Biothiols, Part B: Glutathione and Thioredoxin: Thiols in Signal Transduction and Gene Regulation, Volume 252 (Methods in Enzymology)

Preface Biothiols participate in numerous cellular functions, such as biosynthetic pathways, detoxification by conjugation, and cell division. Studies on oxidative stress in recent years have amply documented the key role of thiols-more specifcally the thiol-disulfide status of the cell--in a wide array of biochemical and biological responses. Awareness of the great importance of biothiols in cellular oxidative injury has grown along with the recognition of free radicals in biological processes. The reactions of thiols with free radicals are not only of interest in free radical chemistry: the most abundant nonprotein thiol in the cell, glutathione, is essential for the detoxification of peroxides as cofactors of various selenium-dependent peroxidases. The high concentration of glutathione in cells clearly indicates its general importance in metabolic and oxidative detoxification processes. In many ways, glutathione may be considered the master antioxidant molecule, a phrase which the late Alton Meister, one of the pioneers in glutathione research and a contributor to this volume, used. Bolstering of glutathione by other thiols, both natural (such as a-lipoic acid) and synthetic (such as Ebselen and several other drugs), has been investigated as a therapeutic approach to the oxidative component of various pathologies. Moreover, the redox changes of several thiol-containing proteins may be involved in key regulatory steps of the enzyme as well as in cell proliferation. The contributions to Volumes 251 and 252 of Methods in Enzymology (Biothiols, Parts A and B) provide a comprehensive and detailed account of the methodology relating to the molecular mechanisms underlying the multiple functions of biothiols, with emphasis on their interaction at the biochemical and molecular biological levels in cellular reactions, with oxidants and other biological and clinical implications of thiols. The contributions in this volume (Part B) include methods on glutathione: distribution, biosynthesis, metabolism, and transport; signal transduction and gene expression; thioredoxin and glutaredoxin; and synthetic mimics of biological thiols and thiols inhibitors. In Part A (Volume 251) methods are included relating to thiyl radicals; chemical basis of thiol/disulfide measurements: monothiols: measurement in organs, cells, organelles, and body fluids; dithiols: oMipoic acid; and protein thiols and sulfides. Credit must be given to the experts in various specialized areas selected to provide state-of-the-art methodology. The topics and methods included in these volumes were chosen on the excellent advice of the volume advisors, Bob B. Buchanan, Enrique Cadenas, Carlos Gitler, Arne Holmgren, Alton Meister, and Helmut Sies, to whom I extend my thanks and most grateful appreciation. LESTER PACKER

xiii

C o n t r i b u t o r s to V o l u m e 2 5 2 Article numbers are in parentheses following the names of contributors. Affiliations listed are current,

MARIE-ODILECHRISTEN(32, 33, 34), Laboratoire de ThOrapeutiqueModerne, SOL VA Y Pharma, 95151 Suresnes Cedex, France

MONIQUE ADOLPHE (34), Laboratoire de Pharmacologie Cellulaire, Ecole Pratique des Hautes Etudes, 75006 Paris, France MICHAEL T. ANDERSON(17), Stanford Medical School, Beckman Center, Stanford, California 94305 FREDRIK ~SLUND (29), Medical Nobel Institute for Biochemistry, MBB, Karolinska Institutet, S-171 77 Stockholm, Sweden K. D. AUMANN(5), Gesellschaftfar Biotechnologische Forschung (GBF), Braunsehweig, Germany E L A I N E S. G . B A R D E S (16), Department of Molecular Cancer Biology, Duke University Medical Center, Durham, North Carolina 27710 ROBERTM. BELL (16), Department of Molecular Cancer Biology, Duke University Medical Center, Durham, North Carolina27710 ERNESTBEUTLER(8), Department of Molecular and Experimental Medicine, Scripps Research Institute, La Jolla, California 92037 MIKAEL BJORNSTEDT(21, 22), Department of Medical Biochemistry and Biophysics, The Medical Nobel Institute for Biochemistry, Karolinska lnstitutet, S-171 77 Stockholm, Sweden RoY A. BORCHARDT(16), Department of Molecular Cancer Biology, Duke University Medical Center, Durham, North Carolina 27710 R. BRIGELIUS-FLOHE(5), Deutsches Institut far Erniihrungsforschung, Postdam-Rehbriicke, Germany BoB B. BUCHANAN(24), Department of Plant Biology, University of California, Berkeley, Berkeley, California 94720 ZHEN-HAI CHEN (14), Department of Pharmacology and Nutrition, School of Medicine, University of Southern California, Los Angeles, California 90033

GEORGE L. DALE (8), Department of Medicine, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73190 PAULETTE DECO'Iq'IGNIES (25), Physiologie V~gOtale Mol~culaire, Universit~ de ParisSud, 91405 Orsay-Cedex, France WULF DROGE (26), Division of Immunochemistry, Deutsches Krebsforschungszentrum, D-69120 Heidelberg 1, Germany H. JANE DYSON (30), Department of Molecular Biology, The Scripps Research Institute, La Jolla, California 92037 L. FACKIR (33), Laboratoire de Chimie-Physique, Universitd R~ne Descartes, 75270 Paris Cedex 06, France L. FLOHE (5), Gesellschafi far Biotechnologische Forschung (GBF), Braunschweig, Germany

Department of Molecular Pharmacologyand Toxicology, University of Southern California, Los Angeles, California 90033 JAMESA. FUCHS(9), Department of Biochemistry, University of_Minnesota, St. Paul, Minnesota 55108 RICHARD S. GLASS (31), Department of" Chemistry, University of Arizona, Tucson, Arizona 85721 RAYUDU GOPALAKRISHNA(14), Department of Pharmacology and Nutrition, School of Medicine, University of Southern California, Los Angeles, California 90033 USHA GUNDIMEDA(14), Department of Pharmacology and Nutrition, School of Medicine, University of Southern California, Los Angeles, California 90033 HENRY JAY FORMAN (7),

ix

X

CONTRIBUTORS TO VOLUME 252

LEONARD A. HERZENBERO (17), Stanford Medical School, Beckman Center, Stanford, California 94305 TOD P. HOLLER (12), Parke-Davis Pharmaceutical, Division of Warner-Lambert Company, Ann Arbor, Michigan 48105 ARNE HOLMGREN (21, 22, 29), Department of Medical Biochemistry and Biophysics, The Medical Nobel Institute for Biochemistry, Karolinska lnstitutet, S-171 77 Stockholm, Sweden PAUL B. HOPKINS (12), Department of Chemistry, University of Washington, Seattle, Washington 98195 TOSHIYUKIHORI (36), Department of Biological Responses, Institute for Virus Research, Kyoto University, Kyoto, Japan STEVAN R. HUBBARD (13), Department of Biochemistry and Molecular Biophysics, Columbia University, New York, New York 10032 RONALD S. HUTCHISON (23), Department of Plant Biology, The Ohio State University, Columbus, Ohio 43210 EMMAMUELLE ISSAKIDIS (25), Physiologie V~g~tale Mol~culaire, Universitd de ParisSud, 91405 Orsay-Cedex, France ROLE D. ISSELS(11), Medizinische Klinik lII, Universitat MEinchen, D-81366 Munich, Germany; and Institut far Klinische Hi~matologie, GSF MEinchen, D-81377 Munich, Germany TAKEO IWAMOTO (7), Department of Biochemistry, Kansas State University, Manhattan, Kansas 66506 SATOSHI IWATA (36), Department of Biological Responses, Institute for Virus Research, Kyoto University, Kyoto, Japan JEAN-PIERRE JACQUOT (25), Physiologie V~g~tale Mol~culaire, Universit~ de Paris-Sud, 91405 Orsay-Cedex, France DEAN P. JONES (1), Department of Biochemistry, Emory University School of Medicine, Atlanta, Georgia 30322 DANIEL JORE (33), Laboratoire de ChimiePhysique, Universit~R~ne Descartes, 75270 Paris Cedex 06, France

BRIAN KETTERER (6), Department of Biochemistry, CRC Molecular Toxicology Research Group, University College and Middlesex School of Medicine, London W1P 6DB, United Kingdom KAROLY KOBREHEL (24), Laboratoire de Technologie des Cdrdales, INRA, 34060 Montpellier Cedex 01, France TAKAHITO KONDO (8), Department of Pathological Biochemistry, Atomic Disease Institute, Nagasaki Univeristy School of Medicine, Nagasaki, Japan SUSHIL KUMAR (22), Department of Medical Biochemistry and Biophysics, The Medical Nobel Institute for Biochemistry, Karolinska Institutet, S-171 77 Stockholm, Sweden LAWRENCE H. LASH (2), Department of Pharmacology, Wayne State University School of Medicine, Detroit, Michigan 48201 MARTINE LEMAIRE (25), Physiologie V~g~tale Mol~culaire, Universit~de Paris-Sud, 91405 Orsay-Cedex, France RuI-MING LIu (7), Departments of Molecular Pharmacology, Toxicology, Pathology, and Pediatrics, University of Southern California, Los Angeles, California 90033 TE-CHUNG LIU (20), Department of Nutrition, Case Western Reserve University, Cleveland, Ohio 44106 M. MAIORINO (5), Department of Biological Chemistry, University of Padua, Padua, Italy THOMAS MEIER (11), Institute far Klinische Hamatologie, GSF Mtinchen, D-81377 Munich, Germany ALTON MEISTER* (3), Department of Biochemistry, Cornell University Medical College, New York, New York 10021 DAVID J. MEYER (6), Department of Biochemistry, CRC Molecular Toxicology Research Group, University College and Middlesex School of Medicine, London WIP 6DB, United Kingdom MYROSLAWAMIGINIAC-MASLOW (25), Physiologie V~g~tale Mol~culaire, Universit~ de Paris-Sud, 91405 Orsay-Cedex, France *Deceased

CONTRIBUTORS TO VOLUME 2 5 2

xi

MASASHI MIZUNO (19), Department of Molec-

HEIKE SCHENK (26), Division of Immuno-

ular and Cell Biology, Universityof California, Berkeley, Berkeley, California 94720 DONALD R. ORT (23), Photosynthesis Research Unit, USDA/ARS, Department of Plant Biology, University of Illinois, Urbana, Illinois 61801

chemistry, Deutsches Krebsforschungszentrum, D-69120 Heidelberg, Germany D. SCHOMBUR6 (5), Gesellschaftfar Biotechnologische Forschung (GBF), Braunschweig, Germany KLAUS SCHULZE-OSTHOFF (26), Division of Immunochemistry, Deutsches Krebsforschungszentrum, D-69120 Heidelberg, Germany PETER SCHURMANN (28), Laboratoire de Biochimie Vegetale, Universit~ de Neuchdtel, CH-2000 Neuch~tel, Switzerland MICHAEL MING SHI (7), Departments of Molecular Pharmacology, Toxicology, Pathology, and Pediatrics, University of Southern California, Los Angeles, California 90033 HELMUT SIES (35), Heinrich-Heine-Universitilt, Instimt far Physiologische Chemie 1. D-40001 Diisseldorf Germany FRANK J. T. STAAL (17), Department of Immunology, Netherlands Cancer Institute, 1066 CX, Amsterdam, Netherlands' THRESSA C. STADTMAN (31), Laboratory of Biochemistry, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892 YUICHIRO J. SUZUKI (18), Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, California94720 MARC THEVENIN (34), Laboratoire de Toxicologie, Facult( de Pharmacie, 75270 Paris Cedex 06, France F. URSINI (5), Department of Chemistry, University of Udine, 1-33100 Udine, Italy

LESTER PACKER (18, 19), Department of Mo-

lecular and Cell Biology, University of California, Berkeley, Berkeley, California 94720 MULCHAND V. PATEL (20), Department of

Biochemistry, State University of New York at Buffalo, Buffalo, New York 14214 ANDREW F. G. QUEST (16), Department of Molecular Cancer Biology, Duke University Medical Center, Durham, North Carolina 27710 PATR1CE RAT (34), Laboratoire de Pharma-

cologie Cellulaire, Ecole Pratique des Hautes Etudes, 75006 Paris, France DONALD J. REED (10), Department of Bio-

chemistry and Biophysics, Environmental Health Sciences Center, Oregon State University, Corvallis, Oregon 97331 T1MOTHY W. ROBINSON (7), Departments of Molecular Pharmacology, Toxicology, Pathology, and Pediatrics, Universityof Southern California, Los Angeles, California 90033 JOAQUfN ROMA (15), Department of Physiol-

ogy, Experimental Toxicology and Neurotoxicology Unit, School of Medicine, University of Valencia, 46010 Valencia, Spain FRANCISCO J. ROMERO (15), Department of

Physiology, Experimental Toxicology and Neurotoxicity Unit, School of Medicine, University of Valencia, 46010 Valencia, Spain A. ROVERI (5), Department of Biological

Chemistry, University of Padua, Padua, Italy MARJORIE RUSSEL (27), Laboratory of Genet-

ics, The Rockefeller University, New York, New York 10021 NORIHITO SATO (36), Department of Biologi-

cal Responses, Institute for Virus Research, Kyoto University, Kyoto, Japan

NATARAJ N. VETTAKKORUMAKANKAV (20),

Department of Biochemistry, State University of New York at Buffalo, Buffalo, New York 14214 JEAN-MICHEL WARNET (34), Laboratoire de Toxicologie, Facult~ de Pharmacie, 75270 Paris Cedex 06, France MICHAEL P. WASHBURN (4), Department of Biochemistry, Michigan State University, East Lansing, Michigan 48824 WILLIAM W. WELLS (4), Department of Biochemistry, Michigan State University, East Lansing, Michigan 48824

xii

C O N T R I B U T O R S TO V O L U M E

252

ELIZABETHWILLOTT(16), Department of Mo-

DIAN PENG XU (4), Department of Biochemis-

lecular Cancer Biology, Duke University Medical Center, Durham, North Carolina 27710 JOSHUAH. WONG (24), Department of Plant Biology, University of California, Berkeley, Berkeley, California 94720 WEN QIN XIE (16), Department of Molecular Cancer Biology, Duke University Medical Center, Durham, North Carolina 27710

try, Michigan State University, East Lansing, Michigan 48824 AKIRA YAMAUCHI(36), Department of Bio-

logical Responses, Institute for Virus Research, Kyoto University, Kyoto, Japan JUNJI YODOI (36), Department of Biological

Responses, Institute for Virus Research, Kyoto University, Kyoto 606-01, Japan

[ 1]

GLUTATHIONE

DISTRIBUTION

IN NATURAL

PRODUCTS

3

[I] G l u t a t h i o n e D i s t r i b u t i o n i n N a t u r a l P r o d u c t s : Absorption and Tissue Distribution

By DEAN P. JONES Introduction G l u t a t h i o n e ( G S H ) is p r e s e n t in m o s t p l a n t a n d a n i m a l tissues f r o m w h i c h t h e h u m a n d i e t is d e r i v e d . M u c h o f this G S H is b i o a v a i l a b l e b e c a u s e cells o f t h e g a s t r o i n t e s t i n a l t r a c t h a v e N a + - d e p e n d e n t a n d N a + - i n d e p e n d e n t u p t a k e m e c h a n i s m s , 1-3 a n d n e t a b s o r p t i o n o f G S H f r o m t h e l u m e n into t h e v a s c u l a r c i r c u l a t i o n o c c u r s in t h e s m a l l intestine. 4 In a d d i t i o n , G S H can f u n c t i o n as an a n t i o x i d a n t in t h e ingesta, c a n m a i n t a i n a s c o r b a t e in a red u c e d a n d f u n c t i o n a l f o r m , 5 c a n d i r e c t l y r e a c t with a n d i n a c t i v a t e toxic e l e c t r o p h i l e s in t h e diet, 6 a n d c a n b e b r o k e n d o w n to y i e l d cysteine. 7 S t u d i e s have shown that dietary GSH enhances metabolic clearance of dietary p e r o x i d i z e d lipids a n d d e c r e a s e s t h e i r n e t a b s o r p t i o n 8'9 a n d t h a t c o n s u m p tion o f f o o d s high in G S H c o n t e n t is a s s o c i a t e d w i t h a b o u t a 50% r e d u c tion in risk o f o r a l a n d p h a r y n g e a l cancer, w T h e r e f o r e , a n i m p r o v e d u n d e r s t a n d i n g o f t h e d i s t r i b u t i o n o f G S H in f o o d s a n d t h e f a c t o r s affecting G S H a b s o r p t i o n a n d d i s t r i b u t i o n c a n b e e x p e c t e d to p r o v i d e t h e basis for i m p r o v e d f o o d s e l e c t i o n a n d p r e p a r a t i o n to r e d u c e risk o f c h r o n i c diseases. 1M. K. Hunjan and D. F. Evered, Biochim. Biophys. Acta 815, 184 (1985). 2 L. J. Dahm, P. S. Samiec, J. W. Eley, E. W. Flagg, R. J. Coates, and D. P. Jones, in "Free Radicals: From Basic Science to Medicine" (G. Poli, E. Albano, and M. U. Dianzani, eds.), p. 506. Birkhaeuser, Basel, 1993. 3 M. T. Vincenzini, F. Favilli, and T. Iantomasi, Biochim Biophys, Acta 1113, 13 (1992). 4 T. M. Hagen and D. P. Jones, Am. J. Physiol. 252, G607 (1987). 5 B. S. Winkler, Biochim. Biophys. Acta 925, 258 (1987). P. S. Samiec, L. J. Dahm, E. W. Flagg, R. J. Coates, J. W. Eley, and D. P. Jones, in "Free Radicals and Antioxidants in Nutrition" (F. Corongiu, S. Banni, M. A. Dessi, and C. RiceEvans, eds.), p. 269. Richelieu Press, London, 1993. 7 N. Tateishi and Y. Sakamoto, in "Glutathione: Transport and Turnover in Mammals" (Y. Sakamoto, T. Higashi, and N. Tateishi, eds.), p. 13. Japan Scientific Societies Press, Tokyo, 1983. 8 D. P. Kowalski, R. M. Feeley, and D. P. Jones, J. Nutr. 120, 1115 (1990). 9 T. Y. Aw and M. W. Williams, Am. J. Physiol. 263, G665 (1992). m E. W. Flagg, R. J. Coates, D. P. Jones, T. E. Byers, R. S. Greenberg, G. Gridley, J. K. McLaughlin, W. J. Blot, M. Haber, S. Preston-Martin, J. B. Schoenberg, D. F. Austin, and J. F. Fraumeni, Am. J. Epidemiol. 139, 453 (1994).

METHODS IN ENZYMOLOGY, VOL. 252

Copyright © 1995 by Academic Press, Inc. All rights of reproduction in any form reserved.

4

GLUTATHIONE

[11

Glutathione Contents of Foods General Considerations

Glutathione is readily oxidized by food processing and preparation, and many foods contain reactive electrophilic compounds that can remove GSH even after food is ingested. 6 Thus, analytically precise measures of GSH in foods only provide an index of GSH delivered to gastrointestinal epithelia. In addition, oxidized forms of GSH, such as oxidized glutathione (GSSG), are also present in foods and are at least partially available as GSH because the intestine contains a GSSG reductase mechanism, tl Consequently, direct measures of GSH probably underestimate GSH availability; whereas, measures of total GSH ( G S H t ) following reduction of food extracts with dithiothreitol probably overestimates GSH that is bioavailable. Many factors affect food contents of nutrients, and established food analysis protocols, such as those of the Association of Official Analytical Chemists, 12 should be used whenever possible. As water content is highly variable in stored foods, dry weight or total nitrogen contents should be determined for all samples. Conditions of growth, seasonal and geographic conditions, plant variety, duration and conditions of storage and processing, and preparation are all expected to affect GSH content of foods, but no systematic studies of the effects of these factors on GSH content are available. The methods that we use to assay GSH contents of foods were developed to provide a database for epidemiological studies of dietary intake of GSH and risk of diseases in humans. 13 We based our food items on the National Cancer Institute's Health Habits and History Questionnaire ( H H H Q ) , 14'15 which includes 98 food items and groups that account for over 90% of calories, fiber, and 18 major nutrients in diets in the United S t a t e s . 14,16 For each individual food item or group, a specific food and method of preparation is selected based on those most commonly reported in the second National Health and Nutrition Survey, a population-based survey conducted in the United States between 1976 and 1980. 11 T. M. Hagen, G. T. Wierzbicka, B. B. Bowman, T. Y. Aw, and D. P. Jones, Am. Z PhysioL 259, G530 (1990). 12 S. Williams (ed.), "Official Methods of Analysis." (1141 pages). Association of Official Analytical Chemists, Arlington, Virginia, 1984. 13 D. P. Jones, R. J. Coates, E. W. Flagg, J. W. Eley, G. Block, R. S. Greenberg, E. W. Gunter, and B. Jackson, Nutr. Cancer 17, 57 (1992). 14 G. Block, C. M. Dresser, A. M. Hartman, and M. D. Carroll, Am. J. Epidemiol. 122,13 (1985). 15 G. Block, A. M. Hartman, C. M. Dresser, M. D. Carroll, J. Gannon, and L. Gardner, Am. J. Epidemiol. 124, 453 (1986). 16 G. Block, C. M. Dresser, A. M. Hartman, and M. D. Carroll, Am. J. EpidemioL 122, 27 (1985).

[ 1]

GLUTATHIONE DISTRIBUTION IN NATURAL PRODUCTS

5

Standard Protocol

For each food item, at least three different brand name products or three different samples (different sources) are purchased from major chain supermarkets, cafeterias, or restaurants. When samples are stored prior to assay, this is done under usual storage conditions, that is, in a refrigerator for perishable foods and in a freezer for frozen foods. Prior to sampling, foods are prepared according to common habits in the United States. Nonedible portions are removed, and foods that are normally cooked are prepared accordingly. In general, steamed or boiled foods are cooked in a small amount of water until tender and then drained. Pan-fried foods, such as pork chops, are fried at medium heat for 5 min or until done (loss of pink or red color in meats). Because GSH can be rapidly lost on homogenization, we developed a standard protocol that provides a correction for GSH loss based on simultaneously assayed samples with known amounts of GSH added. Five 1-g portions are taken from one sample of each food item and treated as follows. One portion is placed in an aluminum tray and dried for at least 24 hr at 105° to obtain a dry weight. The other four portions are added to beakers containing 10 ml of ice-cold homogenization solution A, B, C, or D. Solution A is 0.9% (w/v) NaC1 containing 5 mM EDTA. Solutions B, C, and D are made from solution A by adding 10 ~M GSH, 20/zM GSH, or 5 mM dithiothreitol, respectively. Foods that are solutions or homogenates are mixed on a vortex mixer prior to removal of 1-ml aliquots for addition to test tubes containing 0.5 ml of ice-cold 30% (w/v) trichloroacetic acid. Emulsions (e.g., milk) are extracted 17 to remove lipids before acidification. For other foods, portions are homogenized using a Polytron for a minimum duration to obtain a homogeneous suspension, and 1-ml of aliquots are immediately transferred to ice-cold 30% trichloroacetic acid. Following centrifugation to remove insoluble material, 0.5-ml aliquots are transferred to microcentrifuge tubes containing 0.1 ml of 40 mM iodoacetic acid. 18Each solution is adjusted to pH 8 by addition of i M KHCO3 in I M KOH and incubated for 1 hr at room temperature. One-half milliliter of 1-fluoro-2,4-dinitrobenzene (1.5% in ethanol; v/v) is then added, and samples are incubated overnight at room temperatures in the dark. a8 Samples are then stored at 0o-4 ° for up to 2 weeks prior to assay by highperformance liquid chromatography (HPLC). The HPLC procedure is essentially that described by Reed et al. ~8 using 17 j. Folch, M. Lees, and G. H. Sloane Stanley, J. BioL Chem. 226, 497 (1957). 18 D. J. Reed, J. R. Babson, P. W. Beatty, A. E. Brodie, W. W. Ellis, and D. W. Potter, Anal. Bioehem. 106, 55 (1980).

6

GLUTATHIONE

[11

100

80

60

$ 20

I

0

i

I

10

I

I

20

I

i

30

I

40

i

I

50

Time (rain) FIG. 1. Analysis of GSH in potato by HPLC. The sample (1 g) was homogenized in 10 ml of solution A and derivatized as described. Twenty-five microliters were injected on the HPLC system. The peak at 40.0 min is the S-carboxymethyl-N-dinitrophenyl derivative of GSH. The GSSG derivative is the peak at 44.3 min.

an Ultrasil NH2 column from Beckman (Norcross, GA) or an aminopropyl column from Custom LC (Houston, TX). Initial conditions are 1 ml/min flow with 80% solvent A (80% methanol) plus 20%o solvent B (5 M sodium acetate, pH 4.6, in 80% methanol). Initial conditions are maintained for 10 min followed by a linear gradient to 95% B over 40 min and reequilibration with initial conditions for 10 min prior to analysis of the next sample. Derivatives are detected at 365 nm and quantified relative to standards by integration (Fig. 1). Integral values for portions taken in solutions A, B, and C are plotted as a function of added GSH content (0, 10, and 20/zM), and the negative of the x axis intercept is taken as the GSH concentration (Fig. 2). Values are multiplied by 11 to convert back to sample concentrations and appropriately recalculated to be expressed in terms of millimolar or as mg/g dry weight. Values are converted to milligrams per usual portion size by use of the H H H Q medium portion sizes from the H H H Q database. Total GSH values are estimated from the same graphical representation as the negative of the x axis intercept of a line through the point (0,D) with the same slope as the line through the data from portions A - C (see Fig. 2).

[ 1]

GLUTATHIONE DISTRIBUTION IN NATURAL PRODUCTS

7

600

•~.

~

.=

~2oo

400

IO,BJ _/J

-10

0

10

20

G S H Added (~tNO

F1~. 2. Graphical analysis of the GSH content of bell pepper from multiple samples with a method of additions. Integral values from replicate samples homogenized in solutions A, B, and C were graphed as a function of the respective amounts of GSH in each solution, namely, 0, 10, and 20 ~M. These are identified as points (0,A), (10,B), and (20,C) (filled circles). Extrapolation of the linear least-squares best-fit line back to the x axis provides an estimate of the negative of the GSH content. For most foods, the slope of this line approximated that of the GSH contents of solutions A-C without added food sample (open circles). For some foods, GSH-reactive materials result in less than complete recovery of added GSH (filled squares). Extrapolation of the linear least-squares best-fit line for these points thus allows a correction for incomplete recovery. For estimation of GSHt, the integral value for the sample taken in solution D is plotted as (0,D), and a line is drawn through this point with a slope equivalent to the best fit for the corresponding solutions A-C. The negative of the x intercept is taken as the GSHt concentration. All values are corrected for dilution prior to expression per wet weight, dry weight, or total nitrogen.

Glutathione Content in Food Groups A l t h o u g h k n o w l e d g e of G S H c o n t e n t s of foods r e m a i n s i n c o m p l e t e , studies have p r o v i d e d insight i n t o the r a n g e of values a n d p r o v i d e s o m e useful g e n e r a l i z a t i o n s . O f p a r t i c u l a r i m p o r t a n c e , c o n s i d e r a b l e v a r i a t i o n in G S H c o n t e n t occurs in different foods, a n d e s t i m a t e d daily i n t a k e s of G S H vary c o n s i d e r a b l y b e t w e e n individuals. T h e G S H c o n t e n t s of r e p r e s e n t a t i v e foods are g i v e n in T a b l e I. D a i r y products, eggs, fats, a n d oils, m o s t b e v e r a g e s , a n d m o s t cereal g r a i n p r o d u c t s have very little G S H . I n contrast, fresh fruits a n d v e g e t a b l e s a n d freshly c o o k e d m e a t s are high in G S H . F r o z e n foods g e n e r a l l y have G S H c o n t e n t s c o m p a r a b l e to fresh foods, b u t o t h e r f o r m s of p r e s e r v a t i o n u s u a l l y result

8

GLUTATHIONE

[1]

TABLE I REPRESENTATIVE TOTAL GLUTATHIONE CONTENTS IN COMMONLY CONSUMED FOODS a

Food Vegetables Asparagus (fresh, cooked) Carrots (raw) Carrots (fresh, cooked) Carrots (canned, heated) Potatoes (fresh, cooked) Fruits Oranges (fresh) O r a n g e juice (frozen, reconstituted)

Apples (fresh) Apple juice (bottled) Meats Chicken (fried) Ham (boiled) Beef (ground, fried) Bacon (fried) Cereal, grains, and legumes Pinto beans (canned, heated) Peanut butter Bread, whole wheat Rice Dairy products Milk (whole)

Cheese, American

GSHt (mg/100 g)

(rag/medium portion)

28.3 - 5.4 7.9 -4- 1.5 5.8 - 1.5 0.0 13.6 - 2.5

26.3 5.4 4.3 0.0 12.7

7.3 - 0.8 4.2 _+ 0.1 3.3 _+ 0.5 0.0

10.6 7.9 4.6 0.0

13.1 23.3 17.5 5.0 0.6 2.4 1.2 1.6

-+ 2.8 -+ 6.5 _+ 0.8 -+ 0.7 +-+ -

0.0 0.0

0.1 0.5 0.4 0.1

GSHt

13.4 13.0 14.9 0.8 0.9 0.4 0.6 2.1 0.0 0.0

a Values of GSHt represent

total GSH recovered after reduction by dithiothreitol, expressed as mean _+ S E M for 100 g wet weight.

in substantial or complete loss of GSH. 13 Normal cooking results in variable loss, typically with different consequences for plant and animal products. Plant products lose GSH to forms that are not converted back to GSH with dithiothreitol, whereas animal products have forms that are reducible back to GSH. In addition to the loss with cooking, it is noteworthy that some foods contain substantial amounts of GSH-reactive materials. This is revealed by analyzing the percentage of added GSH that is recovered in the method of additions described above (see Fig. 2, dashed line). 6 Ground beef has a substantial loss of added GSH, whereas beef steak does not. This suggests that disruption of tissue by grinding allows lipid peroxidation to occur and may contribute to an increased load of reactive electrophiles and oxidants in the diet. Additional studies are needed to establish the identity of the

[ 1]

GLUTATHIONE DISTRIBUTIONIN NATURALPRODUCTS

9

reactive species and whether they contribute to diet-related disease processes.

D i s p o s i t i o n of Orally S u p p l i e d R e d u c e d a n d Oxidized G l u t a t h i o n e U p t a k e of G S H by epithelial cells of the gastrointestinal tract can be studied in freshly isolated cells, 19 cultured cells, 2° and in intact preparations where the mucosa is sampled by scraping, a Loss of G S H from the lumen can also be directly measured as a function of time or location along gastrointestinal tract, u However, m e a s u r e m e n t of absorption, which occurs along with degradation and resynthesis, is technically m o r e difficult and must be addressed in terms of the b r o a d e r context of thiol-disulfide balance which includes cysteine and cystine. Cysteine and cystine are also present in the diet and are actively transported in the small intestine. In addition, cysteine is critical for iron absorption. 21 and is transported from the epithelium into the lumen to regulate thiol-disulfide balance. 2t Glutathione and related products are also released into the intestinal lumen in bile. Thus, a variety of intact biological preparations, including cannulated bile duct, t3 lymph fistula, a4 and in v i v o blood sampling, 25 are needed to assess fully the disposition of orally supplied G S H and GSSG. A m e t h o d that we have found to be particularly useful for short-term (up to 1 hr) intestinal absorption studies involves direct m e a s u r e m e n t of lumen to vasculature transit of G S H in an in situ, vascularly perfused small intestinal preparation. 4,26 For this preparation, we use male, SpragueDawley rats (VAF/1) (Sasco, O m a h a , NE) weighing 200-320 g. They are maintained under controlled conditions of t e m p e r a t u r e and humidity on a 12 hr light/dark cycle. Rats are typically given food and water a d l i b i t u m , and experiments are p e r f o r m e d during the late morning or early afternoon when rats are not feeding. Rats are anesthetized with ketamine/xylazine, and heparin is injected into the left femoral vein. The jejunum is isolated and cleared of its contents by flushing with 40 ml of 37 ° saline (0.9%, w/v NaC1). The bile duct, celiac artery, renal arteries, and aorta (proximal to the celiac artery) are ligated 19L. H. Lash, T. M. Hagen, and D. P. Jones, Proc. Natl. Acad. Sci. U.S.A. 83, 4641 (1986). 2oK. Sundqvist, Y. Liu, K. Arvidson, K. Ormstad, L. Nilsson, R. Toftgard, and R. C. Grafstrom. In Vitro Cell Dev. Biol. 27A, 562 (1991). 21M. Layrisse, C. Martinez-Torres, I. Leets, P. Taylor, and J. Ramirez, J. Nutr. 114,217 (1984). 22L. J. Dahm and D. P. Jones, Am. J. Physiol. 267, G292 (1994). 23D. Eberle, R. Clarke, and N. Kaplowitz, J. Biol. Chem. 256, 2215 (1981). 24T. Y. Aw, M. W. Williams, and L. Gray, Am. J. Physiol. 262~G99 (1992). 25B. H. Lauterburg, J. A. Adams, and J. R. Mitchell, Hepatology (Baltimore) 4, 586 (1984). 26T. M. Hagen, C. Bai, and D. P. Jones, FASEB J. 5, 2721 (1991).

10

GLUTATHIONE

[ 11

and the rat is killed by cutting through the diaphragm and vena cava. The aorta is then cannulated distal to the renal arteries, and retrograde perfusion is begun with Krebs-Henseleit buffer (pH 7.4, 37 °) containing 12.5 mM HEPES and equilibrated with 95% 02 and 5% CO2 (v/v). This allows perfusion of the small intestine by flow through the superior mesenteric artery. Perfusate is collected by cannulating the portal vein. The flow rate is usually set at 1 ml/min because this optimizes the ability to measure low transport rates. Under these conditions, the 02 consumption rate is about 60% of maximal, but there is no loss of viability (as measured by lactate dehydrogenase release) or loss of vascular integrity (as measured by polyethylene glycol permeability). Body temperature is maintained by a heated operating tray or a heat lamp. After preperfusion for 5 min to clear the vasculature of blood, the jejunum is instilled with Krebs-Henseleit medium (in mM: 120 NaC1, 4.8 KCI, 2.6 CaCI2, 1.2 MgSO4, i KH2PO4, 2.4 NaHCO3) and other additions as appropriate. In a typical experiment measuring GSH uptake (Fig. 3), 4,26,27 [35S]GSH is placed in the lumen, and samples are taken from the portal vein every minute. Aliquots are derivatized and analyzed by HPLC as described above. Use of this method allows direct measures of the rate of GSH transport as well as the rate of GSH degradation and cysteine absorption. 4 Substitution of choline chloride for NaCI in the Krebs-Henseleit medium allows study of Na+-independent transport. 4 Transport of polyethylene glycol from the lumen to the vasculature provides a measure of loss of vascular integrity. The contribution of GSH synthesis can be virtually eliminated by inclusion of 1 mM buthionine sulfoximine (BSO) in the luminal and perfusate solution. 4 Inhibition of GSH degradation poses more of a technical problem in that 250 /xM acivicin, a concentration that gives over 99% inhibition of y-glutamyltransferase in mucosal homogenates, only gives about 70% inhibition in the in situ vascularly perfused intestine. Inhibition can be increased to more than 95% by increasing the acivicin concentration to 1 mM, but this has the risk of inhibiting GSH transport and other processes because acivicin becomes a nonspecific alkylating reagent at high concentrations. An alternative means to inhibit y-glutamyltransferase is to use serineborate, which gives greater than 99% inhibition with 20 mM each of serine and borate at pH 7.5. When [glycine-3H]GSH is used for tracer studies, we have found that inclusion of 5 or 10 mM glycine in the solutions effectively eliminates only the contribution of GSH synthesis to the measured vascular [3H]GSH. 27 T. M. Hagen, G. T. Wierzbicka, A. H. Sillau, B. B. Bowman, and D. P. Jones, Am. J. Physiol. 259, G524 (1990).

[ 1]

GLUTATHIONE DISTRIBUTION IN NATURAL PRODUCTS

11

;9 0

10 Time (min)

Fie. 3. Uptake of GSH in vascularly perfused small intestine of the rat. The 35S-labeled GSH was placed in the lumen at zero time, and the portal vein effluate was collected and counted by scintillation counting (filled circles). In these experiments, performed in the laboratory by C. Bai, the intestine was pretreated with acivicin to inhibit GSH degradation. Under such conditions, greater than 90% of the total radiolabel is GSH as analyzed by HPLC. Without acivicin treatment, 30% or more of the radiolabel is recovered as cysteine or cystine (4). A comparable concentration of [14C]polyethylene glycol shows little radiolabel appearance in the perfusate (open circles). Additional details are given in Refs. 4, 26, and 27.

Hormonal regulation of GSH transport 26 and intestinal thiol-disulfide regulation22 have been successfully studied using the in situ vascularly perfused intestinal preparation. Additional studies are needed to determine whether GSH absorption or thiol-disulfide regulation are affected by the status of other antioxidants or by exposure to oxidants or reactive electrophiles in the diet. Modulation of glutathione availability and absorption can potentially provide an effective means to reduce morbidity from intestinal distress during chemo- and radiotherapies. Systemic Availability and Tissue Distribution of Absorbed Glutathione Dietary GSH is typically only a small fraction of the total sulfur amino acids in the diet. In addition, the amount of GSH consumed in a day is only a small fraction of the total in the body. Thus, it appears unlikely that a measurable effect of dietary GSH on systemic GSH would be detectable. However, blood plasma concentrations are typically low, and increases in blood plasma concentrations have been detected following oral loads in

12

[ 1]

GLUTATHIONE Control

{Z~ +GSH

6' A

E -to9 O

2,

Liver

Kidney

3.

Heart

Lung

Brain

intestine

Skin

Iml BSO control

•

E

2'

+Amino adds

i--1 +GSH

o

•

0

,

Liver

,

Kidney Heart

,

Lung

Brain Intestine Skin

FIG. 4. Effect of oral supplementation with GSH on tissue GSH concentrations in mice. Top: Mice were given tap water which did not contain BSO but were otherwise treated identically to the mice used for studies shown in the bottom graph. Controls received 0.3 ml saline by gavage i hr prior to sacrifice and tissue sampling. The GSH-treated animals received 100 mg/kg body weight in the saline gavage. Bottom: Mice were treated with BSO in the drinking water for 5 days prior to gavage with either 0.3 ml saline, saline with GSH (100 mg/ kg body weight), or saline with an equivalent amount of constitutive amino acids. Asterisks represent significant differences between GSH-treated and respective controls at p < 0.05.

rats, 4'27 mice, 28 and humans. 29 Although increases in blood plasma G S H are transient following oral consumption, such increases may be important in detoxication of reactive species in the blood 3° or in protection of cell surfaces from oxidative damage. 31 A particularly useful approach to study systemic availability of G S H is to use animals treated with the G S H synthesis inhibitor, BSO. 28 The com28 T. Y. Aw, G. Wierzbicka, and D. P. Jones, Chem.-Biol. Interact. 8 0 , 89 (1991). 29 T. M. Hagen and D. P. Jones, in "Glutathione Centenial: Molecular and Clinical Implications" (Y. Sakamoto, T. Higashi, N. Taniguchi, and A. Meister, eds.), p. 423. Academic Press, San Diego, 1989. 30 C. D. Thomson, M. F. Robinson, J. A. Butler, and P. D. Whanger, Br. Z Nutr. 69, 577 (1993). 31 p. C. Davidson, P. Sternberg, D. P. Jones, and R. L. Reed, Invest. Ophthamol. Vis. Sci. 35, 2843 (1994).

[ 1]

GLUTATHIONE DISTRIBUTION IN NATURAL PRODUCTS

13

pound is an irreversible inhibitor of 7-glutamylcysteine synthetase (glutamate-cysteine ligase), the rate-limiting enzyme in GSH synthesis.3eWith this treatment, mice can be maintained for several days and have substantially decreased tissue GSH concentrations. Substantial ultrastructural effects have been noted, 33,34 but there is no overt pathology. We use outbred albino ICR mice (30-40 g, Sasco) that are maintained on mouse food and water ad libitum. 28 After a 1-week preconditioning period to allow animals to adjust to the animal care facility, 20 mM BSO is included in the drinking water. From the volumes consumed, this amounts to about 80 tzmol BSO per mouse per day. After 5 days food is removed, and, on the sixth day, mice are given an oral load of GSH or amino acids in 0.3 ml of 0.9% (w/v) NaCI by gavage. Control animals receive saline only. One hour after gavage, animals are sacrificed, and 10 ml of ice-cold saline is perfused through the aorta to wash blood from the tissues. Tissue samples are immediately taken, weighed, and placed in ice-cold 30% trichloroacetic acid. After centrifugation to remove protein, aliquots are derivatized and analyzed for GSH content by HPLC as described above. Results from the experiments show that tissue concentrations of GSH can be increased by orally supplied GSH even though GSH synthesis is inhibited, and an equivalent amount of constituent amino acids does not increase tissue GSH concentrations (Fig. 4). Comments Numerous studies have established the importance of GSH in protection against a variety of toxicological and pathological processes. Glutathione is a compound that occurs naturally in the diet and can be readily supplemented or administered therapeutically. Although there are several exciting reports on the possibility that nutritional variations in GSH consumption and oral administrations of GSH can have beneficial effects against specific disease processes, relatively few scientific studies of this nature are available. The described methods provide approaches to more detailed investigation of the nutritional and physiological control of GSH consumption and utilization and should provide an important resource for investigations of the role of dietary GSH in human health.

32 O. W. Griffith and A. Meister, J. Biol. Chem. 254, 7558 (1979). 33 j. Martensson, A. Jain, W. Frayer, and A. Meister, Proc. Natl. Acad. Sci. U.S.A. 87, 5296 (1990). 34j. Martensson, A. Jain, and A. Meister, Proc. Natl. Acad. Sci. U.S.A. 87, 1715 (1990).

14

GLUTATHIONE

[2]

[2] I n t r a c e l l u l a r D i s t r i b u t i o n o f T h i o l s a n d D i s u l f i d e s : Assay of Mitochondrial Glutathione Transport

By LAWRENCEH. LASH Introduction Metabolism of glutathione (GSH) in mammalian cells exhibits many unique characteristics: GSH is synthesized by two ATP-dependent enzymes, rather than by the normal protein synthetic mechanism, and is degraded by a specific enzyme, 7-glutamyltransfcrase (EC 2.3.2.2), which is found selectively on the luminal plasma membranes of epithelial cells, including those of the renal proximal tubule and the small intestinal epithelium. Besides synthesis and degradation, another component of the turnover of cellular GSH is transport across plasma membranes and intracellular membranes. Nutritional and biochemical studies on GSH turnover have shown that the GSH pool within many mammalian cell types, such as liver 1'2 and renal proximal tubule, 3 is divided into two distinct subpools, one comprising the majority of total intracellular GSH (70-85% of total) that turns over relatively rapidly (h/2 of 2 hr) and one that comprises a smaller portion of total intracellular GSH (15-30% of total) that turns over relatively slowly (tl/2 of 30 hr). Investigations employing digitonin fractionation and marker enzymes showed that the larger pool of intracellular GSH is cytosolic in origin, whereas the smaller, slowly turning over pool is predominantly mitochondrial in origin. Much less is known about the properties and regulation of the mitochondrial pool of intracellular GSH as compared with the cytosolic pool because the mitochondrial pool comprises a relatively minor fraction of total cellular GSH and because the cytosolic pool is more easily accessible to analysis and manipulation. However, numerous studies have provided evidence that the mitochondrial pool of GSH is critical for the maintenance of both mitochondrial and cellular function and that several types of chemically induced injury are associated with depletion or oxidation of the mitochondrial, rather than the cytosolic, pool of G S H . 4-9 1 M. J. Meredith and D. J. Reed, J. BioL Chem. 257~ 3747 (1982). 2 F. J. R o m e r o and H. Sies, Biochem. Biophys. Res. Commun. 123, 1116 (1984). 3 R. G. Schnellmann, S. M. Gilchrist, and L. J. Mandel, Kidney Int. 34, 229 (1988). 4 M. C. Beatrice, D. L. Stiers, and D. R. Pfeiffer, J. Biol. Chem. 259, 1279 (1984). s K. L~-Qu6c and D. LS-Qu6c, Arch. Biochem. Biophys. 216, 639 (1982). 6 K. LS-Qu6c and D. L~-Qu6c, J. Biol. Chem. 260, 7422 (1985).

METHODS IN ENZYMOLOGY, VOL. 252

Copyright © 1995 by Academic Press, Inc. All rights of reproduction in any form reserved.

[2]

MITOCHONDRIAL GLUTATHIONE TRANSPORT

15

The inability to detect de n o v o synthesis of GSH within hepatic mitochondria 1° suggested that transport of cytosolic GSH as the intact tripeptide across the mitochondrial inner membrane into mitochondrial matrix must occur to provide the organelle with GSH. Because the fraction of total cellular GSH found in mitochondria approximates the fractional volume of mitochondria within cells such as hepatocytes or renal proximal tubular cells, there is probably little if any gradient of GSH across the mitochondrial inner membrane between cytosol and mitochondrial matrix. The subcellular distribution of GSH cannot be due to simple diffusion, however, but must be actively regulated because: (i) the mitochondrial pool of GSH turns over much more slowly than the cytosolic pool; (ii) the GSH molecule has a net negative charge at physiological pH ranges; and (iii) the mitochondrial matrix is negatively charged relative to the cytosol (i.e., there is a membrane potential across the mitochondrial inner membrane) and there is a pH gradient across the mitochondrial inner membrane (i.e., matrix pH is higher than the cytosolic pH). Indeed, Kurosawa et al.ll and Martensson and Meister 12described transport systems for GSH uptake into rat liver mitochondria. The hepatic mitochondrial system is dependent on membrane potential and the presence of a proton gradient. Two transport systems were distinguished based on kinetics, a high-affinity, low-capacity system and a low-affinity, high-capacity system. 12 Kaplowitz and colleagues 13 have found that activity of the hepatic system is altered by chronic ethanol feeding of rats. Schnellmann 14 found evidence for a GSH transport system in renal cortical mitochondria. Lash and colleagues15 determined that this transport process is electroneutral and appears to involve exchange with dicarboxylic acids, such as malate and succinate. This chapter describes methods to quantitate transport of GSH as the intact tripeptide into rat renal mitochondria. Procedures are described to analyze uptake of GSH into mitochondria using both isolated renal cells and isolated renal cortical mitochondria, although the methods are applicable 7 K. L~-Qu6c and D. L~-Qu6c, Arch. Biochem. Biophys. 273, 466 (1989). 8 H. Sies and K. M. Moss, Eur. J. Biochem. 84, 377 (1978). 9 T. Yagi and Y. Hatefi, Biochemistry 23, 2449 (1984). 10 O. W. Griffith and A. Meister, Proc. Natl. Acad. Sci. U.S.A. 82, 4668 (1985). ~t K. Kurosawa, N. Hayashi, N. Sato, T. Karnada, and K. Tagawa, Biochem. Biophys. Res. Commun. 67, 367 (1990). le j. Martensson and A. Meister, Proc. Natl. Acad. Sci. U.S.A. 86, 471 (1989). 13 j. C. Fernandex-Checa, C. Garcia-Ruiz, M. Ookhtens, and N. Kaplowitz, J. Clin. Invest. 87, 397 (1991). 14 R. G. Schneltmann, Life Sci. 49, 393 (1991). 15 T. B. McKernan, E. B. Woods, and L. H. Lash, Arch. Biochem. Biophys. 288, 653 (1991).

16

GLUTATHIONE

[2]

to measurement of transport into mitochondria from other tissues with appropriate, minor modifications.

Biological Preparations

Preparation of Isolated Renal Cortical Mitochondria Although description of biological preparations is not the primary focus of this chapter, a few points will be made that are relevant to the analysis of GSH transport. Suspensions of isolated renal cortical mitochondria that are suitable for GSH transport studies are obtained from renal cortical homogenates by differential centrifugation methods (e.g., see Johnson and Lardy16 and Lash and Sa1117). These procedures employ homogenization in buffers containing a high concentration of an impermeant solute, such as 225 mM sucrose, and potassium phosphate, magnesium chloride, and an organic buffer, such as Tris-HC1 or triethanolamine hydrochloride. Additionally, a divalent cation chelator such as ethylene glycol bis(/3-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) should be included in the preparation of the mitochondria, but not in the final suspension medium. This removes much of the calcium ions that are released from intracellular stores during homogenization and isolation of the mitochondria. Without chelation of the calcium ions, damage to mitochondrial membranes can occur. The buffer composition is important because it enables isolation of mitochondria that maintain a high degree of structural integrity and selective permeability, thereby making the preparation suitable for transport studies.

Analysis of Mitochondrial Uptake in Intact Renal Cells Two methods can be employed to analyze uptake of GSH into mitochondria with intact renal cells as the biological system. In the first method, the mitochondrial content of GSH is determined after incubations with intact cells by fractionation of cells with digitonin. The digitonin fractionation method (see Lash and Sail 17 for methodological details) involves rapid mixing of cells in a microcentrifuge tube with the top layer that contains buffer and 0.15% (w/v) digitonin in a microcentrifuge tube and centrifugation through a silicone oil-mineral oil layer (6: 1; v/v) into a 40% (v/v) 16D. Johnson and H. Lardy,this series, Vol. 10, p. 94. 17L. H. Lash and J. M. Sail, in "MitochondrialDysfunction"(L. H. Lash and D. P. Jones, eds.), Methodsin Toxicology,Vol. 2, AcademicPress, San Diego, p. 8. 1993.

[2]

MITOCHONDRIAL GLUTATHIONE TRANSPORT

17

glycerol layer. This procedure enables rapid separation of cytosolic and mitochondrial compartments with minimal postisolation artifacts. The second method (see Lash and Sall av for methodological details) enables preparation of mitochondria from suspensions of intact cells, thereby providing isolated mitochondria for direct measurement of transport. A distinct advantage of this method is that cell populations derived from specific regions of a tissue, such as a particular nephron segment in mammalian kidney, can be used as the source of mitochondria. This method is the only one available that gives mitochondria from suspensions of intact cells that exhibit a high degree of morphological and functional integrity. Isolated cells are mixed with digitonin and are centrifuged at a low speed. Further steps beyond simple digitonin treatment are necessary to isolate the mitochondria because digitonin permeabilizes the plasma membranes, thereby releasing soluble components of the cytosol, but it does not alter the intracellular cytoskeleton. To separate mitochondria from intracellular cytoskeletal elements, pellets from the digitonin treatment step are mixed with the protease nagarse, and the suspension is filtered through polycarbonate membranes. The nagarse releases intracellular organelles from the cytoskeletal network, and the polycarbonate membranes are inert toward biological materials. Use of the polycarbonate membranes provides a means to recover intracellular organelles. As with the mitochondria isolated from homogenates, mitochondria isolated by the digitonin-nagarse and filtration method are resuspended in a buffer that should optimize the structural integrity of the mitochondrial inner membrane. This buffer contains an impermeant molecule (i.e., sucrose), potassium chloride, and an organic buffer. This method was originally developed for isolated rat hepatocytes, 18 but has also been applied to the isolation of mitochondria from renal proximal tubular and distal tubular cells from rat. 19 Mitochondria isolated from intact cells can be incubated, processed, and analyzed identically to mitochondria obtained from tissue homogenates (see below).

Assessments of Functional Integrity When any biological preparation is employed for membrane transport studies, it is critical that the functional integrity of the model is validated. Use of an in vitro model that is compromised either morphologically or functionally may alter transport behavior so that nonspecific permeability or collapse of ion gradients across membranes interfere with measurement of carrier-mediated transport. 18 C. Bai, C. W. Slife, T. Y. Aw, and D. P. Jones, Anal. Biochem. 179, 114 (1989). 19 L. H. Lash, Pharmacologist 35, 148 (1993).

18

GLUTATHIONE

[21

Numerous methods are available to assess the functional integrity of suspensions of isolated renal mitochondria (see Lash and Salll7). These methods include spectrophotometric monitoring of matrix swelling with polyethylene glycol or leakage of matrix enzymes and assessment of respiratory control by measurement of rates of state 3 and state 4 oxygen consumption in an oxygraph. Determination of respiratory control allows an assessment of how well integrated, energy-linked processes are maintained. For the two methods employing intact, isolated cells, mitochondrial functional integrity can also be assessed by convenient methods. Because the objective with digitonin fractionation is to measure distribution of GSH into the mitochondrial compartment, it is critical that the viability of the isolated cell suspension is maintained and that the fractionation method produces an adequate separation of mitochondrial and cytosolic compartments. Procedures to assess the quality of the separation use marker enzyme activity distributions. 17 For the isolation of mitochondria from cell suspensions by the digitonin-nagarse treatment and filtration method, the functional integrity of the filtrates, which are essentially suspensions of isolated mitochondria, is evaluated by the same assays as are used with the isolated mitochondria prepared from homogenates.

Measurement of Mitochondrial Glutathione Transport General Principles

The accurate measurement of transport of GSH into or from renal mitochondria requires that GSH degradation and oxidation be minimized and that the mitochondrial compartment be efficiently separated from the extramitochondrial compartment. In experiments with intact renal cells, GSH oxidation can occur by nonenzymatic as well as enzymatic mechanisms.2° Enzymatic oxidation can be catalyzed by the copper-dependent thiol oxidase that is present on the basolateral plasma membrane, z° Oxidation can be prevented or minimized by addition of a metal ion chelator, such as bathophenanthroline disulfonate (BPDS) or ethylenediaminetetraacetic acid (EDTA). BPDS is preferred because it is readily water-soluble and EDTA interferes with the high-performance liquid chromatography (HPLC) assay for GSH (see below). Degradation of GSH is almost completely inhibited by preincubation of cells or mitochondrial fractions with the potent, irreversible inhibitor of y-glutamyltransferase acivicin [L(aS,5S)-a-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid], zl In isolated 20L. H. Lash, D. P. Jones, and M. W. Anders, Rev. Biochem. Toxicol. 9, 29 (1988). 21D. J. Reed,W. W. Ellis,and R. A. Meck,Biochem. Biophys. Res. Commun. 94, 1273(1980).

[2]

MITOCHONDRIAL GLUTATHIONE TRANSPORT

19

renal plasma membrane vesicles, 50% inhibition of y-glutamyltransferase activity is obtained by preincubation with an acivicin concentration of 6 /xM, with essentially complete inhibition (>98%) occurring with an acivicin concentration of 0.25 mM. 22 In contrast, use of acivicin concentrations above 1 mM causes nonspecific alkylation of cellular nucleophiles, often producing cytotoxicity. Reagents and Materials: Biological Materials and Processing of Samples Biological Materials Renal cortical mitochondria (3-6 protein/ml) Isolated renal cells (2-5 × 106 cells/ml) General Materials Plastic Erlenmeyer flasks, 25 ml Acivicin, 2.5 mM in saline (10× solution) Plastic microcentrifuge tubes, 1.5 ml Microcentrifuge, 13,000 g maximum Materials for Isolated Mitochondria Dibutyl phthalate (d of 1.046 g/ml) Mitochondrial buffer: 225 mM sucrose, 10 mM potassium phosphate, pH 7.4, 5 mM MgC12, 20 mM KC1, 20 mM triethanolamine hydrochloride, pH 7.4, 0.1 mM phenylmethylsulfonyl fluoride (PMSF), 2 mM EGTA (included in all but final suspension of mitochondria) Materials for Isolated Renal Cells: Digitonin Fractionation Digitonin (for aqueous solutions; Sigma, St. Louis, MO), recrystallized two times from hot, absolute ethanol; digitonin stock solution (prepared fresh) contains 1.5 mg digitonin/ml N-morpholinoethanesulfonic acid (MES) buffer Silicone oil (d of 1.05 kg/liter) (Aldrich, Milwaukee, WI) White light paraffin oil, viscosity 125/135 (Fisher Scientific, Pittsburgh, PA) Krebs-Henseleit buffer (for suspension of isolated cells): 118 mM NaC1, 25 mM NaHCO3, 4.8 mM KC1, 25 mM HEPES, 1.2 mM MgSO4, 2.6 mM CaCI2, 1.0 mM KH2PO4; adjust to pH 7.2 MES buffer: 19.8 mM EDTA, 19.8 mM EGTA, 250 mM D-mannitol, 19.8 mM MES, pH 7.4 2: L. H. Lash and D. P. Jones, J. BioL Chem. 259, 14508 (1984).

20

OLUTATHIONE

[21

Materials for Isolated Renal Cells: Digitonin/Nagarse Treatment and Filtration Buffer A: 70 mM Tris base, 0.25 M sucrose, pH 7.0 Buffer B: 50 mM HEPES, 0.1 M sucrose, 0.1 M KC1, 1 mM EDTA, pH 7.2 Nagarse (bacterial protease, type XXVII from Sigma); stock solution, prepared fresh, contains 50 mg nagarse/ml buffer B MES buffer (see above) Digitonin (see above); stock solution contains 2.0 mg digitonin/ml MES buffer Bovine serum albumin (BSA; fraction V, Sigma); stock solution, stored at 0°, contains 0.2 g BSA/ml deionized water Polycarbonate membranes (25-mm diameter, 2.0-/zm pore size) with Swinnex filter holders (Millipore, Bedford, MA); filters are presoaked in distilled water for 30 min before being placed in the filter holder and are washed with 2 ml of buffer A before sample filtration

Reagents and Materials: Transport Assays Materials for Transport Method 1: Filtration and Enzymatic Assay Mixed cellulose ester membrane filters, 0.45-/xm pore size, 25-mm diameter Borosilicate glass test tubes, 16 x 125 mm Sulfosalicylic acid, 10% (w/v) Spectrophotometer Triethanolamine base, 25% (v/v) GSSG reductase [EC 1.6.4.2, glutathione reductase (NADPH)], 50, U/ml NADPH, 0.3 mM 5,5'-Dithiobis(2-nitrobenzoic acid) (DTNB), 6 mM

Materials for Transport Method 2: Centrifugation and Analysis by Chromatography Perchloric acid, 70% (v/v) 7-Glutamylglutamate, 15 mM, in 0.3% (v/v) perchloric acid; used as an internal standard for HPLC assay of GSH (see below) Bathophenanthroline disulfonic acid (BPDS), 15 mM, in deionized water Iodoacetic acid, 100 mM (prepared fresh daily) KOH (2 M)-KHCO3 (2.4 M) 1-Fluoro-2,4-dinitrobenzene, 1% (v/v), in absolute ethanol (prepared fresh daily)

[2]

MITOCHONDRIAL GLUTATHIONE TRANSPORT

21

Incubation and Sampling Procedures All incubations are performed at 25 ° to 28 ° for two reasons: biochemical processes in mitochondria often occur too rapidly at physiological temperatures (e.g., 37 °) to permit measurement, and the functional stability of mitochondrial preparations often declines rapidly at higher temperatures. Incubations are performed in 25-ml Erlenmeyer flasks made of polypropylene and are conducted in a Dubnoff metabolic shaking incubator (60 cycles/ min; Precision Scientific, Chicago, IL). In incubations of either whole renal cells or isolated renal mitochondria with GSH or oxidized glutathione (GSSG), it is critical to pretreat the biological material with acivicin for 15 min prior to addition of GSH or GSSG. 7-Glutamyltransferase is present at extremely high activities in brush border membranes of renal cells. Although the plasma membrane contamination of renal mitochondria may be fairly low, the extremely high activities of 7-glutamyltransferase indicate that a small contamination (e.g., 5% of total protein content) may produce a significant amount of GSH degradation. Mitochondria or isolated cells (9 volumes) are mixed with 2.5 mM acivicin (1 volume of 10x solution) and incubated for 15 min at 25 ° or 28 °. The GSH or GSSG transport in mitochondria or cells is measured by one of two methods (see below). In both cases, with either mitochondria or cells, incubations are performed identically in 25-ml Erlenmeyer flasks: Five volumes of acivicin-pretreated mitochondria or cells are mixed with 1 volume of 6x uptake solution (GSH dissolved in mitochondrial or KrebsHenseleit buffer). Total incubation volumes are between 2 and 3 ml. Transport Method 1: Filtration and Enzymatic Assay. Aliquots (0.5 ml) of mitochondrial suspensions from cortical homogenates or from isolated cells are rapidly filtered under vacuum on 0.45-/xm pore size, 25-mm diameter filters. After washing filters twice with 2 volumes of ice-cold incubation buffer, filters are placed in 16 x 125 mm borosilicate glass test tubes with 0.6 ml of distilled water for 30 min on a shaking water bath to release intramitochondrial contents retained on the filters. Samples (0.5 ml) are mixed with 10% (v/v) sulfosalicylic acid (0.25 ml) in 1.5-ml plastic microcentrifuge tubes and are centrifuged in a microcentrifuge at 13,000 g for 2 min. Acid-soluble supernatants are neutralized with triethanolamine base (25%, v/v) and are assayed for total GSH by an enzymatic recycling assay using GSSG reductase and Ellman's reagent (i.e., DTNB). ~3 Nonspecific binding of GSH to filters is assessed by addition of a known amount of GSH to filters without mitochondria, and is typically less than 5% of initial GSH content of mitochondrial samples. 15 Nonspecific binding of GSH to mito23 O. W. Griflith, Anal. Biochem. 106, 207 (1980).

22

GLUTATHIONE

121

chondria is assessed by addition of a known amount of GSH to filters after filtration of mitochondria, and is also typically only 5% of the endogenous amount of GSH normally present in renal mitochondrial samples. 15 Recovery of GSH is assessed by addition of a known amount of GSH to mitochondrial samples just prior to filtration, and is greater than 85%.

Transport Method 2: Centrifugation and Analysis by Chromatography. Aliquots (0.5 ml) of mitochondrial suspensions from cortical homogenates or from isolated cells are layered on 0.45 ml of dibutyl phthalate over 0.55 ml of perchloric acid (10%, v/v) containing 1% (w/v) BPDS and 75 nmol 7-glutamylglutamate (internal standard for HPLC method). Tubes are centrifuged at 13,000 g for 1 min. The supernatant (i.e., top layer) can be sampled to analyze extramitochondrial GSH. The oil layer is gently washed twice with incubation buffer. This step is designed to minimize any carryover of extracellular material into the bottom layer. As much as possible of the oil layer is removed, and a 0.5-ml aliquot of the perchloric acid layer is removed with a syringe. The use of dibutyl phthalate, based on the method described by Fariss and Reed, 24 is done to separate extramitochondrial medium from intramitochondrial medium. The acid-soluble material in the bottom layer of the microcentrifuge tube is analyzed for GSH, GSSG, and related metabolites (e.g., cysteine, glutamate) by ion-exchange HPLC. 24 The method involves derivatization of thiols with iodoacetic acid to form the S-carboxymethyl derivative, and derivatization of amino acids, disulfides, and S-carboxymethyl derivatives with 1-fluoro-2,4-dinitrobenzene to form the corresponding N-dinitrophenyl, N,N-bisdinitrophenyl, and S-carboxymethyl-N-dinitrophenyl derivatives, respectively. The derivatives are then separated on an amine column with a mobile phase of 80% (v/v) aqueous methanol and a linear gradient of acetate ions. Recovery of GSH or GSSG, assessed as described above, is greater than 90%. Studies on Uptake of Glutathione by Renal Cortical Mitochondria from Rats In studies with renal cortical mitochondrial suspensions, Lash and colleagues 15 demonstrated the time- and concentration-dependent uptake of intact GSH across the mitochondrial inner membrane by a cartier-mediated mechanism. A major advantage of using the above-mentioned methods for analysis of GSH uptake into mitochondria is that the methods are specific for measurement of the intact tripeptide rather than for potential degradation products of GSH (i.e., constituent amino acids). This is critical, even 24M. J. W. Fariss and D. J. Reed, this series, Vol. 143, p. 101.

[2]

MITOCHONDRIAL GLUTATHIONE TRANSPORT

23

with pretreatment of cells or mitochondria with acivicin, because 7-glutamyltransferase activity is so prominent in renal subcellular fractions. A time course of intramitochondrial GSH content in renal cortical mitochondria incubated with either buffer or 5 mM (Fig. 1) shows that the organelles rapidly accumulate GSH. In the absence of exogenous GSH, intramitochondrial GSH content decreases by one-third from 4.77 to 3.13 nmol/mg protein during 10 min of incubation at 28°. In the presence of 5 mM exogenous GSH, in contrast, intramitochondrial GSH content increases nearly 3-fold to 13.3 nmol/mg protein after 2 min of incubation, and then slowly declines over the next several minutes. The energetics of mitochondrial GSH transport is complex, but the transport mechanism does not appear to be coupled to the membrane potential, as illustrated in Fig. 2. Uncoupling of mitochondria with the protonophore CCCP (carbonyl cyanide m-chlorophenyl hydrazone) or with 2,4-DNP does not significantly affect GSH uptake, suggesting that the negatively charged GSH molecule is either cotransported with a cation or undergoes countertransport (i.e., exchange) with another anion. In con-

15

12

9

6

0

0

I

I

5

tO

Time (min)

FIG. 1. T i m e course of G S H uptake by isolated rat renal cortical mitochondria. Isolated mitochondria from rat kidney cortex ( 3 - 6 m g protein/ml) were incubated with either buffer (filled circles) or 5 m M G S H (open circles) at 28 ° for the indicated times. The G S H content in mitochondria was m e a s u r e d by HPLC. Results are the m e a n s of experiments from seven mitochondrial preparations.

24

GLUTATHIONE

[2]

100

75 o

50

25

0

FI~. 2. Studies on the energetics of GSH uptake by isolated rat renal cortical mitochondria. Isolated mitochondria from rat kidney cortex (3-6 mg protein/ml) were preincubated for 15 min with the indicated compound and were then incubated for 5 min with 5 mM GSH at 28°. The GSH content in mitochondria was measured by HPLC. Results are the means of experiments from three to six mitochondrial preparations. Asterisks signify results significantly different (p < 0.05) from the corresponding control incubation.

trast, ATP, KCN, and calcium chloride significantly inhibit G S H uptake, suggesting an indirect coupling to mitochondrial energetics. Investigation of the interaction of G S H with mitochondrial anions, by looking at effects on the same side of the mitochondrial membrane (cis effects) and on the opposite side of the mitochondrial membrane (trans effects) (Fig. 3), shows that divalent anions, such as succinate and malate, undergo exchange transport with G S H whereas monovalent anions, such as pyruvate, do not interact with G S H transport. The interaction with L-glutamate is more complex, involving both competitive inhibition and trans-stimulation. Comments Although we have applied these methods to the study of G S H transport in renal mitochondria, they should be equally applicable to mitochondria from other tissues. Analysis of G S H transport with three biological models

[2]

25

MITOCHONDRIAL GLUTATHIONE TRANSPORT 200. []

Cis uptake (%)

[]

Trans uptake (%)

150-

o

~

100

eL

50-

W Pyruvate

Glutamate

Malate

Succinate

Flc. 3. Interactions of mitochondrial GSH transport in rat kidney with mono- and dicarboxylates. Isolated mitochondria from rat kidney cortex (3-6 mg protein/ml) were incubated for 5 min with 5 mM GSH and incubated simultaneously (cis) or preincubated for 15 min to preload the mitochondria (trans) with the indicated mono- or dicarboxylic acid (10 mM). The GSH content in mitochondria was measured by HPLC. Results are the means of experiments from three to five mitochondrial preparations. Asterisks signify results significantly different (p < 0.05) from the corresponding control incubation.

was described. The first method involves studies with suspensions of isolated mitochondria from renal cortical homogenates. The second method involves incubation of suspensions of intact renal cells with GSH and isolation of cytosolic and mitochondrial fractions with digitonin, thus enabling analysis of GSH compartmentation into mitochondria. The final method is an extension of the digitonin method, which involves subsequent treatment with the protease nagarse and filtration on polycarbonate membranes to isolate mitochondria. The unique advantage of this biological model is that intact, viable mitochondria can be isolated from purified preparations of specific cell populations. Critical features of the methods involve the efficient separation of extra- and intramitochondrial space and the ability of the analytical methods to quantitate precisely and unambiguously intact GSH rather than degradation products.

26

GLUTATHIONE

[31

Acknowledgments Research from the author's laboratory was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant 2 R01-DK40725. Lawrence H. Lash is a recipient of a Research Career Development Award from the National Institute of Diabetes and Digestive and Kidney Diseases (Grant 1 K04-DK02090).

[31 Glutathione Biosynthesis and Its Inhibition By A L T O N

MEISTER

Introduction Glutathione (GSH) is synthesized in two steps that are catalyzed, respectively, by 7-glutamylcysteine synthetase (glutamate-cysteine ligase) and GSH synthetase1: u-Glutamate + l-cysteine + ATP ,-~ l-T-glutamyl-L-cysteine + ADP + Pi

(1) L-T-Glutamyl-L-cysteine + glycine + ATP ~-- GSH + ADP + Pi

(2) The reaction catalyzed by T-glutamylcysteine synthetase is feedback inhibited by GSH, and thus the enzyme functions normally at less than its maximal rate. Inhibition by GSH is not allosteric, but is competitive with respect to glutamate. Inhibition is associated with binding of the glutamyl moiety of GSH to the glutamate binding site of the enzyme, and there is evidence that the thiol moiety of GSH binds to another site on the enzyme, possibly to the cysteine binding site. 2'3 Earlier studies on the isolation, properties, catalytic mechanisms, and inhibition of the enzyme and GSH synthetase have been reviewed in this seriesJ ~/-Glutamylcysteine synthetase isolated from rat kidney has a molecular weight of about I00,000 and can be dissociated into subunits that have molecular weights of 73,000 and 27,700. The heavy subunit was isolated after dissociation of the enzyme under nondenaturing conditions in the presence of 50 mM dithiothreitol. The heavy subunit exhibits all of the catalytic activity of the isolated enzyme as well as feedback inhibition by GSH. Although the possibility that the light subunit is not an integral part of the enzyme was considered, studies in which both subunits were cloned i G. F. Selig and A. Meister, this series, Vol. 113, p. 379. 2 p. Richman and A. Meister, J. Biol. Chem. 250, 1422 (1975). 3 C.-S. Huang, L.-S. Chang, M. E. Anderson, and A. Meister, J. Biol. Chem. 268, 19675 (1993).

METHODSIN ENZYMOLOGY,VOL.252

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[3]

GLUTATHIONE BIOSYNTHESISAND ITS INHIBITION

27

and sequenced have indicated that both are essential. Thus, the cDNA for the heavy subunit was isolated from rat kidney and found to code for a protein of 637 amino acid residues (Mr 72,614)-4 The cDNA was expressed in Escherichia coli, and the recombinant heavy subunit was purified.3 The latter is readily distinguishable from E. coli ~/-glutamylcysteine synthetase by making use of the fact that the kidney enzyme is markedly inhibited by cystamine. In contrast, the E. coli enzyme is not inhibited by this reagent. 5 The purified recombinant enzyme as well as the purified heavy subunit obtained from the isolated heterodimeric enzyme were found to exhibit much lower affinity for glutamate than the native enzyme.3 In addition, both the recombinant enzyme and the isolated heavy subunit were found to exhibit a much higher sensitivity to GSH inhibition than the holoenzyme. These findings indicate that the heavy subunit alone would not be significantly active under in vivo conditions. The GSH analog ~/-glutamyl-aaminobutyrylglycine (ophthalmic acid) was found to inhibit the isolated enzyme only slightly, but it inhibited much more effectively after treatment of the isolated enzyme with dithiothreitol. In contrast, ophthalmic acid inhibited the recombinant enzyme and the isolated heavy subunit substantially without dithiothreitol treatment. This supports the conclusions that the light subunit has a regulatory function that affects the affinity of the enzyme for glutamate and GSH, and that feedback inhibition by GSH involves reduction of the enzyme as well as competition between GSH and glutamate for the glutamate binding site of the enzyme. The cDNA for the light subunit of rat kidney ~/-glutamylcysteinesynthetase was isolated, sequenced, and expressed in E. coli. 6 The cDNA was found to code for a protein of 274 amino acid residues (Mr 30,548). Coexpression of the heavy and light subunit cDNAs in E. coli or separate expression of the cDNAs followed by mixing of the expressed proteins led to recombinant holoenzyme preparations that exhibit catalytic and GSH feedback inhibitory properties that are virtually identical to those shown by the holoenzyme isolated from rat kidney. These studies establish that the light subunit is an integral part of the enzyme and also that the light and heavy subunits are separately coded for. Rat kidney 3,-glutamylcysteine synthetase thus differs significantly in some of its properties from E. coli "/-glutamylcysteine synthetase. The E. coli enzyme is composed of a single polypeptide chain. Furthermore, as noted above, it is not inhibited by cystamine. Computer-aided comparison of the amino acid sequences of the heavy subunit and the E. coli enzyme showed a low degree of similarity,4 4N. Yan and A. Meister,J. Biol. Chem. 265, 1588 (1990). 5C.-S. Huang, W. Moore, and A. Meister, Proc. Natl. Acad. Sci. U.S.A. 85, 2464 (1988). 6C.-S. Huang, M. E. Anderson, and A. Meister,J. Biol. Chem. 268, 20578 (1993).

28

GLUTATHIONE

[3]

but greater similarity was found between the light subunit of the kidney enzyme and the E. coli enzyme. 6 It is notable that the rat kidney enzyme and the E. coli enzyme have similar apparent Km values, turnover numbers, and specificity; both enzymes are inhibited by G S H and to about the extent. Neither enzyme is significantly inhibited by the G S H analog ophthalmic acid, but both enzymes are inhibited by ophthalmic acid when the enzymes are treated with dithiothreitol.

Inhibition of 3~-Glutamylcysteine S y n t h e t a s e b y B u t h i o n i n e Sulfoximine Buthionine sulfoximine (BSO) the most widely used inhibitor of G S H biosynthesis. The use of BSO for O

II CH3CH2CH2CH2SCH2CH2CHNH2COOH

il

NH Buthionine sulfoximine such inhibition developed from earlier studies on the inhibition of glutamine synthetase (glutamate-ammonia ligase) by methionine sulfoximine. Methionine sulfoximine, previously known as a convulsant agent, was found to bind to the glutamate binding site of glutamine synthetase and to undergo phosphorylation on the sulfoximine nitrogen atom by ATP. 7'8 Phosphorylation of the sulfoximine nitrogen atom is apparently analogous to the formation of ~/-glutamyl phosphate in the normal catalytic reaction. Binding of methionine sulfoximine phosphate to the active site inhibits the enzyme irreversibly. O f the four diastereomers of methionine sulfoximine, only the (2S, Ss) form is phosphorylated on the enzyme. 9'I° It is only this isomer that produces convulsions. Evidence that T-glutamyl phosphate is also an intermediate in the reaction catalyzed by 3~-glutamylcysteine synthetase led to examination of methionine sulfoximine as a possible inhibitor of the latter enzyme; it was found that "y-glutamylcysteine synthetase is irreversibly inhibited by the same isomer of methionine sulfoximine that inhibits gluta7 R. Ronzio and A. Meister, Proc. Natl. Acad. ScL U.S.A. $9, 164 (1968). s For a review, see A. Meister, Chemtracts: Biochem. Mol. Biol. 3, 75 (1992). 9 A. Meister, in "Glutathione Centennial: Molecular Perspectivesand Clinical Implications," (N. Taniguchi, T. Higashi, Y. Sakamoto, and A. Meister, eds.), Chap. 1, p. 3. Academic Press, New York, 1989. 10j. M. Manning, S. Moore, W. B. Rowe, and A. Meister, Biochemistry 8, 2681 (1969).

[3]

GLUTATHIONE BIOSYNTHESIS AND ITS INHIBITION

29

mine synthetase, n Administration of methionine sulfoximine to experimental animals leads to a decrease in the synthesis of glutamine and also that of GSH. Although such inhibition of GSH synthesis is effective, the usefulness of methionine sulfoximine in experimental studies is limited because it produces convulsions and has lethal effects that appear to be related to the inactivation of brain glutamine synthetase. Studies on the mapping of the active site of glutamine synthetase led to the conclusion that the S-methyl group of methionine sulfoximine binds to the ammonia binding site of the enzyme. 8 Sulfoximines with larger S-alkyl moieties were found not to inhibit glutamine synthetase significantly because they are hindered from binding to the ammonia binding site of this enzyme. However, such compounds effectively bind to and inactivate 7-glutamylcysteine synthetase. Thus, BSO does not inhibit glutamine synthetase significantly and thus decreases GSH levels without affecting the synthesis of glutamine (and does not produce convulsions). In addition to BSO, several other S-substituted homocysteine sulfoximine compounds inactivate 7-glutamylcysteine synthetase without affecting glutamine synthetase. These include S-(n-pentyl)homocysteine sulfoximine, which is similar in action to BSO. 12 The n-hexyl and S-(n-heptyl) compounds have been reported to be toxic. Other inhibitory sulfoximines were also found including S-2-methyl-n-butylhomocysteine sulfoximine and the corresponding 3-methyl, 3,3'-dimethyl, 2-ethyl, and cyclohexyl derivatives. 9 As first shown for methionine sulfoximine, only the (2 S, Ss) diastereomers of these compounds inactivate 7-glutamylcysteine synthetase. L-Buthionine (S,R)-sulfoximine is commercially available; it exhibits about 50% of the effectiveness of the pure L-(S)-isomer. BSO is itself not chemically reactive. L-Buthionine (R)-sulfoximine is useful as a control compound for possible detection of effects of BSO that are unrelated to its effect on GSH synthesis. For example, L-buthionine (R)-sulfoximine does not inhibit proliferation of normal human T lymphocytes, whereas L-buthionine (S,R)-sulfoximine d o e s ) 3 Other aspects of inhibition by BSO and its isomers have been discussed. 14,1sBSO is effective when administered to experimental animals orally or parentally. It is not readily transported across the blood-brain barrier, whereas BSO ethyl ester is more effectively 11 p. G. Richman, M. Orlowski, and A. Meister, J. Biol. Chem. 248, 6684 (1973). 12 O. W. Griffith, this series, Vol. 143, p. 286. ~3M. Suthanthiran, M. E. Anderson, V. K. Sharma, and A. Meister, Proc. Natl. Acad. Sci. U.S.A. 87, 7185 (1990). 14 A. Meister, Pharmacol. Ther. 51, 155 (1991). 15 O. W. Griffith, in "Glutathione Centennial: Molecular Perspectives and Clinical Implications" (N. Taniguchi, T. Higashi, Y. Sakamoto, and A. Meister, eds.), Chap. 20, p. 285. Academic Press, New York, 1989.

