e l i a . I n c l u d e d among t h e s e mechanisms i s t h e h y p o t h e s i s t h a t a l t e r a t i o n s i n N a , K A T P a s e a c t i v i t y have a r o l e i n t h e m i n e r a l o c o r t i c o i d s t i m u l a t i o n of N a t r a n s p o r t . Certain investigators have proposed t h a t Na,K-ATPase i n t h e k i d n e y is i n d u c e d d i r e c t l y by a l d o s t e r o n e , whereas o t h e r s m a i n t a i n t h a t a l d o s t e r o n e - i n d u c e d changes i n r e n a l N a , K - A T P a s e a r e s e c o n d a r y t o an i n c r e a s e d e n t r y o f N a a c r o s s t h e l u m i n a l membrane. R e s o l u t i o n of t h i s i s s u e h a s been d i f f i c u l t s i n c e t h e s e h y p o t h e s e s a r e b a s e d f o r t h e most p a r t on s t u d i e s u s i n g r e n a l whole homogenates o r membrane p r e p a r a t i o n s d e r i v e d from t h e h e t e r o g e n e o u s c e l l t y p e s p r e s e n t i n r e n a l t i s s u e ( f o r r e v i e w , see Marver, 1 9 8 0 ) . I n o r d e r t o examine t h i s i s s u e i n a more d e f i n i t i v e manner, w e have u t i l i z e d an u l t r a m i c r o enzymatic a s s a y f o r 989

Copynght 0 1983 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0- 12-1533 19-0

990

KEVIN J. PETTY et el.

I

0

I0

I

100

mol Pi at StepA ( x l O - l e ) F i g . 1 . S t a n d a r d c u r v e f o r i n o r g a n i c p h o s p h a t e u s i n g the u l t r a m i c r o a s s a y . A t o t a l of (6.75-135) X m o l e s of KHzPOq i n a t o t a l volume of 540 n l were e n t e r e d i n t o the r e a c t i o n scheme i n d u p l i c a t e a l o n g w i t h a p h o s p h a t e - f r e e b l a n k ( P e t t y e t a i . , 1981).

Na,K-ATPase i n i n d i v i d u a l , l y o p h i l i z e d nephron segments ( P e t t y e t a l . , 1 9 8 1 ) . This assay provides t h e a b i l i t y t o d e t e c t picomole q u a n t i t i e s of i n o r g a n i c phosphate i n n a n o l i t e r i n c u b a t i o n volumes ( F i g . 1 ) . S i n c e t h e cort i c a l c o l l e c t i n g t u b u l e (CCT) i s a p u t a t i v e nephron t a r g e t segment f o r a l d o s t e r o n e , o u r p r e s e n t s t u d y f o c u s e s on t h i s p o r t i o n of t h e nephron.

11.

RESULTS AND DISCUSSION

ATPase a c t i v i t i e s were measured i n i n d i v i d u a l CCTs from normal and adrenalectomized r a b b i t s a s w e l l a s adx r a b b i t s s u b j e c t e d t o one of s e v e r a l a c u t e s t e r o i d t r e a t ment regimes. I n a d d i t i o n , t h e r o l e of l u m i n a l Na ent r y i n t h e s t e r o i d a c t i v a t i o n of CCT Na,K-ATPase w a s examined by p r e t r e a t i n g adx r a b b i t s w i t h a m i l o r i d e i n o r d e r t o b l o c k l u m i n a l Na e n t r y i n t o c e l l s of t h e CCT. The results d i s p l a y e d i n T a b l e I show t h a t CCT N a , K ATPase d e c r e a s e d by 86% i n adrenalectomized animals comp a r e d t o normal r a b b i t s . I n j e c t i o n of a p h y s i o l o g i c dose of a l d o s t e r o n e ( 1 0 pg/kg) produced a s l i g h t b u t i n s i g n i f i c a n t i n c r e a s e i n CCT Na,K-ATPase a t 1 . 5 h r

ALDOSTERONE-MEDIATED,Na-DEPENDENTACTIVATION OF Na,K-ATPase

TABLE I.

A T P a s e A c t i v i t i e s i n the R a b b i t CCTa

Group

1. 2. 3. 4. 5.

991

Normal Adx Adx Adx

+ +

aldo aldo spiro dex

Adx + + aldo 6. A d x + 7 . Adx + m i l + aldo 8 . Normal + m i l

Mg-ATPase

10.3 k 1.2 8.8 f 0.6 8.3 f 0.8 8.3

8.2 10.7

10.1

_+

* * +_

0.6 0.8

0.7 1.1

10.7 k 0.4

Na ,K-ATPase 5.6 0.8 1.6 4.9

k

k k k 0.4 k

0.7 0. 3**

0.3** 0.7*

0.2** 0.7 +_ 0.3** 1.2 f 0.4** 5.5 f 0.6*

aGiven i n u n i t s of moles P i / k g d r y w t . / h r , 37OC k SEM. Group 3 was s a c r i f i c e d 1 . 5 hr a f t e r s t e r o i d . Groups 4-7 w e r e s a c r i f i c e d 3 hr a f t e r s t e r o i d . A l d o , a l d o s t e r o n e , 10 pg/kg, i . v . ; s p i r o , s p i r o l a c t o n e SC 2 6 3 0 4 , 1 . 5 m g / k g , i . p . , 30 m i n bef o r e a l d o ; d e x , d e x a m e t h a s o n e , 100 p g / k g , i . v . ; m i l , a m i l o r i d e , 2 mg/kg, i . v . l o a d i n g dose 30 m i n before a l d o , 1 m g / k g / h r maintenance dose. * p = Not s i g n i f i c a n t v e r s u s Group 1 , n = 8-18 t u b u l e s / group. * * p < 0.001 v e r s u s G r o u p s 1 and 4 . (Reproduced w i t h p e r m i s s i o n of the J o u r n a l o f C l i n i c a l I n v e s t i g a t i o n . )

a f t e r t r e a t m e n t . However, 3 h r a f t e r i n j e c t i o n t h e same dose of a l d o s t e r o n e i n c r e a s e d t h e CCT Na,K-ATPase a l m o s t t o t h e l e v e l s e e n i n normal r a b b i t s . The a c t i v a t i o n of CCT Na,K-ATPase a t 3 h r w a s a s p e c i f i c m i n e r a l o c o r t i c o i d phenomenon s i n c e t h e e f f e c t of a l d o s t e r o n e was blocked by s p i r o l a c t o n e SC 26304 and a s i m i l a r a c t i v a t i o n was n o t s e e n i n a d r e n a l e c t o m i z e d r a b b i t s t r e a t e d w i t h dexamethasone ( 1 0 0 pg/kg) o v e r a 3-hr i n t e r v a l . P r e t r e a t m e n t of a d r e n a l e c t o m i z e d a n i mals w i t h a m i l o r i d e b e f o r e a l d o s t e r o n e i n j e c t i o n res u l t e d i n a s i g n i f i c a n t blockade of t h e m i n e r a l o c o r t i The e f f e c t of c o i d a c t i v a t i o n of CCT Na,K-ATPase. a m i l o r i d e was n o t due t o a d i r e c t i n h i b i t o r y i n f l u e n c e of t h e d i u r e t i c on t h e enzyme s i n c e normal r a b b i t s t r e a t e d w i t h a m i l o r i d e showed no decrement i n CCT Na,K-ATPase a c t i v i t y a f t e r a 3-hr t r e a t m e n t p e r i o d . These s t u d i e s i n d i c a t e t h e f o l l o w i n g : (1) Adrenalectomy produces d e c r e a s e s i n CCT Na,KATPase a c t i v i t y t h a t c a n be r e s t o r e d t o normal l e v e l s w i t h i n 3 h r a f t e r i n j e c t i o n of a s i n g l e p h y s i o l o g i c dose of a l d o s t e r o n e i n t o a d r e n a l e c t o m i z e d r a b b i t s . Presumably t h i s i n c r e a s e d enzyme a c t i v i t y enhances t h e c a p a c i t y of t h e CCT t o r e a b s o r b Na.

992

KEVIN J. PETTYetal.

(2) The mineralocorticoid dependence of the aldosterone-induced increase in CCT Na,K-ATPase is demonstrated by the ability of spirolactone SC 26304 to block specifically the response at the level of the mineralocorticoid receptor. Furthermore, an acute physiologic dose of dexamethasone is ineffective in activating CCT Na,K-ATPase in adrenalectomized rabbits, indicating that the acute enzyme activation with aldosterone is not a glucocorticoid-mediated phenomenon. ( 3 ) The ability of amiloride to block the aldosterone-stimulated increase in CCT Na,K-ATPase suggests that this activation is secondary to mineralocorticoidenhanced entry of Na across the luminal membrane.

ACKNOWLEDGMENTS

This work was supported by N I H g r a n t s RO1-AM 21576 and 14677. K . J . P . was supported i n p a r t by funds from t h e Morgan-Good Foundation, D a l l a s , Texas. RO1-AM

REFERENCES

Marver, D. (1980). Aldosterone a c t i o n i n t a r g e t e p i t h e l i a . V i t a m . Horm. (N.Y.) 38, 55-117. P e t t y , K. J., Kokko, J. P . , and Marver, D. (1981). Secondary e f f e c t of aldosterone on Na-K ATPase a c t i v i t y i n t h e r a b b i t c o r t i c a l c o l l e c t i n g tubule. J. C l i n . Invest. 68, 1514-1521.

CURRENT TOPICS IN MEMBRANES AND TRANSWRT, VOLUME

19

Vanadate and Somatostatin Having Divergent Effects on Pancreatic Islet Na,K-ATPase KENJI IKEJIRI' AND SEYMOUR R. LEVIN Wadsworth Veteran 's Administration Hospital and School of Medicine University of California at Los Angeles Los Angeles, California

I.

INTRODUCTION

S t i m u l i of i n s u l i n s e c r e t i o n , such a s g l u c o s e , a r g i n i n e , and o u a b a i n , s u p p r e s s r a t p a n c r e a t i c i s l e t Na,K-ATPase a c t i v i t y (Levin e t a l . , 1 9 7 8 ) . I n c o n t r a s t , c e r t a i n i n s u l i n s e c r e t o r y i n h i b i t o r s s u c h as p h e n y t o i n (diphenylhydantoin), NH4C1, o r diazoxide ( a n o n d i u r e t i c t h i a z i d e ) enhance t h e a c t i v i t y o f t h i s i s l e t enzyme s y s t e m (Levin e t al., 1 9 7 8 ) . P r i o r t o these present experiments, we theorized t h a t o t h e r i n h i b i t o r s of N a , K - A T P a s e might a c t i v a t e s e c r e t i o n by i s l e t s . Vanadate, which had been shown t o i n h i b i t Na,K-ATPase a c t i v i t y ( C a n t l e y e t a l . , 1 9 7 7 1 , was t h e f i r s t s u c h s u b s t a n c e w e t e s t e d . On t h e o t h e r hand, a l t h o u g h a number of mechanisms of a c t i o n s f o r t h e p e p t i d e i n h i b i t o r of i s l e t s e c r e t i o n , 1

Present a d d r e s s : Matsumoto, Japan.

S h i n s h u U n i v e r s i t y , 8-1-1 A s a h i , 993

Copyright 0 1983 by Academic Press, Inc. All rights of reproductioninany form reserved. ISBN 0-12-153319-0

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KENJI IKEJlRl AND SEYMOUR R. LEVIN

s o m a t o s t a t i n ( C l a r o et al., 1 9 7 7 ; Hahn et al., 1 9 7 6 ) , have been r e p o r t e d , none i s uniformly a c c e p t e d . W e post u l a t e d t h a t one b a s i s f o r i t s i n h i b i t o r y e f f e c t on sec r e t i o n might be v i a enhancement of Na,K-ATPase a c t i v i t y , a s w e s a w f o r t h e t h r e e s e c r e t o r y i n h i b i t o r s ment i o n e d above.

11.

MATERIALS AND METHODS

W e a p p l i e d two main methodologies t o s t u d y i n g t h e e f f e c t s of vanadate and s o m a t o s t a t i n upon t h e i s l e t s . F i r s t , w e p e r f u s e d i s l e t s u s i n g t h e method of Lacy and co-workers (1976) a f t e r i s o l a t i n g them w i t h c o l l a g e n a s e a c c o r d i n g t o t h e method of Lacy and Kostianovsky ( 1 9 6 7 ) . W e used 80-100 i s l e t s p e r m i l l i p o r e chamber. The second t e c h n i q u e used was t h e measurement of Na,K-ATPase a c t i v i t y i n a 35,000 g p e l l e t from i s l e t homogenates a s d e s c r i b e d p r e v i o u s l y (Levin et a l . , 1 9 7 8 ) . W e worked with a Na:K r a t i o a t 100:20 i n these present s t u d i e s , whereas most of o u r p r e v i o u s l y r e p o r t e d i s l e t e x p e r i ments used a N a : K r a t i o of 175:0.7 (Levin e t a l . , 1 9 7 8 ) . I n t h e s e c r e t o r y experiments u s i n g v a n a d a t e , w e measured s o m a t o s t a t i n - l i k e immunoreactivity ( S L I ) as w e l l as immunoreactive i n s u l i n ( I R I ) s e c r e t i o n from t h e i s l e t s . Vanadate (10-3 M ) induced I R I and S L I s e c r e t i o n from i s l e t s , when superimposed upon an ongoing p e r i f u Simultaneously p e r i f u s e d s i o n of g l u c o s e ( 1 0 0 mg/dl). c o n t r o l chambers, which d i d n o t r e c e i v e v a n a d a t e , b u t were s i m i l a r l y exposed t o g l u c o s e ( 1 0 0 m g / d l ) , s e c r e t e d no I R I and S L I . I n o t h e r experiments ( I k e j i r i and Levin 1 9 8 1 ) , w e were a b l e t o evoke s e c r e t o r y a c t i v i t y i n t h e absence of background g l u c o s e , o r even i n t h e absence of calcium i n t h e p e r f u s i o n f l u i d , w i t h vanadate (10-3 M I . T h i s vanadate c o n c e n t r a t i o n d i d n o t i n f l u e n c e t h e s t a n d a r d c u r v e f o r immunoassay of I R I o r S L I . When w e measured i s l e t Na,K-ATPase a c t i v i t y w i t h and w i t h o u t vanadate i n t h r e e s e p a r a t e e x p e r i m e n t s , w e found t h a t t h e c o n t r o l a c t i v i t y was 4 . 7 0 . 6 7 SE pmoles Pi/mg p r o t e i n . A c t i v i t y was i n h i b i t e d 28.3, 32.6, 54.3, and 83.6% by 1 0 - 7 , 1 0 - 6 , 10-5, and 10-4 M v a n a d a t e , r e s p e c t i v e l y . A c o n c e n t r a t i o n of 1 0 - 4 M van a d a t e i n t e r f e r e d about 2 0 % w i t h c o l o r development i n t h e F i s k and Subbarow method f o r phosphate a n a l y s i s ( F i s k e and Subbarow, 1 9 2 5 ) . The o t h e r vanadate concent r a t i o n s d i d n o t i n f l u e n c e c o l o r development. Islet MgZ+-ATPase a c t i v i t y was n o t i n f l u e n c e d by vanadate. +_

VANADATE AND SOMATOSTATIN DIVERGENT EFFECTS ON PANCREATIC ISLET

995

W e found t h a t s o m a t o s t a t i n (1 pg/ml) i n h i b i t e d t h e release o f I R I , 4 6 % , i n r e s p o n s e t o g l u c o s e (150 mg/dl)

.

Ouabain (10-3 M c o m p l e t e l y r e v e r s e d t h i s i n h i b i t i o n . Neither somatostatin nor ouabain influenced t h e s t a n d a r d immunoassay c u r v e f o r i n s u l i n . I n 1 2 e x p e r i m e n t s , c o n t r o l i s l e t Na,K-ATPase a c t i v i t y w a s 4 . 0 1 0 . 7 1 SE m o l e s Pi/mg p r o t e i n / h r . In p a i r e d i n c u b a t i o n s , s o m a t o s t a t i n enhanced s p e c i f i c a c t i v i t y 42% ( p < 0.05). Somatostatin d i d not influence Mg2+-ATPase, s p o n t a n e o u s h y d r o l y s i s o f ATP, o r t h e c o l o r i m e t r i c a n a l y s i s of phosphate.

111.