30

6LUTATHIONE

[41

transported. 16 Inhibition of GSH synthesis by administration of BSO is currently under study for treatment of drug- and radiation-resistant tumors (see Meister14). Several other compounds that inhibit y-glutamylcysteine synthetase in vitro have been studied including cystamine, L- and D-3-amino-l-chloro-2pentanone, and L-2-amino-4-oxo-5-chloropentanoate (see Seelig and Meisterl), y-Methylene D-glutamate inactivates the enzyme by binding to an active-site thiol. 17 S-Sulfocysteine and S-sulfohomocysteine inhibit the enzyme by serving as transition-state analogs. 18 16 R. Steinherz, J. M~rtensson, D. Wellner, and A. Meister, Brain Res. 518, 115 (1990). 17 R. P. Simondsen and A. Meister, J. Biol. Chem. 261, 17134 (1986). 18 W. Moore, H. L. Wiener, and A. Meister, J. Biol. Chem. 262, 16771 (1987).

[4] G l u t a t h i o n e By

: Dehydroascorbate

WILLIAM W. WELLS, DIAN PENG Xu,

Oxidoreductases

and MICHAELP.

WASHBURN

Introduction The glutathione:dehydroascorbate oxidoreductases (EC 1.8.5.1) catalyze the following reaction: 2GSH + D H A ~ GSSG + AAH2 Dehydro-L-ascorbic acid (DHA), the first chemically stable oxidation product of L-ascorbic acid (AAH2), is generated in plant or animal cells from L-ascorbic acid by one- or two-electron oxidation reactions in which semidehydroascorbic acid ( A A H . ) may or may not be a required intermediate. The oxidation may proceed through interaction with superoxide free radical (02 ~) or hydroxyl radical (. OH), generated by H202 and 02 ~ interaction in the presence of Fe 3÷. Superoxide is produced by respiration and by a number of enzymes, for example, xanthine oxidase, aldehyde oxidase, and others. Oxidation of many compounds including hydroquinone, flavins, thiols, catecholamines, dialuric acid, herbicides, paraquat, and C C 1 4 produces 02 ~. In addition, D H A may be produced by a number of dioxygenases that use AAH2 as cofactors such as proline 4-hydroxylase.1 1 K. I. Kivirikko and R. Myllyl~i, this series, Vol. 144, p. 96.

METHODSIN ENZYMOLOGY,VOL.252

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[4]

GLUTATHIONE: DEHYDROASCORBATEOXIDOREDUCTASES

31

Regardless of whether D H A is derived by disproportionation of A A H . 2AAH. ~ DHA + AAH2 or whether A A H 2 reacts with H202 chemically or as a result of ascorbate peroxidase (plant) activity, A A H 2 + H202 -'+ 2 H 2 0 + D H A for normal cellular functions, it is essential to regenerate ascorbic acid from the D H A produced and to maintain a highly reduced A A H 2 / D H A set point. The reduction of D H A by glutathione (GSH), especially at an alkaline pH, is a well-recognized chemical reaction. 2 Szent-Gy6rgyi first demonstrated that the oxidized f o r m of vitamin C, D H A , could be regenerated to ascorbic acid by plant and animal tissue extracts that contained GSH. 3 In 1941, C r o o k characterized plant ascorbate oxidase activity and showed that it could be separated from the G S H - d e p e n d e n t D H A reductase catalyzed reaction. 4 The D H A reductase enzyme has been purified and characterized from spinach leaves. 5 The properties of glutathione : dehydroascorbate oxidoreductase, also known as glutathione dehydrogenase (ascorbate), f r o m spinach leaves was reported in this series by Stahl et al. 6 Following the early observations of Szent-Gy6rgyi, further evidence for animal D H A reductases was obtained in various tissues of the guinea pig by Hughes 7 and in h u m a n leukocytes by Christine et aL 8 During a search for alternative functions for thioltransferase, also known as glutaredoxin, this small protein, with thiol/disulfide transhydrogenase ( E C 1.8.4.1, glutat h i o n e - h o m o c y s t i n e transhydrogenase) and electron carrier properties in the ribonucleotide reductase system, 9 was found to have intrinsic D H A reductase activity. 1° In addition, bovine protein disulfide-isomerase (EC 5.3.4.1), but not m a m m a l i a n thioredoxin nor thioredoxin reductase, had D H A reductase activity as measured by the direct spectrophotometric procedure of Stahl e t al. 11 2 H. Borsook, H. W. Davenport, C. E. P. Jeffreys, and R. C. Warner, J. Biol. Chem. 117, 237 (1937). 3 A. Szent-GyOrgyi,Biochem. J. 22, 1387 (1928). 4 E. M. Crook, Biochem. J. 35, 226 (1941). 5 M. A. Hossain and K. Asada, Plant Cell Physiol. 25, 85 (1984). 6 R. L. Stahl, L. F. Liebes, and R. Silber, this series, Vol. 122, p. 10. 7 R. E. Hughes, Nature ( L o n d o n ) 203, 1068 (1964). s L. Christine, G. Thomson, B. Iggo, A. C. Brownie, and C. P. Stewart, Clin. Chim. Acta 1, 557 (1956). 9 A. Holmgren, Proc. Natl. Acad. Sci. U.S.A. 73, 2275 (1976). mW. W. Wells, D. P. Xu, Y. Yang, and P. A. Rocque, J. Biol. Chem. 265, 15361 (1990). it R. L. Stahl, L. F. Liebes, C. M. Farber, and R. Silber, Anal, Biochem. 131, 341 (1983).

32

GLUTATHIONE

I41

Assay Method

Principle A direct 11 and an indirect 12 method of measurement of D H A reductase activity may be chosen depending on the purity of the enzyme preparation. For relatively pure samples of dehydroascorbate reductase of animal cell origin (either thioltransferase or protein disulfide-isomerase), the basic direct spectrophotometric method described by Stahl et al. is preferred. 11 The activity is measured by following the change in absorbance at 265 nm as dehydroascorbic acid is reduced to ascorbic acid in a glutathionedependent manner according to the reaction 2GSH + DHA ~ GSSG + AAH2 In crude preparations of cell extracts, absorbance interference at 265 nm from nucleotides, etc., can be expected. Accordingly, the above reaction can be coupled with regeneration of GSH from oxidized glutathione (GSSG) by measuring the oxidation of NADPH in the presence of excess glutathione disulfide reductase as monitored at 340 nm spectrophotometrically.12

Dehydroascorbate Reductase Assay Reagents 20 mM GSH freshly prepared in Chelex-treated triple-distilled water 20 mM D H A freshly prepared in 10 mM sodium phosphate, 0.5 mM EDTA, pH 5.5 400 mM Sodium phosphate, 2 mM EDTA, pH 6.85 Preparation of Dehydroascorbic Acid. Prepare 20 mM DHA by bromine oxidation of a 20 mM solution of L-ascorbic acid dissolved in 10 mM sodium phosphate, 0.5 mM EDTA, pH 5.5. To 2 ml of the ascorbic acid solution in a graduated glass centrifuge tube, add 10/zl bromine (Aldrich, Milwaukee, WI, 99.99+%), stored over copper wire in a glass bottle sealed with Parafilm. After vortexing at room temperature for 30 sec, the excess bromine is sparged with nitrogen in a ventilation hood until the solution becomes colorless. The nitrogen sparging is continued for 10 min. Chelextreated triple-distilled water is added to adjust the volume to 2.0 ml. Solutions of DHA, prepared according to this procedure, stored on ice, and protected from light, are stable for at least 4 hr. Procedure. The standard reaction mixture contains 200 mM sodium phosphate buffer, pH 6.85, 1.0 mM EDTA, 3.0 mM GSH, and 1.5 mM 12E. I. Anderson and A. Spector, Invest. Ophthalmol.10, 41 (1971).

[41

GLUTATHIONE : DEHYDROASCORBATE OXIDOREDUCTASES

33

DHA with or without enzyme in a total of 500/zl. The increase in absorbance is recorded at 265 nm and 30° after initiating the reaction by the addition of DHA. The reaction, which is linear for up to 1 min is run simultaneously with a cuvette containing all reagents without the enzyme. A similar volume of buffer, used to store the enzyme, is substituted for the enzyme, and the resulting absorbance change is subtracted from that obtained with enzyme present. The millimolar extinction coefficient of ascorbic acid, reported by Daglish, is 14.7.13 Units. A unit of enzyme activity is defined as the amount of enzyme that reduces 1/xmol of L-dehydroascorbic acid in 1 min at 30°. Protein is determined by the bicinchoninic acid protein assay protocol according to the manufacturer's directions (Pierce, Rockford, IL) with bovine serum albumin as standard. Purification Procedures Two mammalian D H A reductases have been identified, m The cytoplasmic reductase, also known as thioltransferase or glutaredoxin, has been purified from numerous sources, including rat liver] 4 pig liver, 15 calf thymus, 16 rabbit bone marrow, 17 human placenta] 8 and human erythrocytes.19 A glutaredoxin homolog encoded by vaccinia virus was reported to be a virion-associated enzyme with thioltransferase and dehydroascorbate reductase activities.2°

Purification of Pig Liver Thioltransferase (Dehydroascorbate Reductase) Liver (2 kg) from 4- to 6-month-old pigs is used as starting material. 15 Except where indicated, all purification procedures are carried out at 4°. The liver is washed of as much blood as possible with buffer containing 10 mM HEPES, pH 7.5, 25 mM KC1, 5 mM MgC12, and 75/~g/ml phenylmethylsulfonyl fluoride (PMSF) and minced into small pieces. The same buffer is used to give a volume of 3200 ml, and the mixture is homogenized in portions with a Tekmar Tissumiser (Tekmar Co., Cincinnati, OH) at 70 V 13 C. Daglish, Biochem. J. 49, 635 (1951). 14 K. Axelsson, S. Ericksson, and B. Mannervik, Biochemistry 17, 2978 (1978). 15 Z.-R. Gan and W. W. Wells, Anal Biochem. 162, 265 (1987). 16 M. Luthman, S. Ericksson, A. Holmgren, and L. Thelander, Proc. Natl. Acad. Sci. U.S.A. 76, 2158 (1979). 17S. Hopper, R. S. Johnson, J. E. Vath, and K. Biemann, J. BioL Chem. 264, 20438 (1989). is K. Larson, V. Ericksson, and B. Mannervik, this series, Vol. 113, p. 520. 19j. j. Mieyal, D. W. Starke, S. A. Gravina, C. Dothey, and J. S. Chung, Biochemisry 30, 6088 (1991). 20 B.-Y. Ahn and B. Moss, Proc. Natl. Acad. Sci. U.S.A. 89, 7060 (1992).

34

GLUTATHIONE

[41

for 3-5 min. The homogenate is passed through two layers of cheesecloth, and a 2.4 M sucrose solution is added to give a final sucrose concentration of 0.24 M. The homogenate is centrifuged at 25,000 g for 40 min. The supernatant is adjusted to pH 5.4 with 5% acetic acid and centrifuged at 20,000 g for 40 min. The resulting supernatant fraction is adjusted to pH 7.0 with 6 N NaOH and is referred to as the high-speed supernatant. Step 1: Heating. The high-speed supernatant is heated by placing in a water bath, maintained at 70 °, with continuous stirring. When the supernatant reaches 62°, the suspension is immediately placed on ice and then centrifuged at 15,000 g for 30 min at 4 °. Step 2: Fractionation with Ammonium Sulfate. Ammonium sulfate is slowly added to the resulting supernatant fraction to give a final concentration of 40% (w/v). After standing on ice for 10 min, the suspension is centrifuged at 15,000 g for 30 min. Ammonium sulfate is added to the supernatant solution to give a final concentration of 65% (w/v), and the resulting suspension is centrifuged at 15,000 g for 30 min after standing on ice for 15 min. Step 3: Sephadex G-75 Gel Filtration. The pellet from the ammonium sulfate precipitation is diluted to a protein concentration of about 150 mg/ ml with water containing 2 mM dithiothreitol and loaded onto a Sephadex G-75 column (5 x 100 cm) which is equilibrated with 10 mM sodium phosphate buffer, pH 6.7. The flow rate is 1.5 ml/min, and the peak fractions are pooled. Thioltransferase activity appears after most of the protein in the Sephadex G-75 column. The salt fraction appears after the thioltransferase peak. Step 4: Sephadex G-50 Gel Filtration. The thioltransferase fractions from the Sephadex G-75 column are pooled (about 450 ml). The soluble proteins are concentrated by 70% ammonium sulfate precipitation. The concentrated proteins are incubated with 5 mM dithiothreitol (40 ml) for 15 min at room temperature, loaded on a Sephadex G-50 column (3 x 110 cm), and equilibrated and eluted with 10 mM sodium phosphate buffer, pH 6.1. Step 5: First CM-Sepharose Chromatography. Thioltransferase fractions (65 ml in 10 mM sodium phosphate buffer, pH 6.1) from the Sephadex G-50 gel filtration are incubated with 5 mM dithiothreitol for 15 min at room temperature, then loaded on a CM-Sepharose column (2.5 × 25 cm) equilibrated with 10 mM sodium phosphate buffer, pH 6.1. The column is first washed with 50 to 100 ml of 10 mM sodium phosphate buffer, pH 6.1, and the enzyme is eluted with 10 mM sodium phosphate buffer, pH 7.0. The thioltransferase fractions (about 30 ml) are pooled and lyophilized. Step 6: Second CM-Sepharose Chromatography. The lyophilized enzyme from the first CM-Sepharose step is incubated in a small volume of 10 mM dithiothreitol for 15 min at 30°. The incubation mixture is loaded on a Sephadex G-25 column equilibrated and eluted with 10 mM sodium phos-

[4]

35

GLUTATHIONE : DEHYDROASCORBATE OXIDOREDUCTASES TABLE I PURIFICATION OF PIG LIVER THIOLTRANSFERASEa

Purification step High-speed supernatant Heat and ammonium sulfate precipitation Sephadex G-75 Sephadex G-50 First CM-Sepharose Second CM-Sepharose

Protein (mg)

Thioltransferase activity (units)

111,750 35,280

10,060 9090

4000 461 81 24

7450 5650 3950 3220

Specific activity (units/rag) 0.09 0.26 1.9 12 49 134

Yield (%)

Purification (-fold)

100 90

1 3

74 56 39 32

21 133 544 1490

a Two kilograms liver from 4- to 6-month-old pigs was used as starting material. Reproduced from Gan and Wells 15 with permission.

phate buffer, pH 6.4. The thioltransferase fractions (1 ml per tube) are directly collected into test tubes which contain 0.1 ml of 20 mM 2-hydroxyethyl disulfide. Thioltransferase fractions are pooled and loaded on a CMSepharose column (1.5 x 28 cm) equilibrated with 10 mM sodium phosphate buffer, pH 6.4. The bound enzyme is first washed with 50 to 100 ml of 10 mM sodium phosphate, pH 6.4, and then eluted by a linear gradient formed by mixing 80 ml of 10 mM sodium phosphate buffer, pH 7.0, with 80 ml of 10 mM sodium phosphate buffer, pH 7.0, containing 0.1 M NaC1. The peak of thioltransferase activity elutes at a concentration of 45 mM NaC1. The thioltransferase fractions from the CM-Sepharose chromatography are lyophilized and reduced by 10 mM dithiothreitol before desalting on a Sephadex G-25 column equilibrated with 20 mM sodium phosphate, pH 7.5, and 20% (v/v) glycerol. The purified enzyme is shown to be homogeneous by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), isoelectric focusing, and high-performance liquid chromatography. The protein has an Mr of 11,740 and, in the reduced form, a pI of 6.4. A summary of the purification is shown in Table I. The recombinant pig liver enzyme has been purified after transformation and expression of the cDNA in Escherichia coli JM105. 21 Thioltransferase purification requires some specific modifications depending on its source. Expression of recombinant D H A reductase results in an 8.6% yield of the soluble protein after three to four simple steps, including heat and ammonium sulfate precipitation, Sephadex G-100 gel filtration, and a final CM-Sepharose ion-exchange chromatography step. 21 For human thioltransferase ( D H A reductase), the procedure of Mieyal et al. is recom2~ y. Yang and W. W. Wells, J. Biol. Chem. 265, 589 (1990).

36

GLUTATHIONE

141

TABLE II KINETIC PARAMETERS FOR MAMMALIAN DEHYDROASCORBATE REDUCTASES a

Parameter kcat (rain-a)d Km(app) (mM) e DHA GSH

Thioltransferaseb

Protein disulfide-isomerase c

374

16

0.26 3.4

2.80 2.9

2.4 × 104 1.8 × 103

1.0 x 102 0.9 × 102

k e a t / g m ( M -1 sec -1)

DHA GSH

The parameters were determined using either constant GSH (3.0 mM) or DHA (1.5 mM). Reactions were run spectrophotometrically in 0.5 ml at 30°, 200 mM sodium phosphate, pH 6.85, 1 mM EDTA, at 265 nm. All data were corrected for nonenzymatic reactions between DHA and GSH. b Recombinant pig liver thioltransferase,z° c Bovine liver protein disulfide-isomerase isolated according to a modification of the procedure of Yang and Wells.el d kcatvalues were calculated by dividing Vmax(app)by the molar concentrations of the two enzymes. e Km(app) values were calculated by nonlinear least-squares fit of the velocity versus substrate concentration data. a

m e n d e d . 19 I m p r o v e d y i e l d s o f h u m a n t h i o l t r a n s f e r a s e a r e o b t a i n e d w h e n t h e b u f f e r s f o r t h e l a t t e r s t e p s a r e s u p p l e m e n t e d w i t h 20% (v/v) glycerol. T h e e n z y m e is s t a b l e for o v e r 1 y e a r if s t o r e d at c o n c e n t r a t i o n s o f 1 0 - 2 0 m g / m l in 20 m M s o d i u m p h o s p h a t e , p H 7.5, a n d 20% (v/v) g l y c e r o l at - 7 0 °. Protein D i s u l f i d e - I s o m e r a s e T h e m i c r o s o m a l D H A r e d u c t a s e , t h a t is, p r o t e i n d i s u l f i d e - i s o m e r a s e , m a y b e p u r i f i e d f r o m b o v i n e liver to n e a r h o m o g e n e i t y b y a m o d i f i c a t i o n o f t h e m e t h o d o f H i l l s o n et aL 22 T h e e n z y m e is f u r t h e r p u r i f i e d b y 85% a m m o n i u m sulfate p r e c i p i t a t i o n f o l l o w e d b y dialysis a g a i n s t a b u f f e r cont a i n i n g 25 m M p o t a s s i u m p h o s p h a t e , p H 8.0, a n d 1 m M E D T A . T h e dialyz a t e is a p p l i e d to a c o l u m n (2.5 × 25 cm) o f D E A E - S e p h a d e x A - 5 0 equilib r a t e d w i t h t h e s a m e buffer. A f t e r w a s h i n g w i t h s e v e r a l b e d v o l u m e s o f t h e e q u i l i b r a t i o n buffer, a l i n e a r g r a d i e n t o f 0 - 0 . 7 M N a C I in t h e s a m e b u f f e r is a p p l i e d w i t h a t o t a l v o l u m e o f 500 ml. z3 T h e activity o f a p r e p a r a 22D. A. Hillson, N. Lambert, and R. B. Freedman, this series, Vol. 107, p. 281. 23 S. Bjelland, B. Foltmann, and K. Wallevik, Anal. Biochem. 142, 463 (1984).

[4]

GLUTATHIONE

CHzOH I

HO-C-H

.,c- 0 -po

H

Cz~C" OfO* + (DHA)+~X.H S-

E" "SH (OH)

: DEHYDROASCORBATE

CHz0H

HO-C-H " O-

37

OXIDOREDUCTASES

ICHz0H

HO-C-H " O

o

C-C W" O S

.c- T_-o

H C=C --

OH OH (AA)

.~,~I +GS-H E,SH

E"S-SG

(OH)

G S H ~ "~#H) ~.~

GSSG

E-S-

\SH (OH)

FIG. 1. Proposed scheme for the DHA reductase mechanism of thioltransferase. Reaction of the nucleophilicCys-22leads to a thiohemiketal intermediate. The reduced product, ascorbic acid (AA), is displaced by glutathione in the active mutant C25S to form a mixed disulfide that is further displaced by GSH to form GSSG and reduced enzyme. Alternatively, an intramolecular disulfide pathway can displace the reduction product, ascorbic acid, followed by exchange with 2 mol of GSH successively. [From W. W. Wells, Y. Yang, T. L. Deits, and Z.-R. Gan, in "Advances in Enzymology and Related Areas of Molecular Biology" (A. Meister, ed.), Volume 66, p. 149. Copyright © 1993 John Wiley & Sons, Inc. Reprinted by permission of John Wiley & Sons, Inc.] tion may be monitored by the insulin reduction assay of Holmgren, 24 the transhydrogenase activity essentially as described by Ibbetson and Freedm a n y or the reconstitution of scrambled ribonuclease assay of Hillson et a t 22 Bovine liver protein disulfide-isomerase concentrated to 10-20 mg/ml in 20 m M sodium phosphate, p H 7.5, and 20% (v/v) glycerol at - 7 0 ° is stable for several months. Analysis of the enzyme preparation by S D S P A G E show that it is free of material with a molecular weight of about 12,000. The exclusion of thioltransferase from the preparations is further verified by the absence of thioltransferase activity using the standard assay with S-sulfocysteine and GSH. 14,15 Properties The D H A reductase properties of thioltransferase and protein disulfideisomerase have been reported. 1° A kinetic study of both enzymes, using D H A prepared as described here, is shown in Table II. Although L-cysteine or cysteamine reduce D H A to A A H 2 chemically, neither thioltransferase nor protein disulfide-isomerase increase the rate of reduction (data not shown). The D H A reductase activity of thioltransferase is inhibited by 24A. Holmgren, J. Biol. Chem. 254, 9627 (1979). 25A. L. Ibbetson and R. B. Freedman, Biochem. J. 159, 377 (1976).

38

GLUTATHIONE

15]

iodoacetamide and other alkylating agents (data not shown) and by platinum complexes. 26 By using site-directed mutagenesis techniques, the essential amino acids at the catalytic center of porcine thioltransferase (dehydroascorbate reductase) were determinedY Seven mutant cDNAs were constructed, subcloned in plasmid pKK233-2, and transfected into E. coli JM105. The proteins were induced by isopropyl-l-thio-/3-D-galactopyranoside treatment at high yields. Activity comparisons showed that Cys-22 is essential for D H A reductase activity and that Cys-25 may participate in the reaction mechanism for the native enzyme; however, the latter is not essential, as substitution of Cys-25 with serine results in increased activity for D H A reductase. The proposed mechanism for the D H A reductase action of thioltransferase is shown in Fig. 1 and discussed elsewhere. 28 Maellaro et al. 29 have reported the purification and partial characterization of a glutathione-dependent dehydroascorbate reductase from rat liver that lacks thioltransferase activity. Acknowledgments This work was supported in part by Research Grants DK-44456 and CA-51972 from the National Institutes of Health. 26 W. W. Wells, P. A. Rocque, D.-P, Xu, Y. Yang, and T. L. Deits, Biochem. Biophys. Res. Commun. 180, 735 (1991). 27 y . Yang and W. W. Wells, J. Biol. Chem. 266, 12759 (1991). 2s W. W. Wells, Y. Yang, T. L. Deits, and Z.-R. Gan, in "Advances in Enzymology and Related Areas of Molecular Biology" (A. Meister, ed.), p. 149. John Wiley & Sons, Inc., New York, 1993. 29 E. Maellaro, B. Del Bello, L. Sugherini, A. Santucci, M. Comporti, and A. F. Casini, Biochem. J. 301, 471 (1994).

[51 D i v e r s i t y o f G l u t a t h i o n e By F.

Peroxidases

URSINI,M. MAIORINO,R. BRIGELIuS-FLoHI~,K. D. AUMANN, A. ROVERI, D. SCHOMBURG,and L. FLOHE

Introduction Selenium was identified as a toxic factor for grazing animals in the first half of the twentieth century and since then has been considered hazardous. 1

1 Committee on Medical and Biological Effects of Environmental Pollutants, "Selenium." National Academy of Science, Washington, D.C., 1976.

METHODSIN ENZYMOLOGY,VOL.252

Copyright© 1995by AcademicPress,Inc. All rightsof reproductionin any formreserved.

[5]

DIVERSITY OF GLUTATHIONE PEROXIDASES

39

The discovery by Schwartz and Foltz 2 that selenium was also an essential micronutrient in higher animals did not markedly change the prevalent view of selenium being a poison. Only long after the identification of the first selenoenzymes in bacteria 3"4 and mammals 5"6 was a Recommended Dietary Allowance gradually established] In fact, the putative biological roles of the selenoenzymes, in particular those of the glutathione peroxidases (GPX), proved instrumental in the understanding of selenium deficiency syndromes in livestock and humans, although the emerging complexity of selenium enzymology still precludes definitive conclusions (for review, see Refs. 8-10). The selenoproteins analyzed so far contain selenocysteine residues as integral parts of the peptide backbones. The selenocysteine is encoded by TGA, H'12 formerly known only as a terminating codon. The selenol group, owing to its low pK and high nucleophilicity, qualifies such selenoproteins for redox catalysis. Correspondingly, most bacterial and mammalian selenoproteins could be characterized as oxidoreductases, with the function of the heavily selenium-loaded protein P remaining unknown. 9"1° The first protein from higher vertebrates shown to be a true selenoprotein was GPX, 5 an unusual peroxidase discovered by Mills in 195713 and shown by Cohen and Hochstein TM to be of pivotal importance for the protection of red blood cells against oxidative damage. In the 1970s it had become common to ascribe the antioxidant effects, if not all biological roles, of selenium in mammals to GPX. In 1982, however, the search for further antioxidant proteins led to the discovery of a new "peroxidation

2 K. Schwarz and C. M. Foltz, J. Amer. Chem. Soc. 79, 3292 (1957). 3 D. C. Turner and T. C. Stadtman, Arch. Biochem. Biophys. 154, 366 (1973). 4 j. R. Andreesen and L. G. Ljungdahl, J. Bacteriol. 116, 867 (1973). 5 L. Floh6, W. A. Gtinzler, and H. H. Schock, F E B S Lett. 32, 132 (1973). 6 j. T. Rotruck, A. L. Pope, H. E. Ganther, A. B. Swanson, D. G. Hafeman, and W. G. Hoekstra, Science 179, 558 (1973). 7 O. A. Levander, in "Selenium in Biology and Medicine" (A. Wendel, ed.), p. 205. SpringerVerlag, Berlin, 1989. 8 L. Floh6, in "Glutathione: Chemical, Biochemical, and Medical Aspects" (D. Dolphin, R. Poulson, and O. Avramovic, eds.), p. 643. Wiley, New York, 1989. 9 T. C. Stadtman, Annu. Rev. Biochem. 49, 93 (1980). lo R. F. Burk and K. E. Hill, Annu. Rev. Nutr. 13, 65 (1993). 11 I. Chambers, J. Frampton, P. Goldfarb, N. Affara, W. McBain, and P. P. Harrison, E M B O J. 5, 1221 (1986). ~2F. Zinoni, A. Birkmann, T. C. Stadtman, and A. B6ck, Proc. Natl. Acad. Sci. U.S.A. 83, 4650 (1986). 13 G. C. Mills, J. Biol. Chem. 229, 189 (1957). 14 G. Cohen and P. Hochstein, Biochemistry 2, 1420 (1963).

40

GLUTATHIONE

[51

inhibiting protein," a5 now known as phospholipid hydroperoxide glutathione peroxidase (PHGPX). In contrast to the classic cytosolic GPX (cGPX), P H G P X is a monomeric protein of 19 kDa and is intriguing in its ability to reduce hydroperoxidases of esterified phospholipids and cholesterol in biomembranes. In 1987 a glycosylated extracellular GPX was isolated from plasma (pGPX) 16 and more recently a new type of GPX apparently prevalent in the gastrointestinal tract (giGPX) 17 was detected; the latter two, like cGPX, are tetrameric proteins. All the glutathione (GSH) peroxidases proved to be selenoproteins, all proved to be distinct gene products, and all proved to be homologous with one another but structurally and phytogenetically unrelated to the selenium-containing bacterial oxidoreductases and the other two sequenced mammalian selenoproteins, the type I thyronine deiodinase TM and protein p.19 In this chapter we compile the structural knowledge on various GPX types to provide a basis for discussion of the possible biological meaning of the structural diversification within this family of selenoenzymes. Molecular Evolution of the Glutathione Peroxidase Superfamily The selenium-dependent peroxidases have long been considered a late achievement of evolution, as they were only detected in vertebrates. This view now has to be revised. Not only is the structural relatedness between the various mammalian GPX types so low that the time point of evolutionary divergence has to be dated to a period long before the origin of mammals, 2°'21 but an obviously typical GPX gene has also been discovered in an invertebrate species, namely, S c h i s o t o s o m a m a n s o n i , z2 Further proteins not containing selenocysteine but being homologous with the mammalian GPX types have been identified in various vertebrate and invertebrate animals, in plants, and even in prokaryotes. Thus, the selenium-dependent 15F. Ursini, M. Maiorino, M. Valente, L. Ferri, and C. Gregolin, Biochim. Biophys. Acta 710, 197 (1982). 16 K. T. Takahashi, N. Avissar, J. Within, and H. Cohen, Arch. Biochem. Biophys. 256, 677 (1987). 17 F. F. Chu, J. H. Doroshow, and S. Esworthy, J. Biol. Chem. 268, 2571 (1993). 18 M. J. Berry, L. Banu, and P. R. Larsen, Nature (London) 349, 438 (1991). 19 K. E. Hill, R. S. Lloyd, J.-G. Yang, R. Read, and R. F. Burk, J. Biol. Chem. 266,10050 (1991). 20 R. Schuckelt, R. Brigelius-Floh6, M. Maiorino, A. Roveri, J. Reumkens, W. Strassburger, F. Ursini, B. Wolf, and L. Floh6, Free Radical Res. Commun. 14, 343 (1991). 21 R. Brigelius-Floh6, K. D. Aumann, H. B16cker, G. Gross, M. Kiess, K. D. KlOppel, M. Maiorino, A. Roveri, R. Schuckelt, F. Ursini, E. Wingender, and L. Floh6, J. Biol. Chem. 269, 7342 (1994). 22 D. L. Williams, R. J. Pierce, E. Cookson, and A. Capron, Mol. Biochem. Parasitol. 52, 127 (1992).

[5]

DIVERSITY OF GLUTATHIONE PEROXIDASES

41

oxidoreductases belong to a superfamily of proteins which in part can hardly or not at all be rated as peroxidases. Unfortunately, it is not straightforward to deduce precisely the molecular evolution of the superfamily from the percentage of misfits between aligned sequences, because the molecular diversity has in part been generated by intron/exon shuffling. The PHGPX enzyme, for instance, contains two partial sequences that are very similar to the corresponding ones in cGPXfl 1 They are interrupted by sequences unrelated or poorly related to cGPX. The highly conserved parts of the sequence are represented in the PHGPX gene by two of the seven exons which again are homologous to parts of the two exons of the cGPX gene. A dendrogram based on mutation statistics, if supposed to have any relevance to molecular evolution, can, however, only be constructed by comparing partial sequences that are unequivocally homologous. For the dendrogram of the GPX superfamily shown in Fig. 1,21-41 we chose the sequences corresponding to the third exon of the PHGPX gene, which meets this prerequisite. Obviously, the GPX superfamily branches early into several molecular clades. Only within 23 D. Lincoln, H. BlOcker, U. Leuner, W. Lehnberg, and G. Graf, "GENMON Version 4.1 Manual." Gesellschaft fiir Biotechnologische Forschung, Braunschweig, Germany, 1989. 24 S. Yoshimura, S. Takekoshi, K. Watanabe, and Y. Fuji-Kuriyama, Biochem. Biophys. Res. Commun. 154, 1024 (1988). 25 y. Ho, A. J. Howard, and J. D. Crapo, Nucleic Acids Res. 16, 5207 (1988). 26 W. A. Gtinzler, G. J. Steffens, A. Grossmann, S.-M. A. Kim, F. Otting, A. Wendel, and L. Floh6, Hoppe-Seyler's Z. Physiol. Chem. 365, 195 (1984). 27 K. Ishida, T. Morino, K. Takagi, and Y. Sukenaga, Nucleic Acids Res. 15~ 10051 (1987). 28 M. Akasaka, J. Mizoguchi, S. Yishimura, and K. Watanabe, Nucleic Acids Res. 17, 2136 (1989). 29 R. A. Sunde, J. A. Dyer, T. Moran, J. K. Evenson, and M. Sugimoto, Biochem. Biophys. Res. Commun. 193, 905 (1993). 30 R. A. Sunde, J. A. Dyer, T. V. Moran, and J. K. Evenson, unpublished; EMBL Acc. No. L 24896, 1994. 31 F. F. Chu and S. R. Esworthy, unpublished, EMBL Acc. No. X 71973, 1994. 32 M. C. Criqui, E. Jamet, Y. Parmentier, J. Marbach, A. Durr, and J. Fleck, Plant Mol. Biol. 18, 623 (1992). 33 D. Holland, G. Ben-Hayyim, Z. Falin, L. Camoin, A. D. Strosberg, and Y. Eshdat, Plant Mol. Biol. 21, 923 (1993). 34 K. Takahashi, M. Akasaka, Y. Yamamoto, C. Kobayashi, J. Mizoguchi, and J. Koyama, J. Biochem. (Tokyo) 108, 145 (1990). 35 j. M. Martin-Alonso, S. Gosh, and M. Coca-Prados, J. Biochem. (Tokyo) 114, 284 (1993). 36 H. Hatta and T. Moriuchi, Biochem. J. 109, 918 (1991). 37 A. C. Perry, R. Jones, L. S. Niang, R. M. Jackson, and L. Hall, Biochem. J. 285, 863 (1992). 38 N. B. Ghyselinck and J.-P. Dufaure, Nucleic Acids Res. 18, 7144 (1990). 39 T. N. Dear, K. Campbell, and T. H. Rabbitts, Biochemistry 30, 10376 (1991). 40 M. J. Friedrich, L. C. DeVeaux, and R. J. Kadner, J. Bacteriol. 167, 928 (1986). 41 E. Cookson, M. L. Baxter, and M. E. Selkirk, Proc. Natl. Acad. Sci. U.S.A. 89, 5837 (1992).

r

Lr] II .IJ

tO E

4..I

oJ F-q

x

C. .iJ

L

E

x" X

m h

X El. L9

X n O

X El. t9

X n (D

X rl t.O

X rl (-9

X n tD

X n C.9

X 13_ ~

X n CD

0J

~

-I O

IT

4-J

0J

C

01

17

CL

13.

.~

.O .O ro

.,4 >

I= ~

C ~I

C~ .r4

-IJ ~0

C ~

O 0'~

o

T

e

O_

rr

~

c

>

IT

03

~

D

tO

T X n

X n (.~

X 13_ CD

X 13_ (.~

X n (-9

tO C

f0 I=

C" .,4

tO n"

~

C

T

0

tO

ffl

~ ~ ~

~

7

~

T X 12.

"r" X 12.

T x 13_

~

~

0J

~ to

4a ro

U to m

~

rr

T X 12.

T X 13_

ZC X 12.

I0 r/-

,-4 O u

10 o

~--

~"

QJ Z

FIG. 1. Dendrogram of the glutathione peroxidase superfamily calculated from conserved amino acid residues of aligned protein sequences according to Ref. 23 applying a gap penalty of 5. The pertinent sequences are as follows: mouse GPX, u rat cGPX, 24'25 bovine GPX, 26 human cGPX, 27 rabbit cGPX, 2s human giGPX, 17 porcine P H G P X , 21'29 rat P H G P X , 3° human P H G P X , 31 Schistosoma m a n s o n i GPX, 22 G P X homolog ( G P X H ) of Nicotiana sylvestris, 32 G P X H from Citrus sinensis, 33 human p G P X , 34 bovine p G P X , 3s rat p G P X , 36 the epididymal G P X homolog ( e G P X H ) of mouse, 37 rat, 38 and Macaca fascicularis, 38 another rat G P X H , 39 the cobalamine-binding protein of Escherichia coli, 4° and a nematode G P X H . 41

[5]

DIVERSITY OF GLUTATHIONE PEROXIDASES

43

a distinct type of the protein does the molecular similarity reasonably correspond to the relatedness of the species. Three major clades can be clearly delineated from the dendrogram (Fig. 1) and can be even more precisely defined when the common molecular characteristics obvious from the sequence alignment (see below and Fig. 3) are considered: (i) the cGPX family with its side branch giGPX; (ii) pGPX together with GPX homologs in which the selenocysteine residue is replaced by cysteine, both characterized as secreted proteins by typical signal peptides, and (iii) the PHGPX family. Whether the common ancester of the GPX superfamily was a selenoprotein or a cysteine-containing homolog cannot be deduced from the available sequences. The only prokaryotic member of the superfamily detected so far, a cobalamine-binding protein of Escherichia coli, 4° does not contain selenocysteine, and despite ongoing efforts, functionally active glutathione peroxidases have not yet been found in prokaryotes. This might indicate that, within the GPX superfamily, the use of T G A as a codon for selenocysteine was not achieved except during eukaryotic evolution. This view, however, conflicts with the observation that selenocysteine in bacterial selenoproteins is also encoded by TGA, 42 although the mechanism for the differential use of T G A as codon for selenocysteine or stop differs in proand eukaryotes. In bacteria selenocysteine incorporation requires a defined stem-loop in the immediate neighborhood of the U G A triplet in the mRNA coding region, 43,44whereas in eukaroytes proper selenocysteine incorporation is guaranteed by a selenocysteinyl insertion sequence (SECIS) in the 3' untranslated tail of the mRNA, its distance from the U G A codon being largely irrelevant. 45 In each case, the formation of a selenoprotein requires the fit of the m R N A structure comprising the U G A codon for selenocysteine and a SECIS and special decoding factors plus the availability of a selenocysteinyl-tRNA with an appropriate anticodon. Despite basic similarities, however, the mechanisms of selenoprotein biosynthesis of pro- and eukaryotes diverged to an extent that a eukaryotic gene encoding a selenoprotein, if expressed in bacteria, inevitably yields a truncated peptide resuiting from chain termination at the U G A codon. The puzzle of how the coevolution of pertinent genes sustained the ability of organisms to build selenoproteins remains to be solved. In view of the multiple ways of being evolutionary deleted and the limited chance of being reinvented, seleno42 A. BOck, K. Forchhammer, J. Heider, and C. Baron, Trends Biochem. Sci. 16, 463 (1991). 43 F. Zinoni, J. Heider, and A. B/)ck, Proc. Natl. Acad. Sci. U.S.A. 87, 4660 (1990). 44 G. E. Garcia and T. C. Stadtman, J. Bacteriol. 173, 2093 (1991). 45M. J. Berry, L. Banu, J. W. Harnex, and P. R. Larsen, E M B O J. 12, 3315 (1993).

44

GLUTATHIONE

[5]

proteins maintained up to mammals should deserve more than curiosity, as their mere existence stresses their biological importance.

S t r u c t u r e a n d F u n c t i o n of G l u t a t h i o n e P e r o x i d a s e s C o m m o n Features

Bovine c G P X has been extensively investigated functionally. 46-49 With the identification of c G P X as a selenoprotein 5° and the determination of its amino acid sequence 26 and three-dimensional (3D) structure, 51 a rather detailed mechanism of action of the enzyme could be proposed 52 which, however, needs to be revised (see below). As far as p G P X and P H G P X are concerned, we still have to rely on functional and structural homology with cGPX. In functional terms the three types of G P X appear very similar. In all cases the reaction catalyzed is identical in principle [Eq. (1)]. The kinetic 2 R S H + R O O H ~ RSSR + H 2 0 + R O H

(1)

pattern is characterized as a Ping-Pong mechanism with indefinite Michaelis constants, indefinite maximum velocities, 46'47'53-55 and negligible, if any, product inhibition. It can be described by the simple Dalziel equation [Eq. (2)]. This kinetic mechanism is routinely explained by a sequence of [Eo! _ ~1 ~2 + - v [ROOH] [RSH]

(2)

bimolecular reactions of the substrates with the enzyme (E) or derivatives (F, G) thereof [Eqs. (3)-(5)]. Comparison of Eqs. (3)-(5) with the Dalziel 46 L. Floh4, O. Loschen, W. A. Giinzler, and E. Eichele, Hoppe-Seyler's Z. Physiol. Chem.

353, 987 (1972). 47W. A. Giinzler, H. Vergin, I. MUller, and L. Floh6, Hoppe-Seyler's Z. Physiol. Chem. 353, 1001 (1972). 48L. Floh6, W. A. Giinzler, G. Jung, E. Schaich, and F. Schneider, Hoppe-Seyler's Z. Physiol. Chem. 352, 159 (1971). 49L. Floh6 and W. A. Giinzler, in "Glutathione" (L. Floh& H. C. BenOhr, H. Sies, H. D. Waller, and A. Wender, eds.), p. 132. Thieme, Stuttgart, 1974. 5oL. Floh6, W. A. Gtinzler, and H. H. Schock, FEBS Lett. 32, 132 (1973). 51O. Epp, R. Ladenstein, and A. Wendel, Eur. J. Biochem. 133, 51 (1983). 52R. Ladenstein, O. Epp, W. A. Giinzler, and L. Floh4, Life Chem. Rep. 4, 37 (1986). 53F. Ursini, M. Maiorino, and C. Gregolin, Bioehim. Biophys. Acta 839, 62 (1985). 54M. Maiorino, A. Roved, C. Oregolin,and F. Ursini, Arch. Biochem. Biophys. 251,600 (1986). 5s R. S. Esworthy, F.-F. Chu, P. Geiger, A. W. Girotti, and J. H. Doroshow, Arch. Bioehem. Biophys. 3ff7, 29 (1993).

[5]

DIVERSITY OF GLUTATHIONE PEROXIDASES

45

E + ROOH

k<) F + ROH

(3)

F + RSH

:> G + H20 k+~

(4)

G + RSH

k:~* E + RSSR

(5)

equation [Eq. (2)] obtained empirically yield the definitions of the Dalziel coefficients: qbl -

%

] k+l

1 -

k+3 + k+5

(6)

(7)

With the questionable exception of rabbit cGPX 56 for which a tendency toward substrate saturation kinetics at extreme GSH levels has been described, Eqs. (3)-(5) would be satisfactory to describe the kinetic mechanisms of all selenoperoxidases analyzed so far. These minimum assumptions, however, are incompatible with substrate-specificity studies. Although distinct differences in the generally low specificity for the hydroperoxide substrates do exist between the different GPX types, they may be explained by a modified accessibility of the catalytic site and, in view of the lacking saturation kinetics, do not justify implication of the intermediate formation of a specific complex between E and ROOH. Despite the lack of saturation kinetics, the pronounced specificity of cGPX for GSH, however, indicates a complex formation between the donor substrate and the oxidized enzyme forms F and G [Eqs. (8) and (10)], before reactions (9) and (11), respectively, can proceed: F + RSH

(F. RSH)

(8)

(F-RSH)

k:~ G + H20

(9)

G + RSH ~ (G.RSH)

k*4

(G-RSH)

~,:5 E + RSSR

(10) (11)

The rate constants k+2 and k+4 are obviously much smaller than k+3 and k+5, respectively, which means that the rate constants for complex formation, k+2 a n d k+4, become rate limiting and approximate k+ 3 and k+5 in the overall reactions [Eqs. (4) and (5)]. The lack of any detectable substrate saturation further implies that regeneration of the ground state E via Eqs. (10) plus 56 p. Morrissey and P. J. O'Brian, Can. J. Biochem. 58, 1012 (1980).

46

GLUTATHIONE

[51

(11) must proceed faster than the formation of the intermediate G according to Eqs. (8) and (9). Deviations of the ratios of rate constants k+2/k'+3, k+4/ k~5, o r k÷2/k+4may be envisaged in certain GPX species. If substantial, they might affect the kinetic pattern without, however, invalidating the basic assumptions explaining the catalytic cycle. Common to all types of glutathione peroxidases to be discussed here is the involvement of selenium in the catalysis. [The so-called nonselenium G P X 57 is a structurally unrelated GSH thioltransferase which mimicks the overall reaction (1) by an entirely different mechanism.] Their biosynthesis strictly depends on the availability of selenium.They become inactivated if their selenocysteines are carboxymethylated by i o d o a c e t a t e 49,58,59 o r if the selenium is eliminated. 6° Further, no relevant peroxidase activity could so far be attributed to any of the sulfur-containing homologs of the superfamily.

Putative Molecular Mechanism of Bovine Cytosolic Glutathione Peroxidase Although bovine cGPX may be considered to be one of the best investigated proteins, some uncertainties with respect to its molecular mechanism remain. These are predominantly due to the kinetics of the enzyme, which make it theoretically impossible to freeze any of the postulated complexes or intermediates (F. GSH), G, and (G. GSH). The groundstate E and the first intermediate F are not easily investigated either because of stability problems. X-ray studies, for instance, aimed at the analysis of the ground state yielded the 3D structure of an enzyme form in which the selenocysteine was oxidized by air to a seleninic acid derivative. 51 This oxidation state most probably can be attributed to a sluggish enzyme derivative which does not become fully active until after prolonged incubation with thiols. 8,61 Removal of the two oxygens from the selenium atom in a molecular model did not, however, essentially alter the 3D structure after energy minimization and molecular dynamics calculations (K. D. Aumann, unpublished). We may thus consider the X-ray structure of Epp et aI.,51 though performed on an overoxidized enzyme, as a reliable basis for our attempts to understand the essential molecular properties of the various enzyme forms implicated in the catalytic cycle. Clearly, the ground state of the enzyme (E) must be characterized by 57 R. F. Burk and R. A. Lawrence, in "Functions of Glutathione in Liver and Kidney" (H. Sies and A. Wendel, eds.), p. 114. Springer-Verlag, Berlin, 1978. 58 R. J. Kraus, J. R. Prohaska, and H. E. Ganther, Biochim. Biophys. Acta 615, 19 (1980). 59 M. Maiorino, C. Gregolin, and F. Ursini, this series, Vol. 186, p. 448. 60 H. E. Ganther and R. J. Kraus, this series, Vol. 107, p. 593. 61 C. Little, R. Olinescu, K. G. Reid, and P. J. O'Brian, J. Biol. Chem. 245, 3632 (1970).

[5]

DIVERSITY OF GLUTATHIONE PEROXIDASES

47

having its selenocysteine residue in the reduced form, that is, as a dissociated selenol. This is obvious (i) from the selective susceptibility of the donor substrate-reduced enzyme by iodoacetate 49 and (ii) from the speed of its reaction with hydroperoxides 46'47which could hardly be expected from any other functional groups of this protein. The selenium is located in the center of a flat impression composed of hydrophobic residues. 51 It is fixed in this position from the interior of the protein core by a glutamine and a tryptophan residue, each forming hydrogen bridges with the selenium (see Figs. 2a and 5a). 51 In the catalytic triad of selenocysteine, glutamine, and tryptophan, a complete dissociation of the selenol is certainly enforced and its nucleophilic attack on the peroxide is facilitated. Because no other redox active residue is available in the immediate neighborhood of the selenol and a free radical mechanism has been ruled out, 6a we have to assume that the selenium has to change its oxidation state from - 2 to 0 on collision with the peroxide. A simple bimolecular reaction between the selenol and the peroxide, mediated by a transition state rather than by a specific enzyme-substrate complex, is also suggested by the speed of the reaction, the broad range of hydroperoxides accepted at comparable rates, 8 and the lack of any structural pecularities reminiscent of a substrate binding pocket. 5~ The next step, the formation of the complex (F. GSH) according to Eq. (8), has to comply with the strict specificity of cGPX for GSH. Further, in this complex the sulfur of GSH has to be directed into a position to enforce a fast, non-rate-limiting intramolecular reaction yielding the intermediate G [Eq. (9)]. Calculations 62a show that these requirements may be met in the following way (see Fig. 2). The glycine carboxylic group of GSH binds electrostatically to the positively charged guanidino group of Arg57; Arg-184 forms a hydrogen bridge with the carbonyl of the cysteine residue of GSH; another hydrogen bridge is built between the amino group of the y-glutamyl residue of GSH and the carbonyl of Gly-53, whereas the carboxylic group remains loosely oriented toward a positively charged area composed of Arg-103 and by Lys-91 of the neighboring subunit (Lys-91B). In this complex the sulfhydryl group of GSH is in an ideal position to react with the oxidized selenium of the active site. In the resulting intermediate G (see Fig. 2b), the glutathione molecule remains essentially bound as in the preceding complex. It should be mentioned that this proposal for G obtained by molecular modeling 63 differs from that originally discussed by Epp e t al. 51 in which the carboxylic groups 62 L. S. Harman, D. K. Carver, J. Schreiber, and R. P. Mason, J. Biol. Chem. 261, 1642 (1986). 62a K.-D. Aumann, D. Schomburg, and L. Flohd, in preparation. 63 L. Flohr, K.-D. Aumann, R. Brigelius-Flohr, D. Schomburg, W. Strassburger, and F. Ursini, in "Active Oxygens, Lipid Peroxides and Antioxidants" (K. Yagi, ed.), p. 299. Japan Scientific Societies Press, Tokyo/CRC Press, Boca Raton, Florida, 1993.

48

GLUTATHIONE

[51

of GSH formed salt bridges with the Arg-57 and Arg-184 residues. By this mode of binding, however, the GSH molecule would be forced into a less relaxed Y-shaped conformation; correspondingly, the energy minimization calculations refused such a solution. Also, in the original proposal the active site selenodisulfide bridge of G was shielded by the glutathionyl residue in a way which would probably prevent any attack of the second GSH molecule. 8 In contrast, the model presented here leaves the selenodisulfide bridge freely accessible. As outlined above, the regeneration of E from G by GSH proceeds faster than the formation of G. This is again achieved by directing the second GSH molecule into an adequate position. Molecular modeling combined with calculation of energy minimization and molecular dynamics yields a proposal for (G. GSH), which again is essentially electrostatic (Fig. 2c). Apart from some hydrogen bridges the second GSH molecule forms with the first one and with some residues of the enzyme including those of the neighboring subunits, the predominant binding forces are the positive charges of Arg-184 now binding to the glycine carboxyl of the second GSH and of Arg-103 plus Lys-91B attracting the T-glutamyl carboxyl of the substrate. In an alternative model (not shown) Arg-185 turns around to compete with Arg-184 for the cosubstrate. In both models the S- of GSH can split the selenodisulfide bridge to generate the product oxidized glutathione (GSSG) and to close the catalytic cycle. Lack of product inhibition finally may be explained by the conformation of GSSG assumed after dissociation of the enzyme-product complex. In an aqueous environment, the four carboxylic groups of GSSG tend to repel one another electrostatically. Thus, a double Y-shaped conformation twisted by 90° around the disulfide bond is favored. Consequently, the product no longer fits into the frame of positive charges surrounding the catalytic site of the enzyme and its affinity is substantially lowered. The mechanism proposed here is consistent with all experimental data available such as X-ray analysis, sl kinetic patterns, 46'4~ inhibition, 49 and substrate specificities.48 It should also be stressed that the residues invoked to play a major role in the catalytic cycle are conserved in all cGPX species sequenced so far (see Fig. 3). It may therefore be stated that the ideas summarized here provide a reasonably realistic proposal for the first example of an enzymatic selenium catalysis. Structural Peculiarities of Individual Glutathione Peroxidases Despite a low incidence of identities in the aligned sequences, molecular models of pGPX and P H G P X could be developed, 63 As the core of the modeled proteins was not changed substantially on simulated energy expo-

blue bails, respectively. For sake of clarity the sizes of these balls are much smaller than the van der Waals radii (30%). Residues belonging to the second subunit of the dimer model are shown in beige. (a) "Empty" ground state of the enzyme (E) essentially corresponding to the X-ray structure. 51 (b) Model for the catalytic intermediate G, in which GSH by its sulfur (yellow) is covalently bound to the selenium. The negatively charged carboxylic groups of GSH noncovalently bound to the arginine and lysine residues are shown as red balls. (c) The complex of G with the second GSH molecule (pink). Note how precisely the sulfur of the second GSH is directed toward the selenadisulfide bridge of G. All computer graphics were obtained by means of the BRAGI program (GBF, Braunschweig, Germany).

FIG. 3. Alignment of available sequences of the GPX superfamily. Pertinent references are given in the legend to Fig. 1. The residues forming the catalytic triad in GPX, selenocysteine (position 52 in the bovine cGPX sequence), glutamine-87, and tryptophan-165 are marked in red and other highly conserved residues in beige. The arginine residues 57, 103, 184, and 185 and lysine-91 (position numbers of bovine cGPX) involved in GSH binding by cGPX are shown in blue. Green defines the signal peptides of pGPX and secreted GPX homologs. Signal peptide processing sites are either taken from the corresponding references or estimated according to the algorithm proposed by von Heijne (Nucl. Acids Res. 14, 4683, 1986). HHH and 13~il3mean a-helices and pleated sheets, respectively, in the bovine cGPX structure.

O

S~

iqil

g.

0

•

Q~IZt

:S:I= :1:

O0 r~lZ

m ~

~

~m~0J :l: I! Q

. f : I~l

,-

i/

Fro. 4. Comparison of the Ccz carbon backbone of a bovine cGPX subunit51 with other members of the cGPX superfamily. (a) Residues of bovine cGPX (red) which are conserved in homologous positions in all members of the superfamily. Note that these residues accumulate around the active-site selenium (orange ball) in the pleated sheets and turns. (b) Overlay of the bovine cGPX structure and a model of human pGPX. Here the pleated sheets are marked in green, and the cz helices in red. Deviations of the pGPX model from the cGPX structure are shown in yellow. (c) Overlay of the PHGPX model with cGPX. Note the substantial deletion of the random loop and of the small c~ helix left of the active center.

FIG. 5. Comparison of the active site structure of bovine cGPX 51 with models of human pGPX and porcine PHGPX. (a) Active site of cGPX with Arg-57 (lower left comer), Arg-103 (lower right

comer) Arg-184 (top), and Arg-185 (upper left comer) in red, Gln-87 in lilac, Trp-165 and Phe-167 in yellow, and selenocysteine in orange. The size of the selenium (big ball) corresponds to its van der Waals radius. Residues supposed to be functionally important are stressed with bigger sticks. (b) Overlay of cGPX in the same colors and human pGPX in pink. Note that Arg residues 57 and 184 are replaced by Gin and His, respectively. (c) Overlay of cGPX with the model of porcine PHGPX (pink). Note that all arginines invoked in substrate binding are missing.

[5]

DIVERSITY OF GLUTATHIONE PEROXIDASES

49

sure in molecular dynamics calculations, the models may be considered fairly realistic. According to the molecular models, the homology of the sequences finds its counterpart in a conservation of the basic structure of the core of the proteins. Figure 4a shows the Ca backbone of a bovine cGPX subunit, 51 which demonstrates where the residues conserved in the entire superfamily are located. Interestingly, conserved residues are particularly frequent in the intimate environment of the active site selenium (or sulfur, respectively). This highly conserved part of the 3D structure is built up by three distant protein loops of the primary sequence, namely, NVA..Sec (or C)..T (positions 47-59 in the bovine cGPX sequence), L.FPCNQF..Q (positions 7787), and WNF (positions 165-167). The conserved clusters comprise the residues Gin, (seleno)Cys, and Trp which appear to represent the catalytic triad, qualifying the enzymes for a more or less efficient redox catalysis depending on the presence of selenium or sulfur (see Fig. 5a for more details). Further highly conserved residues, for example, those in the pleated sheets and Gly-176 forming a turn, are considered responsible for the conservation of the global structure of the molecules, which is obviously indispensible for the formation of the particular catalytic triad. The structural similarity of the two families of cytosolic and plasma GPX is demonstrated in Fig. 4b, showing an overlay of the experimentally determined Ca backbone of bovine cGPX and the model of human pGPX. There is virtually no difference whatsoever in the arrangement of the wellordered secondary structural elements, namely, the four pleated sheets and the four helices. However, a small shortcut in the large loop (right-hand side of Fig. 4) is seen in the pGPX structure. Because this loop forms the subunit interaction site of cGPX, the deviation might slightly affect the affinity of the subunits. Calculations of the subunit interaction forces of pGPX, 63 however, confirm a tetramerization, which is in agreement with the apparent Mr of pGPX. The differences between P H G P X and cGPX are more pronounced (Fig. 4c). Again, the shapes of the core of these enzyme families comprising the pleated sheets and three of the helices appear largely identical. However, the deletions in the P H G P X sequences cause a substantial shortcut of the subunit interaction loop plus an elimination of a small helix (left site) also involved in subunit interaction in cGPX. Thus, both the interfaces responsible for dimerization (loop) and tetramerization (helix) in cGPX are destroyed, which is consistent with the monomeric nature of PHGPX. In the P H G P X model, because of the deletions and the resulting monomeric nature, the active site appears more freely accessible. This could provide a reasonable explanation for the ability of P H G P X to attack more complex hydroperoxy lipids including those integrated into lipid layers.

50

GLUTATHIONE

[51

Comparing the active sites of cGPX with more remote relatives poses rather than answers questions. Figure 5a shows a closeup of the active site of bovine cGPX as derived from the X-ray study of Epp et al. 5a The four arginines in positions 57, 103, 184, and 185 surrounding the catalytic triad and postulated to contribute to the correct orientation of the donor substrates are conserved in all cGPX species sequenced so far (Fig. 3), but one, Arg-185, is replaced by Thr in the most closely related offspring of that family, giGPX. Also, Lys-91B contributing to substrate binding is exchanged by an uncharged amino acid residue (Gin). In the pGPX family, of the five basic residues implicated only Arg-103 and Arg-185 are conserved (Fig. 3), and any isosteric charged substitute for Arg-57 and Arg184 facilitating binding of the first GSH molecule in cGPX is not detectable in the pGPX model, as evident from the overlay shown in Fig. 5b. In the P H G P X family, finally, none of the charged residues is retained in homologous positions (Fig. 3), nor does the P H G P X model suggest any isosteric and similarly charged substitute which could be envisaged to bind GSH (Fig. 5c). For the GPX from Schistosoma mansoni, however, which is the only nonvertebrate GPX sequence known so far, one could envisage a functional substitution of Arg-57 and Arg-184 by lysine residues (Fig. 3). From the alignment and the molecular models one might deduce that the donor substrate specificity declines in the order cGPX > giGPX > pGPX > PHGPX. Also, any attempt to generate a plausible model of the complex (F. GSH), as described for cGPX above, has so far failed in the case of pGPX and PHGPX. Nevertheless, these enzymes have been discovered as functionally active glutathione peroxidases, and despite considerable efforts no physiological donor substrate acting faster than GSH has been found so far. For pHGPX, however, considerable activity has been detected with dithiols like lipoic acid and dithiothreitol (DTT), 64 which are poor substrates for cGPX. Also, the limiting rate constant of the reducing part of the catalytic cycle, that is, probably k+2 of Eq. (8) (see above), has consistently been determined to be one order of magnitude smaller for both pGPX 55 and P H G P X 59 than for cGPX. 47'48'55 This finding is consistent with the view that, in the pGPX and P H G P X family, GSH as donor substrate is less precisely forced into a suitable position to react with the active site selenium. In both cases, it may be reasonably questioned whether the real physiological donor substrates have yet been identified. 64 M. Maiorino, in "Biological Oxidants and Antioxidants; New Development in Research and Health Effects" (L. Packer and E. Cadenas, eds.), p. 69. Hyppocrates Verlag, Stuttgart, 1994.

[5]

DIVERSITY OF GLUTATHIONE PEROXIDASES

51

Apart from the questions related to the physiological donor substrates of pGPX and PHGPX, further peculiarities reveal a functional diversification within the GPX superfamily. The substitution of selenocysteine by cysteine in the catalytic triad of many of the members is incompatible with the assumption of all enzymes being peroxidases in the sense of efficient peroxide removers. Unfortunately, however, few functional studies on the cysteine-containing GPX homologs are available. For the homolog in E. coli, a role as a cobalamine-binding protein has been demonstrated, n° in citrus plants the homolog is induced by salt stress, 33 and the epidydimal homologs respond to androgens. 38

Functional Diversification of Selenoperoxidases Ample evidence reveals that cGPX, in concerted action with the pentose phosphate cycle and glutathione reductase, efficiently metabolizes intracellular H202, particularly in ceils or cellular compartments low in or devoid of catalase/As evident from the early studies of Cohen and Hochstein 14and the k+a values in the range of 108 M -1 s e c 1,46,47 cGPX can also successfully compete with catalase as long as the rate of GSH regeneration can cope with the generation or influx of H202. 8 The broader range of accepted hydroperoxides renders cGPX even more competitive in the defense against oxidative damage. In agreement with early in vitro experiments with isolated membranes, 65 it could also be envisaged that cGPX contributes to the prevention of lipid peroxidation in biomembranes if the latter is initiated by soluble hydroperoxides. To this end, the cGPX system can be rated as a highly efficient and indispensible constituent of cellular devices protecting against oxidative stress. Gastrointestinal GPX, being structurally very similar and functionally indistinguishable from cGPX, has to be seen in the same context. Whether the scope of biological functions of these most typical glutathione peroxidases is definitively described by "defense against soluble hydroperoxides" should, however, be left unanswered. Historically, P H G P X was detected and exclusively discussed as being an indispensible device to prevent unspecific lipid peroxidation within biomembranes. 15 In this respect, it is indeed the substitute for cGPX, to which this function was erroneously attributed. 65 In a concerted action with vitamin E, P H G P X breaks the chain reactions leading to oxidative lipid destruction in the following way: peroxy radicals generated by addition of molecular oxygen on hydrogen abstraction from unsaturated phospholipids are 65 L. Floh6 and R. Zimmerman, in "Glutathione" (L. Floh6, H. C. Bent~hr, H. Sies, H. D. Waller, and A. Wendel, eds.), p. 245. Thieme, Stuttgart, 1974.

52

GLUTATHIONE

[5]

first reduced by vitamin E to the corresponding hydroperoxides; these are reduced by P H G P X with the consumption of GSH; thereby, the decomposition of the hydroperoxides into alkoxy radicals and reinitiation of free radical chains are prevented. 66,67 Again, the scope of P H G P X function appears inadequately described by this narrow model. In vitro, cGPX plus GSH was shown to block the activity of the key enzymes of prostaglandin and leukotriene biosynthesis, the cyclooxygenase6s and the 5-1ipoxygenase. 69 However, in carefully designed experiments taking advantage of the differential response of cGPX and P H G P X to alimentary selenium supply, P H G P X was identified as the more likely candidate to regulate leukotriene biosynthesis in v i v o . 70 Beyond this observation, a preferentially regulatory role of P H G P X is further suggested by its overall low tissue levels, its extremely unusual tissue distribution with prevalence in reproductive and endocrine tissues, 71 susceptibility to posttranslational modifications, 2°,21 change of subcellular location during spermatogenesis, strong gonadotropin dependency of its biosynthesis, 72 and multiple putative hormone-responsive elements in the P H G P X gene. 2a Also, the complete elimination of any GSH-binding residues in the active site of P H G P X and the low specificity for GSH allows one to question its nature as a real glutathione peroxidase. The flat hydrophobic surface surrounding the catalytic site of P H G P X would allow an intimate interaction not only with lipid layers but also with proteins. Thus, oxidation equivalents from some peroxides could be transferred via oxidized selenium to protein thiol groups of functional relevance. It is even more tempting to speculate along these lines in case of pGPX. Although the active site of pGPX with two of the typically four arginine residues being conserved is still reminiscent of a real GPX, an antioxidant function has to be ruled out for this enzyme in view of the extremely low extraceUular GSH levels, the lack of any efficient extracellular GSH regenerating system, and the comparatively low rate constants for GSH. 17 Extracellular GSH levels in the micromolar range could just serve to keep pGPX in a reduced state under undisturbed physiological conditions. An oxidative

66 M. Maiorino, M. Coassin, A. Roveri, and F. Ursini, Lipids 24, 721 (1989). 67 F. Ursini, M. Maiorino, and A. Sevanian, in "Oxidative Stress: Oxidants and Antioxidants" (H. Sies, ed.), p. 319. Academic Press, London, 1991. 68 M. A. Warso and W. E. Lands, J. Clin. Invest. 25, 667 (1985). 69 M. Haurand and L. Floh6, Biol. Chem. Hoppe-Seyler 369, 133 (1988). 70 F. Weitzel and A. Wendel, J. Biol. Chem. 268, 6288 (1993). 71 A. Roveri, M. Maiorino, and F. Ursini, this series, Vol. 233, p. 202. 72 A. Roveri, A. Casasco, M. Maiorino, P. Dalan, A. Calligaro, and F. Ursini, J. Biol. Chem. 267, 6142 (1992).

[6]

GLUTATHIONE TRANSFERASE PURIFICATION

53

burst of polymorphonuclear leukocytes or macrophages could keep the active site selenium oxidized long enough to allow a transfer of the oxidant signal onto thiol groups on distant cellular surfaces.

Conclusion In conclusion, a comparison of the structures of pGPX and PHGPX with that of cGPX should open our eyes to new possible roles of these more recently discovered GPX congeners far beyond the concept of antioxidant defense. The "atypical" members of the GPX family at least will find their biological role more likely in the realm of redox regulation, for example, for the release of lipid mediators 7°,73 and cytokines, TM signal transduction, 73 and NF-~:B-mediated transcriptional control. 75'76 73 S. Orrenius, M. J. Burkitt, G. E. Kass, J. M. Dypbukt, and P. Nicotera, Ann. Neurol. 32, 33 (1992). 74 Fo U. Schade, R. Engel, S. H~irtling, J. Holler, and D. Jakobs, lmmunobiology 187, 283 (1993). 75 T. Okamoto, H. Ogivara, T. Hayashi, A. Mitsui, T. Kawabe, and J. Yodoi, Int. ImmunoL 4, 811 (1992). 76 N. Israel, M. A. Gougerot-Pocilado, F. Aillet, and J. L. Virelizier, Z lmmunol. 149, 3386 (1992).

[6] P u r i f i c a t i o n

By D A V I D

of Soluble Human S-Transferases

Glutathione

J. M E Y E R a n d BRIAN KETTERER

Introduction Soluble glutathione transferases (GSTs) (EC 2.5.1.18) catalyze glutathione-dependent nucleophilic substitution and addition reactions with endogenous and xenobiotic electrophiles as well as the reduction of hydroperoxides, quinones, and quinoneimines. 1They are intracellular dimeric enzymes (molecular weight approximately 50,000), representing four related gene families named alpha, mu, pi, and theta. 2 Both homodimers and heterodimers may occur within but not between gene families. 1 B. Ketterer and L. G. Christodoulides, Adv. Pharrnacol. 27, 36 (1994). 2 B. Mannervik, Y. C. Awasthi, P. G. Board, J. D. Hayes, C. Di Ilio, B. Ketterer, I. Listowsky, R. Morgenstern, M. Murarnatsu, W. R. Pearson, C. B. Pickett, K. Sato, C.-P. D. Tu, M. Widersten, and C. R. Wolf, Biochem. J. 281, 305 (1992).

METHODS IN ENZYMOLOGY, VOL. 252

Copyright © 1995 by Academic Press, Inc. All rights of reproduction in any form reserved.

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General Considerations Glutathione transferases of classes alpha, mu, and pi but not theta have a high affinity for glutathione (GSH) conjugatesJ As a result affinity chromatography with immobilized GSH conjugates, such as amino-linked S-hexylglutathione-agarose 4 or sulfur-linked glutathione-agarose, 5 enables the isolation in one step of a fraction containing alpha, mu, and pi class enzymes. Whereas the former also binds glyoxalase II and minor amounts of uncharacterized proteins of approximately Mr 35,000, the latter retains only GSTs. Subsequent hydroxylapatite and anion-exchange chromatography steps enable the purification of the major human GSTs (AI-1, A1-2, A2-2, Mla-la, Mla-lb, M3-3, and PI-1) as described below. The published purifications of human 0 class GSTs, which do not bind to GSH affinity matrices, namely, GSTTI-1 and GSTT2-2, have been described, 6'7 but because they yield small amounts of unstable enzyme, they are not given in this chapter. The same problems arise with respect to rat theta class GST5-5. In this case the problem has been overcome by the use of a heterologous bacterial expression system. This procedure should be applicable to the human homolog GST-I?I-1 and is described below. All the procedures described give a high yield (50-90%); therefore, detailed analyses of purification are not included. Our laboratory has not isolated GSTM2-2 or the heterodimers GSTM1-2 or GSTM2-3, which are relatively abundant in skeletal muscle. A pr6cis of the method used by Hussey et al. 8 to isolate GSTM2-2 is given below. Readers are referred to the literature for less studied enzymes, such as GSTM4-4 from brain 9 and GSTs omega (co) 1 and 2 from liverJ ° Purification of Glutathione Transferases A I - I , AI-2, A2-2, and M1 from Liver Normal human liver contains the three a class GSTs A1-I, A1-2, and A2-2; GST M1 has two expressing alleles G S T M I * A and G S T M I * B and 3 D. J. Meyer, Xenobiotica 23, 823 (1993). 4 C. Guthenberg and B. Mannervik, Biochem. Biophys. Res. Commun. 86, 1304 (1979). 5 p. C. Simons and D. L. Vander Jagt, this series, Vol. 77, p. 235. 6 D. J. Meyer, B. Coles, S. E. Pemble, K. S. Gilmore, G. M. Fraser, and B. Ketterer, Biochem. J. 274, 409 (1991). 7 A. J. Hussey and J. D. Hayes, Biochem. J. 286, 929 (1992). 8 A. J. Hussey, L. A. Kerr, A. D. Cronshaw, D. J. Harrison, and J. D. Hayes, Biochem. J. 273, 323 (1991). 9 T. Suzuki, D. C. Shaw, and P. G. Board, Biochem. J. 274, 405 (1991). 10H. Ahmad, S. S. Singhal, M. Saxena, and Y. C. Awasthi, Biochim. Biophys. Acta 1161, 333 (1993).

[61

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55

the null allele GSTMI*O. GSTM1 expression occurs in approximately 60% of the population. Of these a small proportion are GSTM1 A,B heterozygotes. GST subunits Mla and Mlb differ in only one amino acid residue conferring a single charge difference, resulting in distinct chromatographic properties. The presence of GSTMI-1 can be most easily tested by measuring soluble GSH transferase activity toward trans-4-phenyl-3-buten-2-one. 11 Materials Equipment. The following equipment is needed: Waring blendor (or similar tissue homogenizer/food processor); ultracentrifuge; low-pressure chromatography column (15-30 cm × 5 cm i.d.); recording spectrophotometer; Pharmacia (Piscataway, N J) FPLC (fast protein liquid chromatography) system or equivalent; Bio-Rad (Richmond, CA) hydroxylapatite (HPHT) column and guard column or low-pressure chromatography column (25-35 cm × 1 cm i.d.); Pharmacia Mono Q anion-exchange column or equivalent; Sephadex G-25 columns (e.g., PD-10 from Pharmacia); and vacuum filtration apparatus to remove particulates from FPLC buffers. Chemicals. Required reagents are as follows: phenylmethylsulfonyl fluoride (PMSF); dithiothreitol (DTT); reduced glutathione (acid); S-hexylglutathione (acid); S-linked glutathione-agarose; polyethylene glycol (molecular weight 15,000-20,000); dialysis tubing; 1-chloro-2,4-dinitrobenzene (recrystallized from ethanol); Tris base (high purity), piperazine hydrate: glycerol (low UV absorbing); and hydroxylapatite (HTP grade) for lowpressure chromatography from Bio-Rad should an HPHT column be unavailable. Water for all solutions should be glass-distilled and deionized. Solutions

Buffer 1 is required for tissue homogenization and affinity chromatography. It contains 0.2 M NaC1, 2 mM dithiothreitol, 2 mM EDTA, 20 mM sodium phosphate, pH 7.0, 25 /~M phenylmethylsulfonyi fluoride. The last is added to the stirred solution from a syringe fitted with a 27-gauge needle containing a 25 mM stock solution in 2-propanol just prior to use Buffer 2, used for elution of GSTs from the affinity matrix, is also prepared just prior to use as follows: to 5 mM S-hexylglutathione in 0.1 mM Tris base is added 5 mM glutathione and the solution adjusted to pH 9.0 at 25° (i.e., pH 9.6 at 4°) Buffer 3, required for hydroxylapatite chromatography, contains 5% (w/v) glycerol, 0.5 mM dithiothreitol, 10 mM sodium phosphate, pH 6.8 11 W. H. Habig and W. B. Jakoby, this series, Vol. 77, p. 398.

56

GLUTATHIONE

[61

Buffer 4 is for the gradient elution from hydroxylapatite and contains 5% (w/v) glycerol, 0.5 mM dithiothreitol, 250 mM sodium phosphate, pH 6.8 Buffer 5, required for anion-exchange FPLC of alpha class GSTs, is composed of 10% (w/v) glycerol, 1 mM dithiothreitol, 30 mM piperazine hydrochloride, pH 9.55. The dithiothreitol is added after adjusting the pH. The buffer is made 0.2 M NaC1 for the gradient elution Buffer 6, for anion-exchange FPLC of mu class GSTs, contains 10% (w/v) glycerol, 1 mM dithiothreitol, 20 mM Tris-HC1, pH 7.8. The dithiothreitol is added after adjusting the pH. The buffer is made 0.3 M NaCI for gradient elution Buffer 7, used for the storage of GSTs, is 10% (w/v) glycerol, 50 mM NaC1, 0.2 mM dithiothreitol, 5 mM sodium phosphate, pH 7.0. All buffers used for FPLC are vacuum-filtered through glass micro fiber filters and 0.2-/.~m pore nitrocellulose.

Tissue Extraction and Affinity Chromatography Safety Note. Care should be taken in the handling and disposal of human tissue extracts in order to avoid exposure to pathogens. Vaccination against hepatitis B is advised. Starting Material. The routine preparation of 2-8 mg amounts of hepatic GSTs is as follows. Normal liver, obtained from either an organ donor unit or mortuary as soon as possible postmortem, is cut into approximately 1.5cm cubes and either used immediately or frozen in liquid N2 and stored at - 7 0 °. The sample chosen for illustration here was HL135, from Tennessee Donor Services (Nashville, TN), which in addition to GSTs A1-1, A1-2, and A2-2 contained the products of both the GSTMI*A and GSTMI*B alleles, namely, GSTMla-la, GSTMlb-lb, and GSTMla-lb. Day 1. Twenty grams of frozen liver is added to sufficient (~200 ml) buffer 1 to cover the blades of a Waring blendor. When partially thawed, the tissue is homogenized for 15 sec at low speed and 10 sec at high speed. The homogenate is centrifuged at 105,000 g for 1 hr and the supernatant poured through precooled glass wool to remove lipid. Meanwhile, 40 ml S-linked glutathione-agarose in a 5-cm diameter column is cooled and equilibrated by passing buffer 1 at 00-4 ° through it at approximately 1.5 ml/min under gravity. The soluble supernatant fraction is applied followed by 100-150 ml of buffer 1 and the effluent disposed of. Thirty milliliters of buffer 2 is applied to the column followed by more buffer I and fractions of approximately 6 ml collected. The elution of GSTs is followed by determining the optical absorption at 280 nm using buffer 2 as a blank (Fig. 1). It may be misleading to measure GST activity at this stage because the

[6]

GLUTATHIONE

TRANSFERASE

PURIFICATION

57

A28o 1,0

A2eo BUFFER 2 0

I

0

I

I

I

t

I

5o

VOL BUFFER 2

I (ml)

FIG. 1. Glutathione-agaroseaffinitychromatography.After application of the soluble fraction from 18 g liverto a 40-mlcolumnof glutathione-agaroseand washing,the GST pool (~25 mg) was eluted with buffer2.

S-hexylglutathione present is a potent inhibitor. The GST pool (-25 ml) is dialyzed twice against 1.5 liters unfiltered buffer 3 at 00-4 °. The affinity column is cleaned with 20 ml of 6 M guanidinium hydrochloride pH 7, and stored in 3 M NaCI; it may occasionally need repacking. The next step usually requires hydroxylapatite FPLC; however, if instead a low-pressure hydroxylapatite column is to be used, it is freshly prepared and equilibrated using approximately 20 g hydroxylapatite HTP in a column 1.5-2 cm in diameter. The column outlet is adjusted to maintain pressure at less than 25 cm of liquid. Day 2. The dialyzed GST pool is concentrated to 4-5 ml by laying the dialysis bag on a bed of cold dry polyethylene glycol, and buffers 3 and 4 for hydroxylapatite FPLC are made ready. Following equilibration with buffer 3, the HPHT column is washed with a 0-100% gradient of buffer 4 (15 ml) at 0.3 ml/min. Pressure should be applied to the HPHT column gradually, to avoid matrix compression and consequent low flow rates; the guard column is particularly sensitive in this respect. The FPLC steps are carried out at room temperature. The concentrated GST pool is applied to the HPHT column (2.5-ml portions using a 3-ml sample loop of PTFE tubing) at 0.3 ml/min, and the flow through, which contains a small amount of GSTPI-1, is collected. The mu and alpha class GST fractions are then eluted with a gradient of 0100% buffer 4 in about 30 ml (Fig. 2). Fractions (~0.7 ml) are collected in polystyrene tubes and kept on ice.

58

[61

GLUTATHIONE

ALPHA CLASS

A2ao

t MU CLASS I

/

/'/

I

/

GSTMla-la I

//

A/

0

0

/ !

10 20 GRADIENT VOL (ml)

|

--

. 10

FIG. 2. Separation of liver GSTs using hydroxylapatite. The GST pool (Fig. 1) was separated into alpha and mu classes using a Bio-Rad HPHT column fitted to an FPLC system at a flow rate of 0.35 ml/min. The flow-through fraction contains small amounts of GSTPI-1 and M3-3.

If low-pressure hydroxylapatite chromatography is used instead of FPLC, the concentration step is omitted and the column is run at 0°-4 ° with a 300 ml gradient at about 0.4 ml/min (day and night of day 2). The pooled GST fractions are then concentrated on day 3. Day 3. The alpha class GSTs are separated as follows. The FPLC Mono Q anion-exchange column is cleaned with 1 column volume of 70% acetic acid and equilibrated with 1 column volume of buffer 5 followed by 1.5 column volumes of filtered 2 M NaC1 and finally 5 column volumes of buffer 5. An effluent pH monitor may be used to ascertain equilibration. The alpha class GST pool is transferred into buffer 5 by applying 2 ml to each of three PD-10 (Sephadex G-25) columns, equilibrated in buffer 5, followed by 0.5 ml buffer 5; 3 ml buffer 5 is then added and the 3 eluates collected into a polypropylene syringe for application to the Mono Q column. Note that this procedure yields a more stringent removal of phosphate ions than would be achieved using the manufacturer's guidelines. The flow through should not contain GST. The AI-1, A1-2, and A2-2 enzymes are eluted with a linear gradient of buffer 5 containing NaC1. To obtain satisfactory separation when using the Mono Q HR5/5 column, less than 6 mg protein should be applied per run. Larger amounts may be separated using the HR10/10 column (Fig. 3). Because buffer 5 has a relatively high pH, after separation the fractions are transferred to buffer 7 using PD-10 columns

I6]

59

GLUTATHIONE TRANSFERASE PURIFICATION GSTA1-2

A28o

GSTA2-2

1.0

GSTA1-1

/

Y 0.1

/ / / / /

z / 0

--

I. /

,z 0

I 5O

I

--O

100

GRADIENT VOL (ml}

FIG. 3. Separation of major c~ class GSTs, The liver c~ class GST fraction (Fig. 2) was separated into GSTs AI-I, A1-2, and A2-2 using anion-exchange FPLC (Mono Q HR10/10) in buffer 5 at flow rate of 1.2 ml/min.

prepared in advance. Immediately after this exchange, the GST fractions are placed on ice. The GSTMI-1 fraction from hydroxylapatite chromatography is usually almost pure. If separation of Mla-la, Mla-lb, and Mlb-lb is required, the mu class GST pool is transferred to buffer 6 using PD-10 columns and the fraction applied to the Mono Q HR5/5 column. The flow-through fraction contains GSTMla-la. Elution with buffer 6 containing 0.3 M NaC1 (Fig. 4) yields first Mla-Mlb followed by Mlb-Mlb. Fractions are collected in plastic tubes on ice and transferred immediately into buffer 7 for storage. The purity and concentration of the GSTs are determined by reversedphase high-performance liquid chromatography (HPLC) and the activity determined (see below). If the GSTs are to be used within 3 weeks, they are kept on ice. For longer storage, small samples are transferred to polypropylene tubes and snap-frozen in liquid nitrogen. However, up to 30% loss of enzyme activity may occur on freezing and thawing. Yield. Liver GST content varies considerably. 12'13A GST-rich, GSTM1positive liver may yield 4-8 mg GSTA1 and A2 and 1-2 mg GSTM1 per 10 g tissue. 12B. van Ommen, J. J. P. Bogaards, W. H. M. Peters, B. Blaauboer, and P. J. van Bladeren, Biochem. Z 269, 609 (1990). 13 B. Ketterer, D. J. Meyer, E. Lalor, P. Johnson, F. P. Guengerich, L. M. Distlerath, P. E. B. Reilly, F. F. Kadlubar, T. J. Flammang, Y. Yamazoe, and P. H. Beaune, Biochem. Pharmacol. 41, 635 (1991).

60

GLUTATHIONE

[6]

[NaCI] (M)

Aa80

1.0

--

0.15

/ /

/ill// J

t~

m

~

0

,

,,/

t

I

10 Gradient vol (ml)

0

FIG. 4. Separation of GSTs Mla-la, Mla-lb, and Mlb-lb. The/z class GST fraction from the hydroxylapatite step (Fig. 2) was separated into allelic forms by anion-exchange FPLC (Mono Q HR5/5) in buffer 6 at a flow rate of 0.3 ml/min.

Purification of Glutathione Transferase P I- 1 from Kidney The human kidney is a good source of GSTPI-I but also contains alpha class GSTs together with small amounts of mu class GSTs, including M33. The procedure is very similar to that for the liver GSTs (see above) with the following modifications. The soluble fraction from 40 g of kidney is applied to the 40-ml affinity column. If the t~ class GSTs are not required, the hydroxylapatite step is omitted and the total GST pool brought to pH 7.4 by dropwise addition of 0.5 M sodium acetate, pH 6.3, and concentrated. If a hydroxylapatite column is used, the flow-throdgh fraction is concentrated. The GSTPI-I is then purified, either from the total GST pool or the hydroxylapatite flow-through fraction, by anion-exchange FPLC in buffer 6 exactly as described for the separation of GSTM-I (Fig. 5). Yield. Ten grams kidney should yield 2 - 4 mg GSTPI-I. Purification of Glutathione Transferase M3-3 from Testis The human testis contains high levels of alpha class GSTs, GSTM3-3, and GSTPI-I, together with smaller amounts of other mu class GSTs. The

[6]

GLUTATHIONE TRANSFERASE PURIFICATION

61

GST P1-1

A28o

/

I

z

/ /

0.2

0,5 z ¢

/

/

,"

GSTM3-3

Z

I

0

I •

l

l

10 GRADIENT VOL (ml)

20

FIG, 5. Purification of GSTP-1, The kidney GST pool was applied to a hydroxylapatite column and GSTPI-1 separated from the flow-through fraction by anion-exchange FPLC exactly as described for liver GSTMI-1 (Fig. 4). Small amounts of pure GSTM3-3 are also obtained.

GSTM3-3 may be purified by minor modification of the method for the purification of hepatic GSTs. The total GST pool, obtained by affinity chromatography, is dialyzed against buffer 3 modified to contain 50 mM rather than 10 mM sodium phosphate. The resulting flow-through fraction then contains GSTPI-1 and GSTM3-3 as major components, together with lesser amounts of other mu class GSTs. After concentration, this fraction undergoes anion-exchange FPLC in buffer 6, exactly as described above. If pure alpha class GSTs are no t required, the hydroxylapatite step can be omitted. In this case, the total GST pool is simply neutralized with 0.5 M sodium acetate (see above), concentrated, transferred into buffer 6, and applied to the Mono Q column (Fig. 6). Yield. Approximately 2 mg GSTM3-3 should be obtained per 10 g testis.

Purification of Glutathione Transferase M2-2 from Skeletal Muscle The concentration of GSTs in skeletal muscle is in the region of 4% that of the liver. However, because skeletal muscle accounts for 40% of body mass, the total hepatic and skeletal muscle GST activities are similar. Skeletal muscle is a very important component of extrahepatic GST activity. Glutathione transferases in skeletal muscle mainly comprise GSTs PI-1

62

[61

OLUTATHIONE GSTM3-3 CO

A2eo /

~

GSTPI-1

0

10

i i"

2O

z

0.15

0

GRADIENT VOL (ml) FIG. 6. Purification of GSTM3-3. The whole testis GST pool was fractionated by anionexchange FPLC (Mono Q HR5/5) under the conditions of Figs. 4 and 5. The major peak between GSTPI-1 and M3-3 is a heterodimer (M2-M3). Without prior hydroxylapatite separation, the GSTPI-1 peak is not pure.

and subunits M1 (in subjects with a G S T M I * G S T M 1 genotype), M2, and M3 occurring in both homo- and heterodimeric forms. Hussey et al. 18 have purified GSTM2-2 (referred to by them as N2N2) from skeletal muscle using similar techniques to those used above. A recommended procedure based on their published work is described below. Solutions

Buffer a is used for homogenization and affinity chromatography on S-hexylglutathione-agarose. The composition is 0.2 M NaC1, 0.5 mM dithiothreitol in 20 mM Tris-HC1 at pH 7.8. Elution from the affinity column is brought about with buffer a containing 5 mM S-hexylglutathione (the pH must be readjusted after addition of S-hexylglutathione) Buffer b is used for gradient elution by FPLC anion exchange using Mono Q columns. It contains 20 mM Tris-HC1 at pH 8.4, 0.5 mM dithiothreitol. Elution is in two stages using a linear gradient from 0 to 0.15 M NaC1 Buffer c for gradient elution from hydroxylapatitite chromatography is 10 mM sodium phosphate, pH 7.0, containing 0.5 mM dithiothreitol. Elution is brought about by a 10-350 mM phosphate gradient Procedure. To purify GSTM2-2 it is advisable to use muscle from subjects with a GSTM1 null phenotype, that is, muscle with activity toward trans-4-phenyl-3-buten-2-one 11 of less than about 0.04 nmol/min/mg protein. This avoids complications arising from the presence of M l a and M l b homodimers and their heterodimers with one another or with GSTM2

[6]

GLUTATHIONE TRANSFERASE PURIFICATION

63

and GSTM3. The purification procedure is carried out at 00-4 ° unless otherwise stated. For a final yield of approximately 1 mg M2-2, 300 g skeletal muscle is homogenized in 900 ml buffer a and clarified soluble supernatant obtained as described above. After dialysis against three changes of 5 liters of buffer a over 24 hr, the GSTs are separated by application to 60 ml S-hexylglutathione-agarose in a 5-cm-diameter column equilibrated in buffer a. After washing with 200 ml buffer a, the GSTs are eluted with buffer a containing 5 m M S-hexylglutathione and monitored at A280. The G S T pool (~40 ml) is dialyzed twice against 2 liters buffer b over 24 hr and concentrated to approximately 5 ml using polyethylene glycol. The sample is applied at 0.3 ml/min to an FPLC Mono Q column equilibrated in buffer b at room temperature and GSTs eluted with a gradient of 0-0.15 M NaC1 in buffer b. Two major peaks absorbing at 280 nm are obtained. The first to elute is a mixture of GSTs PI-1 and M2-2, whereas the second peak contains the GSTM2-3 heterodimer. Fractions from the first peak containing GSTM2-2 and GSTPI-1 are pooled and transferred into buffer c at room temperature and applied to a column of hydroxylapatite (Bio-Rad H P H T or low-pressure column of 1 x 10 cm; see above). The GSTPI-1 is not retained, and GSTM2-2 is eluted with a gradient of sodium phosphate.

Purification of T h e t a Class G l u t a t h i o n e T r a n s f e r a s e s E x p r e s s e d in E s c h e r i c h i a coli

The theta class enzymes have proved difficult to purify from rat and human tissue fully active and in high yield. 6a In the case of rat GST5-5 this has been overcome by its heterologous expression in Escherichia coli, 14 which has facilitated a simplified, high yield procedure. The methods used for purifying theta class GSTs from both rat and human tissues are very similar; therefore, it is anticipated that the simplified procedure will also be successful for the purification of rat GST12-12 and human GSTs TI-1 and T2-2 when bacterially expressed material becomes available. (The c D N A sequence of GSTT1 has already been obtained, is E q u i p m e n t . N e e d e d equipment includes an ultracentrifuge, low-pressure chromatography column 50 x 2.5 cm (i.d.), Pharmacia F P L C system or equivalent, and a Mono Q anion-exchange column. 14R. Thier, J. B. Taylor, S. E. Pemble, W. Griffith Humphreys, M. Permark, B. Ketterer, and F. P. Guengerich, Proc. Natl. Acad. Sci. U.S.A. 90, 8576 (1993). 15S. E. Pemble, K. R. Schroeder, S. R. Spencer, D. J. Meyer, E. Hallier, H. M. Bolt, B. Ketterer, and J. B. Taylor, Biochem. J. 300, 271 (1994).

64

GLUTATHIONE

[6]

Chemicals. The following reagents are required: Matrex gel Orange A (Amicon, Danvers, MA); DNase I; phenylmethylsulfonyl fluoride; dithiothreitol; polyethylene glycol (molecular weight 15,000-20,000); dialysis tubing; piperazine hydrate; and glycerol (low UV absorbing). Solutions

Buffer A used for homogenization contains 25/xM phenylmethylsulfonyl fluoride, 1 mM dithiothreitol, 10 mM sodium phosphate, pH 7.0 Buffer B for dialysis and Matrex gel Orange A chromatography contains 1 mM dithiothreitol, 1 mM EDTA, 5% (w/v) glycerol, 10 mM sodium phosphate, pH 7.0 Buffer C used for gradient elution from Matrex gel Orange A is buffer B made 1 M with respect to KC1 Buffer D for cleaning the Matrex gel Orange A is 6 M guanidinium hydrochloride adjusted to pH 7.0 with Tris base Buffer E used for guanidinium ion removal is 0.4 M sodium phosphate pH 7.0 Buffer F used for anion-exchange FPLC contains 5% (w/v) glycerol, 30 mM piperazine-HC1, pH 9.55, with 1 mM dithiothreitol added after pH adjustment. NaCI is added to 0.2 M for gradient elution. The FPLC buffers are filtered through glass microfiber and 0.2-/zm cellulose nitrate filters TABLE I SPECIFIC ACTIVITIESOF PURIFIED GLUTATHIONETRANSFERASES GST enzyme

Specific activity" (tzmol/min/mg protein)

A1-1 A2-2 M1-1 M2-2 M3-3 P1-1 R a t 5-5

130 130 250 171 b 15 140 220 c

a M e a s u r e d at 37 ° with 1 m M 1-chloro-2,4-dinitrobenzene and 1 m M glutathione at p H 6.5 b Results of H u s s e y et al. 8 c M e a s u r e d at 37 ° with 0.5 m M 1,2-epoxy-3 (pnitrophenoxy)propane and 5 m M glutathione at p H 6.5.

[6]

GLUTATHIONE TRANSFERASE PURIFICATION

65

Procedure. All operations except FPLC are carried out at 0o-4 °. Bacteria are disrupted by sonication in buffer A. DNase I (1000 Kunitz units per 3 liters original culture) is added, and the suspension is centrifuged for 1 hr at 105,000 g. The supernatant is dialyzed against three changes of 1 liter buffer B over 48 hr. The Matrex gel Orange A column is poured (2.5 × 25 cm) and washed with 15 ml buffer D to remove noncovalently bound dye, 30 ml buffer E to remove guanidinium ions, and equilibrated with 2 column volumes of buffer B. The dialyzed soluble fraction is applied to the column at a flow rate of 1-2 ml/min and the flow through discarded. The retained GST is eluted at a similar flow rate using a linear gradient formed from 200 ml buffer B and an equal weight of buffer C. The major peak absorbing at A280 is GST 5-5, which is approximately 85% pure. This fraction is concentrated by placing in dialysis tubing on a bed of dry polyethylene glycol. The Matrex gel Orange A column is cleaned with 15 ml buffer D and stored in 0.05% (w/v) sodium azide. The GST 5-5 is finally purified by anion-exchange FPLC (Mono Q) exactly as described above for the human ot class GSTs using buffer 5 (Fig. 3). The GST 5-5 elutes at about 25 mM chloride ion.

Analysis of Glutathione Transferase Purity and Activity Enzyme purity may be assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), isoelectrofocusing, and reversedphase HPLC. The HPLC method gives the best resolution and quantitative results. The column packing must be of the "wide-pore" type (>300/~) for high yield and around 5-/zm particle size for optimal resolution. Good separations have been described using Dynamax 16,17 and Vydac 12,1s columns. Elution is by a gradient of 0.05% (v/v) trifluoroacetic acid in acetonitrile. The GST subunits elute in the order PI, Mla, Mlb, AI, M3, and A2. Glutathione transferase activity is assayed using 1-chloro-2,4-dinitrobenzene as electrophilic substrate for the alpha, mu, and pi class enzymes and with 1,2-epoxy-3-(p-nitrophenoxy)propane for GST 5-5.11 To obtain accurate initial rates of reaction, it is necessary to dilute the GSTs with a suitable solution such as 15% (w/v) glycerol, 0.5 mM dithiothreitol, 50 mM sodium phosphate, pH 7.0. The specific activities of GSTs purified as described here are given in Table I. 16 D. J. Meyer, E. Lalor, B. Coles, A. Kispert, P. ~lin, B. Mannervik, and B. Ketterer, Biochem. J. 260, 785 (1989). 17 D. J. Meyer, J. M. Harris, K. S. Gilmore, B. Coles, T. W. Kensler, and B. Ketterer, Carcinogenesis 14, 567 (1993). 18j. A. Johnson, K. A. Finn, and F. L. Siegel, Biochem. J. 282, 279 (1992).

66

GLUTATHIONE

and

[71

[7] M e a s u r e m e n t of 7-Glutamyl Transpeptidase 7-Glutamylcysteine Synthetase Activities in Cells

B y HENRY JAY FORMAN, MICHAEL MING SHI, TAKEO IWAMOTO,

RuI-MINO LIU, and TIMOTHY W. ROBISON Introduction There has been an increasing interest in glutathione ( G S H ) as it is the major nonprotein thiol in cells and is a preferred, if not specific, thiol substrate for several enzymes in xenobiotic metabolism and antioxidant defense. Under such stress conditions, cells must maintain glutathione and may even increase glutathione content above the steady state for maximum protection. The intraorgan transport and d e n o v o synthesis of glutathione are two of the possible mechanisms for maintaining or increasing glutathione. I'2 As there is little circulating cysteine and only some epithelial cells have the capacity for uptake of intact glutathione, 3 circulating glutathione, produced principally by the liver, is itself the major source of the required cysteine for d e n o v o glutathione synthesis in other organs. 4'5 In intraorgan transport of glutathionc, "y-glutamyl transpcptidase [yGT; T-glutamyltransfcrasc, {5-glutamyl)-peptide:amino acid 5-glutamyltransfcrase, E C 2.3.2.2], an enzyme located on the outer surface of the cell membrane, catalyzes the first step in the breakdown of extracellular glutathione [Eq. (i)]. The physiological donor of the glutamyl moiety can be L-T-Glutamyl-L-cysteinylglycine + amino acid ---> L-T-glutamylamino acid + L-cysteinylglycine

(I)

glutathione or its thiyl conjugates, such as leukotriene C4 (LTC4). 6 The enzyme can also transfer the "y-glutamyl moiety from other compounds, such as 7-glutamyl-4-methoxy-2-naphthylamide, which has been used in histochemical staining for the enzyme, 7 and can act as a glutaminase. 8 The acceptor can be an amino acid, a dipeptide, another molecule of glutathione, 1M. E. Anderson and A. Meister, this series, Vol, 143, p. 313. 2S. M. Deneke and B. L. Fanburg, Am. J. Physiol. 257, L163 (1989). 3T. M. Hagen, L. A. Brown, and D. P. Jones, Biochem. Pharmacol. 35, 4537 (1986). 40. W. Griffith and A. Meister, Proc. Natl. Acad. Sci. U.S.A. 76, 5606 (1979). 5j. M. Williamson,B. Boettcher, and A. Meister, Proc. Natl. Acad. Sci. U.S.A. 79, 6246 (1982). 6L. Orning, S. Hammarstrom, and B. Samuelsson,Proc. Natl. Acad. Sci. U.S.A. 77, 2014(1980). 7 A. M. Rutenburg, H. Kim, J. W. Fischbein, J. S. Hanker, H. I. Wasserkrub, and A. M. Seligman, J. Histochem. Cytochem. 17, 517 (1969). s G. A. Thompson and A. Meister, J. Biol. Chem. 254, 2956 (1993). METHODS IN ENZYMOLOGY, VOL. 252

Copyright © 1995 by Academic Press, Inc. All rights of reproduction in any form reseiwed.

[7]

'y-GLUTAMYLTRANSPEPTIDASEAND TGCS

67

hippurate analogs, or water. 9 Once formed, both cysteinylglycine and Tglutamyl-amino acids can be further metabolized to supply amino acids to the cell that may then be used for synthesis, including that of glutathioneJ The rate-limiting step in glutathione biosynthesis from the constitutive amino acids is catalyzed by T-glutamylcysteine synthetase (TGCS; glutamate-cysteine ligase; EC 6.3.2.2) [reaction (2)]. Once formed, L-Glutamate + ATP + L-cysteine--> L-T-glutamyl-L-cysteine + ADP + Pi

(2)

T-glutamylcysteine can be linked to glycine in a second ATP-requiring reaction, catalyzed by glutathione synthetase, to produce glutathione. The enzyme also can use other amino acids as the acceptor of the T-glutamyl linkJ ° Principles

y-Glutamyl Transpeptidase The specificity of TGT toward y-glutamyl compounds is quite broad and has allowed the development of several different assays. The most common assay uses L-T-glutamyl-p-nitroanilide with the product detected by absorbance spectrophotometry, n A more sensitive assay was developed by using the fluorescent substrate L-y-glutamyl-7-amino-4-methylcoumarin (y-glutamyl-AMC) [Eq. (3)]. 12 The product, AMC, has quite well-sepaL-y-Glutamyl-AMC + glycylglycine--+ L-y-glutamylglycylglycine+ AMC

(3)

rated excitation-emission spectra from the substrate (Fig. 1). This assay has been modified (as described below) for use with homogenates of cells having quite low TGT activity. Another assay for TGT that has been used for measurement of TGT activity in cells depends on the conversion of LTC4 to leukotriene D4 (LTD4) and separation by high-performance liquid chromatography) 3 This assay was particularly useful in assaying the activity of a T-glutamyl cleaving enzyme related to TGT, which does not recognize the synthetic substrates T-glutamyl-AMC and y-glutamyl-p-nitroanilide.13 As other enzymatic or nonenzymatic reactions may cleave y-glutamyl com9 G. A. Thompson and A. Meister, J. Biol. Chem. 255, 2109 (1993). 10 G. F. Seelig and A. Meister, this series, Vol. 113, p. 379. 11 S. B. Rosalki, Adv. Clin. Chem. 17, 53 (1975). ~z G. D. Smith, J. L. Ding, and T. J. Peters, Anal. Biochem. 100, 136 (1979). 13N. Heisterkamp, E. Rajpert-De Meyts, L. Uribe, H. J. Forman, and J. Groffen, Proc. Natl. Acad. Sci. U.S.A. 88, 6303 (1991).

68

[7]

GLUTATHIONE

ooot

B 1000 -I-glu-AMC ex at 370

-...

T-glu-AMC em at 440 800.

8001

600.

600-

8

iO

m O

E_ 400-

A 400.

200

200. ...... ///

'\ A M C em at 440',,

O.

320

"

~o

............................... ii:::.................................. I

380

3~o

Excitation Wavelength (nm)

400

0 .

.

.

.

~

...................................................................

360' 3;0' 480' 4~0',~0' 4;0' 4~0 s;o Emission Wavelength (nm)

Fic. 1. Fluorescence spectra of 7-glutamyl-AMC and AMC. Solutions of 20/~M 7-glutamylAMC or 20/zM AMC in the assay buffer (see text) were used. Excitation spectra (A) were measured for y-glutamyl-AMC with emission at 400 nm (maximum emission wavelength for y-glutamyl-AMC) and 440 nm (maximum emission wavelength for AMC) and for AMC with emission at 440 nm. Emission spectra (B) were measured for 7-glutamyl-AMC with excitation at 349 nm (maximum excitation wavelength for y-glutamyl-AMC) and 370 nm (maximum excitation wavelength for AMC) and for AMC with excitation at 370 nm.

pounds, the inhibitor acivicin [AT-125; L-(aS,5S)-a-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid] is used to assess specificity of the enzymatic reaction. 14

"y-Glutamylcysteine Synthetase In the most commonly used assay for ~/GCS, A D P generated in reaction (2) is measured by coupling to the pyruvate kinase or lactate dehydrogenase reactions. 1° Other methods determine the Pi produced in reaction (2). 15'16 Unfortunately, both methods compete with other ADP- or Pi-forming 14 L. Allen, R. Meek, and A. Yunis, Res. Commun. Chem. Pathol. Pharmacol. 2?, 175 (1980). z5 A. N. Lestas and J. M. White, Ann. Clin. Biochem. 20, 241 (1983). 16 E. Beufler and T. Gelbart, Clin. Chim. Acta 158, 115 (1986),

[7]

"y-GLUTAMYLTRANSPEPTIDASEAND yGCS

69

systems, particularly ATPases, that are abundant in cells. Development of new assays with greater specificity has been required to enable measurement of activities in cells, where the rate of enzymatic catalysis is sufficient for physiological requirements but where the commonly used assays are too insensitive. One such method is the measurement of the product, 7-glutamylcysteine,iv To avoid the potential loss of -/-glutamylcysteine, which easily oxidizes, another assay using modifications of the method of Huang et al. TM substitutes L-a-aminobutyrate for L-cysteine [Eq. (4)]. 1° Buthionine L-Glutamate + ATP + L-c~-aminobutyrate L-3~-glutamyl-L-a-aminobutyrate + ADP + Pi

(4)

sulfoximine, an inhibitor of yGCS, is used to be certain that formation of product is through catalysis by this enzyme. 19

Methods y-Glutamyl Transpeptidase

The following assays are modifications of the method of Smith et al.12 Procedure 1: E n d Point Assay. A 0.1 ml enzyme solution (cell extract) is added to 0.25 ml of a reaction mixture containing 0.1 M 2-amino-2-methyl1,3-propanediol (ammediol)-HC1 buffer, pH 8.6, 20 mM glycylglycine, 20 /~M 7-glutamyl-AMC, and 0.1% (v/v) Triton X-100. y-Glutamyl-AMC can be prepared as a 1 mM stock solution in methoxyethanol by sonication. The reaction is carried out at 37° for 30 min and terminated by adding 1.5 ml cold 50 mM glycine buffer. The fluorescence is measured at 440 nm with an excitation wavelength of 370 nm. Product formation is calculated by comparison with a standard curve of AMC. The specific activity is confirmed by using acivicin (10/zM) to inhibit the enzymatic reaction. The advantage of the end point assay is that multiple samples can be assayed simultaneously. This is important when activity is low and requires a prolonged incubation to produce a significant amount of product. Fortunately, under such conditions product formation can be linear for hours. Procedure 2: Kinetic Assay o f E n z y m e Activity. When sufficient activity is present, the kinetic assay can be convenient. The reaction is started by adding 0.1 ml enzyme solution to a fluorescence cuvette containing 0.75 ml of the reaction mixture (as in procedure 1) and 0.15 ml of 0.2 M TrisHC1 buffer, pH 7.9, at 37 °. The fluorescence is monitored continuously at J7K. Ohno and M. Hirata, Biochem. Biophys. Res. Commun. 168, 551 (1990). 18C.-S. Huang, W. R. Moore, and A. Meister, Proc. Natl, Acad. Sci. U.S.A. 85, 2464 (1988). 19O. W. Griffith,J. Biol. Chem. 257, 13704 (1982).

70

GLUTATHIONE

[71

440 nm with excitation at 370 rim. The activity of the enzyme is calculated as in procedure 1.

y-Glutamylcysteine Synthetase The activity of yGCS is determined by measuring the formation of yglutamyl-a-aminobutyrate. Each reaction mixture (final volume 100 /zl) contains 100 mM Tris-HCl, pH 8.2, 50 mM KCI, 20 mM MgCI2, 2 mM EDTA, 10 mM ATP, 5 mM glutamic acid, 5 mM a-aminobutyric acid, and cell homogenate. To remove competing acceptors and inhibitors, cell homogenates are concentrated by centrifuging at 5000 g with a Centricon10 column (Amicon, Danvers, MA) at 4°. The mixture is incubated for 0, 15, or 30 rain at 37 ° to assess linearity. After the incubation, each reaction is terminated by adding 20/xl of 50% (w/v) trichloroacetic acid containing 0.5 mM norleucine as an internal standard. Samples are centrifuged at 15,000 g. The supernatants are analyzed using an amino acid analyzer system. A 2-/zl aliquot of each sample is exposed to phenylisothiocyanate in the presence of a base (diisopropylethylamine). The reaction is quantitative, yielding phenylthiocarbamoylamino compounds. Separation of the phenylthiocarbamoyl derivatives is optimized using a gradient buffer system. The buffers are filtered and degased. Solvent A consists of 140 mM sodium

from Tris buffer

0.6-

ID

E c

,;

0.4

o

0

0.2

0.00

1'0

2 1'5

20

"rime (rain) FIG. 2, Amino acid analysis for 7-glutamyl-a-aminobutyrate formation catalyzed by "yglutamylcysteine synthetase. Assays were performed as described in the text using an Applied Biosystems (Foster City, CA) Model 420A/130A amino acid analyzer.

[7]

~/-GLUTAMYLTRANSPEPTIDASEAND 7GCS

71

acetate buffer, pH 6.5, and solvent B is 70% acetonitrile and 30% (by volume) 32 mM sodium acetate buffer, pH 6.1. The derivatives are injected into a PTC C~8 column (Brownlee Scientific, 5 /zm, 220 × 2.2 mm i.d. obtained from Cole Scientific, Calabasas, CA) equilibrated in 95% solvent A and 5% solvent B at 38°. A linear gradient with a flow rate of 0.3 ml/ min is ramped to 32% solvent B in 10 rain, 60% solvent B in another 10 min, and finally 100% solvent B in 5 min. Elution is then maintained with 100% solvent B for 5 min. The elution of derivatives is monitored at 254 nm. The amount of ~/-glutamyl-~-aminobutyrate produced is quantified by comparison to authentic standards derivatized and analyzed as above. The retention times of the standard amino acids glutamate, 3,-glutamyl-a-aminobutyrate, a-aminobutyrate, and norleucine are 5.4, 8.7, 12.6, and 16.6 min, respectively (Fig. 2). The specificity of the method is confirmed using buthionine sulfoximine to inhibit 3~GCS. One unit of enzyme activity is defined as the amount that catalyzes formation of 1/zmol of product per hour at 37°. The method can detect differences of product formation in the nanomole range. Discussion Several studies have shown that yGT-mediated utilization of extracellular glutathione contributes to protection against oxidative injury. 2°-23 The importance of 3,GCS in maintaining glutathione in the face of the challenge of even endogenous generation of H202 has also been demonstrated. 24 Thus, development of assays sensitive enough for detection of low but physiologically significant activities of -/GT and yGCS will become increasingly important in evaluating the role of the enzymes in cellular resistance to toxic stresses.

2o G. L. Jensen and A. Meister, Proc. NatL Acad. Sci. U.S.A. 80, 4714 (1983). 21 M. F. Tsan, J. E. White, and C. L. Rosano, J. Appl. PhysioL 66, 1029 (1989). 22 H. J. Forman and D. C. Skelton, Am. J. Physiol. 259, L102 (1990). 23 M. Chang, M. Shi, and H. J. Forman, Am. J. Physiol. 6~ L637 (1992). 24 j. Martensson, A. Jain, W. Frayer, and A. Meister, Proc. Natl. Acad. Sci. U.S.A. 86, 5296 (1989).

72

GLUTATHIONE

[8]

Thiol Transport

from Human

[81

Red Blood Cells

By TAKAHITO KONDO, GEORGE L. D A L E , a n d ERNEST BEUTLER

Introduction Glutathione (GSH) is present in human red cells at a concentration of approximately 2.5 mM. The antioxidant functions of GSH are well described and have been substantiated by numerous genetic disorders reflecting increased oxidation as a result of abnormal GSH metabolismJ '2 Another role for GSH in the red cell is the detoxification of xenobiotics by covalent conjugation. Both of these roles for GSH result in products that the red cell can either metabolize or extrude into the extracellular space. Specifcally, excess oxidized glutathione (GSSG) is either enzymatically reduced or rapidly transported out of the cell. Similarly, GSH conjugates of xenobiotics are exported out of the erythrocyte. Both transport systems utilize ATP as an energy source for extrusion, and it is these transport processes that are the focus of this chapter. The transport of GSSG was first observed in oxidatively stressed red cells, both normal and glucose-6-phosphate dehydrogenase deficient.3 Transport was shown to be both rapid and energy-dependent. Subsequently, Lunn et aL 4 demonstrated that a modest basal level of GSSG transport occurred in native red cells at a rate sufficient to account for the previously unexplained turnover of intracellular GSH. In 1981, Board 5 demonstrated that red cells treated with 1-chloro-2,4-dinitrobenzene consumed nearly all of their intracellular GSH in conjugating this xenobiotic but, more importantly, the cells also extruded the GSH-2,4-dinitrophenyl adduct in an energy-dependent process. Further progress in delineating the nature of the GSSG transport process was made by utilizing inside-out vesicles (IOV) prepared from red cells to decipher the molecular requirements for the transport process. 6 This work not only revealed many of the parameters associated with GSSG transport but also complicated the issue by demonstrating that two transport pro1 E. Beutler and G. L. Dale, in "Coenzymes and Cofactors: Glutathione--Chemical, Biochemical and Medical Aspects, Part B" (D. Dolphin, R. Poulson, and O. Avramovic, eds.), p. 291. Wiley, New York, 1989. 2 X. Shan, T. Y. Aw, and D. P. Jones, PharmacoL Ther. 47, 61 (1990). 3 S. K. Srivastava and E. Beutler, J. Biol. Chem. 244, 9 (1969). 4 G. Lunn, G. L. Dale, and E. Beutler, Blood 54, 238 (1979). 5 p. G. Board, FEBS Lett. 124, 163 (1981). 6 T. Kondo, G. L. Dale, and E. Beutler, Proc. Natl. Acad. Sci. U.S.A. 77, 6359 (1980).

METHODSIN ENZYMOLOGY.VOL.252

Copyright© 1995by AcademicPress.Inc. All rightsof reproductionin any formreserved.

[8]

THIOL TRANSPORT FROM HUMAN RED BLOOD CELLS

73

cesses were apparently present in the red cell. O n e system was a lowaffinity, high-Vm~x transport system, and the other was a high-affinity, 1OWVm~x transport. Subsequent work has d e m o n s t r a t e d that the transport of G S H adducts also occurs through two separate transport systems. W h e t h e r the two systems are the same as for G S S G is still an unresolved question. The high-affinity, low-Vma~ transport system for G S H adducts is competitively inhibited by GSSG, whereas the low-affinity, high-Vmax transport is not. 7 A further advance in this field came with purification of a G S S G d e p e n d e n t A T P a s e f r o m h u m a n red cell m e m b r a n e s . 8 Additionally, a G S H conjugate-dependent A T P a s e has b e e n purified 9 which does not utilize GSSG. Characterization of these transport proteins will o p e n the way for cloning of the relevant genes. This is particularly important to resolve a long-standing discussion as to the relationship between the G S S G and G S H - a d d u c t transport systems and the P-glycoprotein found in multiple drug-resistant t u m o r cells lines. 1°

Thiol T r a n s p o r t f r o m W h o l e Cells O x i d i z e d Glutathione Transport f r o m Oxidatively Stressed R e d Ceils

The demonstration that oxidatively stressed red cells extruded G S S G in an energy- and t e m p e r a t u r e - d e p e n d e n t process 3 served as the foundation for this field and has resulted in the subsequent elucidation of the transport processes for G S S G as well as G S H adducts that are detailed below. Red cells are purified f r o m h u m a n blood by cellulose filtration u and washed four times with 0.9% (w/v) saline. The washed red cells are susp e n d e d at a 25% hematocrit in phosphate-buffered saline (PBS; 0.15 M NaC1, 10 m M NaH2PO4, p H 7.4) containing 14 m M glucose and 1.3 m M Na2CrO4. The sodium chromate is added to inhibit glutathione reductase ~2 and thereby allow accumulation of G S S G in an energy-replete cell. The mixture is placed in a W a r b u r g flask along with 200/.d of 30% (w/v) H202 in the center well of the flask. T h e system is then sealed, and the reaction 7 T. P. M. Akerboom, G. Bartosz, and H. Sies, Biochim. Biophys. Acta 1103, 115 (1992). T. Kondo, K. Miyamoto, S. Gasa, N. Taniguchi, and Y. Kawakami, Biochern. Biophys. Res. Commun. 162, 1 (1989). 9 R. Sharma, S. Gupta, S. V. Singh, R. D. Medh, H. Ahmad, E. F. LaBelle, and Y. C. Awasthi, Biochem. Biophys. Res. Cornrnun. 171, 155 (1990). 10I. C. West, Trends Biochern. Sci. 15, 42 (1990). u E. Beutler, in "Red Cell Metabolism: A Manual of Biochemical Methods." Grune & Stratton, New York, 1975. 12S. K. Srivastava and E. Beutler, Biochem. J. 114, 833 (1969).

74

GLUTATHIONE

[8]

proceeds with shaking for 2 hr. Samples are collected at intervals over a 4-hr period; red cells are pelleted, and the supernatants are saved for analysis. The GSSG extruded into the extracellular medium is quantitated enzymatically,I1 and corrections are made for hemolysis during the incubation. 3,~2 The rate of GSSG transport from human red cells under these conditions is approximately 65 nmol/hr/ml cells, a2

Oxidized Glutathione Transport from Native Red Cells The sensitivity required to measure transport from red cells that have not been oxidatively stressed so as to increase the level of GSSG is achieved by incubating the cells with [3H]glycine. This labels preexisting GSH through an exchange reaction catalyzed by glutathione synthetase. In a series of experiments utilizing energy-replete, unstressed red cells, the transport rate out of red cells was found to be 6.7 nmol/hr/ml of red cells. No transport of GSH was observed. The transport rate for GSSG also suggested that the half-life for intracellular GSH would be 4.7 days if the turnover of the peptide were strictly a function of transport. The value of 4.7 days agrees closely with previously measured turnover of total glutathione of 4 days. Human red cells are filtered through cellulose 11 and washed with 0.9% saline. The cells are suspended at a 20% concentration (v/v) in 1% (w/v) bovine serum albumin (BSA), 8 mM glucose, 62 mM NaC1, 40 mM NaH2PO4, 35 mM TES, pH 7.4 (buffer A), with 15/.,Ci of [3H]glycine/ml erythrocytes. The mixture is incubated at 37° for 4 hr, at which time the red cells are washed four times with 0.9% saline. To demonstrate transport the labeled red cells are resuspended at a 20% hematocrit in buffer A and incubated in a shaking water bath at 37°. The incubation is allowed to proceed for 150 min during which 3-ml aliquots are taken for analysis. Each aliquot is centrifuged and the supernatant saved for determination of [3H]GSSG. If hemolysis is noted in the supernatants, correction for release of intracellular metabolites must be taken into account. 4 The [3H]GSSG transported at each time point is quantitated by mixing 2 ml of the reaction supernatant with 14 ml of 0.5 mM glycine, 0.5 mM GSSG, pH 7, and each sample is applied to a 10 × 95 mm column of Dowex-l-X8 (formate form) preequilibrated with water. The column is then eluted with a 100 ml linear gradient of 0 to 1 M ammonium formate; 3.5-ml fractions are collected. A typical chromatographic profile is shown in Fig. 1. The relevant fractions are pooled, and total GSSG radioactivity associated with each time point is determined. To calculate the actual rate of GSSG transport from red cells, the specific

[8]

THIOL TRANSPORT FROM HUMAN RED BLOOD CELLS

75

o-o-o-o

18i

Q)~

14"

~"

10-

o

a~ 6-

/'/\

-loo

to

°~O~o "~o. o

o-o ._._~=Q=Q=Q=Q=Q=

.~ Q= Q=O~

E

\

-o

\O~o

6o

"8

2o

~

-o

FRACTION

FIG. 1. Chromatography of [3H]glutathione. Erythrocytes were incubated with [3H]glycine to label the glutathione pool. A portion of the cells were lysed, incubated with tert-butyl hydroperoxide to oxidize all of the GSH to GSSG, and then chromatographed on Dowex-1 formate. The column was eluted with a 0 to 1 M ammonium formate gradient. Radioactivity is represented by circles (©); the filled circles (Q) are GSSG (nmol/ml). The radioactivity that appeared before fraction 1 is from residual [3H]glycine. (Reprinted from Lunn et al. 4 with permission.) activity of the G S S G after the [3H]glycine incubation must be determined. A n aliquot of the labeled cells is incubated with an excess of tert-butyl hydroperoxide to oxidize intracellular G S H to GSSG. 13 The cells are then precipitated with an equal volume of 4% (w/v) perchloric acid at 0 °. The precipitated protein is pelleted by centrifugation at 1500 g for 15 min, and the supernatant is neutralized with 1 M K2CO3.11 A n aliquot is diluted 4fold with water and then applied and eluted from a Dowex-1 column as described above but without carrier GSSG. The quantitation of G S S G in each fraction is p e r f o r m e d enzymatically 11 and the specific activity calculated as counts per minute (cpm) per micromole of GSSG.

Glutathione-Adduct Transport from Red Cells W h e n red cells are exposed to a xenobiotic such as 1-chloro-2,4-dinitrobenzene (CDNB), enzymatic conjugation of glutathione with C D N B is a3 S. K. Srivastava, Y. C. Awasthi, and E. Beutler, Biochem. J. 139, 289 (1974).

76

GLUTATHIONE

[81

catalyzed by glutathione S-transferase with a resulting product of glutathione-S-2,4-dinitrobenzene (GS-DNP). The transport of the GSH adduct out of the red cell can be readily demonstrated. 5 Red cells are filtered through a-cellulose/microcellulose columns and washed as detailed above. To derivatize erythrocyte GSH, the washed cells are suspended in 4 volumes of 0.9% saline with 1.2 mM CDNB for 15 min at 37°. The cells are then washed twice with buffer A. Under these conditions, approximately 70% of the red cell GSH is conjugated. Transport experiments are performed in buffer A at approximately a 25% hematocrit. The cells are incubated at 37 ° with shaking and samples taken over a 2-hr period. Supernatants are collected by centrifugation, and GSH conjugate present in the supernatants is quantitated spectrophotometrically; the extinction coefficient at 340 nm for the GSH-CDNB conjugate is 9.6 mM -1. Again, corrections for lysis occurring during the transport experiments must be made. The maximum transport rate for GSH adducts is approximately 450 nmol/hr/ml red cells, 5 which is considerably higher than that for GSSG. 12 Alternatively, GS-DNP transport is studied after incubating erythrocytes with 45/zCi of [3H]glycine (25/zCi/nmol) for 2 hr at 37°, followed by washing with saline to remove most of the residual [3H]glycine. Before the transport experiment, cells containing labeled GSH are incubated with 0.5 mM CDNB at 37 ° for 1 hr. After the incubation, the cells are washed three times in 20 volumes of the incubation medium to remove excess CDNB and further incubated for 1 hr at 37°. After incubation, aliquots of the supernatant fraction of the incubation mixture are diluted with 10 volumes of 0.5 mmol/liter GS-DNP and 0.5 mmol/liter glycine, pH 7.0, and applied to a 10 × 95 mm column of Dowex-1 (formate form). Elution and fractionation are the same as those for the GSSG-transport experiment. To calculate the actual rate of GS-DNP transport from red cells, the specific activity of the GS-DNP after the incubation with [3H]glycine must be determined as described above for GSSG transport. Transport Studies with Inside-Out Vesicles Inside-out vesicles (IOV) serve as a useful tool for characterization of systems that transport substances from the inside to the outside of erythrocytes. The concentration of activators, inhibitors, as well as substrates and the temperature and pH of the medium are easily modified. IOV from human erythrocytes were first prepared by Steck and Kant TM using ultracentrifugation methods in a dextran density gradient. Alterna14T. L. Steck and J. A. Kant, this series, Vol. 31, p. 172.

[8]

THIOL TRANSPORT FROM HUMAN RED BLOOD CELLS

77

Km2=7"m lM

~o

y

Krn1=0.1mM

¢

0

I 10

I 20

I 30

I 40

I 50

(G~G,mM) "1 FIG. 2. L i n e w e a v e r - B u r k plot of GSSG transport. Inside-out vesicles were incubated with various concentrations of GSSG, 2 m M A T P , 10 m M MgCI2 in PBS for 2 hr at 37 °. (Reproduced from K o n d o et al. 6)

tively, IOV can be prepared from human erythrocytes using an affinity column of cellulose coupled to concanavalin A. 15 The purity of IOV prepared by the two methods is similar, although with either method there is some contamination with resealed right-side-out vesicles and unsealed vesicles. The existence of two different transport systems for GSSG was first observed using IOW.6 Both systems are dependent on the concentrations of ATP and GSSG (Fig. 2). As mentioned above, the rate of GSSG transport under physiological conditions is sufficient to account for the turnover rate of glutathione in erythrocytes, and larger amounts of GSSG are effluxed when the cells are exposed to oxidative stress. The presence of two transport processes for GSSG provides a reasonable explanation for these previous findings: the high-affinity, IoW-Vma~transport may be important to maintain the steady-state levels of glutathione, and the low-affinity, high-Vmax transport may serve as an emergency pathway for the elimination of GSSG during oxidative stress. Furthermore, pyrimidine nucleotide triphosphates such as CTP and UTP have been found to inhibit competitively the highaffinity, low-Vmax GSSG transport. 6 This inhibition may explain the high levels of intracellular GSH observed in hereditary hemolytic anemia associ15 T. Kondo, this series, Vol. 171, p. 217.

78

GLUTATHIONE

[8]

TABLE I TRANSPORTOF GLUTATHIONES-CoNJUGATESIN HUMANERYTHROCYTESa Erythrocyte Parameter

Reticulocyte-rich

Transport of GS-DNP (nmol/ml RBC/hr) GSH (nmol/ml RBC) ATP (nmol/ml RBC) -r-Glutamylcysteine synthetase (units/g Hb) Glutathione S-transferase (units/g Hb)

468 _+ 38 2.3 ± 0.1 1.9 ± 0.1 1.16 ± 0.11 6.8 -+ 1.7

Reticulocyte-poor 282 2.2 ± 1.8 ± 0.89 ± 6.7 -

42 0.1 0.2 0.16 1.9

a Erythrocytes were separated into two groups by centrifugation on the basis of specific gravity. Values are expressed as means ± SD of four different experiments. Extracellular GS-DNP was corrected by hemolysis. RBC, Red blood cell; Hb, hemoglobin. Data from T. Kondo, K. Yoshida, Y. Urata, S. Goto, S. Gasa, and N. Taniguchi, J. Biol. Chem. 268, 20366 (1993). ated with e r y t h r o c y t e pyrimidine 5 ' - n u c l e o t i d a s e deficiency, which is invariably associated with increased a m o u n t s o f pyrimidine nucleotides. 16 Active transport of G S - D N P can also be m e a s u r e d using this technology. T h e a p p a r e n t g m for G S - D N P is 940 ~ M at an A T P c o n c e n t r a t i o n of 2 m M , and that for A T P is 7 6 0 / z M at a G S - D N P c o n c e n t r a t i o n of 1 m M . 17 W h e n red cells are separated into two g r o u p s on the basis of specific gravity using P e r c o l l - H y p a q u e centrifugation, 18 the transport rate is higher in the reticulocyte-rich fraction than in the r e t i c u l o c y t e - p o o r fraction b o t h in native red cells and I O V f r o m the fractions, suggesting that the transport system for G S - D N P is inactivated or lost during reticulocyte m a t u r a t i o n (Table I). Oxidized Glutathione-Stimulated ATPase from Human R e d Cell M e m b r a n e s T h e existence o f A T P - d e p e n d e n t transport systems suggests the presence of A T P a s e s which are stimulated b y G S S G . E s t i m a t i o n o f the activity o f G S S G - s t i m u l a t e d A T P a s e o f h u m a n red cell m e m b r a n e s is difficult b e c a u s e erythrocytes are very rich in various types o f A T P a s e activities. A G S S G - s t i m u l a t e d A T P a s e was first d e m o n s t r a t e d in the p l a s m a m e m b r a n e fraction of rat h e p a t o c y t e s attached to polycationic b e a d s J 9 Utilization of 16T. Kondo, Y. Ohtsuka, M. Shimada, Y. Kawakami, Y. Hiyoshi, Y. Tsuji, H. Fujii, and S. Miwa, A m . I. Hematol. 26, 37 (1987). 17T. Kondo, M. Murao, and N. Taniguchi, Eur. J. Biochem. 125, 551 (1982). 18L. Vettore, M. C. DeMatteis, and P. Zampini, A m . J. Hematol. 8, 291 (1980). 19p. Nicotera, M. Moore, G. Bellomo, F. Mirabelli, and S. Orrenius, J. Biol. Chem. 260, 1999 (1985).

[8]

THIOL TRANSPORT FROM HUMAN RED BLOOD CELLS

79

such polycationic beads also allowed the demonstration of enzyme activity in red cell membranes, and the activity can also be demonstrated in erythrocyte membranes solubilized in detergents.

Assay Attachment of Red Cell Membranes to Polycationic Beads. Erythrocyte membranes are ionically attached to polyethyleneimine-coated beads (AffiGel 731, Bio-Rad, Richmond, CA) as described by Jacobson and Branton. 2° Two grams of beads are washed three times with 0.2 M NaC1, twice with 150 mM Tris-HCl buffer, pH 7.4, and once with buffer A (7 volumes of 310 mM sucrose and 3 volumes of 49 mM sodium acetate, pH 5.0). Eight milliliters of a 50% (v/v) suspension of beads is added slowly to 4 ml of washed erythrocytes suspended in buffer A. After washing with buffer A twice, hemoglobin is removed from the erythrocytes attached to the beads by washing three times in 30 volumes of 10 mM Tris, pH 7.4, at 0°. The cell membranes are further disrupted by sonication for 5 sec. The membrane/bead suspension, washed with Tris-HC1, pH 7.4, is resuspended in the same buffer and kept at 0 ° until use. Oxidized Glutathione-Stimulated ATPase Activity Using Membrane/ Bead Suspension. One hundred microliters of membrane/bead suspension (20-30 /xg protein) is incubated in a 250-/~1 system containing 100 mM Tris-HC1, pH 7.4, 0.5 mM EDTA, 10 mM MgCl2, 1 mM ATP. The GSSG is added at the concentration indicated. No GSSG is present in the blank system. After preincubation for 5 min at 37°, the reaction is initiated by the addition of 1 mM ATP. The reaction is terminated after 30 rain by the addition of 250/~l of ice-cold 8% (w/v) trichloroacetic acid. The tubes are centrifuged in the cold, and inorganic phosphate (Pi) is extracted from the supernatant into benzene/isobutanol (1:1, v/v) as a molybdate complex 2~ and assayed colorimetrically. The activity is quantitated by Pi released by the addition of GSSG and is expressed as nanomoles of Pi released per minute per milligram protein. Membrane protein is removed from the polycationic beads as described by Cohen et a1.,22 and protein concentration is determined according to the method of Redinbaugh and Turley. 23 Figure 3 depicts the GSSG-stimulated ATPase present in red cell membranes attached to polycationic beads. The activity of the enzyme is esti20 B. S. Jacobson and D. Branton, Science 195, 302 (1977). 21 E. Beutler and W. Kuhl, Biochim. Biophys. Acta 601, 372 (1980). ~2 C. M. Cohen, D. I. Kalishi, B. S. Jacobson, and D. Branton, J. Cell Biol. 75, 119 (1977). 23 M. G. Redinbaugh and R. B. Turley, Anal. Biochem. 153, 267 (1986).

80

GLUTATHIONE

[8]

'7,

E ~. 0.8

~ 0.6 e

~ OA

> 0.2 ~

f o

Kin2=2.0raM ,'o

'

s'o

,;o

(GSSG concentration)l,(mM) 1 FIG. 3. Lineweaver-Burk plot of GSSG-stimulated ATPase activity. The nanomoles Pi released per minute per milligram protein of erythrocyte membranes attached to polycationic beads is expressed as a function of GSSG concentration. [From T. Kondo, Y. Kawakami, N. Taniguchi, and E. Beutler, Proc. Natl. Acad. Sci. U.S.A. 84, 7373 (1987).]

mated as a function of GSSG concentration ranging from 25 /zM to 10 mM. Lineweaver-Burk analysis of the data gives a biphasic plot with two apparent Km values of 0.13 and 2.0 m M GSSG, having apparent Vma~values of 6.4 and 38.5 nmol of Pi released per minute per milligram protein, respectively. Oxidized Glutathione-Stimulated A TPase Using Solubilized Membranes. When the activity of the GSSG-stimulated ATPase is estimated using solubilized red cell membranes, the assay mixture consists of 150/zl of enzyme solution in a 250-/xl system containing 100 m M Tris-HCl, pH 7.4, 0.5 mM EDTA, 10 mM MgCI2, 1 m M ATP, and GSSG. To estimate activities of the distinct GSSG-stimulated ATPases, 50/zM and 5 mM concentrations of GSSG are used to detect the low-Kin and high-Kin enzymes, respectively. After 5 min of preincubation followed by incubation for 30 min at 37 °, the reaction is terminated by the addition of 250/zl of 8% (w/v) trichloroacetic acid as described above. When the protein concentration in the assay system is less than 20/zg, 100/zg bovine serum albumin is added after termination of the reaction. In some studies, [3,-32p]ATP with a specific activity of 1.5 pmol per disintegration per minute (dpm) is used to quantitate [32P]Pi release. Glutathione S-Conjugate-Stimulated A TPase of Human Red Cell Membranes. S-(2,4-Dinitrophenyl)glutathione (GS-DNP) is prepared enzymati-

[8]

THIOL TRANSPORT FROM HUMAN RED BLOOD CELLS

81

2.0

200

2o01/

o

,00 o ._E 6_o100

d~ [] 0

10

20

30 Fraction No.

40

FIG. 4. Affinity chromatography of two GSSG-stimulated ATPases. Erythrocyte membranes solubilized in 0.25% Triton X-100, 10 mM imidazole hydrochloride, pH 7.4, 0.2 mM EDTA, 50/xM 2-mercaptoethanol, 0.5 m M phenylmethylsulfonyl fluoride were chromatographed on S-hexylgluthathione-Sepharose 6B. Elution was with a 0-2 mM S-hexylglutathione gradient. Protein concentrations are represented by filled circles, activities of GSSG-stimulated ATPases by open bars, and GS-DNP-stimulated ATPase by hatched bars.

cally using glutathione S-transferase as described by Wahllander and Sies. 24 The [3H]GS-DNP is prepared using [3H]GSH and 1-chloro-2,4-dinitrobenzene according to the same method used for the unlabeled compound. The activity of the GS-DNP-stimulated ATPase is measured at 37° in a 250-/xl system containing 100 mM Tris-HCl, pH 7.4, 0.5 mM EDTA, 10 mM MgCI2, l m M [y-32p]ATP, 0.1 mM GS-DNP, and 150/zl enzyme solution. As a blank system, no GS-DNP is added to the assay mixture. Released [3aP]Pi is measured in a liquid scintillation counter after extraction with benzene/isobutanol. The activity is expressed as the amount of P~ released by the addition of GS-DNP.

Purification of Oxidized Glutathione-Stimulated A TPase from Human Red Cells Unless otherwise indicated, all procedures are performed at 4°. Hemoglobin-free red cell membranes are prepared by lysing 100 ml of washed red cells in 10 volumes of 10 mM Tris-HCl, pH 7.4, at 0°, and washing five times in the same buffer. White ghosts are suspended in 20 volumes of 0.5 mM Tris-HC1, pH 8.0, for 12 hr to remove most of the extrinsic proteins such as spectrin. Spectrin-depleted ghosts are collected by centrifugation 24 A. Wahllander and H. Sies, Eur. J. Biochem. 96, 441 (1979).

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at 15,000 g for 30 min and are solubilized in 0.5% (w/v) Triton X-100 in 10 m M imidazole hydrochloride, pH 7.4, containing 0.2 mM EDTA, 50 ~ M 2-mercaptoethanol, 0.5 mM phenylmethylsulfonyl fluoride (buffer B), for 20 min. Insoluble proteins are removed by centrifugation at 10,000 g for 90 rain. The extract is diluted to adjust the concentration of Triton X100 to 0.25% and then applied to a column of S-hexylglutathione-Sepharose 6B (1 × 6 cm). The column is washed with 50 ml of 0.5 NaC1 in buffer B containing 0.25% Triton X-100, and bound material is eluted with a 60-ml linear gradient of 0-2 m M S-hexylglutathione at a flow rate of 8 ml/hr. Fractions of 1 ml are collected, and the enzyme activities and protein concentration of fractions are determined. As shown in Fig. 3, the presence of of two distinct GSSG-stimulated ATPases is observed by elution from S-hexylglutathione-Sepharose 6B. The specific activity of the low-Km enzyme is 1200 nmol of Pi released/ min/mg protein, and the protein purified from 100 ml erythrocytes is 0.09 mg. The specific activity of the high-Kin enzyme is 980 nmol of Pi released/ min/mg protein, and the protein purified is 0.1 mg. The low-affinity, but not the high-affinity transport system is activated by treatment with dithiothreitol and inhibited by the addition of p-chloromercuribenzoate, iodoacetamide, or N-ethylmaleimide. Mixed disulfides other than GSSG such as Glu-Cys-SSG and Cys-SSG partially stimulate the enzyme, but GSH has no effect. GS-DNP-stimulated ATPase is purified from red cell membranes using the method as described above for GSSG-stimulated ATPase. 25'26 The elution of GS-DNP-stimulated ATPase is identical to that for the highKr~ form of GSSG-stimulated ATPase (Fig. 4). The enzyme has an apparent Km value of 0.14 m M for ATP and 0.038 m M for GS-DNP. These results show the presence of two different GSSG-stimulated ATPases in human red cell membranes with properties resembling those of the GSSG-transport systems. It seems reasonable to conclude that the enzymes play a role in the outward transport of thiols in human red blood cells.

25 T. Kondo, K. Yoshida, Y. Urata, S. Goto, S. Gasa, and N. Taniguchi, Z BioL Chem, 268, 20366 (1993). 26 T. Kondo, Y. Kawakami, N. Taniguchi, and E. Beutler, Pro~ N a ~ Aca& ScL U.~A. 84, 7373 (1987).

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Glutathione

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Mutants

By JAMES A. FUCHS

Introduction This chapter concentrates on Escherichia coli mutants altered in the synthesis, reduction, or utilization of glutathione as a model system but briefly discusses some mutants in yeast and humans. Although some contradictory results have been reported for mutants altered in glutathione metabolism, these differences can probably be explained by a combination of the redundancy of functions in E. coli that reduce thiols and a strong selection pressure to compensate for mutations that decrease the cellular concentration of reduced glutathione. As an example, we find that a null gshA mutant strain made by P1 transduction is unable to grow at 42 ° unless supplied with glutathione but the same strain after being maintained for some time under laboratory conditions grows at 42 ° (J. A. Fuchs, unpublished). The first observation that there may be selection pressure to alter glutathione metabolism under laboratory conditions came from a study by Owens and Hartman ~ who found that laboratory strains of E. coli and Salmonella typhimurium characterized excreted up to 30% of their glutathione into the medium, whereas fresh isolates excreted at least 19-fold less. Because the excretion could be eliminated by azide, it was energy dependent. Although 50% of the glutathione in the medium was oxidized, reconstruction experiments 1 indicated that most of the oxidation must have occurred in the aerobic medium after excretion and in sample preparation. As another example, Russel and Holmgren 2 isolated a grx mutant that appeared to have a very different phenotype than that isolated by Kren et aL 3 However, when a double mutant 4 was constructed that was defective in glutaredoxin and thioredoxin, the strain obtained was found to have mutated in several other genes in addition to grx and trxA, presumably to compensate for the primary mutations. Thus, one must be careful in maintenance of mutant strains as well as in selections to prevent accumulation of secondary mutations. This chapter discusses how E. coli mutants defective in the various i R. A. Owens and P. E. Hartman, J. Bacteriol. 168, 109 (1986). 2 M. Russel and A. Holmgren, Proc. Natl. Acad. Sci. U.S.A. 85, 990 (1988). 3 B. Kren, D. Parsell, and J. A. Fuchs, J. Bacteriol. 170, 308 (1988). 4M. Russel, P. Model, and A. Holmgren, J. Bacteriol. 172, 1923 (1990).

METHODS IN ENZYMOLOGY, VOL. 252

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steps of glutathione synthesis, reduction, and utilization are isolated and characterized and then briefly discusses similar mutants in yeast as well as in humans.

gshB M u t a n t s The first Escherichia coli mutants found to be defective in the synthesis of glutathione were simultaneously isolated and characterized by two groups. 5'6 In an attempt to isolate a mutant in thioredoxin or thioredoxin reductase, Fuchs and Warner 5 isolated a mutant in gshB (gene encoding glutathione synthase) by selecting for ceils with a decreased capacity to reduce ribonucleotide diphosphates to deoxyribonucleotide diphosphates. In the gshB mutant, the activity of one of the subunits of ribonucleotide reductase, the enzyme that carries out the reaction, was significantly decreased. The defect was corrected by restoring glutathione synthase, indicating that gshB was responsible for the decreased activity. The gshB mutant contained no detectable glutathione but contained an increased pool of yglutamylcysteine, approximately the same amount as the glutathione pool in the parent. The gshB mutant had an increased sensitivity to the oxidant diamide. 7 Diamide led to a rapid conversion of small molecular weight thiols to disulfides in the mutant strain but not the parental strain, suggesting that the mutant cell is able to keep y-glutamylcysteine reduced by a system other than glutathione reductase (y-glutamylcysteine is not a substrate for glutathione reductase). However, that system is insufficient to keep small molecular weight thiols reduced when challenged by an external oxidant. The gene encoding gshB was cloned by complementation of a gshB, trxB double mutant which requires glutathione for growth. 8 The cloned gene, inactivated by insertion of a kanamycin cassette, was integrated into the E. coli chromosome by homologous recombination. The gene was then used to map the locus to 63.4 rain on the genetic map and to a region between 3100 and 3120 kilobase pairs on the physical map. s Apontoweil and Berends 6 also isolated a gshB mutation by selection of diamide-resistant ceils followed by detection of colonies that failed to react with sodium nitroprusside. Their studies found that the gshB mutant grew slightly slower than its parent and was devoid of detectable glutathione; however, the mutant was not extensively characterized.

5j. A. Fuchs and H. R. Warner, J. Bacteriol. 124, 140 (1975). 6p. Apontoweil and W. Berends, Biochem. Biophys. Acta 399, 10 (1975). 7 K. A. Hibberd, P. B. Berget, H. R. Warner, and J. A. Fuchs, J. BacterioL 133, 1150 (1978). 8T. Daws, C.-J. Lim, and J. A. Fuchs, J. Bacteriol. 171, 518 (1989).

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gshA M u t a n t s In the same selection used to isolate a $shB mutant, Apontoweil and Berends 6 obtained three gshA mutants. The gshA mutants were found to have an increased sensitivity to a large number of chemical agents, suggesting that the mutation may have altered membrane permeability. However, Apontoweil and Berends 6 did not find that the gshA mutants had increased sensitivity to X rays. The gshA mutation was mapped by P1 transduction to the pheA-naIB region" and shown to be closely linked to srl: :Tnl0. ~°

O t h e r gshA a n d gshB M u t a n t s In an attempt to isolate a mutant consitutive for an adaptive response to alkylating agents, Sedgwick and Robins ix isolated mutants with decreased glutathione content. Because a thiol compound is required for activation of N-methyl-N'-nitro-N-nitrosoguanidine and glutathione is a major source of thiols, this phenotype was not unexpected. In fact, Apontoweil and Berends 6 had earlier observed the phenotype in gshA mutants. Greenberg and Demple ~2 took advantage of the phenotype to isolate an insertional inactivated allele of the gshA gene that allows gshA to be easily moved between strains. The gshA : :TnlOkan mutant was used to investigate the role of glutathione in protection against oxygen during X-irradiation in E. coll. Although Apontoweil and Berends 6 found no protection by glutathione, Morse and DahP 3 using a different gsh allele and nonisogenic strains found that glutathione was the key to the oxygen effect. The study by Greenberg and Demple 12 was consistent with those of Apontoweil and Berends 6 and found no effect of glutathione on the oxygen effect present during X-irradiation. Using 6°C0 as a source of y-irradiation, H a r r o p et al.a4 measured the oxygen enhancement of killing in gshA and gshB mutants and found that the mutants did affect the oxygen enhancement. The fast chemical repair of O2-dependent damage in the strains was measured using a fast mixing and irradiation method, namely, the gas explosion technique. The chemical repair rates in the various E. coil strains varied approximately in proportion to the 02 concentration that gives half-maximal 02 effects (K value), and both the rates of chemical repair and the K values correlated 9p. Apontoweil and W. Berends, Mol. Gen. Genet. 141, 91 (1975). ~oB. L. Hailer and J. A. Fuchs, J. Bacteriol. 159, 1060 (1984). ~1B. Sedgwick and P. Robins, Mol. Gen. Genet. 180, 85 (1980), 12j. T. Greenberg and B. Demple, J. Bacteriol. 168, 1026 (1986). 13M. L. Morse and R. H. Dahl, Nature (London) 271, 661 (1978). 14H. A. Harrop, K. D. Held, and B. D. Michael, Int. J. Radiar Biol. 59, 1237 (1991).

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approximately with the levels of nonprotein sulfhydrals in the various strains. The use of E. coli gsh mutants as a model for the role of glutathione in oxidant stress in mammalian hepatocytes was evaluated by Romero and Canada.15 They concluded that the lack of a correlation of GSH content with either oxidant-induced growth inhibitory or lethality effects, the presence of catalase in the cytoplasm in E. coli rather than in peroxisomes as in mammals, and the absence of glutathione peroxidase, made E. coli a poor model for mammalian oxidant stress. Accumulation of K ÷ and glutamate plays a primary role in maintaining osmotic balance in E. coli, as illustrated by the high concentrations of the ions present in cells growing in medium of high osmolarity. McLaggan et al. 16 found that two 3,-glutamyl peptides and glutamine also accumulated during growth at high osmolarity. A mutant deficient in glutathione owing to an insertion in the gshA gene was unable to grow above 1.4 osM, grew more slowly at intermediate osmolarities, and took longer to start growing following osmotic upshock. The involvement of glutathione in osmoregulation was independent of the effect of glutathione on K ÷ retention. Selenite toxicity in Salmonella (a species closely related to E. coli) involves the ability of selenite to act as an oxidizing agent. 17The gsh mutants were found to exhibit a biphasic killing curve as a function of selenite concentrations. At low concentrations, the mutants were more sensitive to selenite than the parent strain, presumably owing to the lack of sulfhydrals, but at higher concentrations the mutants were more resistant than the parent strain. Under these conditions the lack of sulfhydrals appeared to protect as sulfhydrals are needed to form more reactive oxidizing species. Gardner and FridovichTM investigated the effect of glutathione on the superoxide-sensitive [4Fe-4S]-containing aconitase of Escherichia coli. A gshA mutant grew slower than did the parental strain in a defined medium, and this effect was more pronounced when succinate, as opposed to glucose, was supplied as the carbon source. This suggested that the citric acid cycle was compromised in the gshA strain. Aconitase activity was approximately 25% lower in the gshA cells compared to the parental strain growing on either glucose or succinate. Addition of glutathione to the medium stimulated growth of the gshA strain on succinate and also elevated the aconitase activity even in the presence of chloramphenicol, which was added to block protein synthesis. In the presence of 4.2 atm of 02, however, aconitase 15 M. J. Romero and A. T. Canada, Toxicol. Appl. PharmacoL U l , 485 (1991). 16D. McLaggan, T. M. Logan, D. G. Lynn, and W. Epstein, J. Bacteriol. 172, 3631 (1990). 17 G. F. Kramer and B. N. Ames, Mutat. Res. 201, 169 (1988). 18p. R. Gardner and I. Fridovich, Arch. Biochern. Biophys. 301, 98 (1993).

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activity decreased in b o t h m u t a n t and parent, with the recovery of the mutant strain being much slower than that of the parent strain. Because both aconitase and the R1 subunit of ribonucleotide reductase have decreased activity in the absence of glutathione, 5 it is reasonable to conclude that other enzymes are likewise affected by the absence of glutathione in the cell.

g s h M u t a n t s in E s c h e r i c h i a coli B Although most experiments with glutathione mutants, like most other experiments in E. coli, have b e e n conduced with K strains, some work has also been conducted with B strains. T h e B strains were isolated independently from K strains and have a different modification and restriction system, making transfer of genes between t h e m difficult and thus allowing t h e m to diverge during evolution. Murata et al. 19 isolated an E. coil B mutant that was resistant to methylglyoxal and found that the mutant excreted 50-fold the normal level of glutathione when given methylglyoxal. The mutant had high levels of the glutathione synthesizing enzymes as well as glyoxal I and II. Murata and K i m u r a 2° isolated a gsh mutant in E. coli B by starting with the strain that excreted glutathione. The strain was mutagenized and then plated on a plate seeded with 107 cells of a strain that was deficient in the synthesis of cysteine but was able to use glutathione as a source of cysteine. Mutants that failed to form halos of the seeded ceils were collected and analyzed for glutathione synthase (gshII) and T-glutamylcysteine synthetase activity (gshI). The mutants had properties similar to gsh mutants in E. coli K12 strains. The gshII gene was cloned by selecting for resistance to tetramethylthiuram disulfide in a gshII mutant 21 and then was sequenced. 22

gor (gshC) M u t a n t s Mutants defective in glutathione reductase (gor) were isolated by different methods. Tuggle and Fuchs 23 employed the same selection used to obtain gshA mutants, assuming that these mutants would be sensitive to diamide. Davis et al. 24 isolated gor mutants without selection using transpo19K. Murata, K. Tani, J. Kato, and I. Chibata, J. Gen. Microbiol. 120, 545 (1980). 20K. Murata and A. Kimura, J. Gen. Microbiol. 18, 10047 (1992). 21K. Murata, T. Miya, H. Gushima, and A. Kimura, Agric. Biol. Chem. 45, 2131 (1981). 22H. Gushima, S. Yasda, E. Soeda, M. Yokoa, M. Kondo, and A. Kimura, Nucleic Acids Res. 12, 9299 (1984). 23C. K. Tuggle and J. A. Fuchs, J. Bacteriol. 162, 448 (1985). 24N. K. Davis, S. Greer, M. C. Jones-Mortimer, and R. N. Perham, J. Gen. Microbiol. 12, 631 (1982).

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son mutagenesis followed by direct assays of glutathione reductase activity in colonies on plates. The gor mutation was mapped between 77 and 78 min on the E. coli map and was found to be 32% cotransducible with xyl with T4GT7 phage but not with P1 phage. 24 The gor gene was linked t o pit by P1 transduction, and a clone with an insert of 25.5 kb was found to contain the gor gene approximately 10 kb away from the pit gene. 25 Greer and Perham 26 also isolated a clone that contained the gor gene and determined the DNA sequence of gor. In a selection for hydrogen peroxide-sensitive E. coli cells, Barbado et al.27 found that approximately 20% of the mutants were gor, but the mutants were not further characterized. In accordance with the observation that y-glutamylcysteine in a gshB mutant is reduced although it is not a substrate for glutathione reductase, the characterization of a gor mutant 23 indicated that glutathione reductase was not required in vivo to keep glutathione reduced. Escherichia coli has alternate systems that can keep the redox state of the cell almost fully reduced without glutathione reductase. The addition of an amount of diamide to the gor mutant strain that failed to alter significantly the ratio of reduced glutathione to total glutathione in the parent oxidized most of the cellular glutathione in the gor mutant. Thus, glutathione reductase is not essential for E. coli and does not seem necessary to keep glutathione reduced, but it is important in conditions that are more oxidizing than in an aerobically growing culture of E. coli. 23 Kunert et aL 2s using a cloned gor gene in a gor mutant to investigate the sensitivity of E. coli to methyl viologen found that sensitivity increased with glutathione reductase content. They further reported that the activity of glutathione reductase is essential to maintain a high glutathione content. This result is surprising as glutathione content appears to be controlled by the level of cysteine in the cell. Perhaps the amount of glutathione excreted is affected by reduction. grx M u t a n t s

Glutaredoxin in E. coli can couple the oxidation of glutathione to the reduction of disulfide bonds. Mutants deficient in glutaredoxin were ob25 C. M. Elvin, N. E. Dixon, and H. Rosenberg, Mol. Gen. Genet. 204, 477 (1986). 26 S. Greer and R. N. Perham, Biochemistry 25, 2736 (1986). 27 C. Barbado, M. Ramirez, A. Blanco, J. Lopez-Barea, and C. Pueyo, Curr. Microbiol. 8, 251 (1983). 28 K. J. Kunert, C. F. Cresswell, A. Schmidt, P. M. Mullineaux, and C. H. Foyer, Arch. Biochem. Biophys. 282, 33 (1990).

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tained in a selection by Kren et al. 3 based on the assumption that a double mutant defective in the genes encoding thioredoxin reductase and glutaredoxin would have the same phenotype as double mutants defective in other steps of the glutaredoxin-dependent and the thioredoxin-dependent pathways of reduction of protein thiols. These types of double mutants were all unable to reduce SO]- and thus had a requirement for a reduced sulfur source. Because the double mutants were viable at least at temperatures of 37° or less, these double mutants had sufficient ribonucleotide reductase activity for DNA synthesis? Two mutants defective in glutaredoxin were isolated and characterized. Both failed to grow without a reduced sulfur source. Introduction of a trxB gene on a plasmid failed to restore growth with sulfate, indicating that the grx gene product was required for the reduction of sulfate. Strains containing a grx mutant and a nonmutant trxB gene on a plasmid formed filaments when grown at 42°, indicating they were defective in reduction of ribonucleotides. Introduction of a plasmid-borne grx gene into the strains restored all phenotypes to the parental type. Russel and Holmgren z produced a grx null mutant by gene replacement of the chromosomal gene with a cloned grx gene that had been interrupted by the gene encoding kanamycin. The mutant had a phenotype that did not differ from its parent. Because a null mutant would be expected to have a more severe phenotype than a point mutant, this mutant strain appeared to differ from those isolated by Kren et al. 3 owing to either secondary mutations or strain differences. The null grx mutation was mapped at 18.5 min of the E. coli map. 2 A derivative strain that also contained a null trxA mutation was constructed.4 This double mutant had a cystine requirement as well as having a cell separation defect and formed colonies that were mucoid. The latter two phenotypes were found to be independent of the trxA and grx mutations. In addition, Russel et al. 4 found that the double mutant had accumulated an additional mutation in cysA. They observed that in the absence of cystine a trxA, grx strain is inviable owing to accumulation of 3'-phosphoadenosine 5'-phosphosulfate (PAPS). Cystine suppresses the synthesis of PAPS. Growth of a double mutant without the cystine added to the medium leads to a strong selection for a cysA mutation. It was proposed that there is an additional system to reduce disulfides required for the synthesis of deoxyribonucleotides. The difference in phenotypes observed by Kren et al. 3 and Russel et al. 4 appears to be due to accumulation of secondary mutations. The large overlap of functions of enzyme systems involved in reduction of disulfides may allow secondary mutations in regulation to compensate for various primary mutations.

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gsh M u t a n t s in Yeast Kistler et al.29 isolated glutathione-deficient (gsh) mutants of the yeast Saccharomyces cerevisiae after U V treatment using N-methyl-N'-nitro-Nnitrosoguanidine (MNNG) as was used in E. coli as a selective agent. Mutant isolates showed a 2 : 2 segregation of the G S H - and G S H ÷ phenotypes, indicating that the mutant was recessive. Complementation analysis indicated that all gsh mutants belong to one complementation group. Ohtake and Yabuuchi 3° cloned the gshl gene by complementation and used gene disruption to show that the resulting mutant had the same properties as the original gsh mutant. The cloned gene was sequenced and found to have 46% similarity with the rat kidney gshl gene and 26% with the E. coli gshl gene. Southern analysis was used to show that gshl is located on the X chromosome. Mutoh et aL 31 cloned the gene for the large subunit of glutathione synthase (EC 6.3.2.3) of Schizosaccharornyces pombe from a S. pombe genomic D N A library by complementation of cadmium hypersensitivity of a glutathione synthase-deficient mutant of S. pombe. The strain harboring the cloned gene expressed more glutathione synthase in spite of having extra copies of only one subunit of the enzyme. Lisowsky 32 isolated a new temperature-sensitive nuclear mutant affecting the biogenesis of functional mitochondria and characterized the mutant as having a complete block of mitochondrial translation at the restrictive temperature. Transformation of the mutant with plasmids from gene banks identified a chromosomal D N A fragment which can restore growth at the restrictive temperature. Sequence analysis identified the gene as the yglutamylcysteine synthetase of yeast. Disruption of the yeast y-glutamylcysteine synthetase caused a drastic reduction of growth on glucose medium. The insertion mutants were not able to grow on plates with glycerol as the sole carbon source, indicating the dependence of mitochondria on glycerol. Crosses between the temperature-sensitive mutant and the insertion mutant indicated that the mutations are in different genes and that the cloned yeast gene acts as an extragenic suppresser when present on a high copy number plasmid.

29M. Kistler, K.-H. Summer, and F. Eakardt, Mutat. Res. 173, 117 (1986). 3oy. Ohtake and S. Yabuuchi, Yeast7, 953 (1991). 31N. Mutoh, C. W. Nakagaawa, S. Ando, K. Tanabe, and Y. Hayashi, Biochem. Biophys. Res. Commun. 181, 430 (1991). 32Z. Lisowsky, Curt. Genet. 23, 408 (1993).