DISCUSSION

I n h i b i t i o n o f i s l e t Na,K-ATPase by v a n a d a t e m i g h t be a s s o c i a t e d w i t h d e p o l a r i z a t i o n of t h e i s l e t s as i n duced by a number o f s e c r e t a g o g u e s ( M e i s s n e r e t a l . , 1 9 8 0 ) . T h i s r e m a i n s t o be examined. The a c t u a l secret o g e n i c a c t i v i t y might b e i n d u c e d by a n o x i d a t i o n s t a t e o t h e r t h a n t h a t i n Na3-VO4. Vanadate c o u l d a l s o a c t by enhancing a d e n y l a t e c y c l a s e a c t i v i t y (Krawietz et a l . , 1979; Schwabe e t al., 1979) o r by p r o m o t i n g g l u c o s e o x i d a t i o n and t r a n s p o r t ( S c h e c h t e r and K a r l i s h , 1 9 8 0 ) . The l a t t e r i s less l i k e l y s i n c e w e i n d u c e d s e c r e t i o n i n t h e a b s e n c e o f e x t r a c e l l u l a r g l u c o s e ( I k e j i r i and L e v i n , 1 9 8 1 ) . W e h a v e shown t h a t g l u c o s e can i n h i b i t C a 2 + ATPase ( L e v i n e t a l . , 1978) and t h i s s y s t e m may b e i n v o l v e d i n i s l e t s e c r e t o r y a c t i v i t y (Kasson and L e v i n , 1 9 8 1 ) . S i n c e v a n a d a t e c a n i n h i b i t Ca2+-ATPase i n some t i s s u e s ( O ' N e a l e t a l . , 1979) , t h i s s u b s t a n c e c o u l d a c t on i s l e t s t h r o u g h i n h i b i t i o n o f CaZ+-ATPase. S o m a t o s t a t i n w i l l i n h i b i t i n s u l i n and g l u c a g o n sec r e t i o n from p a n c r e a t i c i s l e t s . T h i s p e p t i d e h a s been shown t o h y p e r p o l a r i z e monolayer c u l t u r e i s l e t s (Pace et a l . , 1977. This i s c o n s i s t e n t with a c t i v a t i o n of an e l e c t r o g e n i c pump by s o m a t o s t a t i n . Though o u a b a i n can enhance g l u c o s e - i n d u c e d i n s u l i n s e c r e t i o n (Hales and M i l n e r , 1 9 6 8 ) , i t s a c t i o n may b e , i n p a r t , v i a pathways o t h e r t h a n i n h i b i t i o n of Na+K+ATPase. Thus, Gagerman e t a l . (1979) found t h a t o u a b a i n had s t i m u l a t o r y e f f e c t s upon i s l e t CAMP f o r m a t i o n u n d e r c e r t a i n conditions. I n v e s t i g a t o r s h a v e found t h a t e l e c t r o l y t e f l u x e s d u r i n g n u t r i e n t - i n d u c e d s e c r e t i o n do n o t n e c e s s a r i l y p a r a l l e l t h o s e which might b e e x p e c t e d by s i m p l y i n h i b i t i n g Na,K-ATPase. Thus, f o r example, K a l k h o f f and Siegesmund (1981) h a v e found r e d u c e d N a + c o n t e n t ( w i t h

996

KENJI IKEJlRl AND SEYMOUR R. LEVIN

reduced K+ c o n t e n t ) i n s i n g l e i s l e t 5 c e l l s d u r i n g glucose-induced i n s u l i n s e c r e t i o n . A c a s c a d e of e v e n t s , i n v o l v i n g i n h i b i t i o n o f Na,K-ATPase (Levin e t a l . , 1978) , a l t e r e d N a / C a exchange ( S i e g e 1 e t a l . , 1980) , and changes i n N a c h a n n e l s (Pace, 1979) o c c u r i n t h e I t i s t h u s n o t unexpected t h a t n e t secretory process. i o n flux may n o t e n t i r e l y r e f l e c t t h e e x p e c t e d e f f e c t s upon any i n d i v i d u a l system. Understanding which system o r s y s t e m s predominate i n normal i n s u l i n s e c r e t i o n may g i v e v a l u a b l e c l u e s a b o u t c r i t i c a l d e f e c t s i n d i s e a s e s t a t e s i n which i n s u l i n release i s i m p a i r e d , as i n some forms o f d i a b e t e s mellitus.

ACKNOWLEDGMENTS

T h i s work was supported by g r a n t s from t h e Veterans Administ r a t i o n (5218-Ol), N a t i o n a l I n s t i t u t e s of H e a l t h (AM 21031-031, American Diabetes A s s o c i a t i o n , K r o c Foundation, and Upjohn Laboratories.

REFERENCES

C a n t l e y , L. C . , Josephson, L . , Warner, R . , Yanagisawa, M . , Lechene, C . , and G u i d o t t i , G. (1977). Vanadate i s a p o t e n t Na+K+ATPase i n h i b i t o r found i n ATP d e r i v e d from muscle. J. B i o l . Chem. 252, 7241-7423. C l a r o , A . , G r i l l , V., E f e n d i c , S . , and L u f t , R. (1977). S t u d i e s on t h e mechanisms of s o m a t o s t a t i n a c t i o n on i n s u l i n r e l e a s e I V . E f f e c t of s o m a t o s t a t i n on c y c l i c AMP l e v e l s and phosphodiesterase a c t i v i t y i n i s o l a t e d rat pancreatic islets. Acta Endocrinol. (Copenhagen) 85, 379-388. F i s k e , C . H . , and Subbarow, Y . (1925). The c o l o r i m e t r i c determin a t i o n of phosphorus. J. B 01. Chem. 66, 375-400. Gagerman, E . , Hellman, B . , and T a l j e d a l , I.-B. (1979). E f f e c t s of ouabain on i n s u l i n r e l e a s e , adenosine 3',5'-monophosphate i n p a n c r a t i c i s l e t s . Endocrinology 104, 1000-1002. Hahn, H. J . , G o t t s c h l i n g , H. D . , and Woltanski, P. (1976). E f f e c t of s o m a t o s t a t i n on i n s u l i n s e c r e t i o n and CAMP c o n t e n t of i s o l a t e d p a n c r e a t i c r a t i s l e t s . Metab. C l i n . Exp. 27, Suppl. 1, 1291-1294. Hales, C. N . , and M i l n e r , R. D (1968). The r o l e of sodium and potassium i n i n s u l i n s e c r e t i o n from rabbit p a n c r e a s e . J. P h y s i o l . (London) 194, 725-743.

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I k e j i r i , K . , and Levin, S . R. ( 1 9 8 1 ) . Vanadate i n d u c e s i n s u l i n and s o m a t o s t a t i n s e c r e t i o n from r a t p a n c r e a t i c i s l e t s . Clin. R e s . 2 9 , 409A. Kalkhoff, R. K . , and Siegesmund, K . A. ( 1 9 8 1 ) . F l u c t u a t i o n s o f calcium, phosphorus, sodium, p o t a s s i u m , and c h l o r i n e i n s i n g l e a l p h a and b e t a c e l l s d u r i n g g l u c o s e p e r i f u s i o n o f r a t i s l e t s . J. C l i n . Invest. 6 8 , 517-524. Kasson, B . , and Levin, S . ( 1 9 8 1 ) . C h a r a c t e r i z a t i o n of p a n c r e a t i c i s l e t Ca2+ ATPase. B i o c h i m . B i o p h y s . A c t a ( i n p r e s s ) . Krawietz, W . , Werdan, K . , and Erdmann, E. (1979). S t i m u l a t o r y e f f e c t o f vanadate on t h e a d e n y l a t e c y c l a s e o f c a r d i a c t i s s u e . B i o c h e m . P h a r m a c o l . 2 8 , 2517-2520. Lacy, P. E . , and Kostianovsky, M. ( 1 9 6 7 ) . Method f o r t h e i s o l a t i o n o f i s l e t s of Langerhans from t h e r a t p a n c r e a s . D i a b e t e s 11, 35-39. Lacy, P. E . , F i n k e , E. H . , Conant, S . , and N a b e r , S. ( 1 9 7 6 ) . Long term p e r i f u s i o n of i s o l a t e d r a t i s l e t s i n v i t r o . D i a b e t e s 2 5 , 484-493. Levin, S. R . , Kasson, B. G . , and D r i e s s e n , J. F. (1978). Adenos i n e t r i p h o s p h a t a s e s of r a t p a n c r e a t i c i s l e t s : Comparison w i t h t h o s e of r a t kidney. J. C l i n . I n v e s t . 6 2 , 692-701. Meissner, H . P., P r e i s s l e r , M . , and Henquin, J. C. ( 1 9 8 0 ) . Poss i b l e i o n i c mechanisms of t h e e l e c t r i c a l a c t i v i t y induced by g l u c o s e and t o l b u t a m i d e i n p a n c r e a t i c b e t a c e l l s . I n t . Congr. Ser.--Excerpts Med. 500, 166-171. O ’ N e a l , S. G., Rhoads, B. Bb, and Racker, E . (1979). Vanadate i n h i b i t i o n o f s a r c o p l a s m i c r e t i c u l u m Ca2+ATPase and o t h e r ATPase. B i o c h e m . B i o p h y s . R e s . Commun. 8 9 , 845-850. Pace, C. S. (1979). A c t i v a t i o n o f Na c h a n n e l s i n i s l e t c e l l s : Metabolic and s e c r e t o r y e f f e c t s . Am. J. P h y s i o l . 2 3 7 ( 2 ) , E130-E135. Pace, C. S . , Murphy, M . , Conant, S . , and Lacy, P. ( 1 9 7 7 ) . Somatos t a t i n i n h i b i t i o n of glucose-induced e l e c t r i c a l a c t i v i t y i n c u l t u r e d r a t i s l e t c e l l s . Am. J. P h y s i o l . 2 3 3 , C167-Cl71. S c h e c h t e r , Y . , and K a r l i s h , S. (1980). I n s u l i n l i k e s t i m u l a t i o n o f g l u c o s e o x i d a t i o n i n r a t a d i p o c y t e s by vanadyl ( I V ) i o n s . N a t u r e ( L o n d o n ) 2 8 4 , 556-558. Schwabe, V . , P u c h s t e i n , C . , Hannemann, H . , and S o c h t i g , E. (1979) A c t i v a t i o n o f a d e n y l a t e c y c l a s e by vanadate. N a t u r e (Lond o n ) 2 8 4 , 556-558. S i e g e l , E . G . , Wollheim, C. B . , Renold, A. E . , and S h a r p , G. W. G. ( 1 9 8 0 ) . Evidence f o r involvement o f Na/Ca exchange i n glucose-induced i n s u l i n r e l e a s e from r a t p a n c r e a t i c i s l e t s . J. C l i n . Invest. 6 6 , 996-1003.

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CURRENT TOPICS IN MEMBRANES AND TRANSPORT. VOLUME 19

Phosphorylation of a Kidney Preparation of Na,K-ATPase by the Catalytic Subunit of CAMP-Dependent Protein Kinase SVEN M

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H

Biomedicinska Ceturum Uppsala Universitets Uppsala , Sweden

I.

INTRODUCTION

The CAMP-dependent p r o t e i n k i n a s e c h e m i c a l l y modif i e s many c e l l u l a r p r o t e i n s by r e v e r s a l p h o s p h o r y l a t i o n o f s e r i n e o r t h r e o n i n e r e s i d u e s ( f o r review, see Weller, 1 9 7 9 ) . Some of t h e s e p r o t e i n s are key enzymes i n m e t a b o l i s m and t h e i r a c t i v i t i e s are f i n e l y r e g u l a t e d v i a a kinase-phosphatase system. A b a s i c q u e s t i o n s t i l l remains i n connection w i t h t h e Na,K-ATPase: Is t h i s enzyme under hormonal c o n t r o l v i a CAMP and t h e CAMP-dependent p r o t e i n k i n a s e ? One d i r e c t way t o t e s t t h i s h y p o t h e s i s would b e t o i n v e s t i g a t e w h e t h e r t h e Na,K-ATPase i s a s u b s t r a t e f o r t h e p r o t e i n k i n a s e . P r e v i o u s r e s u l t s i n d i c a t e d t h e p r e s e n c e of a n endogenous p r o t e i n k i n a s e t h a t w a s a b l e t o p h o s p h o r y l a t e t h e me@rane p r o t e i n s of a b r a i n N a , K - A T P a s e p r e p a r a t i o n (Mardh and Z e t t e r q v i s t , 1 9 7 2 ) . Moreover, a h i g h l y pur i f i e d p r e p a r a t i o n of t h e c a t a l y t i c s u b u n i t of s k e l e t a l m u s c l e p r o t e i n k i n a s e was found t o p h o s p h o r y l a t e a r a b b i t k i d n e y p r e p a r a t i o n o f N a , K - A T P a s e (Mardh, 1 9 7 9 ; C a r l s s o n and Mardh, 1 9 7 9 ) . Copynght B 1983 by Academic Press. Inc. 999

All rights of reproduction in any form reserved. ISBN 0-12-153319-0

WEN MARDH

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F i g . 1 . P o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s o f Na,K-ATPase. A b o u t 75 p g of Na,K-ATPase w a s p h o s p h o r y l a t e d b y a b o u t 5 pg o f the c a t a l y t i c subunit of the p r o t e i n kinase for 30 see ( p o s i t i o n 2 ) and for 5 min ( p o s i t i o n 3 ) i n the p r e s e n c e o f 2 mV MgCl2 and 100 U M [y-32P]ATP. E l e c t r o p h o r e s i s was p e r f o r m e d i n a 6 % p o l y a c r y l a m i d e g e l . P a n e l a s h o w s the g e l a f t e r s t a i n i n g w i t h Coomassie B r i l l i a n t B l u e . P a n e l b shows an a u t o r a d i o g r a m o f the gel.

11.

EXPERIMENTAL PROCEDURES

Na,K-ATPase w a s p r e p a r e d from t h e o u t e r m e d u l l a of p i g k i d n e y (Jfbrgensen, 1974; Msrdh, 1 9 7 9 ) . Its a c t i v i t y w a s a b o u t 1 0 pmoles/mg/min a t 3OoC i n t h e p r e s e n c e of 1 mM ATP, 2 mM MgCl2, 120 mM N a C 1 , and 1 0 mM KC1 i n 30 mM T r i s - H C 1 b u f f e r (pH 7 . 4 ) . C a t a l y t i c s u b u n i t of s k e l e t a l m u s c l e CAMP-dependent p r o t e i n k i n a s e w a s p r e p a r e d a s d e s c r i b e d (Beavo e t a l . , 1 9 7 4 ) . P r o t e i n w a s d e t e r m i n e d by t h e Lowry a s s a y (Lowry e t a l . , 1 9 5 1 ) .

PHOSPHORYLATIONOF A KIDNEY PREPARATION

*i

1001

1

1.0

20 fraction no

30

40

Isolation of [32P]phosphoserine from Na ,K-ATPase.

111.

RESULTS

I n c u b a t i o n of Na,K-ATPase and t h e c a t a l y t i c subu n i t o f t h e p r o t e i n k i n a s e i n t h e p r e s e n c e of [32P]MgATP r e s u l t e d i n a p h o s p h o r y l a t i o n of a p r o t e i n of a molecul a r w e i g h t i d e n t i c a l t o t h a t of t h e a - s u b u n i t of t h e ATPase as j u d g e d from p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s i n sodium d o d e c y l s u l f a t e ( F i g . 1 ) . The p h o s p h o r y l a t e d p r o t e i n w a s c u t o u t of t h e qe; and t h e n h y d r o l y z e d i n 2 N H C 1 f o r 2 0 h r a t 1 0 0 ° C (Mardh and Z e t t e r q v i s t , 1 9 7 2 ) . By chromatography o f t h e h y d r o l y z a t e on Dowex 50-X8 [ 3 2 P ] p h o s p h o s e r i n e w a s i s o l a t e d ( F i g . 2 ) . The r a t e and e x t e n t of p h o s p h o r y l a t i o n of s e r i n e r e s i d u e ( s ) ( a c i d - s t a b l e phosphate) w a s i n v e s t i g a t e d i n a t i m e d e p e n d e n t s t u d y ( F i g . 3 ) . A f t e r 15 min t h e e x t e n t of a c i d - s t a b l e phosphate w a s about 65%of t o t a l ATPase s i t e s , which w a s measured as maximal amount of t h e phosphoenzyme i n t e r m e d i a t e (EPmax) i n t h e p r e s e n c e o f N a + . A s a t e s t f o r a p o s s i b l e p h y s i o l o g i c a l r o l e of t h e a c i d - s t a b l e p h o s p h o r y l a t i o n of t h e N a , K - A T P a s e which w a s c a t a l y z e d by t h e p r o t e i n k i n a s e , p h o s p h o r y l a t e d and unphosphorylated ATPase w a s incorporated i n t o s e p a r a t e b a t c h e s of l i p o s o m e s ( F i g . 4 ) . Liposomes w i t h phosphor y l a t e d N a , K - A T P a s e e x h i b i t e d a n i n c r e a s e i n t h e r a t e of

1002

SVEN MARDH

5

10

15

MINUTES Fig. 3. Time dependence o f a c i d - s t a b l e p h o s p h o r y l a t i o n . Na ,K-ATPase was p h o s p h o r y l a t e d a t 3OoC b y the c a t a l y t i c s u b u n i t of the p r o t e i n k i n a s e f o r v a r i o u s times. T h e r e a c t i o n m i x t u r e c o n t a i n e d 100 pM [y-32P]ATPf 2 mM MgC12 i n 30 mM T r i s - H C 1 b u f f e r (pff 7 . 4 ) -

22Na u p t a k e a f t e r t h e a d d i t i o n of ATP. Half-times were 8 min and 1 2 min, r e s p e c t i v e l y , f o r liposomes w i t h phosp h o r y l a t e d and unphosphorylated ATPase.

IV.

CONCLUSION

The c a t a l y t i c s u b u n i t of t h e --dependent prot e i n k i n a s e was a b l e t o t r a n s f e r t h e y-phosphorus group of ATP t o a 1 2 0 K p r o t e i n of t h e Na,K-ATPase p r e p a r a t i o n . The a c c e p t o r of t h e phosphorus group was a s e r y l r e s i d u e i n t h e p r o t e i n . The s t o i c h i o m e t r y of t h i s a c i d - s t a b l e phosphate r e l a t i v e t o t h e number of a c y l phosphate s i t e s i n d i c a t e d a c l o s e one-to-one r a t i o . When r e c o n s t i t u t e d i n t o liposomes, p h o s p h o r y l a t i o n of t h e ATPase by t h e p r o t e i n k i n a s e i n c r e a s e d t h e r a t e of 2 2 N a u p t a k e i n t o t h e liposomes. E x t r a p o l a t i o n from t h e p r e s e n t r e s u l t s s u g g e s t s t h a t an i n c r e a s e i n i n t r a c e l l u l a r CAMP and conc o m i t a n t a c t i v a t i o n of t h e CAMP-dependent p r o t e i n k i n a s e may l e a d t o an i n c r e a s e d t r a n s p o r t of monovalent c a t i o n s

1003

PHOSPHORYLATIONOF A KIDNEY PREPARATION

I

I

I

I

I

10

20

30

40

MINUTES

R e c o n s t i t u t i o n i n t o l i p o s o m e s ; u p t a k e of 22Na. U n p h o s p h o r y l a t e d Na,K-ATPase ( ) or Na,K-ATPase which had been p h o s p h o r y l a t e d b y the p r o t e i n k i n a s e ( 0 ) was i n c o r p o r a t e d i n t o the l i p o s o m e s . 22Na content o f the l i p o s o m e s was measured a t v a r i o u s t i m e s a f t e r a d d i t i o n of A T P .

via a protein kinase-catalyzed phosphorylation of a component of the Na,K-ATPase system, possibly its a-subunit. Before knowing the physiological significance of the acid-stable phosphorylation of the Na,K-ATPase, however, its reversal via a protein phosphatase reaction must be kinetically characterized.