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B e r t h e - C o r t i et al. 33 d e t e r m i n e d glutathione ( G S H ) c o n t e n t in S a c c h a r o m y c e s c e r e v i s i a e as a function o f g r o w t h conditions and used the genotoxicity of M N N G , which is activated by G S H , as a quantitation of G S H . Levels of G S H varied greatly with g r o w t h conditions in a m a n n e r similar to that f o u n d for E. coli. gsh Mutants in Humans

T h e first h u m a n m u t a n t s deficient in the synthesis of glutathione were d e t e c t e d as patients with hemolytic anemia. ~,35 T h e patients had a defect in the gene e n c o d i n g a specific e r y t h r o c y t e glutathione synthase. This defect has also b e e n r e p o r t e d for an additional three families. 36'37 A s e c o n d type of defect that causes a generalized defect in glutathione synthase was reported. 38 T h e individual excreted large a m o u n t s o f 5-0xoproline. Sixteen patients with the defect have b e e n reported. 39 In all cases, patients were mentally r e t a r d e d and h a d n u m e r o u s neurological symptoms. T h e sympt o m s could be detected in the n e o n a t a l state. A defect in the gene e n c o d i n g ~/-glutamylcysteine synthetase was rep o r t e d for two siblings with hemolytic anemia. 4°m T h e patients h a d significant excretion of dibasic and m o n o b a s i c m o n o c a r b o x y l i c a m i n o acids and exhibited n e u r o m u s c u l a r degeneration. A family with a generalized defect in glutathione reductase has b e e n reported. 42 T h e individuals h a d p r o b l e m s with cataracts and nonspecified o p h t h a l m o l o g i c a l problems. O t h e r r e p o r t e d cases of low glutathione reductase activity a p p e a r to be due to insufficient riboflavin in the diet. 43 33L. Berthe-Corti, R. Hulsch, U. Nevries, and F. Eckardt-Schupp, Mutagenesis 7, 25 (1992). 34M. Oort, J. A. Loos, and H. K. Prins, Vox Sang. 6, 370 (1961). 35H. K. Prins, M. Oort, J. A. Loos, C. Ziarcheer, and T. Becker, Blood 27, 145 (1966). 36D. N. Mohler, P. W. Majerus, V. Minnich, C. E. Hess, and M. D. Garrick, N. Engl. J. Med. 283, 1253 (1970). 37E. Buetler, T. Gelbart, and C. J. Pegelow, Clin. Invest. 77, 38 (1986). 38E. Jellum, T. Kluge, H. C. Borresen, O. Stokke, and L. Eldjarn, Stand. J. Clin. Lab. lnvest. 26, 327 (1970). 39A. Larsson, in "Glutathione: Metabolism and Physiological Function" (J. Vino, ed.), p. 360. CRC Press, Boca Raton, Florida, 1990. 40p. N. Konrad, F. H. Richards, W. N. Valentine, and D. E. Paglia, N. Engl. J. Med. 286, 557 (1972). 41 F. H. Richards, M. R. Cooper, L. A. Pearce, R. J. Cowan, and C. L. Spurr. Arch. Int. Med. 14, 535 (1974). 42j. A. Loos, H. D. Roos, R. Weening, and J. Houwerzijl, Blood 48, 53 (1976). 43G. W. Lrhr, K. G. Blume, H. W. Rfidiger, and H. Arnold, in "Genetic Variability in the Enzymatic Reduction of Oxidized Glutathione" (L. Flohe, H. BenOhr, H. Sies, H. O. Waller, and A. Wendel, eds.), p. 165. Thieme, Stuttgart, 1974.

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Conclusion Although E. coli serves as an excellent model for mutants defective in glutathione metabolism in general, additional phenotypes are observed in simple eukaryotes owing to the increased complexity of the cell structure and the fact that glutathione appears to serve many functions including its role in keeping cellular proteins in a reduced state. Additional functions for glutaredoxin appear in multicellular organisms that are more important than the functions present in E. coli.

[ 10] C y s t a t h i o n i n e By DONALD J. REED Introduction Cystathionine is a thioether-containing amino acid without a known function other than serving as an intermediate in the only known transsulfuration pathway in mammals for the biosynthesis of a nonessential amino acid, cysteine. Transsulfuration has been known for more than 50 years, first as a nutritional phenomenon and more recently as a potentially important factor in vascular disease. Methionine is capable of providing cysteine equivalents by the utilization of the sulfur atom of methionine and the carbon skeleton and amino group of serine. The capacity for transsulfuration was initially observed as a decreased requirement for dietary methionine when cystine was present in the diet. Principally as a result of work by Binkley and duVigneaud, 1 the role of the cystathionine pathway in the transsulfuration process was elucidated. Methionine is converted via Sadenosylmethionine by demethylation to homocysteine (Fig. 1).2 Homocysteine condenses with serine in a reaction catalyzed by cystathionine synthase to form the thioeter cystathionine. Cystathionine is subsequently cleaved by y-cystathionase to cysteine plus ammonia and ot-ketobutyrate. This pathway is a major source of cysteine in the liver during dietary limitation of cyst(e)ine or when a high rate of glutathione biosynthesis is required by t h e l i v e r 3 and a limited number of other cell t y p e s . 4 In mammals, such as rats, the liver is the main site of cysteine biosynthe1 F. Binkley and V. du Vigneaud, J. Biol. Chem. 144, 507 (1942). 2 j. D. Finkelstein and S. H. Mudd, J. Biol. Chem. 242, 873 (1967). 3 D. J. Reed and S. Orrenius, Biochem. Biophys. Res. Commun. 77, 1257 (1977). 4 A. E. Brodie, J. Potter, and D. J. Reed, Eur. J. Biochem. 123, 159 (1982).

METHODS IN ENZYMOLOGY, VOL. 252

Copyright © 1995 by Academic Press, Inc. All tights of reproduction in any form reserved.

[ 1 0]

CYSTATHIONINE

93

CQOH I NH2-C-H I

c.~ ~

coo. I CH2

METHIONINE

I

c~-~ C~Hz

A.de

H 5-METHYLTETRA_~,.~[

H HO

t '%'"-"~C"3)3"C"2C00H

OH /~ACCEPTOR

\\

coo. c

CHz

HOMOCYSTEINE

Ade

.

HO

NH2--IC-H CIHz S I

CH2

OH

$-ADENOSYLHOMOCYSTEINE

"

I

H-C-- NH2 I

COOH CYSTATHIONINE

~-

COOH

COOH I

I

NH2-- IC-H CH2 I

SH CYSTEINE

C=O I

-'1"

CH2 I

-I-

NH3

CH3 CI-KETOBUTYRATE

FIG. 1. Cystathionine pathway and associated enzymes for the biosynthesis of L-cysteine. Enzymes catalyzing the steps are as follows: (1) methionine adenosyltransferase (EC 2.5.1.6), (2) S-Adenosylmethionine methyltransferases (EC 2.1.1.?), (3) adenosylhomocysteinase (EC 3.3.1.1.), (4) betaine-homocysteine S-methyltransferase (EC 2.1.1.5), (5) 5-methyltetrahydrofolate-homocysteine S-methyltransferase (methionine synthase) (EC 2.1.1.13), (6) cystathionine/3-synthase (EC 4.2.1.22), and (7) cystathionine 7-1yase (EC 4.4.1.1).

94

GLUTATHIONE

[101

sis which occurs via the cystathionine pathway. Normally, maintenance of a high glutathione content in the liver is associated with a very dynamic rate of secretion into plasma that is followed by an extensive and rapid extracellular degradation of both reduced glutathione (GSH) and oxidized glutathione (GSSG). These events support the concept that liver glutathione is a physiological reservoir of cysteine. This concept, originally proposed by Tateishi et al. 5 and Higashi et al., 6 involves two glutathione pools that are physiologically separate, one with a fast turnover in the cytosol and another, representing about 10% of the total liver glutathione, with a slow turnover in the mitochondria. 7 Evidence has led to the conclusion that cysteine may be an essential amino acid during premature growth s and even during surgical shock owing to the absence or a loss of function of the cystathionine pathway and transsulfuration of methionine sulfur. 9

Methods Sample Preparation Principle. Accurate measurement of cystathionine and related thiols and disulfides in biological specimens relies in part on sample preparation, namely, the rapid termination of metabolic processes in fluids, cells, and tissues and the prevention of thiol oxidation and thiol-disulfide interchange during the assay procedure. To satisfy these criteria, biological specimens are treated promptly with perchloric acid (PCA; 1 M or 10% v/v) containing a metal chelator such as 1 m M bathophenanthrolinedisulfonic acid (BPDS). Reagents

Perchloric acid (PCA), 70% (double distilled, G. F. Smith Chemicals) Perchloric acid (PCA), 10% (v/v) in metal-free water (Milli-Q Reagent Water System, Millipore, Bedford, MA) containing 1 m M bathophenanthrolinedisulfonic acid (BPDS) (G. F. Smith Chemicals) y-Glutamylglutamate (7-Glu-Glu; Vega Biochemicals) 15 mM, in 0.3% PCA, stored at - 1 0 °, stable at room temperature for several days, used as an internal standard 5 N. Tateishi,T. Higashi, A. Naruse, K. Nakashima,H. Shiozaki,and Y. Sakamoto,J. Nutr. 107, 51 (1977). 6T. Higashi, N. Tateishi, A. Naruse, and Y. Sakamoto,J. Biochem. 82, 117 (1977). 7M. J. Meredith and D. J. Reed, J. Biol. Chem. 257, 3747 (1982). 8 F. V. Pallardo, J. Sastre, M. Areseni, F. Rodrigo, J. M. Estrela, and J. Vina. Biochem. J. 274, 891 (1991). 9j. Vina, A, Girnenez, I. R. Puertes, E. Gasco, and J. R. Vian, Br. J. Nutr. 68, 421 (1992).

[101

CYSTATHIONINE

95

Bathophenanthrolinedisulfonic acid (BPDS, 15 mM), in metal-free water Standard Procedure. Cells, subcellular fractions in suspension, or cell culture media are treated by adding 50/~l y-Glu-Glu (15 raM), 50/~l BPDS (15 raM), and 100/~l 70% PCA. Following mixing with a vortex mixer, freezing, thawing, and centrifugation, a portion of the 10% PCA extract (0.5 ml) is derivatized and analyzed by high-performance liquid chromatography (HPLC) by the method of Reed et al. ~° as modified previously. T M O t h e r Methods. Stabler et al. t3 have examined the elevation of serum cystathionine levels in patients by a new capillary gas chromatographicmass spectrometric assay and measured cystathionine in the serum of normal subjects and patients with clinically confirmed deficiencies of cobalamin and folate. Ohmori et al.24 have described a sensitive determination of cystathionine and assays for cystathionine /3- and y-lyase, as well as cystathionine flsynthase, using HPLC. Cystathionine was cleaved to 2-ketobutyric acid, cysteine, and ammonia by cystathionase. 2-Ketobutyric acid was converted to 2-ethyl-2-hydroxy-6,7-dimethoxyquinoxaline (EHDQ) by reaction with 1,2-diamino-4,5-dimethoxybenzene. When E H D Q was measured in a mobile phase of pH 2.1 using HPLC with UV detection, 250 pmol of L-cystathionine in 250 ~l of the reaction mixture could be determined. Because E H D Q has a strong fluorescence in a mobile phase of pH 6.5 at 447 nm, on excitation at 365 nm, as little as 2.5 pmol of cystathionine in 250 /~l of the reaction mixture could be determined by HPLC with fluorimetric detection. Cystathionase activity was assayed on the basis of the same principle by determining cystathionine in as little as 63 ng of rat liver by fluorimetric detection. Cystathionine/3-synthase activity was measured by the same method by determining cystathionine formed in only 113 ng wet weight of rat liver. Using these methods, both cystathionine fl- and y-lyase activities in S a c c h a r o m y c e s cerevisiae were determined, because quinoxaline derivatives from pyruvate and 2-ketobutyrate could be measured simultaneously by HPLC. Watanabe et al. 15 have developed qualitative analyses of sulfur amino 10D. J. Reed, J. R. Babson, P. W. Beatty, A. E. Brodie, W. W. Ellis, and D. W. Potter, Anal. Bioehem. 106, 55 (1980). 11 M. W. Fariss and D. J. Reed, this series, Vol. 143, p. 101. 12 D. J. Reed, Methods Toxieol. 1B, 421 (1994). 13 S. P. Stabler, J. Lindenbaum, D. G. Savage, and R. H. Allen, Blood 81, 3404 (1993). 14 S. Ohmori, K. Nakata, K. Nishihara, S. Yamamoto, M. Kawase, and S. Tsuboi, J. Chromatogr. 574, 35 (1992). 15 H. Watanabe, Y. Fujita, K. Sugahara, H. Kodama, and S. Ohmori, Biol. Mass Spectrom. 20, 602 (1991).

96

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[ 1O]

acids in the urine of a patient with cystathioninuria using liquid chromatography/atmospheric pressure ionization mass spectrometry. Standard sulfur amino acids and various cystathionine metabolites in the urine of a patient with cystathioninuria were analyzed using liquid chromatography/ mass spectrometry with an atmospheric pressure ionization interface system. Very intense quasi-molecular ions ([M + HI ÷) of synthetic cystathionine, N-monoacetylcystathionine, perhydro-l,4-thiazepine-3,5-dicarboxylic acid, S-(3-hydroxy-3-carboxy-n-propyl)cysteine, S-(2-carboxyethyl)cysteine, S-2-hydroxy-2-carboxyethyl)homocysteine, S-(carboxymethyl)homocysteine, N-acetyl-S-(3-hydroxy-3-carboxy-n-propyl)cysteine, and N-acetylS-(2-carboxyethyl)cysteine were observed by this method. Quasi-molecular ions ([M + H] +) of the sulfur amino acids were observed in the urine sample of the patient with cystathioninuria, and N-acetyl-S-(3-hydroxy-3carboxy-n-propyl)- and N-acetyl-S-(2-carboxyethyl)cysteine as N-substituted sulfur amino acids were also identified in the urine of the same patient. Sugahara et al. 16 have determined cystathionine and perhydro-l,4-thiazepine-3,5-dicarboxylic acid in the urine of a patient with cystathioninuria using column liquid chromatography-mass spectrometry. The concentrations found for cystathionine and perhydro-l,4-thiazepine-3,5-dicarboxylic acid were 1.289 + 0.099 and 0.310 _+ 0.067 mg/ml, respectively. Nutritional Biochemistry of Cystathionine and Related Amino Acids Methionine is an essential amino acid for mammals and must be derived entirely from the diet. In contrast, cysteine is synthesized by mammals, with the cysteine sulfur being derived from methionine and the carbon and nitrogen of cysteine from serine via the cystathionine pathway that functions principally in the liver. The dependence on methionine sulfur implies that cysteine remains a nonessential amino acid only if methionine intake is sufficient to meet the total sulfur amino acid requirement. Conversely, the dietary requirement for methionine is reduced, but not eliminated, when cyst(e)ine intake is adequate. 17 Another sulfur compound, the tripeptide glutathione, is present in many food and feed products and is capable of delivering 100% of its cysteine content to the animal as bioavailable cysteine. Rats, mice, dogs, and pigs have relatively low dietary requirements for sulfur amino acids compared to chicks and catsJ 7 In all the species, cyst(e)ine has been found capable of supplying 50% of the total requirement (w/w) for growth. Although transsulfuration is a biologically efficient process, unless interfered with by inhibition, a higher concentration of methio16K. Sugahara, J. Ohta, M. Takemura, and H. Kodama, I. Chromatogr. 579, 318 (1992). 17 D. H. Baker, this series, Vol. 143, p. 297.

[ 10]

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97

nine alone is needed to furnish the same sulfur content as a 1 : 1 mixture (w/w) of a methionine-cyst(e)ine combination owing to the molecular weight difference between methionine and cysteine. Excess dietary methionine is generally considered to be the most toxic of the (dietary) essential amino acids. A level twice the requirement is generally well tolerated, but at 3-fold or above levels toxicity results) 7 Fasting or nitrogen-free diet feeding to cause depletion of the sulfur amino acids from tissue pools leads to a greater depletion of sulfur amino acids compared to other essential amino acids. Excess dietary cyst(e)ine is well tolerated even though intracellular concentrations of cysteine are of the order of 30-200 ~M, values that are among the lowest observed for any of the protein amino acids.5 Higher concentrations of cysteine are potentially dangerous to cells because cysteine forms a thiazolidine derivative with pyridoxal phosphate and thereby may deplete cells of that coenzymeJ s Multiple pathways of cysteine catabolism function efficiently to prevent toxic accumulation of cysteine. Glutathione, a physiological reservoir of cysteine, is present in liver cells and other cells in a nontoxic, metabolically active form that participates in an interorgan process to supply cysteine rapidly to all cells in the body. Methionine metabolism is initiated by the formation of S-adenosylmethionine (AdoMet), a sulfonium compound in which each of the carbons attached to sulfur is activated toward nucleophilic attack. In most cases, AdoMet reacts by transfer of the S-methyl group to one of several possible acceptors, with methyl transfer reactions as a group thought to consume about 95% of the AdoMet f o r m e d . 19 Pallardo and co-workers 8 have examined physiological changes in glutathione metabolism in fetal and newborn rat liver. Isolated hepatocytes from fetal, newborn, and adult rats were utilized to examine glutathione metabolism. The GSH/GSSG ratio decreased 15- to 20-fold through the fetal-neonatal-adult transition. The change in the GSH/GSSG ratio was mainly due to an increase in GSSG. It was observed that all enzyme activities involved in the glutathione redox cycle increased during that transition but the relative increases in glutathione peroxidase and glutathione Stransferase were 3-5 times those of glutathione reductase or glucose-6phosphate dehydrogenase. Synthesis of GSH from methionine as a sulfur source was 6 times lower in fetal than in adult hepatocytes. When Nacetylcysteine was used as a sulfur donor to bypass the cystathionine pathway, the rates of GSH synthesis were similar in fetal and adult cells. It was concluded that this was a result of cystathionase activity being very low in is O. W. Grfffith, this series, Vol. 143, p. 366.

19S. H. Mudd and J. R. Poole, Metab. Clin. Exp. 24, 721 (1975).

98

GLUTATHIONE

[10]

fetal cells. The low activity is reflected in the blood amino acid pattern, where the concentration of cysteine rises from 8 to 52/zM from fetuses to adult rats. It was concluded that cysteine may be an essential amino acid for the premature animal. Stabler e t a l J 3 have observed elevation of serum cystathionine levels in patients with cobalamin and folate deficiency. Homocysteine can be methylated to form methionine by the cobalamin and folate-dependent enzyme methionine synthase (5-methyltetrahydrofolate-homocysteine Smethyltransferase). Serum levels of total homocysteine are elevated in greater than 95% of patients with either cobalamin or folate deficiency. Homocysteine can condense with serine to form cystathionine in a pyridoxal phosphate-dependent reaction catalyzed by cystathionine fl-synthase. Cystathionine is subsequently cleaved to cysteine and ot-ketobutyrate by the pyridoxal phosphate-dependent enzyme T-cystathionase. The normal range for serum cystathionine was 65 to 301 nmol/liter (median of 126 nmol/ liter) for 50 normal blood donors. In 30 patients with clinically confirmed cobalamin deficiency, values for cystathionine ranged from 208 to 2920 nmol/liter (median of 816 nmol/liter) and 26 (87%) had levels above the normal range. In 20 patients with clinically confirmed folate deficiency, cystathionine concentrations ranged from 138 to 4150 nmol/liter (median of 1560 nmol/liter) and 19 (95%) had values above the normal range. Five homozygotes for cystathionine/3-synthase deficiency had high values for serum total homocysteine and low or low-normal values for serum cystathionine that ranged from 30 to 114 nmol/liter even though they were on treatment with pyridoxine and had partially responded. It was concluded that levels of cystathionine are elevated in the serum of most patients with cobalamin and folate deficiency and that they are useful in the differential diagnosis of an elevated serum total homocysteine level. Oxidative Stress Effects Because elevated circulating homocyst(e)ine is a risk factor for occlusive ascular disease, Dudman et al. 2° have measured circulating lipid hydroperoxide levels in human hyperhomocysteinemia. They explored whether elevated plasma homocyst(e)ine is associated with increased plasma lipid hydroperoxides that might trigger vascular disease. Plasma containing high levels of homocyst(e)ine was obtained from four patients with a homozygous deficiency of cystathionine /3-synthase activity and also from four heterozygotes with a deficiency of the enzyme after an oral methionine 20 N. P. Dudman, D. E. Wilcken, J. Wang, J. F. Lynch, D. Macey, and P. Lundberg, Arterioscler. Thromb. 13, 1253 (1993).

[ 101

CYSTATHIONINE

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load. The mean plasma nonprotein-bound homocyst(e)ine level in all subjects was more than 11-fold higher than the mean normal fasting value. Levels of high-density lipoprotein (HDL) cholesteryl ester hydroperoxides (CEOOH), normalized against the concentration of free cholesterol in HDL, were not elevated compared with values for fasting healthy donors. An inverse dependency was observed between plasma total homocyst(e)ine and HDL CEOOH. The ubiquinol-10/ubiquinone-10 ratio in HDL, which is expected to fall during oxidative stress, increased with plasma homocyst(e)ine. Because HDL contains the majority of detectable plasma lipid hydroperoxides, of which CEOOHs are the most abundant, Dudman et al. z° suggest that an elevated plasma homocyst(e)ine level does not enhance oxidative stress, increase the levels of lipid hydroperoxides in plasma, or generate vascular damage by this mechanism. Rusakow et al. 21 examined Oz-induced changes thiols in mice. Because glutathione is an important antioxidant, they hypothesized that changes in lung and systemic availability of GSH and its precursor amino acid, cysteine, are induced by exposure to hyperoxia and that the changes could be modulated by toxic Oz metabolites. In relatively Oz-resistant Swiss-Webster mice, lung GSH increased during O2 exposure, whereas liver GSH and liver and plasma cysteine all decreased. Pair-feeding studies suggested that nutritional deprivation alone did not cause the decrease in plasma cysteine. In lung, sulfur amino acids were not decreased by O2 exposure. Cystathionine increased 6-fold, and y-cystathionase was not inhibited. These findings suggest that hyperoxia increases transsulfuration pathway activity and that cystathionase rate-limits this process in lung. In comparative studies, lung GSH increased in Oz-resistant high-Cu,Zn-superoxide dismutase (SOD) transgenic mice but not in genetically similar, nontransgenic controls (CBYB/6 × B6D/2) during hyperoxic exposure. In addition, liver GSH and plasma cysteine decreased in nontransgenic control but not in highSOD mice, whereas lung cystathionine increased similarly in both groups. Thus, superoxide or its secondary products can modulate, at least in part, the changes in cysteine and GSH. Nonetheless, regardless of strain or SOD status, hyperoxic exposure consistently caused thiol and sulfur amino acid changes, including increased lung cystathionine and GSSG, demonstrating a strong association between these dynamic changes and oxidant stress. Vina and co-workers9 have examined the impairment of cysteine synthesis from methionine in rats exposed to surgical stress. The activity of liver cystathionase (EC 4.4.1.1) was decreased after 3 days of stress induced by surgery. The rate of L-cysteine synthesis from L-methionine was significantly higher in isolated hepatocytes from controls than in hepatocytes from rats 21 L. S. Rusakow, C. W. White, and S. P. Stabler, J.

Appl. Physiol. 74, 989 (1993).

100

OLUTATHIONE

[ 1 O]

suffering from surgical stress. The half-life of L-[2(n)-3H]methionine was significantly higher in rats subjected to surgical stress than in controls. The plasma L-methionine/L-cystine ratio was higher in stressed rats than in controls. L-Cystine was increased significantly in the surgically stressed rats compared with the controls. From these observations, it was concluded that the data were consistent with the hypothesis that the observed inhibition of cystathionase is physiologically important and that L-cysteine might be considered as an essential amino acid in cases of surgical stress. Takeuchi and co-workers 22have examined glutathione levels and related the enzyme activities in vitamin B6-deficient rats fed a high methionine and low cystine diet. Vitamin B6-deficient rats were fed a vitamin B6-deficient diet containing 0.56% (w/w) methionine and 0.075% cystine for 8 weeks. Controls were fed an identical diet supplemented with 10 mg/kg pyridoxine hydrochloride. Glutathione concentrations in each organ examined were similar in control and vitamin B6-deficient rats, and the values were comparably lower after intraperitoneal injection of diethyl maleate, a glutathione S-transferase substrate that causes depletion of glutathione. However, buthionine sulfoximine, an inhibitor of glutathione biosynthesis, caused a significantly greater decrease in glutathione levels in the liver and lungs of vitamin B6-deficient rats relative to controls. Glutathione peroxidases activity in the liver of vitamin B6-deficient rats was higher than in control animals; however, glutathione transferase activity in tissues other than liver of vitamin B6-deficient rats was higher than in the controls. The holoenzyme activities of cystathionine/3-synthase and cystathionine ~/-lyase in the liver of vitamin B6-deficient rats were markedly reduced. These findings indicated that although the activities of enzymes that synthesize cysteine from methionine were decreased by vitamin B6 deficiency, the level of synthesis and supply of cysteine in vitamin B6-deficient rats were sufficient to maintain the same glutathione level as in controls, and that glutathione utilization in the liver was accelerated by vitamin B6 deficiency. Effects of Propargylglycine in Laboratory Animals Abeles and Walsh 23 synthesized and tested a number of acetylenic enzyme substrates and observed the irreversible inactivation of the pyridoxal phosphate-dependent rat liver enzyme y-cystathionase. The irreversible inactivation is accompanied by covalent labeling of the enzyme to the extent of I m01 of labeled inactivator per 80,000 daltons, equivalent to two subunits. Subsequently, an inactivator-enzyme adduct was isolated. 24 The 22 F. Takeuchi, S. Izuta, R. Tsubouchi, and Y. Shibata, J. Nutr. 121, 1366 (1991). 23 R. H. Abeles and C. T. Walsh, J. Am. Chem. Soc. 95, 6124 (1973). 24 W. Washtien and R. H. Abeles, Biochemistry 16, 2485 (1977).

[ lOl

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inactivator, DL-2-amino-4-pentynoic acid (propargylglycine, PPG), has been shown to rapidly inactivate the rat liver cystathionase in vivo when administered to mice and leads to a rapid decrease in the hepatic activity of the enzyme. In this manner, experimental cystathionuria has been induced by a single intraperitoneal administration of propargylglycine to mice. An extensive decrease in the hepatic cystathionase activity occurs within 1 hr and is maintained at a low level of enzyme activity for I day. It is accompanied by a considerable increase in urinary excretion of cystathionine, glycine, and alanine. The strict specificity and high efficiency of propargylglycine to inactivate y-cystathionase appears to ensure quantitative information concerning the actual metabolic significance of the target enzyme. This concept is supported by the fact that the concentration of cystathionine in liver, kidney, and brain increased rapidly on the administration of L-propargylglycine. Glutathione in liver diminished in parallel with the increase in cystathionine, reaching 40% of the original level when the cystathionine level was at a maximum. Following the restoration of the hepatic cystathionase activity, the cystathionine level declined gradually with concomitant increase in the glutathione level. These findings indicate that, in liver, the cystathionine pathway supplied more than half of the cysteine store for the biosynthesis of glutathione. Unlike the case with liver, this metabolic correlation between cystathionine and glutathione is not explicit in kidney and brain, suggesting that the supply of cysteine for the maintenance of glutathione in these tissues is from a source other than the cystathionine pathway. New studies continue to support the concept of liver glutathione being the physiological reservoir for cysteine as it is applied to the other cell types in mammals. Acetylenic substrate analogs are useful in the inactivation of enzymes which abstract carbon-bond hydrogens as protons at a position adjacent to the acetylenic linkage. Specificity of the interactions is a very attractive feature, as it is not based solely on structural similarity to substrate, but also on the mechanism of the catalytic process. It is important to point out that the acetylenic linkage is made chemically reactive as a result of specific enzymatic action on the inhibitor molecule. Inactivators are very useful as active site-directed reagents for enzymes and serve as very specific agents for in vivo inactivation of these specific target enzymes. Propargylglycine is such a potent irreversible inhibitor that only 6.6 × 10 -5 M inactivator is necessary to cause the loss of enzymatic activity of y-cystathionase with a half-life of 2 min at 25 °. In vivo, a single intraperitoneal administration of L-propargylglycine (5 txmol/mouse) leads to almost complete inactivation of y-cystathionase activity within 1 hr. e5 25S. Shinozuka, S. Tanase, and Y. Morino, Eur. J. Biochem. 124, 377 (1982).

1 02

GLUTATHIONE

[ 1 0]

Houghton and Cherian 26 have examined the effects of inhibition of cystathionase activity on glutathione and metallothionein levels in the adult rat. Rats were fed a diet containing methionine (0.66% (w/w)) and cystine (0.20%) for 1 week before receiving three consecutive daily intraperitoneal injections of propargylglycine, at various doses (2.5-375/xmol/kg). They observed that when hepatic cystathionase was inhibited greater than 90% (->50/~mol propargylglycine/kg), renal and hepatic metallothionein and hepatic glutathione were unaltered except at the highest dose. In contrast, renal glutathione was increased 2-fold. The authors concluded that renal and hepatic glutathione pools appear to be altered differently and that renal glutathione may be preferentially maintained even when hepatic glutathione is decreased. Cho and co-workers 27 have examined propargylglycine infusion effects on tissue glutathione levels, plasma amino acid concentrations, and tissue morphology in parenterally fed, growing rats. Amino acid solutions currently used for total parenteral nutrition (TPN) contain little cysteine or cystine. Some premature human infants have low liver activities of y-cystathionase and presumably require preformed cysteine or cystine. Growing animals tend to have higher liver y-cystathionase activity, which makes them unsuitable as models to study effects of cystathionine precursors. Because propargylglycine inhibits y-cystathionase specifically, rats infused with PPG as part of a TPN regimen were evaluated as a potential model. Two groups of rats (120-160 g) were infused for 15 days with TPN regimens, one without and one with PPG (40 ~mol/day). A third group received the TPN control regimen, with methionine added at toxic levels. Propargylglycine treatment significantly decreased plasma cysteine and taurine concentrations and significantly increased plasma cystathionine concentration without affecting methionine concentration. Propargylglycine treatment significantly decreased brain, muscle, liver, intestine, and stomach glutathione concentration without affecting erythrocyte or heart glutathione concentrations. Electron microscopic examination showed no abnormalities in heart and kidney of PPG-treated rats. Hepatocyte glycogen was lower in TPNfed controls than in orally fed rats and was further reduced in TPN-PPGfed animals. Growing rats infused with low doses of PPG show promise as an animal model to study a numbers of important issues concerning human sulfur amino acid metabolism. Acknowledgment The author generally acknowledges the support of NIEHS Grants ES-01978 and ES-00210. 26 C. B. Houghton and M. G. Cherian, I. Biochem. Toxicol. 6, 221 (1991). 27 E. S. Cho, J. Brown-Hovanec, Tomanek, and L. D. Stegink, I. Nutr. 121, 785 (1991).

[11]

PROMOTION OF CYST(E)INEUPTAKE

[ 1 II P r o m o t i o n By

of Cyst(e)ine

103

Uptake

THOMAS MEIER and ROLF D. ISSELS

Introduction Besides its involvement in protein synthesis, the amino acid cysteine has a central function in the cellular metabolism of glutathione. The tripeptide glutathione, which can be defined as the intracellular "package form" of cysteine, is limited in regard to its biosynthesis by the availability of cysteine. Therefore, under conditions where the supply of cellular cysteine is not sufficient, a promotion of cysteine uptake by other chemical compounds would be expected to increase glutathione levels. The importance of glutathione for cellular metabolism is well known. 1 Glutathione can protect cells against oxidative stress, heavy metal ions, and various toxic compounds. Many studies show that depletion of cellular glutathione levels leads to sensitization of cells to radiation and antineoplastic agents 2 and vice versa. The increase of cellular glutathione often corresponds to an increased resistance to these therapeutical modalities. In most eukaryotic cell types cysteine has to be taken up from the extraceUular medium, but some cells (e.g., hepatocytes) are able to synthesize cysteine from methionine via the cystathionine pathway. For promotion studies of cysteine uptake different approaches can be taken. Direct supplementation of cells with cysteine is critical because cysteine exhibits a high degree of toxicity. 3 Several nontoxic cysteine derivatives have been proposed as delivery systems for cysteine. Administration of thiazolidines, ring form cysteine derivatives, can promote glutathione biosynthesis. 4 After entering the cells thiazolidines become intracellularly cleaved and release cysteine. Several cysteine esters can act as cysteine delivery systems and elevate intracellular cysteine and glutathione. -~In addition, administration of extracellular monoethyl or diethyl esters of glutathione rather than glutathione itself can also enhance intraceUular glutathione levels. 6 There is no evidence that intact glutathione can be taken up by mammalian cells] 1A. Meister and M. E. Anderson, Annu. Rev. Biochem, 52, 711(1983). 2 A. Meister, PharrnacoL Ther. 51, 155 (1991), 3y. Nishiuch, M. Sasaki, M. Nakayasu, and A. Oikawa, In Vitro 12, 635 (1976). 4j. M. Williamson,B. Boettcher, and A. Meister, Proc. Natl. Acad. Sci. U.S.A. 79, 6246 (1982). _sM. Butterworth, D. G. Upshall, M. Hobbs, and G. M. Cohen, Biochem. Pharmacol. 45, 1769 (1993). 6E. J. Levy, M. E. Anderson, and A. Meister, Proc. Natl. Acad. Sci. U.S.A. 90, 9171 (1993). 7M. E. Anderson and A. Meister, this series, Vol. 143, p. 313.

METHODS IN ENZYMOLOGY, VOL. 252

Copyright © 1995 by Academic Press, Inc. All rights of reproduction in any form reserved.

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GLUTATHIONE

[ 11 ]

However, extracellular or membranous metabolism of glutathione may form products such as ~-glutamylcysteine, cysteinylglycine, or cysteine which may be transported into cells and utilized for glutathione biosynthesis. 7 For a promotion of cysteine uptake the cellular transport systems are of critical importance. In aqueous solutions rapid autoxidation of most of the free thiol cysteine to cystine occurs. Therefore under physiological conditions only 10-20% of the total of free cystine is present as the reduced form. 8 After entry into a cell a prompt subsequent reduction of cystine to cysteine may be expected. Cystine and cysteine appear to be taken up by different transport systems, and no direct interaction between the systems has yet been found. In general, the transport systems vary considerably among tissues. Three transport systems are known and are explained in more detail. The uptake of cystine can be mediated by the transport system xc- in exchange with glutamate. 9 The uptake system is Na÷-independent and further shared by homocysteate and a-aminoadipate. Some cells express high activity for this anion-exchanging transport (e.g., fibroblasts), in other cells it is strongly inducible (e.g., macrophages), whereas other cell types lack this transport (e.g., lymphocytes). 1°-12 Cysteine is transported into cells by a transport system totally different from xc-. The system ASC (alanine, serine, cysteine) transports neutral amino acids and is Na÷-dependent. 13 Alanine, serine, and cysteine share this transport system, which is ubiquitously distributed among cell types. Exracellular cysteine can also spontaneously react with other thiol compounds to form mixed disulfides under certain conditions. The disulfides can be taken up by the transport system L, which was demonstrated for the disulfide of cysteamine and cysteine to be Na÷-independentJ 4 More detailed descriptions of the amino acid transport systems for cyst(e)ine uptake are reviewed elsewhere. 8'~5 To study the promotion of cyst(e)ine uptake of mammalian cells in vitro we use Chinese hamster ovary (CHO) cells as a model system. The cells have a limited capability to utilize extracellular cystine. 16 The uptake of cystine can be investigated when extracellular cystine is replaced by the 8 S. Bannai, Biochim, Biophys. Acta 779, 289 (1984). 9 M. Makowske and H. N. Christensen, J. Biol. Chem. 257, 5663 (1982). 10T. Ishii, H. Sato, K. Miura, J. Sagara, and S. Bannai, Ann. N. Y. Acad. Sci. 663, 497 (1992). 11 H. Watanabe and S. Bannai, J. Exp. Med. 165, 628 (1987). 12 H. Gmunder, H.-P. Eck, and W. Droge, Eur. J. Biochem. 201, 113 (1991). 13H. N. Christensen, M. Liang, and E. G. Archer, J. Biol. Chem. 242, 5237 (1967). 14R. L. Pisoni, J. G. Thoene, and H. N. Christensen, J. Biol. Chem. 260, 4791 (1985). 15 H. N. Christensen, Physiol. Rev. 70, 43 (1990). 16 R. D. Issels, A. Nagele, K. G. Eckert, and W. Wilmanns, Biochem. Pharmacol. 37, 881 (1988).

[1 ll

PROMOTION OF CYST(E)INE UPTAKE

105

70

r

60

~'

5o 40

30 ~,

20

ffl

oi

0

10

20

30

40

50

60

Time (rain)

FIG. 1. Time course (0-60 min) of the uptake of 0.1 mM [3SS]cystine by CHO cells in McCoy's 5A medium at 37°. The medium contained the following supplements: (Q) 0.4 mM cysteamine; (11) 0.8 mM N-acetylcysteine; ( ~ ) no additional thiol (control). Data are means _+ SD of three independent experiments. [From R. D. Issels, A. Nagele, K. G. Eckert, and W. Wilmanns, Biochem. PharmacoL 37, 881 (1988).]

radioactively labeled compound. Addition of several thiols to the medium of CHO cells can promote uptake of cystine as shown in Fig. 1. A pronounced increase in [35S]cystine uptake compared to untreated control cells is observed when the thiols cysteamine and N-acetylcysteine are added to the medium. The promotion of cystine uptake has also been studied for other thiols. A selection of thiols that have been investigated are summarized in Table I. Dihydrolipoic acid, WR-1065, and Mesna promote cystine uptake; in contrast, a-lipoic acid, N,N-diacetylcystine, and WR-2721 show no effect. The common molecular structure shared by such compounds that promote cystine uptake in CHO cells is the free SH group. Modification of the free thiol group by chemical reaction to form a disulfide bond or the addition of a phosphate moiety to the thiol group abolishes the potential of the compound to promote cystine uptake in CHO cells. Utilization of Cystine for Glutathione Biosynthesis Addition of extracellular thiols to the medium has been demonstrated to influence the intracellular glutathione levels of CHO cells. 17As shown for cysteamine and N-acetylcysteine, intracellular glutathione content increases during thiol exposure (see Table II). Total cellular glutathione of C H O cells 17 R. D. lssels, S. Bourier, J. E. Biaglow, L. E. Gerweck, and W. Wilmanns, Cancer Res. 45, 6219 (1985).

106

[ 11]

GLUTATHIONE TABLE 1 CYSTINE UPTAKE IN CHO CELLS

Thiol"

Promoting cystine uptake N-Acetylcysteine Mesna WR-1065

Cysteamine Dihydrolipoic acid Not promoting cystine uptake WR-272t

a-Lipoic acid

Formula

HOOCCHCHzSH

I

CH3OCNH SO3CH2CH2SH H2NCH2CH2CHzNHCH2CH2SH H2NCHzCHzSH SH SH HOOCCH2CH2CH2CH2CHCHeCH2 H2NCH2CH2CH2NHCH2CH2SPO3 HOOCCH2CH2CH2CH2CH-- CH2----~CH 2

~s__s j N,N-Diacetylcystine

Ref.

d

c

HOOCCHCH2SSCH2CHCOOH CH3OCNH

NHCOCH3

a Mesna, 2-Mercaptoethane sulfonate; WR-1065, N-(2-mercaptoethyl)-l,3-diaminopropane; WR-2721, S-2-(3-aminopropylamino) ethylphosphorothioic acid. b R. D. Issels, A. Nagele, K. G. Eckert, and W. Wilmanns, Biochem. Pharmacol. 37, 881 (1988). c T. Meier and R. D. Issels, Biochem. Pharmacol., submitted for publication. a R. D. Issels and A. Nagele, Cancer Res. 49, 2082 (1989).

increases about 2-fold after exposure to the thiols. Buthionine sulfoximine (BSO), an irreversible inhibitor of glutathione biosynthesis, when added to the medium immediately prior to the addition of cysteamine or Nacetylcysteine could completely block the increase of glutathione. This demonstrates that the increase in glutathione content mediated by cysteamine and N-acetylcysteine is dependent on cellular glutathione biosynthesis. The utilization of radioactively labeled cystine from the medium for glutathione biosynthesis is shown in Fig. 2. More than 90% of the 35S radioactivity in the soluble fraction of the cell lysates could be recovered in the peak of glutathione. These data show that the cysteine moiety of the newly synthesized glutathione is derived from the extracellular pool of cystine. The promoted uptake of cystine in the presence of cysteamine or Nacetylcysteine is dependent on amino acid transport systems of CHO cells. Addition of L-alanine, L-serine, and L-leucine reduced the uptake rate of

[11]

PROMOTION OF CYST(E)INE UPTAKE

107

TABLE II GLUTATHIONE CONTENT OF C H O CELLS AFTER EXPOSURE TO CYSTEAMINE AND N - A C E T Y L C Y S T E I N E a

Treatment

GSH + GSSG

Controls +BSO 0.4 mM Cysteamine +BSO" 0.8 mM N-Acetylcysteine +BSO ~

26.3 _+ 5.6b 17.5 _+ 0.9 61.0 +_ 14.0 16.7 _+ 0.4 41.4 _+ 18.9 16.0 ± 1.4

"After exposure to thiols for 120 rain at 37°. b Means ± SD of three or more independent experiments. c Addition of 1 mM DL-buthionine (S,R)-sulfoximine (BSO) occurred 10 min prior to the addition of cysteamine or N-acetylcysteine. [Modified from R. D. lssels, A. Nagele, K. G. Eckert, and W. Wilmanns, Biochem. Pharmacol. 37, 881 (1988).] cystine, whereas L-glutamate or DL-homocysteate showed no effect. Therefore, part of the uptake p r o m o t e d by cysteamine or N-acetylcysteine occurs most likely in the form of cysteine, which is mainly transported by the ASC system in most cell types. Besides uptake via the A S C system, uptake of mixed disulfides (e.g., cysteine-cysteamine, cysteine-N-acetylcysteine) via system L is also likely. In conclusion, the p r o m o t i o n of cystine uptake induced by extracellular thiols in our model system needs two different steps: first, the formation of cysteine and cysteine mixed disulfides by reaction of the added thiol with extracellular cystine and, second, the uptake of cysteine and/or the mixed disulfide via cellular transport systems. I n Vitro A s s a y of T h i o l - P r o m o t e d C y s t i n e U p t a k e

T w o different experimental protocols are described that can be used to study adherent or n o n a d h e r e n t cell types. A d h e r e n t Cells

The protocol is optimized for C H O cells and might need slight modifications when other cell types are used. A r o u n d 5 × 105 exponentially growing C H O cells are seeded in polystyrene tissue culture petri dishes (35 m m

108

111]

GLUTATHIONE

A

S-12,/,- dinitrophenyl) glutnthione (GS-DNP) 0.01 E3/.0nm

~_a-"

L

I- chloro-2,4-dinitrobenzene

I

B

,

~.

A

.

50000

5 E

@

M

r

i

0

2

i

i

[

1

4

6

8

10

i

12

i

14

i

16

Time {rain}

FIG. 2. Recovery of [3SS]cystine incorporated into S-(2,4-dinitrophenyl)glutathione (GSDNP). (A) Typical elution profile of GS-DNP at 340 nm after HPLC on a/zBondapak C18 column. After cysteamine exposure of CHO cells for 60 min at 37 ° in McCoy's 5A medium containing 0.1 mM [35S]cystine, total GSH of the acid-soluble fraction was transferred to 1chloro-2,4-dinitrobenzene by glutathione S-transferase, forming GS-DNP as described. (B) Measurement of 35S radioactivity in fractions of the eluate after HPLC separation. More than 90% of the total 35S radioactivity applied to the column could be recovered in the peak of GS-DNP. [From R. D. Issels, A. Nagele, K. G. Eckert, and W. Wilmanns, Biochem. Pharmacol. 37, 881 (1988).]

diameter) containing 2 ml of complete McCoy's 5A medium. Twenty-four hours after plating the cell number is 1.5-2 × 106 per dish with a confluency of about 50%. The cells are washed twice with warm phosphate-buffered saline (PBS), and 0.5 ml of the uptake medium is added. The uptake medium consists of McCoy's 5A medium without cysteine, cystine, methionine, and glutathione and is supplemented with 0.1 mM L-[35S]cystine (50-120 mCi/ mmol, Amersham, Braunschweig, Germany). Concentrated stock solutions of thiols are freshly prepared in ice-cold (PBS) which has been previously

[1 I I

PROMOTION OF CYST(E)INE UPTAKE

109

gassed with nitrogen for about 30 min. The thiol stock solution is added in small volumes (5-20/xl) to reach the final concentration. The dishes are immediately placed on a temperature-controlled water bath, the bottom of the dishes being submersed in the water. After the incubation period, the radioactive medium is removed and the cell layer washed three times with ice-cold PBS. The cells are trypsinized (0.25% v/v trypsin) for 2 min at room temperature, and the cell number of an aliquot is determined. The rest of the suspension is centrifuged (500 g, 5 min, at 4 °) and the cell pellet is carefully resuspended with 0.5 ml of 1 N perchloric acid. After incubation for 10 min on ice, the solution is centrifuged at high speed. The acidinsoluble pellet, which is solubilized in 0.5 ml of 0.1 N NaOH for 12 hr, and the acid-soluble supernatant are mixed with a appropriate scintillation cocktail and separately counted in a/3-counter. An aliquot of the NaOH solution can be used for protein determination by the method of Lowry. N o n a d h e r e n t Cells

The cystine uptake in nonadherent cells is determined by using a modification of the method described by Novogrodsky et al.18 Mitogen-activated peripheral blood lymphocytes (PBL) (107 cells/ml) are suspended in the same radioactive uptake medium as described for the adherent cells. The suspension is incubated with shaking in a temperature-controlled water bath, and the thiol stock solution is added in a small volume. Aliquots (100 ixl) are removed at given time points and layered on 0.1 ml of a mixture of mineral oil and dibutyl phthalate (1:4, v/v). After centrifugation in a Beckman Microfuge for 15 sec the Beckman reaction vial is frozen in liquid nitrogen. Then the tip of the vial containing the cell pellet is cut off with a razor. The pellet is solubilized with 1 ml Biolute-S (Zinsser, Frankfurt, Germany) overnight, then mixed with an appropriate scintillation cocktail and counted in a/3-counter. Determination of Intracellular Glutathione Total gluathione including the reduced (GSH) and oxidized (GSSG) form is quantified using the high-performance liquid chromatography (HPLC) method of Reed et aL 19 Briefly, the cell pellet is extracted with 0.5 ml of 1 N perchloric acid, and y-glutamyl glutamate is added as an internal standard. The extract is incubated for 120 rain with iodoacetic i8 A. Novogrodsky, R. E. J. Nehring, and A. Meister, Proc. Natl. Acad. Sci. U.S.A. 76, 4932 (1979). 19 D. J. Reed, J. R. Babson, P. W. Beatty, A. E. Brodie, W. W. Ellis, and D. W. Potter, Anal. Biochern. 106, 55 (1980).

110

GLUTATHIONE

[ 1 1]

acid at pH 8.5 to form S-carboxymethyl derivatives of free thiol groups. Subsequently, the amino groups are derivatized with 1-fluoro-2,4-dinitrobenzene (Sanger's reagent). Aliquots of the reaction mixture are injected onto a/xBondapak amine column (4 × 250 mm, Waters, Eschborn, Germany) and eluted with a sodium acetate gradient (flow rate of 2 ml/min) in a water-methanol-acetic acid solvent at pH 4.5. The dinitrophenyl derivatives are detected at 365 nm. The GSH and GSSG derivatives are quantified in relation to the internal standard. M e a s u r e m e n t of [a~S]Cystine Incorporated into Glutathione Recovery of [3SS]cystine incorporated into GSH is determined by HPLC and scintillation counting as described. 2° The incubation of cells in [35S]cystine-containing McCoy's 5A medium is essentially the same as described for the uptake method (see above). The cell pellet is extracted with icecold I N perchloric acid containing i mM EDTA (50/zl). The supernatant is neutralized with 22 /xl of 1.57 M K3PO4, vortexed, and the resulting precipitate of KCIO4 pelleted after 4 min on ice. The GSSG is reduced by adding 6/zl of a mixture of 20/zl of 100 mM glucose 6-phosphate (in water), 20 /zl of i mM N A D P H (in I% NaHCO3), 20 /zl of 5 mg/ml glucose6-phosphate dehydrogenase (from yeast, 140 U/rag, in water), 20/zl of glutathione reductase (from yeast, suspension of 120 U/ml), and finally 40 /xl of (i mg/ml) glutathione S-transferase. After i0 min at 37°, 5/zl of lchloro-2,4-dinitrobenzene (i0 mM in methanol) is added and the reaction mixture further incubated for 30 min at 37 °. Then the mixture is acidified with 5 /zl of i M H3PO4, centrifuged, and injected onto a /xBondapak C18 column (4 x 250 mm, Waters). The S-(2,4-dinitrophenyl)glutathione conjugate (GS-DNP) is eluted with 0.1 mM H3PO4/methanol (60:40, v/v, flow rate of 1.5 ml/min, Rt (retention time) of 4.65 rain, detection at 340 nm) and the radioactivity in the collected GS-DNP fractions counted as described. It should be noted that the possible 2,4-dinitrophenyl derivatives of cysteine and cysteamine are eluted at a n R t of 3.65 rain. Methological Considerations Investigations of reduced thiols in vitro require some precautions. Autoxidation reactions of reduced thiols during storage and dilution procedures occur spontaneously but can be avoided by simple techniques. During thiol exposure of ceils the autoxidation reaction consumes oxygen and produces reactive oxygen species (e.g., hydrogen peroxide) which may exert cytotoxic effects on the cells. 21 An enhanced toxicity of thiols in CHO cells 20K. G. Eckert and P. Eyer, Biochem. PharmacoL 35, 325 (1986). 21R. D. Issels, J. E. Biaglow,L. Epstein, and L. E. Gerweck, Cancer Res. 44, 3911 (1984).

[1 1]

PROMOTION OF CYST(E)INEUPTAKE

11 1

at higher t e m p e r a t u r e s was d e m o n s t r a t e d to be due to the generation of activated oxygen species. 22 Therefore, depending on the sensitivity of the cell system used, the addition of protection factors (e.g., catalase, heavy metal chelators) may be necessary. 23Besides the oxidation of the thiol group, other oxidation reactions of the thiol molecule m a y also occur. As demonstrated for WR-1065, which p r o m o t e s cystine uptake in C H O and Chinese hamster ovarian carcinoma (OvCa) cells, a increase in cellular glutathione was observed only for O v C a cells. 24In C H O cells the incubation with WR-1065 leads to a significant depletion of intracellular glutathione. Exposure of C H O cells to WR-1065 together with aminoguanidine, an inhibitor of copper-dependent amine oxidases, increased cellular glutathione as observed for other thiolsY Aminoguanidine prevents the enzymatic oxidation of the polyamine moiety of WR-1065 and the production of b r e a k d o w n products which are detoxified by the conjugation with glutathione. 26 Biological I m p l i c a t i o n s Exposure of ceils to several cytostatic agents leads to a depletion of intracellular glutathione. We have d e m o n s t r a t e d that activated ifosfamide (4-hydroperoxyifosfamide), an alkylating anticancer drug, depletes intracellular glutathione of P B L in v i t r o . 27 Also ifosfamide had differential effects on the capacity of lymphocyte subsets to lyse their target ceils which correlate with intracellular glutathione levels. 28 Reconstitution of glutathione levels and lymphocyte functions was achieved by addition of extracellular thiol compounds, (e.g., 2-mercaptoethanesulfonate (Mesna)). 28,29 Exposure of lymphocytes to Mesna in v i t r o increased the uptake of radioactive labelled cystine f r o m the m e d i u m Y Studies investigating the effects of Mesna in v i v o demonstrate p r o m o t i o n of cysteine uptake in PBL. 3° These findings suggest that the mechanisms of cyst(e)ine uptake and elevation of glutathione as demonstrated for C H O cells may also be relevant for other cell systems. z2 R. D. Issels and A. Nagele, this series, Vol. 186, p. 696. 2_'~j. E. Biaglow, R. W. Issels, L. E. Gerweck, M. E. Varnes, B. Jacobson, J. B. Mitchell, and A. Russo, Radiat. Res. 100, 298 (1984). 24R. D. Issels and A. Nagele, Cancer Res. 49, 2082 (1989). 25T. Meier and R. D. Issels, Biochem. PharmacoL, submitted for publication. 26A. Nagele, T. Meier, and R. D. Issels, Prog. YharmacoL Clin. Pharmacol. 8, 309 (1991). 27T. Meier, A. Allenbacher, E. Mueller, G. Multhoff, C. Botzler, M. Wiesnet, and R. D. Issels, Anti-Cancer Drugs 5, 403 (1994). 2s G. Multhoff, T. Meier, C. Botzler, M. Wiesnet, A. Allenbacher, W. Wilmanns, and R. D. lssels, Blood 4, (1995). 29R. D. Issels, T. Meier, E. MUller, G. Multhoff, and W. Wilmanns, MoL Aspects Med. 14, 281 (1993). 30B. Stofer-Vogel, T. Cerny, A. Kiipfer, E. Junker, and B. H. Lauterburg, Br. J. Cancer 68, 590 (1993).

112

GLUTATHIONE

[ I I]

Conclusions Exposure of CHO cells to thiols in vitro leads to enhanced intracellular glutathione levels via an increased uptake of cyst(e)ine. The use of such model systems where effects on intracellular glutathione play a major role may lead to a beter understanding of the fundamental processes which are influenced by the availability of cyst(e)ine and intracellular thiol metabolism.

[ 121

OVOTHIOLS

115

[ 12] Ovothiols By T O D

P. HOLLER

and

PAUL

B. HOPKINS

Discovery a n d D i s t r i b u t i o n Investigation of the molecular mechanism by which hydrogen peroxide is formed during the cyanide-insensitive respiratory burst that immediately follows sea urchin egg fertilization led T u r n e r et al.l to the observation that a cytosolic cofactor was able to confer cyanide-insensitive N A D ( P ) H oxidase activity on the enzyme ovoperoxidase. The isolated cofactor was identified by spectroscopic techniques 2 as the highly methylated 4-mercaptohistidine (3, in Fig. 1), which was eventually named ovothiol C. The positions of the methyl groups were determined by desulfurization of the cofactor with Raney nickel and comparison of the product with authentic samples of N-methylated histidines. These assignments were further demonstrated by nuclear Overhauser enhancement studies and total synthesis. 3 A survey conducted by T u r n e r el al. 4 of the eggs or ovaries of other marine invertebrates revealed two additional methylated mercaptohistidines that were given the names ovothiol A (1 in Fig. 1) and ovothiol B (2 in Fig. 1). A group of researchers in Naples, Italy, independently reported the isolation of a series of mercaptohistidines from marine invertebrates, s which were formulated as isomeric to the ovothiols with respect to the position of ring methylation. Comparison of ovothiol C with a sample of the purportedly isomeric compound isolated in Italy showed them to be identical, 3'4 strongly suggesting that the other mercaptohistidines reported by the Italian group are also members of the ovothiol family. Ovothiols A and B have also been identified in the eggs of trout and salmon, 6 and ovothiol A has been isolated from a marine a l g a l i E. Turner, C. Somers, and B. M. Shapiro, J. Biol. Chem. 260, 13163 (1985). E. Turner, R. Klevit, P. B. Hopkins, and B. M. Shapiro, J. Biol. Chem. 261, 13056 (1986). 3T. P. Holler, A. Spaltenstein, E. Turner, R. E. Klevit, B. M. Shapiro, and P. B. Hopkins, J. Org. Chem. 52, 4420 (1987). 4 E. Turner, R. Klevit, L. J. Hager, and B. M. Shapiro, Biochemistry 26, 4028 (1987). 5 A. Palumbo, G. Misuraca, M. d'Ischia, F. Donaudy, and G. Prota, Comp. Biochem. Physiol. 78B, 81 (1984). E. Turner, L. J. Hager, and B. M. Shapiro, Science 242, 939 (1988). 7 S. Selman-Reimer, R. J. Duhe, B. J. Stockman, and B. R. Selman, J. Biol. Chem. 266, 182 (1991).

METHODS IN ENZYMOLOGY,VOL. 252

Copyright © 1995 by AcademicPress, Inc. All rights of reproduction in any form reserved 7

116

[12]

SIGNAL TRANSDUCTIONAND GENE REGULATION CH3NH2 • HCI ++ CH20(aq) NaCN

a ~

O CH~N,,~H - ["~rr~

O CH3N,,~ H ~,,,NH 2

~ b

i 1 4

S

CH3 N C

5

CH3 N-~./CH2 OH

N~,SCH2C6H4CH 3 6

d

N,~.SCIq2C6H4CH3 7

CH3 N -CH2C1

CHACO2

N f~.SCHeC6HzCH3

N

H

SCHeC6HaCH3

8

9

CH3 . N~--~$CH2C6H4CH3

h

II N

N(CH3)2 SCHzC6H4CH3

10

11 CH3 N ~

CH3 ~q~CO2H ~

~ ~RIR2 N~SCH2C6H4CH 3

RI=H, R2=CH3 10: RI=R2=H 11:RI=R2=CH3

9:

~

CO2H

' ' - ' ~ -I~ SH I~R1R2 N

i 1: RI=R2=H 2: R~=H, R2----CH3 3:RI=R2---CH3

Fro. 1. Chemical synthesis of racemic ovothiols. Reagents: a, (i) H20, (ii) HC(O) OC(O)CH3. b, H2S, (CH3CH2)3N; c, (i) (CH3)3SiCI, (CH3CH2)3N, (ii) CHsC6H4CHzCI; d, CH20, sodium acetate/acetic acid; e, SOC12; f, (i) CHsCONHCH(CO2CH2CH3)2, Nail, (ii) HC1 (aqueous); g, (i) CHaCON(CH3)CH(CO2CH2CH3)z, Nail, (ii) HC1 (aq); h, CH20, NaBH3CN; i, (i) Hg(OCOCF3)2, CF3CO2H, (ii) H2S.

[ 121

OVOTHIOLS

117

Ovothiols from Natural Sources Detection in Tissue

A high-performance liquid chromatography (HPLC) method that allows detection of 0.1-1.0 nmol of ovothiol from tissue has been developed by Turner et al. 4 Tissue samples are homogenized in 4 volumes of water. A 1-ml sample of the homogenate is adjusted to pH 8, mixed with 1/zmol of dithiothreitol (DTT), incubated 10 rain, then admixed with 2.5 tzmol of tritiated [2 x 106 counts/rain (cpm)//xmol], iodoacetic acid for 30 min. Proteins are removed from the sample by precipitation with 10% trichloroacetic acid, followed by centrifugation in a microcentrifuge for 3 rain. The supernatant is loaded onto a 1-ml column of Dowex 50-X8, 100-200 mesh, ammonium form, previously equilibrated with 0.5 M formic acid. The column is washed with 10 ml of 0.5 M formic acid and then eluted with 10 ml of 0.5 M ammonium formate, pH 4.5 (0.5 M ammonium hydroxide adjusted to pH 4.5 with 88% aqueous formic acid). The concentrated eluate is lyophilized twice from water, resuspended in water, and loaded onto a 15-cm Altex ultrasphere ODS C18 column equipped with a standard precolumn. The column is washed for 10 min with a buffer containing 20 mM phosphoric acid and 5 mM sodium heptanesulfonate adjusted to pH 4.5 with aqueous ammonia, and then eluted with a gradient of 0 to 50% methanol in the same buffer. Ovothiols are detected by UV absorbance at 210 nm and scintillation counting. Identification is made by comparison to authentic samples. Isolation

Underivatized ovothiol C has been isolated in 100-mg quantities as described below. Ovothiol A may be obtained in about 10-mg quantities by isolation from cultured marine algae] but milligram quantities of ovothiol B have not been obtained from natural sources. Sea urchins (Strongylocentrotus purpuratus) are gathered intertidally and induced to spawn by intracoelomic injection of 0.50 M KC1. The eggs are collected in 0.45-/zm filtered seawater. The jelly coat is removed from 300 ml of settled eggs by resuspension in 3 liters of artificial seawater (0.5 M NaC1, 10 mM KCI, 2.5 mM NaHCO3, 25 mM EGTA, pH 8.0). The supernatant containing the jelly coat is decanted, and the eggs are resuspended in 600 ml of artificial seawater. Homogenation is accomplished with 10 strokes in a Teflon pestle tissue homogenizer. The homogenate is spun at 15,000 g for 60 rain, and the supernatant is boiled for 10 rain. Precipitated material is removed by centrifugation at 15,000 g for 10 min, and this supernatant is lyophilized to dryness. The residue is redissolved in 80 ml of water and brought to 800 ml with

118

SIGNAL T R A N S D U C T I O N AND GENE REGULATION

[ 12]

absolute ethanol. The solution is evaporated to dryness and extracted a second time, with 80 ml of 90% (v/v) ethanol. Lyophilized material from the second extraction is dissolved in 10 ml of water and applied to a 30-ml column of Dowex 1-X8 (200-400 mesh, hydroxide form) anion-exchange resin. The column is developed with a 300-ml linear gradient of 0 to 0.4 M ammonium formate, pH 7.0 (prepared by neutralization of 0.4 M formic acid with concentrated ammonia). Ovothiol C is identified in the fractions by its UV spectrum (see Fig. 2). This crude material is further purified by HPLC. Samples are adjusted to pH 2.5 with HC1 and loaded onto a Waters (Milford, MA) SP-5PW sulfopropyl cation-exchange column. After washing the column for 10 min with 40 mM formic acid, pH 2.5 (adjusted with concentrated ammonia), the ovothiol C is eluted with a gradient of 40 mM to 2 M formic acid, pH 2.5. Two peaks containing ovothiols are observed. The reduced form elutes at approximately 1.2 M, and the oxidized material, which normally constitutes the majority of the material, elutes at approximately 1.4 M formic acid, pH 2.5. As an alternative to the HPLC separation, acidified material can be applied to a column of Dowex 50-X8, 100-200 mesh, equilibrated with 0.5 M formic acid, and eluted with a gradient of 0.5 M formic acid to 0.5 M ammonium formate, pH 4.5. This material retains some organic impurities, detected by nuclear magnetic resonance (NMR) spectroscopy. 4

¢J

o.2

b

U L 0 0 •~

0.1

250

3~

350

Wovelength (nm) F~c. 2. Ultraviolet spectra of ovothiol C. (a) The UV spectrum of approximately 0.2 mM oxidized ovothiol C. (b) Spectrum taken after addition of a slight molar excess of dithiothreitol. (Reprinted with permission, from Turner et al. 2)

[ 121

OVOTHIOLS

i 19

Chemical Synthesis Chemical syntheses of the ovothiols, in racemic or optically active form, have been described. 8 A synthesis of racemic ovothiols amenable to multigram scale is presented in Fig. 1. The initial phase involves the synthesis of a protected mercaptoimidazole moiety. This general procedure can be used to synthesize a variety of imidazole-4-thiols which might serve as ovothiol analogs for a variety of purposes. 9 In the initial step (Fig. 1) formaldehyde, methylamine, and sodium cyanide are condensed to form an unstable intermediate which is promptly formylated with formic acetic anhydride. The nitrile amide 4 is quite stable and can be purified by distillation at reduced pressure, Addition of gaseous H2S to the nitrile is performed in a mixture of ethanol and triethylamine at room temperature. The crystalline thioamide 5 is cyclized to the imidazole-4-thiol (6) using trimethylsilyl chloride and triethylamine in methylene chloride at - 7 8 °, and the thiol group is protected in situ as the p-methylbenzyl thioether. Elaboration of the amino acid side chain begins with hydroxymethylation using aqueous, acetate-buffered formaldehyde. This particular hydroxymethyl compound (7) can be crystallized from ethyl acetate, avoiding chromatography at this early stage of the synthesis. Addition of 7 in small portions to ice-cold, freshly distilled thionyl chloride, followed by stirring at room temperature and evaporation in vacuo, provides the chloromethyl derivative as the hydrochloride (8). This material is used directly in the next step. Reaction of 8 with 2 equivalents of the sodium salt of diethyl acetamidomalonate in tetrahydrofuran (THF), keeping the reaction temperature below 30 °, followed by hydrolysis for 12 hr in boiling 6 N HC1 and concentration at low pressure affords crude Sprotected ovothiol A (9). This material is diluted with water, brought to pH 7 with aqueous ammonia, and applied to a column of Amberlite XAD4. The column is washed exhaustively with water to remove the glycine, then eluted with batches of 10, 20, 35, and 50% aqueous methanol. Fractions containing the desired product are pooled and evaporated to a yellow solid, which becomes white on trituration with ethanol. Substitution of the sodium salt of diethyl N-methylacetamidomalonate in this procedure afforded Sprotected ovothiol B (10). Reductive methylation of S-protected ovothiol A with aqueous formaldehyde and sodium cyanoborohydride afforded Sprotected ovothiol C (11). The p-methylbenzyl protecting group is removed from each of the Sprotected ovothiols by reaction with mercuric trifluoroacetate in ice-cold T. P. Holler, F. Ruan, A. Spaltenstein, and P. B. Hopkins,J. Org. Chem. 54, 4570 (1989). 9 A. Spaltenstein, T. P. Holler, and P. B. Hopkins, J. Org. Chem. 52, 2977 (1987).

120

SIGNAL TRANSDUCTION AND GENE REGULATION

1121

trifluoroacetic acid containing anisole. The final product (1-3) is most easily desalted and stored as the disulfide, which is obtained by adding a trace of CuC12 to an aqueous solution of the thiol and bubbling oxygen through the mixture. The oxidized material is desalted by loading the aqueous solution onto a column of Dowex 50W-X8, hydrogen form, washing with water, and eluting with 1 M ammonium hydroxide. Ninhydrin-reactive fractions are pooled and lyophilized. Salt-free ovothiol A can then be obtained by reduction of the disulfide in a minimum of water treated at 25 ° with 2 to 3 equivalents of dithiothreitol. For applications compatible with the presence of both reduced and oxidized dithiothreitol, the resulting solution may be used. These contaminants may be removed by chromatography on Dowex 50-X8 ion-exchange resin, ammonium form, containing oxygen-free (N2 purged) aqueous ammonium formate, pH 2.5, then eluted with oxygen-free 1 M ammonium formate, pH 4.0. The eluted ovothiol may be detected by ultraviolet spectroscopy or Ellman's assay. Samples of racemic ovothiol A disulfide synthesized by this method can be obtained, while supplies last, from one of the authors (P.B.H.).

Physical Properties

Spectroscopic Properties The aH NMR spectra of the ovothiols vary somewhat with reduction state and pH of the solution; however, the method remains a useful one for determining purity. The spectra of ovothiol disulfides in approximately neutral DzO include a singlet at approximately ~ 7.8 (imidazole C-2 H), a singlet at approximately 8 3.7 (imidazole N-CH3), and three multiplets at approximately 8 3.3 (C-o~ H), 2.8 (C-/3 H), and 2.3 (C-/3' H). The spectra of ovothiols B and C contain, in addition to the resonances listed above, a singlet at approximately 8 2.8 which arises from the aliphatic N-methyl groups. It has been our experience that XH NMR spectroscopy in aqueous solution is not useful for the determination of the thiol/disulfide ratio of ovothiols, because only a single set of resonances is observed, even at 500 mHz. This is presumably due to fast chemical exchange of the two species. As noted below, ultraviolet spectroscopy can be used for this analysis. The ultraviolet spectra of the ovothiols provide both a useful handle during their detection and isolation and a means of determining their redox state. The UV spectra of reduced and oxidized ovothiols, as seen in Fig. 2, have an isosbestic point at 288 nm, an absorbance ratio of 1.63 at 260 nm, and an absorbance ratio of 2.8 at 236 nm. The oxidized material has

[ ] 2]

OVOTHIOLS

121

significant absorption above 320 nm, imparting a yellow color to concentrated solutions; the reduced form lacks this absorption and is colorless. Ultraviolet spectroscopy provides a particularly simple method for the analysis of the ovothiol content and thiol/disulfide ratio in unknown sampies. The absorbances at 288 and 240 nm of a solution (35 tzg sample/ml) in phosphate buffer, pH 7.0, are measured in a 1-cm cuvette. The weight percent ovothiols in the original sample are Weight percent ovothiol = 100(A240 - A288) Weight percent ovothiol disulfide --- 100(3A28s - A240) Weight percent (ovothiol + disulfide) = 200A288 For example, a sample of ovothiol in phosphate buffer, pH 7, giving an A288 of 0.24 and an Ae40 of 0.55 is 31% ovothiol [100(0.55 - 0.24)]. The same sample is 17% disulfide, or a total of 48% total ovothiol/disulfide. Alternatively, as mentioned below, Ellman's reagent can be used for the analysis of the thiol content of ovothiols. Thiol pKa

Knowledge of the pK, of the thiol group is important in understanding the reactivity of the ovothiols. To avoid the complications of making this determination in the presence of the a-amine and carboxylic acid of the natural substances, Holler and Hopkins ~°titrated 1,5-dimethyl-4-mercaptoimidazole (DMI), a model compound which lacks them. Two observed pKa values at 2.3 and 10.3 were combined with the observation that the Smethyl derivative of DMI has a single pKa of 6.0 to produce the diagram in Fig. 3. At neutral pH, DMI, and by inference the ovothiols, exists almost exclusively as the zwitterion in which the imidazole nitrogen is protonated and the thiol is present as the thiolate. Interestingly, the histidine ring nitrogen is sufficiently basified by the neighboring thiolate to persist in the protonated form to about pH 10, some 4 units higher than the histidine itself. Redox Properties

The midpoint redox potential for the conversion of ovothiol C disulfide to the thiol form has been estimated to be 84 mV positive compared to glutathione.11 The thiol form is rapidly oxidized by hydrogen peroxide. Turner et al. 6 measured a second-order rate constant of 2.0 M -1 s -1 at 30° and pH 7.2 for the reaction of ovothiol C with hydrogen peroxide. Ovothiol disulfides are reduced to the thiol forms by glutathione and, more rapidly, to T. P. Holler and P. B. Hopkins, J. Am. Chem. Soc. U0, 4837 (1988). 11 B. M. Shapiro and E. Turner, Biofactors J. 1, 85 (1988).

122

SIGNAL TRANSDUCTION AND GENE REGULATION

[ 1 2]

CH 3 N~jCH3

(Im - SH)

pKim =

~ K s r

t = 6.7

CH3

CH3

N~CH3

N ~..~CH3 pKzw = 3"7 N+

(~NLs.

SH

(ImH + - SH)

/¢~

X

~ p K

pK 1 = 2 . 3 ~

(Ira - S') 2 = 10.3

OH3

(ImH ÷ - S') FIG. 3. Acid-base equilibria of an ovothiol analog. The pKa values depicted were calculated from the two macroscopic pKa values measured on titration of 1,5-dimethyl-4-mercaptoimidazole (DMI) and the assumption that the pKa at upper left [(ImH+-SH) to (Im-SH)] is identical to that measured on titration of S-methyl-DMI. (Reprinted with permission from Holler and Hopkins. 1° Copyright 1988 American Chemical Society.)

by dithiothreitol. Reduced ovothiols can be determined, as can many other thiols, 12 using Ellman's reagent [5,5'-dithiobis(2-nitrobenzoic acid), DTNB]. The reactivity of the ovothiols in one-electron reduction reactions has been investigated by Holler and Hopkins. 1°'13'14 The first demonstration that the mercaptoimidazole moiety is an effective one-electron donor used ferricytochrome c as a generic one-electron acceptor. The ovothiol analog DMI (see Fig. 3) reacts at physiological pH with a second-order rate constant of 30 M -1 sec -~. The pH-rate profile revealed that the active species is the DMI anion, which reacts with a rate constant of roughly 104 M -I sec-k The ability of ovothiol A to scavenge photochemically generated 12p. C. Jocelyn, this series, Vol. 143, p. 44. 13T. P. Holler and P. B. Hopkins, AnaL Biochem. 180, 326 (1989). 14T. P, Holler and P. B. Hopkins, Biochemistry 29, 1953 (1990).

[131

XAS

STUDIES OF PROTEIN

KINASE

C

123

tyrosyl radicals 12 far surpasses that of glutathione, more closely resembling the reactivity of ascorbic acid and the soluble tocopherol derivative Trolox. This reactivity difference, which is also observed when using Fremy's salt or Banfield's radical, 13 is presumably due to the more favorable free energy of conversion of an aromatic thiol to thiyl radical relative to its aliphatic analog. The presumed thiyl radical intermediate in these reactions seems, in model compounds at least, to be unreactive with oxygen. Biological Significance Ovothiols are present at millimolar concentrations in the eggs of marine invertebrates, persist in the first 2 weeks of embryo development, but are absent from the somatic tissues of adults. This suggests some special role in early development that is no longer necessary in later life. The fact that high concentrations of hydrogen peroxide are generated and used just outside the egg in early fertilization events, and the differences in redox chemistry of the ovothiols compared to glutathione, have led Turner et al. 6 to propose that the ovothiols replace glutathione peroxidase as a hydrogen peroxide scavenger. The ready participation of ovothiols and their analogs, but not glutathione, in one-electron redox chemistry has led Holler and Hopkins 14 to suggest that there may also be a role for the ovothiols in protection of the embryo from free radical-induced damage as well. When the low thiol pKa and relative abundance of the ovothiols are taken into consideration along with the redox properties mentioned above, it becomes clear that the ovothiols have the capability of protecting the egg and the early embryo from damage that might result from a wide range of electrophilic species.

[13] C h a r a c t e r i z a t i o n of the Cysteine-Rich Zinc-Binding Domains of Protein Kinase C by X-Ray Absorption Spectroscopy B y STEVAN R. HUBBARD

Introduction Protein kinase C (PKC) plays a key role in the regulation of cellular signaling events in eukaryotic organisms from yeast to humans, catalyzing the transfer of the terminal phosphate of adenosine triphosphate (ATP) to specific serine or threonine residues of substrate proteins (for review, METHODS IN ENZYMOLOGY. VOL. 252

Copyright © 1995 by Academic Press, lnc, All rights of reproduction in any form reserved,

124

SIGNAL TRANSDUCT1ON AND GENE REGULATION

[ 13]

see Ref. 1). Protein kinase C forms a family of closely related isozymes which are activated at the inner surface of the plasma membrane by diacylglycerol, a product of receptor-mediated phosphatidylinositol hydrolysis. Phosphatidylserine is an essential cofactor for activation, and calcium is required for a subset of isozymes. The single polypeptide chain of PKC consists of an NH2-terminal regulatory domain, in which the divergence among isozymes is primarily found, and a COOH-terminal catalytic domain, which shows sequence similarity to the catalytic domains of other protein kinases. Within the regulatory domain are two adjacent, highly similar sequences of approximately 50 amino acids containing strictly conserved cysteines (six) and histidines (two) in the pattern H-XI2-C-X2-C-Xlo_14-C-X2-C-X4-H-X2-C-X7-C (C6H2) , where the intervening X residues are, in general, more variant. The C6H2 regions of PKC have been shown to be essential for the binding of phorbol esters, which can substitute for diacylglycerol.2 The C6H2 motif is also found in Raf, Vav, chimaerin, diacylglycerol kinase, and the Caenorhabditis elegans unc-13 gene product. Because a cytosolic protein such as PKC is not expected to contain disulfide bonds, the conserved cysteines in the C6H2 regions are likely involved in metal ion coordination. My colleagues and I investigated this proposition using X-ray absorption spectroscopy (XAS), and a report of our findings has been published. 3 Here, I present in greater detail the experimental methods used to show that PKC is a metalloprotein which contains four zinc ions coordinated primarily by cysteine thiolates. Description of X-Ray Absorption Spectroscopy Techniques In XAS, a metal-containing sample is illuminated by a monochromatic beam of X-rays of sufficient energy to stimulate core electron transitions. Absorption is measured by monitoring either the diminution of the X-ray beam as it traverses the sample or the fluorescence emanating from the sample arising from electron relaxation. Two types of XAS techniques were employed in the PKC study. The first, called X-ray fluorescence (XF), was used to identify which if any metal atoms were present in the protein sample and to quantitate the amount. Here, the sample is irradiated by monochromatic X-rays with a fixed energy higher than the absorption edges of the metals of interest, and the energy spectrum of fluorescent photons i S. Stabel and P. J. Parker, Pharmacol. Ther. 51, 71 (1991). 2 y. Ono, T. Fujii, K. Igarashi, T. Kuno, C. Tanaka, U. Kikkawa, and Y. Nishizuka, Proc. Natl. Acad. Sci. U.S.A. 86, 4868 (1989). 3 S. R. Hubbard, W. R. Bishop, P. Kirschmeier, S. J, George, S. P. Cramer, and W. A. Hendrickson, Science 254, 1776 (1991).

[ 13]

XAS STUDIESOF PROTEINKINASEC

125

is recorded. Each atomic element has fluorescence emission lines of characteristic energy which can be used to identify its presence. The second technique, called extended X-ray absorption fine structures (EXAFS), was used to obtain information regarding the chemical environment of the metal atom, namely, the type and number of ligating atoms and metal-ligand distances. In this case, absorption is measured as a function of incident X-ray energy over a range that typically extends from several 100 eV below an absorption edge to 500-1000 eV above. Above the edge, backscattering of the ejected photoelectron by atoms in the near vicinity of the metal atom leads to a modulation of the absorption cross section, which appears as oscillations in the absorption spectrum. 4 The XAS experiments are optimally performed at synchrotron X-ray sources, owing to their high flux and tunability. In a preliminary XF experiment done on beam line X-19A at the National Synchrotron Light Source (NSLS), Brookhaven National Laboratory, we identified stoichiometric amounts of zinc in a PKC sample. At a later date, we collected EXAFS spectra and repeated the XF measurements.

X-Ray Fluorescence Experiment

Sample Preparation The preparation of the PKC sample used for both the XF and EXAFS experiment has been described) Briefly, Sf9 insect cells (Spodoptera frugiperda, fall armyworm ovary) were infected with a recombinant baculovirus containing the cDNA for the rat PKC/3I isozyme. The protein was purified to near homogeneity in three column chromatography steps. To remove any metal ions not tightly bound to the protein, i mM EGTA was included in all column buffers. The sample was fully active as determined by an in vitro kinase assay. The PKC/3I from three preparations was pooled and concentrated in a Centricon-30 (Amicon, Beverly, MA) to approximately 25 mg/ml. The sample used for a control in the XAS experiments was a concentrated pool of fractions that elutes just prior to PKC /3I on the last column.

Data Collection and Processing The detector used for the XF and EXAFS experiments was a liquid nitrogen-cooled array of 13 germanium elements with an energy resolution 4S. P. Cramer and K. O. Hodgson,Prog. Inorg. Chem. 25, 1 (1979).

126

SIGNAL TRANSDUCTION AND GENE REGULATION

[ ] 3]

of approximately 200 eV. 5 For the XF experiment, the signal from one germanium element, which is proportional to the energy of the incident photon, was fed into a Canberra multichannel analyzer (MCA). A pulse height analysis is performed by the MCA, and a histogram of detected photons versus channel number (energy) is displayed and stored. Fluorescence was monitored in the direction normal to the incident X-rays in the horizontal plane. For our experiment, the silicon (220) double-crystal monochromator was set to pass X-rays of 11.2 keV. We were able to detect fluorescent photons with energies from approximately 3.5 to 10.0 keV, which includes Ks emission lines for calcium (atomic number Z = 20) through germanium (Z = 32) and L~ emission lines for tin (Z = 50) through iridium (Z = 77). Zinc standards of 0, 0.5, 1.0, and 2.0 mM were prepared from a zinc atomic absorption standard solution (Sigma, St. Louis, MO); volumes were measured gravimetrically. A Lucite EXAFS cell (outer dimensions of 25.4 by 3.7 by 2.4 mm) with a Mylar window and a sample volume of approximately 70 t~l was used for all samples. The cell was rinsed extensively with an EDTA solution and deionized water between samples. The XF data at room temperature were accumulated for 5 rain for each sample. Raw data in the form of fluorescence counts versus MCA channel number (1024 points per spectrum) were converted to counts versus energy with a two-point linear calibration using the chromium K~ peak (see below) and the incident beam scatter peak (11.2 keV). Each of the energy-calibrated spectra was interpolated onto the same uniformly spaced energy grid and normalized by the integrated counts in the scatter peak to correct for differences in total incident flux. Difference spectra were obtained by subtracting the control spectrum from the PKC/~I spectrum and the 0 mM zinc spectrum from the other zinc standards spectra. The counts in the zinc K~ peaks of the difference spectra were summed. Results

The XF data for the PKC/3I and control samples are shown in Fig. 1. The chromium and copper K~ peaks (5.41 and 8.04 keV) are present in the spectrum of an empty sample cell and derive from the Mylar window; Kapton was later found to be a more suitable window material. The two peaks unique to the PKC ~I spectrum are consistent with zinc K~ and K B emissions (8.63 and 9.57 keV) and are highlighted in the difference spectrum (Fig. 1, inset), The small peak in the difference spectrum at copper K~ 5 S. P. Cramer, O. Tench, M. Yocum, and G. N. George, Nucl. Instrurn. Methods Phys. Res. Sect. A 266, 586 (1988).

[13]

X A S STUDIES OF PROTEIN KINASE C 70

. . . .

i . . . .

i . . . .

I . . . .

L , q l l l l l l

i

I J L~l

.............................. Zn K~

60×

a . . . .

127 Ii

,li

Scatter peak

50E

40O

"~ 30>, -

(

I1)

---~ 100

Cr KcL

-

ZnKcz ~

C

/

/

~ . . . .

3.0

i

,

4.0

,

,

,

I

. . . .

5.0

i

. . . .

6.0

i

. . . .

7,0

i

. . . .

8.0

t

. . . .

9.0

i

. . . .

i

,

,

10.0 11.0 '12.0

Energy (keY) FIG. 1. Energy-calibrated XF spectra of the PKC/3[ (solid fine) and control (dotted line) samples prior to normalization by incident X-ray flux. Inset: Normalized difference spectrum. (Reprinted with permission from Hubbard et al. 3 Copyright © 1991 AAAS.)

is probably not significant owing to the sizable copper Ks peak in the control spectrum. The concentration of zinc in the PKC/3I sample was determined by a comparison of the summed counts in the zinc Ks peaks of the protein and standards spectra. Based on a linear regression analysis of the zinc standards (fit correlation of 0.991), the zinc concentration in the PKC/3I sample was 1.22 + 0.03 raM. The standard deviation was estimated by examining the effects of reasonable changes in energy calibration, spectra normalization, and peak integration on the concentration calculation. The concentration of PKC BI in the sample was obtained by measuring the total protein concentration by amino acid analysis and the PKC 13I purity by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE) and densitometry. Duplicate measurements using a Beckman 6300A amino acid analyzer were made for each of two separate aliquots taken from the XAS sample. The value obtained for the total protein concentration was 29.2 + 1.2 mg/ml. An SDS-polyacrylamide gel (12.5%) stained with Coomassie blue was scanned in two dimensions on a Molecular Dynamics 300A computing densitometer. Several lanes with differing amounts of loaded protein were analyzed; a lane containing just sample buffer served for baseline measurements. According to the densitometry, PKC/3I comprised 76 + 4% of the total stained protein (the most significant contaminant comprised 4%). As a positive control, a homogeneous preparation of ribonuclease H was also run on the gel, for which a purity value of 98% was obtained. Because Coomassie blue, like other protein dyes, shows

128

SIGNAL TRANSDUCTION AND GENE REGULATION

[ 13]

some amino acid composition dependence in protein staining, the systematic error in the purity measurement decreases with increasing protein purity. The combined measurements gave a PKC/3I concentration of 22.2 + 1.5 mg/ml or 0.289 mM (Me 76,781). The zinc ion to PKC/3I stoichiometry is therefore 4.2 _+ 0.3. The error estimate was obtained by applying the standard formula for propagating errors assumed to be independent. Thus, the XF data indicate that four zinc ions are tightly bound to PKC/3I.

Extended X-Ray Absorption Fine Structure Experiment

Data Collection and Processing To prevent X-ray-induced protein damage, the PKC/3I sample was cooled to approximately 15 K using a liquid helium cryostat. Twenty-one scans through the zinc K-absorption edge (9.66 keV), from 9.25 to 10.50 keV, were recorded with step sizes of 1.0 eV in the edge region and 2.0 or 3.0 eV in the EXAFS region. The duration of each scan was 35 min. Two scans for baseline subtraction were taken of a cell containing sample buffer. A zinc foil placed downstream of the sample position was used to calibrate the energy for each scan. Transmission EXAFS data were taken at room temperature for a zinc model compound, (1,10-phenanthroline)bis(4-toluenethiolato)Zn2+, in which zinc is coordinated by two sulfur and two nitrogen atoms. 6 Data from the seven active germanium elements of the detector array were weighted by the size of the edge jump and averaged together. No time-dependent changes in the scans, indicative of sample deterioration, were observed. A small edge-like feature in the far EXAFS region of the PKC/3I spectrum, which was attributed to a tungsten contaminant in the cryostat, limited the usable data to below 10.2 keV. In addition, the baseline spectrum showed a zinc K-edge feature with an edge jump 14% of that in the PKC/3I spectrum. This was also due to a contaminant in the cryostat, which was not present in the preliminary setup of the experiment. The baseline spectrum was smoothed, with the edge feature retained, and subtracted from the PKC/3I spectrum. Tests showed that the EXAFS results were not overly sensitive to this subtraction. A cubic spline was fit to the region above the edge to extract the EXAFS oscillations. Conversion from energy E (keV) to photoelectron wave number k (/~-~) was done with an estimate for the threshold energy, E0, of 9.670 keV {k = [0.262(E E0)]}v2. EXAFS spectra were smoothed with a Gaussian function of width 0.1/~-i. 6 T. L. Cremers, D. R. Bloomquist, R. D. Willet, and G. A. Crosby, Acta Crystallogr., Sect. B: Struct. Crystallogr. Cryst. Chem. 36, 3097 (1980).

[13]

X A S STUDIES OF PROTEIN KINASE C

129

Curve fitting analyses were done with the nonlinear least-squares program OPT, 7 using curved wave-derived theoretical backscattering amplitudes and phases computed with the program FEFF. 8 The single-scattering expression used to obtain the calculated EXAFS spectrum x¢(k) is given by

e_2 2abk2e_2Rab/X(k) sin[2kRab + aab(k)] xc(k) = S ~ NbAab(k,Rab) ~ where Nb is the number of b-type backscattering atoms at a distance Rab from the absorber atom a, k is the photoelectron wave number, Aab(k, Rab) is the total backscattering amplitude including intrinsic losses, aab(k) is the total phase shift function, Crab is the root mean square deviation of Rab (Debye-Waller-like factor), h(k) is the electron mean free path, and S is a k-independent scale factor relating experimental and calculated spectra, s

Results The zinc K-edge absorption spectrum for the PKC flI sample is shown in Fig. 2a, and the extracted EXAFS oscillations are shown in Fig. 2b. The observed absorption spectrum is a superposition of the absorption features for each of the four zinc sites in the protein. The initial curve fitting analyses clearly indicated that the EXAFS oscillations were primarily due to backscattering from sulfur atoms with a zinc-sulfur distance of approximately 2.3/~ (Fig. 2c). It was more difficult to establish: (1) the average number of coordinating sulfur atoms, (2) the presence of a lower Z ligand (e.g., nitrogen), and (3) the strength of evidence for thiolate-bridged zinc sites. Fitting results for several plausible coordination models are given in Table I. The 3.5S/0.5N model represents a coordination scheme in which two zinc ions are bridged by a single thiolate, one zinc ion having a total of four sulfur ligands and the other having one nitrogen and three sulfur ligands. The 2S/2S model was included to test a four-sulfur model with the same number of independent parameters as for the 3S/1N model (a 3S/1S model was also tested). The model consisting of three sulfur atoms and one nitrogen atom (3S/1N) gave the best fit, and the derived zinc-sulfur and zincnitrogen distances, 2.32 and 2.08 ~ , and mean-square distance deviations are chemically reasonable. Distinguishing between a nitrogen and oxygen backscatterer is problematic, especially when its contribution to the EXAFS is minor. A 3S/10 model would have given an equally good fit, but a nitrogen was favored because of the conserved C6H2 histidines. 7 Z. K. Eccles, Thesis, Stanford University, Stanford, California (1977); modifications to the program were made by G. N. George and S. P. Cramer. 8 j. j. Rehr, J. Mustre de Leon, S. I. Zabinsky, and R. C. Albers, J. Am. Chem. Soc. 113, 5135 (1991).

1 3 0

TRANSDUCTION

S I G N A L

(a)

A N D

G E N E

(:l) ~ "

0.25 ~. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

(J)

i

.~..,

[13]

R E G U L A T I O N

,

.

~: x~ k5 ¢~ ~'-~"

9.6

9.7

9.8 9.9 10.0 Energy (keY)

10.1

(b)

10.2

(c) 8.0

~

4.0

2.o

X 0.0 ~<~1.o

I.U -4.0-8.02.00

•

,

•

,

4.0

•

,

.

,

."

,

6.0

.

f

.

8.0

,

.

,

•

i

10.0

k (A-I)

o.o

2.o

4.0

6.0

R (A)

Flo. 2. (a) Baseline-subtracted zinc K-edge absorption spectrum for the PKC/31 sample. (b) Fit of the 3S/1N model (Table I) to the k3-weighted, smoothed EXAFS data; the nonsmoothed experimental spectrum is shown by the solid line and the calculated spectrum by the dashed line. (c) Fourier transform of the experimental and calculated k3-weighted EXAFS data over the k-range 2.0 to 11.4 ~ i, phase corrected with Zn-S phases. An R-space window of 1.6 /~ approximately centered on the Zn-S peak was used in the backtransformation, that yields the first shell contribution to the EXAFS. (Reprinted with permission from Hubbard et al. 3 Copyright © 1991 AAAS).

A discussion of various zinc c o o r d i n a t i o n m o d e l s for P K C involving nonbridged, singly bridged, and d o u b l y bridged zinc ions has b e e n presented. 3 In light o f the X A S results, k n o w n zinc c o o r d i n a t i o n chemistry, and s e q u e n c e c o n s e r v a t i o n arguments, the m o s t plausible m o d e l is one in which there are two n o n b r i d g e d zinc sites per C6H2 region, with an average c o o r d i n a t i o n e n v i r o n m e n t of three sulfur a t o m s and o n e nitrogen atom. This m o d e l was subsequently confirmed b y the three-dimensional N M R structure of a single cysteine-rich domain. 9 Discussion T h e c o m b i n a t i o n of X F and E X A F S techniques e n a b l e d us to show that P K C tightly binds four zinc ions which are c o o r d i n a t e d primarily by 9 U. Hommel, M. Zurini, and M. Luyten,

N a t u r e Struct. Biol.

1, 383 (1994).

[131

X A S STUDIES OF PROTEIN KINASE C

131

TABLE I EXTENDED X-RAY ABSORPTION FINE STRUCTURE CURVE FITrlNG RESULTS FOR PROTEIN KINASE C/3I

Model 3S/IN 3.5S/0.5N 2S/2S 4S

Atom a

NA b

S N S N S S S

3 1 3.5 0.5 2 2 4

R (z~)c

2.32 2.08 2.32 2.09 2.30 2.35 2.32

[2.31] [2.03] ]2.311 [2.001 [2.30] [2.31] 12.31]

ty2 (/~k2 X 103)

3.1 2.6 4.2 1.5 3.1 6.2 5.0

[2.5] [4.11 [3.5] I1.51 [10.6] [1.7] [4.7]

Fit indexa 3.1 [144] 5.2 11561 10,6 [1731 11,9 [184}

" S, Sulfur; N, nitrogen. b The coordination number N A was held fixed during the fittings. "The first values given in the table are from fitting the k3-weighted, Fourier-filtered EXAFS data isolating the first shell contributions, and the values in brackets are from fitting the k3-weighted, smoothed EXAFS data. The estimated errors in the distances are _+0.02 for Zn-S and +_0.04 ~ for Zn-N, based on fittings of the EXAFS spectrum for the zinc model compound. ,t The fit index is defined as [E(X~ - Xc)2k6]ln, where ×e and Xc are the experimental and calculated EXAFS and n is the number of data points in the fitting range. In the fittings, the overall scale factor was held fixed at 0.9. Adjustments to the threshold energy, AE0, were held fixed at the values derived from the Zn model compound fittings (performed over the same k ranges as the PKC/31 fittings). The AE0 values were -4.2 eV for the Fourier-filtered spectrum fittings (k = 3.0 to 11.0 ~ 1) and -6.4 eV for the smoothed spectrum fittings (k = 2.0 to 11.4 ~-l), (Reprinted with permission from Hubbard et al.3 Copyright © 1991 AAAS).

s u l f u r a t o m s . S e q u e n c e c o n s e r v a t i o n a n d a n e g a t i v e test r e s u l t for i n o r g a n i c sulfide a r g u e t h a t t h e c y s t e i n e t h i o l a t e s in t h e C6H2 r e g i o n s o f t h e r e g u l a t o r y d o m a i n s e r v e as l i g a n d s . T h e z i n c i o n s m o s t l i k e l y p l a y a s t r u c t u r a l r a t h e r t h a n a c a t a l y t i c role, s t a b i l i z i n g t h e c o n f o r m a t i o n of t h e C6H2 r e g i o n req u i r e d for i n t e r a c t i o n w i t h m e m b r a n e p h o s p h o l i p i d s , d i a c y l g l y c e r o l , o r both. A n a l t e r n a t i v e t e c h n i q u e to X F t h a t is o f t e n u s e d to c h a r a c t e r i z e m e t a l l o p r o t e i n s is a t o m i c a b s o r p t i o n s p e c t r o s c o p y . T h i s t e c h n i q u e h a s b e e n app l i e d to P K C , 1° a n d t h e d e r i v e d z i n c s t o i c h i o m e t r y is c o n s i s t e n t w i t h o u r X F r e s u l t . A t o m i c a b s o r p t i o n s p e c t r o s c o p y is a m o r e s e n s i t i v e t e c h n i q u e t h a n X F , r e q u i r i n g less c o n c e n t r a t e d p r o t e i n s a m p l e s , b u t it p r o v i d e s n o i n f o r m a t i o n r e g a r d i n g t h e c h e m i c a l n a t u r e o f t h e m e t a l site. T h e a d v a n t a g e s

~0A. F. G. Quest, J. Bloomenthal, E. S. G. Bardes, and R. M. Bell, J. Biol. Chem. 267, 10193 (1992).

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of the X A S t e c h n i q u e s are the s i m u l t a n e o u s d e t e c t i o n of a v a r i e t y of m e t a l s a n d the ability to o b t a i n d e t a i l e d i n f o r m a t i o n o n m e t a l c o o r d i n a t i o n . Acknowledgments I thank Robert Bishop, Paul Kirschmeier, Stephen Cramer, and Simon George for their roles in the XAS work and gratefully acknowledge the contributions of Wayne Hendrickson.

[14] Modifications of Cysteine-Rich Regions in Protein Kinase C Induced by Oxidant Tumor Promoters and

Enzyme-Specific Inhibitors B y RAYUDU GOPALAKRISHNA, ZHEN-HAI CHEN, a n d USHA GUNDIMEDA

Introduction P r o t e i n k i n a s e C ( P K C ) is a c t i v a t e d n o t o n l y b y n o r m a l cellular signals such as C a 2+ a n d diacylglycerol, b u t also b y t u m o r p r o m o t e r s such as p h o r b o l e s t e r s J P h o r b o l esters also i n d u c e the d o w n r e g u l a t i o n of P K C . 2 V a r i o u s o x i d a n t s have b e e n s h o w n to b e t u m o r p r o m o t e r s J -5 W o r k f r o m o u r l a b o r a t o r y , 6-8 as well as f r o m others 9-11 has s h o w n the a c t i v a t i o n a n d i n a c t i v a t i o n of P K C b y oxidative modification. A m o n g various n a t u r a l p r o d u c t s s c r e e n e d , c a l p h o s t i n C, hypericin, a n d c h e l e r y t h r i n e have e m e r g e d as specific i n h i b i t o r s for P K C . 12-14 T h e s e agents are c u r r e n t l y e m p l o y e d as 1y. Nishizuka, Science 258, 607 (1992). 2 S. Stabel and P. J. Parker, Pharmacol. Ther. 51, 71 (1991). 3 p, Cerutti, Science 227, 375 (1985). 4 T, J. Slaga, A. J. P. Klein-Szanto, L. L. Triplett, P. Yotti, and J. E. Trosko, Science 213, 1023 (1981). 5 L. Srinivas and N. H. Colburn, Carcinogenesis 5, 515 (1984). 6 R. Gopalakrishna and W. B. Anderson, FEBS Lett. 225, 233 (1987). 7 R. Gopalakrishna and W. B. Anderson, Proc. Natl. Acad. Sci, U.S.A. 86, 6758 (1989). s R, Gopalakrishna and W. B. Anderson, Arch. Biochem. Biophys. 285, 382 (1991). 9 G. E. N. Kass, S. K. Duddy, and S. Orrenius, Biochem. J. 260, 499 (1989). 10R. Larsson and P. Cerutti, Cancer Res. 49, 5627 (1989). 11E. J. Palumbo, J. D. Sweatt, S. Chen, and E. Klann, Biochem. Biophys. Res. Commun. 187, 1439 (1992). 12E. Kobayashi, H. Nakano, M. Morimoto, and T. Tamaoki, Biochem. Biophys. Res. Commun. 159, 548 (1989). 13I. Takahashi, S. Nakanishi, E. Kobayashi, H. Nakano, K. Suziki, and T. Tamaoki, Biochem. Biophys. Res. Commun. 172~993, (1990). 14j. M. Herbert, J. M. Augereau, J. Gleye, and J. P. Maffrand, Biochem. Biophys. Res. Commun. 172, 993, (1990).

METHODSINENZYMOLOGY,VOL.252

Copyright@1995byAcademicPress,Inc. Allrightsofreproductionin anyformreserv~:l.

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pharmacological tools to study the PKC signal transduction pathway. Our studies have demonstrated an involvement of site-specific oxidations or alkylating mechanisms in the action of these agents, a5'~6This further emphasized the functional role for unique thiol-rich regions in both regulatory and catalytic domains of PKC. As PKC is currently targeted for the development of new anticancer drugs, the knowledge of mechanisms concerning how the natural products induce modifications of thiol-rich regions in PKC may be beneficial in the design and devleopment of more specific PKC inhibitors. It is important to take into consideration the structural differences in the cysteine-rich regions in the regulatory and catalytic regions while designing procedures for PKC modification. The C1 constant region present within the regulatory domain of PKC (a,/3, and y isoenzymes) contains two sequences referred to as Cysl and Cys2, each containing six cysteine residues. 17-19The cysteine residues in the C1 region totally coordinate four atoms of zinc, 2°-22 and the zinc-thiolate structure is required for binding of phorbol ester and diacylglycerol.23The C3 constant region present within the catalytic domain has a putative ATP-binding site and one cysteine residue. The C4 constant region present in the catalytic domain has four to six cysteine residues as well as another putative ATP-binding site. 17-t~ The thiol-rich regions in both domains are extremely sensitive to oxidative modification. A selective oxidative modification of the regulatory domain results in a Ca2+/lipid-independent activation of the kinase with a loss of phorbol ester binding. 7 Alternately, a selective oxidative modification of the kinase domain results in the generation of a modified form of PKC which exhibits only phorbol ester binding. 8 Some oxidants such as H202 induce irreversible oxidative modification, 7 whereas others such as metaperiodate induce reversible oxidative modification. 8Furthermore, PKC (the 15 R. Gopalakrishna, Z. H. Chen, and U. Gundimeda, FEBS Lett. 314, 149 (1992). 16 R. Gopalakrishna, Z. Chert, K. Yoolee, and U. Gundimeda, Proc. Am. Assoc. Cancer Res. 35, 615 (1994). 17 p. j. Parker, L. Coussens, N. Totty, L. Rhee, S. Young, E. Chen, E. Stabel, M. Watefield, and A. Ullrich, Science 233, 853 (1986). 18 U. Kikkawa, K. Ogita, Y. Ono, Y. M. S. Shearman, T. Fuji, K. Ase, K. Sekiguchi. K. Igarashi, and Y. Nishizuka, F E B S Lett. 223, 212 (1987). 19 G. M. Housey, C. A. O'Brian, M. D. Johnson, P. Kirschmeier, and I. B. Weinstein, Proc. Natl. Acad. Sci. U.S.A. 84, 1065 (1987). 20 S. R. Hubbard, W. R. Bishop, P. Kirschrneier, S. J. George, S. P, Cramer, and W. A. Hendrickson, Science 254, 1776 (1991). 21 S. Ahmed, R. Kozma, J. Lee, C. Monfries, N. Harden, and L. Lim, Biochem. J. 2,80, 233 (1991). 22 A. F. G. Quest, J. Bloomenthal, E. S. G. Bardes, and R. M. Bell, J. Biol. Chem. 267, 10193 (1992). 23 R. M. Bell and D. Burns, J. BioL Chem. 266, 4661 (1991).

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phorbol ester receptor) was shown to be regulated by lipid peroxidation, 24 redox-cycling quinones, 9 and oxidant-generating systems. I° Although the importance of the cysteine residues in the regulatory domain is recognized, 24 the functional roles for cysteine residues within the catalytic domain are not known. Here we describe methods to induce specific modifications of cysteinerich regions in the regulatory and catalytic domains of PKC by oxidant tumor promoters and PKC-specific inhibitors involving oxidations or alkylating mechanisms. These procedures have been standardized using a,/3, and y isoenzymes of PKC, and the discussion is limited to these isoenzymes.

General Procedures

Reagents Buffer A: 20 mM Tris-HCl, pH 7.5/1 mM EDTA/0.1 mM dithiothreitol (DTT)/1/zM leupeptin/0.15/zM pepstatin A Buffer B: 20 mM Tris-HC1, pH 7.5/1 mM EDTA/0.1 M NaCI/0.1 mM DTT/0.15/~M pepstatin A Buffer C: 20 mM Tris-HC1, pH 7.5/1 mM EDTA/1 mM DTI'/0.5 mM phenylmethylsulfonyl fluoride (PMSF)/25 /zM leupeptin/0.15 /zM pepstatin A / l % Nonidet P-40 (v/v) BSA solution: Fatty acid-free bovine serum albumin (BSA) solution (10 mg/ml) in 20 mM Tris-HC1 buffer, pH 7.5, is treated with 1 mM PMSF for 2 hr at room temperature and then dialyzed versus same buffer to remove unreacted PMSF

Purification of Protein Kinase C Free from Endogenous Calpain Oxidatively modified proteins exhibit increased susceptibility to proteolysis compared to native proteins. If trace amounts of proteases are present in the preparations of enzyme used for oxidative modification experiments, even though oxidative activation of the enzyme might have occurred, it may not be noticed as the modified enzyme may be rapidly degraded, resulting in an apparent inactivation of enzyme. Because calpain proteolytically activates PKC and prefers oxidatively modified proteins, it is important to remove any contaminating calpain from the purified preparations of PKC. Apparently homogeneous preparations of PKC, obtained by other published methods are, in our hands, contaminated to varying degrees with calpain I. This can be overcome by passing the PKC preparations through 24K. B. Delcos and P. M. Blumberg, CancerRes. 42, 1227 (1982).

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a small (1-ml) phenyl-Sepharose column equilibrated and eluted with 20 mM Tris-HCl, pH 7.5/0.1 M NaC1/1 mM EDTA. Under these conditions calpain I remains tightly bound to the phenyl-Sepharose, whereas PKC came in the unbound protein fraction. Such preparations of PKC free from calpain retain full activity for at least 20 min when incubated at room temperature in the presence of I mM Ca 2+. Because of the increased susceptibility of oxidatively modified proteins to proteolysis, it is important to include protease inhibitors such as leupeptin (1 txM) and pepstatin A (0.2 ~M) in the PKC preparations during oxidative modification of the purified enzyme. Calpain-derived PKC (M-kinase) that contains only the catalytic domain can be employed to study selective modification of cysteine residues in this domain. Calpain is purified by Ca2+-dependent hydrophobic chromatography from bovine brain] Calpain-digested rat brain PKC is prepared as described previously]

Oxidative Modification of Protein Kinase C Occurring during Storage Protein kinase C is known to be altered during storage. In storage at - 2 0 ° or at 4 °, PKC gradually loses its dependency on phospholipids and expresses its activity in a Ca2+/lipid-dependent manner. However, this can be partially reversed by adding freshly prepared thiol agents such as DTT (1 mM) or 2-mercaptoethanol (10 mM), suggesting that thiol oxidation may be involved in the process. We prefer to use PKC within 3 weeks after purification by storing at 4°. Regardless of the method of storage, it is important to take into consideration the ability of the enzyme to expose the hydrophobic site in the presence of Ca 2+ as a marker for the native state of PKC (c~,/3, and 7 isoenzymes). 15

Calcium-Dependent Hydrophobic Chromatography The PKC samples (2 ml) are applied to a 1-ml phenyl-Sepharose column previously equilibrated with buffer A containing 0.5 M NaC1 and allowed to bind Ca2+-independently to the resin by washing with the same buffer. Then the column is washed with buffer A containing 1 mM CaCI2 in place of 1 mM EDTA. The CaZ+-dependently bound PKC is then eluted with 5 mM EGTA. 15

Protein Kinase C Modification by Various Agents Because PKC is purified and stored in the presence of thiol agents, it is important to remove the agents from the PKC preparation prior to enzyme modification studies. This can easily be accomplished by dialysis

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or when the sample is limited (<3 ml) by using PD-10 Sephadex (Pharmacia, Piscataway, N J) gel-filtration columns. The PD-10 columns are also useful for exchanging buffers in the PKC preparation which are better suited for oxidation or alkylation studies. Tris and HEPES buffers are well suited for oxidation studies, whereas for alkylating agents such as acrolein and chelerythrine, HEPES buffer is not suited. Among the four buffers tested (Tris, HEPES, imidazole, and phosphate), Tris buffer is found to be the best for both oxidizing and alkylating agents. All PKC modifications are carried out in polystyrene tubes to avoid nonspecific binding of enzyme to the tubes. In previous studies, PKC inhibitors such as calphostin C and chelerythrine or cysteine modifying agents such as N-ethylmaleimide were added directly to the kinase assay or to the phorbol 12,13-dibutyrate (PDBu) binding incubation, to determine their effects on PKC. 12-1<18 Because the PKC assay mixture contains various other components, it is difficult to determine the factors that influence the action of these agents. It is also difficult to distinguish whether the agents are inducing an inhibition or a permanent inactivation of PKC. Furthermore, ligands that bind to catalytic sites (ATP, Mg 2÷, and substrate protein) and ligands for the regulatory domain (Ca 2+, phosphatidylserine, diolein, and phorbol esters) can protect appropriate sites in the enzyme from modification. The PKC preparation is initially treated with various agents under defined conditions, and then these agents along with other low molecular weight compounds are removed from the treated PKC sample. Thus, at a later step during the determination of kinase activity and PDBu binding, these agents are no longer present. When samples are incubated with a high concentration (1 mM) of Ca 2÷, it is important to add EDTA (5 mM final concentration) to the treated samples first, followed by the addition of PMSF-treated (BSA). Because the PKC protein used is a small amount, prior to subjecting the sample to the centrifuge column technique, the addition of BSA (200/xg/ml) minimizes nonspecific adsorption of PKC to the Sephadex gel matrix. The oxidants or other agents used to modify PKC can be removed conveniently by employing the centrifuge column technique. 25 Sephadex G-50 (fine) is packed to a 4-ml bed volume in a 5-ml syringe or 1-ml bed volume in a 3-ml syringe and then equilibrated with 4 bed volumes of buffer B with or without DTT. The syringes are centrifuged at 2000 g using a swinging-bucket rotor in a bench-top centrifuge for 5 min. Immediately, the samples from which low molecular weight compounds are to be removed are applied to the top of the shrunken gel in a volume of 0.5 ml for a 4-ml 25R. Gopalakrishna and B. Nagarajan, Biochem.

Med.

22, 70 (1978).

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bed volume of gel or 0.125 ml for 1-ml bed volume of gel. The centrifugation is then repeated to collect the samples into centrifuge tubes.

Assessing Modifications of Catalytic Domains Phosphotransferase Activity The Ca2+/lipid-dependent phosphotransferase activity using protein substrates such as histone H1 depends on the functional integrity of not only the catalytic site, but also on the regulatory site where Ca 2+ and lipids bind. Protamine phosphorylation by PKC is independent of Ca z+ and lipids. Therefore, PKC assay using protamine sulfate as the substrate may be used to assess the modification of the catalytic domain independently of the changes occurring in the Ca 2+/lipid binding region. However, this protamine phosphorylation may be influenced by a polycation-regulated region in the PKC. The study of kinetics of PKC modifications by various agents require determining PKC activity and phorbol ester binding in a larger number of samples in the presence and absence of various cofactors. The PKC assay using the multiwell filtration approach is well suited for this purposeY The unique aspect of this method is that both incubations and filtrations are carried out in the same multiwell plate. Briefly, PKC reaction samples containing 20 mM Tris-HC1, pH 7.5/10 mM MGC12/0.33 mM CaCI2/0.1 mM ATP/histone H1 (0.1 mg/ml)/0.04/~M leupeptin and 25/xl of PKC sample in a total volume of 125/zl are incubated in the wells of a multiwell filtration plate at 30° for 5 min. Histone H1 is precipitated and filtered with 25% (w/v) trichloroacetic acid. The filters are punched into scintillation vials, and the radioactivity associated with the filters is counted. The PKC activity is expressed as units, where one unit of enzyme transfers 1 nmol of phosphate to histone H1 per minute at 30°.

Assessing Modifications of Regulatory Domains Phorbol Ester Binding Because the zinc-coordinating cysteine-rich region is required for phorbol ester binding, 23 this binding assay may indicate the functional integrity of the cysteine-rich region in the regulatory domain. [3H]Phorbol 12,13dibutyrate (PDBu) is used as a ligand for the determination of phorbol ester binding using the multiwell filtration approach. 26 For optimal PDBu binding to purified PKC the conditions discussed in method 1 as described previously are used. 26 Briefly, 25/~1 of purified PKC is incubated with 20 mM Tris-HCl, pH 7.5/0.6 mM CAC12/0.15 tzM leupeptin/0.06 ~M pepstatin A/20 nM [3H]PDBu [20 Ci/mmol; 200,000 disintegrations/min (dpm)]/BSA 26 R. Gopalakrishna, Z. H. Chen, U. Gundimeda, J. C. Wilson, and W. B. Anderson, Anal. Biochem. 206, 24 (1992).

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(0.1 mg/ml)/bovine y-globulin (0.1 mg/ml) in the wells of the 96-well filtration plate with fitted Durapore membrane disks at 30 ° for 10 min. The ligand-bound PKC is adsorbed to DEAE-Sephadex beads, and the beads then are filtered and washed in the same multiwells with 20 mM TrisHCI, pH 7.5. The radioactivity associated with the DEAE-Sephadex beads retained on the filter is counted. To determine the optimal PDBu binding to a PKC fraction isolated by DEAE-cellulose chromatography from the crude cell extracts, the conditions standardized with the cytosolic receptor (method 2) are used. 26 The major differences in method 2 compared to method 1 are that the concentration of phosphatidylserine is increased by 5-fold, 10 mM Mg 2+ is included during incubation for PDBu binding, and while washing the P K C - P D B u complex adsorbed to DEAE-Sephadex beads, 0.1 mM CaC12 and 5 mM MgC12 are included in the wash buffer.

Isolation of Oxidatively Modified Protein Kinase C from Cells It is important to use protease inhibitors during the isolation of oxidatively modified PKC from cells and to measure the enzyme as quickly as possible. It is difficult to achieve a reliable determination of PKC activity in crude extracts. In the crude extracts, the observed activity of PKC is due to the net phosphorylation of the PKC substrate protein by the kinase and its dephosphorylation by protein phosphatases, particularly type 2A which prefers histone H1 phosphorylated by PKC. It is known that catalytic subunits of both protein phosphatases type 1 and 2A contain a critical cysteine residue which is susceptible to modification by oxidants and alkylating agents. Therefore, it is possible that a selective inactivation of protein phosphatase could lead to overestimation of PKC in the crude extracts. The following simple chromatographic column can be used to separate the native PKC from the oxidatively modified PKC and protein phosphatases. Cells are grown in 150-cm2 flasks in minimum essential medium (MEM) containing 10% fetal calf serum. At confluency, the medium is changed and the cells are left overnight in the incubator. The cells then are treated with various concentrations of oxidants or PKC inhibitors for the indicated periods. The treated cells are Dounce homogenized in 7 ml of buffer C containing detergent. The homogenates are centrifuged at 13,000 g for 15 min, and the supernatants are used to isolate PKC. To test whether the oxidative modification is reversible, cell homogenization is carried out as mentioned above in buffer C without DTT and the homogenate is divided into two aliquots. To one aliquot is added DT-I? to a final concentration of 2 raM. The samples with and without DTT are separately processed while maintaining the difference in DTI? in all buffers used for PKC isolation by DEAE-cellulose chromatography.

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Detergent-solubilized cell extract (5 ml, 15-25 mg protein) is applied to a 1-ml column of DEAE-cellulose (DE-52, 0.8 × 2 cm) previously equilibrated with buffer A. After washing the column with 5 ml of buffer A, the bound native form of PKC is eluted with 2.5 ml of 0.1 M NaC1 in buffer A (peak A). The oxidatively modified and activated PKC is then eluted with 2.5 ml of 0.25 M NaC1 in buffer A (peak B). The native Ca2+/ lipid-stimulated PKC present in the peak A fraction is free from protein phosphatase 2A, whereas the oxidatively modified PKC present in the peak B fraction is contaminated with protein phosphatase 2A. Therefore, it is important to add protein phosphatase inhibitors such as okadaic acid (1 /xM) or microcystin LR (100 nM) to this fraction in order to reliably measure the kinase activity of oxidatively activated PKC. Alternatively, the PKC assay using polypeptide substrates, which are not good substrates for protein phosphatase 2A, may help overcome the interference from protein phosphatase 2A. The PKC activity present in these fractions is measured in the presence of Ca2+/lipids or 1 mM EGTA. Specialized Conditions for Individual Agents

Hydrogen Peroxide for Irreversible Oxidation Often, H202-induced oxidations are mediated by the iron-catalyzed formation of hydroxyl radicals (Fenton reaction). Although traces of iron contamination in the buffers may be sufficient for this purpose, adding excess iron can assure a high rate of oxidations of proteins. To PKC (0.5 to 1 units) are added 25 ~M FeC12, 1 mM E G T A or 0.05-5 mM CaCI~, 25 t~M leupeptin, and various amounts of other ligands (phosphatidylserine) from bovine brain, diolein, phorbol 12-myristate 13-acetate (PMA), ATP, and Mg 2+ in a total volume of 0.25 ml. These samples are brought to room temperature or 30° in a water bath. Oxidative modification is initiated by adding various concentrations of H202, and the samples are further incubated for a desired time. To these samples is added 12 ~1 of 0.2 M dithiothreitol to prevent further oxidative modification. The samples are placed in ice; both PKC activity and PDBu binding are then determined. Although both regulatory and catalytic domains are susceptible to oxidative modification, H202 at low (4 mM) concentrations decreased PDBu binding and increased CaZ+/lipid-independent activity. 7 Because oxidation can also affect the catalytic site, it is difficult to obtain an appreciable activation of PKC by limited oxidation without protecting the catalytic site with ATP/Mg 2+. The extent of oxidative activation of PKC varied depending on the PKC preparation. The stored preparations of bovine brain PKC exhibited an increase in Ca 2+/lipid-independent activity with oxidative

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modification, to a maximal level 80% of that exhibited by unmodified PKC activated with Ca 2÷ and lipids. However, freshly purified bovine PKC as well as fresh or stored preparation of rat brain PKC exhibited only 3050% of this activation by oxidative modification. The commercial type IIIS preparation of histone (a mixture of histone H1 and high mobility group proteins) served as a better substrate than pure histone H1 for oxidatively activated PKC. A synthetic polypeptide has been reported to serve as a substrate for oxidatively activated PKC. 11

Metaperiodate for Reversible Oxidation of Vicinal Thiols Sodium metaperiodate acts as a tumor promoter and also as a mitogen to some cell types. 5 Periodate has been used to modify protein vicinal thiols in a variety of enzymes. 8 It was suggested that by virtue of having a tetrahedral structure, periodate can bind to vicinal thiols present within the binding site for phosphorylated compound. Therefore, such binding sites may be protected by either phosphate or phosphorylated ligand. To the PKC sample in the presence of 1 mM Ca z+ or EGTA along with other factors such as phosphatidylserine, periodate (100 tzM) is added and incubated at room temperature for 5 rain. Then the samples are subjected to the centrifuge column technique to remove periodate. It is important to remove periodate before assaying PKC activity, especially when higher (>100/zM) concentrations of periodate are used. The residual periodate can oxidize vicinal diols of ribose residue, and then the oxidized ATP generated can bind to the ATP-binding site of PKC and inactivate the enzyme. Periodate at micromolar concentrations modifies the regulatory domain of PKC as determined by the loss of Ca2+/lipid-stimulated kinase activity and also by the loss of phorbol ester binding. However, it results in a 8-fold increase in CaZ+/lipid-independent activity, with nearly 30 to 45% of the initial Ca2+/lipid-stimulated activity of the unmodified control enzyme. However, at higher concentrations (>100 /zM) periodate also modifies the catalytic domain, resulting in complete inactivation of PKC. The oxidative modifications induced by periodate are completely reversible by a brief (few minutes) treatment with 1 mM DTT. Among the various phospholipids and other regulators (unsaturated fatty acids and diolein), only phosphatidylserine protects the regulatory domain from periodate oxidation. In the presence of phosphatidylserine, the catalytic site is selectively modified by periodate, resulting in the formation of a form of PKC that exhibited only phorbol ester binding but not kinase activity. In intact cells treated with 1 to 3 mM concentrations of periodate, there is an activation and inactivation of PKC which is completely reversed by DTT. To reverse the changes that have occurred in PKC, it is important

[14]

141

MODIFYING CYSTEINES IN PROTEIN KINASE C OH

0

H~CO

0

OH

C 3

0

OH

HO

CH3

yy,ocO,yOyOH

0

0

Calphostin C

~,.'~

OH

OH

0

OH

Hypericin

F~G. 1. Structures of PKC-specific inhibitors that induce a site-specific oxidation of the regulatory domain.

to add DTT as soon as the cells are homogenized. Periodate is required at higher (1 to 3 raM) concentrations to modify PKC in intact cells, which may be due to a limited uptake of this agent by cells. It is possible that at these concentrationsperiodate can also modify vicinal diols of sugars present on the cell surface or in the cell, and the resultant oxidized sugarcontaining compounds can bind to and permanently inactivate PKC.

Calphostin C and Hypericin: Agents for Inducing Site-Specific and "CageType" Oxidations Calphostin C is a perylenequinone metabolite (Fig. 1) isolated from the fungus Cladosporium cladosporioides.12 It is one of the specific inhibitors of PKC, and it inhibits both phorbol ester binding and phosphotransferase activity of PKC, presumably by binding to the regulatory domain in a light-dependent manner, t2,27 Calphostin C and related compounds such as cercosporine as well as hypericin and related compounds have been well studied in photochemistry.28,29 These agents are well-known generators of singlet oxygen (type II mechanism).28,29 Purified PKC (2 units) is incubated with calphostin C (0.1 to 1 /zM) along with 1 mM Ca 2+ and various other factors in a total volume of 0.5 ml in polystyrene tubes at room temeprature under ordinary fluorescent light for 3 min. Then, 25 /zl of BSA solution (10 mg/ml) and 25 /~1 of 0.1 M EDTA are added. Calphostin C and other low molecular weight compounds are removed from the treated PKC samples by subjecting them 27 R. F. Burns, F. D. Miller, R. L. Meriman, J. J. Howbert, W. F. Heath, E. Kobayashi, I. Takahashi, T. Tamaoki, and H. Nakano, Biochem. Biophys. Res. Commun. 176, 288 (1991). 28 N. Duran and P.-S. Song, Photochem. Photobiol. 43, 677 (1986). 29 C. Thomas, R. S. MacGill, G. C. Miller, and R. S. Pardini, Photochern. PhotobioL 55, 47 (1992).

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to the centrifuge column technique. Calphostin C mediated inactivation of PKC (a,/3, and y isoenzymes) is strictly Ca2+-dependent. The maximal inactivation of PKC by calphostin C or hypericin is varied. Although light is important for PKC inactivation induced by these agents, the variable extent of PKC inactivation is unlikely to be caused by different degrees of exposure to light. Depending on the duration and methods of storage, PKC preparations responded to the drugs to a variable extent. The PKC in freshly purified preparations from rat brain is inactivated by calphostin C above 95%, whereas a preparation of PKC purified and stored for 3 months at - 2 0 ° is inactivatd only 50 to 70% by calphostin C. The Ca2+-independent weak hydrophobic region on PKC is not affected by storage, whereas a fraction (40%) of the PKC molecules in the stored preparation lost the ability to expose the strong hydrophobic site in the presence of Ca 2+. Only the fraction of PKC exhibiting the Ca2+-induced hydrophobic site is inactivated by calphostin C, whereas the fraction of PKC that did not exhibit a Ca2+-induced hydrophobic site is resistant to calphostin C inactivation. Conceivably, the Ca2+-induced hydrophobic site may play a role in the initial binding of the drug to the site followed by a site-specific oxidation of the amino acid residues present within the vicinity. The experiment to demonstrate the oxygen requirement for PKC inactivation induced by calphostin C and hypericin are carried out in microcentrifuge tubes with fitted rubber stoppers. As inactivation induced by these agents is strictly Ca2÷-dependent, this can be exploited for initiating and arresting the reactions of these agents with PKC. To two sets of PKC samples are added various concentrations of calphostin C or hypericin along with 1 mM EGTA. One set of samples is left in normal atmospheric air, whereas in the other set of samples air is displaced with nitrogen gas (anaerobic). To initiate PKC modification, CaC12 is injected to both sets of samples to obtain 1 mM free Ca 2+. The samples are incubated for 3 min in the presence of light, and then EGTA is added to a final concentration of 5 mM. The agents are removed from the treated samples by using the centrifuge column technique, and both PKC activity and PDBu binding are determined in the treated samples. Photoexcitation of calphostin C and hypericin in apolar solvents is known to produce singlet oxygen. 28'29 Therefore, it is possible that the singlet oxygen generated by the binding of the drugs to hydrophobic sites in the presence of light can modify zinc thiolates. Because the zinc thiolate structure is required for phorbol ester binding, the loss of such binding suggests this cysteine-rich region is affected. Furthermore, the C1 region also contains one tryptophan and two methionine residues, which can also be oxidized by singlet oxygen. The formation of covalently bound reactive intermediates by photoreaction of calphostin C under anaerobic conditions

[14]

MODIFYING CYSTEINES IN PROTEIN KINASE C

OCH3 H3C--~~ O C H 3 o

143

÷ H3C',~N~O

/

Chelerythrine

Sanguinarine

FIG. 2. Structures of PKC-specific inhibitors that alkylate cysteine residues in the catalytic domain.

cannot be excluded. Nevertheless, the covalent binding of the drug may not be the causative factor in oxygen-dependent inactivation of PKC. Treatment of various cell types with calphostin C (0.2 to 0.5 /xM) in the presence of regular fluorescent light at room temperature resulted in a permanent inactivation (70-90%) of PKC in both membrane and cytosol within 1 hr. Therefore, cells can be pretreated for a limited time (1 hr) with the agents, and such treated cells can be used to test the role of PKC in signal transduction for several hours (12-15 hr) until new PKC is synthesized in the cell. The limited treatment with the agent minimizes unwanted side effects and improves the specificity of the drug for PKC. Both calphostin C and hypericin, when incubated in the dark, decreased PKC activity only in the membrane.

Chelerythrine and Sanguinarine: Agents that Alkylate Cysteine Residues in Catalytic Domain Chelerythrine, a benzophenanthridine alkaloid (Fig. 2), has been shown to be a specific inhibitor for PKC and presumably acts by binding to catalytic domain.14 It requires nearly a 200-fold higher concentration to inhibit other protein kinases. 14 Although chelerythrine has become popular as a PKC inhibitor, this along with other benzophenanthridine alkaloids such as sanguinarine and nitidine were studied extensively to understand the mechanism of argemone oil toxicity years ago. 3° Because these agents are known to inactivate some enzymes by reacting with critical thiols, 31,32 we have designed procedures to test wether the agents permanently inactivate PKC by alkylating critical cysteinyl residues in PKC. 3o S. N. Sarkar, Nature (London) 162, 265 (1948). 3J H. G. Cohen, E. E. Seifen, K. D. Straub, C. Tiefenback, and F. R. Stermitz, Biochem. Pharmacol. 27, 2555 (1978). 32 D. Walterova, J. Ulrichova, V. Preininger, V. Simanek, J. Lenfeld, and J. Lasovsky, J. Med. Chem. 24, 1100 (1981).

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Purified PKC free from thiol agents (2 units) is treated with either chelerythrine or sanguinarine along with various cofactors in a 0.5-ml volume for 10 min at room temperature. After removing the excess amount ofinhibitors, both PKC activity and PDBu binding are determined in the treated samples. In the presence of 1 mM Ca 2÷ both chelerythrine and sanguinarine decreased PKC activity with IC50 values of 13 and 2.5/~M, respectively. However, such IC50 values varied depending on the enzyme concentration and presence of histone H1, ATP, and Mg 2÷. Phorbol ester binding is also affected at higher concentrations (>20/zM) of the drugs, when incubated with PKC in the absence of cofactors. However, PDBu binding is not affected if these agents are added directly to the PDBu binding assay mixture where other cofactors are also present. The enzyme activity is not recovered by subjecting the inactivated PKC to extensive dialysis, DEAE-cellulose chromatography, or phenyl-Sepharose chromatography. Unlike the case for agents such as calphostin C and hypericin, for chelerythrine-mediated inactivation there is no requirement for Ca 2÷ or molecular oxygen. However, the presence of Ca 2+ (1 mM) decreased 2- to 3-fold the concentrations of these agents required to induce inactivation of PKC. Sulfur nucleophiles such as DTT and 2-mercaptoethanol (5 mM) protected the enzyme from the inactivation induced by chelerythrine and sanguinarine. However, once the enzyme is inactivated by these agents, the thiol agents failed to restore the enzyme activity. A sulfhydryl-modifying agent, N-ethylmaleimide, exhibited a similar type of Ca2+-independent inactivation of PKC preferentially at the catalytic site but not at the phorbol ester binding site. However, the concentrations (IC50 75/xM) required to inactivate PKC are 5- to 6-fold higher than that of chelerythrine. Chelerythrine and sanguinarine formed florescent adducts with PKC, which were prevented by prior treatment with N-ethylmaleimide. Therefore, it is likely that chelerythrine or sanguinarine may react through the electrophilic iminium carbon with the critical cysteine residue(s) present within the catalytic region. The pKa of the group reacting with chelerythrine is between 6 and 6.5. Thus, although the typical pK~ value of protein sulfhydryls is often 8.5, the observed lower pKa for reacting cysteine residue(s) may suggest the presence of a basic residue within the vicinity forming an ion pair as described for other enzymes. 33 Although these agents can react with low molecular weight thiols with a lower affinity similar to electrophiles such as N-ethylmaleimide, the hydrophobicity of the benzophenanthridine alkaloids allows them to bind initially with a higher affinity to a hydrophobic site on the enzyme, which may facilitate alkylation of thiolate present within the vicinity. However, one should be aware, when using these kind of agents as specific PKC inhibitors, that they are capable of inactivating other enzymes having similar reactive cysteine residues. 33 H. F. Gilbert, Adv. Enzyrnol. 63, 69 (1990).

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145

In intact cells treated with chelerythrine and sanguinarine, an irreversible inactivation of PKC (both kinase activity and PDBu binding) occurs. By treating various confluent cells with 10 to 30 t~M chelerythrine or 2.5 to 10/xM sanguinarine, it is possible to inactive 70 to 90% of the cellular PKC within 1 hr. Therefore, when pretreating cells for a limited time, cellular PKC can be lowered for a prolonged time (10 hr), until it is resynthesized. This information can be exploited to improve specificity and to decrease cytotoxicity when these agents are used as PKC signal transduction tools.

Nitric Oxide-Induced Reversible Inactivation: Mixed-Type Mechanism Nitric oxide (NO) is not a strong oxidant. Nevertheless, it can produce some effects on enzymes by inducing an initial S-nitrosylation. S-Nitrosylated vicinal thiols may readily oxidize to form protein disulfides. Nitric oxide either in the form of donors such as S-nitrosocysteine, S-nitroso-Nacetylpenicillamine, or nitroprusside (in the presence a thiol) or in the form of gas can be used to modify cysteine residues in PKC. The NO-generating agents or NO itself (< 100 tzM) inactivated PKC histone phosphotransferase activity in a Ca 2+- and oxygen-dependent manner, which is readily reversed by a brief (minutes) incubation with 1 mM DTT. 34 However, at this low concentration NO has no effect on the phorbol ester binding. Higher (>100 txM) concentrations of NO or NO-generating agents induced an inactivation of kinase activity which is not reversed with DTT treatment. Moreover, the higher concentrations also partially affected phorbol ester binding. It is important to take into consideration other active species formed from NO during PKC thiol modifications. It is possible that in an oxygen atmosphere appreciable amounts of NO2 may be formed at high (>100/~M) concentrations of NO. NO2 is a strong oxidant and can induce irreversible cysteine modifications; it can also effect the zinc-thiolate site in the phorbol ester binding region. Furthermore, in intact cells, chronic generation of NO induces a permanent inactivation of PKC. This is probably due to conversion of NO to strong oxidants such as as peroxynitrite.35

Comments on Differential Reactivity of Cysteine Residues Present within Regulatory and Catalytic Domains to Oxidants and Alkylating Agents Cysteine residues present within the regulatory and catalytic domains may react at a different rate with oxidizing and alkylating agents. Based on the current models of four zinc atoms binding to the regulatory domain, 34 R. Gopalakrishna, Z. Chen, and U. Gundimeda, J. BioL Chem. 268, 27180 (1993). 3s j. S. Beckman, T. W. Beckman, J. Chen, P. A. Marshall, and B. A. Freeman, Proc. Natl. Acad. Sci. U.S.A. 87, 1620 (1990).

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all the cysteine residues in this domain may exist in the zinc thiolate form, whereas in the catalytic domain cysteine residues may exist as free thiols or thiolates. Because N-ethylmaleimide, nitric oxide, chelerythrine, and sanguinarine preferentially modify the catalytic region, it is possible that the thiolates in this region may readily react with alkylating (arylating) agents. As thiolates in the regulatory domain coordinate zinc binding, they may not readily react with alkylating agents, However, anionic oxidants such as hydroxyl radicals, peroxynitrite, superoxide, and hypochlorite at lower concentrations may preferentially react with positively charged zinc thiolate followed by the oxidation of thiolates involved in metal coordination. However, at higher concentrations these oxidants may also react with cysteine residues present within the catalytic region.

Acknowledgments This work was supported in part by Research Grant RT 389 from the Tobacco-Related Diseases Research Program, University of California, Research Grant 93B from the American Institute for Cancer Research and U.S. Public Health Service Grant CA 62146 from the National Cancer Institute. We thank Eduardo Gomez, Steven Shao, and Hiroshi Yamauchi for excellent technical assistance.

[15] G l u t a t h i o n e

and Protein Kinase C in Peripheral Nervous Tissue

B y FRANCISCO J. ROMERO a n d JOAQUfN R o M A

Introduction It has long been known that the redox state of thiols in peripheral neural structures might play an important role in electrophysiological function. 1 Former studies suggested that the integrity of certain sulfhydryl groups in nerve fibers would be essential for conduction. 2 This classic work showed data strongly suggesting that the blockade of SH groups resulted in a loss of excitability and a reduction of the resting potential, and proposed for the first time the role of SH groups in the relationship between structure and function in nerve. Most of these points were confirmed later by other 1 N. S. Kosower and E. M. Kosower, Int. Rev. Cytol. 54, 109 (1978). 2 H. M. Smith, J. Cell. Comp. Physiol. 51, 161 (1958).

METHODSIN ENZYMOLOGY,VOL.252

Copyright© 1995by AcademicPress,Inc. All fightsof reproductionin any formreserved.

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authors for different electrophysiological parameters, for example, spike duration and amplitude of action potential. 3-6 The histochemical localization of glutathione (GSH) in peripheral nerve showed that, unlike what happens in central nervous tissue (strong staining of the central nervous system neuropil with little or no observable fluorescence in neuronal somata), the sciatic nerve and the sensory cell bodies of the lumbar dorsal root ganglia exhibited prominent staining. This heterogeneous distribution of GSH and related detoxification enzymes were proposed as the basis for a selective cellular and/or regional expression of neurotoxicity.7 Moreover, the fact that the peripheral nerves normally considered for these studies are covered with Schwann cells makes it difficult to ascribe the content of antioxidant to the axons or to the myelin covers, unless this previous type of histochemical approach is used for all of the antioxidants considered. An example of this difficulty is the wide range of GSH concentrations measured in peripheral nerve when freshly homogenizing the tissue or after freeze-clamping (a process that normally includes the whole Schwann cell in the homogenate) (see Table I). A systematic determination of the GSH content, as well as other antioxidants, in different experimental models would certainly improve the knowledge about this metabolically active tissue. Different experimental neuropathies have been described, and in most the role of oxidative stress, and therefore of glutathione and other antioxidants, has been suggested. Based on previous work on isoniazid-induced neuropathy,8'9 Cavanagh was one of those proposing that the maintenance of neurons with long axons would indeed involve glutathione, as well as other antioxidants such as a-tocopherol. 1° Phenytoin (DPH) has been reported to be one of the most common causes of drug-induced polyneuropathy. The development of DPH-induced clinical and electrophysiological peripheral nerve or neuromuscular dysfunction is well established in humans, 11-~3 as well as in experimental studies, where a decrement in motor nerve conduction velocity and also 3 F. Huneeus-Cox, H. L. Fernandez, and B, H. Smith, Biophys. J. 6, 675 (1966). 4 j. K. Marquis and H. G. Mautner, J. Membr. Biol. 15, 249 (1974). 5 p. Shrager, Ann. N.Y. Acad. Sci. 264, 293 (1975). P. Shrager, J. Gen. Physiol. 69, 183 (1977). 7 M. A. Philbert, C. M. Beiswanger, D. K. Waters, K. R. Reuhl, and H. E. Lowndes, Toxicol. Appl. PharmacoL 107, 215 (1991). 8 W. A. Jones and G. P. Jones, Lancet 1, 1073 (1953). 9 j. B. Cavanagh, Z Neurol. Neurosurg. Psychiatry 30, 26 (1967), l0 j. B. Cavanagh, Lancet 2, 1284 (1984). it E. L. So and J. K. Penry, Epilepsia (N.Y.) 22, 467 (1981). J2 T. R. Swift, J. A. Gross, L. C. Ward, and B. O. Crout, Neurology 31, 826 (1981). ~3R. Danner. Acta Neurol. Scand. 65, 668 (1982).

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TABLE I GLUTATHIONE AND PROTEIN KINASE C ACTIVITY IN PERIPHERAL NERVOUS TISSUE Source

Rat

Mouse

Parameter Glutathione Control Control Control Histochemical regional distribution Phenytoin (150 mg/kg) Phenytoin (150 mg/kg) + H7 (20 rng/kg, 30 min prior to phenytoin) Ethanol (3 g/kg) tert-Butanol (0.6 g/kg) Buthionine sulfoximine (3 × 900 mg/kg body weight, 16, 15, and 3 hr before death) Control Diabetic (Day 21 after alloxan, 200 mg/kg)

Dog

Activity

Ref.

122-397J 94.46 k 5.83 l 3.71 t 5.98 t

a, b c d e d d

3.44 t 5.53 t 86 ~

f d a

7.09 ~ 7.78 t 834 k

g g g

Protein kinase C activity (microsomal fraction) Mouse Control H7 (30 mg/kg) Calphostin C (30/zg/kg)

4.45 m 3.37 m 3.32 m

a p. B. Carroll, B. M. Thornton, and D. A. Greene, Diabetes 35, 1282 (1986). b F. J. Romero, E. Monsalve, C. Hermenegildo, F. J. Puertas, V. Higueras, E. Nies, J. Segura-Aguilar, and J. Romfi, Neurochem. Res. 16, 157 (1991). c A. Raya, J. Romfi, C. Hermenegildo, and F. J. Romero, Biochem. Soc. Trans. 21, 92S (1993). d A. Raya, J. Gallego, F. Bosch-Morell, J. Rom~i, and F. J. Romero, Free Radical Biol. Med. 18, in press (1995). e M. A. Philbert, C. M. Beiswanger, D. K. Waters, K. R. Reuhl, and H. E. Lowndes, Toxicol. Appl. Pharmacol. 107, 215 (1991). IA. Raya, C. Hermenegildo, F. J. Romero, and J. Rom~i, Eur. J. Neurosci. 5(Suppl.), 299 (1992). g C. Hermenegildo, A. Raya, J. Rom~i, and F. J. Romero, Neurochem. Res. 18, 893 (1993). h M. Chvapil, J. M. Kern, R. Misiorowski, and P. R. Weinstein, Exp. Neurol. 78, 765 (1982). i C. Hermenegildo, V. Felipo, M. D. Mifiana, F. J. Romero, and S. Grisol/a, Diabetes 42, 257 (1993). J Micrograms/gram nerve. k Nanomoles per gram wet weight. t Nanomoles per milligram protein. m Nanomoles per milligram protein × minute.

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in the amplitude of evoked muscle action potentials was found. 14-16 Several hypotheses have been proposed to explain DPH-induced neurotoxicity. These include (i) decrease in Na + conductance, 17"a8 (ii) blockade of Ca2+ channels, 19-22 and (iii) alterations of Na+,K+-ATPase activity. 23-25 It has been reported that the protein kinase C (PKC) enzyme family has a key role in the modulation of ion channel activity, including that of Ca2+ and Na ÷ channels (for an extensive review, see ref. 26). In addition, it has been demonstrated that the Na+,K+-ATPase is phosphorylated by PKC. 27 The effect of phosphorylation on the activity of the enzyme is controversial. It has been reported that PKC activators decrease the activity of Na+,K ÷ATPase, 28'29and it has also been suggested that they increase this activity? ° Nonetheless, what seems clear is that the activity of the Na+,K+-ATPase may be regulated by a protein kinase 31'3e and that this could be P K C . 28-3°'33-35 We have reported that inhibition of PKC with H7, a PKC inhibitor, prevents the DPH-induced decreases of the amplitude of indirectly evoked muscle action potentials and also of the Na+,K+-ATPase activity in rat peripheral nervous tissue, thus suggesting a possible direct 14p. M. LeQuesne, V. Goldberg, and F. Vajda, J. Neurol. Neurosurg. Psychiatry 39, 995 (1976). 15D. J. Marcus, T. R. Swift, and T. F. McDonald, Muscle Nerve 4, 48 (1981). 16j. Rom~, A. M. Cuervo, F. Maci~in, A. Raya, J. Gallego, J. E. Llopis, and F. J. Romero, NeurotoxicoL TeratoL 12, 627 (1990). z7 F. N. Quandt, Mol. Pharmacol. 34, 557 (1988). ~8j. R. Schwarz and G. Grigat, Epilepsia (N.Y.) 30, 286 (1989). 19j. H. Pincus and S. Lee, Arch. Neurol. 26, 239 (1973). 20 j. H. Pincus and H. M. Weinfeld, Brain Res. 296, 313 (1984). 21 R. O. Messing, C. L. Carpenter, and D. A. Greenberg, J. Pharmacol. Exp. Ther. 235, 407 (1985). 22 j. H. Pincus and A. Kiss, Exp. Neurol. 94, 777 (1986). 23 D. M. Woodbury, J. Pharmacol. Exp. Ther. 115, 74 (1955). 24 M. D. Rawson and J. H. Pincus, Biochem. Pharmacol. 17, 573 (1968). 25 D. Guillaume, T. Grisar, and A. V. Delgado-Escueta, J. Neurochem. 47, 904 (1986). 26 M. S. Shearman, K. Sekiguchi, and Y. Nishizuka, Pharmcol. Rev. 41, 211 (1989). 27 j. M. Lowndes, M. Hokin-Neaverson, and P. J. Berties, Biochim. Biophys. Acta 1052, 143 (1990). 28 A. M. Bertorello and A. Aperia, Am. J. Physiol. 256, F370 (1989). 29 L. A. Vasilets, G. Schmalzing, K. Madefessel, W. Hasse, and W. Schwarz, J. Membr. Biol. 118, 131 (1990). 3o C. J. Lynch, P. B. Wilson, P. F. Blackmore, and J. H. Exton, J. BioL Chem. 2,61, 14551 (1986). 31 L. A. Yeh, L. Ling, L. English, and L. Cantley, J. Biol. Chem. 258, 6567 (1983). 32 L. Ling and L. Chantley, J. Biol. Chem. 259, 4089 (1984). 33 H. Oishi, B. Zheng, and I. F. Kuo, J. BioL Chem. 265, 70 (1990). 34 A. M. Bertorello, A. Aperia, S. I. Walaas, A. C. Nairn, and P. Greengard, Proc. Natl. Acad. Sci. U.S.A. 88, 11359 (1991). 35 A. M. Civan, A. Oler, K. Peterson-Yantorno, K. George, and T. G. O'Brien, J. Membr. Biol. 121, 37 (1991).

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DPH-mediated activation of PKC as the mechanism involved in phenytoininduced neuropathy. 36 Table I shows that the decrease of GSH content in sciatic nerve of phenytoin-treated rats is absolutely prevented by a previous treatment with H7. 36a Raya et aL 37 have showed a modification in motor nerve conduction velocity before ethanol-induced complete blockade of muscle action potential. By the time of the complete blockade of muscle action potential (67.5 _+ 9.7 min after 3 g/kg ethanol injection), a 60% decrease in GSH content of sciatic nerve was detected. This depletion does not appear when tert-butanol is used instead of ethanol (Table I), thus suggesting the involvement of ethanol metabolites and not a nonspecific action of the alcohols (A. Raya, F. J. Romero, and J. Rom~i, unpublished). The GSH decrease has also been observed in central nervous system. 3s These metabolic changes might contribute to the electrophysiological modifications observed, without excluding a possible impairment of Na+,K+-ATPase during acute ethanol intoxication, as has been proposed during chronic ethanol intake. 39 The peripheral nerve electrophysiological modifications during experimental diabetes have been associated to two main types of mechanisms: vascular modifications such as the hyperglycemia-induced generation of endoneural h y p o x i a , 4°-42 and metabolic changes such as the generation of o x i d a t i v e s t r e s s 43'44 (both mechanisms are not strictly independent of one another, as has been already brought forward4S'46). It has also been proposed that the early abnormalities in experimental diabetic neuropathy are largely 36 A. Raya, J. Gallego, C. Hermenegildo, F. J. Puertas, F. J. Romero, V. Felipo, M.-D. Mifiana, S. Grisolia, and R. Rom~i, Toxicology 75, 249 (1992). 36a A. Raya, J. Gallego, F. Bosch-Morell, J. Rom~, F. J. Romero, Free Radical BioL Med. 18, in press (1995). 37 m. Raya, C. Hermenegildo, F. J. Romero, and J. Rom~, Eur. J. Neurosci. 5(Suppl.), 299 (1992). 3s M. Uysal, G. Kutalp, G. Ozdemirler, and G. Ayka~c, Drug Alcohol Depend. 23, 227 (1989). 39 C. Guerri, R. Wallace, and S. Grisolia, Eur. J. Biochem. 86~ 581 (1978). 40 p. A. Low, K. Ward, J. D. Schmelzer, and S. Brimijoin, Am. J. Physiol. 248, E457 (1985). 41 p. A. Low, T. D. Logerlund, and P. G. McManis, Int. Rev. Neurobiol. 3L 355 (1989). 42 N. E. Cameron, M. A. Cotter, and P. A. Low, Am. J. Physiol. 261~ E1 (1991). 43 p. A. Low, R. R. Tuck, and M. Takcuchi, in "Diabetic Neuropathy" (P. J. Dyck, P. K. Thomas, A. K. Asbury, and A. I. Winegrad, eds.), p. 266. Saunders, Philadelphia, Pennsylvania, 1987. 44 j. B. Bayes, Diabetes 40, 405 (1991). 45 M. Kihara, J. D. Schmelzer, J. F. Poduslo, G. L. Curan, K. K. Nickander, and P. A. Low, Proc. Natl. Acad. Sci. U.S.A. 88~ 6107 (1991). 46 j. R. Williamson, K. Chang, M. Frangos, K. S. Hasan, Y. Ido, T. Kawamura, J. R. Nyengaard, M. van der Enden, C. Kilo, and R. G. Tilton, Diabetes 42, 801 (1993).

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attributable to a diminished Na+,K+-ATPase activity in peripheral nerve 41,47 The demonstration of changes in nerve conduction velocity as well as in nerve blood flow during experimental diabetes requires longer times of evolution than required for the changes in Na+,K+-ATPase activity in peripheral nerve, the latter modification being one of the earliest observed in experimental diabetic neuropathy.47The elevated levels of lipid peroxidation and glycoxidation products in diabetic humans and animals that support the idea of a diabetes-induced oxidative stress 44 provide no evidence, however, to what extent these parameters of increased oxidative stress reflect an increase production of free radicals and/or impaired free radical scavenger function.46 In this respect, we have shown that the sciatic nerve of alloxaninduced diabetic mice has decreased glutathione peroxidase activity when compared with control animals, as early as 7 days after alloxan injection.4s However, the glutathione redox state of peripheral nerve in streptozotocininduced diabetic rats, 14 days after streptozotocin treatment, was reported not to be involved in the myo-inositol-related Na +,K+-ATPase defect, since no GSH alteration could be established.49 On the other hand, signs of oxygen free radical generation have been demonstrated in long-term studies of experimental diabetes (over 4 weeks after streptozotocin treatment)4s.5!~ and more recently, also a GSH decrease in peripheral nerve, 4 weeks after STZ, that was prevented with lipolic acid treatment, s°~ Because PKC phosphorylates Na+,K+-ATPase,27 the beneficial effects of PKC inhibitors demonstrated for nervous tissue in vitro s~-53 have been confirmed in vivo when studying Na+,K+-ATPase activity in sciatic nerve of diabetic mice. 54,55 However, data from our laboratory demonstrate that the complete recovery of Na+,K+-ATPase activity by prolonged treatment with PKC inhibitors (over 14 days)ss had no effect on motor nerve conduction velocity, which remained decreased to levels seen in diabetic animals (C. Hermenegildo, F. J. Romero, and J. Romfi, unpublished). 47 D. A. Greene, A. A. F. Sima, M. A. Pfeifer, and J. W. Albers, Annu. Rev. Med. 41, 303 (1990). 48 C. Hermenegildo, A. Raya, J. Rom~, and F. J. Romero, Neurochem. Res. 18, 893 (1993). 49 p. B. Carroll, B. M. Thornton, and D. A. Greene, Diabetes 35, 1282 (1986). 5o p. A. Low and K. A. Nickander, Diabetes 40, 873 (1991). 50~ M. Nagamastu, K. Nickander, J. D. Schmelzer, A. Raya, D. A. Whittrock, H. Trilschler, and P. A. Low, Diabetes Care, in press. 5~ V, Felipo, M.-D. Mifiana, and S. Grisolia, J. Biol. Chem. 265, 9599 (1990). 52 M.-D. Mifiana, and S, Grisolla, Proc. Natl. Acad. Sci. U.S.A. 87, 4335 (1990). 53 F. J. Romero, J. Llopis, V. Felipo, M.-D. Mifiana, J. Rorn~i, and S. Grisolia, F E B S Lett. 303, 19 (1992). 54 C. Hermenegildo, V. Felipo, M.-D. Mifiana, and S. Grisol/a, J. Neurochem. 58, 1246 (1992). 55 C. Hermenegildo, V. Felipo, M.-D. Mifiana, F. J. Romero, and S. Grisolia, Diabetes 42, 257 (1993).

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In view of the accumulating evidence regarding the interactions between GSH and other thiols and P K C , 56-58 a n accurate and precise procedure for the routine determination of both in peripheral nervous tissue is certainly required. The GSH procedure is detailed here. No modification is required for the determination of PKC in peripheral nervous tissue relative to the procedure by Hannun et al., 59 provided enough peripheral nervous tissue protein is added to the reaction mixture, even under conditions of PKC inhibition (see Table I). 55 Assay of Glutathione in Sciatic Nerve The determination of GSH in sciatic nerve is based on the procedure by Brigelius et al.6° with the precautions regarding sample preparation that are mentioned below, modified from Akerboom and Sies. 61 Sample Preparation

Samples are prepared by means of the freeze-stop technique. After surgical exposure of the sciatic nerve, a section of 1-4 cm (depending on the animal size, e.g., mouse, rat) is obtained and the frozen sample weighed. To avoid individual variability the results should be referred to protein content of the sample. For this purpose, the sample (1 single sciatic nerve section in the case of the rat, and 4-6 sections for the mouse) is homogenized in 0.75-1.0 ml of prechilled 0.1 M potassium phosphate buffer, pH 7.0, containing 1 mM EDTA. A quick procedure is desired in order to obtain a homogeneous suspension from which a 0.6-ml aliquot is separated and added to 0.1 ml of 2 M perchloric acid containing 1 mM EDTA; from the remainder of the material another aliquot for protein determination is collected. The use of perchloric acid is also advantageous in peripheral nervous tissue because it can be easily removed by precipitation at neutral pH as the potassium salt. 61 The acid extracts are centrifuged at 5000 g for 5 min (room temperature), in all cases long enough to obtain a clear transparent sample. The samples should be processed quickly in order to avoid autoxidation at acidic pH, although for GSH determinations autoxidation represents 56 R. Gopalakrishna and W. B. Anderson, F E B S Lett. 225, 233 (1987). s7 G. E. N. Kass, S. K. Duddy, and S. Orrenius, Biochem. Z 260, 499 (1989). 58 R. Gopalakrishna, Z.-H. Chen, and U. Gundimedia, this volume [14]. s9 y. A. Hannun, C. R. Loomis, and R. M. Bell, J. Biol. Chem. 260, 10039 (1985). 60 R. Brigelius, C. Muckel, T. P. M. Akerboom, and H. Sies, Biochem. Pharmacol. 32, 2529 (1983). 61 T. P. M. Akerboom and H. Sies, this series, Vol. 77, p. 373.

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always less than 0.2% (w/v) GSH per hour. 61 Neutralization is performed with 40% (w/v) K2HPO4 containing 0.3 M N-morpholinopropanesulfonic acid (MOPS).

Principle and Procedure The reaction between 1-chloro-2,4-dinitrobenzene (CDNB) and GSH, catalyzed by glutathione S-transferase (ED 2.5.1.18), is monitored by following the formation of S-dinitrophenylglutathione at 240 nm and using a molar extinction coefficient of 9.6 mM -1 cm-1.6° The specificity of the reaction is provided by the enzyme. Pipette into a plastic disposable microcuvette or a glass microcuvette the following: 0.5 ml of buffer (0.2 M potassium phosphate buffer, pH 7.0, containing 2 mM EDTA), 0.1-0.35 ml neutralized sample (no difference has been observed when MOPS is omitted in the neutralization solution), 10/.d of 10 mM CDNB, and water up to 1 ml. After mixing, record the absorbance at 340 nm and start the reaction by adding 5 tzg of purified equine liver glutathione S-transferase (commercially available). The enzyme should have a specific activity higher than 600/~mol/mg protein × min, in order to (i) reduce the volume of the addition and (ii) proceed at a relatively high velocity at 300-37 °, and it should be prepared free of GSH by dialysis (most of the commercially available enzymes are stabilized with GSH). Acknowledgments Supported in part by Grant PM92-0146 from DGICYT and Grant 94/1629 from FISS to F. J. R.

[16] E x p r e s s i o n o f P r o t e i n K i n a s e CT R e g u l a t o r y Domain Elements Containing Cysteine-Rich Zinc-Coordinating Regions as Glutathione S-Transferase Fusion Proteins

By A N D R E W F. G . QUEST, ELAINE S. G . BARDES, W E N Q I N XIE, ELIZABETH WILLOTT, R O Y A . BORCHARDT, a n d ROBERT M . BELL Introduction Protein kinase C (PKC) is a family of serine/threonine protein kinases consisting of 10 or more members, whose activities are physiologically METHODSIN ENZYMOLOGY,VOL.252

Copyright© 1995by AcademicPress,Inc. All rightsof reproductionin any formreserved.

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[ 1 6]

regulated by lipids. 1-6 Members of the PKC family are divided into three groups based on cDNA-derived protein sequence similarities: calciumdependent (cPKCs), non-calcium-dependent (nPKCs), and atypical (aPKCs) PKC enzymes.4 Each isoform possesses two functionally distinct segments, a COOH-terminal kinase domain and an NH2-terminal regulatory domain. 7,8 Within the regulatory domain lie several regions involved in PKC activation that may promote distinct interactions of PKC with lipids. 3'4'6'9'1° These are the peudosubstrate (Pseudo) motif within the first variable region (V1), the two cysteine-rich motifs in the first conserved region (C1), and the calcium-dependent lipid binding domain (CaLB) within the second conserved region (C2) (see Fig. 1). Electrostatic interactions have been suggested to occur between the pseudosubstrate domain, present in the NH2-terminal variable V1 region of PKCs, and acidic phospholipids, based on the observation that polycationic peptides mimicking this region are able to bind to such lipid membranes.11'12 The 50-amino acid cysteine-rich motifs Cysl and Cys2 within the C1 region bind tumor-promoting phorbol esters 8'9 and are thereby implicated in the physiological activation of PKC by sn-l,2-diacylglycerol (DAG). The C2 region is present in calcium-dependent PKC isoforms, but absent in noncalcium-dependent isoforms. This and other observations have implicated the C2 region, and in particular the CaLB domain, in mediating calciumdependent lipid interactions of the cPKC regulatory domain and cytosolic phospholipase AzJ 3 Each of the two highly conserved cysteine-rich segments of about 50 amino acids possesses six conserved cysteines (C) and two conserved histidines (H) in a pattern H - X 1 2 - C - X 2 - C - X 1 0 - 1 4 - C - X 2 - C - X 4 - H - X 2 - C - X T - C

1 y. Nishizuka, Science 225, 1365 (1984). 2 y. Nishizuka, Science 233, 305 (1986), 3 y. Nishizuka, Nature (London) 334, 661 (1988). 4 y. Nishizuka, Science 258, 607 (1992). 5 R. M. Bell, Cell (Cambridge, Mass.) 45, 631 (1986). 6 R. M. Bell and D, J. Burns, J. Biol. Chem. 266, 4661 (1991). 7 M.-H. Lee and R. M. Bell, J. Biol. Chem. 261, 14867 (1986). s y. Ono, T. Fujii, K. Igarashi, T. Kuno, C. Tanaka, U. Kikkawa, and Y. Nishizuka, Proc. Natl. Acad. Sci. U.S.A. 86, 4868 (1989). 9 D. J. Burns and R. M. Bell, J. Biol. Chem. 266, 18330 (1991). 10A. F. G. Quest and R. M. Bell, in "Protein Kinase C: The Molecular Mechanism of Protein Kinase Regulation by Lipids" (J. F. Kuo, ed.), p. 64. Oxford Univ. Press, New York, 1994. 11 M. Mosior and S. McLaughlin, Biophys. J. 60, 149 (1991). 12 M. Mosior and S. McLaughlin, Biochemistry 31, 1767 (1992). 13j. D. Clark, L. L. Lim, R. W. Kriz, C. S. Ramesha, L. A. Sultzman, A. Y. Lin, L. Milona, and J. L. Knopf, Cell (Cambridge, Mass.) 65, 1043 (1991).

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PKC-GLUTATHIONE S-TRANSFERASE FUSION PROTEINS

REGULATORY DOMAIN Vl NH2 ~ ' PseudoCysl

C1 V2 C2 ~ ~ ~ Cys2 CaLB

155

CATALYTIC DOMAIN V3 ~

C3

V4

C4

V5 '~ COOH

GST-VI i GST-VI C y s l GST-VICysICys2

i t

GST-VlCyslCys2CaLB

GST-Cysl GST-Cys2 GST-Cysl Cys2 GST-Cys2CaLB GST-CaLB

Fro. 1. Schematic representation of PKCy and fusion proteins characterized in these studies. PKC~/is shown at the top divided into several variable (V1-V5) and constant (C1C4) regions based on sequence similarity with other PKC family members. The amino-terminal regions V1, C1, V2, and C2 constitute the regulatory domain, V3 contains an element referred to as the hinge region, and the carboxyl-terminal elements C3, V4, C4, and V5 are parts of the catalytic domain. Lipid interactions critical for PKC activation occur within the regulatory domain. For sections of the regulatory domain with known functions or characteristic sequence motifs additional abbreviations are used: Pseudo, pseudosubstrate motif; Cysl, first cysteinerich motif; Cys2, second cysteine-rich motif; and CaLB, calcium-dependent lipid binding domain. In our studies we focused on elements of the regulatory domain that were expressed as fusion proteins with glutathione S-transferase (GST). Abbreviations for the respective fusion proteins are shown at left, and elements of the PKCT regulatory domain present are indicated to the right as bars. GST-V1, GST-V1Cysl, GST-V1CyslCys2, GST-V1CyslCys2CaLB, GST-Cysl, GST-Cys2, GST-CyslCys2, GST-Cys2CaLB, and GST-CaLB are carboxyl-terminal fusion proteins of GST with the first variable region (amino acids 1-37), the first variable region and cysteine-rich region of PKC7 (amino acids 1-91), the first variable region and both cysteine-rieh regions of PKC~/(amino acids 1-173), the entire PKCy regulatory domain up to and including the calcium-dependent lipid binding domain (amino acids 1234), the first PKCT cysteine-rich region (amino acids 27-9I), the second PKC~/cysteine-rich region (amino acids 92-173), the first and second PKC~/cysteine-rich regions (amino acids 27-173), the second cysteine-rich region and the calcium-dependent lipid binding domain (amino acids 92-234), and the calcium-dependent lipid binding domain (amino acids 183234), respectively [numbered as in J. L. Knopf, M.-H. Lee, L. A. Sultzman, R. W. Kriz, C. R. Loomis, R. M. Hewick, and R. M. Bell, Cell (Cambridge, Mass.) 46, 491 (1986)].