REFERENCES

Beavo, J. A . , B e c h t e l , P. J . , and Krebs, E. G . ( 1 9 7 4 ) . P r e p a r a t i o n of homogeneous c y c l i c *-dependent p r o t e i n k i n a s e ( s ) and i t s s u b u n i t s f r o m r a b b i t s k e l e t a l muscle. I n "Methods i n Enzymology" (J. G. H a r d m a n and B. W. O'Malley, e d s . ) , V o l . 38, Academic Press, New York. P a r t C, pp. 299-308. Carlsson, T . , and M z r d h , S. ( 1 9 7 9 ) . R e g u l a t i o n of t h e Na-pump by t h e c a t a l y t i c s u b u n i t of p r o t e i n k i n a s e . A c t a Chem. S c a n d . Ser. B 33, 607-608.

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Jgkgensen, P. L. (1974). P u r i f i c a t i o n and c h a r a c t e r i z a t i o n of Na,K-ATPase. B i o c h i m . B i o p h y s . d c t a 3 5 6 , 36-52. Lowry, 0. H . , Rosebrough, B. J., F a r r , A. L . , and Randall, R. J. (1951). P r o t e i n measurement w i t h t h e F o l i n phenol r e a g e n t . J. Biol. Chem. 1 9 3 , 265-275. Mardh, S. (1979). Phosphorylation by t h e c a t a l y t i c s u b u n i t of p r o t e i n k i n a s e of a p r e p a r a t i o n o f kidney Na,K-ATPase. In "Na,K-ATPase: S t r u c t u r e and K i n e t i c s " (J. C. Skou and J. G. Ndrby, e d s . ) , pp. 359-370. Academic P r e s s , N e w York. Mardh, S. , and Z e t t e r q v i s t , 6. (1972). P h o s p h o r y l a t i o n of bovine b r a i n Na,K-stimulated ATP phosphohydrolase by adenosine [32P]triphosphate s t u d i e d by a rapid-mixing t e c h n i q u e . B i o c h i m . B i o p h y s . d c t a 255, 231-238. Weller, M. (1979) " P r o t e i n Phosphorylation." Pion L t d . , London

.

CURRENT TOPICS IN MEMBRANES AND TRANSPORT, VOLUME 19

Modulation of Na,K-ATPse Activity in Rat Brain by Adenosine 3 ' ,5'-Monophosphate RUSSELL B. LINGHAM' AND AMAR K. SEN Department of Pharmacology Faculty of Medicine University of Toronto Toronto, Ontario, Canada

I.

INTRODUCTION

The N a , K - A T P a s e enzyme r e g u l a t e s t h e a c t i v e t r a n s I n addip o r t of N a + and K+ a c r o s s t h e c e l l membrane. t i o n , t h e sodium pump i s i n t i m a t e l y i n v o l v e d i n s e v e r a l physiological processes. These i n c l u d e ( i ) the r e l e a s e and u p t a k e of n e u r o t r a n s m i t t e r s (Meyer and Cooper, 1 9 8 1 ) ; ( i i ) t h e g e n e r a t i o n of t h e N a + and K+ g r a d i e n t s a c r o s s t h e c e l l membrane n e c e s s a r y f o r t h e m a i n t e n a n c e of t h e membrane r e s t i n g p o t e n t i a l ; ( i i i ) t h e c o n t r o l of v a s c u l a r (Lang and B l a u s t e i n , 1980) and v i s c e r a l ( S c h i e d e t a1 , 1979) smooth muscle t o n e ; ( i v ) t h e t r a n s p o r t of g l u c o s e a c r o s s c e l l membranes (Moore, 1 9 7 3 ) ; and ( v ) t h e s e c r e t i o n of f l u i d i n s e v e r a l e p i t h e l i a l t i s s u e s ( S t e w a r t and Sen, 1 9 8 1 ) . The sodium pump is a l s o a f f e c t e d by e t h a n o l ( R a n g a r a j and K a l a n t , 1978) , as w e l l a s by i n s u l i n (Moore, 1973) and t h y r o i d hormone (Edelman and I s m a i l - B e i g e , 1 9 7 3 ) . I t is c l e a r t h a t c h a n g e s i n N a , K - A T P a s e enzyme act i v i t y w i l l a f f e c t s e v e r a l p h y s i o l o g i c a l p r o c e s s e s , and

.

R-esent address: Department of Pharmacology, University of Texas a t Houston, Health Sciences Center, P.O. Box 20708, Houston, Texas 77025. 1005

Copyright 0 1983 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN O-I2-1533190

1006

RUSSELL B. LINGHAM AND AMAR K. SEN

it i s a p p a r e n t t h a t c e l l u l a r mechanisms must e x i s t which can c l o s e l y modulate Na,K-ATPase a c t i v i t y . D e s p i t e t h e f a c t t h a t t h e enzymatic p r o p e r t i e s of N a , K - A T P a s e are known i n c o n s i d e r a b l e d e t a i l , v e r y l i t t l e i s known a b o u t t h e c e l l u l a r mechanisms t h a t r e g u l a t e enzyme a c t i v i t y . One form o f c o n t r o l r e s i d e s i n t h e cell u l a r mechanisms t h a t r e g u l a t e p r o t e i n s y n t h e s i s and deg r a d a t i o n (Lingham et al., 1 9 8 0 ) . A second form of c o n t r o l of t h e N a , K - A T P a s e enzyme i s mediated by c y c l i c AMP (CAMP). W e have p r e v i o u s l y r e p o r t e d t h a t t h e N a , K - A T P a s e enzyme i n r a t b r a i n c o u l d be i n h i b i t e d a p p r o x i m a t e l y 30% by CAMP (Sen et a l . , 1 9 7 6 ) . W e now r e p o r t t h a t t h e mechanism o f CAMP i n h i b i t i o n i s mediated t h r o u g h a c t i v a t i o n o f a membrane-bound CAMP-dependent p r o t e i n k i n a s e (CAMP-PK) which, i n t u r n , p h o s p h o r y l a t e s a n e u r o n a l membrane s u b s t r a t e p r o t e i n . P h o s p h o r y l a t i o n of t h e s u b s t r a t e p r o t e i n l e a d s t o a decrease i n t h e o v e r a l l N a , K - A T P a s e a c t i v i t y . The d e c r e a s e i n Na,K-ATPase a c t i v i t y a p p e a r s t o be l o c a l i z e d t o t h e Na+-dependent p h o s p h o r y l a t i o n s t e p o f t h e N a , K - A T P a s e enzyme.

11.

RESULTS AND DISCUSSION

W e examined t h e i n t e r a c t i o n of CAMP w i t h t h e Na,KA T P a s e enzyme by u t i l i z i n g a t e c h n i q u e d e s c r i b e d by Corbin et a l . ( 1 9 7 7 ) . W e t r e a t e d r a t b r a i n synaptosomal membranes w i t h e i t h e r CAMP ( 1 0 P M ) p l u s N a C l ( 2 5 0 m) o r N a C l ( 2 5 0 m) a l o n e . The r e s u l t s are shown i n F i g . 1. T r e a t i n g synaptosomal membranes w i t h CAMP p l u s NaCl produced a s i g n i f i c a n t d e c r e a s e i n CAMP-dependent P K a c t i v i t y (compare B and D i n F i g . 1) w i t h o u t a f f e c t i n g t h e CAMP-independent PK a c t i v i t y . The decrease i n CAMP-PK

a c t i v i t y w a s accompanied by a s i g n i f i c a n t i n c r e a s e i n Na,K-ATPase a c t i v i t y ( T a b l e I ) . W e b e l i e v e t h a t t h e i n crease i n Na,K-ATPase a c t i v i t y w a s due t o t h e removal of

the dissociated c a t a l y t i c subunit of the protein kinase from synaptosomal membranes. I f t h i s w a s indeed t h e case, one would e x p e c t a c o r r e l a t i o n t o e x i s t between t h e p r o t e i n k i n a s e a c t i v i t y and N a , K - A T P a s e a c t i v i t y i n membranes c o n t a i n i n g t h e p r o t e i n k i n a s e holoenzyme, b u t n o t i n membranes l a c k i n g most of t h e c a t a l y t i c s u b u n i t The r e s u l t s are shown i n F i g . 2 . It of t h e CAMP-PK. c a n be s e e n t h a t o u r e x p e c t a t i o n w a s c o r r e c t . The i n t e r a c t i o n between t h e CAMP-PK and t h e N a , K A T P a s e enzyme was examined f u r t h e r by making u s e of a The CAMP-PKI w a s a b l e t o CAMP-PK i n h i b i t o r (CAMP-PKI).

MODULATIONOF Na,K-ATPaseACTIVITY IN RAT BRAIN

1007

A = NaCl Supernatant B = NaCl Pellet C = CAMP + NaCl Supernatant D = CAMP + NaCl Pellet

-CAMP +CAMP

18 16 14

-

12 10 8-

64-

A

B

C

i D

1 . E f f e c t of NaCl o r CAMP p l u s NaCl t r e a t m e n t of r a t b r a i n s y n a p t o s o m a l membranes on p a r t i c u l a t e or s o l u b i l i z e d p r o t e i n k i n a s e a c t i v i t y . R a t b r a i n s y n a p t o s o m a l membranes w e r e t r e a t e d w i t h CAMP p l u s NaCl or NaCl a l o n e . The v a l u e s g i v e n a r e the mean 2 SEM f r o m 11 e x p e r i m e n t s . p < 0.05 w i t h r e s p e c t t o c o r r e s p o n d i n g v a l u e i n a b s e n c e of CAMP.

i n c r e a s e t h e Na,K-ATPase a c t i v i t y i n membranes c o n t a i n i n g t h e p r o t e i n k i n a s e holoenzyme b u t w a s w i t h o u t e f f e c t i n membranes l a c k i n g t h e c a t a l y t i c s u b u n i t of t h e p r o t e i n k i n a s e ( F i g . 3 ) . S i n c e t h e CAMP-PKI i n t e r a c t s s p e c i f i c a l l y w i t h t h e c a t a l y t i c s u b u n i t of t h e CAMP-PK, t h e r e s u l t s can be e a s i l y e x p l a i n e d . The n e x t s t e p was t o e l u c i d a t e t h e e x a c t mechanism by which t h e CAMP-PK i n h i b i t e d t h e N a , K - A T P a s e enzyme. The N a , K - A T P a s e enzyme, t h e membrane-bound CAMP-PK, and t h e s u b s t r a t e p r o t e i n f o r t h e CAMP-PK w e r e p r e p a r e d from r a t b r a i n . Upon r e c o n s t i t u t i o n of t h e CAMP-PK and t h e s u b s t r a t e p r o t e i n w i t h t h e N a , K - A T P a s e enzyme, w e were

RUSSELL B. LINGHAM AND AMAR K. SEN

1008

TABLE I.

E f f e c t of CAMP P l u s N a C l o r N a C l A l o n e T r e a t m e n t of R a t B r a i n S y n a p t o s o m a l Membranes o n Na,K-ATPase A c t i v i t y

Group NaC1-treated s y n a p tosomal membranes CAMP + N a C 1 - t r e a t e d s yn a p t o s o m a l

Na,K-ATPase a c t i v i t y ( y m o l e s Pi/mg/hr)

47.5 k 4.2a

56.5 k 4 . 3

b

Percentage o f control

(10)

100

(10)

1 2 0 . 3 t 2.8%

membranes a

V a l u e s a r e mean t SEN. T h e v a l u e s in p a r e n t h e s e s i n d i c a t e the number o f e x p e r i m e n t s . bThese v a l u e s a r e s i g n i f i c a n t l y d i f f e r e n t f r o m the N a C l t r e a t e d s y n a p t o s o m a l membranes, p < 0.001.

able to demonstrate a decrease in Na,K-ATPase activity in the presence, but not absence, of CAMP (Table 11). Preliminary experiments indicate that, when the activated protein kinase and the substrate protein were reconstituted with the Na,K-ATPase enzyme, there was a decrease in the Na+-dependent phosphorylation step of the Na,K-ATPase, whereas the K+-dependent dephosphorylation step was unaffected. The results support the tennet that the Na,KATPase enzyme present in rat brain plasma membranes may be modulated by a membrane-bound CAMP-dependent protein kinase. The CAMP-PK does not interact directly with the Na,K-ATPase enzyme, but rather phosphorylates a substrate protein, which leads to an overall decrease in Na,K-ATPase activity. The Na,K-ATPase enzyme is involved in several physiological processes. Since changes in Na,K-ATPase activity will affect the cellular processes subserved by the enzyme, it is imperative that, for normal cell function, dramatic changes in enzyme activity and ion fluxes be avoided. We would like to suggest that the modulation of Na,K-ATPase activity by CAMP to the extent of 20-30% reported in this and a previous communication is physiologically significant.

MODULATIONOF Na,K-ATPaseACTIVITY IN RAT BRAIN

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1009

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I

10 20 3040 50 60 70 80 Na-K ATPase Activity (umoles Pi/mg ProVhr)

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- -*8,o

o'61

k0.082

p
0.4

1

1

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20 30 40 50 60 70 80 Na-K ATPase Activity (umoles P i h g Protlhr) 10

F i g . 2 . C o r r e l a t i o n b e t w e e n the membrane-bound p r o t e i n k i n a s e a c t i v i t y r a t i o and Na,K-ATPase a c t i v i t y i n i n d i v i d u a l experiments: ( A ) C o r r e l a t i o n t h a t e x i s t s f o l l o w i n g the NaCl t r e a t ment (r = 0.710, p < 0 . 0 2 ) ; ( B ) c o r r e l a t i o n t h a t e x i s t s f o l l o w i n g t h e CAMP p l u s NaCl t r e a t m e n t ( r = 0 . 0 8 2 , p < 0 . 9 0 ) .

1010

RUSSELL 6. LINGHAM AND AMAR K. SEN

a

5

0 L

c

CAMP + NaCl WASHED MEMBRANES NaCl WASHED MEMBRANES

0

T

pg CAMP-dependent Protein Kinase Inhibitor F i g . 3 . E f f e c t o f CAMP-dependent p r o t e i n k i n a s e inhibitor on Na,K-ATPase a c t i v i t y . Na,K-ATPase a c t i v i t y was a s s a y e d i n the p r e s e n c e o f 20 or 80 Ug of CAMP-dependent p r o t e i n k i n a s e i n h i b i t o r . T h e v a l u e s g i v e n a r e the mean ?r SEM o f 4 s e p a r a t e e x p e r i m e n t s .

Effect of the CAMP-PK and Substrate Protein (SP) on Na,K-ATPase Activity

TABLE 11.

Na,K-ATPase specific activity (pmoles Pi/mg/hr)

a Group ~~

-~ ~

-CAMP ~

Enzyme (El Enzyme + cAMP-PK Enzyme + SP Enzyme + CAMP-PK

+CAMPb

~

+

SP

224.0 212.3 214.3 233.3 (100%)

233.3 234.2 221.4 192.0 (82.2 k 2.7%ICrd

a

T h e E a l o n e , E + P K , E + S P , or E + PK + SP w e r e recombined a s described b y Racker e t a l . (1979). & c o n c e n t r a t i o n = 1. o U M . C ~ A M Pd e c r e a s e d the Na,K-ATPase a c t i v i t y b y 1 8 % . These v a l u e s a r e the mean k SEM o f three s e p a r a t e reconstitution ex-

periments. dThese v a l u e s , i n the p r e s e n c e o f CAMP, a r e s i g n i f i c a n t l y d i f f e r e n t f r o m those i n the a b s e n c e of CAMP, p < 0.05.

MODULATION OF Na,K-ATPaseACTIVITY IN RAT BRAIN

1011

ACKNOWLEDGMENTS

The a u t h o r s would l i k e t o acknowledge t h e h e l p o f T h i s work M r . Douglas Pon d u r i n g t h e l a t e r s t a g e s o f t h i s work. w a s s u p h o r t e d by t h e Medical Research Council o f Canada ( M R C , MT-2485), t h e N a t i o n a l I n s t i t u t e o f H e a l t h (1-ROl-AM20425-01), and t h e O n t a r i o Heart Foundation ( T l - 5 1 ) .

REFEWNCES

B i h l e r , I . , and Sawh, P. C . ( 1 9 7 9 ) . J. M o l . C e l l . C a r d i o l . 11, 404-414. C o r b i n , J. D . , Sugden, P. M . , L i n c o l n , T. M . , and Keely, S. L. ( 1 9 7 7 ) . J. B i o l . Chem. 252, 3854-3861. Edelman, I . S . , and Ismail-Beige, F. ( 1 9 7 3 ) . Recent Prog. Horm. R e s . 30, 235-257. Lang, S., and B l a u s t e i n , M. P. ( 1 9 8 0 ) . C i r c . R e s . 46, 463-470. Lingham, R. B . , S t e w a r t , D. J., and Sen, A. K. ( 1 9 8 0 ) . Biochirn. Biophys. A c t a 601, 229-234. Meyer, E. M . , and Cooper, J . R. ( 1 9 8 1 ) . J. Neurochem. 36, 467475. Moore, R . D. (1973). J . P h y s i o l . (London) 232, 23-45. Racker, E . , Violand, S . , O ' N e a l , S . , Alfonzo, M., and T e l f o r d , J . ( 1 9 7 9 ) . Arch. Biochem. Biophys. 198, 440-447. R a n g a r a j , N . , and K a l a n t , H. ( 1 9 7 8 ) . Biochem. Pharmacol. 2 7 , 1139-1144. S c h i e d , C. R . , Honeyman, T. N . , and Fay, F. S. ( 1 9 7 9 ) . Nature (London) 277, 32-36. Sen, A. K., Murthy, R . , S t a n c e r , H . , Awad, G . , Godse, B . , and G r o f , P. ( 1 9 7 6 ) . I n "Membranes and Disease" (L. Bolis, J . Hoffman, and A. L e a f , e d s . ) , pp. 109-122. Raven Press, New York. S t e w a r t , D. J . , and Sen, A. K. ( 1 9 8 1 ) . Am. J. P h y s i o l . 240, C207-C214.