(C6H2), where X's are variable residues. TM Based on sequence comparisons between phorbol ester-binding proteins, the two conserved histidines and six conserved cysteines have been predicted to be required for phorbol ester binding. ~5In some transcription factors, cysteine-rich protein structures that 14 S. R. Hubbard, W. R. Bishop, P. Kirschmeier, S. J. George, S. P. Cramer, and W. A. Hendrickson, Science 254, 1776 (1991). 15 S. Ahmed, R Kozma, J. Lee, C. Monfries, N. Harden, and L. Lim, Biochem. J. 280, 233 (1991).

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coordinate zinc are essential for the ability of the factor to recognize and bind specific D N A sequences. 16 The cysteine-rich regions of PKC show striking similarity to such zinc-coordinating sequences identified in transcription factors. 3,9 Indeed, quantitative analysis of native, calcium-dependent PKC isozymes and proteolytic fragments derived from P K C a revealed that the lipid-binding regulatory domain of PKC contains four atoms of tightly bound zinc. ~7 However, the precise region of PKC in which zinc is coordinated and the importance for phorbol ester binding remained unknown. The close proximity of several lipid interaction sites and the complexity of lipid interactions required for PKC activation have hampered the assessment of individual contributions toward PKC activation. Thus, to analyze how zinc coordination and phorbol ester binding within a cysteine-rich region are related ~8'I9 and, further, to determine how the known lipid interaction sites may participate to forge the regulatory properties of PKC, 2° we have utilized the p G E X expression plasmid for protein expression in Escherichia coli. PKCy, a representative of the group of cPKC isoforms, was chosen for reasons related to the anticipated multiplicity of lipid interactions in the regulatory domain as explained elsewhere. 2° Elements of the regulatory domain of PKCy were expressed as fusion proteins with glutathione S-transferase (GST) which allowed affinity purification, essentially to homogeneity, in one step. Crucial to the success of this approach was obtaining expression conditions which yielded large quantities of soluble products that allowed easy purification of functional proteins. The insights gained from studies with such fusion proteins have been detailed in the aforementioned references. Here we describe studies that were initially undertaken to optimize expression conditions for the cysteine-rich regions in X L I B E. cull and subsequent modifications thereof for the expression of the entire regulatory domain in BL21 E. coli. Segments of the regulatory domain obtained in this manner are shown in Fig. 1. E x p e r i m e n t a l Procedures In this section we describe all the methods required including the final protocol used for expression of G S T - P K C y fusion proteins in both X L I B and BL21 E. coli host cells. In the section following we then discuss the experiments done to establish the conditions that are now routinely used 16j. E. Coleman,Annu. Rev. Biochem. 61, 897 (1992). 17A. F. G. Quest, J. Bloomenthal, E. S, G. Bardes, and R. M. Bell, Z Biol. Chem. 267, 10193 (1992). 18A. F. G. Quest, E, S. G. Bardes, and R. M. Bell, Z Biol. Chem. 269, 2953 (1994). 19A. F. G. Quest, E. S. G. Bardes, and R. M. Bell, Z Biol. Chem. 269, 2961 (1994). 20A. F. G. Quest and R. M. Bell, J. Biol. Chem. 269, 20000 (1994).

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for protein expression in our laboratory and highlight some of the problems encountered. Purification of Protein Kinase C'y

The PKCy enzyme is purified from infected Spodoptera frugiperda (Sf9, fall armyworm ovary) cells as described. 17'21 Purified enzyme preparations are stored at - 7 0 ° in storage buffer (20 mM Tris-HC1, pH 7.5, 0.5 mM EGTA, 0.5 mM EDTA, 10 mM 2-mercaptoethanol, 10% (v/v) glycerol, and 0.05% Triton X-100). Protein Kinase C Domains Expressed as Glutathione S-Transferase Fusion Proteins in Escherichia coli

To generate large quantities of PKC subdomains, in particular the cysteine-rich phorbol ester-binding regions, the subdomains are expressed in E. coli as COOH-terminal fusion proteins with glutathione S-transferase from Schistosoma japonicum. DNA encoding regions of interest are amplified by polymerase chain reaction (PCR) from a PKCy template sequence22 using specific oligomer primers which are synthesized on an Applied Biosystems (Foster City, CA) Model 380B oligonucleotide synthesizer. The primers employed have been described in detail elsewhere. 18-2° The PCR is carried out for 40 cycles, each at 94° for 1 min, 55° for 1 min, and at 72° for 2 min (Perkin-Elmer Cetus, Norwalk, CT). The PCR products are digested with EcoRI and BamHI restriction enzymes, purified on 2% low-melt SeaPlaque agarose (FMC Bioproducts, Rockland, ME) gels, isolated from the gel pieces by treatment with Gel'ase (Epicenter Technologies, Madison, WI), and ligated into BamHI/EcoRI double-digested pGEX2T plasmid (Pharmacia LKB Biotechnology, Piscataway, NJ). The XL1B E. coli cells (Stratagene, La Jolla, CA) are transformed with ligated plasmids and grown overnight at 37 ° on Luria-Bertiani (LB) agar plates under ampicillin selection (50 tzg/ml). Double-stranded DNA obtained from positive (Magic Minipreps DNA purification system; Promega, Madison, WI) is checked by restriction analysis and cycle sequencing (using the reagent kit from GIBCO-BRL Life Technologies, Gaithersburg, MD) following recommendations of the thermocycler manufacturer (Perkin-Elmer Cetus). Standard methods in molecular biology not discussed here are as described. 23 Escherichia coli with plasmids containing the required inserts are grown in LB medium overnight at 37° under ampicillin selection (50 txg/ml) until zl D. J. Burns, J. Bloomenthal, M.-H. Lee, and R. M. Bell, J. Biol. Chem. 265, 12044 (1990). 22 D. A. Persons, W. O. Wilkison, R. M. Bell, and O. J. Finn, Cell (Cambridge, Mass.) 52, 447 (1988). 23 j. Sambrook, E. F. Fritsch, and T. Maniatis, in "Molecular Cloning: A Laboratory Manual" 2rid ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1989.

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the stationary phase is reached. A fresh culture is then inoculated at 37 ° with the cells (1-2 ml per 100 ml LB medium) and grown to an absorbance of 0.45-0.5 measured at 600 nm (Shimadzu UV-160 spectrophotometer). The cells are then transferred to 20 °, and growth is continued until the culture reaches an absorbance of 0.7. At that density ZnC12 is added to the medium (final concentration 1 tzM), and protein expression is induced by addition of isopropyl,/3-D-thiogalactopyranoside (IPTG; 50 mg/ml stock solution) to a final concentration of 0.2 mM in the growth medium. After 16 hr cells are harvested by centrifugation and cell pellets frozen at - 7 0 °. In some cases where proteolytic degradation of fusion proteins is a problem (GST-V1, GST-V1Cysl, GST-Cys2CaLB, and GST-VICyslCys2CaLB; see Fig. 3), protease-deficient E. coli BL21 (Novagen, Madison, WI) are transformed with plasmid D N A isolated from XL1B E. coli. For protein expression, BL21 E. coli cells are grown as described for XL1B cells. However, because these cells grow much faster they are harvested 5-8 hr after induction of protein expression (see Fig. 4). Purification of Glutathione S-Transferase and Fusion Proteins

To recover GST or GST fusion proteins, E. coli cell pellets are lysed by cavitation (French press) and extracted in detergent at a ratio of 1 ml extraction buffer [50 mM HEPES, pH 8, 1 mM dithiothreitol (DTF), 0.1% Triton X-100, 10% ethylene glycol, 1 mM phenylmethylsulfonyl fluoride, 10/xg/ml benzamidine, 2/zg/ml antipain, 1/~g/ml leupeptin] per cell pellet from 15 ml of E. coli culture medium. Insoluble cell debris is removed by centrifugation at 15,000 g. Solubilized cell components are applied three times to an equal volume of glutathione-agarose (Sigma, St. Louis, MO) matrix equilibrated in sodium phosphate buffer at room temperature (1 mM sodium phosphate, pH 7.3, 15 mM NaC1). After washing with 10 column volumes of phosphate buffer to remove nonspecifically bound proteins, GST fusion proteins are then specifically eluted with 5 mM reduced glutathione (Sigma) in 50 mM HEPES, pH 8, with 10% ethylene glycol. All fusion proteins are stored at - 7 0 ° in elution buffer. Note that, in contrast to rat brain PKC or the individual PKC isozymes, GST fusion proteins with PKCy domains are never stored in the presence of chelators. 4[3-[3H]Hydroxyphorbol 12,13-dibutyrate Binding Assay

Binding of 4/3-[3H]hydroxyphorbol 12,13-dibutyrate, [20-3H(N)] ([3H]PDBu) is measured by the vesicle assay in the presence of 125 nM PDBu as described. 17'24Nonspecific binding determined in the presence of 40/zM unlabeled/3-PDBu is subtracted. 24j. G. Ebling, G. R. Vandenbark, L. J. Kuhn, B. R. Ganong, and R. M. Bell, Proc. Natl. Acad. Sci. U.S,A. 82~815 (1985).

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159

Protein Quantitation The PKC and GST fusion proteins are quantitated by the amido black dye binding assay2s using fatty acid-free bovine serum albumin as a standard.

Zinc Measurements Zinc is quantitated by atomic absorption spectroscopy as previously described in detail. ~7'26 All zinc stoichiometries illustrated are calculated based on such zinc measurements and protein quantitation by the amido black dye binding assay,2s assuming a molecular mass of 80 kDa for intact PKC, 27.7 kDa for GST, 34.3 kDa for GST-Cysl, 35.5 kDa for GST-Cys2, 42.2 kDa for GST-CyslCys2, 31.2 kDa for GST-V1, 36.6 kDa for GSTVICysl, 41.9 kDa for GST-Cys2CaLB, and 50.9 kDa for GST-V1CyslCys2CaLB fusion proteins and an atomic mass of 65 for zinc. These and all other molecular masses used for calculations of zinc stoichiometry and PDBu binding values are summarized in Table I. The observed standard error of stoichiometries calculated for several determinations of samples separated from adventitious zinc by gel filtration 17,26 reflects the error of the measurements involved and lies in the vicinity of 10% of the averaged values.

Gel Electrophoresis If necessary, protein samples are precipitated prior to electrophoresis in chloroform-methanol, 27 analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) 28 on 12.5% minigels (Bio-Rad, Richmond, CA), and protein bands visualized by Coomassie Blue (Sigma, St. Louis, MO) staining.

Glutathione S- Transferase Activity The GST activity is measured as previously described. 29 Experiments to Define Expression Conditions in XL1B and BL21 Escherichia coli Cells Experimental systems previously used to study domains critical for phorbol ester binding of PKC or n-chimaerin were hampered either by 2s W. Schaffner and C. Weissmann, A n a l Biochern. 56, 502 (1973). 26 A. F. G. Quest, E. S. G. Bardes, J. Bloomenthal, R. A. Borchardt, and R. M. Bell, Methods Neurosci. 18, 138 (1993). 27 D. Wessel and U. I. Fluegge, Anal. Biochem. 138, 141 (1984). 28 U. K. Laemmli, Nature (London) 227, 680 (1970). :9 p. C. Simons and D. L. VanderJagt, Anal. Biochem. 82, 334 (1977).

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TABLE I ZINC STOICHIOMETRIES AND PHORBOL 12,13-DmUTYRATE BINDING DETERMINEDFOR PROTEINKit C~, GLUTATHIONES-TRANSFERASE,AND FUSIONPROTEINSa

Protein/fusion protein b

PKCy GST XLIB strain GST-V1 GST-VICysl GST-V1CyslCys2 GST-V1CyslCys2CaLB GST-Cysl GST-Cys2 GST-CyslCys2 GST-Cys2CaLB GST-CaLB BL21 strain GST-V1 GST-V1Cysl GST-V1CyslCys2 GST-V1CyslCys2CaLB GST-Cysl GST-Cys2 GST-CyslCys2 GST-Cys2CaLB GST-CaLB

PKC), amino acids present c 1-701 --

Molecular mass a (kDa)

Zn stoichiometrye (mol/mol) (±S.D.)

80 27

1-37 1-91 1-173 1-234 27-91 92-173 27-173 92-234 183-234

31.2 36.6 44.5 50.9 34.3 35,5 42,2 41.9 32.7

1-37 1-91 1-173 1-234 27-91 92-173 27-173 92-234 183-234

31.2 36.6 44.5 50.9 34.3 35.5 42.2 41.9 32.7

PDBu bindin (mmol/mol) ( ±

3.6 -+ 0.4 0

0.7 1.1 1.5 1.3 1.7 2.6

0.16 ± 0.01 ± 0.1 (0.7 +-- 0.1) ± 0.4 (1.4 ± 0.2) --- 0.7 (2.3 ± 0.3) ± 0.4 (l.8 ± 0.2) ± 0.1 (1.8 ± 0.1) ± 1.7 (3.8 ± 0.3) 1.2 ± 0.1 0.04 ± 0.06

NDg 0.5 ± 0.1 1.0 ± 0.1 1.1 ± 0.4 (1.9 ± 0.2) 0.5 ± 0.l 1.2 ± 0.l 1.2 ± 0.1 1.0 ± 0.1 ND

423 ± 11 0

7 30 12 78 143 104 41

1 --- 1 ± 8 (17 ~_ ± 18 (62 -~ ± 3 (18 -~ --- 33 (116 -" ± 26 (193 _~ ± 52 (167 _~ ± 6 (51 _~ 0.1 ± 0.2

1± 1 10 ± 1 28 ± 18 (40 _~ 30 ± 7 (38 -~ 9 ± 1 185 ± 5 26 + 5 57 ± 3 3 ± 3

a Fusion proteins between the PKC3, regulatory domain and GST were expressed in XLIB or ] strains of E. coli. In come cases similar values to those shown were reported elsewhere~8'2°bL included here for comparison. b The experimental data shown summarize the zinc stoiehiometries (moles zinc/mole proteinl [3H]PDBu binding (millimoles/mole protein) determined for PKC3' purified from baculo~ infected Sf9 cells, GST from XL1B E. coli, and GST-V1, GST-VICysl, GST-V1CyslCys2, ( V1CyslCys2CaLB, GST-Cysl, GST-Cys2, GST-CyslCys2, GST-Cys2CaLB, and GST-( fusion proteins. ¢ Abbreviations used here and numbering of amino acids present are explained in the legen Fig. 1. a Molecular masses shown were used in calculations of both the zinc stoichiometry and PDBu bin e For each zinc stoichiometry determination sample concentrations of zinc were measured times. Protein concentrations were measured in triplicate. The errors indicated are eithe standard deviations (S.D.) calculated for the zinc determination values averaged (n -> 3) o: less than three determinations, the standard deviation of the measurements averaged (n = 8) highest zinc stoichiometries determined for individual fusion proteins are shown in parentt Data for fusion proteins with no adjacent values in parentheses are single determinations.

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the instability of the domains when expressed individually in baculovirusinfected Sf9 cells9 or by their insolubility when expressed as fusion proteins in E. coll. 3° Using a plasmid pGEX2T (Pharmacia LKB Biotechnology) for protein expression in E. coli, PKC~/regions of interest were then expressed as COOH-terminal fusion proteins with GST. Initial experiments in XLIB cells revealed that GST alone was readily expressed in large quantities as a soluble active protein following standard protocols at 37°, as was easily assessed by measuring GST activity.29 In contrast, the fusion proteins of GST with segments of PKC'y were expressed but could only be retrieved from cell homogenates as insoluble aggregates that required harsh conditions, such as the presence of sodium dodecyl sulfate (SDS) and urea, to be solubilized. Protein obtained under such conditions was inactive and could not be subsequently affinity purified (data not shown). Various parameters w e r e t e s t e d 31,32 that could have improved the solubility of expressed fusion proteins, which was monitored very simply by measuring glutathione S-transferase activity. Results with two such parameters, namely, the inclusion of 10% (v/v) glycerol in the culture medium and reduction of the temperature at which protein expression was induced, are shown (Fig. 2). Clearly, for all three fusion proteins tested, namely, GSTCysl (Fig. 2A), GST-Cys2 (Fig. 2B), and GST-CyslCys2 (Fig. 2C), reduction of the culture temperature to 20° before induction enhanced the recovery of soluble protein in cell lysates up to 10-fold. In comparison, the addition of glycerol had no significant beneficial effects on protein recovery. All other parmeters tested, including the addition of zinc chloride to the growth medium, did not improve the recovery of soluble fusion proteins (data not shown). In contrast, supplementation of medium with 10/zM zinc chloride slightly reduced cell growth, as evidenced by increasing doubling times of cells in culture, and at 1 mM concentrations zinc chloride completely inhibited E. coli growth (data not shown). To be sure not to inhibit cell growth, an addition of only 1/xM ZnC12 was chosen. This addition is 30 S. Ahmed, R. Kozma, C. Monfries, C. Hall, H. H. Lim, P. Smith, and L. Lirn, Biochern. J. 272, 767 (1990). 31 C. Schein, BioTechniques 7, 1141 (1989). 32 C. Schein, BioTechniques 8, 308 (1990).

f T o determine PDBu binding values, samples were measured in quadruplicate. Protein values were measured in triplicate. The errors indicated are either the standard deviations (S.D.) calculated for PDBu binding values averaged (n -> 3) or, for less than three determinations, the standard deviation of the measurements averaged (n = 4). Highest PDBu binding values determined for individual fusion proteins are shown in parentheses. Data for fusion proteins with no adjacent values in parentheses are single determinations. g ND, Not determined.

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SIGNAL TRANSDUCTION AND GENE REGULATION

[ 161

50

Cys1 )

n +Glycerol • -Glycerol

40 30 20

10 ND

0

_~ so u

B

ND

(GST-Cys2)

40 3o ,c

° o

-7

2o

-

10 0

~E

0 so ] C

(GST-CyslCys2)

40 30, 20,

10. 20

33 Temperature

37

(*C)

FIG. 2. Optimization of expression conditions for three GST-PKC3, fusion proteins in XL1B E. coli. The GST fusion proteins GST-Cysl (A), GST-Cys2 (B), and GST-CyslCys2 (C) were expressed in XLtB cells following the procedure that eventually became established (see text), except that in some cases (white bars) glycerol was present at a final concentration of 10% (v/v) in the expression medium and the temperature at which expression was induced by addition of IPTG was varied as indicated. Recovery of soluble fusion protein after cell lysis and centrifugation was quantitated by measuring GST activity. Note that in all three cases reduction of the expression temperature to 20° greatly improved the recovery of soluble fusion proteins. In contrast, the presence of glycerol had no beneficial effects in this respect.

p r o b a b l y n o t n e c e s s a r y b e c a u s e r e g u l a r m e d i u m c o n t a i n s a b o u t 2 0 / z M zinc (22 _+ 5 / z M ) , as was l a t e r d e t e r m i n e d in c o n j u n c t i o n with e x p e r i m e n t s using BL21 cells (see Fig. 4). Thus, t r a n s f o r m e d E. coli w e r e initially g r o w n at 37 ° b u t s u b s e q u e n t l y t r a n s f e r r e d to 20 ° p r i o r to t h e i n d u c t i o n o f p r o t e i n synthesis b y the a d d i t i o n o f I P T G to a final c o n c e n t r a t i o n o f 0.2 m M in t h e p r e s e n c e o f l / z M ZnCI2

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163

once cultures reached an absorbance of 0.7 (see Experimental Procedures). Under such conditions, fusion proteins were present as soluble proteins in E. coli homogenates and could be readily purified in a single step to homogeneity by affinity chromatography. Following this procedure roughly 1 mg of pure GST-PKC'y fusion proteins containing short segments of the regulatory domain (Cysl, Cys2, CyslCys2) was obtained from each 25-30 ml of induced E. coli culture. Conditions established with fusion proteins containing the cysteine-rich regions essentially allowed expression of fusion proteins containing all segments of the PKC~/regulatory domain in XLIB cells (Fig. 3A). However, in some cases, in particular when the V1 or CaLB region was present, affinity-purified proteins migrated with apparent A

1

2

3

4

5

6

7

8

9

10

11

Mr (x10-3) -

106

"

80

-

49.5

-

32.5

-

27.5/GST

-

18.5

- D y e front

B -

106

"

80

-

49.5

-

32.5

-

27.5/GST

-

18.5

- D y e front

FIG. 3. Analysis by SDS-PAGE of fusion proteins expressed in either XLIB or BL21 cells. PKCy (lane 1) or GST-PKC3~ fusion proteins (1/zg) were resolved by SDS-PAGE on 12.5% gels and subsequently stained with Coomassie blue. Fusion proteins isolated from both XL1B (A) and BL21 (B) cells are shown: GST (lane 2), GST-V1 (lane 3), GST-V1Cysl (lane 4), GST-V1CyslCys2 (lane 5), GST-V1CyslCys2CaLB (lane 6), GST-Cysl (lane 7), GST-Cys2 (lane 8), GST-CyslCys2 (lane 9), GST-Cys2CaLB (lane 10), and GST-CaLB (lane 11). Expression in XL1B cells yielded mainly intact fusion proteins in several cases (lanes 7, 8, 9, and I0), but others were severely degraded (lanes 3, 4, 5, 6, and 11). In the latter cases expression in BL21 improved the yield of nondegraded fusion protein. The Mr values of prestained marker proteins (Bio-Rad, Richmond, CA) are indicated at right together with the dye front.

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SIGNAL T R A N S D U C T I O N A N D GENE R E G U L A T I O N

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molecular weights lower than anticipated, suggesting that both the V1 and CaLB sequences were susceptible to proteolysis. To circumvent these problems, an alternative host strain, with reduced protease activity, was sought for protein expression. BL21 cells, an E. coli strain deficient in ornp T and Ion proteases, were transformed with plasmids isolated from the XL1B cells. Protein expression was induced and cells were harvested as described above. A major difference from previous experiments with XL1B was that maximum levels of protein expression were obtained after only 5-8 hr, as will be discussed (Fig. 4). Figure 3B shows an analysis of the same fusion proteins as discussed above purified from BL21 cells. Clearly, alteration of the host cell dramatically improved the recovery of intact fusion proteins, especially in cases where either V1 or CaLB were adjacent to GST (Fig. 3, lanes 3, 5, 6, and 11). However, the yield of larger fusion proteins like GST-V1CyslCys2CaLB was about 4-fold lower (1 mg per 100 ml E. coli culture; see Fig. 4B) than that for GST-Cys2. A particularly difficult fusion protein to obtain intact was GST-V1CyslCys2CaLB. Thus, the time dependence of expression in BL21 cells was optimized using this fusion protein as an example. Expression was monitored as a function of several parameters, namely, by measuring GST activity in E. coli lysates (Fig. 4A), fusion protein and PDBu binding recovery after affinity purification (Fig. 4B), the specific PDBu binding activity (Fig. 4C) and zinc stoichiometry (Fig. 4E). Cell growth (doubling times at 37°: BL21, 24 min; XL1B, 54 min) and protein expression were more rapid in the BL21 cells than in XL1B E. coli. After 5-8 hr both GST activity (Fig. 4A) and specific PDBu binding activity (Fig. 4C) reached constant levels, whereas this occurred to a lesser extent for protein recovery from the affinity column (Fig. 4B) and total PDBu binding in lysates (Fig. 4A). This could have indicated that at longer time intervals more degradation was occurring. However, gel analysis of GST-V1CyslCys2CaLB purified at different time points suggested that there was no difference in this respect. Surprisingly, an inverse relationship was noted with the zinc stoichiometry of purified protein, suggesting that, as time progressed, either zinc became limiting or it was not effectively incorporated into the protein. Our previous studies indicate that although 4 zinc atoms are coordinated within the regulatory domain of P K C , 17 n o t all appear equally important for PDBu binding. 18,19 This may explain why an increase in specific PDBu binding activity was observed (Fig. 4C), despite the decrease in zinc stoichiometry (Fig. 4E). In any case, these studies revealed that zinc stoichiometries for fusion proteins isolated from BL21 cells may vary considerably depending on the time point postinduction at which cells are harvested. Fusion proteins were routinely expressed in BL21 cells for 5-8 hr postinduction, and representative samples of such preparations are shown in Fig.

[16]

PKC-GLUTATHIONE S-TRANSFERASE FUSION PROTEINS

lOO A

165 1000

lOOO "_ @ Protein B o POSu binding

80

80o -~

800

E=

40

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6OO

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~ 0

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,

'

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o.

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0 Time

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,~ ~t.o

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Flo. 4. Expression of GST-V1CyslCys2CaLB in BL21 cells. Fusion protein expression was induced at 20° as described in the text and followed by measuring several parameters as a function of time after induction with IPTG. The GST activity after centrifugation of BL21 cell lysates is shown in (A), protein (@) and PDBu binding activity (O) recovered by affinity purification from lysates of 100 ml BL21 cell culture in (B), specific PDBu binding activity of affinity-purified fusion protein in (C), and zinc stoichiometry of affinity-purified fusion protein in (E). Fusion protein (1/xg), purified at time points of 2, 5, 8, or 20 hr postinduction, is shown after S D S - P A G E and Coomassie blue staining in (D) (lanes 2, 3, 4, and 5, respectively). Prestained molecular weight marker proteins are shown in lane 1, corresponding, from top to bottom, to Mr values of 106,000, 80,000, 49,500, 32,500, 27,500 and 18,500. Note that although the yield of affinity-purified fusion protein and total PDBu binding activity as well as the specific PDBu binding activity increase with time postinduction, a decrease in zinc stoichiometry was seen.

166

SIGNAL TRANSDUCTION AND GENE REGULATION

[161

3. Preparations of fusion proteins obtained both from XLIB and BL21 cells were additionally characterized with regard to PDBu binding and zinc stoichiometry. These results are summarized in Table I. Most notably, zinc levels were below the anticipated values for all fusion proteins except those containing Cysl, Cys2, and CyslCys2 (XLB1 strain), but even there considerable variation was observed. The PDBu binding values were also highest in these proteins. Thus, although proteins produced in strain XLB1 were less degraded (Fig. 3), both zinc stoichiometries and PDBu binding values were lower in all cases except for PDBu binding to GST-Cys2 (Table I), possibly owing to the more rapid production of GST fusion proteins in BL21 cells, in comparison to XL1B cells. Zinc stoichiometries were highest for the GST-V1CyslCys2CaLB fusion protein at early time points during expression (1-2 hr postinduction) and decreased subsequently (Fig. 4E), being paralleled by a decline in the rate of protein production as fusion protein accumulated within the BL21 cells (Fig. 4A,B). This may indicate that intracellular levels of free (i.e., not protein-bound) zinc, rather than the rate of protein expression, became more critical as time progressed. In general, we found that values for zinc stoichiometries were much lower than expected mainly in fusion proteins with predicted stoichiometries greater than 2. The expression of 1 mg of fusion protein of Mr 35,000-40,000 consumed roughly 25 nmol of zinc per coordinated zinc atom. At a total concentration of 20/zM zinc, roughly 500 nmol of zinc was present in 25 ml expression medium, the volume from which the equivalent of 1 mg of fusion protein was obtained. Even the coordination of 4 atoms of zinc per fusion protein molecule should not have depleted the medium of zinc. Thus, zinc incorporation into fusion proteins appeared to be limited by the rate at which E. coli took zinc up from the medium. In BL21 cells, which grow faster and express fusion proteins more rapidly, such a limitation would be expected to affect zinc stoichiometries more seriously, as was indeed the case (Table I). Concluding Remarks We have described here how conditions that enable the expression and isolation of large quantities of segments of the PKC'y regulatory domain as soluble GST fusion proteins were initially established using XL1B as an E. coli host. Affinity-purified fusion proteins, containing the cysteine-rich region of PKC referred to as Cys2 and deletions thereof, have provided excellent molecular tools to define the PKC regulatory domain elements involved in zinc coordination and PDBu (or diacylglycerol) binding, as well as the relationship between these two properties. 18,19 Furthermore, using PDBu binding as a sensitive method of quantitation, GST-PKC fusion

[161

PKC-GLUTATHIONE S-TRANSFERASE FUSION PROTEINS

167

proteins have enabled us to analyze contributions of several cofactor binding sites in the regulatory domain toward phosphatidylserine-, diacylglycerol-, and calcium-dependent regulation of PKC3, activity.2° For the latter studies, fusion proteins containing almost the entire regulatory domain of PKC~/were employed, which were difficult to obtain in large quantities owing to protein degradation (Fig. 3A). These problems were overcome by using protease deficient BL21 E. coli as host cells for protein expression (Fig. 3B). Unfortunately, however, some proteins expressed in these cells had lower PDBu binding activities than when expressed in XL1B (GST-Cysl, GST-CyslCys2), and zinc stoichiometries were generally lower, too (see Table I). This may have been a result of the more rapid expression kinetics in BL21 cells, as the zinc binding stoichiometry was observed to decline as a function of time after induction of protein expression with IPTG (Fig. 4). Thus, although more rapid expression in conjunction with a reduction of proteolytic activity served well to increase the yield of intact fusion proteins in BL21 cells, the proteins obtained were not necessarily improved in terms of functional (PDBu binding) and structural (zinc coordination) criteria. Our results suggest that expression conditions need to be optimized individually for different proteins and that protein yield and SDS-PAGE analysis are not sufficient to assess the quality of the protein product. Depending on the final objectives, additional criteria are essential, and particular attention should be paid to the rate at which proteins are being expressed. Despite these limitations the procedures described here should yield the quantities of fusion proteins necessary for future nuclear magnetic resonance (NMR) and crystallographic studies of PKC structure. Furthermore, these procedures have already been employed successfully in our laboratory to express raf-1 kinase, another protein kinase with a cysteinerich domain that coordinates zinc, as well as different segments thereof, as soluble GST fusion proteins. 33 Thus, these methods should also provide helpful guidance to the successful expression of other metal ion-coordinating thiol proteins. Acknowledgments Research was supported by National Institutes of Health Grant GM 38737.

33 S. Ghosh, W. Q. Xie, A. F. G. Quest, G. M. Mabrouk, J. C. Strum, and R. M. Bell, Jr. Biol. Chem. 269, 10000 (1994).

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[l 7]

[ 17] R e d o x R e g u l a t i o n o f A c t i v a t i o n o f NF-KB Transcription Factor Complex: Effects of N-Acetylcysteine

By FRANK J. T.

S T A A L , MICHAEL T. ANDERSON, and LEONARD A. HERZENBERG

Introduction NF-KB is a heterodimeric transcription factor complex composed, in its classic form, of two DNA-binding subunits: p50 and p65 (reviewed in Ref. 1). NF-KB was first identified as a DNA-binding activity specific for the KB motif in the immunoglobin kappa (K) enhancer. Cloning of the NF-KB p50 and p65 genes revealed a family of NF-KB/rel proteins that participate in a variety of transcriptionally regulated processes, such as lymphocyte differentiation, responsiveness to cytokines, and embryonic development in Drosophila. NF-KB is present in many cell types in an inactive form in the cytoplasm, bound to a cytoplasmic retention molecule called I-KB (for inhibitor of NF-KB). 2 Nuclear translocation of NF-KB is achieved after stimulation of the cells by many different inducers (cytokines, phorbol esters, viral proteins, oxidants) and presumably involves phosphorylation of I-KB which disrupts its interaction with the p65 NF-KB subunit. 3 The NF-KB complex is then free to migrate to the nucleus and transactivate its various target genes. Studies from several laboratories have revealed that the signal transduction pathways leading to activation of NF-KB are redox regulated. 4-7 That is, antioxidants such as N-acetyl-L-cysteine (NAC) can inhibit activation, whereas oxidants such as hydrogen peroxide (1-[202) can directly activate NF-KB. The N A C inhibition of NF-KB activation first implicated a redoxsensitive step in the NF-KB signal transduction pathway. 4 Further studies have found that a wide variety of compounds which share the ability to modulate intracellular oxidant levels also inhibit NF-KB activation. These 1 G. P. Nolan and D. Baltimore, Curr. Opin. Gen. Dev. 2, 211 (1992). 2 R. Sen and D. Baltimore, Cell (Cambridge, Mass.) 47, 921 (1986). 3 S. Ghosh and D. Baltimore, Nature (London) 344, 678 (1990). 4 F. J. T. Staal, M. Roederer, L. A. Herzenberg, and L. A. Herzenberg, Proc. Natl. Acad. Sci. U.S.A. 87, 9943 (1990). 5 T. Kalebic, A. Kinter, G. Poli, M. E. Anderson, A. Meister, and A. S. Famci, et al., Proc. Natl. Acad. Sci. U.S.A. 88, 986 (1991). 6 S. Mihm, J. Ennen, U. Passara, R. Kurth, and W. Droge, A I D S 5, 497 (1991). 7 R. Schreck, P. Bieber, and P. Baeuerle, E M B O J. 10~ 2247 (1991).

METHODS IN ENZYMOLOGY,VOL 252

Copyright © 1995 by AcademicPress, Inc. All rights of reproduction in any form reserved.

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results, coupled with the observations that oxidants such as H202 are by themselves sufficient to activate NF-KB, 7 suggests oxidants play key roles within NF-KB signaling pathways. Here we describe detailed methods on the appropriate cell culture conditions to study the influence of oxidants and antioxidants, the preparation of nuclear protein extracts, and the use of gel retardation assays to detect the presence of activated NF-KB in the nucleus. In addition, we briefly describe a reporter gene-based system to assay whether the DNAbound NF-KB is transcriptionally active. Methods and Procedures Principle

The electrophoretic mobility shift assay (EMSA) or gel retardation assay is a powerful method for quantitative and qualitative analysis of p r o t e i n - D N A interactions. The technique is based on the principle that the electrophoretic mobility of a nucleic acid (e.g., a radiolabeled oligonucleotide) through a polyacrylamide gel is impeded when a protein is bound to it. Thus, free (labeled) oligonucleotide and oligonucleotides that have bound protein can be discerned based on their relative migration in the gel. This principle was originally described by Garner and Rezvin s and by Fried and Crothers 9 and extended to the identification of mammalian transcription factors by Singh et a l l ° With the EMSA small amounts of a D N A (or RNA)-binding protein can be detected in a heterogeneous mixture of proteins such as a nuclear extract. Cells and Stimulation Conditions

A variety of cells can be used for measurement of NF-KB. Either constitutive or inducible EMSA-shifted NF-KB complexes are detectable in lymphoid, epithelial, and fibroblast cell lines as well as in primary cells. It is important that the cells are viable, proliferating in logarithmic phase, and grown at the right density. For instance, Jurkat T cells grown at high density (2 x 10 6 cells/ml) can have about 25% lower glutathione (GSH) levels than cells grown at low density. This density-induced change in GSH can confound redox effects of the compounds actually studied. It is therefore good experimental practice to perform the experiments under standardized culture conditions. M. M. Garner and A. Revzin, Nucleic Acids Res. 9, 3047 (1981). 9 M. G. Fried and D, M. Crothers, Nucleic Acids Res. 9, 6505 (1981). ~0H. Singh, R. Sen, D. Baltimore, and P. A. Sharp, Nature (London) 319, 9 (1986).

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Cells are incubated with appropriate stimuli (e.g., cytokines, phorbol esters) that activate NF-KB and the redox-regulating compounds (antioxidants, iron chelators, or oxidants) that may inhibit or increase activation. Both the stimuli and inhibitors should be added from stock solutions that are at least 100x concentrated, pH-neutralized, and sterilized before use. If organic solvents [such as dimethyl sulfoxide (DMSO)] are used, it is important to include a control adding the vehicle only. As a first estimate, it is useful to explore the kinetics of activation and inhibition of NF-KB over a time range from 0.5 to 8 hr; subsequently, one can establish a rough titration curve of the redox-active compound.

Preparation of Nuclear Protein Extract Several methods to prepare nuclear extracts are available, most of which involve salt extraction of whole cell or nuclear homogenates. The original procedure was described by Dignam, Lebovitz, and Roeder and has been modified by others. 11 The nuclear extract method described here works well with 1 x l06 to 2 x 107 cells and is an adaptation from the method described by Schaffner and co-workers. 12 We have compared this method to the original large-scale method and obtained equivalent results for the NF-xB, Oct-l, AP-1, and NF-AT transcription factors (although total protein yield is, of course, lower). 1. After stimulation, the cells are harvested, washed once, and then resuspended in 1.0 ml of cold Tris-buffered saline (TBS). To harvest adherent cells, do not use trypsin but use phosphate-buffered saline (PBS)E D T A (2.5 mM) instead. Subsequent steps are done in the cold room at 4 °. 2. Transfer sample to a 1.5-ml Eppendorf tube. 3. Centrifuge 15 sec at 14,000 rpm. 4. Remove supernatant using a drawn out Pasteur pipette and suction. 5. Resuspend (cellular) pellet in 400/zl of buffer A [10 m M HEPES, pH 7.8/10 mM KC1/2 mM MgC12/0.1 mM EDTA/0.5 mM dithiothreitol (DTT), supplemented with the protease inhibitors phenylmethylsulfonyl fluoride (PMSF, 0.5 mM), antipain (1/zg/ml), leupeptin (0.3/xg/ml), and pepstatin (0.5/zg/ml); DTT and the protease inhibitors are added freshly from stock solutions.]. 6. Incubate cells on ice for 15 min. 7. Add 25 tzl of 10% Nonidet P-40 (NP-40) solution. This solution should be added to the inner part of the Eppendorf cap. Cap the tubes in 11H. Ohlsson and T. Edlund, Cell (Cambridge, Mass.) 45, 35 (1986). 12 E. Schreiber, P. Matthias, M. M. Mueller, and W. Schaffner, Nucleic Acids Res, 17, 6419 (1989).

[17]

REDOX REGULATIONOF NF-rB

171

pairs, invert the tubes, and immediately vortex 10 times for 1 sec. This procedure allows reproducible extraction of nuclear proteins, whereas direct addition of NP-40 can give problems. 8. Centrifuge extract 30 sec at 14,000 rpm. Remove as much as possible of the supernatant (if necessary, centrifuge a second time to remove all liquid). A small white nuclear pellet should be visible. 9. Resuspend pellet in 40/~l of buffer C (50 m M HEPES, pH 7.8/50 mM KC1/300 mM NaC1/0.1 mM EDTA/0.5 mM DTT/10% (v/v) glycerol; add protease inhibitors as for buffer A). Resuspension is usually somewhat difficult. It is important to assure that clumps are at least somewhat dispersed. 10. Salt-extract nuclear proteins by rotating on a rotator for 30 min. 11. Centrifuge 10 min at full speed (14,000 rpm). 12. Transfer supernatant (nuclear extract) to a new Eppendorf tube. Discard pellet (cell debris). The extracts can be stored at - 7 0 ° or used immediately for determination of protein concentration and gel retardation.

Electrophoretic Mobility Shift Assay The amount of protein obtained can be determined using the Bradford assay (Bio-Rad, Richmond, CA). We routinely use 200/.d of reagent with 2.0 ~l of extract and a standard curve of different known concentrations of bovine serum albumin (BSA). We then determine the 560 nm absorbance on a 96-well enzyme-linked immunosorbent assay (ELISA) plate reader, from which the protein concentration in the extract can be calculated. Two micrograms of nuclear extract per condition should be sufficient for detection of NF-KB and other transcription factors by EMSA. Optimal binding of NF-KB is dependent on the final salt concentration in the binding reaction, the concentration of DTY, and the amount of nonspecific competitor DNA. In our hands, optimal binding occurs under conditions of 70 mM salt, 5 mM D ] T , and 2.0/~g poly(dI-dC) as competitor. Poly(dI-dC) is dissolved in Tris-EDTA buffer (TE)/100 mM NaCl, heated to 90°, and slowly cooled (over 30-45 min) to room temperature before use. It may be necessary to vary both the amount of extract and amount of nonspecific competitor to optimize the sensitivity of the assay for detection of NF-KB within different cell types. The radiolabeled probe (see below) is added last; 10,000 counts/ min (cpm) should be sufficient for detection after overnight exposure on film. Binding reactions are done at room temperature for 45 min in a final volume of 20-50 tzl. Subsequently, samples are electrophoresed on a 4.5% polyacrylamide gel with 0.25× TBE (Tris-borate-EDTA buffer, pH 8.3) as running buffer

172

SIGNAL TRANSDUCTION

AND

OENE

[17]

REGULATION

for 1.5-2 hr at 100 V. After electrophoresis, the gel is disassembled, transferred onto two sheets of Whatman (Clifton, N J) filter paper, and dried. A typical 1.5-mm gel requires about 1 hr of drying under vacuum at 80°. The dried gel is exposed to film overnight at - 7 0 ° and developed. For most purposes this exposure time should yield a good signal. If necessary, longer exposures should be used.

Labeling of Probe Many different KB probes can be used. We use a double-stranded probe made by annealing two single-stranded synthetic oligonucleotides with the following sequences. T C GAGT C AGAGGGGAC T T T C C GAG C AGT C T C C C CT G A A A G G C T C A G C The unlabeled probe is stored at 100 ng/tA at - 2 0 °, and 0.5 IA is used to end-label the probe with Klenow D N A polymerase. Mix 0.5/zl probe, 10 /zl GTC 2 mM dNTP mixture (2 mM dATP, 2 mM dGTP, 2 mM dq-TP),

Slirnulafion 20 n t M ~ NAC

~o

.,,~, --

I

I

~

+

-I

.%~

+

_ I~

+

_ I

~,o~

+ I

J

NFr.B

Free Probe

F~G. 1. Inhibition of TNF-a- and PMA-induced NF-KB binding activity by NAC. 293 cells were stimulated with TNF-a, PMA, or both in the presence or absence of 20 mM NAC. Nuclear protein extracts were made and EMSAs performed as described in the text. First and last lanes contain free probe without nuclear protein added. The top arrow indicates the inducible NF-KB band; the bottom arrow marks unbound probe. (Reproduced from Staal e t al. 4 with permission from the publishers.)

[17]

REDOX REGULATIONOF NF-KB

173

30/zl doubly distilled water, 5/zl of 10× Klenow buffer (0.5 mM Tris-C1, pH 7.5, 0.1 M MgC12, 10 mM DTT, 0.5 mg/ml BSA), 1/xl Klenow DNA polymerase (100 U/ml), and 4/zl of [a-32p]dCTP (1 mCi/ml). Incubation is for 30 min at room temperature, after which the reaction is stopped by addition of 50/zl TE. Labeled probe and unincorporated [a-32p]dCTP are separated using a spin column packed with Bio-Rad P6DG or Sephadex G-25. The columns are made using a 1-ml insulin syringe (centrifuge 2 rain at 1600 rpm (500 g), packed volume should equal about 0.9 ml). Two microliters of the labeled probe is counted using a scintillation counter. A good probe should have at least 25,000-50,000 counts/sec (cps).

Analysis of Results Typical results of a gel retardation assay are shown in Fig. 1. The presence of NF-KB under stimulated [tumor necrosis factor (TNF), phorbol 12-myristate 13-acetate (PMA)] conditions is visible as a set of two sharp bands, which presumably correspond to the (p50)2 homodimer (lower band) and the p65-p50 heterodimer (upper band). Clearly, if stimulation is done in the presence of 20 mM NAC, no activated NF-~B can be found. Similar

20 mM ....~ NAC

I

I

--

I

+

I

-

I

-

I

+

I

+

I

-

I

+

1 Oct-1

Free Probe

FIG. 2. N-Acetylcysteine does not inhibit the Oct-1 transcription factor. T h e same nuclear protein extracts from Fig. 1 were probed with a Oct-1 probe, T h e top arrow indicates the constitutive Oct-I band, the bottom arrow the u n b o u n d probe. ( R e p r o d u c e d from Staal et aL 4 with permission.)

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SIGNAL TRANSDUCTION AND GENE REGULATION

[ 17]

results can be obtained with many different cell lines and stimulation conditions. Quantitative results can be obtained by scanning the gel with a phosphorimager (radioanalytic imaging system). It is important to ascertain the specificity of the NF-KB inhibition by antioxidants such as NAC. Therefore, we usually use the same nuclear protein extracts to probe another transcription factor that is not redoxsensitive. The ubiquitous Oct-1 transcription factor can be useful for this purpose. As shown in Fig. 2, conditions that effectively inhibit activation of NF-KB do not affect Oct-1 expression, demonstrating that NAC selectively inhibits NF-xB. Finally, the presence of activated NF-KB in a nuclear extract does not necessarily mean that NF-KB is transcriptionally active (binding does not imply function). Functional NF-KB can be readily assayed using reporter genes, the transcription of which is directed by an NF-KB-dependent promoter. We have used Jurkat T cells stably transfected with a plasmid containing three KB motifs fused to the lacZ (/3-galactosidase) gene (Jurkat kB5.2). 4 Cells can be stimulated under the appropriate conditions and assayed for/3-galactosidase activity by biochemical means in lysates ~3 or by the fluorescence-activated cell sorting (FACS)-Gal assay using a flow cytometer. TMIt is beyond the scope of this chapter to describe these methods in detail, but readers are referred to protocols given elsewhereJ 5 Acknowledgment Supported in part by NIH CA-42509.

13 M. Roederer, F. J. T. Staal, P. A. Raju, L. A. Herzenberg, and L. A. Herzenberg, Proc. Natl. Acad. Sci. U.S.A. 87, 4884 (1990). 14 G. P. Nolan, S. N. Fiering, J. F. Nicolas, and L. A. Herzenberg, Proc. Natl. Acad. Sci. U.S.A. 85, 2603 (1988). 15 M. Roederer, S. N. Fiering, and L. A. Herzenberg, Methods (San Diego) 2, 248 (1991).

[ 18]

D N A - P R O T E I N INTERACTIONS

[18] R e d o x R e g u l a t i o n o f D N A - P r o t e i n by Biothiols

175

Interactions

By YUICmRO J. SUZUKIand LESTZRPACKER Introduction Redox regulation of DNA-protein interactions plays an important role in mechanisms of actions of many mammalian transcription factors for controlling gene transcription. NF-KB DNA-binding activity is stimulated by reducing agents (dithiothreitol, 2-mercaptoethanol, thioredoxin) and abolished by exposure to sulfhydryl-modifying agents (diamide, N-ethylmaleimide). 1-~ Important sulfhydryl groups such as Cys-62 in the p50 subunit of NF-~B for DNA-binding activity have been identified.6 Similarly, the DNA-binding activity of the Fos-Jun heterodimeric complex (AP-1) has been shown to be regulated by a redox mechanism.7 A ubiquitous protein, redox factor-1 (Ref-1), appears to participate in regulation of AP-1. 8"9 Steroid receptors such as glucocorticoid receptoP ° and progesterone receptoP 1 also require reduced sulfhydryl groups for DNA-binding activity. Hence, the identification of reductants which are capable of influencing the D N A protein interactions has physiological and pharmacological importance as they can enhance the activities of transcription factors, and in turn either positively or negatively modulate gene expression. Here we describe a simple and rapid methodology to screen effects of reductants on DNA-protein interactions. This methodology utilizes the knowledge that DNA-binding proteins usually require the presence of reducing agents such as dithiothreitol (DTI') to exhibit DNA-binding activity. Thus, creating a nonreducing environment by omitting DTT from the I M. B, Toledano and W. J. Leonard, Proc. Natl. Acad. Sci. U.S.A. 88, 4328 (1991). 2 j. A. Molitor, D. W. Ballard, and W. C. Greene, New Biol. 3, 987 (1991). 3 T. Okamoto, H. Ogiwara, T. Hayashi, A. Mitsui, T. Kawabe, and J. Yodoi, Int. Immunol. 4, 811 (1992). 4 j. R. Matthews, N. Wakasugi, J.-L. Virelizier, J. Yodoi, and R. T. Hay, Nucleic Acids Res. 20, 3821 (1992). 5 T. Hayashi, Y. Ueno, and T. Okamoto, J. Biol. Chem. 268, 11380 (1993). 6 j. R. Matthews, W. Kaszubska, G. Turcatti, T. N. C. Wells, and R. T. Hay, Nucleic Acids Res. 21, 1727 (1993). 7 C. Abate, L. Patel, F. J. Rauscher III, and T. Curran, Science 2,49, 1157 (1990). 8 S. Xanthoudakis and T. Curran, EMBO J. 11, 653 (1992). 9 S. Xanthoudakis, G. Miao, F. Wang, Y.-C. E. Pan, and T. Curran, EMBO J. 11, 3323 (1992). 10 C. M. Silva and J. A. Cidlowski, J. Biol. Chem. 264, 6638 (1989). H S. Peleg, W. T. Schrader, and B. W. O'Malley, Biochemistry 28, 7373 (1989).

METHODS IN ENZYMOLOGY, VOL. 252

Copyright © 1995 by Academic Press, Inc. All rights of reproduction in any form reserved.

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[ 18]

general protocol allows the isolation of DNA-binding proteins whose activity cannot be detected, yet a potential for DNA-binding activity is retained in the presence of appropriate reducing factors. Briefly, nuclear extracts are isolated by a miniextraction procedure without the use of DTT, 12,13 followed by binding reactions with interesting reductants for electrophoretic mobility shift assays. The results can be obtained within 24 hr.

Preparation of Nuclear Miniextracts 1. Pellet 1 × 106 cells by centrifuging for 10 rain at 1200 rpm in microcentrifuge. 2. Remove supernatant. Wash pellet with 1 ml of cold phosphatebuffered saline (PBS). 3. Pellet ceils by centrifuging for 15 sec at 14,000 rpm in microcentrifuge. 4. Remove supernatant and resuspend pellet in 0.4 ml of buffer A [10 mM HEPES, pH 7.8, 10 mM KC1,2 mM MgCI2, 0.1 mM EDTA, 0.1 mM phenylmethylsulfonylfluoride (PMSF), 5/zg/ml antipain, 5/zg/ml leupeptin, 1 mM DTT*]. 5. Incubate on ice for 15 min to swell ceils. 6. Add 25/M of 10% Nonidet P-40 and vortex vigorously for 15 sec. 7. Centrifuge homogenate for 30 sec at 14,000 rpm in microcentrifuge. 8. Remove supernatant and resuspend nuclear pellet in 50/~1 of buffer C [50 mM HEPES, pH 7.8, 50 mM KC1, 300 mM NaC1, 0.1 mM EDTA, 0.1 mM PSMF, 10% (v/v) glycerol, 1 mM DTT*]. 9. Mix vigorously for 20 rain at 4°. 10. Centrifuge for 5 rain at 14,000 rpm in microcentrifuge at 4 °. 11. Harvest supernatant containing the nuclear proteins. 12. Determine protein concentrations. 13. Samples can be stored in aliquots at - 8 0 ° until use.

Electrophoretic Mobility Shift Assay The simplest and the most user-friendly method to study DNA-protein interactions is the electrophoretic mobility shift assay (EMSA; band-shift assay, gel-shift assay, gel retardation assay). This technique was first devel12E. Schreiber, P. Matthias, M. M. Mueller,and W. Schaffner,Nucleic Acids Res, 17, 6419 (1989). 13F. J. T. Staal, M. Roederer, L. A. Herzenberg, and L. A. Herzenberg, Proc. Natl. Acad. Sci. U.S.A. 87, 9943 (1990). * The DTT is omittedfrom buffersA and C in order to create a nonredueingenvironment.

[ 18]

DNA-PROTEIN INTERACTIONS

177

oped simultaneously by two groups in 1981.14'15 There already exist excellent reviews which describe the theory and step-by-step instructions for the EMSA. 16,17 Thus, users should refer to those sources for detailed instructions. The following is one example of a protocol for performing the EMSA. Binding reaction mixtures (20 ~1) containing the following components are incubated for 20 min at 25°: 10 m M Tris-HC1 (pH 7.5), 50 m M NaCI, 0.2 m M E D T A , 2% (v/v) glycerol, nuclear protein extract, poly(dI-dC) • poly(dI-dC) (nonspecific competitor), 32p-labeled D N A fragment, and DTT. Titrations of nuclear protein extract, nonspecific competitor, and 32p-labeled D N A fragment may be necessary to determine the optimal conditions for the assay. D T T is omitted to create a nonreducing environment. Titration with D T T may also provide useful information in determining the redox characteristics of D N A - p r o t e i n interactions. Reducing factors of interest are also included in the binding reaction. Proteins are separated by electrophoresis through a native polyacrylamide gel, and detection can be made by autoradiography following standard procedures.

E x a m p l e : E n h a n c e m e n t of NF-KB DNA Binding b y Biothiols NF-KB is a mammalian transcriptional activator that is involved in the transmission of signals from the cytoplasm to the nucleus, and it participates in activation of genes involved in the immune, inflammatory, or acute phase responses, such as various cytokines and surface receptors as well as viruses including human immunodeficiency virus (HIV). is Studies also point to the importance of this transcription factor in neurological disorders 19 and atherosclerosis. 2° The DNA-binding activity of NF-~cB is regulated through redox mechanisms. Treatment of the activated form of NF-KB molecule with N-ethylmaleimide, diamide] ,2 or hydrogen peroxide has been shown to eliminate D N A binding. Inversely to the inhibition by oxidants, thiol reductants such as D T T and reduced thioredoxin enhance the DNA-binding activity. 4 The present study demonstrates that other biothiols including dihydrolipoate, N-acetylcysteine, and L-cysteine also enhance the D N A NF-KB interactions. ~ M. M. Garner and A. Revzin, Nucleic Acids Res. 9~ 3047 (1981). t5 M. Fried and D. M. Crothers, Nucleic Acids Res. 9, 6505 (1981). 16G. H. Goodwin, in "Gel Electrophoresis of Nucleic Acids: A Practical Approach" (D. Rickwood and B. D. Haines, eds.), p. 225. IRL Press, Oxford, 1990. ~7j. Carey, this series, Vol. 208, p. 103. 18p. Baeuerle, Biochim. Biophys. A c t s 1072, 63 (1991). 19B. Kaltschmidt, P. A. Baeuerle, and C. Kaltschmidt, Mol. Aspects Med. 14, 171 (1993). 20 F. Liao, A. Andalibi, F. C. deBeer, A. M. Fogelman, and A. J. Lusis, J. Clin. Invest. 91, 2572 (1993).

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REGULATION

[ 18]

Materials and Methods Jurkat T (acute human leukemia) cells are grown in RPMI 1640 medium supplemented with 10% fetal calf serum, 1% penicillin/streptomycin, and 1% glutamine. Cells are plated at a density of 1 × 106 cells per well in 1.0 ml. Recombinant Escherichia coli-derived human tumor necrosis factor-a (TNF-a; 25 ng/ml; Genentech Inc., South San Francisco, CA) is added, and cells are incubated for 1 hr in an atmosphere of 5% (v/v) .CO2 in air humidified at 37 °. Nuclear extracts are prepared from 1 × 106 cells as described above without the use of DT]?. The binding reaction mixtures (20 /zl) for E M S A containing 1.5 ~g protein of nuclear extract, 0.5 /~g poly(dI-dC)-poly(dI-dC), 32p-labeled oligonucleotide probe for NF-KB (see sequences below), 50 m M NaC1, 0.2 m M EDTA, 2% (v/v) glycerol, and 10 mM Tris-HCl (pH 7.5) are incubated for 20 min at 25 °. In some experiments, binding reaction mixtures also contain DTT (Sigma, St. Louis, MO), dihydrolipoate (Asta Medica, Frankfurt, Germany), Noacetylcysteine, L-cysteine, ascorbate, a-tocopherol, NADH, N A D P H , or pyruvate (Sigma). Proteins are separated by electrophoresis through a native 6% polyacryl-

4-

4-

4-

4-

4-

4-

4-

4-

4-

4-

NF-rB

,~-Freepr~ FIG. 1. Effects of thiol and nonthiol reductants on NF-rB DNA-bindingactivity.Nuclear extracts (NE) from nonstimulatedor TNF-c~-stimulatedcells were prepared without the use of dithiothreitol (DTT). Various reductants were added in binding reaction mixtures for the electrophoretic mobility shift assay before the addition of 3~P-labeled NF-KB-specific oligonucleotide probe. DHLA, Dihydrolipoate;NAC, N-acetylcysteine.

[181

DNA-PROTEIN INTERACTIONS

179

amide gel in a running buffer of 12.5 mM Tris-borate (pH 8.0) containing 0.25 mM EDTA, followed by autoradiography. 5' G A T C C G A G G G G A C T T T C C G C T G G G G A C T T T C C A G G 3' 3' GCT C C C C T G A A A G G C G A C C C C T G A A A G G T CCCTAG 5'

Results and Discussion

In EMSA experiments which were performed without the use of DTT, no observable NF-KB bands were detected in nuclear extracts from either nonstimulated or TNF-o~-stimulated Jurkat T cells (Fig. 1, lanes 2 and 5, respectively). Although no changes were observed in nonstimulated samples (Fig. 1, lanes 3 and 4), the addition of thiol reductants, DTT or dihydrolipoate (10 ~M-1 raM), to the binding reaction mixtures caused concentration-dependent restorations of NF-KB DNA-binding activity in nuclear extracts from TNF-o~-stimulated cells (Fig. 1, lanes 6-11). Other thiols, namely, N-acetylcysteine (Fig.l, lane 12) and e-cysteine (Fig. 1, lane 13), also enhanced the NF-KB DNA-binding activity. In contrast, nonthiol reductants such as ascorbate (Fig. 1, lane 14), ot-tocopherol (Fig. 1, lane 15), NADH, NADPH, and pyruvate (data not shown) did not enhance the DNA-binding activity. The present example demonstrates that reduced thiols including DTT, dihydrolipoate, N-acetylcysteine, and L-cysteine enhance the DNA-binding activity of NG-KB. The order of potencies of the thiols in enhancing DNAbinding activity generally agrees with the order of redox potentials (see Table I). In contrast, nonthiol reductants, including those with very negative redox potentials such as pyruvate, NADH, and NADPH (see Table I), do

TABLE I REDOX POTENTIALSFOR THIOLS AND NONTHIOLSa System

Eo (V)

Pyruvate/acetate + CO2 Dithiothreitol, ox/red NAD(P)+/NAD(P)H + H + Lipoate, ox/red L-Cystine/cysteine

-0.70 -0.33 -0.32 -0.29 -0.22

Midpoint potentials at pH 7.0; ox/red, oxidized form/reduced form.

" E o,

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[19]

not enhance DNA-NF-KB interactions. Therefore, biothiols may regulate gene transcription by enhancing the DNA-binding activity of NF-KB transcription factor. Acknowledgments Research was supported by the National Institutes of Health (CA47597) and ASTA Medica. This work was done during the tenure of a research fellowship from the American Heart Association, California Affiliate, to Y. J. S.

[ 191 S u p p r e s s i o n of Protooncogene c-los Expression Antioxidant Dihydrolipoic Acid

By

by

MASASHI M I Z U N O a n d LESTER PACKER

Introduction Cells in a growth-arrested quiescent state require a set of growth factors for initiation of cell proliferation. 1 Competence factors include serum, tumor promoters such as phorbol 12-myristate 13-acetate (PMA), plateletderived growth factor (PDGF), and so on. When quiescent cells are stimulated by these factors, certain genes called immediate early genes are immediately transcribed without de novo protein synthesis. The protooncogene c-fos is one of the immediate early genes, and the product derived from the gene is thought to participate in signal transduction after stimuli. It is induced very rapidly and transiently in response to extracellular stimuli. 2 For example, in the case of PMA stimulation, the expression of cofos is rapidly increased, with m R N A levels reaching a maximum within the first hour after stimulation and decreasing to constitutive levels thereafter. A substantial amount of evidence suggests that the generation of free radicals, such as superoxide anion and hydroxyl radicals, may be involved in tumor promotion. A number of studies indicate that various antioxidants inhibit tumor promotion. Additionally, several lines of evidence indicate that generation of reactive oxygen species (ROS) is one of the earliest events involved in stimulation of cell growth in response to growth factors or in growth-promoting conditions. It has demonstrated that hydrogen peroxide and superoxide anion radical induce expression of c-fos m R N A A. B. Pardee, R. Dubrow, J. L. Ham/in, and R. F. Kletzien, Annu. Rev. Biochem. 47, 715 (1978). z M. E. Greenberg and E. B. Ziff, Nature (London) 311, 433 (1984).

METHODS IN ENZYMOLOGY, VOL. 252

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SUPPRESSION o r c-los BY DIHYDROLIPOIC ACID

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and D N A synthesis in quiescent BALB/3T3 cells,3 and it has been reported that low concentrations of oxidants act as a mitogenic signal. 4 It has also been reported that ROS are produced by cultured cells when they are stimulated by PMA. 5 Furthermore, the antioxidant retinoic acid suppresses expression of the protooncogene c-los in cells stimulated by PMA and serum. 6 This evidence indicates the possibility that ROS are mitogenic and mediate growth-associated gene expression. oL-Lipoic acid, one of the disulfide compounds, is an essential substance synthesized in the mammalian liver and other organs which serves in physiological systems as a cofactor for ot-ketoacid dehydrogenase reactions (e.g., in the pyruvate dehydrogenase complex), o~-Lipoic acid exerts antioxidant actions in vivo and in vitro] Moreover, the reduced form of o~-lipoic acid, dihydrolipoic acid (DHLA), acts as a potent antioxidant and scavenges superoxide, hydroxyl, peroxyl, and other radical species, s Because the formation of ROS plays an important role in the expression of m R N A after extracellular stimuli, antioxidants might suppress expression of protooncogenes. A method is presented here that utilizes D H L A to investigate the suppressive effect of antioxidants on the expression of the protooncogene c-fos induced by PMA. RNA Isolation and Northern Analysis The difficulty in R N A isolation is that most ribonucleases (RNases) are very stable and active enzymes. It is important that cells are lysed under chemical conditions that promote RNase denaturation. The following steps should be taken to avoid contamination by RNase: (1) all water and salt solutions should be treated with diethyl pyrocarbonate (DEPC); (2) glassware should be heated to 250 ° for 4 hr, and plasticware should be disposable; (3) gloves should be worn at all times during the extraction procedure and when handling materials and equipment. Caution: DEPC is a suspected carcinogen and should be handled carefully in a hood. Cell Culture

Jurkat T cells, a human T-cell leukemia cell line, are grown in RPMI 1640 medium supplemented with 10% fetal calf serum (FCS), 1% glutamine, 3 M. Shibanurna, T. Kuroki, and K. Nose, Oncogene 3, 17 (1988). 4 D. Crawford, I. Zbinden, P. Amstad, and P, Cerutti, Oncogene 3, 27 (1988). 5 M. Shibanurna, T. Kuroki, and K, Nose, Biochern. Biophys. Res. Cornrnun. 144, 1317 (1987). 6 K. J. Busam, A. B. Roberts, and M. B. Sporn, J, Biol. Chem. 267, 19971 (1992). 7 j. Peinado, H. Sies, and T. P. Akerboom, Arch. Biochem. Biophys. 273, 389 (1989). Y. J. Suzuki, M. Tsuchiya, and L. Packer, Free Radical Res, Cornmuns. 15, 255 (1991).

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[191

1% sodium pyruvate, and antibiotics including 100 units/ml of penicillin and streptomycin in 5% (v/v) CO2 in air at 37°. At approximately 70% confluency (7 x l0 s to 9 x 105 cells/ml), cells are suspended at a density of 1 x 106 cells/ml in medium in which the concentration of FCS is reduced from 10 to 1%, and the mixtures are incubated for 16-18 hr in the presence or absence of 0.2 or 0.02 mM D H L A in dimethyl sulfoxide (DMSO) solution. Then PMA (Sigma, St. Louis, MO) is added (0.5/xM) to the medium to stimulate the protooncogene c-los, and the cell suspensions are incubated.

Extraction o f Total R N A Total RNA is prepared essentially according to the acidic guanidiumphenol-chloroform method of Chomczynski and Sacchi9 with slight modification.

Reagents Denaturing solution (solution D): 4 M guanidium thiocyanate, 25 mM sodium citrate (pH 7.0), 0.5% Sarkosyl, 0.1 M 2-mercaptoethanol DEPC-water: Add 0.2 ml DEPC to 100 ml water, shake vigorously, let stand for at least 30 rain, and then autoclave to inactive the remaining DEPC. Water-saturated phenol: Dissolve phenol cyrstals at 68° and saturate with DEPC-water

Procedure 1. Harvest cell suspensions at indicated times and centrifuge for 10 min at 500 g. Remove the supernatant under vacuum. 2. Lyse the cell pellet in 1 ml of denaturating solution per 107 cells. 3. Add 0.1 ml of 2 M sodium acetate (pH 4.0), 1 ml of phenol (water saturated), and 0.2 ml of chloroform-isoamyl alcohol mixture (49:1, v/v) to the solution D homogenate, mixing by inversion after the addition of each reagent. Shake vigorously for 10 sec and cool on ice for 15 min. 4. Centrifuge at 10,000 g for 20 rain at 4 °. Transfer the upper aqueous phase to a new centrifuge tube that has been prechilled on ice. 5. Mix with an equal volume of 2-propanol and place at -20 ° for at least 1 hr to precipitate RNA. 6. Centrifuge at 10,000 g for 20 rain at 4° and discard the supernatant. Dissolve the pelleted RNA in 0.3 ml of solution D and transfer to a 1.5ml Eppendorf tube. 9p. Chomczynskiand N. Sacchi,Anal. Biochem. 162, 156 (1987).

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SUPPRESSION OF

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BY DIHYDROLIPOIC ACID

183

7, Precipitate the R N A with 0.3 ml of 2-propanol at - 2 0 ° for 1 hr. Centrifuge at 10,000 g for 20 rain at ambient temperature and remove the supernatant from the R N A pellet. 8. Wash the R N A pellet with 70% (v/v) ethanol (prepared with D E P C water). Centrifuge 10 rain at 10,000 g and discard ethanol solution, then dry under vacuum for 15 min. Note: R N A is protected from RNase by guanidium thiocyanate prior to Step 8. The R N A is no longer protected now. Take special care that RNase contamination does not occur under any circumstance. 9. Dissolve the R N A in 200/~1 of DEPC-water. Dilute 10/xl of R N A solution to 1 ml and measure the optical density at 260 nm to determine its concentration: 1 OD260 = 40 ~g/ml RNA.

Analysis of RNA Purified R N A is subjected to Northern blot analysis to investigate the effect of D H L A on the expression of the protooncogene c-los when ceils are stimulated with PMA.

Reagents 20× MOPS: 0.4 M 3-(N-morpholino)propanesulfonic acid (MOPS), pH 7.0, 0.I M sodium acetate, 20 mM ethylenediaminetetraacetic acid (EDTA) 20x SSPE: 0.2 M sodium phosphate (pH 7.4), 2.98 M sodium chloride, 20 mM E D T A disodium salt Hybridization buffer: 1 M sodium chloride, 50 mM Tris-HC1 buffer (pH 7.5), 10% dextran sulfate, 1% sodium dodecyl sulfate (SDS), 100 ~g/ml denatured salmon sperm D N A R N A loading buffer: 720/~1 formamide, 80/xl of 20x MOPS, 260/.d formaldehyde, 150/~1 of 50% glycerol, 10 tzl bromphenol blue dye (saturated with DEPC-water), and 280/xl DEPC-water

Procedure 1. Adjust the volume of R N A sample (20 ~g) to 5 ~1 with D E P C water; add 16 ~1RNA loading buffer and I ~1 of 1 mg/ml ethidium bromide. 2. Electrophorese 20 ~g of total R N A in a 1.2% agarose gel containing 0.66 M formaldehyde using 1 × MOPS as running buffer. 3. Transfer the R N A from the gel to a nylon membrane (Gene Screen Plus, N E N - D u Pont, Boston, MA) by upward capillary transfer in 10× SSPE. 4. Place RNA-side down on a UV transilluminator (254 nm wavelength) for 3 rain.

184

[19]

SIGNAL TRANSDUCTION AND GENE REGULATION

5. Label the 40-mer D N A probe for c-fos and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, Oncogene Science, NY) at 5' DNA fragment termini with [y-32p]dATP (ICN Biomedicals, Costa Mesa, CA) using T4 polynucleotide kinase (Amersham, Arlington Heights, IL). 6. Purify radiolabeled D N A probe from unincorporated probe through Sephadex G-25 column. 7. Prehybridize the membrane for at least 2 hr at 65° in hybridization buffer and hybridize in the same solution with 5 x 10 6 t o 10 7 counts/min (cpm) 32p-labeled D N A probe at 65° for 20 to 24 hr. 8. Briefly rinse the membrane four times with 2x SSPE, 0.1% SDS at room temperature, and wash with the same solution prewarmed to 65 ° for 30 min at 65°. 9. Carry out a wash using 2x SSPE, 0.1% SDS for 10 min at room temperature followed by a final incubation in 0.2x SSPE for 5 min at room temperature. 10. Blot excess liquid from the membrane, cover it with plastic wrap, and then expose to X-ray film (Amersham).

Caution: Formamide is a teratogen. Formaldehyde is toxic. Ethidium bromide is a mutagen and potential carcinogen. All operations involving these reagents should be carried out with care. Exposure to UV light is harmful to the eyes and skin. Eyes and skin need protection when using a UV light source.

28S c-fos 18S 28S

18S GADPH 0

15

30

45

60

75

90

120 (min)

FI6. 1. Kinetics of c-fos mRNA expression. Cells were incubated in RPMI 1640 supplemented with 1% FCS for 18 hr with DMSO at a concentration of 2/~l/ml. Thereafter, cells were exposed to 0.5/~M PMA and analyzed at the times indicated. Total RNA was extracted and Northern analyses performed hybridizing with a 32P-labeled c-fos or GAPDH DNA probe.

[19l

SUPPRESSION OF

c-los

185

BY DIHYDROLIPOIC ACID

J 28S

c-los

18S

28S

•~

18S

GAPDH

F]G. 2. Dose-dependent inhibition of c-fos m R N A expression by DHLA. Cells were incubated in the indicated concentration of D H L A in 1% FCS/RPMI 1640 for 18 hr prior to stimulation by 0.5 /zM PMA for another 1 hr. As a control, cells were incubated with the same volume of DMSO solution. Total R N A was extracted and Northern analyses performed hybridizing with a 32p-labeled c-los or G A P D H D N A probe.

Application The kinetics of c-fos accumulation after treatment of cells with PMA was studied as shown in Fig. 1. A rapid and transient increase in c-fos mRNA was observed after PMA stimulation in the absence of DHLA. The level of c-los mRNA became detectable 30 min after the addition of PMA, peaked at 60 min, and returned rapidly to constitutive levels by 2 hr. G A P D H expression, which served as a positive control for a constitutively expressed gene, showed no changes under the different experimental conditions. To investigate whether the onset of c-los mRNA induction by PMA is altered by DHLA, cells were cultured for 18 hr in the presence or absence of D H L A (0.02 or 0.2 raM) before 0.5 /xM PMA was added for another 1 hr (Fig. 2). Suppression of PMA-induced c-fos mRNA levels was already seen at 0.02 mM DHLA. At 0.2 mM DHLA, the level of c-fos mRNA was,

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SIGNAL T R A N S D U C T I O N A N D G E N E R E G U L A T I O N

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further decreased. This experiment suggests that effects of antioxidants can be estimated at the molecular biological level by using the detection of immediate early genes such as c-los mRNA after PMA stimulation. Acknowledgments Research was supported by National Institutes of Health (CA 47597) and ASTA Medica (Germany).

[201 D i h y d r o l i p o a m i d e

By M U L C H A N D

Dehydrogenase:

Activity Assays

N. V E T T A K K O R U M A K A N K A V , and TE-CHUNG LIU

S. P A T E L , N A T A R A J

Introduction Dihydrolipoamide dehydrogenase (DHLipDH; EC 1.8.1.4) catalyzes the reoxidation of the protein-bound dihydrolipoamide moiety as per the reaction shown. Protein-dihydrolipoamide + NAD ÷ .~ protein-lipoamide + N A D H + H ÷ The enzyme has been extensively characterized from a variety of eubacteria, archaebacteria, and eukaryotes, and the properties of the enzyme are similar among members of these groupsJ -3 This flavoprotein disulfide oxidoreductase catalyzes the above reaction as part of the pyruvate, a-ketoglutarate, and branched-chain a-keto acid dehydrogenase multienzyme complexes as well as in the glycine cleavage multienzyme complex.4'5 The dihydrolipoamide acyltransferase component of these a-keto acid dehydrogenase complexes and hydrogen carrier protein (H-protein) component of the glycine cleavage system are the physiological substrates for this enzyme. The enzyme has a redox disulfide and a tightly but noncovalently bound FAD cofactor, both of which participate in the transfer of electrons from dihydrolipoamide to NAD ÷ (reviewed by Williams1'2). The DHLipDH func1 C. H. Williams, Jr., in "Chemistry and Biochemistry of Flavoenzymes" (F. Muller, ed.), Vol. 3, p. 121. CRC Press, Boca Raton, Florida, 1992. 2 C. H. Williams, Jr., in "The Enzymes" (P. D. Boyer, ed.), Vol. 13, 3rd ed., p. 87. Academic Press, New York, 1976. 3 D. J. Carothers, G. Ports, and M. S. Patel, Arch. Biochern. Biophys. 268, 409 (1989). 4 S. J. Yeaman, Biochem. J. 257, 625 (1989). 5 M. S. Patel and T. E. Roche, FASEB J. 4, 3224 (1990).

METHODS IN ENZYMOLOGY,VOL. 252

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tions as a dimer and is inactive as a monomer because the active site is composed of amino acid residues from both subunits. 1'2 The reaction catalyzed by DHLipDH is a Ping-Pong Bi-Bi mechanism that can be divided into two half-reactions: (i) the reduction of the oxidized enzyme [E] to the reduced form [EH2] and (ii) the transfer of electrons from the [EH2] form to the nicotinamide cofactor.6'7 Crucial to the characterization of this flavoenzyme has been the ease with which the enzyme can be assayed in the forward and backward reactions as well as by electron transfer to electron-accepting dyes. The simplest assay procedure is monitoring the NAD+-dependent oxidation of free OLdihydrolipoamide. Dihydrolipoamide + NAD ÷ ,~ lipoamide + NADH + H + The aim of this chapter is to describe the commonly used procedures to monitor the enzyme activity, both from prokaryotic cells as well as from eukaryotic cells. The forward, or physiological, reaction is widely used to monitor the activity of DHLipDH and is described in detail. We first describe the preparation of crude cell exracts to assay DHLipDH activities from prokaryotic and eucaryotic cells, followed by the actual assay procedures.

Preparation of Crude Cell Extracts

Bacterial Cell Extracts Bacterial cell extracts can be prepared for detection of DHLipDH activity by any of the routine cell lysis procedures. These include lysis by osmotic shock, which is done by suspending frozen cells in 50 mM potassium phosphate (pH 7.4) and subsequent treatment with DNase I to reduce the viscosity of the preparation (Haloferax volcanii), s lysis by sonication (6 to 10 30-sec cycles in an ice-cooled sonication cell at 50% pulse efficiency),9 and lysis by French press treatment (Escherichia coli). After the bacterial cells have been lysed, the preparation is clarified by centrifugation at 6000 g for 10 min to remove cell debris and cells that 6 V. Massey, Q. H. Gibson, and C. Veeger, Biochem. J. 77, 341 (1960). 7 V. Massey and C. Veeger, Biochim. Biophys. Acta 48, 33 (1961). s N. N. Vettakkorumakankav, M. J, Danson, D. W. Hough, J. A. Young, M. Davison, and K. J. Stevenson, Biochem. Cell Biol. 70, 70 (1992). 9 S. R. Adamson and K. J. Stevenson, Biochemistry 20, 3418 (1981).

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[20]

escaped lysis. The crude extract at this stage can be used for the assay of D H L i p D H activity.

Eukaryotic Cell Preparations If membranes or eukaryotic cell organelles are to be assayed for D H L i p D H activity, the enzyme must be solubilized by treatment with a detergent or sonication before activity can be assayed. Cells (cultured) are washed three times in phosphate-buffered saline (PBS; 130 mM NaC1, 10 mM Na2HPO4, 10 mM NaH2PO4, pH 7.4) and resuspended in hypotonic extraction buffer (20 mM potassium phosphate, pH 7.4; 1% (w/v) Triton X-100) containing 2 mM EDTA, 2mM EGTA, and protease inhibitors [0.2 mM phenylmethylsulfonyl fluoride (PMSF), 0.5 tzg/ml leupeptin, 0.7/zg/ ml pepstatin]. The cell suspensions are incubated on ice for 30 min, subjected to three cycles of freezing and thawing, and finally placed on ice for a further 30 rain to ensure complete solubilization of the enzyme. The whole cell extracts can be used for the determination of D H L i p D H activity. Mitochondrial extracts for the assay of D H L i p D H activity are prepared as follows. The tissues are minced and homogenized (10%, w/v) in a buffer containing 70 mM sucrose, 220 mM mannitol, 2 mM HEPES, and 1 mM EDTA, pH 7.4 (for preparation of mitochondria from liver cells), or 250 mM sucrose, 10 mM Tris, 1 mM 2-mercaptoethanol, pH 7.4, for the preparation of mitochondria from kidney or heart cells. 1° The homogenization of the tissue is performed by six or seven passes through a motor-driven Fisher Dynamix homogenizer. Unbroken cells and nuclei are removed by centrifugation at 650 g for 10 min and the pellet discarded. The floating lipid on the surface of the supernatant may be removed by pipetting. The supernatant is then centrifuged at 12,000 g at 4° for 10 rain to sediment the mitochondria. The mitochondrial pellet is resuspended in half the original volume of homogenization buffer. The suspension is passed once through a hand-held homogenizer and the preparation centrifuged at 12,000 g for 10 rain. The pellet is resuspended in one-fourth the original volume of the homogenization buffer and centrifuged again. The pellet is finally resuspended in phosphate-buffered saline containing the protease inhibitors described above (the amount of solution used to resuspend the pellet is 1 ml/g of tissue used). Aliquots (1 ml) can be stored frozen at - 7 0 ° until required. When required for enzyme assays, the aliquots are thawed and extracted once in the hypotonic extraction buffer with protease inhibitors as described for cell extracts or sonicated to ensure complete lysis. The extract is clarified by centrifugation at 20,000 g for 30 min at 4°. The extract at this stage may be used for activity assays. 1° 10 D. Carothers, Ph.D. Thesis, Case Western Reserve University, Cleveland, Ohio (1987).