This Page Intentionally Left Blank

CURRENT TOPICS IN MEMBRANES AND TRANSPORT, VOLUME 19

Stimulation and Inhibition by Plasma of Ouabain-Sensitive Sodium Efflux in Human Red Blood Cells A. R. CHIPPERFIED Department of Physiology The University Dundee DDl4HN, United Kingdom

I.

INTRODUCTION

The f i r s t e x p e r i m e n t s on t h e e f f e c t of plasma on a c t i v e t r a n s p o r t i n human r e d b l o o d c e l l s ( R B C s ) w e r e d e s i g n e d t o see w h e t h e r t h e r e might b e a c a r d i a c g l y c o s i d e - l i k e s u b s t a n c e i n r a t b l o o d . The r e a s o n i n g w a s t h a t any such s u b s t a n c e m i g h t b e p r e s e n t a t h i g h e r l e v e l s i n a o u a b a i n - r e s i s t a n t s p e c i e s . Moreover, t h e r e were r e a s o n s t o b e l i e v e i n m o d i f i e r s of pump a c t i v i t y ( R o z e n g u r t and Heppel, 1 9 7 5 ; Beauge and Glynn, 1 9 7 7 ) . I n f a c t , human and r a t plasma had i d e n t i c a l e f f e c t s - namely, t o raise a c t i v e N a e f f l u x by 20-50% ( C h i p p e r field, 1978).

1013

Copyright 0 1983 by Academic hess. h c . All rights of reproductionin any form resewed. ISBN 0-12-153319-0

v r n q r n a r - m ~ m ~ m a m ~

booooooooooooo

+I +I +I +I +I +I +I +I

~?????????????

+I +I +I

3 0 0 0 0 0 0 0 0 0 0 0 0 0

t l +I +I

...........

, . * 0 0 0 0 0 0 0 0 0 0 0 0 0 0

N W d ' 0 0 0 0 0 N d O

???????????

r l r - m N N ~ ~ m m O m

N ,

r l q rlq

??

1

+I +I +I +I +I +I +I +I +I +I +I

N ,

0 0 0 0 0 0 0 0 0 0 0

1 < N,

H

U

H

0 0

N .

H

H

+I +I

. . .1

~ r - ~ m ~ ~ m m ~ m r n m - i + m ~ ~ ~ o m m r n m m ~ r n r - ~ m

0 0 0 0 0 0 0 0 0 0 0 0 0 0

N W N

H

a

T r a c e r 24Na e f f l u x i n f r e s h l y drawn RBC was measured b y s t a n d a r d m e t h o d s ( P r i e s t l a n d and W h i t t a m , 1 9 6 8 ) i n media c o n t a i n i n g 142 mM NaCl, 8 mM KCI, 8 mM g l u c o s e , and 8 mM T r i s C 1 (pH 7.5) plus 0.1 mM o u a b a i n and p l a s m a a s r e q u i r e d . R a d i o a c t i v i t y was measured u s i n g the Cerenkov phenomenon w i t h i n t e r n a l s t a n d a r d i z a t i o n t h r o u g h o u t . E x p t . I : f i n a l E x p t . 11: Ca + Mg , 1 mM; Pi a s 10 mM T r i s [ h e p a r i n ] , 0.7 units/ml; [EDTA], 0.5 mM. P i (pH 7.5)-

STIMULATION AND INHIBITIONOF OUABAIN-SENSITIVESODIUM EFFLUX

11.

1015

RESULTS AND DISCUSSION

The s t i m u l a t o r y e f f e c t of plasma a t low c o n c e n t r a t i o n s i s i l l u s t r a t e d i n T a b l e I . The s t i m u l a t i o n w a s c o n s i s t e n t b u t modest, and t h e p o s s i b i l i t y t h a t it might be f a l s e h a s t o be c o n s i d e r e d . F i r s t , i t s h o u l d be p o i n t e d o u t t h a t N a e f f l u x i n t h e p r e s e n c e o f plasma followed t h e s t a n d a r d p a t t e r n w i t h r e g a r d t o t i m e c o u r s e and s e n s i t i v i t y t o o u a b a i n , and t h a t e x t e r n a l pH w a s una f f e c t e d by plasma. Experiment I ( T a b l e I ) shows t h a t heparin, t h e usual anticoagulant, is not t h e stimulatory a g e n t . There w a s s t i m u l a t i o n w i t h e i t h e r h e p a r i n o r EDTA as a n t i c o a g u l a n t o r w i t h none (serum) ; c o n v e r s e l y , hepar i n w a s i n e f f e c t i v e . Experiment I1 shows t h a t C a , Mg, and P i ( n o t normally i n t h e i n c u b a t i o n media) made no d i f f e r e n c e . The e f f l u x w a s r e d u c e d , b u t t h i s i s a t t r i b u t e d t o i n h i b i t i o n by P i . The p o s s i b i l i t y t h a t d i v a l e n t i o n s c o u l d be i n v o l v e d i n a d i f f e r e n t way, i . e . , because plasma s e q u e s t e r s an i n h i b i t o r y i o n , i s r u l e d o u t by Expt. I: s t i m u l a t i o n was s e e n e v e n i n t h e p r e s ence o f 0 . 5 m~ EDTA, which was i n e f f e c t i v e by i t s e l f . Experiment I11 i s concerned w i t h changes i n c e l l volume. A s a r u l e , t h e media were r e n d e r e d h y p e r t o n i c t o t h e ext e n t t h a t plasma w a s added and i n h y p e r t o n i c media a c t i v e t r a n s p o r t i s r a i s e d (Poznansky and Solomon, 1 9 7 2 ) . The d e g r e e of h y p e r t o n i c i t y s h o u l d be t o o small t o produce t h e s t i m u l a t i o n observed and Expt. I11 b e a r s t h i s o u t . A t h i g h e r plasma l e v e l s , t h e r e w a s i n h i b i t i o n of t h e o u a b a i n - s e n s i t i v e Na e f f l u x ( T a b l e 11). T h i s app e a r e d t o b e v a r i a b l e i n t h a t t h e e f f l u x c o u l d b e above o r below t h e c o n t r o l w i t h o u t plasma. On r a i s i n g t h e t o n i c i t y w i t h NaCl o r s u c r o s e t o c o r r e s p o n d w i t h plasma, t h e r e s u l t s w e r e again variable. However, t h e e f f e c t s of N a C l and s u c r o s e w e r e i n d i s t i n g u i s h a b l e and d i s t i n c t from plasma. P l a s m a e f f e c t s on t h e Na pump i n human RBCs a r e f a r less s t r i k i n g t h a n t h e I - f o l d i n c r e a s e i n pumping obs e r v e d i n 3T3 c e l l s (Rozengurt and Heppel, 1 9 7 5 ) . On t h e o t h e r hand, t h e s t i m u l a t i o n i n RBCs i s t o o g r e a t t o be a c c o u n t e d f o r by enhanced N a e n t r y due t o HCO3 (Weith, 1 9 6 9 ) , and n e i t h e r s t i m u l a t i o n n o r i n h i b i t i o n conform t o t h e i n c r e a s e i n p a s s i v e N a e f f l u x caused by plasma. Thus i n RBCs t h e r e a p p e a r t o b e more d i r e c t e v i d e n c e t h a t t h e N a pump may b e i n f l u e n c e d by something b e s i d e s i o n s and ATP t h a n i n more complex c e l l s l i k e 3T3. However, t h e e f f e c t of plasma c l e a r l y depends on t h e cond i t i o n s , may be subject t o v a r i a b i l i t y , and, s i n c e K i n f l u x i s s t i m u l a t e d by less t h a n Na e f f l u x ( C h i p p e r f i e l d , 1 9 7 8 ) , depends on t h e f l u x measured.

A. R. CHIPPERFIELD

1016

TABLE 11.

I n h i b i t i o n of Na E f f l u x i n Human RBC by Plasma

Ouabain-sensitive Na e f f l u x ( r a t e c o n s t a n t , hr-', mean SE _+

Control

f 0.009

0.204 0.220 0.344 0.360 0.298 0.198

f. 0.004

f 0.023 f.

0.003

f. 0.008

f 0.005

With plasma 0.221 0.189 0.285 0.306 0.265 0.256

f 0.004 f 0.004 f 0.003 0.013 f 0.016 2 0.006 _+

%

Plasma ( v/v)

14 15 15 15 11.5

10

a

Na e f f l u x , of c o n t r o l

%

108 86 83 85 89 129

a

N a e f f l u x was measured a s i n Table I: s t i m u l a t i o n a t 3-5% plasma was observed i n a l l experiments.

ACKNOWLEDGMENT

Supported by t h e S c o t t i s h H o s p i t a l s Endowments Research T r u s t , Grant HERT 519.

REFERENCES

Beaug6, L. A , , and Glynn, I. M. ( 1 9 7 7 ) . A m o d i f i e r o f (Na++K+)ATPase i n commercial ATP. Nature (London) 268) , 355-356. C h i p p e r f i e l d , A. R. ( 1 9 7 8 ) . S t i m u l a t i o n of a c t i v e t r a n s p o r t i n human e r y t h r o c y t e s by human plasma. J . P h y s i o l . (London) 276, 29P. Poznansky, M . , and Solomon, A. K. ( 1 9 7 2 ) . E f f e c t of c e l l volume on potassium t r a n s p o r t i n human r e d c e l l s . Biochim. Biop h y s . Acta 274, 111-118. P r i e s t l a n d , R . N . , and Whittam, R. (1968). The i n f l u e n c e o f ext e r n a l sodium i o n s on t h e sodium pump i n e r y t h r o c y t e s . Biochem. J. 2 0 9 , 369-374. Rozengurt, E . , and Heppel, L. A ( 1 9 7 5 ) . Serum r a p i d l y s t i m u l a t e s o u a b a i n - s e n s i t i v e 86F?b+ i n f l u x i n q u i e s c e n t 3T3 c e l l s . Proc. Natl. Acad. Sci. USA 72, 4492-4495. Weith, J . 0. ( 1 9 6 9 ) . E f f e c t s o f b i c a r b o n a t e and t h i o c y a n a t e on f l u x e s of N a and K , and on glucose metabolism of a c t i v e l y t r a n s p o r t i n g human r e d c e l l s . Acta P h y s i o l . Scand. 75, 313-329.

CURRENT TOPICS IN MEMBRANES AND TRANSPORT. VOLUME 19

Inhibition of the Na Pump by Cytoplasmic Calcium in Intact Red Cells A. M.B R O W Department of Physiology and Pharmacology University of Noningham Medical School Notringham, United Kingdom

V. L. LEW Physiology Laboratory University of Cambridge Cambridge, United Kingdom

I.

INTRODUCTION

I n t r a c e l l u l a r C a i s known t o i n h i b i t t h e N a pumpm e d i a t e d i o n f l u x e s and t h e N a , K - A T P a s e . However, t h e e x t e n t of pump i n h i b i t i o n u n d e r p h y s i o l o g i c a l c o n d i t i o n s remains u n c e r t a i n , due p a r t l y t o t h e d i f f i c u l t y i n c o n t r o l l i n g i n t r a c e l l u l a r i o n i z e d C a ( [Ca2+] i ) i n i n t a c t c e l l s . T h i s problem h a s now been overcome i n red cells w i t h t h e use of t h e d i v a l e n t c a t i o n ionophore A23187.

11.

MATERIALS AND METHODS

Red c e l l s from f r e s h o r 4-day-old bank b l o o d were washed and l o a d e d w i t h 2 4 N a as d e s c r i b e d by Flatman and Lew ( 1 9 8 1 ) . I n some e x p e r i m e n t s , t h e 24Na l o a d i n g 1017

Copyright 0 1983 by Academic Press. Inc. All rights of reproduction in any form reserved. ISBN 0-12- I533 19-0

1018

A.

M.BROWN AND V. L. LEW

medium c o n t a i n e d 1 0 m a d e n o s i n e , 10 mM p y r u v a t e , and 50 m phosphate i n o r d e r s i m u l t a n e o u s l y t o i n c r e a s e t h e c e l l ATP c o n t e n t . A f t e r washing, t h e 4Na-loaded c e l l s were i n c u b a t e d a t 1 0 % h e m a t o c r i t , 37OC, i n a high-K medium (see Lew and Brown, 1 9 7 9 ) c o n t a i n i n g 1 0 m inos i n e ("normal-ATP" c e l l s ) or 1 0 mM adenosine + 1 0 mkf p y r u v a t e ("high-ATP" c e l l s ) , 0.25-0.5 mM MgC12, v a r i o u s c o n c e n t r a t i o n s of C a C 1 2 ( w i t h t r a c e r 45CaC12) and ouabain (10-3 M ) where r e q u i r e d . Following A23187 add i t i o n , t h e Ca i o n d i s t r i b u t i o n r e a c h e d a new s t e a d y s t a t e i n about 10 min and t h e c e l l Ca l e v e l s t h e n remained roughly c o n s t a n t f o r a t l e a s t 4 0 min. During t h i s t i m e , 24Na e f f l u x (Flatman and Lew, 19811, c e l l 45Ca (Lew and Brown, 1 9 7 9 ) , and c e l l ATP (Brown, 1 9 8 2 ) were measured a t 10-min i n t e r v a l s . [Ca2+]i w a s d e t e r mined as d e s c r i b e d p r e v i o u s l y ( L e w and Brown, 1 9 7 9 ) .

111.

RESULTS AND DISCUSSION

F i g u r e 1 shows t h a t i n normal-ATP c e l l s , [Ca2+ I i caused marked i n h i b i t i o n of t h e o u a b a i n - s e n s i t i v e Na e f f l u x (which a t t h e s a t u r a t i n g l e v e l s of e x t e r n a l K used h e r e measures o n l y N a - K exchange through t h e Na pump) although t h e k i n e t i c s were complex and depended on t h e A23187 c o n c e n t r a t i o n . Q u a l i t a t i v e l y s i m i l a r eff e c t s of [CaZ+]i and A23187 were a l s o o b t a i n e d w i t h t h e high-ATP c e l l s 50% i n h i b i t i o n r e q u i r i n g a b o u t 80 pM and 250 pM [Caa+]i w i t h 10 p~ and 1 pM A23187, r e s p e c t i v e l y . T h i s e f f e c t o f A23187 d i d n o t i n v o l v e a d i r e c t a c t i o n on t h e N a pump s i n c e t h e ionophore had no e f f e c t on t h e o u a b a i n - s e n s i t i v e ATPase a c t i v i t y of r e d c e l l membrane fragments. A s w e l l as i n h i b i t i n g t h e Na pump f l u x e s , i n c r e a s e d c e l l Ca caused marked f a l l s i n ATP c o n t e n t b o t h i n normal-ATP and i n high-ATP c e l l s , and i n each c a s e t h e mean c e l l ATP over t h e Na f l u x measurement p e r i o d showed [Caz+]i and A23187 dependencies s i m i l a r t o t h o s e s e e n f o r t h e N a f l u x e s ( t h e changes i n c e l l ATP were a p p r o x i m a t e l y l i n e a r w i t h t i m e o v e r t h i s p e r i o d ) . The i n s e t i n F i g . 1 shows t h a t f o r b o t h normal-ATP and high-ATP c e l l s , t h e i n h i b i t i o n of t h e o u a b a i n - s e n s i t i v e N a e f f l u x seemed t o c o r r e l a t e w i t h t h e mean c e l l ATP c o n t e n t , g i v i n g i n each c a s e a s i n g l e c u r v e f o r b o t h ionophore c o n c e n t r a t i o n s . However, ATP a l o n e could n o t have been t h e mediator of pump i n h i b i t i o n s i n c e i n t h e high-ATP c e l l s , f u l l pump i n h i b i t i o n was observed w i t h c e l l ATP l e v e l s h i g h e r even t h a n i n t h e Ca-free normal-ATP c e l l s .

INHIBITION OFTHE Na PUMP BY CYTOPLASMIC CALCIUM

1019

'I

.= 0 4

-2

Cell ATP(mmoles/litre cells)

f

L

0

-

1

LOO

800

1200 1600 INTRACELLULAR IONISED CALCIUM, ICa2'li IpM]

F i g . 1 . O u a b a i n - s e n s i t i v e Na e f f l u x as a f u n c t i o n o f ioni z e d i n t r a c e l l u l a r calcium ( [ C a z + ] i ) a t s t e a d y s t a t e i n n o r m a l ( 0 ) 0.6 pM A23187; ( 0 ) 10 pM A23187. ATP human r e d c e l l s : Inset: O u a b a i n - s e n s i t i v e Na e f f l u x as a f u n c t i o n o f mean c e l l ATP f o r normal-ATP r e d c e l l s ( 0 ,0 ) and high-ATP r e d c e l l s , 0): ( @ ) 0.6 U M A 2 3 1 8 7 ; ( m ) 1 W A23187; ( 0 , U ) 10 p M A23187. In e a c h p a r t o f the f i g u r e , the c u r v e s show the o u a b a i n s e n s i t i v e Na e f f l u x , $ N ~ ,p r e d i c t e d f r o m E g . ( 1 ) (see t e x t ) w i t h = 0.85 hr-1 for the n o r m a l ATP Km = 800 pM and K i = 100 pM. ) 10 pM A23187; c e l l s and 0.37 hr-1 f o r the high-ATP c e l l s : ((----- ) 0.6 pM or 1 pM A23187.

(m

$Ex

W e n e x t i n v e s t i g a t e d t h e h y p o t h e s i s t h a t Ca i n h i b i t s t h e Na e f f l u x by g e n e r a t i n g a c o m p e t i t i o n between CaATP and MgATP a s had been s u g g e s t e d b e f o r e f o r t h e Na,K-ATPase ( E p s t e i n and Whittam, 1 9 6 6 ; Robinson, 1 9 7 4 ) . Assuming a s i m p l e c o m p e t i t i o n g i v e n by t h e e q u a t i o n :

ON,

max - +Na [MgATP]/([MgATP] + K , I ~ + ( [ C a A T P ] /1 k.)))

'

-

0

(1) where Km and K i a r e t h e d i s s o c i a t i o n c o n s t a n t s of MgATP and CaATP, r e s p e c t i v e l y , a t t h e pump, w e s e a r c h e d f o r v a l u e s f o r K, and K i which would b e s t f i t t h e observed r e l a t i o n s between normalized Na pump f l u x ( $ N ~ / max) $ N ~,

1020

A.

M. BROWN AND V. L. LEW

c e l l ATP, and [ C a 2 + ] i . [MgATP] and [CaATP] were c a l c u l a t e d from t h e d a t a f o r [ C a 2 + ] i I c e l l ATP, and [Mg2+]i assuming d i s s o c i a t i o n c o n s t a n t s f o r CaATP and MgATP of 2 1 7 !JM and 8 3 V M , r e s p e c t i v e l y . The b e s t f i t w a s obt a i n e d w i t h Km Of 400-800 V M and K i o f 20-100 p M , and t h i s i s shown by t h e c u r v e s i n F i g . 1. S i n c e f r e e ATP and MgATP a r e i n c o n s t a n t p r o p o r t i o n a t a g i v e n Mg2+, and s i n c e [Mg2+]i w a s k e p t r e a s o n a b l y c o n s t a n t t o p r e v e n t d i r e c t e f f e c t s on t h e N a pump (Flatman and Lew, 19811, t h e p r e s e n t d a t a do n o t d i s t i n g u i s h between ATP and MgATP a s t h e pump s u b s t r a t e w i t h which CaATP i s competing The r e a s o n a b l e f i t o b t a i n e d w i t h t h i s model sugg e s t s t h a t under t h e c o n d i t i o n s o f h i g h Mg and ATP normally p r e v a i l i n g i n s i d e c e l l s , CaATP can i n h i b i t t h e N a pump by d i s p l a c i n g MgATP ( o r f r e e ATP) from a l o w a f f i n i t y s i t e on t h e i n n e r s u r f a c e o f t h e pump. T h i s r e a c t i o n i s known t o c o n t r o l t h e r a t e of inward K release from an occluded s t a t e , a s t e p i n pump t u r n o v e r which may become r a t e - l i m i t i n g ( P o s t e t al. , 1 9 7 2 ) . Taking t h e normal i n t r a c e l l u l a r c o n c e n t r a t i o n s of [Mg2+]i and ATP t o be 0.4-1.5 mM and 3-6 mM, r e s p e c t i v e l y , then t h e analysis presented here p r e d i c t s t h a t d u r i n g c e l l e x c i t a t i o n t h e pump might be i n h i b i t e d by as much as 25%, assuming t h e maximum [Ca2+]i t o b e 1 0 pM. During r e s t , however, when [ C a 2 + ] i 1s below 1 ! J M , i n h i b i t i o n would be less t h a n 4 % . Thus, under p h y s i o l o g i c a l c o n d i t i o n s , t h e r e might be t r a n s i e n t p a r t i a l i n h i b i t i o n of t h e N a pump i n some e x c i t a b l e c e l l s w i t h d e p o l a r i z i n g e f f e c t s where t h e c o n t r i b u t i o n of e l e c t r o g e n i c pump c u r r e n t s had been s i g n i f i c a n t .

.

ACKNOWLEDGMENT

W e g r a t e f u l l y acknowledge support from t h e MRC and Wellcome Trust.

REFERENCES

Brown, A. M.

(1982).

ATP and ATPase determinations i n r e d blood

cells. In "Red C e l l Membranes : A Methodological Approach" (J. C. E l l o r y and J. D. Young, e d s . ) pp. 223-238. Academic Press I New York.

INHIBITION OF THE Na PUMP BY CYTOPLASMIC CALCIUM

1021

E p s t e i n , F. H . , and Whittam, R . ( 1 9 6 6 ) . The mode of i n h i b i t i o n by calcium of cell-membrane adenosine-triphosphatase activity. B i o c h e m . J . 99, 232-238. F l a t m a n , P. W . , a n d Lew, V. L. (1981). The magnesium dependence of sodium-pump-mediated sodium-potassium and sodium-sodium J. P h y s i o l . (London) exchange i n i n t a c t human r e d cells. 3 1 5 , 421-446. Lew, V. L . , and Brown, A. M. ( 1 9 7 9 ) . E x p e r i m e n t a l c o n t r o l and a s s e s s m e n t of f r e e and bound calcium i n t h e c y t o p l a s m of i n t a c t mammalian r e d c e l l s . In " D e t e c t i o n and Measurement of F r e e C a 2 + i n C e l l s " (C. C. Ashley a n d A . K . Campbell, e d s . ) , pp. 423-432. E l s e v i e r / N o r t h H o l l a n d , Amsterdam. P o s t , R. L . , Hegyvary, C . , and K u m e , S . ( 1 9 7 2 ) . A c t i v a t i o n by a d e n o s i n e t r i p h o s p h a t e i n t h e p h o s p h o r y l a t i o n k i n e t i c s of sodium and potassium i o n t r a n s p o r t a d e n o s i n e t r i p h o s p h a t a s e . J. Biol. C h e m . 2 4 7 , 6530-6540. Robinson, J. D. ( 1 9 7 4 ) . N u c l e o t i d e and d i v a l e n t c a t i o n i n t e r a c t i o n s w i t h t h e ( N a + + K+) -dependent ATPase. B i o c h i r n . B i o p h y s . A c t a 341 , 232-247.

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CURRENT TOPICS IN MEMBRANES AND TRANSWRT. VOLUME 19

Involvement of Cdmodulin in the Inhibition of Na,K-ATPase by Ouabain LJONEL G. LELJEW,' M. T. PIASCIK, J. D. POTTER, E. T. WALLICK, AND A. SCHWARlZ Department of Pharmacology and Cell Biophysics College of Medicine Cincinnati. Ohio

I.

INTRODUCTION

I t w a s p r e v i o u s l y shown t h a t t h e s e n s i t i v i t y t o o u a b a i n of t h e membrane-bound Na,K-ATPase from m u r i n e plasmocytoma c e l l s w a s a l t e r e d by a d d i t i o n o f C a 2 + p l u s membrane p r o t e i n s ( L e l i B v r e e t al., 1979) o r t r o pomyosin (Charlemagne e t a l . , 1 9 8 0 ) . Membranes t r e a t e d w i t h EDTA (IC50 f o r o u a b a i n ca. 0 . 4 - 0 . 7 PM) r e c o v e r t h e o r i g i n a l r e s i s t a n c e (IC5 : 1 2 0 k 2 0 V M ) This a r t i c l e a f t e r a d d i t i o n o f tropomyosin and C a +. d e a l s w i t h t h e i n v o l v e m e n t of c a l m o d u l i n i n t h e a l t e r a t i o n o f t h e s e n s i t i v i t y of Na,K-ATPase t o o u a b a i n .

s

' P r e s e n t a d d r e s s : INSEIU Paris, Bance.

U127, H o p i t a l L a r i b o i s i e r e , 75010

1023

Copyright 0 I983 by Academic Press, Inc. All rights ofreproduction in any form reserved. ISBN 0-12-153319-0

1024

11.

LIONEL G. LELIEVRE eta/.

METHODS

MOPC 173 murine plasmocytoma c e l l s ( s t r a i n MF2S) were used. The plasma membranes w e r e i s o l a t e d i n t h e p r e s e n c e o f EDTA from t h e microsomal f r a c t i o n ( L e l i B v r e et a l . , 1 9 7 9 ) by i s o p y c n i c c e n t r i f u g a t i o n t h r o u g h a sucrose gradient. I n t h e l i g h t f r a c t i o n (L f r a c t i o n : d e n s i t y 1 . 1 4 g/cm3) t h e y i e l d of membrane p r o t e i n w a s 1%and o f Na,K-ATPase a c t i v i t y 30%. C o n t r a c t i l e p r o t e i n s and bovine b r a i n c a l m o d u l i n were p r e p a r e d as p r e v i o u s l y d e s c r i b e d (Dedman et al., 1977; L i n e t al., 1 9 7 4 ) . The amount o f membrane-bound c a l m o d u l i n w a s est i m a t e d from a s t a n d a r d p h o s p h o d i e s t e r a s e a c t i v a t i o n c u r v e a f t e r s o l u b i l i z a t i o n of t h e membranes i n Lubrol WX and i n c u b a t i o n a t 95'C. The N a , K - A T P a s e a c t i v i t y w a s a s s a y e d as p r e v i o u s l y d e s c r i b e d ( L e l i e v r e e t a l . , 1 9 7 9 ) . The e f f e c t of c o n t r a c t i l e p r o t e i n s and/or c a l modulin on t h e o u a b a i n s e n s i t i v i t y w a s s t u d i e d a f t e r a p r e i n c u b a t i o n of L f r a c t i o n f o r 30 min a t 4 O C w i t h t h e a p p r o p r i a t e p r o t e i n s i n a pH 6.8 b u f f e r c o n t a i n i n g 2 mM EGTA, 250 m M s u c r o s e , 30 m M i m i d a z o l e - c h l o r i d e , and CaC12 a d j u s t e d t o a c a l c u l a t e d free Ca2+ c o n c e n t r a t i o n a t pH 6.8 ( P o t t e r and G e r g e l y , 1 9 7 5 ) . A l i q u o t s were t h e n t r a n s f e r r e d t o i n c u b a t i o n t u b e s f o r Na,K-ATPase assays.

111.

RESULTS AND DISCUSSION

The Na,K-ATPase i n t h e L f r a c t i o n w a s h a l f maxim a l l y i n h i b i t e d by 1.5 5 0 . 2 p~ o u a b a i n . A f t e r a d d i t i o n of tropomyosin (Tm) and C a 2 + , t h e IC50 i n c r e a s e d t o 1 2 0 t 20 pM ( F i g . 1 ) . T h i s s h i f t w a s dependent b o t h upon t h e Tm and t h e f r e e C a 2 + c o n c e n t r a t i o n s . A t sat u r a t i n g ( C a 2 + ) , w i t h 1 mg of L f r a c t i o n , 3 3 0 pmoles Tm were r e q u i r e d t o s h i f t 1 0 0 % o f o u a b a i n - s e n s i t i v e Na,K-ATPase form t o t h e less s e n s i t i v e form. To s t u d y t h e e f f e c t of f r e e ( C a 2 + ) on t h e a l t e r a t i o n of s e n s i t i v i t y , t h e r a t i o of Tm t o membrane p r o t e i n w a s k e p t c o n s t a n t (330 pmole/mg) From 0 . 1 piV t o 3 pM C a 2 + , t h e s h i f t w a s p a r t i a l b u t was complete a t calcium concenF i f t y p e r c e n t of t h e enzyme t r a t i o n s h i g h e r t h a n 3 pM. p o p u l a t i o n w a s changed t o a more o u a b a i n - r e s i s t a n t form a t 0.65 ? 0 . 1 1 p~ C a 2 + . The s h i f t t o i n s e n s i t i v i t y c o u l d b e a b o l i s h e d by p r e t r e a t i n g w i t h an e x c e s s of t r o p o n i n I (TnI) a t s a t u r a t i n g ( C a 2 + ) ( F i g . 1 ) . The e f f e c t of TnI appeared t o be s p e c i f i c s i n c e p u r e

.

CALMODULIN IN INHIBITION OF Na,K-ATPaseBY OUABAIN

1025

Ouabain concentrations( pM 1

F i g . 1 . Dose-response curve o f Na ,K-ATPase a c t i v i t y versus ouabain concentration. Inhibitory e f f e c t o f troponin I on the s h i f t t o i n s e n s i t i v i t y . One m i l l i g r a m o f L f r a c t i o n was preincubated with TnI (100 pg) and Ca2+ (100 !AM) prior t o addition of 660 pmoles bovine cardiac Tm ( m ) . Controls: L f r a c t i o n p l u s TnI and Ca2+ (01,p l u s Ca2+ (A}, plus Tm ( t ) , plus both CaZ+ and Tm ( 8 ) . F o r comparison: membranes prepared i n the absence of EDTA ( 0 )(Lelibvre e t a l . , 1 9 7 9 ) .

troponin C, troponin T, and actin did not prevent the decrease in ouabain sensitivity induced by Tm. The specific activity of the enzyme was not affected by the addition of the contractile proteins. The requirement for Ca2+ and the specific inhibitory effect of TnI suqgest that a membrane-bound troponin C- or calmodulinlike protein is involved in the sensitivity of this Na,K-ATPase to ouabain. The contamination o f Tm by calmodulin was found to be negligible. To deplete the L fraction of calmodulin, membranes were washed three times in 1 m~ EDTA, 250 m~ sucrose, 30 mM imidazoleHC1 buffer (pH 6.8) and then three times in the same buffer supplemented with 1 m~ EGTA. This treatment reduced the calmodulin content from 3.1 to 1.8 pg/mq. There was a parallel loss of membrane proteins and enzyme activity (55 + 5 % ) but no change in the ouabain dose-response curve of Na,K-ATPase. Pure Tm was ineffective at saturating (Ca2+) in inducing any shift to insensitivity of the calmodulin-depleted membranes (Table I) . When these membranes were "reconstituted" first with calmodulin at saturating (Ca2+) prior to ad-

LIONEL G. LELIEVRE eta/.

1026

TABLE I.

Role of Calmodulina

b

C almodul in

(pg protein) Calmodulin depleted L fraction (1 mg)

0 100 0 1.8-100

(pg protein) 0 O

20- 30 20-30

d

IC50

wc

1.1-1.4 1.2-1.4 1.2-1.4 100-140

a

Preincubations and a s s a y s were performed i n t h e presence of 100 pM Ca2+. bTwo d i f f e r e n t l o t s o f bovine b r a i n c a l m d u l i n w e r e used on two membrane p r e p a r a t i o n s . COuabain c o n c e n t r a t i o n r e q u i r e d t o i n h i b i t 50% of t h e Na ,K-ATPase a c t i v i t y . d20-30 pg = 0.33-0.5 pmole bovine (or r a b b i t ) c a r d i a c Tm.

d i t i o n of Tm, t h e enzyme w a s less s e n s i t i v e t o ouabain (Table I ) . T h i s i n s e n s i t i v i t y was s i m i l a r t o t h a t of u n t r e a t e d membranes p l u s s a t u r a t i n g l e v e l s of b o t h Ca2+ and Tm. Calmodulin d i d n o t modify t h e s p e c i f i c a c t i v i t y of Na,K-ATPase. When added t o L f r a c t i o n , calmodulin d i d n o t a f f e c t t h e enzyme a c t i v i t y , t h e dose-response c u r v e , o r t h e s h i f t induced by Tm. Thus, i n murine plasmocytoma c e l l s , membrane-bound calmodulin i s i n v o l v e d i n t h e s e n s i t i v i t y of Na,K-ATPase t o ouabain. Arguments i n f a v o r of t h i s r o l e are t h e following: 1. The Ca2+ dependence of t h e s h i f t i s s i m i l a r t o t h e Ca2+ dependence of changes i n t h e c i r c u l a r d i c h r o i s m and t y r o s i n e f l u o r e s c e n c e of calmodulin (Dedman e t al.,

2. 3.

4.

197733).

TnI s p e c i f i c a l l y i n h i b i t s t h e s h i f t induced by Tm. T h i s i s c o n s i s t e n t w i t h t h e o b s e r v a t i o n s t h a t TnI can s p e c i f i c a l l y b i n d t o calmodulin. P a r t i a l removal of calmodulin from t h e membranes r e n d e r s Tm i n e f f e c t i v e i n s h i f t i n g t o i n s e n s i t i v i t y . Calmodulin which i s removed (1.3 pg/mg membranes) i s r e s p o n s i b l e f o r t h i s phenomenon. Addition of calmodulin t o d e p l e t e d membranes res t o r e d t h e a b i l i t y of Tm and C a 2 + t o i n d u c e t h e s h i f t . The low dose of calmodulin used h e r e ( 1 . 8 pg/mg membrane) f o r r e c o n s t i t u t i o n i s s i m i l a r t o t h a t observed e a r l i e r (Dedman e t al., 1977b).

CALMODULIN IN INHIBITIONOF Na,K-ATPase BY OUABAIN

1027

REFERENCES

Charlemagne, D., Lgger, J., Schwartz, K . , Geny, B., Zachowski, A . , and L e l i b v r e , L. (1980) Biochem. Pharmacol 29 , 297- 300. Dedman, J. R . , P o t t e r , J. D., and Means, A . R. ( 1 9 7 7 a ) . J. Biol. Chem. 252, 2437-2440. Dedman, J . R . , P o t t e r , J. D . , J a c k s o n , R. L . , Johnson, D. J . , and Means, A. R. (197713). J. Biol. Chem. 252, 8415-8422. L e l i b v r e , L . , Zachowski, A . , Charlemagne, D . , L a g e t , P., and P a r a f , A. (1979). Biochim. Biophys. Acta 557, 399-408. L i n , Y. M . , L i u , Y. P . , and Cheung, W. Y. (1974). J. Biol. Chem. 249, 4943-4954. P o t t e r , J. D . , and Gergely, J. ( 1 9 7 5 ) . J. Biol. Chem. 250, 46284633.

.

.