[20]

DIHYDROLIPOAMIDE DEHYDROGENASE

189

Preparation o f Cultured Skin Fibroblasts f r o m Patients Deficient in E n z y m e Activity

Control and patient skin fibroblasts are normally cultured in minimum essential medium supplemented with 20% (v/v) fetal calf serum, 100 Ixg/ ml streptomycin, 60 tzg/ml penicillin, and 1.5/zg/ml amphotericin B Y The cells are harvested and lysed by resuspension in phosphate-buffered saline containing 0.5% Triton X-100 and sonicationJ 2

Purification of D i h y d r o l i p o a m i d e D e h y d r o g e n a s e The purification of D H L i p D H from several sources has been reported. General purification strategies include ammonium sulfate fractionation of crude extracts, heat treatment, and chromatography on a hydroxyapatite matrix. 13 The aforementioned procedure has been modified and used to purify the enzyme from several other sources, 14'15including some extremophiles, s Because D H L i p D H is extremely heat stable, a heat treatment step may be routinely included in the purification strategy. The yield of purified enzyme from most sources is approximately 30%. The specific activity of the purified human enzyme 14 is 407 units/rag protein.

S y n t h e s i s of D i h y d r o l i p o a m i d e T o a chilled suspension of DL-lipoamide (200 mg in 4 ml of methanol and 1 ml of water), 1 ml of a cold solution of sodium borohydride (200 mg/ml) is added and stirred on ice for 1 hr. The reaction is acidified to p H 1-2 with 1 M HCI and extracted three times with chloroform. The extracts are pooled and evaporated to dryness on a rotary evaporator. The resulting white residue is resuspended in 10 ml benzene, and 4 ml n-hexane is added. The solution is set on ice until white precipitates are formed. The precipitates are filtered on a Whatman 3MM paper and dried under vacuum. The melting point of DL-dihydrolipoamide is between 50° and 55 °. The reduction of lipoamide to dihydrolipoamide is not complete, and therefore the reagent may be separated from the oxidized form by high performance liquid chromatography (HPLC) on a 0.39 × 15 cm Waters (Milford, MA) C18, Pico-Tag column. The solvent system used is 10 m M u L. Ho, C.-W. C. Hu, S. Packman, and M. S. Patel, J. Clin. Invest 78, 844 (1986). LaT,-C. Liu, H. Kim, C. Arizmendi, A. Kitano, and M. S. Patel, Proc. Natl. Acad. Sci. U.S.A. 90, 5186 (1993). 13C. H. Williams, Jr., J. Biol. Chem. 240, 4793 (1965). 14H. Kim, T.-C. Liu, and M. S. Patel, J. BioL Chem. 2.66, 9367 (1991) 15H. Kim and M. S. Patel, J. Biol. Chem. 267, 5128 (1992).

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[201

sodium acetate, 30/zM EDTA, pH 6.3 (buffer A), and acetonitrile containing 30/zM E D T A (buffer B). The dihydrolipoamide is separated from the lipoamide by a linear gradient of 100% buffer A to 50% buffer B, and the elution of the compounds is monitored at 214 rim. The flow rate is 1 ml/min, and the gradient is established over 50 min (L. David Arscott and C. H. Williams, Jr., personal communication, 1993). The purified, dried dihydrolipoamide may be stored at - 2 0 ° until neededJ 6 Determination of Enzymatic Activity The forward reaction catalyzed by D H L i p D H is the oxidation of dihydrolipoamide and the reduction of NAD +. This can be monitored spectrophotometrically at 340 nm. Different concentrations of dihydrolipoamide have been used by various laboratories for routine assays of the forward reaction. The following final concentrations of various components produce satisfactory activities in our laboratory: 100 mM potassium phosphate, pH 8.0, 1.5 mM EDTA, 3.0 mM DL-dihydrolipoamide, 3.0 mM NAD ÷. Dihydrolipoamide solution is prepared in 95% (v/v) ethanol and appropriately diluted in the final reaction mixture to achieve the final concentration of 3 raM. Typically, 120 mM solutions of both dihydrolipoamide and N A D ÷ are prepared, and 25/zl of each is used in the assay (total volume of I ml). It is noted that in crude cell extracts, the presence of alcohol dehydrogenase can interfere with D H L i p D H activity assays. To avoid such an interference, a solution of dihydrolipoamide dissolved in acetone can be usd. 17 Several laboratories have used lower concentrations of dihydrolipoamideY 8 usually around 0.4 raM. We have found, however, that maximum enzyme activity can be realized on using higher dihydrolipoamide concentrations specified aboveJ 9 The reaction is initiated by the addition of the enzyme preparation. The progress of the reaction is monitored by following the increase in absorbance at 340 nm. An extinction coefficient for N A D H of 6.22 × 103 M -I cm -~ is used in the calculation of enzyme activity. The product of the forward reaction, NADH, is known to inhibit D H L i p D H J '2° Therefore, it is important that only the initial rates are used for determining enzyme activity, as product inhibition is apparent about 1.5 rain after initiation of the reaction. It is important to determine experimentally the appropriate 16 L. J. Reed, M. Koike, M. E. Levitch, and F. R. Leach, J. Biol. Chem. 232, 143 (1958). 17j. R. Sokatch, V. McCuUy, and C. M. Roberts, I. Bacteriol. 148, 647 (1981). 18M. J. Danson and R. N. Perham, Bioehern. J. 159, 677 (1976). 19 H. Kim, Ph.D. Thesis, Case Western Reserve University, Cleveland, Ohio (1992). 20 L. P. Hager and I. C. Gunsalus, J. A m . Chem. Soc. 75, 5767 (1953).

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concentrations of enzyme in the assay mixture, where the change in absorbance at 340 nm is linear over a period of 2-3 min and within 0.050.15 absorbance units. T o minimize the inaccuracies resulting from the inhibition of D H L i p D H activity by N A D H , an N A D + analog, acetylpyridine adenine dinucleotide, can be used to monitor the enzyme activity by following increase in absorbance at 363 nm. 13 The original assay using this compound, as reported by Williams 13 is described below. In a total reaction volume of 1 ml are mixed 50 m M sodium phosphate, p H 7.6, 1 mg bovine serum albumin, 1 m M E D T A , 0.9 m M acetylpyridine adenine dinucleotide, and 0.1 m M dihydrolipoamide. The assay is initiated with the addition of an enzyme preparation. An extinction coefficient of 9.1 × 103 M -1 cm -~ for reduced acetylpyridine adenine dinucleotide is used for calculations of enzyme activity. 21 A n o t h e r concern that has been reiterated is the fact that the dihydrolipoamide used in the enzymatic assays is a mixture of the D and L forms. Only the L- or the (R)-enantiomer is the natural substrate for the enzyme, and biphasic kinetics are observed using the mixture of substrates. 22 Therefore it is necessary to interpret kinetic parameters cautiously when using this mixture. One unit of enzyme activity is defined as the amount of enzyme required for the production of 1/zmol of N A D H or reduced acetylpyridine adenine dinucleotide in 1 min at 25 °. The specific acivity is expressed as units per milligram protein or per mole ravin cofactor (there is 1 mol F A D bound per mole of the subunit enzyme). The amount of enzyme-bound F A D can be quantitated from the absorption at 455 nm using an extinction coefficient of 11,300 M -a cm -~ for enzyme-bound F A D J 3 The D H L i p D H activity (expressed as units/mg protein) from various tissues using the dihydrolipoamide/NAD + assay are illustrated in Table I to provide the reader with a ready source of comparison.

Reverse R e a c t i o n The reverse reaction catalyzed by D H L i p D H is the reduction of lipoamide with N A D H as the reducing agent. The progress of the reaction can be monitored by the disappearance of N A D H at 340 nm. This assay has been previously described and is less common than the assay in the physiological direction. 6 The problem with this assay, especially with the E. coli D H L i p D H , is that N A D H inhibits D H L i p D H at high concentrations, as 2t j. M. Siegel, G. A. Montgomery, and R. M. Bock, Arch. Biochern. Biophys. 82, 288 (1959). 22L. D. Arscott and C. H. Williams, Jr., in "Flavins and Flavoproteins" (K. Yagi, ed.), p. 527. W. deGruyter, New York, 1994.

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SIGNAL TRANSDUCTION AND GENE REGULATION

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TABLE I SPECIFIC ACTIVITIES OF DIHYDROLIPOAMIDE DEHYDROGENASE IN CELLULAR PREPARATIONS FROM DIFFERENT SPECIES AS MONITORED BY FORWARD REACTION

Species Rat

Haloferax volcanii Escherichia coli

Pig heart Bovine liver Human liver Human enzyme (recombinant)

Preparation

Specific activity (units/mg protein)

Heart (homogenate) Kidney (homogenate) Skeletal (homogenate) Brain (homogenate) Crude extract Purified enzyme Purified enzyme Crude extract Crude extract Purified enzyme Crude extract Purified enzyme Purified enzyme

0.723 0.589 0.161 0.287 0.1 16.1 120.0 1.33 0.066 70.4 0.096 76.0 407.0

Ref. a

8 13 b c d 14

a N. N. Vettakkorumakankav and M. S. Patel, unpublished observations, 1993. b T. Hayakawa, M. Hirashima, S. Ide, M. Hamada, K. Okabe, and M. Koike, J. Biol. Chem. 241, 4694 (1966). c C. J. Lusty, J. Biol. Chem. 238, 3443 (1963). d S. Ide, T, Hayakawa, K. Okabe, and M. Koike, J. Biol. Chem. 242, 54 (1967). it c a n c o n v e r t t h e e n z y m e to t h e i n a c t i v e E H 4 f o r m w h i c h is a c c o m p a n i e d b y a p o s s i b l e d i m e r to m o n o m e r t r a n s i t i o n . 23,24

Diaphorase Activity T h e N A D H - 2 , 6 - d i c h l o r o p h e n o l i n d o p h e n o l d i a p h o r a s e activity can b e m o n i t o r e d b y t h e d i s a p p e a r a n c e of 600 n m a b s o r b a n c e , i n d i c a t i v e of t h e r e d u c t i o n o f t h e d y e , a n d has b e e n p r e v i o u s l y d e s c r i b e d . 13 T h e d e t a i l s o f this a s s a y a r e as follows. T h e final v o l u m e o f t h e r e a c t i o n is 3 ml, a n d t h e a s s a y c o n t a i n s 50 m M s o d i u m p h o s p h a t e , p H 7.2, 2 m g b o v i n e s e r u m a l b u m i n , i m M E D T A , 4 0 / x M 2 , 6 - d i c h l o r o p h e n o l i n d o p h e n o l , a n d 0.2 m M N A D H . T h e a s s a y is i n i t i a t e d b y t h e a d d i t i o n o f t h e e n z y m e p r e p a r a t i o n a n d t h e c h a n g e in a b s o r b a n c e at 600 n m f o l l o w e d at 25 °. A n e x t i n c t i o n coefficient for t h e o x i d i z e d d y e o f 21 x 103 M -I c m -I is u s e d for t h e calculations of enzyme activityY 23j. F. Kalse and C. Veeger, Biochim. Biophys. Acta 159, 244 (1968). 24j. Visser and C. Veeger, Biochim. Biophys. Acta 159, 265 (1968). 25E. P. Steyn-Parve and H. Beinert, Z Biol. Chem. 233, 843 (1958).

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P r o p e r t i e s of Purified E n z y m e The purified D H L i p D H has been extensively characterized from several sources. 1 The properties of all the enzymes are somewhat similar, with an enzyme subunit molecular mass of approximately 50,000 daltons. The oxidized enzyme shows the characteristic absorption spectrum of free F A D with pronounced shoulders at 480 and 430 nm because F A D is associated with the protein. The ratio of the peak at 273 nm to that at 455 nm, which is an excellent indicator of the purity of the preparation, is 5.3.~ The catalytic mechanism of all the enzymes are similar in that they follow a Ping-Pong mechanism. The reaction, as mentioned before, can be divided into two half-reactions (i) the reductive half-reaction where the oxidized enzyme is reduced to the two-electron form and (ii) the oxidative half-reaction where the two electrons are passed to the nicotinamide cofactor, N A D +. The rates of the individual half-reactions can be measured in a stopped-flow spectrophotometer by following absorbance changes at 530 nm (the characteristic absorbance of the two-electron form with a charge transfer between the thiolate anion and the F A D cofactor). 6,7 Dihydrolipoamidedehydrogenase is extremely stable to a wide variety of denaturants including urea. It is, however, very sensitive to high concentrations of guanidinium hydrochloride, as the presence of that denaturant leads to the dissociation of the F A D cofactor from the enzyme. It is also remarkably resistant to the action of several proteases owing to the compactly folded globular structure. 26 The enzyme is extremely heat stable (in fact, heat treatment has been used as a purification step), being stable for at least 5 min at 75°. 15 The optimum p H for D H L i p D H activity is p H 8 for the physiological reaction. 13 The enzyme is sensitive to arsenite, copper, and cadmium (after reduction with N A D H or dihydrolipoamide) 1,27 and to copper without prior reduction. 2s Trivalent arsenicals have been used extensively as probes for the presence of vicinal thiol groups (formed as a result of reduction of the enzyme with N A D H or dihydrolipoamide). ~,9 The enzyme is also sensitive to other reagents that modify cysteines in proteins (e.g., iodoacetamide) because of the indispensible role played by the redox disulfide in catalysis. 1 Finally, D H L i p D H has been characterized extensively, and the steady-state kinetic parameters have been determined (Table II). The primary structure of D H L i p D H has been deduced from the nucleotide sequence of the cloned genes from a variety of different sources includ-

26R. G, Mathews, L. D. Arscott, and C. H. Williams,Jr., Biochim. Biophys. Acta 370,26 (1974). 2~S. Ide, T. Hayakawa, K. Okabe, and M. Koike, J. Biol. Chem. 242, 54 (1967). 28C. Thorpe and C. H, Williams, Jr., Biochemistry 14, 2419 (1975).

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TABLE II STEADY-STATEKINE37CPARAMETERSOF DIHYDROL1POAMIDEDEHYDROGENASEFROM DIFFERENT SOURCES

Source

Parameter

Value

Ref.

Azotobacter vinelandii

Km (dihydrolipoamide) Km (NAD +) Turnover number~

b

Rat liver

Km (dihydrolipoamide)

390/xM 325/xM 420 sec-1 490/xM 520 heM 345 sec-1 300/zM 200/.~M 550 sec-~ 690/xM 320 gM 421 sec-1

Pig heart

Human (recombinant)

Km (NAD +) Turnover number Km (dihydrolipoamide) Krn (NAD ÷) Turnover number K~ (dihydrolipoamide) Km (NAD ÷) Turnover number

1

1

15

a The turnover numbers were obtained under conditions of maximum velocity. b j. Benen, W. V. Berkel, N. Dieteren, D. Arscott, C. H. Williams, Jr., C. Veeger, and A. DeKok, Eur. Z Biochem. 207, 487 (1992).

ing t h e e u b a c t e r i a E. coli 29 a n d A z o t o b a c t e r vinelandii3°; the a r c h a e b a c t e r i u m Haloferax volcanii31; a n d f r o m m a m m a l s (e.g., p i g h e a r t , 3z human33). T h e r e a r e e x t e n s i v e s e q u e n c e similarities a m o n g t h e e n z y m e s , 1'3 e s p e c i a l l y in t h e n u c l e o t i d e c o f a c t o r b i n d i n g r e g i o n s a n d in t h e active site region. T h e h i g h - r e s o l u t i o n 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 s of t h e e n z y m e f r o m A z o t o b a c ter vinelandii 34 a n d P s e u d o m o n a s putida 35 a r e a v a i l a b l e , a n d , again, t h e r e a r e e x t e n s i v e s i m i l a r i t i e s in t h e o v e r a l l t e r t i a r y s t r u c t u r e of t h e e n z y m e f r o m t h e s e sources.

Summary We have described the most commonly used assay procedures determin a t i o n o f t h e D H L i p D H activities f r o m p r o k a r y o t i c a n d e u k a r y o t i c cells. W e h a v e also d e s c r i b e d t h e p r o c e d u r e s for t h e p r e p a r a t i o n o f tissue e x t r a c t s 29p. E. Stephens, H. M. Lewis, M. G. Darlison, and J. R. Guest, Eur. J. Biochem. 135, 519 (1983). 3oA. H. Westphal and A. DeKok, Eur. J. Biochem. 172, 299 (1988). 31 N. N. Vettakkorumakankav and K. J. Stevenson, Biochem, Cell Biol. 70, 656 (1992). 32 G. Otulakowski and B, H. Robinson, J. Biol. Chem. 262, 17313 (1987). 33G. Pons, C. Raefsky-Estrin, D. J. Carothers, R. A. Pepin, A. A. Javed, B. W. Jesse, M. K. Ganapathi, D. Samols, and M. S. Patel, Proc. Natl. Acad. Sci. U.S.A. 85, 1422 (1988). 34A. Mattevi, A. J. Schierbeek, and W. G. J. Hol, J. Mol. Biol. 220, 975 (1991). 3s A. Mattevi, G. Obmolova, J. R. Sokatch, C. Betzel, and W. G. J. Hol, Proteins; Struct. Funct. Genet. 13, 336 (1992).

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to determine the enzymatic activity. We have briefly alluded to the problems inherent in these assay procedures and suggested some precautions for using the assays to determine D H L i p D H activity. W e have also included a statement about the purification procedures and described some of the properties of this enzyme, which might help the reader in planning studies. We have, by no means, a t t e m p t e d to be comprehensive in this regard; therefore, the reader should consult the appropriate references (and references therein). Acknowledgments The authors express gratitude to Prof. Charles H. Williams, Jr., University of Michigan, and Dr. Gail Willsky of the Department of Biochemistry, State University of New York at Buffalo, for critical reading of the manuscript and for helpful suggestions. Work described in this chapter is supported in part by U.S. Public Health Service Grant DK42885.

[2 1]

THIOREDOXIN

AND THIOREDOXIN

REDUCTASE

199

[21] T h i o r e d o x i n a n d T h i o r e d o x i n R e d u c t a s e By ARNE HOLMGREN and MIKAEL BJORNSTEDT Introduction Thioredoxin (Trx) is a small (Mr 12,000) multifunctional and ubiquitous protein characterized by having a redox-active disulfide/dithiol within the conserved active site sequence: -Trp-Cys-Gly-Pro-Cys-. 1-4 Oxidized thioredoxin (Trx-S2) has a disulfide, and reduced thioredoxin [Trx-(SH)2] has a dithiol. This chapter summarizes current methods to determine Trx and thioredoxin reductase (TR). Elsewhere in this volume the genetics of Trx is summarized by Russel 5 and structural studies of Trx in solution by nuclear magnetic resonance ( N M R ) are described by Dyson. 6 A previous volume in this series contains a chapter by H o l m g r e n 7 on the use of N A D P H , TR, and Trx for specific reduction of disulfide bonds in proteins. Buchanan and co-workers s review aspects of the control of photosynthetic enzymes by specific chloroplast thioredoxins and the enzyme ferredoxin-thioredoxin reductase. Thioredoxin reductase specifically reduces Trx-S2 to Trx-(SH)2 using N A D P H 1-3 [reaction (1)]. The Trx-(SH)2 f o r m is a powerful protein disulfide reductase [reaction (2)]. Thus, Trx, TR, and N A D P H , collectively called the thioredoxin system, operate as a powerful N A D P H - d e p e n d e n t protein disulfide reductase system. Note that the redox potential (E~ value) TR

Trx-S2 + N A D P H + H + .

-' Trx-(SH)2 + N A D P *

(1)

spontaneous

Trx-(SH)2 + protein-S2 ~

Trx-S2 + protein-(SH)2

(2)

of H + + N A D P H / N A D P + at p H 7.0 and 25 ° is - 3 1 5 m V and that the E{~ of E s c h e r i c h i a coli Trx-(SH)z/Trx-S2 is - 2 7 0 inV. 1-3 Reaction (1), which I A. Holmgren, Annu. Rev. Biochem. 54, 237 (1985). 2 A. Holmgren, J. Biol. Chem. 264, 13963 (1989). 3 F. K. Gleason and A. Holmgren, FEMS Microbiol. Rev. 54, 271 (1988). 4 H. Eklung, F. K. Gleason, and A. Holmgren, Proteins 2, 13 (1991). 5 M. Russel, this volume [27]. 6 H. J. Dyson, this volume [30]. 7 A. Holmgren, this series, Vol. 107, p. 295. s j. H. Wong, K. Kobrehel, and B. B. Buchanan, this volume [24].

METHODS IN ENZYMOLOGY, VOL. 252

Copyright © 1995 by Academic Press, Inc All rights of reproduction in any form reserved.

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THIOREDOXINAND GLUTAREDOXIN

[2 1]

is fully reversible, may thus be used to determine the E6 value of a particular Trx. For example, in Escherichia coli Trx the mutation of Pro-34 to His changed the E~ value of Trx from -270 to -235 i n V . 9 Thioredoxin has been isolated and characterized from a wide variety of prokaryotic and eukaryotic species. 1-4 Mammalian thioredoxins show about 25% sequence identity to the well-characterized E. coli protein with 108 residues. <6 In particular, mammalian thioredoxins with 104 or 105 residues contain two conserved half-cysteine residues in addition to the two redox-active residues located in the classic active site. The human Trx contains three such extra cysteine residues. These structural or noncatalytic half-cystine residues, Cys-61, Cys-68, and Cys-72 in human Trx, can undergo oxidation, which leads to inactivation and dimerization. 1° This is a process of specific interest in reactions of the Trx system with selenite and selenodiglutathione (GS-Se-SG) that is treated elsewhere in this volume by Bj/~rnstedt et al. 11 Redox regulation of human Trx is linked to Cys-72 in Trx; this residue is exposed at a hydrophobic surface area located at the active site. 4'12 One classic function of the thioredoxin system is to act as a hydrogen donor for ribonucleotide reductase, which is essential for DNA synthesis.13 That enzyme contains pairs of redox-active disulfides which are oxidized in the reaction. 13 In addition, thioredoxin can regulate different systems via thiol redox control. ~-3'14 Redox control processes involve changes in the activity of an enzyme, a receptor, or a transcription factor via dithiol/ disulfide interchange reactions, m'14 Thioredoxin is secreted from human virus-transformed ~5 as well as normal lymphocytes,16 and the expression increases on antigen stimulation of lymphocytes. Adult T-cell leukemiaderived factor (ADF) produced by both human T-lymphotropic virus type 1 (HTLV-1) 17 and Epstein-Barr virus-transformed B lymphocytes~s 9 G. Krause, J. Lundstr6m, J. Lopez-Barea, C. Pueyo de La Cuesta, and A. Holmgren, J. Biol. Chem. 266, 9494 (1991). 10X. Ren, M. Bjrrnstedt, B. Shen, M.-L. Ericson, and A. Holmgren, Biochemistry 32, 9701 (1993). 11 M. Bj6rnstedt, S. Kumar, and A. Holmgren, this volume [22]. 12j. D. Forman-Kay, G. M. Clore, P, T. Wingfield, and A. M, Gronenborn, Biochemistry 30, 2685 (1991). 13 p. Reichard, Science 260, 1773 (1993). 14A. Holmgren, Trends Biochem. Sci. 6, 26 (198l). 15j. Yodoi and T. Tursz, Adv. Cancer Res. 57, 381 (1991). 16M.-L. Ericson, J. Hrrling, V. Wendel-Hansen, A, Holmgren, and A. Rosrn, Lymphokine Cytokine Res. 5, 201 (1992). 17y . Tagaya, Y. Maeda, A. Mitsui, N. Kondo, H. Matsui, J. Hamuro, N. Brown, K.-I. Arai, T. Yokota, H. Wakasugi, and J. Yodoi, E M B O Z 8~ 757 (1989). 18N. Wakasugi, Y. Tagaya, H. Wakasugi, A. Mitsui, M. Maeda, J. Yodoi, and T. Tursz, Proc. Natl. Acad. Sci. U.S.A. 87, 8282 (1990).

[21]

THIOREDOXIN AND THIOREDOXIN REDUCTASE

20l

has been identified as a human thioredoxin. Thioredoxin domains in protein disulfide-isomerase (PDI) and other luminal endoplasmic reticulum proteins have been shown to be the catalytically active site for native disulfide formation during protein folding. 19 The E; value of the active site dithiols/disulfides in PDI is much higher, -185 + 10 mV compared to -270 mV for wild-type Trx. z° A factor in this is the active site exchange of Pro to His (see above) which contributes -35 inV. The structural basis of the residual - 5 0 mV is not yet explained. Other Trx domains in larger proteins have been described by Hennecke in functions relating to regulation of genes for nitrogen fixation in Rhizobium strains. 21.22 Thioredoxin reductase is a FAD-containing enzyme with general structural and functional similarities to glutathione reductase and lipoamide dehydrogenase.2'23 The E. coli enzyme has an Mr of 70,000 and is a dimer; the crystal structure of this enzyme been refined to 2.0 ~ resolution, z425 The enzyme mechanism involves the transfer of reducing equivalents from NADPH to a disulfide bond in the enzyme within the sequence -Cys-AlaThr-Cys-, via FAD. The TR subunit contains FAD and NADPH binding domains with relative orientations different from that of glutathione reductaseY The redox-active disulfide in E. coli TR is within the NADPH domain. Results suggest that TR must undergo a large conformational change during enzyme catalysis in order for Trx-S2 to be reduced by the dithiol of the reduced enzyme. Thus, a large-scale rotation of the NADPH domain may be a unique part of the mechanism for TRY Cloning of TR from E. coli 26 has formed the basis of overexpression and site-directed mutagenesis experiments. Escherichia coli TR is specific for its homologous Trx-S2 but also shows reactivity with other prokaryotic Trx-S2 molecules, such as that from Anabaena. 2-4,27 In contrast, mammalian TR is larger (M, 116,000) and will reduce Trx-S2 from other species as well as low molecular weight compounds, for example, selenite, II GS-Se-SG, II alloxan, 28 vitamin ~ J. Lundstr6m and A. Holmgren, J. Biol. Chem. 265, 9114 (1990). 2o j. Lundstr6m and A. Holmgren, Biochemistry 32, 6649 (1993). 21 H. Loferer, M. Bott, and H. Hennecke, EMBO J. 12, 3373 (1993). 22 H. Loferer and H. Hennecke, Trends Biochem. Sci. 19, 169 (1994). 23 C. H. Williams, Jr., "Chemistry of Flavoenzymes" (F. Miiller, ed.), Vol. 3, p. 121. CRC Inc., Boca Raton FL. 1992. 24 j. Kuriyan, T. S. R. Krishna, L. Wong, B. Guenther, A. Pah[er, C. H. Williams, Jr., and P. Model, Nature (London) 352, 172 (1991). 25 G. Waksman, T. S. R. Krishna, C. H. Williams, Jr., and J. Kuriyan, J. Mol. Biol. 236, 800 (1994). 26 M. Russel and P. Model, J. Biol. Chem. 263, 9015 (1988). 27 M. Nikkola, F. K. Gleason, J. A. Fuchs, and H. Eklund, Biochemistry 32, 5093 (1993). 2s A. Holmgren and C. Lyckeborg, Proc. Natl. Acad. Sci. U.S.A. 77, 5149 (1980).

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THIOREDOXINAND GLUTAREDOXIN

[2 1]

K, a and 5,5'-dithiobis (2-nitrobenzoic acid) (DTNB), 29 but not oxidized glutathione (GSSG). T h i o r e d o x i n a n d T h i o r e d o x i n R e d u c t a s e f r o m E s c h e r i c h i a coli Thioredoxin f r o m E. coli was originally isolated as a hydrogen donor for ribonucleotide reductase. I It is the best characterized Trx protein, with a three-dimensional structure determined to high resolution by both crystallography 3° and NMR. 6 The E. coli Trx structure contains a twisted/3 sheet c o m p o s e d of five strands (~31-fl5) flanked by four a helices (al--Og4). 1'4 In Trx-S2 and Trx-(SH)2 the active center disulfide/dithiol is located in a loop making a unique protrusion of the three-dimensional structure between a /3 strand (/32) and the long a2 helix. 3° F r o m the similarity of primary structures, all cellular thioredoxins have this c o m m o n fold. 4 A p a r t from being a potent reductant of protein disulfides, 7 Trx has also been shown to take part in the reduction of sulfate (3'-phosphoadenosine 5'-phosphosulfate; P A P S ) 3l and both free and peptide-bound methionine sulfoxideJ 2 Escherichia coli Trx is required for the assembly of the filamentous viruses f 1 and M13. 33'34Furthermore, reduced E. coli Trx is an essential subunit of phage T7 D N A p o l y m e r a s e Y Escherichia coli B Trx is purified as described by H o l m g r e n and Reichard. 36 Escherichia coli Trx and TR, as well as glutathione reductase and lipoamide dehydrogenase, can be purified from the same bacterial extract. 37 Thioredoxin has been isolated by i m m u n o s o r b e n t chromatography, as which has been combined with 2 ' , 5 ' - A D P - S e p h a r o s e affinity chromatography to isolate TR. 39 Many recombinant expression systems for E. coli Trx are available. A useful system for high-level expression is the plasmid pBHKS, which has also been used for 15N-labeling of Trx for multidimensional N M R . 4° Escherichia coli Trx as well as T R and corresponding antibodies are commercially available f r o m I M C O (S-10031 Stockholm, Sweden). e9A. Holmgren, J. Biol. Chem. 252, 4600 (1977). 30S. K. Katti, D. M. LeMaster, and H. Eklund, J. Mol. Biol. 212, 167 (1990). 31M. L.-S. Tsang, J. Bacteriol. 146, 1059 (1981). 32N. Brot, L. Weissbach, J. Werth, and H. Weissbach, Proc. Natl. Acad. Sci. U.S.A. 78, 2155 (1981). 33M. Russel and P. Model, Proc. Natl. Acad. Sci. U.S.A. 82, 29 (1985). 3~C.-J. Lim, B. Haller, and J. A. Fuchs, J. Bacteriol. 161, 799 (1985). 35H. E. Huber, S. Tabor, and C. C. Richardson, J. Biol. Chem. 262, 16224 (1987). a6A. Holmgren and P. Reichard, Eur. J. Biochem. 2, 187 (1967). 37C. H. Williams, G. Lanetti, L. D. Arscott and J. K. McAllister,J. Biol. Chem. 242, 5226 (1967). 3s B.-M. Sj6berg and A. Holmgren, Biochim. Biophys. Acta 315, 176 (1973). 39V. P. Pigiet and R. R. Conley, J. Biol. Chem. 252, 6367 (1977). 40K. Chandrasekhar, G. Krause, A. Holmgren, and H. J. Dyson, FEBS Lett. 284, 178 (1991).

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203

Purification of Mammalian Thioredoxin a n d Thioredoxin Reductase The first steps in the purification of mammalian Trx and TR are common. 41,42 Compounds that interfere in the assay systems, mainly reduced glutathione (GSH) or other low molecular weight thiols, are removed by the initial batch steps. The tissue (rat liver, calf thymus, or human placenta) is homogenized in 50 ram Tris-HCl, pH 7.5, I mM EDTA (TE buffer) and centrifuged. The supernatant is adjusted to pH 5.0 with continous stirring by dropwise addition of ice-cold 1.0 M acetic acid. The precipitate formed is removed by centrifugation and the supernatant adjusted to pH 7.5 with 1.0 M NH4OH. Dilute the neutralized supernatant to 15 mg protein/ml with TE buffer and heat to 60° in 1-1iter batches under continous stirring in a boiling water bath. This is a modification of earlier methods using 65 ° as a report shows much improved recovery of the human enzyme at 60°.43 The solution is then rapidly cooled to 4° in an ice bath with continuous stirring and the precipitate removed by centrifugation. Add ammonium sulfate to 85% saturation and collect the precipitate by centrifugation. The precipitate is dissolved in a small volume of TE buffer and dialyzed extensively against the same buffer before chromatography on DEAE-celIulose. Thioredoxin reductase activity, tested by the DTNB assay described below, is eluted in a main peak around 0.13 M NaCl. Thioredoxin activity is located around 0.15 M NaCl. Depending on the starting material, the relative separation of Trx and TR varies, and the samples may either be pooled or a separate TR portion made.

Sephadex G-50 and CM-Cellulose Chromatography of Thioredoxin To avoid aggregation of thioredoxin and the apperance of multiple peaks, the concentrated pool from the DEAE-cellulose chromatography is incubated with excess dithiothreitol (DTT, 2mM) under N2, for 30 min at room temperature. The reagent may also be included in the elution buffers (1 mM DTT) but will interefere with the assays. The reduced protein is applied to a column of Sephadex G-50 equilibrated with TE-buffer or 60 mM NFLHCO3; the latter is useful for lyophilization. The Trx and TR are well separated. Fractions containing Trx are pooled, lyophilized, and dissolved in a small volume (5 ml) of water, adjusted to pH 7 with 0.1 M NHaOH. After incubation with excess D T r (2 mM) for 30 min in room temperature under N2, 0.1 M sodium acetate, pH 5.16, is added to a final 41N.-E. EngstrOm,A. Holmgren,A. Larsson,and S. Stiderhhll,Z BioL Chem. 249,205 (1974). 42M. Luthmanand A. Holmgren,Biochemistry 21, 6628 (1982). 43j. E. Oblong, P. Y. Gasdaska, K. Sherrill, and G. Powis,Biochemistry 32, 7271 (1993).

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THIOREDOXIN AND GLUTAREDOXIN

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concentration of 10 mM. The sample is then applied to a column of CMcellulose equilibrated with 10 mM sodium acetate, pH 5.16. The column is eluted with a linear gradient of 10 and 100 mM sodium acetate, pH 5.16. The Trx-containing fractions appearing around 60 mM sodium acetate are combined, adjusted to pH 7.5 with 1 M NH4OH, and lyophilized, Thioredoxin is normally homogeneous after this step. If not, a rechromatography on Sephadex G-50 in 60 mM NH4HCO3 serves to remove traces of impurities or aggregated material. The Trx activity is measured by its ability to reduce insulin disulfides in the presence of mammalian TR. 42

2',5'-ADP-Sepharose and (oJ-Aminohexyl)Agarose Chromatography of Thioredoxin Reductase Thioredoxin reductase fractions from Sephadex G-50 or DEAE-cellulose are dialyzed against TE buffer and applied to a column of 2',5'-ADPSepharose. After washing the column with the same buffer, TR is eluted with 0.20 M potassium phosphate, pH 7.5, 1 mM EDTA. After concentration by ultrafiltration and dialyzis against TE buffer, the TR fraction is applied to a column of (o~-aminohexyl)agarose and eluted with a linear gradient of sodium chloride, 0 to 0.8 M, in TE buffer. The TR elutes around 0.20 M NaC1. The enzyme is concentrated and dialyzed against TE buffer and stored frozen at - 2 0 °. Thioredoxin reductase is usually electrophoretically pure after these steps. If not, an additional 2',5'-ADP-Sepharose step using enzyme applied in 10 mM potassium phosphate-1 mM EDTA, pH 7.0, and elution with 0,20 M potassium phosphate-1 mM EDTA, pH 7.4, • or a gradient of 0-0.30 M potassium phosphate serves to remove impurities.

Spectrophotometric Assay of Mammalian Thioredoxin Reductase Activity

Principle. Mammalian TR reduces 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) by NADPH. 29 DTNB + NADPH + H +

TR

~"2TNB + NADP +

The product in the reaction, TNB (5'-thionitrobenzoic acid), is yellow and has an absorbance maximum at 412 nm with an extinction coefficient of 13,600 M -1 cm -~.

Reagents NADPH, 40 mg/ml in water EDTA, 0.2 M

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THIOREDOXIN AND THIOREDOXIN REDUCTASE

205

Potassium phosphate buffer, 1.0 M, pH 7.0 DTNB, 25 mg/ml in 99.5% ethanol BSA (bovine serum albumin), 20 mg/ml Procedure. A reaction mixture is made from 50/zl of NADPH, 0.5 ml of EDTA, 1.0 ml of potassium phosphate buffer, 0.8 ml of DTNB, and 100 /xl of BSA. Finally, water is added to a final volume of 10 ml. Add 500 tzl of the reaction mixture to two cuvettes. To the sample cuvette a maximum of 100 ~1 of the protein sample is added, and an equal volume of buffer is added to the reference cuvette. The reaction is followed at 412 nm. Calculation of Activity: Definition of Units. One unit of mammalian thioredoxin reductase is defined as 1 txmol TNB formed per minute: 1 U = &44~2 during the first minute/27.2. Another convenient way of defining activity is by using A412units. One A412unit is an initial increase of the absorbance by 1.00 per minute in the test system with a final volume of 500 ~1. The activity of 1 mg of homogeneous mammalian TR, with an Mr of 116,000, is found to be 1200 A412 units. Values up to 2000 have been reported 43 (A. Holmgren and M. Bj~Srnstedt, unpublished results, with human placenta enzyme).

Determination of Mammalian Thioredoxin Reductase Activity in Biological Material

Principle. Mammalian TR has a broad substrate specificity and reduces thioredoxins from different species including E. coli. 4a'43 The rat liver enzyme has Km values of 2.5 and 35/zM for rat liver and E. coli Trx, respectively.42 No apparent differences have been observed in the activity of human or calf TR with human or bovine Trx. 1° The activity of Trx-(SH)2 is detected by its ability to reduce protein disulfides in insulin] '42 In this assay an excess of Trx, typically 5-10 #M, is added to the reaction mixture together with the cell extract. The Trx-dependent formation of SH groups in insulin is compared to a standard curve with known concentrations of mammalian TR. It is an advantage to use E. coli Trx because this molecule is very stable and shows no aggregation due to air oxidation, which can complicate the assay. Reagents HEPES buffer, 1.0 M, pH 7.6 EDTA, 0.2 M NADPH, 40 mg/ml Insulin, 10 mg/ml DTNB, 4 mg/ml in 99.5% ethanol (store at - 2 0 °)

206

THIOREDOXIN AND GLUTAREDOXIN

[2 lI

Guanidine hydrochloride, 6 M in 0.2 M Tris-HC1, pH 8.0 Escherichia coli Trx, 60/zM Mammalian TR, 3 A412 units/ml (20 nM), freshly diluted in TE buffer with 100/xg/ml BSA Procedure. Cells are homogenized by either repeated freezing in dry ice/ethanol and thawing or by sonication. Protein is measured by the method described by Bradford. 44 A reaction mixture is made by mixing 200 tzl of HEPES buffer, 40/xl of EDTA, 40/.d of NADPH, and 500/zl of insulin. To each test tube is added 40/zl of the reaction mixture. After this step all test tubes are kept in an ice bath. Mammalian TR is added (0, 10, 20, 30, 40, 50, and 70/zl to make a standard curve), and cell extract (the amount depends on the type of cell but usually 20 tzg of protein is sufficient) is added to the sample tubes. All samples are made in duplicate. Thioredoxin from E. coli (10/xl) is added. Finally, water is added to a final volume of 120/zl in all tubes. The samples are transferred to a water bath at 37 ° and incubated for 20 min. The reaction is stopped by addition of 500/zl of 0.4 mg/ml DTNB/6 M guanidine hydrochloride in 0.2 M Tris-HC1, pH 8.0, and the absorbance at 412 nm is recorded. The absorbance of each individual sample is compared to the standard curve for mammalian TR. As a control for SH groups in the cell extracts, protein from each sample is kept on ice during the incubation. Immediately prior to addition of DTNB/guanidine hydrochloride, Trx and reaction mixture is added to the cell extract. A second control is a sample of cell extract containing everything except Trx. This sample is incubated and treated the same way as all other samples. The absorbances of the controls are subtracted from the absorbances of the samples.

Determination of Thioredoxin Activity in Biological Material

Radioimmunoassay Reagents Borate buffer, 0.1 M, pH 8.5 Potassium phosphate buffer, 50 mM, pH 7.5, containing 0.25% glycerin Phosphate-buffered saline (PBS) w i t h / % BSA Sephadex G-50 Dithiothreitol, 0.5 mM Human, calf thymus, or rat thioredoxin, 1.0 mg/ml 125I-Labeled Bolton-Hunter reagent, 0.5 mCi 44 M. M. Bradford, A n a l Biochem. 72, 258 (1976).

[9.I]

THIOREDOXIN AND THIOREDOXINREDUCTASE

207

Rabbit antithioredoxin antibody diluted to A280 of 0.03 Sheep anti-rabbit immunoglobulin Procedure. Trx (5/zg) in 10/zg borate buffer is reduced, 10 min at 37°C, with 1.5/zl of 0.5 mM DTT in N2 atmosphere. Trx is iodinated as described by Bolton and HunterJ 5 The iodinated Trx is separated from unbound iodine by gel filtration on a 20-ml Sephadex G-50 column equilibrated with 50 mM potassium phosphate buffer, pH 7.5, containing 0.25% glycerin. Fractions of 0.5 ml are collected, and the radioactivity of the fraction is measured in a -/-counter. Thioredoxin appears in a broad peak usually around fraction 20. Two major peaks within the broad peak can appear, representing dimeric and monomeric Trx. To measure Trx, iodinated Trx 0.2 pmol is added to 100/zl of PBS-BSA and 100/zl of sample in different dilutions. Rabbit anti-Trx antibody (100 tzl) is added, and the samples are incubated for 4 hr at 37°. Sheep anti-rabbit immunoglobulin diluted 1:5 (100 ~1) is added, and the samples are incubated for 16 hr at 4°. After centrifugation for 90 min at 20,000 g, the supernatants are carefully withdrawn and the ratio of bound to free Trx is calculated using a ~/-counter. The result is compared to a standard curve for 0-100 pmol of Trx.

Enzyme Assay Principle. The principle that Trx, after reduction by TR, will reduce insulin disulfides, is the same as that described above for the determination of TR activity in biological material. Reagents HEPES buffer, 1.0 M, pH 7.6 EDTA, 0.2 M NADPH, 40 mg/ml Insulin, 10 mg/ml DTNB, 4 mg/ml in 99.5% ethanol Guanidine hydrochloride, 6 M in 0.2 M Tris-HC1, pH 8.0 Mammalian TR, 100 A412units/ml Mammalian Trx DTT activation buffer composed of 50 mM HEPES, 1 mM EDTA, Bovine serum albumin 1 mg/ml, and 2 mM dithiothreitol Procedure. Cells are homogenized and the protein concentration measured by the method described by Bradford.44 Cell extracts, 20 txg per sample in a volume of 68/xl, are incubated at 70° for 10 rain. DTT activation buffer (2/xl) is added and the samples incubated at 37° for 15 min to reduce 45A. E. Bolton and W. M. Hunter, Biochem. J. 133, 529 (1973).

208

TH1OREDOXIN AND GLUTAREDOXIN

[2 ] ]

and activate Trx, which may contain aggregated material with inter- or intramolecular disulfides. A reaction mixture composed of 200 ~1 of HEPES buffer, 40/xl of EDTA, 40/xl of NADPH, and 500/xl of insulin is made. To test tubes containing 40/zl of reaction mixture is added the preincubated cell extracts and water to a final volume of 120/xl. The reaction is started by addition of mammalian TR (10/zl) and an equal volume of water to a control sample for each cell extract. The samples are incubated at 37° for 20 min. The reaction is terminated by the addition of 500 tzl of 0.4 mg/ml DTNB/6 M guanidine hydrochloride in 0.2 M Tris-HC1, pH 8.0, and the absorbance at 412 nm is measured. Calculation of Results. From the absorbance of each sample is subtracted the absorbance of the respective control sample lacking TR. Note that DTT contains SH groups which limit the amount used in the activation step or samples from column fractions with DTT-containing buffers. The absorbance is compared to a standard curve for Trx. An advantage is to use E. coli Trx-S2 because it is stable and its activity is unaffected by oxidation; it lacks structural half-cystine residues which can redox regulate the activity of mammalian thioredoxins. Concluding Remarks Thioredoxin and thioredoxin reductase have a long evolutionary history as a dithiol-disulfide oxidoreductase system. Of special interest is the finding of Trx in multiple systems for redox regulation of cellular functions particularly related to transcription factors, control of cell growth, and the immune system. Thioredoxin domains are incorporated in larger proteins such as PDI, which is involved in protein folding and disulfide formation. Thioredoxin reductase from mammalian cells is of special interest because the enzyme has almost doubled its subunit size compared to the wellknown E. coli enzyme. Mammalian thioredoxin reductase, with its broader substrate specificity, is likely to be involved in multiple signaling systems for redox control of cellular processes. Cloning of the mammalian thioredoxin reductase and studies of its structure, distribution, and regulation will be an exciting area for future research. Acknowledgments This research was supported by grants from the Swedish Cancer Society (961), The Swedish Medical Research Council (13X-3529), Inga-Britt och Arne Lundbergs Stiftelse, Knut och Alice Wallenbergs Stiftelse, and the Karolinska Institute.

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SELENITE AND SELENODIGLUTATHIONE

[22] S e l e n i t e a n d S e l e n o d i g l u t a t h i o n e : Thioredoxin Systems

209

Reactions with

By M I K A E L BJ(bRNSTEDT, SUSH1L KUMAR, and ARNE HOLMGREN Introduction

Selenium is an essential trace element for higher eukaryotes, l-3 Chemically, selenium is closely related to sulfur. Some selenium compounds are efficient oxidants of SH groups, 4'5 The most established physiological function of selenium is its presence in the detoxifying enzyme glutathione peroxidase as a selenocysteine residue in the active site. 6 Selenite (SeO32 ) and other selenium compounds have strong inhibitory or, at low concentrations, stimulatory effects on the growth of normal as well as tumor cells by yet incompletely characterized mechanisms.7"8 The first step in the metabolism of inorganic selenium compounds such as selenite is stepwise reduction by thiols. Glutathione (GSH) is the most abundant thiol in mammalian cells, and it has therefore been suggested that a main pathway for selenium metabolism is a reduction by GSH. 7 A first step in the metabolism would thus be the formation of selenodiglutathione (GS-Se-SG, also called selenotrisulfide) according to reaction (1) origi2H ÷ + 4GSH + S e O f - --+ GSSG + GS-Se-SG + 3H20

(1)

nelly demonstrated by Painter. 9 Further reduction of GS-Se-SG will lead to the formation of selenide (Se 2 ), a highly reactive form of seleniumJ ~ Selenide can undergo oxidation and form elemental selenium [see reactions (2)-(6)]. It is also an important precursor to selenoproteins or methylated selenium compounds. 1~ Selenide can bind to metal ions, which is an important reaction in the detoxification of heavy metals. ~ i K. Schwartz and C. M. Foltz, J. Am. Chem. Soc. 79, 3292 (1957). T. C. Stadtman, Science 183, 915 (1974). 3 j. E. Spallholz, J. L, Martin, and H, F. Ganther, "Selenium in Biology and Medicine." AV1 PUN., Westport, Connecticut, 1981. 4 H. E. Ganther, Biochemistry 7, 2898 (1968). 5 C. C. Tsen and A. L. Tappel, J. Biol. Chem. 233, 1230 (1958). 6 T. C. Stadtman, Annu. Rev. Biochem. 49, 93 (1980). 7 R. J. Shamberger, Muter Res. 154, 29 (1985). s D. Medina and C. J. Oborn, Cancer Res. 44, 4361 (1984). E. P. Painter, Chem. Rev. 28, 179 (1941). 10M. BjOrnstedt, S. Kumar, and A. Holmgren, J. Biol. Chem. 267, 8030 (1992). ii j. A. Milner, J. Agric. Food. Chem, 32, 436 (1984).

METHODS IN ENZYMOLOGY, VOL. 252

Copyright © 1995 by Academic Press, Inc. All rights of reproduction in any fi)rm reserved.

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THIOREDOXIN AND GLUTAREDOXIN

[221

Selenite and GS-Se-SG are efficient oxidants of thioredoxin (Trx) and inhibit the functions of the thioredoxin system. 1°,12 In addition, these selenium derivatives will also oxidize noncatalytic or structural cysteine residues which are characteristic of mammalian thioredoxins. This process has been implicated in the regulation of Trx enzyme activity via the formation of intra- or intermolecular disulfides.13 Incubation of selenium compounds with thioredoxin reductase (TR) or TR plus Trx results in redox cycling and a large nonstoichiometric oxidation of NADPH. ma2 Our studies with ferricytochrome c, selenite, and the thioredoxin system suggest the existence of and the participation in recycling of an intermediate in oxidation state -2, such as selenideJ 2 This intermediate acts as a charge-transfer catalyst transferring electrons from NADPH via the thioredoxin system to oxygen. The stoichiometry of the oxygen-dependent reaction with selenite or GSSe-SG can best be explained on the basis of the following reaction sequences [reactions (2)-(9)]. These have been used to describe the reactions of GSSe-SG with glutathione reductase 7 (GR) or TRJ ° GS-Se-SG + NADPH + H ÷ TRor6R> GS-SeH + GSH + NADP +

(2) GS-Se- + H20 ---> Se ° + GSH + OH-

(3)

GS-SeH + NADPH + H20

T~or6R H2Se + GSH + NADP + + OH(a) GS-Se- + GSH ---> HSe- + GSSG (5) HSe- + (O) ---> Se ° + OH(6) HSe- + 2GSH + (O) ---> HSe- + GSSG + H20 (7) SH S

/

HSe- + R

\SH

+ (O) --~ HSe- +

J

+ H20

(8)

kS

R is the active site of thioredoxin or mammalian thioredoxin reductase. Preparation a n d Purification of Selenodiglutathione Mix a 10-/~l aliquot of 0.i M selenite (dissolve Na2SeO3, SeO2, or H2SeO3 in water) with 60/zl of 0.1 M HCI and add 40/zl of freshly prepared 0.I M GSH. The order in which the different components are mixed is 12 S. Kumar, M. Bj0rnstedt, and A. Holmgren, Eur. J. Biochem. 207, 435 (1992). 13X. Ren, M. Bj6rnstedt, B. Shen, M. L. Ericson, and A. Holmgren, Biochemistry 32, 9701 (1993).

[22]

211

SELENITE AND SELENODIGLUTATHIONE

E c

c'q

GS-Se-SG

GSSG

8 1

c t~ zc/

o ,<

1 5

10

15

20

25

Time after injection, rain

FI6. 1. Separation of GS-Se-SG and GSSG by HPLC. [Reprinted from Ref. 10, by permission].

important because addition in another order will lead to the formation and precipitation of elemental selenium. After incubation at room temperature for 1 min, separate the reaction products [oxidized glutathione (GSSG) and GS-Se-SG] by high-performance liquid chromatography (HPLC) on a 30-cm (10 txm) Cls column with an inner diameter of 4.1 mm. Distilled and Millipore (Bedford, MA) filtered water (adjusted to pH 2 with HC1) is used as the mobile phase with a flow rate of 2 ml/min. The separation is followed at 263 nm. This method is a modification 1° of the procedure described by Nakagawa e t aL 14 The reaction mixture made by mixing SeO32- and GSH will be eluted in one small and two major well-separated peaks (Fig. 1); the third peak 14T. Nakagawa, E. Aoyama, N. Kobayashi, H. Tanaka, M. Chikuma, H. Sakurai, and M. Nakayama, Biochem. Biophys. Res. Commun. 150, 1149 (1988).

212

THIOREDOXINAND GLUTAREDOXIN

[22]

is pure GS-Se-SG, as detected by using 75Se. Control experiments show that GSSG and any unreacted SeO32- elutes well before the GS-Se-SG peak. Thus, a pure preparation of GS-Se-SG in good yield is obtained by this method. The concentration of GS-Se-SG can be determined using the extinction coefficient of 1.87 mM -1 cm -~ at 263 nm15; the accuracy of this has been checked by amino acid analysisJ° The Cx8 column must be cleaned as recommended by the supplier immediately after separation because the low pH will cause damage to the resin which may lead to severe interference with the separation procedure described above. Storage and Stability of Selenodiglutathione Selenodiglutathione is stable for months if it is kept dark in a high concentration at low pH and 4 °. Avoid freezing and thawing, which leads to decomposition. Reactions with Mammalian Thioredoxin Reductase The reactions between GS-Se-SG or selenite and mammalian TR are performed at room temperature and in the presence of air in 50 mM TrisHCI-1 mM EDTA (pH 7.5) containing 200/zM NADPH and 10-100 nM mammalian TR (from calf thymus, rat liver, or human placenta, purified essentially as described by Luthman and Holmgren16). The reaction is started by the addition of GS-Se-SG or selenite to the sample cuvette and an equal volume of buffer only to the reference cuvette. The oxidation of NADPH is followed at 340 nm. The consumption of NADPH can be quantified using the extinction coefficient of 6.22 mM -1 cm -1. A low concentration of selenite (1-10/xM) initially imparts a lag phase to the reaction followed by a nonstoichiometric oxidation of NADPH (Fig. 2). However, the same concentration of GS-Se-SG results initially in a fast but stoichiometric oxidation of NADPH followed by a continued and nonstoichiometric reaction. These phenomena are independent of buffer (Tris-HC1 or phosphate) and the presence or absence of albumin (0.1-0.2 mg/ml) or EDTA (1-10 mM). The nonstoichiometric oxidation of NADPH is a characteristic of mammalian TR and is not seen with yeast or rat liver glutathione reductase.10 A high concentration of selenite in the assay system does not influence the enzymeJ 2 The rate of the reaction is proportional to the amount of enzyme in the reaction mixtureJ 2 No oxidation of NADPH occurs with selenite or GS-Se-SG and Escherchia coli TR. 1°'12 However, 15H. E. Ganther, Biochemistry 10, 4089 (1971). 16M. Luthmanand A. Holmgren,Biochemistry 21, 6628 (1982).

[22]

SELENITE AND SELENODIGLUTATHIONE

60 ~

213

100nMCT-TR+3pHC_T-Trx k. 1,00nHE.cotiTR.3pHIrx / / " 1 0 0 n M CT-TR

50

3o

c-

10

ot~TR

~ 0

5 TINIE

10 (rain)

Flo. 2. Selenite-dependent nonstoiehiometric oxidation of NADPH by the calf thymus (CT) or E. coli thioredoxin systems. ]Reprinted from Ref. 12, by permission].

inclusion of 1-5 ~ M E. coli Trx results in oxidation of N A D P H J °,~2 Addition of E. coli or m a m m a l i a n Trx (1-5 /~M) to the assay system further increases the rate of the reaction seen with m a m m a l i a n TR.12 Use of sodium selenate (SeO4 2-) in place of selenite as a substrate will not give oxidation of N A D P H . u

Anaerobic Reaction with Mammalian Thioredoxin Reductase Anaerobic reactions are p e r f o r m e d in special cuvettes (covered by rubber septa) which are continuously evacuated and flushed with oxygenfree argon. To avoid contamination with 02, the argon gas is washed by p r e t r e a t m e n t with freshly p r e p a r e d solutions of 2% and 0.2% sodium dithionite in 0.1 M N a O H . The composition of the assay mixture is the same as described above. The contents of the reaction mixture in the cuvettes and GS-Se-SG or selenite in a separate tube are flushed with argon for at least 5 rain before starting the reaction. After the argon treatment, the reaction is started by injecting oxygen-free GS-Se-SG or selenite solu-

214

THIOREDOXIN AND GLUTAREDOXIN

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tions into the sample cuvette and an equal volume of buffer into the reference cuvette using a Hamilton syringe. With selenite, 12the reaction starts with the same rate as seen aerobically, but stops after oxidation of 3 molar equivalents of NADPH (i.e., after consumption of three pairs of electrons). Admission of air into the assay system restarts a nonstoichiometric oxidation of NADPH. This is consistent with the following overall stoichiometry of the reaction: SeO32- + 3NADPH + 3H + --> Se 2- + 3NADP + + 3H20

(9)

Thus, anaerobically selenide (Se 2+) will be generated, which is known to be highly sensitive to autoxidation by oxygenJ7 The formation of selenide (aerobically or anaerobically) can be proved by the addition of cyanide, which stops the oxidation of NADPH in above reactions instantaneously. With GS-Se-SG] ° the anaerobic reaction starts with oxidation of a stoichiometric amount of NADPH followed by a very slow but continuous reaction. This is due to the generation of GS-Se- by cleavage of GS-SeSG [reaction (2)] followed by a slow formation of HSe- [reaction (4)]. The subsequent admission of air results in a fast and nonstoichiometric oxidation of NADPH (with a slight initial lag phase), consistent with the redox cycling of selenide as described in reaction (8). Formation of Electron Transfer Catalyst: Detection by Cytochrome c Reduction Mammalian thioredoxin reductase and NADPH will reduce cytochrome c with selenium working as an electron transfer catalyst. This can be followed at 550 nm in a spectrophotometer using a modification12 of the method of Bromberg and Pick. TMThe reaction is performed aerobically at 25 ° in 65 mM potassium phosphate, pH 7.0, containing 200/zM NADPH, i00/~M ferricytochrome c, 170 mM sucrose, 2 mM sodium azide, I mM EGTA, and 10/zM FAD. An aliquot of 20/zM SeO32- can be used to start the reactions. The change in A550 following the addition of selenite can be quantified for the reaction of ferricytochrome c using the extinction coefficient of 2.1 × 104 M -I cm -1. The reduction is fast in the presence of oxygen, but slower under anaerobic conditions12 (for anaerobic procedures, see above). Selenium anions formed in the reaction between selenite and thiols are known to reduce cytochrome cJ 2"19 The highly efficient oxygendependent reduction of ferricytochrome c by selenite and the thioredoxin 17 j. A. Milner, Fed. Proc. 44, 2568 (1985). a8 y . Bromberg and E. Pick, J. Biol. Chem. 2~0, 13539 (1985). 19 O. A. Levander, V. C. Morris, and D. J. Higgs, Biochemistry 12, 4591 (1973).

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SELENITE AND SELENODIGLUTATHIONE

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system suggests the existence and recycling of a selenium intermediate in oxidation state - 2 , such as selenide. In the literature the formation of this intermediate via reactions of higher concentrations of GSH with selenite in the presence of glutathione reductase has been described, where the result is precipitation of elemental selenium leading ultimately to the end of the reaction. This is not the case in reactions between the selenium compounds at physiological concentrations and the thioredoxin system. Here, the nonstoichiometric reaction is continuous and is explained by the recycling of HSe- as an intermediate. In these reactions HSe- acts as a charge transfer catalyst transferring electrons from NADPH via the thioredoxin system to the final acceptor oxygen [reaction (8)]. Specificity of Mammalian Thioredoxin Reductase for Selenium over Sulfur Analogs Sodium selenite or selenious acid (20/~M) shows similar reactivity with the enzyme. Sodium selenate up to 100/zM is inert. Replacement of selenite with sulfite (20-100/zM) does not result in oxidation of NADPH, showing the specificity of the reaction for selenium. Organic forms of selenium such as selenocystine and selenomethionine have been tested. Selenomethionine (100/zM) is not a substrate, but L-selenocystine (16/zM) shows a nonstoichiometric reactivity, which is steady and linear like GS-Se-SG. Substitution of L-selenocystine by L-cystine (up to 160/zM in the assay mixture) will not result in oxidation of NADPH. L-Selenocystine (2-20/zM) also shows a nonstoichiometric oxidation of NADPH if E. coli Trx is present together with E. coli TR. The rate of the reaction is proportional to the concentration of selenocystine. However, with the sulfur analog L-cystine, the E. coli thioredoxin system will only oxidize an equimolar amount of NADPH equivalent to regular disulfide reduction. This demonstrates that the nonstoichiometric oxidation of NADPH is a unique property of the selenium atom. Oxidation of Reduced Thioredoxin by Selenodiglutathione Reduced thioredoxin [Trx-(SH)2] from E. coli shows a 3-fold decrease in tryptophan fluorescence emission on oxidation to Trx-S2.20 This property can be used as a sensitive method to study the kinetics of the reactions of selenium and disulfide compounds or any oxidant with Trx-(SH)2.1° To study such interactions, E. coli thioredoxin is reduced in 50 mM Tris-HC11 mM EDTA, pH 7.5, at 37° for 15 min, with at least a 10-fold molar excess 20A. Holmgren,J. Biol. Chem. 247, 1992 (1972),

216

[22]

THIOREDOXIN AND GLUTAREDOXIN

of dithiothreitol (DTT) and desalted on a column of Sephadex G-25 (PD10, 9 ml) equilibrated with ice-cold and N2-saturated 50 mM Tris-HCl-1 mM EDTA, pH 7.5, as described elsewhere. 1° The oxidation of Trx-(SH)2 is followed by recording the decrease in tryptophan fluorescence emission at 350 nm using excitation at 280 nm. Addition of 200 nM GS-Se-SG to 200 nM Trx-(SH)2 from E. coli results in a fast oxidation of Trx-(SH)2 (Fig. 3). The reaction follows a sigmoidal progress curve not analyzed in detail. The reaction between Trx-(SH)2 and insulin is comparatively slower and follows an apparent second-order reaction with a second-order rate constant (K2) of 1.5 × 105 M -1 sec -l. In contrast, GSSG is a very poor oxidant of Trx(SH)2. The oxidation of Trx-(SH)z by S e e 3 2- is linear and follows apparent zero-order kinetics with an estimated rate constant of 1.6 × 10 -9 M sec -1. However, this reaction is complicated because of the formation of selenium intermediates which also are substrates for Trx-(SH)2. Inhibition of Thioredoxin Function Selenite and its reduced forms [reactions (1)-(9)] inhibit 1°'12 protein disulfide reduction by the thioredoxin system [reactions (10)-(12)]. Reac-

GSSG

200

insulin 150 "O

"O

8 -8

100

~

\ GS-Se-SG

50

I 10

I~!! 20

30

l

i

1

40

50

60

Time, s

FIG. 3. Oxidation of E. coli Trx-(SH)2 by an equimolar concentration of GS-Se-SG, insulin, or GSSG. [Reprinted from Ref. 10 by permission].

[22]

SELENITE AND S E L E N O D I G L U T A T H I O N E

217

Trx-S2 + N A D P H + H + ~ Trx(SH)2 + N A D P + (10) Trx-(SH)2 + protein-S2 --~ Trx-S2 + protein-(SH)2 (11) Net: N A D P H + H + + protein-S2 ~ N A D P ÷ + protein-(SH)2 (12) tions involving mammalian T R (which is active also with E. c o l i Trx 2~) are particularly sensitive to inhibition. This is consistent with selenite reacting both directly with the enzyme and also with Trx-(SH)2 thereby competing with and blocking the protein disulfide reduction. ~2 Selenite and its reduced forms also inhibit reduction of CDP catalyzed by E. c o l i ribonucleotide reductase ~° using the thioredoxin system as a hydrogen donor, as this reaction involves reduction of redox-active disulfides in the enzyme? 2

O x i d a t i o n of Active Site a n d S t r u c t u r a l Thiol G r o u p s in H u m a n T h i o r e d o x i n T o study thiol oxidation, human Trx 13 is incubated for 15 rain at 37 ° with a 20-fold molar excess of D T T and desalted on a column of Sephadex G-25 (NAP-5) equilibrated with ice-cold and N2-saturated 50 m M TrisHCI-1 mM E D T A , p H 7.5. The reduced Trx thus prepared is stable in liquid nitrogen for at least 3 months. Repeated freezing and thawing lead to oxidation of SH groups and must be avoided. The reduced protein is incubated with GS-Se-SG or SeO32-. The SH groups are measured by 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) as described elsewhere. 13 Addition of GS-Se-SG results in a fast initial oxidation of SH groups followed by a slow but complete oxidation of all SH groups.

Inhibition of H u m a n T h i o r e d o x i n b y S e l e n o d i g l u t a t h i o n e t h r o u g h O x i d a t i o n of S t r u c t u r a l Thiol G r o u p s The structural SH groups in human Trx have to be reduced for the protein to be active. 13 Completely reduced human Trx with five SH groups is a highly active protein disulfide reductase. However, exposure to a small amount (0.1 molar equivalent) of GS-Se-SG strongly inhibits its activity. We compared the activity of wild-type human Trx with three mutant proteins which lack either Cys-61, Cys-72, or both, after exposure to GS-SeSG. The mutant proteins were constructed as described by Ren et al. ~3 The proteins were reduced as described previously, and activity was tested as the ability to reduce insulin disulfides, a3 The reaction mixture consists of 21A. Holmgren, J. Biol. Chem. 252, 4600 (1977). 22L. Thelander and P. Reichard, Annu. Rev. Biochem. 48, 133 (1979). 23A. Holmgren, J, Biol. Chem. 254, 9113 (1979).

218

THIOREDOXIN AND GLUTAREDOXIN

[221

100 mM potassium phosphate, pH 7.0, 2 mM EDTA, 0.4 mM NADPH, and 0.16 mM insulin. Mutant and wild-type Trx was added to a final concentration of 2 /zM. The reduction of insulin disulfides was started by the addition of human placenta TR to a final concentration of 12 nM, and the reaction was followed at 340 nm. Incubation of wild-type human Trx with 0.1 molar equivalent of GSSe-SG for 3 hr resulted in a decreased insulin disulfide reduction activity, and the reaction progress curve showed a marked lag phase. The double mutant (C61S/C72S) and the C72S protein were not inhibited and showed the same reactivity as the fully reduced proteins. The oxidized C61S Trx showed a slower reaction. Oxidation of wild-type Trx by GS-Se-SG for 24 hr resulted in zero SH groups per molecule and a pronounced lag phase in the insulin disulfide reduction assay. The data clearly show that Trx will regain activity by autoreduction and that the redox status of the Cys-72 residue is of critical importance for the function of human Trx. This residue is obviously a target for oxidants like GS-Se-SG. The mechanism involves the formation of intra- or intermolecular disulfides.13

Formation a n d Separation of Dimer H u m a n Thioredoxin Fully reduced human Trx is incubated for 24 hr with 0.1 molar equivalent of GS-Se-SG at 25 ° and injected on a fast protein liquid chromatography (FPLC) Superdex column. Immediately prior to injection, a 20-100 molar excess of iodoacetic acid is added to the mixture to block any remaining SH groups and to block further oxidation and dimerization. The column is eluted (flow rate 1.0 ml/min) with 50 mM Tris-HCl, pH 7.5-i mM E D T A 500 mM NaCl, and fractions of 1.0 ml are collected. Most of the GS-SeSG treated protein elutes as a dimer, and only a small peak of monomer appears in the 12-kDa area. After incubation of the reaction mixture containing GS-Se-SG with a 20-fold molar excess of DTT, a single peak of monomeric Trx appears, in the 12-kDa area. This proves that the two molecules in the dimer are coupled by a disulfide bridge. We found, by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, that C72S Trx does not form dimers, showing the importance of this residue for dimer formation and inactivation by GS-Se-SG. t3

Conclusions Selenite and GS-Se-SG are efficient substrates for mammalian TR or TR plus Trx from E. coli, causing oxygen-dependent and nonstoichiometric oxidation of NADPH. Addition of mammalian Trx to the mammalian

[221

SELENITEAND SELENODIGLUTATHIONE

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reductase results in an increased reaction rate, showing that the reactive intermediates of the selenium compounds are substrates for both proteins of the mammalian thioredoxin system. This property of the thioredoxin system can be used during purification and characterization of Trx or T R from other sources. The oxidation of Trx-(SH)2 by GS-Se-SG is at least 4 times faster than the most rapid reaction known so far, namely, that between Trx-(SH)2 and insulin disulfides. 23 The reactions between the thioredoxin system and the selenium compounds lead to the formation of selenide (Se 2-, H S e - ) and may be the metabolic reaction leading to reduced selenium for synthesis of selenocysteine residues in proteins. The selenium anion then serves as an electron transfer catalyst to oxygen acting as the final acceptor under aerobic conditions, and it is regenerated by redox cycling. The functions of the thioredoxin system as a hydrogen donor for ribonucleotide reductase, which is essential for D N A synthesis, and also as a general protein disulfide reductase are effectively inhibited by selenium c o m p o u n d s ) °'12 The mechanisms for these effects are (i) N A D P H depletion, (ii) competitive inhibition of T R by inhibition of its reduction of Trx$2, and (iii) oxidation of catalytic and structural cysteines in human Trx inactivating the molecule. The combined effect of these inhibitory mechanisms on the human thioredoxin system may provide an explanation of the inhibitory effects of selenium compounds on mammalian cell growth and proliferation. Acknowledgments This investigation was supported by grants from the Karolinska Institute, the Swedish Cancer Society(961), the Swedish Medical Research Council (13X-3529),and Knut och Alice Wallenbergs Stiftelse,Sushil Kurnar was on leave from Industrial ToxicologyResearch Centre (CSIR), Lucknow,India, and was supported by a guest scientist fellowship from the Swedish Cancer Society.

220

THIOREDOXIN

AND GLUTAREDOXIN

123]

[23] M e a s u r e m e n t o f E q u i l i b r i u m M i d p o i n t P o t e n t i a l s o f Thiol/Disulfide Regulatory Groups on ThioredoxinActivated Chloroplast Enzymes B y RONALD S. HUTCHISON a n d DONALD R . O R T

Introduction The illumination-dependent activation of at least seven different chloroplast enzymes involves the reversible thiol/disulfide exchange between thioredoxin and the photosynthetic enzyme. Two different forms of thioredoxin mediate the light-dependent activation of these enzymes in chloroplasts. The reduction of thioredoxin is coupled to photosynthetic electron transfer by ferredoxin through ferredoxin/thioredoxin reductase.1 The function of the ferredoxin-ferredoxin/thioredoxin reductase system in chloroplast enzyme activation can be replaced in in vitro studies by thiol donors such as reduced dithiothreitol (DWWred). The thermodynamics of the thiol/disulfide exchange between thioredoxin and the regulatory sulfhydryl groups of the various light-regulated chloroplast enzymes is of increasing interest with the emergence of evidence that differences in the redox properties among the enzymes may play an important role in the regulation of photosynthesis. The electron-sharing equilibria among thioredoxin and its target enzymes qualitatively explains the differential responses to light intensity and the kinetics of activation and deactivation of the different thioredoxin-regulated enzymes.2 However, the thermodynamics of the thiol/disulfide conversions can be significantly influenced by changes in substrate and cofactor concentrations as well as by changes in the chloroplast stromal pH. A great deal more quantitative information about these dependencies is necessary before a comprehensive understanding of light activation will be possible. In this chapter we present an experimental strategy and protocol for measuring the reversible exchange of a protein intramolecular disulfide between thioredoxin f and chloroplast target enzymes. These data can in turn be used to determine the equilibrium redox midpoint potentials that control the change in catalytic activity of these enzymes. Here we use the titration of fructose-l,6-bisphosphatase (FBPase), a nuclear-encoded protein located in the chloroplast stroma, to illustrate the procedures and 1 B. B. Buchanan, Arch. Biochem. Biophys. 288, 1 (199l). 2 D. M. Kramer, R. R. Wise, J. R. Frederick, D. M. Aim, J. D. Hesketh, D. R. Ort, and A. R. Crofts, Photosynth. Res. 26, 213 (1990).

METHODS IN ENZYMOLOGY, VOL. 252

Copyright © 1995 by Academic Press, Inc. All rights of reproduction in any form reserved.

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considerations involved with determination of equilibrium redox midpoint potentials of the thiol/disulfide conversions controlling chloroplast enzyme activation. Chloroplast FBPase is a tetramer composed of four identical 45-kDa subunits forming a holoenzyme of 180 kDa. Each monomer subunit has a regulatory site near the center of the polypeptide chain containing the sequence... Arg-CyslV4-Val-Val-Asn-Val-Cys179-Gly .... 3 Presented are two different titration methods, one based on the measurement of enzyme activity and one based on fluorescent detection of protein thiols by bromobimane labeling, that can be usefully employed in the redox determinations.

Experimental Methods Isolation o f Thioredoxin f and Fructose-l,6-Bisphosphatase Thioredoxin Purification. The isolation procedure for spinach chloroplast thioredoxin is derived from that of Crawford et al. 4 All procedures were conducted at 4°. Batches of destemmed frozen leaf tissue (totaling 2.5 kg) are homogenized for 3 rain using a standard blender in a solution of 30 mM Tris-HC1 (pH 7.9), 3 mM MgC12, 1% polyvinylpolypyrrolidone (w/v), 14 mM 2-mercaptoethanol, 0.5 mM aminocaproic acid, and 2 mM benzamidine hydrochloride. The homogenate (final volume of 6 liters) is filtered through cheesecloth and then adjusted to pH 4.5 with 2 N formic acid. The solution is stirred for 10 min followed by centrifugation for 10 rain at 13,000 g. The dark green pellet is saved for purification of FBPase (see below). The precipitate from a 90% ammonium sulfate (w/v) fractionation of the yellow supernatant is collected by centrifugation (10 rain at 13,000 g) and resuspended in a minimal volume of the homogenization buffer lacking polyvinylpolypyrrolidone. The sample is dialyzed for 6 hr against three 4-liter batches of solution containing 30 mM Tris-HCl (pH 7.9), 3 mM MgCI2, and 14 mM 2-mercaptoethanol. The sample is subsequently clarified by centrifugation (28,000 g for 20 min) before loading on a 5 × 20 cm hydroxyapatite column. The column is sequentially washed with l-liter solutions of 30 mM Tris-HCl (pH 7.9), 3 mM MgCI2, and 14 mM 2-mercaptoethanol containing 0, 10, 50, and 100 mM potassium phosphate. After the addition of 0.2 mM phenylmethylsulfonyl fluoride, the 50 and 100 mM potassium phosphate fractions containing thioredoxin activity (assayed by ability to activate FBPase as described below), are combined and concentrated to 130 ml by ultrafiltration (Ami-

3F. Marcus and P. B. Harrsch,Arch. Biochem. Biophys. 279, 151 (1990). 4N. A. Crawford,B. C. Yee, S. W. Hutcheson,and B. B. Buchanan,Arch. Biochem. Biophys. 244, l (1986).

222

THIOREDOXIN AND GLUTAREDOXIN

[231

con, Danvers, MA, YM5 membrane). The solution is again clarified by centrifugation and thereafter loaded on a 5 × 100 cm Sephadex G-100 column that has been equilibrated with 30 mM Tris-HC1 (pH 7.9), 3 mM MgCle, 200 mM NaC1, and 14 mM 2-mercaptoethanol. The column is eluted with the same buffer, and 20-ml fractions are assayed for thioredoxin activity. Ammonium sulfate is added to the active fractions to 35% of saturation, and the solution is clarified by centrifugation and applied to a 2.5 × 12 cm column containing phenyl-Sepharose CL-4B equilibrated with 30 mM TrisHC1 (pH 7.9), 3 mM MgC2, 30% NH4(SO4)2 (w/v), and 14 mM 2-mercaptoethanol. Thereafter, the column is rinsed with 200 ml of this buffer before a 300-ml linear descending gradient of ammonium sulfate (30 to 0%) is applied followed by a final wash of the column with 200 ml of 30 mM Tris-HCl (pH 7.9), 3 mM MgC12, and 14 mM 2-mercaptoethanol. Initial separation of the two forms of chloroplast thioredoxin occurs here, with thioredoxin f eluting at the end of the ammonium sulfate gradient and during the beginning of the final wash. The thioredoxin f-containing fractions are combined and concentrated to approximately 6 ml by ultrafiltration as above. Further purification of thioredoxin f is achieved on a 2.5 × 90 cm Sephadex G-75 SF column equilibrated with 30 mM Tris-HC1 (pH 7.9), 3 mM MgCI:, 200 mM NaC1, and 14 mM 2-mercaptoethanol and eluted with the same buffer. Active fractions are dialyzed overnight against 10 mM sodium acetate (pH 4.6) and 3 mM MgCle and then applied to a 1 x 5 cm S-Sepharose column. The column is eluted with an ascending linear gradient of 0 to 500 mM NaC1 in 10 mM sodium acetate (pH 4.6). Thioredoxin fcontaining fractions are combined and desalted by equilibrium dialysis in 30 mM Tris-HC1 (pH 7.9) and 3 mM MgC12. The sample, containing about 0.5 mg thioredoxin f, is concentrated to approximately 2 ml by ultra filtration and stored at - 2 0 °. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-~PAGE) shows that greater than 90% of the protein present is thioredoxin f. We have also used thioredoxin f isolated from E. coli that had been transformed with the cloned spinach thioredoxin fgene, 5 and it performs identically to that which we isolated from leaves. Fructose 1,6-Bisphosphatase Purification. The purification procedure for chloroplast FBPase from spinach or tomato leaves is derived from that of Nishizawa et al. 6 The dark green pellet obtained during the thioredoxin 5 We are grateful to Prof. Schurmann for providing thioredoxin f isolated from transformed E. coli as described by F. Aguilar, B. Brunner, L. Gardetsalvi, E. Stutz, and P. Schurmann, Plant Mol. BioL 20, 301 (1992). 6 m. Nishizawa, 13. C. Yee, and B. B. Buchanan, in "Methods in Chloroplast Molecular Biology" (M. Edelman, R. B, Hallick, and N.-H. Chua, eds.), p. 707. Elsevier Biomedical, New York, 1982.