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1041

INDEX Reconstituted human red cell ghosts, sidedependent ion effects on ouabain binding to, 229-232 Red blood cell ghosts Na,K-ATPase of. 677-681 resealed, 247 -249,677 -681 Red blood cell membrane Na,K-ATPase. relation between band 3 and, 481 -483 Red blood cells abnormal Na/K pump and, 973 -975 anion-coupled Na efflux mediated by Na/K pump in, 693 -695 component arrangement in, 483 ion transport in, 602 magnesium dependence of sodium-pumpmediated Naf transport in, 653 -657 sodium-pumpcatalyzed ATP-ADP exchange in, 671 -674 sodium pump inhibition by cytoplasmic calcium in, 1017- 1020 ouabain-sensitive sodium efflux in, 1013- 1016 trypsin digestion effect on kinetic behavior of Na/Kpumpin, 697-701 Renal Na,K-ATPase, see also Na,K-ATPase subunit distribution of sulfhydryl groups and disulfide bonds in, 153 - 155 thyroid hormone in, 813 Resealed red blood cell ghosts Na-ATPase of, 677-681 ouabain binding and Na,K-ATPase in, 247 - 249 a-L-Rhamnose, 262 Rough endoplasmic reticulum, Na,K-ATPase synthesis in, 715 Rubidium. binding to Na,K-ATPase, 224 Rubidium active transport, isoproterenol and propranolol effects on, 873 Rubidium fluxes in absence of ATP or of P,, 428 -436 in presence of ATPor P,, 436-446 steady-state solution for, 445 Rubidium ion occlusion of, 626-2633 release from Na,K-ATPase, 629 retention by Na,K-ATPase, 628 Rubidium ion/ADP binding, to potassium-like form of NaKATPase, 223 -227 Rubidium ion concentration, Rb binding and, 208 Rubidium movements, in vesicles reconstituted with Na,K-ATPase, 425-447 Rubidium-rubidium exchange, 428 ATP concentration and, 442 computer simulations of, 446

phosphate concentration and, 44 1 vanadate,-sensitive, 43 1-432 Rubidium-86, equilibration of countertransport of into Rb-free or Rb-loaded vesicles, 433 Rubidium-86 uptake obesity and, 970-971 ouabain-sensitive, 428-429,872,891-894 in submandibular gland slices, 987

S Sarcolemma dissociation half-lives from, 903 -904 Scatchard analysis of, 905 -906 Sarcolemma membranes, digitalis binding to, 903 -906 Scatchard plots of binding sites per enzyme molecule, 296 of eosin binding to Na,K-ATPase, 454 of Rb binding without ATP, 225 of solubilized Na,K-ATPase and sarcolemma, 905 -906 SDS-gel electrophoresis, of Na,K-ATPase, 136 Sheep, large and small reticulocytes in, 803 -806 Sheep Purkinje fibers, sodium pump inhibition or contraction in, 885 -889 Sodium,, interaction with potassium,, 23 1 -232, see also Na abbreviations Sodium, potassium ion-transport adenosine triphosphatase, see Na,K-ATPase Sodium activation sites, identity of in ATPase with K activation sites inp-nitrophenylphosphate, 581 -585 Sodium concentration ouabain binding and, 170- 172 and type I ouabain-Na,K-ATPase complex dissociation, 176 Sodium-dependent phosphorylation, of rat brain Na,K-ATPase. 553 -555 Sodium dodecyl sulfate, Na,K-ATPase and, 70 Sodium efflux, anion-coupled, 693 -695 Sodium enzyme, 593 -594, see also Na,KATPase Sodium flux, and ATP-free or ATP-activated Na,K-ATPase in liposomes, 639 -642 Sodium ion(s) interaction with Na,K-ATPase, 561 -563 interactions with sodium pump, 649-651 K+-independentactive transport of by Na,KATPase, 659-662 kinetic analysis of effects on Na,K-ATPase, 591 -594 in lens transparency, 959

Index

Adenosine triphosphate dissociation, rate constant for, 527 - 528 Absorbance ratio. o f a - + @-submits,408 Adenosine triphosphate effect, pH and, 330-333 Acid hydrolysis, plasma sample susceptibility to, Adenosine triphosphate hydrolysis 920 in basolateral plasma membrane vesicles, Acid-stable phosphorylation, time dependence of, 703 - 705 1002, see also Phosphorylation as function of enzyme time, 332 Active transport, Na,K-ATPase molecular Na+ transport and, 361 structure and, 84 phosphoenzyme formation in, 513 -534 Actodigin genin, 260 Adenosine triphosphate phosphorylation, [W] Adamantane diazarine, incorporation into presteady state of, 557 -559 Na,K-ATPase, 129 Adenosine triphasphate +Na+, activation of KAdenine nucleotides, in sodium-sodium and phosphatase reaction by, 504 -505 sodium-potassium exchanges, 683 -685 ADP-ATP exchange, see also Adenosine Adenosine diphosphate, phosphoenzyme diphosphate; Adenosine triphosphate dephospholylation by, 5 18 -520 characteristics of, 607-609 Adenosine diphmphate-adenosinetriphosphate in internally diayzed squid giant axons, exchange, see ADP-ATP exchange 665 - 668 Adenosine diphosphate sensitivhy, of native and ouabain-sensitive, 677-681 oligomycin-treated Na,K-ATPase, 569 - 572 in reaction sequence, 501 -502 Adenosine 3 ' ,5 '-monophosphate, rat brain Na,Ksodium-ATPase and, 610-612 ATPase activity modulation by, 1005- 1009 sodium-pumpcatalyzed, 671 -674 Adenosine triphosphate sosium-sodium exchange and, 609-610 binding to cytoplasmic side of membrane, 320 ADP-sensitive phosphoenzyme, conformational cleavage by Na,K-ATPase, 343 transition between potassium-sensitive conformation at E,K, 392 phosphoenzyme and, 477 -479 dissociation kinetics of, 525 -528 Adrenal steroids, in Na,K-ATPase regulation, in inside-out vesicles with Na/K pump. 734 - 739 687-691 Albers-Post reaction, 486-490,523,528,602 interaction with Na,K-ATPase, 561 -563 Aldehyde, structure of, 260 ligand binding sites for, 491 -493 Aldosterone, Na,K - ATPase biosynthesis presteady state hydrolysis of by Na,K-ATPase. enhancement by, 809-811 577 - 580 Alkali cation transport, regulation of, 713 Adenosine triphosphate binding a-subunit of Na,K-ATPase to E,K, 391 -392 absorbance ratio of, 408 to E,Na, 392-393 amino acid composition of, 406 fluorescent probe of, 451 -455 antigenic properties of, 781-785 to Na,K-ATPase, 41 1 ATP- and Mg+-boundconformations of. Adenosine triphosphate binding site, affinity 390 - 393 labeling studies of, in canine kidney Na,Kcarbohydrate composition of, 407 ATPase, 367 -369 cation binding and conformational transition

A

1029

1030 a-subunit of Na,K-ATPase, continued

INDEX

ATP analogs, equilibriumbinding to high-affinity site, 283 related to, 386-388 ATPase, see also Na,K-ATPase cation-stabilizedconformations of, 380 -386 identity of Na activation sites in, 581 -585 hydrophobic labeling of, 385 - 386 structure of, 753 immunoreactivityof, 791 -794 two kinds of cation-binding sites for, 209 inhibitor-stabilizedconformations of, 395 ATPase activity intrinsic protein fluorescence of, 382 -383 K+ dependencechanges and, 475 labeling of lipidembedded and surface domains membrane disruption effect on, 776 of, 127 preincubation with PLP, 334 Mg-bound forms of protein in, 393 strophanthidin-sensitive, ioK-independent, molecular weights of, 407 775 -777 nucleotide binding region in, 384-385 ATP binding, to catalytic and regulatory sites, and pH dependence of tryptophan fluorescence 21 1-214 responses, 384 ATP binding sites, number per enzyme molecule, phospholipidcomposition of,405 291 -297 principal conformations of, 377 -396 ATP concentration,and inhibition of Na,Kprotein structure of, 378-380 ATPase in absence of, 205 -209 protein weight ratio for, 408 ATP hydrolysis, pH effect on, 329 relaxed or native conformation of, 386 ATP interactions, sodium pump and, 643 -646 sulfhydryl groups of, 383 -384 Axolemma Na,K-ATPase inhibition, by brain synthesis of, 760 extract factor, 770 tryptic and chymotryptic digestion in, 381 -382 a-subunit components, of kidney Na,K-ATPase B induced by heat treatment, 135 - 138 a(+)-subunit, immunoreactivityof, 791 -795 Balanus nubilis, 708 Amiloride, in murine erythroleukemia cell Barnacle muscle treatment, 798 -799 ouabain-dependentcurrent-andNa efflux in, Amino acid composition, of a-and @-subunits, 709 406 stoichiometryof electrogenic sodium pump in, Aminotriazole, in rat nephron Na,K-ATPase 707 -7 10 activity, 815 Basolateral plasma membrane vesicles, sodium Amphiphilic agents, in cataract formation, 959 movement and ATP hydrolysis in, 703 -705 AMPP (NH)P analog, Mg effect and, 285 -286, @-subunit 293 absorbance ratio for, 408 Analytical polyacrylamidegel electrophoresis,of amino acid composition of, 406 Na,K-ATPase, 91 -95 antigenic properties of, 781 -785 Anemic sheep, Na/K pumps and passive K+ carbohydratecomposition of, 407 transport in, 703-807 labeling of, 190 Anthroylouabainbinding site, fluorescein 5 'molecular weights of, 407 isothiocyanatebinding site and, 320 phospholipidcomposition of, 405 Antibodies protein weight ratio for, 408 binding to proteolytic fragments, 788 synthesis of in bound polysomes, 756 characterization and use of, 787-789 Binding, nucleotide, see Nucleotide binding; see cross-reactivity of, 788 also Adenosine triphosphate binding, etc. Aryldiazonium ouabain, chemical structure of, Binding energy 272, see also Ouabain in coupled vectorial process, 3, 13 - 16 Astrocyte Na,K-ATPase, inhibition by brain specificity rules of, 15 extract factor, 770 Bound adenosine triphosphate, dissociation ATP, see also Adenosine triphosphate kinetics for, 525 -528, see also Adenosine [WIATP, binding to Na,K-ATPase, 147 triphosphate [IZP]ATP,labeling with, 127 Brain, functional differences between two Na,KATP-ADP exchange, see ADP-ATP exchange ATPases of, 765 -777

INDEX

1031

Brain extracts inhibitory factor in, 769-774 norepinephrine K+-NPPase stimulationinhibition by, 935 Brain Na,K-ATPase regulation by norepinephrine and endogenous inhibitor, 931 -936 separation of, 767 -769 Bufalin, 260 Bufo marinus, 844 Butanedione, inactivation of ATP and ADP by, 416

C Ca2+,Mg2+-ATPase, selective purification of, 105- 106 Calcium binding and release, in coupled vectorial process, 5-7 Calcium ion ATPase, relative inlubition of by Folch partitioning, 949 Calcium ion flux, in terminal differentiation of murine erythroleukemia cells, 797-801 Calmodulin, in Na.K-ATPase inhibition by ouabain, 1023- 1026 CAMP, interaction with Na-K-ATPase, 1006 CAMP-dependent protein kinase, phosphorylation of kidney Na,K-ATPase by catalytic subunit of, 999- 1003 CAMP-dependent protein kinase inhibitor, Na,KATPase activity and, 1010 Canine kidney Na,K-aTPase, affinity labeling studies of ATP binding site of, 367 -369 Carbachol, Na/K pump activation by, 985-986 Carbohydrate composition, of a-and &subunits, 457 Cardiac glycosides, see also Cardiotonic steroids; Digitalis; Ouabain binding site labeling for, 188 - 194 binding to Na,K-ATPase, 235 biphasic action of, 913 catecholamine and, 871 -880 in heart muscle contraction, 857 interaction with Na,K-ATPase, 167,247 -249 mediated effects of, 871 - 880 in monovalent cation active transport inhibition, 859 - 860 positive inotropic effects of, 871 Na,K-ATPase as receptor enzyme for, 891 sodium pump stimulation by, 913-915 unitary vs. pluralistic views of, 875-880 Cardiac Na,K-ATPase, as molecular point of attack of cardiontonic steroids, 251 -255

Cardiac receptor site study, PROPHET and MMS-X computer graphics in, 257-263 Cardiotonic steroid binding, to Na,K-ATPase, 167- 194 Cardiotonic steroids, see also Cardiac glycosides binding of, 252 cardiac Na,K-ATPase as molecular point of attack of, 251 -255 covalent labeling results for derivatives of, I89 dipole moment vector of, 253 ellectrical field of, 254 stereoelectronic interaction with Na,K-ATPase, 25 1 -255 Na,K-ATPase inhibition by. 600 structure-activity relationships for, 177 - 183 Cataract formation, loss of Na,K-ATPase activity during, 959-964 Cataractous lenses, cation and sulfhydryl group contents of, 962 Cat atria and ventricles, ouabain contractile effects in, 897 -902 Cation binding, conformational transition and, 386-388 Cation movements, in Rb uptake and Rb/Rb exchanges, 434-436 Cations, sidedness of, 643 -646 Cation transport norepinephrine stimulation of, 874 regulation of, 713 C,,E,-solubilized Na,K-ATPase, 75 -78 Cerebral cortex, ouabain binding in, 935 Chemical equilibrium condition, 44 Chemical reaction, nonelectrolyte solution without, 23-26 Chemical specificity, in coupled vectorial process, 4 Chlormadinone acetate, in Na,K-ATPase inhibition, 833 Cholesterol hexane extraction in removal of, 165 role of in Na,K-ATPase, 163 - 166 Cleland diagram, of Na,K-ATPase reaction sequence, 489 Conformational changes, magnesium-induced, 403-421 Conformational equilibria, glutaraldehyde treatment in alteration of, 471 Conformational forms, in reaction sequence, 497 -498 Conformational states, of Na,K-ATPase, 595 - 598 Conformational transitions cation binding and, 386-388

1032

INDEX

Conformational transitions, conrinued of Na,K-ATPase, 426-428 pH effect on rate of, 328-330 scheme for, 427 Conformations, inhibitor-stabilized, of a-subunit, 395 Controlled proteolysis, specific chemical labeling and, 127 Cortical collecting tubule, aldosterone-mediated Na-dependent Nd,K-ATPase activation in, 989 -992 Coupled vectorial process, 1 - 16 binding energy in, 3 , 3 - 16 calcium binding and release in, 5 -7 coupling rules in, 2 potassium binding and dissociation in, 9 simple cycle in, 4 specificity rules in, 4 - 9 substrate specificity in, 10- 13 Covalent labeling ofdigitalis binding site, 271 -275 of ouabain binding site, 183 - 194 CrATPaanalogs, 287,361 -364 Cultured heart cells 4s Ca uptake by, 866 - 867 inotropic effect and contractility studies in, 862 - 870 ouabain effect on contractility of, 864-865 Cyanogen bromide cleavage, 88 Cyanogen bromide digestion, 90-91,94 Cyanogen bromide fragments apparent length of, 93 electrophoretic separation of, 92 preparative resolution of, 95 -96 Cysteine, Na,K-ATPase cleavage at, 100 Cytoplasmic calcium, Na pump inhibition by, 417-420

D 4lI' DAMN-digitoxin, cardiotinic steroids and, 179 [3HH]Dansylphosphatidylethanolamine,157 - 158 DCCD, enzyme modification by, 214 De Donder affinity, 37 Dehydroergosterol, 157 Deoxycholate, 72 Dephosphorylation of E,-phosphoenzymes, 305 -306 of phosphoenzyme by EDTA and ATP, 515-518 substrate concentration and, 5 17

Destabilizations, in coupled vectorial process, 13-16 Digitalis, see also Cardiac glycosides; Ouabain endogenous, 835-837 inotropic mechanism of, 835 in Na,K-ATPase inhibition, 826 Na,K-ATPase system as pharmacological receptor for, 827 positive inotropic action of, 825 - 838,907 Digitalis action molecular mechanism of, 25 1 -255 specificity of, 255 Digitalis binding, differences between Na,KATPase and sarcolemma membranes, 903 -906 Digitalis binding site, ouabain derivatives in covalent labeling of, 271 -275 Digitalis glycosides, positive inotropic effect of, 825-838,907 Digitalis-induced inotropy , monovalent cation transport and mechanisms of, 857 -880 Digitalis receptor, pharmacological and biochemical studies of, 907-910 Digitalis structure, biological roles of, 257 0-D-Digitose, 262 Digitonin, 73 Digitoxigenin, 178,260 Digoxin C-17 side group and first sugar of, 258 in Na/K pump stimulation, 836 Digoxin-like factor, isolation of, 917-921 Dimeric enzyme models, in kinetic analysis, 505 -506 Dimeric reaction process, 491 Dimethyl sulfoxide, in murine erythroleukemia cell treatment, 797 Disulfide bonds, subunit distribution of, in renal Na,K-ATPase, 153- 155 5.5 '-Dithio-bis(2-nitrobenzoic acid), 355 - 357 Divalent cations interaction with fluorescein-labeled Na,KATPase, 457-462 of Na,K-ATPase, 595-598 Dog kidney Na,K-ATPase, lithium-catalyzed ouabain binding to, 241 -244 Dog kidney outer medulla membranes, 113- 116 DTNB, 355 -357

E E-AMPP(NH)P-Mg-AMPP(NH)P, formation of, 293

1033

INDEX Eel electroplax, Na.K-ATPase selective purification fro, 103- 105 Eel enzyme, conversion of E,P to E,P in, 345 Effective dipole moment vector, variability of, 253 Electric eel Na.K-ATPase phosphoprotein, 343 - 346 Electrochemical potential difference, of permeant ions, 30-31.40 Electrodes, phases of, 32 Electrogenic sodium pump, stoichiometry of in barnacle muscle, 707-710, see also Sodium Pump Electrolyte solution with chemical reactions, 40-49 Electrolyte solution without chemical reactions, 26 - 35 Electroneutrality condition, 42 Electrophorous Na,K-ATPase, vanadate inhibition of, 528-530 Endodigin, 835-837.945-950 Endogenous digitalis-like factor Na/K pump stimulation and, 913-915 immunochemical approaches to isolation of, 9 I7 -92 1 Endogenous glycoside-like substances, 843 - 852 Endogenous inhibitor, brain Na,K-ATPase regulation of, 931 -936 Enzymatic activity, divalent cation effects and, 597 Eosin, binding to Na,K-ATPase, 451 -454 Eosin fluorescence, Na/K ratios and, 325, 335 Essential hypertension disorders in molecular assemblies for Na transport in, 95 1 -952 sodium-potassium cotransport system in. 953 -956 N-Ethylmaleimide Na,K-ATPase inhibition by, 417 in phosphorylation from ATP in presence of Na+ and Mgz+,349-352 sulfhydryl group reactivity to, 383 Extracellular sodium sodium ion activity and, 605

F Fluorescein. fluorescence energy transfer from, 151

Fluorescein isothiocyanate-reactive site, in active site structure of Na.K-ATPase, 149- 151 Fluorescein-labeled Na,K-ATPase, interaction of divalent cations with, 457 -462

Fluorescence changes, magnesium and manganese effects in, 460 Fluorescence isothiocyanate reactive labeling, stoichiometry of, 150 Fluorescence polarization. of DNS-PE and DNSPS, 157- 161 Fluorescence quenching, Mg2+and Mn2+effects in, 159-461 Fluorescent lipid probes of ATP binding to Na,K-ATPase, 451 -455 Na,K-ATPase lipid regions examined with. 157- 161 5 ’p-Fluorosulfonylbenzoyladenosine,as ATP binding site affinity probe, 367 Flux measurements. in rubidium movement studies, 426 Folch partitioning, inhibition of Na,K-ATPase and Ca’+-ATPase by, 949 Foxglove plant, cardiac glycosides from, 825 Free fatty acids, in Na.K-ATPase from rabbit kidney and outer medulla, 164 Frog bladder, ouabain binding to. 846 FSBA, see 5 ‘p-Fluorosulfonylbenzoy ladenosine Fundulus heteroclitus, 727

G y-aubunits, antigenic properties of, 781 -785 Genins example of, 261 total dipole movement of, 259 Gibbs-Duhem relation, 25.33 Glucocorticoids, in Na,K-ATPase regulation, 737 -739 Glutaraldehyde treatment, in Na+- and K+-rich media, 472 Glycoside actions, intracellular sodium enhancement in, 225-238 Glycoside-like substances endogenous, 843 -852 source and purification of, 850 Glycosides, first sugar of, 259, see also Cardiac glycosides Group-specific modifications, Mg2+effects on, 4 16 -420 Guinea pig atria and vesicles, ouabain contractile effects on, 897 -902 Guinea pig brain extract, gel filtration chromatography of, 928 Guinea pig . -brain microsomes, inhibition of [3H]ouabain binding to, 847 I

1034

INDEX

H Halobacterium halobium, 55 Heart cells in culture, 862 -870 vanadate inhibitory and stimulatory effects on sodium pump, 939-942 Heart muscle cardiac glycosides in contraction of, 857 ['H Iouabain binding to, 891 -894 positive inotropic effect on, 861 -862 Helmholtz free energy variation, 26-28 Hemoglobin, as membrane transport system, 56 Hormones, in long-term Na,K-ATPase regulation, 729-739 Horse kidney Na,K-ATPase high-performance gel chromatography of, 107- I09 molecular weights of, 110 Humoral sodium-potassium pump inhibitor, in experimental low-renin hypertension, 923 -924 Hydrolysis cycles, for Na,K-ATPase with one active substitute site, 587- 590 Hypertension, see Essential hypertension

I Inorganic phosphate, oxygen exchange with water, 371 -374 Inotropy cardiac glycoside-induced, 857 -860, 871 half-life of, 904-905 and monovalent cation active transport inhibition, 859 - 860 sodium pump inhibition and, 861 -862 Inside-out vesicles, Na/K pump in, 687-691 Insulin binding, to purified Na,K-ATPase, 977 -982 Internal adenine nucleotides, in sodium-pumpcatalyzed Na-Na and Na-K exchanges, 683 -685 Intracellular sodium enchancement of ouabain binding to Na,KATPase, 235-238 in enzyme phosphorylation, 603 51110donaphthylazide,labeling with, 127 Ion fluxes, in cell proliferation control, 797 Ions, permeant and impermeant, 29-30 Ion translocating steps, 599-617 Ion transport occluded forms and, 613-614 in red blood cells, 602

Isometric contractile force, in rat right ventricular strips, 908 Isoproterenol, in rubidium active transport, 873

K K', see also Potassium; Potassium ion K+-dependentphosphatase activity, inhibition by ADP, 358 K form pH effect on distribution between Na form and. 326 - 328 transformation to Na form, 330, 337 Kidney Na,K-ATPase different a-subunit components induced by heat treatment, 135-138 phosphorylation by catalytic subunit of CAMPdependent protein kinase, 999- 1003 Kidney outer medulla, Na,K-ATPase enriched, 113-116 Kinetic analysis dimeric enzyme models in, 505 -506 of Na+ and K+effects on Na,K-ATPase, 591 -594 reaction mechanism and, 485 -506 K+/K+exchange, in reaction sequence, 502-503 K-phosphatase reaction, activation of, 504 -505

L Lamb kidney, a-and @subunit immunoreactivity in, 792 Lamb kidney Na,K-ATPase, structural studies on, 131 - I33 Lens, transparency of, 959-964 Ligand binding, to Na/K pump, 430 Ligand binding sites K + , 496-497 MgZ+,493-494 Na2 ,495 phosphate, 494-495 reaction mechanism and, 491 -497 Ligand effects occluded state and, 447 physiological implications of, 447 with substrate site of Na,K-ATPase, 281 -307 Ligands fluorescence changes induced by, 479 stoichiometrical binding of to Na,K-ATPase, 145 - 148 Lipids, fatty acid analysis of, 164 Lithium-catalyzed ouabain binding, 241 -244, see also Ouabain binding +

INDEX

1035

Low-renin hypertension, humoral Ka/K pump inhibitor in, 923 -934, see also Essential hypertension Lubrol, Na,K-ATPase and, 73 -74

M Magnesium AMPP (NH)P analog of, 285 -286 as cofactor for Na,K-ATPase activity, 653 in sodium-pump-medicated Na-K and Na-Na exchanges in human red cells, 653 -657 Magnesium, interaction with C%, 231 interaction with Na,, 230 Magnesium binding sites, 493 -494 Magnesium-dependent ouabain binding, lithium effect in, 244 Magnesium-induced conformational changes, in Na,K-ATPase, 403 -421 Magnesium ion in group-specific modifications, 416-420 nucleotide binding and, 285-287,412-416 Manganese ion, fluorescene quenching induced by, 459 MDCK cells, biosynthesis of Na,K-ATPase in, 753-761 Membrane-bound Na,K-ATPase, electron microscope analysis of two-dimensional crystals of, 123- 125 Membrane couplings, between flow and chemical reactions, 21 Membrane disruption, Na +-independent ATPase activity and, 776 Membrane equilibrium, 2 1 - 50 electrolyte solution without chemical reactions, 26-35,40-49 nonelectrolyte solution without chemical reactions, 23 -26 phases in, 21 -22 Membrane potential, defined, 31 Membrane transport parameters, in anemic sheep, 805

Membrane tansport systems bacteriorhodopsin as, 55 examples and models of, 55 -57 2-Metcaptoethy1, in brain extract activity, 771 Mg-ATPase activity, ouabain-sensitive, 114. see also Magnesium; Magnesium ion Mineralocorticoids, in Na.K-ATPase regulation, 735 -737 MMS-X computer graphics, in cardiac steroid receptor site study, 257 -263

Molecular assemblies for Na transport disorders in, 951 -952 Molecular weights, of a! and 0 subunits, 407 Monovalent cations binding to Na,K-ATPase, 203 enzyme conformation and nucleotide binding in relation to, 288 -291 Monovalent cation transport, digitalis-induced inotropy and, 857 - 880 Murine erythroleukemia cells amiloride inhibition in, 798-799 Na+ and Ca2+fluxes in terminal differentiation of, 797 -801

N Na+, see also Sodium; Sodium ion Na-ATPase, see also Na,K-ATPase ADP-ATPexchange and, 610-612 transport correlates of, 603 -606 ['HI-NAB ouabain, photoaffmity labeling by, 190- 192 NAB-ouabain 11, 178 Na+ concentration, Na+ binding and, 206 Na form, see also Sodium; Sodium ion; Sodium Pump pH effect on distribution between K form and, 326-328 transformation to K form, 330,337 Na,K-ATPase (sodium and potassium ion transport adenosine triphosphatase) active site structure of, 149- 151 ADP binding to, 296 adrenal steroids in regulation of, 734-739 aldosterone-mediated, Na-dependent activation of in cortical collecting tubule, 989-992 01- and &subunits of, 54-55,208,781-785, see also a-subunit; 0-subunit alternative cleavages of, 100- 101 AMP-PNP binding to, 412-415 analytical polyacrylamide gel electrophoresis of, 91 -95 anionic and nonionic detergents in purification of, 69 antibodies to, 787 -789 antigenic properties of subunits of, 781 -785 Asp-Pro cleavage of, 97 ATP affinity change in, 323 ATP analogs and, 361 -365 ATP binding to, 291 -299,411 ATP concentration and, 327 binding of monovalent cations to, 203 -214 biosynthesis of, 714-717,753-761

1036

Na, K-ATPase , continued biosynthesis enhancement in toad urinary bladder, 809-81 1 calmodulin in inhibition of by ouabain, 1023 - 1026 CAMPinteraction with, 1006 cardiac glycoside binding to, 235-238 cardiac steroid receptor site for, 257-263 cardiotonic steroid binding to, 167- 194 cardiotonic steroid inhibition of, 600 as catalyzer for Mg2+-andK+-dependent exchange, 371 chlormadione acetate inhibition of, 833 cholesterol and other neutral lipids in, 163 - 166 C,,E,-solubilized enzyme and, 75 -78 cleavage method of large subunit of, 83 - 101 components of, 54 - 55 conformational changes necessary for transport of, 315-321 conformational transitions of, 426 -428 consecutive phosphoentermediates of, 573 -576 cyanogen bromide cleavage and digestion of, 88-91 cyanogen bromide fragments of, 92 -96 defined, 54 deoxycholate and, 72 detergents and sources of enzymes in preparation of, 68 detergent solubilization of, 67 -69 digitalis as inhibitor of, 826 digitalis binding to, compared with sarcolemma membrane binding, 903 -906 digitonin and, 73 discontinuous stacking system in slab gel for urea-dodecyl sulfate-polyacrylamide gels, 88 discrimination between Na+ and K + , 323-329 dissociation half-lines from, 903 -904 divalent cations and conformational states of, 595 - 598 dog kidney outer medulla membranes enriched in, 113-116 electron microscope analysis of hvo-dimensional crystals of membrane-bound form of, 123- 125 electrophoresis of proteins from purification stagesof, 104 endogenous inhibitor of, 945 -950 eosin binding to, 451 -454 N-ethylmaleimide inhibitor of, 417 fluorescent probe of ATP binding to, 451 -455 free Mg*+requirement for, 403

INDEX function of, 67,707, 1005 glucocorticoids in regulation of, 737 -739 half-of-the-sites reactivity of, 219 -222 hormones in long-term regulation of, 729-739 horse kidney, 107- 110 hydrolysis cycles for, 587 -590 incubation with Mgz+ + Na+ [y-]zP]ATP, 300 insulin binding to, 977 -982 interaction with nucleotides, 282 -297 interaction with vanadate, 298 -300 from kidney outer medulla, 163 K+-independentactive transport of Na+ by, 659 -662 kinetics of, 3 17 -3 18 ligand interactions with substrate site of, 281 -307 lubrol and, 73-74 magnesium and CDTA in inhibition of, 418 magnesium-induced conformational changes in, 403-421 mineralocorticoids in regulation of, 735 -737 modification with DCCD, 213-214 modification with NEM, 350 molecular adaptation in, 324 molecular structure of, 84 motion in proteins of, 377 multiple interacting ligand sites of, 47 I from nerve terminals, 830 in obesity, 739-742 occluded-ion forms of, 625-637 octaethylene glycol dodecyl monoether and, 74-78 octyl glucoside and, 73 oligomycin-treated, 569-572 optical density of, 75 organization of transmembrane segments of, 127-130 ouabain binding to, 146- 147, 897, see also Ouabain ouabain dissociation from, 174- 176 [12P]phosphoserineisolation from, 1001 phosphorylation of, 300-307 photolysis of, 268 photoaffinity labeling of ouabain binding site of, 265-269 polyacrylamide gel electrophoresis of, 98, loo0 potassium binding to, 223 presteady state hydrolysis of ATP by, 577-580 protein and phospholipid patterns of, 405 proteins of, in freeze-fracture electron microscopy, 119- 121

+

INDEX Na,K-ATPase, continued protein structure in a-subunit of, 378 - 380 proteolipid associated with, 132 purification methods for, 57-59,67-70, 73, 87, 103-105 pyridoxal 5-phosphate as inhibitor of reversibility in, 333 -338 radiation inactivation of, 139- 143 radiochemical reagents in labeling of, 83 reaction mechanism and ion translocating steps in, 599-617 as receptor enzyme for cardiac glycoside effects, 891 red cell membrane. 481 -483 reduction and alkylation of, 87 regulation in response to Na' loading and K + depletion. 723-729 regulation through biosynthesis and turnover, 713-742 rubidium binding to, 224 rubidium movements in vesicles reconstituted with, 425-447 Scatchard analysis of, 225,296,454,905 -906 SDS-polyacrylamide gel electrophoresis of, 136 selective purification of, 103 - 105 separation of, in brain, 767-769 smallest active unit of. 292 sodium-dependent phosphorylation of. 353 -354,553 -555 sodium-dodecyl sulfate and, 70 sodium ion, K + , and ATP interaction with, 561 -563 sodium ions in phosphorylation of, 353 -354, 553 - 555 specific fluorescein isothiocyanate-reactive site for. 149-151 stoichiometrical binding of ligands to less than 160 kilodaltons of, 145- 148 stripping, concentration. and transferring protein in relation to, 89 structurally different nucleotide binding sites in, 355 structure and function of, 53-62.67, 315-321,697,705. 1005 subunit association for, 59-61, seealso asubunit; P-subunit substrate site of,281 subunit characterization in, 71,404-406, 781 -785 sulfhydryl groups of, 349-352 thyroid hormones in regulation of, 730-734 topological mapping . .. - of a-subunit of, 61 -62 tritiation of, 88 -89

1037 triton and, 72 - 73 trypsin in elucidation of structure of, 697 tryptic and proteolytic fragmentation of, 275-276 tryptic digestion in large chain of, 90, 97 -99 turnover of, 717-723 two configurations of, 477 ultrastructure of in plasma membrane vesicles, 119-121 [48V]vanadatebinding to, 221 Na.K- ATPase actvity aminotriazole and. 815 in brain, 765-777 CAMP-dependent protein kinase inhibitor and, 1010 decrease of in obesity, 969-972 lipid function in, 157 loss of in cataract formation, 959-964 monoexponential decay of, 38 1 vs. ouabain concentration, 1025 in rat nephronsegments, 813-816 thyroid hormone and, 813 [-thyroxine in, 815 Na,K-ATPase conformational changes, 403 -42 I NaKATPase inhibition absence of ouabain-like activity in, 927 -930 by Folch partitioning, 949 Na,K-ATPase ligands. fluorescence changes induced by, 479 Na,K-ATPase lipids, fluorescent examination of, 157- I61 Na,K-ATPase ouabain complex. ATP binding to, 220 Na,K-ATPase reaction, see also Reaction sequence alternative sequences in hydrolytic steps of, 503 - 504 biphasic substrate-velocity plots for. 2 19 kinetic analyses of, 485 -506 uncertainties, alternates, and anomalies in, 49 1 - 506 Na,K-ATPase subunits, see also a-subunit; 8subunit; y-subunit antigenic properties of, 781 -785 molecular weights of, 71 Na:K ratios, vs. eosin fluorescence, 335 Na+/Na+exchange, in reaction sequence. 501 -502 NAP-strophanthidin, 178 Natriuretic hormone, 844 Na+ vs. K+ titration curve, pH increase and, 326 - 327 NEM, see N-Ethylmaleimide

1038

INDEX

[I4C]NEM,free sulfhydryl groups with, 154 NEM-modified Na,K-ATPase, binding of monovalentcations to, in presence of ATP andMg*+,209-211 Nephron, sodium reabsorption in, 813 Nerve terminals, Na,K-ATPase from, 830-831 K+-p-Nitrophenylphosphataseactivity, ouabain inhibition of, 932 31P(lSg)NMP kinetic analysis, of '80 exchange reaction between Pi and HzOcatalyzed by Na,K-ATPase, 371 -374 Nonelectrolyte solution with chemical reactions, 35-39 Nonelectrolyte solution without chemical reaction, 23-26 Norepinephrine brain Na,K-ATPase regulation by, 931 -936 in cation transport stimulation, 874 Norepinephrine K+NPPase stimulation, inhibition bv brain extract. 935 NPT-ouabain, chemical structure of, 272, see also Ouabain Nucleotide binding Mg*+effects in, 412-416 monovalent cations and, 288-291 phosphorylation and, 410-412 stoichiometry of, 410-412 Nucleotide binding sites inhibition effects on, 35 -68 in Na,K-ATPase, 355-359 structural differences in, 355 -359 Nucleotides, Na,K-ATPase interaction with, 282-297

0 Obesity abnormal Na/K pump in, 973 -975 Na,K-ATPase activity decrease in, 969 -972 Na,K-ATPase content in, 739-742 Na/K pump and, 965-967 %Rbuptake and,970 -97 1 Occluded-ion forms, 625 -637 Occluded potassium ion, 633 -634 Occluded rubidium ions, second route to, 63 I -632 Occluded sodium ions, 634 -637 Occluded state, physiological implicationsof, 447 Occlusion, dissipative fluxes and, 388-390 Octaethylene glycol dodecyl monoether, 74-78 Octyl glucoside, 73 Oligomycineffects, on dephosphorylationof 344 -phosphoenzvmes, -

Oligomycin-treatedNa,K-ATPase, ADP sensitivity of, 569-572 Onsager matrix, 46-47 Ouabain, see also Cardiac glycosides; Cardiotonic steroids; Digitalis aryldiazonium, 272 in anuran membrane preparations, 845 binding to frog bladder, 846 binding to Na,K-ATPase,' 897 calmodulin in Na,K-ATPase inhibition by, 1023 - 1026 contracted force effects of, in mammalian atria and vesicles, 897 -902 in cultured heart cell contractility, 864 dissociation from Na,K-ATPase, 174- 176 in E,P to $P conversion, in cell enzyme, 345 heterogeneity of binding kinetics for, 766 K+-p-nitrophenylphosphataseinhibition by, 934 K+ uptake rate in, 870 in lamb kidney Na,K-ATPase labeling, 133 maximal inotropic action of, 834 Na, Ca content and, 868 negative inotropic effects of, 891 -901 NPT, 272-273 photolabeled, 127 positive inotropic effects of, 899-901,909 pump stimulation by, 833 in rabbit renal outer medulla enzyme inhibition, 848 in rat ventricle, 834 and rubidium uptake in guinea pig atria, 872 in sodium pump stimulation, 873 -874 Ouabain binding in cerebral cortex, 935 factors affecting, 168- 176 intracellular enhancementof, 235 -238 lithium activation curve for, 243 lithium-catalyzed, to canine kidney Na,KATPase, 241 -244 Mg*+ + P i - ~ ~ p p ~ r t173-174 ed, and Na,K-ATPase, 146- 147,247-249 Na+ and Mgz+ ATP-stimulated, 169 Na+ concentration and, 170- 172 piperoxane and, 935 prazosin and, 935 quantitative evaluation of, 891 -894 rate and affinity of, 168 to reconstituted human red cell ghosts, 229-232 typeI, 169-173,182 type 11, 173- 174 I['HlOuabain binding in contracting guinea pig left atria, 893

+

INDEX

1039

[3H] Ouabain binding, continued inhibition of, 847 Ouabain binding site covalent labeling of, 183 - 194 localization in, 186- 187 photoaffinity labeling of, 265 -269 photolysis at, 187 - 188 selectivity in, 185 - 186 specificity of cardiotonic steroid molecule binding in, 184 - 185 Ouabain concentration displacement of, 9 10 Na,K-ATPase activity and, 1025 Ouabain derivatives, to covalently label digitalis binding site, 271 -275 [SHIOuabaindissociation half lives, for Na,KATPase and sarcolemma membrane, 905 Ouabain-EP interaction, inhibition of, 346 Ouabain-induced fluorescence quenching, Mg2+ and Mn Z + in, 461 Ouabain-like activity, absence of in guinea pig brain extract. 927-930 Ouabain-sensitive ATP-ADP exchange, 677 - 68 1 Ouabain-sensitive ATPase activity, in dog kidney outer medulla, 115 Ouabain-sensitive 86Rb uptake, 428 -429 Ouabain-sensitive sodium efflux, plasma stimulationand inhibition of, 1013 - 1016 Ouabain sensitivity dose-response curves for, 278-280 of %Rbuptake, 237 species differences in, 277 -278

P Pancreatic islet Na,K-ATPase, vanadate and somatostatin effects on, 993 -996 Parallel pathways, in phosphoenzyme formation, 5 13 -534 P-E-ouabain complex, formation of, 299 PH ATP and, 329-333 in distribution between K form and Na form, 326 and rate of conformational transition, 328 -330 Phosphate activity, inhibition by sodium, 12 Phosphate binding sites, 494 -495 Phosphate substrate sites, 497 Phosphatidylserine, loss of in lens, 963 Phosphoenzyme decay inorganic phosphate release and, 5 16 patterns of, 523

Phosphoenzyme decomposition, simulation of, 520-525 Phosphoenzyme formation ADP and EDTA in, 520 parallel pathways in, 5 13 -534 simulation of, 520-525 time course of, 520-525 Phosphoenzymes conformational transition between ADPsensitive and potassium-sensitive, 477 -479 dephosphorylation by ADP, 518-520 dephosphorylation by ATP and EDTA, 515-518 K+ and ADP sensitivities in, 351 oligomycin effects on dephosphorylation of, 344 protein conformations of, 393 -394 E,-Phosphoenzymes, dephosphorylation of, 305 - 306 Phosphoenzyme sensitivity sequence, of Na,KATPase to ADP andK+, 557-559 Phosphointermediates, of Na,K-ATPase, 573-576 Phospholipid composition, of a-and &subunits, 405 Phosphorus-containing species, quantitative analysis of. 947-948 Phosphorylation acid-stable, 1002 alternative pathways of, 353 -354 kinetic models of Na-dependent, 553 -555 Mg role in, 302 -303 Na and K roles in, 303 -307 of Na,K-ATPase, 300-307 nucleotide, 4 10-4 12 Phosphorylation-dephosphorylation sequence, for sodium ion, 575 [32P]Phosphoserine, isolation from Na,K-ATPase, 1001 Photoaffinity labeling. of crude pig kidney microsomes by [3H]NAB-ouabain, 191 - 192 Pig kidney microsomes, photoaffinity labeling of, 191 - 192 Pig kidney Na,K-ATPase radiation inactivation of, 140 sodium ion discharge from, 565 - 567 ,Pig kidney outer medulla, [48V]vanadatebinding to, 221 Ping-pong mechanism, in reaction sequences, 500-501 Plasma effect, on human red blood cell active transport, 1013- 1016 PLP, see Pyridoxal5-phosphate

INDEX

1040

PNPPase activity, K+ dependence in, 473 Polarization, fluorescence, 157- 161 Polyacrylamide gel electrophoresis, of Na,KATPase, 91 -95 Positive inotropic action, see also Inotropic action of ouabain, 909 two-site hypothesis for, 907-910 Post-Albers reaction, see Albers-Post reaction Potassium, binding to Na,K-ATPase, 223 Potassium activation sites, in p-nitrophenylphosphate, 581 -585 Potassium binding and dissociation, in coupled vectorial process, 8-9 Potassium chloride, [3Hlouabaindisplacement activity of, 929 Potassium flux and ATP-free or ATP-activated Na,K-ATPase in liposomes, 639-642 ouabain inhibition of, 279 Potassium ion displacement by Na+, 207 fluorescencequenching due to, 458 interaction with Na,K-ATPase, 561 -563 kinetic analysis of effects on Na,K-ATPase, 591 -594 kinetic properties of cross-linked enzyme in presence of, 474 inlens transparency, 959 Na,K-ATPase in presence of, 323 occlusion of, 626-630,633 -634 in phosphorylation process, 303 - 307 Potassium ion binding sites, 496-497 Potassium ion depletion, Na,K-ATPase regulation and, 723-729 Potassium ion independent active transport, of sodium by Na,K-ATPase, 659 -662 Potassium ion transport, Na/K pumps and, 803 -807, see also Potassium transport Potassium-potassium exchanges, 539 - 542 biochemical correlates of, 614-616 kinetic parameters for, 541 through Na/K pump, 444 Potassium transport, see also Potassium ion transport aspartate gate model of, 3 19 ATP-driven kineticsof, 317-319 PROPHET computer graphics, in cardiac steroid receptor site study, 257 -263 Propranolol, in rubidium active transport, 873 Protein conformations, of phosphoenzymes, 393 - 394 Protein weight ratio, in a-and @-subunits,408 Protomers, association of, 60

Pump kinetics, 602 -603, see also Sodium pump Pump-mediated fluxes, cardiotonic steroid inhibition and, 600 Pump protein, occlusion and dissipative fluxes through, 388-390 Pump stoichiometry, 600-601 Purified Na,K-ATPase, insulin binding to, 977 -982, see also Na,K-ATPase Purkinje fibers, sodium pump inhibition and, 885 - 889 P,water exchange, in reaction sequence, 502 -503 Pyridoxal 5-phosphate ATPase activity following preincubation with, 334 modification of, 333 -338 pH effect and, 338

R Rabbit cortical tubule, ATPase activities in, 991 Rabbit kidney Na,K-ATPase, labeling of, 274 Rabbit kidney outer medulla lipid and free fatty analysis in Na,K-ATPase of, 164- 165 Na,K-ATPase from, 404 Radiation inactivation, of Na-ATPase and KpNNPase activities, 139- 143 Rat atria and vesicles. ouabain contractile effects in, 897 -902 Rat brain Na,K-ATPase modulation of adenosine 3 ' ,5 '-monophosphate, 1005-1009 Na-dependent phosphorylation of, 553 -555 Rat brain synaptosomal membranes, NaCl or CAMPeffect on, 1007 - 1008 Rat kidney tubular cells, sodium movement across vesicles from, 703 -705 Rat Na,K-ATPase, strophanthidin inhibition of, 768 Rat nephron segments, Na,K-ATPase activity in, 813-816 Rat submandibular gland, cholinergic stimulation of sodium pump in, 985-987 Reaction mechanisms, 599-617 Albers-Post, 486-490,523,528, 602 dimeric, 491 Reaction sequence ADPlATP and Na+/Na+exchanges, 501 -502 Cleland diagram of, 489 conformational forms in, 497 -498 pathways in, 499-500 ping-pong sequences in, 500-501 P,water and K+/K+exchanges in, 502-504

1041

INDEX Reconstituted human red cell ghosts, sidedependent ion effects on ouabain binding to, 229-232 Red blood cell ghosts Na,K-ATPase of. 677-681 resealed, 247 -249,677 -681 Red blood cell membrane Na,K-ATPase. relation between band 3 and, 481 -483 Red blood cells abnormal Na/K pump and, 973 -975 anion-coupled Na efflux mediated by Na/K pump in, 693 -695 component arrangement in, 483 ion transport in, 602 magnesium dependence of sodium-pumpmediated Naf transport in, 653 -657 sodium-pumpcatalyzed ATP-ADP exchange in, 671 -674 sodium pump inhibition by cytoplasmic calcium in, 1017- 1020 ouabain-sensitive sodium efflux in, 1013- 1016 trypsin digestion effect on kinetic behavior of Na/Kpumpin, 697-701 Renal Na,K-ATPase, see also Na,K-ATPase subunit distribution of sulfhydryl groups and disulfide bonds in, 153 - 155 thyroid hormone in, 813 Resealed red blood cell ghosts Na-ATPase of, 677-681 ouabain binding and Na,K-ATPase in, 247 - 249 a-L-Rhamnose, 262 Rough endoplasmic reticulum, Na,K-ATPase synthesis in, 715 Rubidium. binding to Na,K-ATPase, 224 Rubidium active transport, isoproterenol and propranolol effects on, 873 Rubidium fluxes in absence of ATP or of P,, 428 -436 in presence of ATPor P,, 436-446 steady-state solution for, 445 Rubidium ion occlusion of, 626-2633 release from Na,K-ATPase, 629 retention by Na,K-ATPase, 628 Rubidium ion/ADP binding, to potassium-like form of NaKATPase, 223 -227 Rubidium ion concentration, Rb binding and, 208 Rubidium movements, in vesicles reconstituted with Na,K-ATPase, 425-447 Rubidium-rubidium exchange, 428 ATP concentration and, 442 computer simulations of, 446

phosphate concentration and, 44 1 vanadate,-sensitive, 43 1-432 Rubidium-86, equilibration of countertransport of into Rb-free or Rb-loaded vesicles, 433 Rubidium-86 uptake obesity and, 970-971 ouabain-sensitive, 428-429,872,891-894 in submandibular gland slices, 987

S Sarcolemma dissociation half-lives from, 903 -904 Scatchard analysis of, 905 -906 Sarcolemma membranes, digitalis binding to, 903 -906 Scatchard plots of binding sites per enzyme molecule, 296 of eosin binding to Na,K-ATPase, 454 of Rb binding without ATP, 225 of solubilized Na,K-ATPase and sarcolemma, 905 -906 SDS-gel electrophoresis, of Na,K-ATPase, 136 Sheep, large and small reticulocytes in, 803 -806 Sheep Purkinje fibers, sodium pump inhibition or contraction in, 885 -889 Sodium,, interaction with potassium,, 23 1 -232, see also Na abbreviations Sodium, potassium ion-transport adenosine triphosphatase, see Na,K-ATPase Sodium activation sites, identity of in ATPase with K activation sites inp-nitrophenylphosphate, 581 -585 Sodium concentration ouabain binding and, 170- 172 and type I ouabain-Na,K-ATPase complex dissociation, 176 Sodium-dependent phosphorylation, of rat brain Na,K-ATPase. 553 -555 Sodium dodecyl sulfate, Na,K-ATPase and, 70 Sodium efflux, anion-coupled, 693 -695 Sodium enzyme, 593 -594, see also Na,KATPase Sodium flux, and ATP-free or ATP-activated Na,K-ATPase in liposomes, 639 -642 Sodium ion(s) interaction with Na,K-ATPase, 561 -563 interactions with sodium pump, 649-651 K+-independentactive transport of by Na,KATPase, 659-662 kinetic analysis of effects on Na,K-ATPase, 591 -594 in lens transparency, 959

1042

Sodium ion(s), continued occlusion of, 634-637 in phosphorylation process, 303 -307 Sodium ion binding sites, 495 Sodium ion concentration, for half-maximum activation of hydrolysis at different ATP concentrations, 33 1 Sodium ion discharge, from pig kidney Na,KATPase, 565-567 Sodium ion flux, in terminal differentation of murine erythroleukemia cells, 797 - 801 Sodium ion loading, Na,K-ATPase regulation and, 723 -729 Sodium movement, in basolateral plasma membrane vesicles, 703 - 705 Sodium-potassium cotransport system in essential hypertension, 953 -956 kinetic analysis of, 954-956 Sodium-potassium enzyme, 594, see also Na,KATPase Sodium-potassium exchanges internal adenine nucleotides in, 683 -685 sodium-sodium exchange and, 612-613 Sodium-potassium pump, see also Sodium pump abnormality of in erythrocytes from obese subjects, 973-975 anion-coupled sodium efflux mediated by, 693 -695 cardiac glycoside stimulation of, 913-915 dietary influence on, 966-967 humoral inhibitor of, 923 -924 in inside-out vesicles using ATP synthesized at membrane, 687-691 obesity and, 965-967,973-975 passive K+ transport and, 803 -807 trypsin digestion vs. kinetic behavior of, 697 - 701 Sodium pump, see also Sodium-potassium pump ATP interactions with, 643-646 in barnacle muscle, 707 -710 carbachol activation and, 986 cholinergic stimulation of, in rat submandibular gland, 985 -987 consecutive model of, 547-549 electrogenic. 707-710 Na+ or K + ion trapping in, 625 in Na+ transport through cellular membranes, 36 1 orientation of in vesicles, 430 ouabain binding and, 171 reaction mechanism evaluation by steady-state kinetics, 537-544

INDEX

reserve capacity of, 237 sidedness of sodium interactions with, 649-651 sodium-sodium exchange catalyzed by, 677 vanadate effect on, 939-942 Sodium pump-ATP interactions, sidedness of, 643-646 Sodium pump-catalyzed ATP-ADP exchange, in red blood cells, 671 -674 Sodium pump inhibition in contraction of sheep Purkinje fibers, 885 - 889 by cytoplasmic calcium in intact RBC, 1017- 1020 intracellular acidification and, 889 and positive inotropic effect of heart muscle, 861 -862 Sodium pump-mediated sodium transport, magnesium dependence of, 653 -657 Sodium pump stimulation, ouabain in, 873-874 Sodium reabsorption, in mammalian nephron, 813 Sodium/sodiumexchange, 539 -542 ADP-ATPexchange and, 609-610 characteristics of, 606-607 internal adenine nucleotides in, 683 -685 phloretin-sensitive, 95 1 sodium-potassium exchange and, 612-613 Sodium transport aspartate gate model of, 3 19 ATP-drivenkinetics of, 317-319 ATP hydrolysis and, 361 disorders in molecular assemblies for, 951 -952 magnesium dependence of, 653 -657 Somatostatin, and pancreatic islet Na,K-ATPase, 993 -996 Squalus acanthias, 73,349 Squid giant axons ADP-ATP exchange in, 655 -658 internal dialysis technique for, 643, 665-668 Steady-state kinetics, reaction mechanism evaluation by, 537-544 Stereoelectronicinteraction, between Na,KATPase and cardiotonic steroids, 251 -255 Strophanthidin inhibition, of rat Na,K-ATPases, 768 Strophanthidin-sensitiveion-independent ATPase activity, 775-177 Substrate concentration, dephosphorylation and, 517 Substrates and products, order of addition and release of. 542 -544

INDEX

1043

Substrate sites, high- and low-affinity types, 492 Tryptic digestion Substrate specificity, in coupled vectorial process, divalent cation effects on. 596 10- 13 and kinetic behavior of Na/K pump in red blood cells, 697 -701 Subunit stoichiometry,406-409, see also asubunit; @-subunit of Na,K-ATPase, 90 Sulfhydryl groups, subunit distribution of, in Tryptophan, Na,K-ATPase cleavage at, 100 Type I enzyme-ouabain complexes, 174, 182 renal Na,K-ATPase, 153- 155 Type I1 cardiac glycoside binding, structureactivity results for, 181 T T,, see Triiodothyronine T4, see I-Thyroxine Thimerosal cation activation of Na,K-ATPase after treatment with, 465 -468 modification with, 387 Thyroid hormones, in Na,K-ATPase activity or regulation, 730-734, 813 !-Thyroxine, in Na,K-ATPase activity, 815 Transitions, conformational, see Conformational transitions Transmembrane segments, organization of, 127-130 Transport systems, membrane, 55 -57 Trichloroaceticacid-resistantbinding, to Na,KATPase, 136- 138 Triiodothyronine, Na,K-ATPase biosynthesis and, 809-81 1 Triparanol, cataract formation and, 959 -962 Triton, Na,K-ATPaseand, 72 -73 Trypsin, ouabain-sensitiveK influx and, 700

V Vanadate ATPase inhibition by, 773 electrophorousNa,K-ATPase inhibition by, 528 - 530 flux inhibition by, 389 inhibitory and stimulatory effects on sodium pump in heart cells, 939-942 in insulin binding to Na,K-ATPase, 982 as Na,K-ATPase inhibitor, 939 Na,K-ATPaseinteraction with, 298 - 300 and pancreatic islet Na,K-ATPase, 993 -996 VanadateIATP accessibility, half-of-sites reactivity examined by, 219-222 Vanadate,-sensitive &Rb/Rbexchange, 43 1-434 ATP effect on, 437 Vanadate,-sensitive Rb uptake, phosphate effects on, 439 Van't Hoff's law, 25.34

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