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DETERMINING SULFHYDRYL MIDPOINT POTENTIALS

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isolation procedure described above is resuspended in a minimal volume of buffer containing 25 mM H E P E S - N a O H (pH 7.5) and 0.25 mM EDTA, adjusted to pH 6.5 with NaOH, and thereafter stirred for 30 rain at 4 ° followed by a 20-min centrifugation at 13,000 g. The precipitate of a 40 to 90% ammonium sulfate fraetionation of the supernatant is collected and resuspended in 50 mM sodium acetate (pH 5.5). The solution is clarified by centrifugation (28,000 g for 20 rain) and applied to a 5 x 100 cm Sephadex G-100 column. Fractions are eluted from the column in 50 mM sodium acetate (pH 5.5), and those containing significant amounts of FBPase activity [measured as release of inorganic phosphate (Pi), see below] are loaded onto a 2.5 x 20 cm column of DEAESephacel beads in 50 mM sodium acetate (pH 5.5). Following washing with 150 mM NaCI in 50 mM sodium acetate (pH 5.5), FBPase activity is eluted from the column with a 500-ml linear gradient of 150 to 750 mM NaCI in 50 mM sodium acetate (pH 5.5). The 6-ml active fractions ( - 1 2 ) are applied to a 1.5 x 4 cm hydroxyapatite column that is sequentially eluted with 50ml aliquots of potassium phosphate (0, 100, 200, 300, and 400 mM) in 50 mM sodium acetate. The active fractions are concentrated by ultrafiltration (Amicon YM30 membrane), rediluted with 50 mM sodium acetate (pH 5.5), and finally reconcentrated to 5 ml and stored at - 2 0 °. The isolation procedure yields about 15 mg of FBPase. Analysis by S D S - P A G E shows that greater than 95% of the protein present is FBPase. Measurement of Fructose-l,6-Bisphosphatase Activity

The FBPase activity (fructose 1,6-bisphosphate ~- fructose 6-phosphate + Pi) is measured by monitoring the FBPase-dependent release of inorganic phosphate using a procedure modified 7 from that published by Chifflet et al. 8 Color development is initiated by the addition of 0.1 ml of reagent I (see below) to an equal volume FBPase reaction mixture consisting of 100 mM Tricine-NaOH (pH 7.9), 10 mM MgCl2, 1.7 mM EDTA, 2.4 mM fructose 1,6-bisphosphate, and 7/xg FBPase. After 5 rain of incubation at room temperature, 0.1 ml of reagent II (see below) is added and, at least 15 min later, 9 the absorbance at 700 nm is measured. The amount of phosphate released is calculated from a standard calibration curve with inorganic phosphate. Reagent I is prepared immediately before use by adding the following in order: 5 ml of 10 N HC1, 10 ml of 5% (w/v) ammonium molybdate, 30 ml water, and 5 ml of 15% (w/v) sodium ascorbate. Reagent II contains 2% (v/v) acetic acid, 2% (w/v) sodium citrate, and 2% (w/v) sodium meta7 E. M. Larson, personal communication, 1994. S. Chifflet, A. Torriglia, R. Chiesa, and S. Tolosa, Anal. Biochem. 168, 1 (1988). 9 The color has fully developed within 15 min and is stable for several hours,

224

THIOREDOXIN

AND

[231

GLUTAREDOXIN

arsenite. When the assay is used during the thioredoxin isolation procedure, thioredoxin-containing leaf extracts replace purified thioredoxin f in a 10rain preincubation with 10 mM DTT red. When used during the FBPase isolation, the reaction is initiated by the addition of FBPase-containing leaf extracts, and the reaction pH is raised to 8.8, circumventing the reduction dependence of FBPase activity.6

Redox Titration of Fructose-l,6-Bisphosphatase Activity Figure 1A shows the dependence of chloroplast FBPase activity on the ambient redox potential. Such data can be used to estimate accurately the equilibrium midpoint potential of the catalytic activation of the enzyme provided that several vital experimental criteria are met. A ._>

E "-t

E

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100

80 60 40 20

B ~) ¢.)

100

I

,

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,

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,

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o

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E E

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,

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E h (mv) FIG. 1. Equilibrium redox titrations of thiol/disulfide regulatory groups on chloroplast FBPase. Purified FBPase was incubated for 1.5 hr (A) or 3 hr (B) at pH 7.9 in the presence of thioredoxin f a n d 10 mM DTT in various thiol/disulfide ratios. (A) Dependence of FBPase activity, measured as phosphate release from fructose 1,6-bisphosphate, on the ambient redox potential (Eh). (B) Level of fluorescence emission at 485 nm after reacting FBPase for 10 rain with 10 mM monobromobimane. The solid lines are the theoretical curves determined from the nonlinear least-squares fit of the data to the Nernst equation with the value of n fixed at 2. The calculated midpoint potential of the thiol/disulfide regulatory group of FPBase (Era,7.9) for the two titration methods agree closely.

[231

DETERMINING SULFHYDRYL MIDPOINT POTENTIALS

225

Stability of Ambient Redox Potential. It is important that the thiol/ disulfide ratio of the redox buffer not change over the course of the experiment. Oxygen is the major threat to the stability of the ambient redox poise in the relatively reducing potential range in which the titrations are performed even though DTT red is not very susceptible to direct oxidation by 02.1° The FBPase (7 /zg) is incubated at 25° in 0.1 ml of solution containing 100 mM Tricine-NaOH (pH 7.9), 0.2 tzg thioredoxin f, and 10 mM Dq-T ~1 in various thiol/disulfide ratios. The incubation solution has been purged with argon, and samples are kept under a constant stream of humidified argon throughout the incubations. The dipolar ionic buffer Tricine is an effective chelator of trace metals that might otherwise catalyze the oxidation of thiols by oxygen. The stability of the redox poise under these conditions for incubations of 5 hr is confirmed using Ellman's reagent 12 to monitor the concentration of DTT tea. However, it should be noted that in titrations of other enzymes, such as phosphoribulokinase, in which the thiol/disulfide midpoint potential is more oxidizing than FBPase, we find that 10 mM DTT is not adequate to maintain a satisfactorily stable poise throughout the full range of the titration. For those values far below the maximum buffering range of the DTT redox couple, where the concentration of the DTT thiol is very low, we use 80 mM total DTT to ensure a stable redox poise at the most oxidizing potentialsJ 3 Establishing that Equilibrium Has Been Achieved. Although the thioldisulfide exchange rates between thioredoxin and the target enzymes must be relatively rapid under physiological conditions in order to explain observed rates of light activation, this situation cannot be assumed for in vitro titrations. Although the pH sensitivity of thiol/disulfide exchange is well known, more cryptic factors associated with enzyme substrates and cofactors may play important roles in determining the equilibration kinetics. Changes in the protein which affect the local electrostatic environment around the regulatory thiol/disulfide or changes in the physical accessibility due to rearrangements within the protein could have large and unforeseen effects on thiol/disulfide exchange rate. In our experiments, we monitor the time course of enzyme activation in order to ensure that redox equilibrium has been attained. Under the conditions cited for the FBPase titration reported in Fig. 1A, maximum activity is achieved within 1.5 hr, and we observe no loss of activity after 3 hr of incubation at 25°.13 Without thioredoxin f, the rate of activation m W. W. Cleland, Biochemistry 3, 480 (1964). 1~Ultrapure DTT red is purchased from Boehringer Mannheim (Indianapolis, IN) and DTT °x from Sigma (St. Louis, MO), Both are used without further purification. ~2G. L. Ellman, Arch. Biochem. Biophys. 82, 70 (1959). 13 R. S. Hutchison, Ph.D. Thesis, University of Illinois, Urbana/Champaign, 1994.

226

THIOREDOXIN AND GLUTAREDOXIN

[231

of FBPase by direct thiol exchange with DTT is exceedingly slow, and equilibrium is not attained during the lifetime of the protein under these incubation conditions. We find13 that the effect of thioredoxin f on the rate of FBPase activation is saturated at about 10/zg/ml thioredoxin f, which differs from the unsaturable, first-order dependence of FBPase reduction rate on thioredoxin f concentration observed by Clancey and Gilbert. 14 In the presence of thioredoxin f, we observe no dependence on the rate of FBPase activation on the total concentration of DTT present during the incubation.

Redox Titration of Fructose-l,6-Bisphosphatase by Monobromobimane Labeling of Protein Thiols The thioredoxin f-dependent reduction of the regulatory disulfide on FBPase can be measured using the thiol-labeling reagent monobromobimane (mBBr) that reacts rapidly and exclusively with accessible thiol groups, producing a fluorescent, chemically modified protein. 15 Figure 1B shows an equilibrium redox titration of FBPase using mBBr to quantitate the amount of thiol present at different ambient redox potentials. Two hundred micrograms FBPase, 0.4 txg of thioredoxin f, and 10 mM DTT in various thiol/disulfide ratios are incubated for 3 hr in 0.1 ml of 100 mM Tricine-NaOH buffer (pH 7.9) as described above. Monobromobimane is added to 10 mM (from a 250 mM stock solution in acetonitrile). After 10 rain, unreacted mBBr is extracted with 0.1 ml of methylene chloride, leaving the protein-mBBr complex in the aqueous phase. The fluorescence emission intensity of each sample is measured in a SLM Aminco SPF-500 spectrofluorometer (Urbana, IL) at 485 nm with 395 nm excitation light. Fluorescence emission originating from mBBr labeling of protein thiols not involved in the thioredoxin-mediated disulfide/thiol interconversion can be estimated from a sample incubated as above with 10 mM DTT °x. This background fluorescence level is less than 10% of the fluorescence emission from samples incubated in 10 mM DTT red. It should be noted that the fluorescence originating from mBBr labeling of nonregulatory protein thiols may not be as low for all other thioredoxin-regulated chloroplast enzymes as we observed for FBPase. Were an enzyme to have accessible nonregulatory thiol groups of similar redox potential to the regulatory thiols, significant background fluorescence would be anticipated. Samples incubated with 10 mM D T r red were considered to be fully reduced, and the fluorescence intensity of such samples was assigned the value of 100% in Fig. lB. 14 C. J. Clancey and H. R. Gilbert, J. Biol. Chem. 262, 13545 (1987). x5 N. S. Kosower and E. M. Kosower, this series, Vol. 143, p. 76.

[23]

DETERMINING SULFHYDRYL MIDPOINT POTENTIALS

227

There is one additional concern that should be addressed for techniques such as this, in which the measurement technique perturbs the equilibrium. By reacting exclusively and irreversibly with the thiol, mBBr could "pull" the reaction in the direction of thiol formation and thereby produce erroneous results. This is not likely to be a problem in the in vitro titration of chloroplast enzymes as performed here, however, because conditions can be set to ensure that the reaction of mBBr with protein thiols is much more rapid than the thioredoxin-mediated reduction of the protein disulfide. Under the conditions used here for FBPase, the half-time for mBBr labeling is less than 1 min, whereas the half-time for the reductive activation for FBPase is more than 30 min. 13

Analysis of Redox Data Curve Fitting. The data in Fig. 1 were fit to the Nernst equation in the form: 59 [disulfide] Eh --- Em,x + ~'~ log [dithiol] where Eh denotes that the redox potential values are referenced to the standard hydrogen half-cell. Em~ is the symbol for the midpoint potential under conditions when the components of the redox reaction are present at less than unit activities and the ambient pH value (x) for the determination is greater than zero16; in this case the pH was 7.9. There are 2 electrons involved in the thioredoxin-mediated thiol/disulfide interconversion for FBPase, so we have evaluated the equation for n = 2. In Fig. 1A the [disulfide] was calculated as 100% minus the percent maximum FBPase activity at any given Eh, and the [dithiol] was then the percent maximum FBPase activity at that Eh. In Fig. 1B, the [disulfide]/[dithiol] ratio was calculated similarly with the percent maximum fluorescence intensity for mBBr-labeled FBPase replacing FPBase activity in the calculation. The data points were computer fit to the Nernst equation using an iterative nonlinear least-squares analysis. We used Fig P, which is a versatile commercial data analysis package (Biosoft, Cambridge, UK) replete with convenient plotting and graph drawing capabilities. The solid lines in Fig. 1 are the theoretical curves determined from the nonlinear least-squares fit to the Nernst equation with the value of n fixed at 2.

16p. L. Dutton, this series, Vol. 54, p. 411.

228

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THIOREDOXIN AND GLUTAREDOXIN

Determination o f Eh. The absolute values determined for the midpoint potential of the regulatory sulfhydryls on FBPase in Fig. 1 are dependent on the Eh values assigned to various ratios of DTTrea/DTT°x at pH 7.9. The initial value for the midpoint potential of DTT at pH 7 reported by Cleland 1° of -332 mV is well supported by measurements employing diverse techniques. 17,18Because the redox reaction of DTT involves protons, the Em of the redox buffer will have a substantial pH dependence which must be taken into account in Eh calculations. The reduced form of DTT is a very weak acid, with the pKa values of the thiols reported to be 9.2 for the first ionization and 10.1 for the secondJ 9 Because DTT °x is a very strong acid, the redox chemistry of DTT will involve one proton per electron at pH values below approximately 8.2 (at higher pH values ionization of the first thiol o n D T T red will lower the proton to electron ratio below 1). Thus for pH values below 8.2, the Em of DTT can be calculated according to Em~ = Em~7 - 0.059 (pH x - pH 7)

We have used the value of Lees and Whitesides is for the Era, 7 for DTI" of -327 mV at 25 °, giving a value for Era,7. 9 of --380 mV. It should be noted that Cleland's original value for Em,8.1 of -366 mV 1° (implying a - 3 0 mV per unit pH dependence) has not been substantiated by subsequent measurements and should not be used for calculations of Eh.

17 D. M. R o t h w a r f and H. A. Scheraga, Proc. Natl. Acad. Sci. U.S.A. 89, 7944 (1992). is W. J. Lees and G. M. Whitesides, J. Org. Chem. 58, 642 (1993). 19 G. M. Whitesides, J. E. Liburn, and R. P. Szajewski, J. Org. Chem. 42, 332 (1977).

[24] By

Thioredoxin

and Seed Proteins

JOSHUA H. WONG, KAROLY KOBREHEL,

and BOB B.

BUCHANAN

Introduction Storage proteins, which are formed and stored within seeds, serve as the main source of nitrogen and as a primary source of carbon for the germination and growth of seedlings. Storage proteins are synthesized after pollination and accumulate during seed maturation. The proteins are mobilized, proteolytically degraded, and utilized when conditions are favorable METHODS1NENZYMOLOGY,VOL. 2.52

Copyright© 1995by AcademicPress,Inc. All rightsof reproductionin any formreserved.

[24]

THIOREDOXIN AND SEED PROTEINS

229

for germinationJ '2 In the case of cereals, the bulk of the storage proteins are water insoluble after the grains mature. In most other types of seeds, the proteins remain soluble and enclosed within a membrane, thereby creating a structure known as a protein body. 3 In some cereals, the protein body m e m b r a n e is disrupted during maturation and drying of the grain, thereby exposing the storage proteins to endogenous proteases of the endosperm. 3,n However, in typically nonglutenous cereals such as corn, rice, sorghum, and millet, storage proteins remain in protein bodies in the mature grains (see references in Ref. 5). Disulfide bonds are one of the main stabilizing forces in storage proteins. The formation of disulfide bonds appears to function in cereals to provide increased structural stability on the one hand and decreased solubility on the other. Both features provide protection against proteolysis. The group includes typically large, insoluble storage proteins (60-100 kDa) as well as generally less abundant low molecular weight soluble proteins (5-30 kDa),4 rich in disulfide bonds, with four or more cystines per molecule. Examples include a wide variety of exogenous a-amylase and protease inhibitors. Except for the ot-amylase/subtilisin inhibitor of barley and other cereals, which inhibits endogenous a-amylase, 6,7 the physiological function of these small disulfide proteins within the seed remains a mystery. It has long been considered that proteins of this type play a bioprotection role by blocking digestive enzymes of invading pests. 8 Several investigators have shown that small disulfide proteins of seeds, like the larger insoluble storage proteins, are degraded during germination? a° Our laboratory has uncovered evidence that both protein groups undergo reduction in conjunction with the degradationJ ~ The NADP+/ thioredoxin system (NTS) appears to play a role in the reduction of critical disulfide groups of seed proteins (S-S ~ 2SH) and, thereby, as seen below, trigger germination. The thioredoxin is reduced by N A D P H via NADP+/ J A. M. Mayer and A. Poljakoff-Mayber, "The Germination of Seeds." 4th Ed,, p. 160. Pergamon, New York, 1989. 2 F. M. Ashton, Annu. Rev. Plant Physiol. 27, 95 (1976). 3 E. Weber and D. Neumann, Biochem. Physiol. Pflanz. 175, 279 (1980). 4 p. R. Shewry and B. J. Miflin, Adv. Cereal Sci. Technol. 7, 1 (1985). s D. B. Bechtel, R. L. Gaines, and Y. Pomeranz, Cereal Chem. 59, 336 (1982). 6j. Hejgaard, I. B. Svendsen, and J. Mundy, Carlsberg Res. Commun. 48, 91 (1983). 7 R. J. Weselake, A. W. MacGregor, R. D. Hill, and H. W. Duckworth, Plant Physiol. 73, 1008 (1983). s C. A. Ryan, Biochem. Plants 6, 351 (1981). 9 p. Hwang and W. Bushuk, Cereal Chem. 50, 147 (1973). 10j. E. Kruger and B. A. Marchylo, Cereal Chem. 62, 1 (1985). ~K. Kobrehel, J. H. Wong, A. Balogh, F. Kiss, B. C. Yee, and B. B. Buchanan, Plant Physiol. 99, 919 (1992).

230

THIOREDOXINAND GLUTAREDOXIN

[241

thioredoxin reductase (NTR), a flavoprotein [Eq. (1)]. The reduced thioredoxin, in turn, reduces disulfide groups on target proteins [Eq. (2)]. The evidence suggests that thioredoxin preferentially reduces intramolecular disulfide bonds relative to intermolecular counterpartsJ 2 Thioredoxin hox + N A D P H + H + -NTR thioredoxin hred + N A D P + (1) (--S--S--) (--SH HS--) Thioredoxin hred + proteinox --~ thioredoxin hox + proteinr~d (--SH HS--) (--S--S--) (--S--S--) (--SH HS--)

(2)

Thioredoxin h, a 12-kDa protein with a catalytically active disulfide group, seems to be ubiquitous in plant cells and to be localized in the cytosol, endoplasmic reticulum, and mitochondriaJ 3-15 At the onset of germination, thioredoxin h appears to be reduced by N A D P H generated by the oxidative pentose phosphate pathway of the cereal endospermJ 1 Once reduced, the thioredoxin, in turn, reduces the large storage proteins (leading to their solubilization), as well as the small disulfide inhibitor proteins (leading to loss of their ability to inhibit target enzymes). On reduction, both the large storage proteins 11A6,17 and the small disulfide enzyme inhibitor proteins ~8,19 show increased susceptibility to proteolysis. There is some evidence t h a t glutathione maintained in reduced form (GSH) by N A D P H and glutathione reductase may also function in the reduction of these proteins [Eqs. (3) and (4)], particularly after thioredoxin has initiated the reduction process. 11 GSSG + N A D P H + H +~ 2GSH + NADP ÷ (--S--S--) (--SH)

(3)

2 G S H + proteinox --~ GSSG + proteinred (--SH) (--S--S--) (--S--S--) (--SHHS--)

(4)

Much of the evidence for the role of thiols in seed germination has come from studies in our laboratory. For these studies, we have adapted a relatively simple, yet specific method for measurement of the thiol redox 12S. Shin, J. H. Wong, K. Kobrehel, and B. B. Buchanan, Planta 189, 557 (1993). 13T. C. Johnson, K. Wada, B. B. Buchanan, and A. Holmgren, Plant Physiol. 85, 446 (1987). 14j. Bodenstein-Lang, A. Buch, and H. Follmann, FEBS Lett. 258, 22 (1989). 15F. Marcus, S. H. Chamberlain, C. Chu, F. R. Masiarz, S. Shin, B. C. Yee, and B. B. Buchanan, Arch. Biochem. Biophys. 287, 195 (1991). 16j. H. Wong, K. Kobrehel, C. Nimbona, B. C. Yee, A. Balogh, F. Kiss, and B. B. Buchanan, Cereal Chem. 70, 113 (1993). 17I. Besse, unpublished findings from our laboratory, 1995. ~8j. Jiao, B. C. Yee, K. Kobrehel, and B. B. Buchanan, J. Agric. Food Chem. 40, 2333 (1992). 19j. Jiao, B. C. Yee, J. H. Wong, K. Kobrehel, and B. B. Buchanan, Plant Physiol. Biochem. 31, 799 (1993).

[24]

THIOREDOXIN AND SEED PROTEINS

231

status of proteins. The technique is a modification of an earlier procedure developed for analysis of chloroplast enzymes regulated by thioredoxin. 2° In that study, the chloroplast proteins of interest, because of their low abundance, were immunoprecipitated to permit quantitation of the reduction of the disulfide groups by light. An uncharged probe, m o n o b r o m o b i m a n e (mBBr), was used to bind sulfhydryl groups specifically and covalently, thereby rendering the p r o t e i n m B B r derivative fluorescent. 2°'21 The fluorescent m B B r - p r o t e i n products can be separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis ( S D S - P A G E ) and then visualized and quantified. The method can be applied equally well in vitro (both with purified proteins ls'19`2L22 and with extracts composed of many proteins 11,16)or in vivo (with intact seedsU). In either case, the redox status of the seed proteins can be assessed. A description of the procedure is presented below, using wheat as an example. Also, as seen below, the procedure can be applied equally well to disulfide proteins purified from a variety of sources.

S o u r c e s of Materials a n d C h e m i c a l s Materials. Either seeds or semolina of durum wheat (Triticum durum) or seeds or flour of bread wheat (Triticum aestivum) may be used. In our case, the respective cultivars are Desf. cv. M o n r o e or cv. Scout 66. Escherichia coli thioredoxin and N T R are either purchased from American Diagnostica (Greenwich, CT) or isolated from cells overexpressing each protein. 23'24 Wheat thioredoxin h and N T R are isolated from germ, following the procedures developed for spinach leaves. 25 Glutaredoxin (a gift of Prof. A. Holmgren, Karolinska Institutet, Stockholm) is isolated from E. coli cells genetically modified to overexpress the protein. 26 Chemicals. Reagents for S D S - P A G E are purchased from Bio-Rad Laboratories (Richmond, CA). M o n o b r o m o b i m a n e (mBBr) or Thiolyte is obtained from Calbiochem (San Diego, CA). Aluminum lactate and methyl green are products of Fluka Chemicals (Buchs, Switzerland).

20N. A. Crawford, M. Droux, N. S. Kosower, and B. B. Buchanan, Arch. Biochem. Biophys. 271, 223 (1989). 21N. S. Kosower and E. M. Kosower, this series, Vol. 143, p. 76. 22K. Kobrehel, B. C. Yee, and B. B. Buchanan, J. Biol. Chem. 266, 16135 (1991). ~3F. de la Motte-Guery, M. Mijiulac-Maslow, P. Decottiguies, M. Stein, P. Minard, and J. P. Jacquot, Eur. J. Biochem. 196, 287 (1991). 24M. Russel and P. Model, J. Biol. Chem. 263, 9015 (1988). 25F. J. Florencio, B. C. Yee, T. C. Johnson, and B. B. Buchanan, Arch. Biochem. Biophys. 266, 496 (1988). z6A. Holmgren, J. Biol. Chem. 264, 13963 (1989).

232

THIOREDOXINAND GLUTAREDOXIN

[241

Isolation of Protein Fractions Chemicals and Materials

Durum wheat semolina or bread wheat flour, 5 g Tris-HC1 buffer, pH 7.5, 50 mM Ethanol, 70% (v/v) Acetic acid, 0.1 M Procedure. For isolation of the soluble and insoluble proteins, semolina or flour (0.2 g) is extracted sequentially with 1 ml of the following solutions for the indicated times at 25°: (1) 50 mM Tris-HC1 buffer, pH 7.5 (20 min), for the albumins and globulins; (2) 70% ethanol (2 hr) for the gliadins; and (3) 0.1 M acetic acid (2 hr) for the glutenins. 1° Alternatively, the glutenins can be isolated by extraction with sodium myristate. In that case, 80 mg myristate is added to 1 g of flour together with 8 ml distilled water. 27 Similar conditions can be used to extract total proteins in one step. 28During extraction, samples are placed on an electrical rotator and, in addition, occasionally agitated with a vortex mixer. After extraction with each solvent, samples are centrifuged (12,000 rpm for 5 min at 4°) in an Eppendorf microcentrifuge, and supernatant fractions are saved for analysis. In between each extraction, pellets are washed with 1 ml of water and collected by centrifugation as before; the supernatant wash fractions are discarded. By convention, z9 the fractions are designated as follows: (1) albumin/globulin; (2) gliadin; and (3) glutenin. If desired, the albumins and globulins can be separated by dialysis against distilled water; the albumins remain soluble and the globulins precipitate. Following centrifugation (10 min, 10,000 g at 4°), the supernatant fraction containing the albumins is decanted and the globulins are redissolved in 50 mM Tris-HCl buffer, pH 7.5.

Reduction and in Vitro Labeling of Proteins In Vitro Labeling o f Proteins with Monobromobimane Reagents

Tris-HC1 buffer, pH 7.9 at 20 °, 100 mM Thioredoxin, E. coli, 0.1 mg/ml in 30 mM Tris-HC1, pH 7.9 NTR, E. coli, 0.1 mg/ml in 30 mM Tris-HCl, pH 7.9 NADPH, 20 mM in 30 mM Tris-HCl, pH 7.9 27K. Kobrehel and R. Alary, J. ScL Food Agric. 48, 441 (1989). 28K. Kobrehel and B. Matignon, Cereal Chem. 57, 73 (1980). 29T. B. Osborne and C. G. Voorhees,Am. Chem. J. 15, 392 (1893).

[241

THIOREDOXIN AND SEED PROTEINS

233

Dithiothreitol (DTT), 100 mM Glutathione, reduced, 20 mM in 30 mM Tris-HC1, pH 7.9 Glutathione reductase (purified from spinach leaves25), 0.1 mg/ml in 30 mM Tris-HC1, pH 7.9 Glutaredoxin, E. coli, 0.1 mg/ml in 30 mM Tris-HC1, pH 7.9 Monobromobimane (mBBr) or Thiolyte, 20 mM dissolved in acetonitrile and stored protected from light SDS, 10% (w/v) 2-Mercaptoethanol (2-ME), 100 mM Target proteins, 5-30/zg, in extraction solvent; alternatively, as seen below, 5-10 tzg pure disulfide proteins may be used Procedure. Both steps of the reaction are carried out at room temperature. As indicated, 7 txl NTR (0.7/~g) and 10/~1 thioredoxin (1 tzg) (both from E. coli unless specified otherwise) along with 10 t~l N A D P H are added to 70 tzl of 100 mM Tris-HC1 buffer, pH 7.9, and 30 txg of target protein. When thioredoxin is reduced by DTT, the N A D P H and NTR are omitted and 5 txl DTT is added to a final concentration of 1 mM. Reduction by reduced glutathione is performed similarly, but at a final concentration of 2 mM. After incubation for 20 min, 5/zl of mBBr (80 nmol) is added, and the reaction is continued for another 15 min. To stop the reaction and derivatize excess mBBr, 10/zl of 10% SDS and 10 tzl of 100 mM 2-ME are added, and the samples are then applied to the gels. For reduction by glutaredoxin, the thioredoxin and NTR are replaced by 10 Ixl E. coli glutaredoxin (1/xg), 10/xl glutathione reductase (1.4/xg), and 10 txl NADPH.

Germination and in Vivo Labeling of Proteins Germination of Wheat Seeds Materials

Seeds of durum wheat Plastic petri dishes Whatman (Clifton, N J) No. 1 filter paper Growth chamber Procedure. Duplicate sets of 20 to 30 seeds are placed in a plastic petri dish on three layers of Whatman No. 1 filter paper moistened with 5 ml of deionized water. Petri dishes are then transferred to a darkened growth chamber. Germination is carried out for up to 4 days at room temperature, with 5 ml of deionized water being added daily to each petri dish. On the fifth day, the germinated seedlings are harvested.

234

THIOREDOXIN AND GLUTAREDOXIN

[24]

In Vivo Labeling of Proteins with Monobromobimane Chemicals and Materials Endosperm of germinated seedlings Liquid N2 mBBr, 2.0 mM, freshly diluted in 100 mM Tris-HC1 buffer, pH 7.9 Procedure. At the indicated times, the dry seeds or germinated seedlings (selected on the basis of similar radicle or shoot length) are taken from the petri dish and the embryos or germinated axes are removed with a razor blade. Five endosperm from each lot are weighed and then ground in liquid N2 with a mortar and pestle. One milliliter of 2.0 mM mBBr in 100 mM Tris-HCl buffer, pH 7.9, is added just as the last trace of liquid N2 disappears. The thawed mixture is ground for another minute and transferred to a microcentrifuge tube of 1.5 ml capacity. The volume of the suspension is adjusted to 1 ml with the appropriate mBBr or buffer solution. Protein fractions of albumin/globulin, gliadin, and glutenin from endosperm of germinated seedlings are extracted as described above for semolina and flour. The extracted protein fractions are stored at - 2 0 ° until use for electrophoresis. A buffer control (minus mBBr) is included for each time point. Analysis of Labeled Samples by Polyacrylamide Gel Electrophoresis

Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis of Monobromobimane-Labeled Samples Analysis by S D S - P A G E of the mBBr-derivatized samples of each protein fraction is performed in 15% polyacrylamide gels at pH 8.5 as described by Laemmli. 3° Before sample application, an aliquot (20/xl) of 50% (v/v) glycerol containing 0.01% (w/v) bromphenol blue is added to each mBBr-labeled sample. Gels of 1.5 mm thickness are developed for 16 hr at a constant current of 9 mA.

Native Gel Electrophoresis for Separating Different Gliadins Reagents and Equipment A gel solution in 100 ml final volume contains the following: 6.0 g acrylamide, 0.3 g bisacrylamide, 0.024 g ascorbic acid, 0.2 mg ferrous sulfate heptahydrate, 0.25 g aluminum lactate, and 85% lactic acid (certified ACS); the gel solution is adjusted to pH 3.1 with lactic acid 30 U. K. Laemrnli, Nature (London) 227, 680 (1970).

[24]

THIOREDOXINAND SEEDPROTEINS

235

Catalyst solution, hydrogen peroxide, 3% Tank buffer, aluminum lactate buffer, 0.5 g/liter, is adjusted to pH 3.1 with lactic acid Tracking dye, methyl green, 0.6% (w/v) in 80% (w/v) sucrose in tank buffer Electrophoresis equipment, Hoefer Model SE600 (Hoefer Scientific Instrument, San Francisco, CA) Procedure. To resolve the different types of gliadins, native polyacrylamide gel electrophoresis is performed in 6% gels of 3 mm thickness (a procedure designed to separate gliadins into the four types, a,/3, y, and ~0) as described by Bushuk and ZiUman 31 and modified for vertical slab gels by Sapirstein and Bushuk. 32 A gel stock solution can be prepared beforehand by mixing acrylamide, bisacrylamide, and aluminum lactate, adjusting to pH 3.1 with lactic acid, and then storing at 4 °. Before use the ascorbic acid (0.024 g) and ferrous sulfate (0.2 rag) are added to 100 ml of the gel stock solution, which is then degassed for 2 hr at 4 °. Because polymerization occurs essentially instantaneously after adding the hydrogen peroxide catalyst, it is best to set up and precool the gel plate (16 × 18 cm) assembly, with the 3-mm spacers, in a cold room. It is also best to pour the gel solution as quickly as possible through a funnel, mounted on a stand, that fits into the space between the plates with the comb already in place. Before sample application an aliquot (20 /xl) of the tracking dye is added to each sample. The duration of electrophoresis is approximately 4 hr, with a constant current of 50 mA. The temperature of the tank buffer ( - 2 - 3 liters) is controlled by circulating water at 21 ° during electrophoresis. (Caution: As gliadins are positively charged at this pH, it is imperative to reverse the polarity of the two electrodes, i.e., by attaching the negative electrode of the gel box to the positive outlet of the power supply and vice versa for the positive electrode.) Electrophoresis is terminated when the lower mobility band of the methyl green tracking dye migrates to about ! cm from the end of the gel. Monobromobimane Removal and Fluorescence Photography Reagents and Equipment

12% (w/v) Trichloroacetic acid 40% Methanol/10% acetic acid (v/v)

31W. Bushuk and R. R. Zillman, Can. J. Plant. Sci. 58, 505 (1978). 32H. D. Sapirstein and W. Bushuk, Cereal Chem. 62, 372 (1985).

236

THIOREDOXIN AND GLUTAREDOXIN

[24]

Spectroline transilluminator, (Spectronics Corp., Westbury, NY), Model TS-365, 365 nm ultraviolet Polaroid Land camera Polaroid Positive/Negative Landfilm, type 55 Wratten gelatin filter No. 8 (cutoff of 460 nm) Procedure. Following electrophoresis, gels are placed in 200 ml trichloroacetic acid and soaked for 4 to 6 hr with one change of solution to fix the proteins; gels are then transferred to 200 ml of 40% methanol/10% acetic acid for 8 to 10 hr (with two to three changes of solution) to remove excess mBBr. The fluorescence of mBBr, both free and protein bound, is visualized by placing gels on a light box fitted with an ultraviolet light source (365 nm). Gels are photographed with Polaroid Positive/Negative Landfilm, type 55, through a yellow Wratten gelatin filter No. 8 (cutoff of 460 nm) (exposure time ranges from 25 to 60 sec at f4.5). The negatives are used for densitometric scanning after being processed and dried (see below).

Protein Staining/Destaining/Photography Reagents Coomassie Brilliant Blue R-250, 0.25% (w/v) 40% methanol/10% acetic acid (v/v) Coomassie Brilliant Blue R-250 (0.1 g dissolved in 10 ml of 95%, v/v, ethanol) Trichloroacetic acid, 12% Procedure. The SDS gels are stained with 100 ml of 0.25% Coomassie Brilliant Blue R-250 in 40% methanol/10% acetic acid for 1 to 2 hr and destained overnight with several changes (200 ml) of the above solvent without the dye. 2° Aluminum lacate native gels are stained overnight in a filtered solution containing 0.1 g Coomassie Brilliant Blue R-250 in 240 ml of 12% trichloroacetic acid. Gels are destained overnight in 200 ml of 12% trichloroacetic acid. 1l Protein-stained gels are photographed with Polaroid type 55 film to produce prints and negatives. Prints are used to determine band migration distances and loading efficiency as well as to quantitate protein SH. Quantitation of Fluorescence [Reduction)

Scanning of Gels The Polaroid negatives of fluorescent gels and prints of wet proteinstained gels are scanned with a laser densitometer (Pharmacia-LKB, Piscataway, N J, UltroScan XL). Intensities of fluorescent bands are quantified by evaluating peak areas after integration with GelScan XL software. The

[24]

237

THIOREDOXIN AND SEED PROTEINS Proteln

Fluorescence 2

3

I 5

4

~ 1

I 5

Protein

Fluorescence I 6

7

8

9

10

I

I

6

10

kDa 97.4 66.2 w

45.0

31.0

21.5

14.4

Gliadins:

Glutenins:

1. 2. 3. 4. 5,

6. Control 7. GSH 8. GSH/GR/NADPH 9. NGS 10. NTS

Control GSH GSH/GR/NADPH NGS NTS

FIG. 1. Relative effectiveness of the NADP+/thioredoxin (NTS), NADP+/glutaredoxin (NGS), and the glutathione/glutathione reductase system (GSH/GR/NADPH) in the reduction of seed proteins (SDS, polyacrylamide gel electrophoresis).

intensity of fluorescent bands, when totally reduced by 2 mM DTT at 100° for 3 min and derivatized with mBBr, is proportional to the amount of added protein (up to 30/zg). It is assumed that the intensity of fluorescence of the proteins under analysis is also a direct measure of percent reduction of sulfhydryl groups. Specific Applications The techniques described above have been used successfully in determining the reduction of seed storage proteins and purified disulfide proteins from seeds as well as other sources. The following examples serve to illustrate use of the technique.

238

[241

THIOREDOXIN AND GLUTAREDOXIN

Effectiveness of Thiols in Reduction of Seed Proteins. By employing the in vitro labeling technique described above, our laboratory has obtained evidence that the NADP+/thioredoxin system reduces wheat storage proteins--gliadins and glutenins--much more effectively than the glutaredoxin or glutathione systems (Fig. 1). T M Similar results have been obtained in an unpublished survey of extracted prolamines and glutelins from several nonglutenous cereals. The system works equally well for pure disulfide proteins from seeds (Fig. 2) as well as other s o u r c e s . 12'19,2°,22 Change in Redox Status of Proteins during Germination. Using the in vivo labeling technique described above, our laboratory has obtained evidence for a change in redox state of wheat proteins during germination.11 We observed a progressive increase of 2- to 3-fold in reduction of the albumin/globulin, gliadin, and glutenin fractions up to the second day of germination; thereafter, reduction declined (Fig. 3). This pattern of redox change was coincident with the proteolytic degradation (or mobilization) of storage proteins. A similar series of events has been observed with barley DSG-1

BBTI

•

,g"

DTT GSH

~ + , ÷1i t

DTT ÷

_

GSH II +

_

C ~l +

DTT II

.

GSH ,

+

C ,

+

I

+ and - refer to thioredoxin (E.coli) C = Control (no reductant) •~ = Thioredoxin

Fie. 2. Dithiothreitol-reduced thioredoxin as a reductant for seed a-amylase and trypsin inhibitor proteins. DSG-1, Wheat a-amylase inhibitor; BBTI, soybean Bowman-Birk trypsin inhibitor (SDS, polyacrylamide gel electrophoresis).

[241

THIOREDOXIN AND SEED PROTEINS

5

I

I

,

o

.~a

i

a

•

,

,

I

239 I

t

o,/

i

i

~ ~ 3

~

o

0

0

1

2

3

4

Day

FIG. 3. In vivo reduction status of protein fractions during germination. [Data obtained by analysis of SDS-polyacrylamide gels.]

(C. Marx, J. H. Wong, and B. B. Buchanan, unpublished results); also in unpublished experiments, the reduction of disulfide bonds of gliadins and glutenins by the NTS was found to facilitate their degradation by proteases (I. Besse, and B. B. Buchanan, unpublished findings). Change in Redox Status of Thiol-Containing Proteins during Seed Maturation. Samples (wheat ears) are harvested at different stages of seed maturation up to complete maturity. Immediately after harvest, samples are put into liquid N2, and grains are dehuUed manually in a mortar in the presence of liquid N2. In vivo labeling with mBBr is performed under the conditions described for the in vivo labeling of germinated seeds. Results to date provide evidence that the storage proteins are synthesized in the reduced form and become oxidized during maturation and drying. Redox Changes during Technological Transformations. An extension of these experiments has revealed that the NADP+/thioredoxin system reduces selected flour proteins and strengthens the dough network formed

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with flour of poor cooking quality, as measured by farinograph 16and baking tests. 33 These results suggest that addition of the NADP+/thioredoxin system improves products prepared from wheat and other cereals. Monobromobimane is currently being used as a probe to identify the major sulfhydryl/disulfide redox changes taking place in proteins during dough strengthening. Samplings are taken at different stages of the dough making process. Dough samples are put into liquid N2, and protein is fractionated and labeled with mBBr as described above.

33 K. Kobrehel, C. Nimbona, B. B. Buchanan, D. Bergmann, J. H. Wong, and B. C. Yee, "Gluten Proteins 1993," p. 381. Assoc. of Cereal Research, Detmold, Germany.

[251 A n a l y s i s a n d M a n i p u l a t i o n o f T a r g e t E n z y m e s Thioredoxin Control

By J E A N - P I E R R E

for

J A C Q U O T , E M M A N U E L L E ISSAKIDIS,

PAULETTE DECOTTIGNIES, MARTINE LEMAIRE,

and

MYROSLAWA MIGINIAC-MAsLOW

Introduction Several systems are able to control the activity, stability, and correct folding of enzymes through dithiol/disulfide isomerization reactions including the enzyme protein disulfide-isomerase, the glutathione-dependent glutaredoxin system, and the thioredoxin systems. 1-3 Plants contain two different thioredoxin systems, one extrachloroplastic in which thioredoxin is reduced through NADPH and the flavoenzyme NADPH-thioredoxin reductase (EC 1.6.4.5), and the other chloroplastic in which the reducing system is photoreduced ferredoxin and the iron-sulfur enzyme ferredoxinthioredoxin reductase. 4'5 To study thioredoxins and their reducing systems, it is necessary to use a target enzyme, the properties of which can be modified after reaction with reduced thioredoxin. Several proteins with this property have been isolated (including fructose-l,6-bisphosphatase, phosphoribulokinase, glyceraldehyde-3-phosphate dehydrogenase, glu1 R. B. Freedman, FEBS Lett. 97, 201 (1979). z A. Holmgren, Annu. Rev. Biochem. 54, 237 (1985). 3 H. Eklund, F. K. Gleason, and A. Holmgren, Proteins: Struct. Funct, Genet. 11, 13 (1991). 4 B. B. Buchanan, Annu. Rev. Plant Physiol. 31, 341 (1980). 5 T. C. Johnson, K. Wada, B. B. Buchanan, and A. Holmgren, Plant Physiol. 85, 446 (1987).

METHODSIN ENZYMOLOGY,VOL.252

Copyright © 1995by AcademicPress,Inc. All rightsof reproductionin any formreserved.

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cose-6-phosphate dehydrogenase, and CF1-ATPase). One of the best documented, easy to use, and most versatile enzymes, however, is the NADP +malate dehydrogenase (EC 1.1.1.82). This chapter deals with the purification of the protein from the leaves of C4 plants (in which the enzyme is present at a much higher level relative to C3 plants), the optimal conditions for its reductive activation, and activity measurements. Other procedures have been published for the purification of the protein from C4 or C3 plants. 6'7 A system is described for the expression and purification of the enzyme from Eseherichia coli cells and for its modification to make it even more versatile. Purification and Assay of Corn or Sorghum Leaf NADP+-Malate Dehydrogenase Purification Procedure Materials and Chemicals

Corn or sorghum plants grown on vermiculite, watered twice a week with demineralized water, for 3 weeks in a greenhouse (illumination of 60 W/m 2, 15 hr/day) Tris-HCl, 1 M, pH 8.0, 1 liter stock solution EDTA (Na salt), 1 M, 50 ml stock solution K2-K phosphate buffer, pH 7.2, 0.5 M stock solution (1 liter) 2-Mercaptoethanol Phenylmethylsulfonyl fluoride (PMSF), 50 mM (in 2-propanol) Polyvinylpyrrolidone (PVP 40), 40 g MgCI2, 1 M, 100 ml stock solution NaCI, 150 g Ammonium sulfate (Merck, Darmstadt, Germany), 1 kg Aca 44 (IBF, Villeneuve la Garenne, France), 1.5 liters, swollen DEAE-Sephacel (Pharmacia, Piscataway, N J), 80 ml, swollen Matrex red A, (Amicon, Exernon, France), 25 ml, swollen Dialysis tubing Pair of scissors, Waring blendor (1 gallon capacity), magnetic stirrer, UV monitoring system, peristaltic pump, fraction collector, temperature-controlled recording spectrophotometer Cold room (5 °) Extraction and Purification. Leaves (1 kg) are harvested, deribbed, cut into small pieces, and blended in 2 liters extraction buffer [30 mM Tris6 A. Agostino, P. Jeffrey, and M. D. Hatch, Plant Physiol. 98, 1506 (1992). 7 K. Fickenscher and R. Scheibe, Biochim. Biophys. Acta 749, 249 (1983).

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[25]

HCI, pH 8.0, 1 mM EDTA, 5 mM MgCI2, 14 mM 2-mercaptoethanol, 0.2% (w/v) PVP 40, 100/zM PMSF]. The resulting slurry is filtered through a double layer of nylon net (48 mesh) and centrifuged for 20 min at 10,000 g to eliminate cell debris. Ammonium Sulfate Fractionation. Solid ammonium sulfate (226 g/liter supernatant) is slowly added to the protein solution to bring it to 40% of saturation. After 30 min of stirring at 5 °, precipitating proteins are eliminated by centrifugation (20 min at 10,000 g). Then 120 g of solid ammonium sulfate is added per liter supernatant, which brings it to 60% of saturation. The NADP+-malate dehydrogenase (NADP+-MDH) precipitates at this step. After 30 min of stirring, it is recovered in the protein pellet obtained by centrifuging the extract for 20 min at 10,000 g. The proteins are dissolved in around 100 ml of extraction buffer from which PVP and MgC12 have been omitted and dialyzed overnight against 5 liters of 20 mM potassium phosphate buffer, pH 7.2, containing 1 mM EDTA. The latter buffer (PE) is then used throughout the purification. The sample is cleared after dialysis by spinning for 1 hr at 50,000 g. DEAE-Sephacel Chromatography. The clear supernatant is applied to a DEAE-Sephacel column (2.5 × 15 cm) previously equilibrated in PE buffer at a flow rate of 80 ml/hr. After extensive washing (3 bed volumes), the proteins are eluted with a 500-ml linear gradient of NaC1 (0-600 mM) in PE buffer. Fractions (6 ml) are collected and assayed for activity (see below). The enzyme elutes at a concentration of about 300 mM NaC1. The fractions showing N A D P + - M D H activity are pooled and concentrated by ultrafiltration in an Amicon cell fitted with a YM10 membrane to a final volume of 20 ml. Aca 44 Chromatography. The concentrated sample is applied to an Aca 44 column (5 x 70 cm) previously equilibrated with PE buffer. The filtration is performed at a flow rate of 60 ml/hr, and 7-ml fractions are collected and assayed for activity. The active fractions are pooled and used for the Matrex red A step. Matrex Red A Chromatography. The N A D P + - M D H contained in the solution is then bound to a Matrex red A column (1 x 10 cm) at a flow rate of 25 ml/hr. Unbound material is washed off the column by 5 bed volumes of PE buffer, and the proteins specifically retained (NADP+-dependent enzymes) are eluted using a 400-ml linear gradient of NaC1 (0-2 M) in PE buffer. The plant NADP+-MDH is eluted at a concentration of 1.5 M NaCI. After this step of purification, the enzyme is at least 80% homogeneous and can be used in this form for most purposes. If a fully purified sample is desired a final purification step by hydrophobic interaction chromatography as described below can be added.

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Hydrophobic Interaction Chromatography. The NADP+-MDH can be further purified on a TSK phenyl 5PW column (0.75 x 7.5 cm; LKB). The column is equilibrated with 1.8 M ammonium sulfate in 0.1 M potassium phosphate, pH 7.2, and the proteins eluted with a 30-rain linear decreasing gradient of ammonium sulfate (1.8-0 M) in 0.1 M potassium phosphate, pH 7.2, followed by a 10-min wash with 0.1 M potassium phosphate, pH 7.2, at a flow rate of 1 ml/min. The NADP+-MDH sample is then dialyzed and concentrated using a C30 microconcentrator (Amicon). Dialysis and Storage. The NADP+-MDH sample recovered after Matrex red A chromatography has a very high concentration of salt. Dialyzing it by ultrafiltration at that step can result in denaturation and heavy precipitation. It is therefore recommended to dialyze it overnight against 5 liters of PE buffer, before concentrating it by ultrafiltration as described above. The enzyme can be conveniently stored in aliquots of 200 tzl at a concentration of 1 mg/ml at - 7 0 °. The protein concentration of the sample can be determined from its absorbance at 280 nm (1 A280 unit = 0.8 rag/ ml). The specific activity of the fully activated enzyme is about 400 U/rag protein at 30° (1 U = 1/zmol NADPH oxidized/rain).

Measurement of Activity The NADP+-MDH is totally dependent on activation by reduced thioredoxins for activity. Hence, it requires a preactivation step before the activity can be measured (this is detailed below). Once activated, the activity of the enzyme can be detected as follows.

Reagents Tris-HC1, pH 8.0, 1 M (stock solution 1 liter) Oxaloacetate* (OAA), 75 mM (stock solution 1 ml) NADPH, 15 mM (stock solution 1 ml) Assay Procedure. One cuvette (1 ml) contains 100 tzl Tris-HCl, pH 8, 10/xl stock OAA, 10 txl stock NADPH, and 880 txl water. The reaction is initiated by the addition of activated NADP+-MDH. It is followed spectrophotometrically by the decrease of absorbance at 340 nm (molar extinction coefficient for NADPH of 6200 M -1 cm-X).

Activation Methods for NADP+-Malate Dehydrogenase As explained above, the NADP+-MDH purified from plant leaves requires a preactivation step before its activity can be measured. This can

* Warning: Oxaloacetateis very unstable in solutions above pH 7. It shouldbe dissolvedin water, kept frozen at -20 °, and used within2 weeks.

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be achieved in two ways: either with thioredoxin chemically reduced by dithioreitol (DTT) or with thioredoxin reduced photochemically through light and the ferredoxin-thioredoxin system. Therefore the activation of N A D P * - M D H can be used to detect either thioredoxin (any type when DTT is the reductant) 8 or ferredoxin-thioredoxin reductase (FTR) (reconstituted light activation system). 9

Dithiothreitol- Thioredoxin-Linked Activation Reagents Tris-HC1, pH 8.0, 1 M (stock solution 1 liter) Dithiothreitol (DTT), 100 mM (stock solution 1 ml) N A D P + - M D H , 1 mg/ml Activation Conditions. The reaction mixture contains 10/xl of 1 M TrisHCI, 10 /xl of 100 mM DTT, 5 /xl NADP+-MDH, 5-20 /xl thioredoxin fractions, and water to 100 txl. The activation is started by the addition of thioredoxin. The activation rates are followed by removing 20-/zl samples at 5-rain intervals and injecting them into the spectrophotometer cuvette containing the reaction medium. The rates are proportional to the thioredoxin content of the fractions. Under these conditions, as little as 0.1 txg thioredoxin can be detected per 20-txl sample. Most thioredoxins have a good reactivity with NADP+-MDH. There are, however, at least two cases where the enzyme is poorly activated, namely, with human thioredoxin 1° and with thioredoxins from Dictyosteliurn.ll

Light-Dependent Activation The assay is carried out essentially as described in elsewhere this series, xz with some simplification.

Reagents Tris-HC1, pH 8.0 (stock solution 1 liter) Pea thylakoids, freshly isolated, 2.5 mg chlorophyl/m113 8 p. Schfirmann, K. Maeda, and A. Tsugita, Eur, J. Biochem. 116, 37 (1981). 9 M. Droux, M. Miginiac-Maslow, J. P. Jacquot, P. Gadal, N. A. Crawford, N. S. Kosower, and B. B. Buchanan, Arch. Biochem. Biophys. 256, 372 (1987). 10j. p. Jacquot, F. de Lamotte, M, Fontecave, P. Schfirmann, P. Decottignies, M. MiginiacMaslow, and E. Wollman, Biochem. Biophys. Res. Comrnun. 173, 1375 (1990). ll B. Wetterauer, M. V6ron, M. Miginiac-Maslow, P. Decottignies, and J. P. Jacquot, Eur. J. Biochem. 209, 643 (1992). ~2N. A. Crawford, B. C. Yee, M. Droux, D. E. Carlson, and B. B. Buchanan, this series, Vol. 167, p. 415. 13 M. Miginiac-Maslow, J. P. Jacquot, and M. Droux, Photosynth. Res. 6, 201 (1985).

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Spinach ferredoxin, 2 mg/m114 Spinach thioredoxin m, 1 mg/ml 8 N A D P + - M D H , 1 mg/ml Activation Conditions. In a colorless E p p e n d o r f tube are mixed 10/xl of 1 M Tris-HC1, 10 /~1 pea thylakoids, 5 /zl ferredoxin, 5 /xl N A D P +M D H , 20 txl thioredoxin, 5-20/xl ferredoxin-thioredoxin reductase (FTR) fractions, and water to 100/xl. The activation reaction is initiated by placing the mixture in an illuminated water bath (100 W/m2). Every 5 min a sample of 30 txl is removed and injected into the spectrophotometer cuvette for activity measurement. When using freshly isolated thylakoids, there is no need for anaerobiosis, and water can serve as the electron donor, which makes the system easier to use. (Warning: The thylakoid preparation is not stable and should be kept in the dark on ice and used within 5 hr at most.) With this assay, as little as 0.3 tzg F T R can be detected per 30-/xl sample.

Cloning, O v e r e x p r e s s i o n , a n d Purification of R e c o m b i n a n t NADP +Malate D e h y d r o g e n a s e The purification procedure using plant leaves is time-consuming and requires large amounts of plant material. Thus, it is often more convenient to overproduce the protein in E. coli.

Expression Systems for NADP÷-Malate Dehydrogenase One prerequisite for expressing a eukaryotic protein in prokaryotes is to have a full-length c D N A available. This was realized in our laboratory with a c D N A obtained from a sorghum library. 15 Several plasmids are available for expression of nonfused proteins (including pKK, p P R O K , pTrc, t6 and pET17). All the plasmids have either an NcoI or an NdeI site which allows the introduction of the sequence at the level of the initiator methionine. Depending on the sequence to be introduced, several strategies can be employed for the cloning. The easiest and most versatile way of cloning is indeed the use of the polymerase chain reaction (PCR) as described below. 14S. G. Mayhew, Anal. Biochem. 42, 191 (1971). 1.~C. Cr6tin, P. Luchetta, C. Joly, P. Decottignies, L. Lepiniec, P. Gadal, M. Sallantin, J. C. Huet, and J. C. Pernollet, Eur. J. Biochem. 192, 299 (1990). f6E. Amman and J. Brosius, Gene 40, 183 (1985). 17F. W. Studier, A. H, Rosenberg, J. J. Dunn, and J. W. Dubendorff, this series, Vol. 185, p. 60.

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Reagents 10X Taq polymerase buffer Mix of the four deoxynucleoside triphosphates (dNTPs), 5 mM Upstream and downstream oligonucleotides, 5 t~M stock BioTaq polymerase (5 units/tzl) (Bioprobe, Montreuil-Sous-Bois, France) Oligonucleotide primers For the design of the oligonucleotides it is recommended to use the following rules: both oligonucleotides should hybridize over a 15- to 18base length. Appropriate restriction sites and additional bases (2) can be introduced upstream of the codon for the first amino acid and downstream of the termination codon. A typical example is given below (designed for the use of pET 3d vector). N-terminal protein sequence: S V D A A K C-Terminal protein sequence: L P G E V Ter Upstream oligonucleotide: 5' CCCC A T G GCC GTC GAC GCC GCC A A G 3' NcoI Downstream oligonucleotide: 5' CCGGATC CTA CAC TTC TCC CGG TAG 3' BamHI

This upstream olignonucleotide has been chosen for cloning convenience in the pET 3d vector. The use of the cloning series with the NcoI site sometimes results in the need for changing the first amino acid downstream of the initiator methionine (in this case, Set to Ala) to keep the proper reading frame. This modification does not change the properties of the protein. It can be circumvented by the use of pET12a series in which the upstream cloning site is NdeI. 17 Polymerase Chain Reaction. The reaction mixture contains 10/xl of 10x Taq polymerase buffer, 4 /xl of 5 mM dNTPs, 20 /xl of upstream oligonucleotide stock, 20/xl of downstream oligonucleotide stock, and plasmid containing the nucleotidic sequence (100 ng DNA). The reaction mixture is placed in 1.5-ml Eppendorf tubes tightly closed with a plastic clip (avoid using mineral oil), and DNA is first denatured by heating for 5 min at 95°. The mixture is then cooled on ice, spun briefly, and 0.5/zl BioTaq polymerase added. Twenty cycles are performed in a PCR machine (Biomed Thermocycler 60, Braun, Theres, Germany) (1 min at 94 °, 2 min at 50 °, 3 min at 72°). The resulting product is then purified by agarose gel electrophoresis (1%), extracted with phenol, and concentrated by precipitation with ethanol. Following digestion with the appropriate restriction enzymes (the addition of the two external bases next to the restriction sites is essential for this purpose), the product is ligated to the plasmid previously digested under the same conditions. The resulting construction is then used to transform E. coli cells. DNA minipreparations are realized with random chosen

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ampicillin-resistant colonies (a resistance conferred by the plasmid) and checked by digestion with the restriction enzymes used above. All the procedures used for digestion, ligation, transformation, and D N A preparations are quite standard and are described by Sambrook et aL TM The above cloning procedure has proved successful in our laboratory for a number of sequences including NADPH-thioredoxin reductase, t9 thioredoxins, fructose-l,6-bisphosphatase (FBPase) (J.-P. Jacquot, E. Issakidis, P. Decottignies, M. Leymaire, and M. Miginiac-Maslow, unpublished results). Therefore, it seems to be applicable to a broad variety of cases. The ehloroplastic thioredoxin-dependent enzymes are nuclear-encoded. Hence, their full-length cDNA sequence comprises a transit peptide which is processed after protein import into the chloroplast. In the case of N A D P ÷MDH, the transit peptide is cleaved by the bacteria, and hence the sequence can be cloned either as the full-length form or in a short form deprived of the sequence coding for the transit peptide. 2° Expression and Purification o f R e c o m b i n a n t Protein Materials

Incubator shaker, Series 25, New Brunswick Instruments (Edison, NJ) Centrifuge Luria-Bertani (LB) medium, sterile, 5.5 liters containing ampicillin (50 tzg/ml) Stock solutions as for the plant enzyme purification All cultures are performed at 37 °. After transformation of the appropriate E. coli strain (BL21 for the pET plasmid), one colony is used to inoculate 3 ml of LB medium. After 8 hr, the culture is transferred to 400 ml of LB medium and left to grow overnight (15 hr). This culture is in turn used to inoculate 4.8 liters of LB medium distributed in six l-liter Erlenmeyer flasks (each containing 400 ml) and three 2-liter Erlenmeyer flasks (each containing 800 ml). After 3 hr of culture (OD600of 0.6), 100/~M isopropyl-/3-o-thiogalactopyranoside (IPTG) is added. After an additional 5 hr, the cells are collected by centrifugation (5000 g, 10 min). The pellets can then be stored at - 7 0 °. Protein purification is carried out essentially as for the plant enzyme. However, the overproduction of the enzyme by transformed bacteria allows ts j. Sambrook, E. F, Fritsch,and T. Maniatis,in "MolecularCloning:A LaboratoryManual." Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. 1989. f9j. p. Jacquot, R. Rivera-Madrid,P. Marinho, M. Kollarova,P. LeMarrchal, M. MiginiacMaslow, and Y. Meyer, J. MoL Biol. 235, 1357 (1994). 20j. p. Jacquot, E. Keryer, E. Issakidis,P. Decottignies,M. Miginiac-Maslow.J. M. Schmitter. and C. Crrtin, Eur. J. Biochem. 199, 47 (1991).

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[9,5]

some simplification of the procedure. Bacterial extracts are much cleaner than plant extracts, and consequently MgCI2, 2-mercaptoethanol, and PVP can be omitted. The cell pellets are thus resuspended in 50 ml of extraction buffer consisting of 30 mM Tris-HC1 buffer, pH 7.4, 1 mM EDTA, and 100 /zM PMSF. The bacteria are disrupted by three passages in a French pressure cell (60 MPa). The cell debris are removed by centrifugation (30 rain, 20,000 g). Ammonium Sulfate Fractionation. Solid ammonium sulfate (19.4 g per 100 ml supernatant) is slowly added to the protein solution to bring it to 35% of saturation. After 30 min of stirring at 5°, precipitating proteins are eliminated by centrifugation (20 min at 10,000 g). Approximately 15.1 g of solid ammonium sulfate is added per 100 ml supernatant, which brings it to 60% of saturation. The NADP+-malate dehydrogenase precipitates at this point. After 30 min of stirring, it is recovered in the protein pellet obtained by centrifuging the extract for 20 rain at 10,000 g. The proteins are dissolved in approximately 50 ml of extraction buffer and dialyzed overnight against 5 liters of 20 mM potassium phosphate buffer, pH 7.2, containing 1 mM EDTA. The latter buffer (PE) is then used throughout the purification. The sample is cleared after dialysis by spinning for 1 hr at 50,000 g. DEAE-Sephacel Chromatography. Chromatography on DEAE-Sephacel is carried out as for the plant protein. The fractions containing NADP ÷MDH activity (major protein peak) are pooled and directly loaded onto the affinity Matrex red A column. Matrex Red A Chromatography. Matrex red A chromatography is performed as for the plant protein. The enzyme is almost homogeneous after this step (90-95%). Up to 40 mg protein can be obtained and used for most studies without further purification. Dialysis and Storage. Dialysis and storage are as for the plant protein. N-Terminal Sequencing. To check that the recombinant NADP +-MDH is identical to the plant protein, it is necessary to analyze the amino-terminal sequence. The sample is desalted by reversed-phase high-performance liquid chromatography (HPLC) on a Vydac C4 column (4.5 × 150 ram) (Touzart et Matignon, Vitry-sur-Seine, France), eluted with a 20-rain gradient of 35-70% acetonitfile in 0.1% trifluoroacetic acid. This step is also useful to remove the minor contaminants still present in the preparations. The solvents are evaporated in a Speed-Vac concentrator (Savant) and the protein subjected to Edman degradation using an Applied Biosystems (Foster City, CA) Model 476A sequqencer, with on-line HPLC detection of phenylthiohydantoin amino acids. The amino-terminal sequence of the recombinant NADP+-MDH is identical to that of the plant enzyme. The fact that the recombinant protein is produced in the mature form indicates

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that the transit peptide can be cleaved by the E. coli cells. In addition to the major form of the protein, some additional forms, shortened by a few amino acids, are obtained. This microheterogeneity, although less marked, is also observed in the case of the plant protein. Modification of NADP+-Malate Dehydrogenase for Use in NADPH/ NADPH-Thioredoxin Reductase Assay The major drawback for the utilization of NADP+-MDH is that its activation is strongly inhibited by NADP ÷.21 As a consequence, in its native form the wild-type enzyme cannot be used to detect the functioning of the NTR system, as the latter generates oxidized NADP ÷. One way to circumvent this problem is to design an NADP+-MDH molecule, the activation of which is not inhibited by NADP +. This can be achieved by site-directed mutagenesis followed by expression of the recombinant protein in E. coli cells and subsequent purification of the protein. Site-Directed Mutagenesis

The procedures used for site-directed mutagenesis of NADP÷-MDH have been detailed by Issakidis et al. 22 They are based on the use of the method described by Kunkel et al. in this seriesY The technique is based on the use of an uracil-containing single-stranded DNA molecule as a template. Modification is carried out by hybridizing a mutagenic oligonucleotide to the template. Wild-type DNA is then counterselected by transformation of an ung + E. coli strainY First, the cDNA of choice must be cloned into an M13 phagemid which is used to infect E. coli strain RZ1032 (dut-, ung-) in order to generate and purify uracil-substituted single-stranded DNA template. Stock Solutions

10× Kinase buffer: Tris-HCl, 500 mM, pH 7.5; MgCI2, 100 mM; DTT, 20 mM 10× Annealing buffer: Tris-HCl, 200 raM, pH 7.5; EDTA, 20 mM, pH 8.0; NaC1, 500 mM 10× Synthesis buffer: Tris-HCl, 100 raM, pH 7.5; MgCI2,50 raM; DTT, 20 mM ATP 10 mM Mix of the four dNTPs (5 mM each) 21R. Scheibe and J. P. Jacquot, Planta 157, 548 (1983). 22E. Issakidis,M. Miginiac-Maslow,P. Decottignies,J. P. Jacquot, C. Cr6tin, and P. Gadak J. Biol. Chem. 267, 21577 (1992). a3T. A. Kunkel,J. D. Roberts, and R. A. Zakour, this series, Vol. 154, p. 367.

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T4 kinase (->1 U//zl) Klenow fragment of E. coli D N A polymerase I (->1 U/Izl) T4 ligase (->1 U//zl) Phosphorylation of Mutagenesis Oligonucleotide. Mix the following in an Eppendorf tube: 5/~1 water, 1/zl of 10× kinase buffer, 1/zl oligonucleotide (0.1 /zg//zl), 2 /~I of 10 mM ATP, and ] /zl T4 kinase (->1 U//zl). Incubate for 30 min at 37 °. Inactivate the kinase by heating (10 min, 70°). Spin and store on ice.

Annealing of Phosphorylated Oligonucleotide with Single-Stranded Uracil-Containing Template; Polymerization/Ligation. Mix the following in an Eppendorf tube: 2/zl uracil-containing single-stranded D N A (100 ng//zl), 1/zl phosphorylated oligonucleotide, 1 /zl of 10x annealing buffer, and 6 /zl water. Place the mixture at 70° in a dry bath; then let the temperature decrease to 30 ° on the bench. Keep on ice. Add the following to the annealing mixture: 2/xl water, 2/zl of 10x elongation buffer, 2/zl of a mix containing the four dNTPs (5 mM each), 2/zl of 10 mM ATP, 1/xl T4 ligase (->1 U//zl), and 1 tzl of the Klenow fragment of E. coil D N A polymerase (->1 U//zl). Vortex, spin, place for 5 min on ice, and then 5 min at room temperature. Incubate for 90 min at 37 °. After transformation of an E. coil (ung +, dut ÷) strain (DHSc~), six plaques are picked at random and mutant phagemids are identified by sequencing. This procedure yields from 60 to 100% mutants. The mutant sequences are transferred to one of the expression systems described earlier.

NADP+-Malate Dehydrogenase C405A/C417A as Tool for NADPHThioredoxin Reductase/Thioredoxin h Detection Sorghum leaf NADP+-malate dehydrogenase contains 8 cysteine residues, 4 of which constitute two regulatory disulfide bridges located at the N and C termini of the protein as shown in Fig. 1. It has been found that the mutation of either C405 or C417 to Ala or the combined mutation C405A/C417A yields a protein the activation of which is no longer sensitive

C64 C6g

C405 C417

FIG. 1. Schematic drawing of sorghum NADP+-malate dehydrogenase subunit showing the sequence extensions (in black) which are not present in the NAD+-dependent forms, and the positions of the N-terminal (left) and C-terminal (right) bridges. The cysteine numbering includes the 40 amino acids of the transit peptide, which is cleaved in the mature protein.

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to NADP + inhibition. 24 Hence, the mutant protein can be used in a convenient test for the detection of NADPH-thioredoxin reductase (NTR) or thioredoxin h as described below.

Purification of Mutated NADP+-Malate Dehydrogenase The mutated protein, overproduced in E. coli, is purified essentially by following the same procedure as for the wild-type protein. However, it binds more strongly to the Matrex red A affinity column. As a consequence, a 500-ml linear gradient of NaC1 (0-4 M) in PE buffer must be applied, the protein being eluted at 2.7 M NaC1. This very high salt concentration must be removed by two successive dialysis steps (against 5 liters PE buffer) before concentration, in order to avoid protein precipitation. The resulting mutant protein, where the two C-terminal cysteines have been replaced by alanines, can be activated in the presence of NADP + and hence is suitable for the detection of the NTR/thioredoxin h system.

Use of Mutated NADP+-Malate Dehydrogenase for Detection of NA D PH- Thioredoxin Reductase Reagents Tris-HC1, pH 8 (stock solution 1 liter) Thioredoxin h, 1 mg/m125 N A D P H 10 mM (stock solution 1 ml) Mutated N A D P + - M D H 1 mg/ml Activation Conditions. For activation, 10 /zl of 1 M Tris-HCl, 10 /xl NADPH, 5/xl N A D P + - M D H , 20/xl thioredoxin h, 5-20/xl NTR fractions, and water to 100/xl are mixed in an Eppendorf tube, the reaction being started by the addition of NTR fractions. Aliquots (20/xl) are removed every 5 rain and injected into the spectrophotometer cuvette for the measurement of N A D P + - M D H activity. As little as 0.1 /xg of NTR can be detected per 20-/xl sample with this procedure. In addition, because the overal volume for activation is 100/xl instead of 1 ml for the standard 5,5'dithiobis(2-nitrobenzoic acid) (DTNB) reduction assay, the quantities of thioredoxin to be added are much lower (in a homolgous NTR/thioredoxin h assay, a more or less standard Km value is 2/xM). To reach this concentration, 24/zg should be used per ml versus 2.4/xg in a 100-/xl assay mixture. 24 E. lssakidis, M. Saarinen, P. Decottignies, J. P. Jacquot, C. Crdtin, P. Gadal, and M. MiginiacMaslow, J. BioL Chem. 269, 3511 (1994). 2s p. Decottignies, J. M. Schmitter, S. Dutka, J. P. Jacquot, and M. Miginiac-Maslow, Eur..I. Biochem. 198, 505 (1991).

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Properties of Mutant C405A/C417A. The mutated protein still requires preactivation by reduced thioredoxin to be active, with activation kinetics similar to those of the wild-type protein (full activation with 10-15 min of preincubation). However, it exhibits a slight spontaneous activity (5% of the activity of the fully activated protein). As a consequence, controls without thioredoxin must be run in all cases. The mutant protein is also more susceptible to denaturation by repeated freeze/thawing and should be stored in small aliquots at - 7 0 °. Use of Site-Directed Mutagenesis for Identification of ThioredoxinControled Disulfide Bridges In our hands, site-directed mutagenesis proved very useful for the identification of the thioredoxin-dependent regulatory disulfides of NADP +MDH. It has also been applied successfully to identify the regulatory disulfide of phosphoribulokinase. 26 It should be applicable to identify other thioredoxin-regulated sites. As a rule, the identification of the disulfide should consist of the replacement of two single cysteines and the combined replacement of both by isosteric amino acids (serines or alanines), giving identical results. In the case where one of the cysteines is involved in substrate binding or catalysis, one of the single mutations (and the double one) might result in an inactive protein which must then be monitored during purification by immunological methods (dot-blots). The thioredoxin sensitivity of the mutant proteins should be checked using different reduction systems (DTT or a specific reductase), as thioredoxin-independent illegitimate disulfides (reducible by DTT, but not by thioredoxin) might be formed during protein folding in E. coli or repeated freeze/thawing of the pure protein. Candidate cysteines for mutagenesis of thioredoxinregulated chloroplastic enzymes can generally be identified from sequence comparisons with equifunctional but thioredoxin-independent enzymes: they appear in sequence extensions or insertions 27 and are strictly conserved among the various plant sources.

26 S. Milanez, R. J. Mural, and F. C. Hartman, J. Biol. Chem. 266, 10694 (1991). 27 R. Scheibe, Bot. Acta 103, 327 (1990).

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[26] E f f e c t s o f T h i o r e d o x i n o n A c t i v a t i o n o f T r a n s c r i p t i o n Factor NF-KB

By KLAUS

SCHULZE-OsTHOFF, H E I K E SCHENK, a n d W U L F D R O G E

Introduction Studies from different laboratories have shown that the regulation of the intracellular redox state may represent a versatile physiological control mechanism in gene expression. Low molecular weight thiols, such as cysteine or glutathione, control various cellular functions. 1-3 In lymphocytes reducing conditions promote cell proliferation, cytotoxic T-cell activity, and other effector functions. Other processes are facilitated or potentiated under prooxidant conditions or in situations of oxidative stress. 4-1° Reactive oxygen intermediates and lipid peroxides, for instance, have been shown to increase the expression of cytokines,4-6 adhesion molecules, 7,8 and protooncogenes. 9"~° These observations suggest that lymphocyte functions in general and gene expression in particular may be controlled by a delicate balance between prooxidant and antioxidant conditions and that a shift in either of these directions may selectively induce certain sets of genes. The induction of gene expression is in most cases regulated at the transcriptional level by the activation of transcription factors that bind to specific D N A sequences in the promoter region of target genes. The inducible or tissue-specific transcription factors act in conjunction with the basal transcriptional machinery and can dramatically increase the rate of transcription directed by R N A polymerase II. u An important activator of expression of numerous immunoregulatory genes is the transcription factor ~H. Gmtinder, H.-P. Eck, B. Benninghoff, S. Roth, and W. DrOge, Cell ImmunoL 12, 32 (1990). 2 W. Drfge, H.-P. Eck, and S. Mihrn, ImmunoL Today 14, 211 (1992). 3 W. DrOge, H.-P, Eck, and S. Mihrn, Med. lmmunol. 26, 357 (1993). 4 S. Roth and W. Dr6ge, Cell. Immunol. 108, 417 (1987). 5 S. Roth and W. Drfge, Eur. J. lmmunoL 21, 1933 (1991). 6 L. E. DeForge, A. M. Preston, E. Takeuchi, J. Kenney, L. Boxer, and D. R. Rernick, .I. Biol. Chem. 268, 25568 (1993). 7 S. K. Lo, K. Janakidevi, L. Lai, and A. B. Malik, Am. J. Physiol. 264, L406 (1993). s N. Marui, M. K. Offermann, R. Swerlick, C. Kunsch, C. A. Rosen, M. Ahmad, R. W. Alexander and R. M. Medford, J. Clin. Invest. 92, 1866 (1993). 9 D. Crawford, J. Zbinden, P. Amstad, and P. A. Cerutti, Oncogene 3, 27 (1988). 10 K. Nose, M. Shibanuma, K. Kikuchi, H. Kageyama, S. Sakiyama, and T. Kuroki, Eur. J. Biochem. 201, 99 (1991). ~J R. G. Roeder, Trends Biochem. Sci. 16, 402 (I991).

METHODS IN ENZYMOLOGY, VOL. 252

Copyright © 1995 by Academic Press, lnc All rights of reproduction in any form reserved.

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NF-KBJ 2 In noninduced cells, NF-xB resides in the cytoplasm as an inactive complex bound to the inhibitory subunit IKB. On cell stimulation, IKB rapidly dissociates from the inactive complex and allows NF-KB to be translocated into the nucleus and to bind to its D N A sequence. Evidence has been accumulated indicating that this key step is essentially dependent on redox processes. First, NF-KB is rapidly activated in cells treated with hydrogen peroxide or tert-butyl hydroperoxide. 13-15 Second, activation of NF-KB by all stimuli known so far, including cytokines, phorbol esters, and viral transactivator proteins, is commonly inhibited by thiols and other antioxidant compounds. 16-2° However, NF-xB is not directly activated by prooxidants in cell-free systems, implicating the involvement of intermediate redox-sensitive molecules. A signal generated by these molecules may be counteracted by cellular antioxidants. Besides the glutathione system, an important antioxidant is the protein oxidoreductase thioredoxin, zI-23 Here we describe some experimental procedures to evaluate the effects of thioredoxin on NF-KB activation. These methods are suitable to analyze the effects of other thiols as well and may also be used for other transcription factors.

Effects of Recombinant Thioredoxin on NF-t
[26]

THIOREDOXIN AND ACTIVATION OF N F - K B

255

protein fractions are then used to treat HeLa or other cell types and to study the effect on NF-KB D N A binding. Procedure to Express Thioredoxin in Escherichia coli Recombinant human thioredoxin is expressed as a fusion protein containing a tag of 6 histidine residues and a short linker sequence attached to the N-terminus. Owing to the high affinity of the histidine sequence for nickel ions, the protein can easily be purified by nickel-chelate chromatography. An expression kit (QIAexpress) containing the pQE vector, suitable bacterial strains, and the affinity resin can be obtained from Qiagen (Chatsworth, CA). In the vector system expression of thioredoxin is driven from the bacteriophage T5 promoter under control of the Iac operator. Expression is induced in logarithmic cultures of E. coli by the addition of 1 mM isopropylthiogalactopyranoside (IPTG). After 3 hr bacteria are harvested by centrifugation, resuspended in 2-3 volumes of sonication buffer (50 mM sodium phosphate, 300 mM NaC1), pH 8.0, and lysed by sonication. After centrifugation lysates obtained from a l-liter bacterial culture are treated with RNase A (10/xg/ml) and DNase I (5 tzg/ml) and loaded batchwise on a nickel affinity resin (10 ml Ni-NTA resin, Qiagen). Contaminating proteins are removed by extensive washings in sonication buffer, pH 8.0, followed by a washing step in sonication buffer, pH 6.0. Thioredoxin is then eluted from the column with sonication buffer, pH 6.0, containing 250 mM imidazole. The purified fraction is reduced by the addition of 2 mM dithiothreitol (DTT), dialyzed in a nitrogen atmosphere against phosphatebuffered saline (PBS), and stored at - 7 0 °. Dithiol-dependent reducing activity can be assessed by the classic insulin reduction assay. 26 A l-liter bacterial culture usually yields about 8-12 mg of purified thioredoxin. Analysis of Transcription Factor Activation Transcription factors consist of a DNA-binding region and a transactivating domain which interacts with core transcription factors binding in the vicinity of the TATA box element and increases R N A polymerase IIcontrolled m R N A synthesis. Even if a certain gene contains consensus DNA sequences specific for a given transcription factor, this does not necessarily imply that these elements are functional and transactivate gene expression. Consequently, determining the activity of transcription factors generally involves both the measurement of its DNA-binding activity and the assessment of its transactivating effects. DNA-binding activities are routinely determined by gel retardation assays, which provide a quantitative 26 A. Holmgren, J. Biol. Chem. 254, 9627 (1979).

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[261

measure to analyze the effect of thioredoxin on a particular DNA-binding activity. The method is based on the observation that the electrophoretic mobility of an oligonucleotide is altered when a protein is bound to it. For example, binding of active NF-KB in cell extracts to a radiolabeled DNA oligonucleotide containing a KB motif reduces the mobility of the DNA in a nondenaturing polyacrylamide gel and thus results in a complex that can be distinguished electrophoretically from the unbound probe. The transactivating activity is usually determined with the help of reporter gene assays. A regulatory sequence of interest, namely, a DNAbinding site of a transcription factor, is linked to a reporter gene, such as chloramphenicol acetyltransferase, luciferase, or other stable heterologous enzymes. After transfection into appropriate cells, the reporter gene product provides an accurate and quantitative measure of gene expression directed by this element. Procedure to Determine Effects o f Recombinant Thioredoxin on NF-KB Activity

HeLa cells are treated with serial dilutions of recombinant thioredoxin ranging from 2 to 20/zg/ml, which is approximately 0.13 to 1.3/zM. After 1-2 hr, NF-KB activation is induced by the addition of tetradecanoyl phorbol acetate (TPA, 25 ng/ml) or tumor necrosis factor (1 ng/ml). After further incubation for 1 hr, cell monolayers are washed twice with cold PBS and scraped off with a rubber policeman. In pilot experiments, nuclear extracts can be prepared following the protocol described in Refs. 27 and 28. Because NF-KB in its activated form is neither found in the cytoplasm nor activated artificially during the extraction procedure, total cell extracts may also be used in routine tests, which allows NF-KB to be analyzed in relatively small amounts of cell material. For example, 1-3 × 106 cells are lysed at 4° with 60/zl of a high-salt buffer containing 20 mM HEPES, pH 7.5, 400 mM NaC1, 1 mM MgC12, 0.5 mM EDTA, 0.1 mM EGTA, 20% (v/v) glycerol, 5 mM DTT, and 1% nonidet p-40 (NP-40). To prevent proteolysis of NF-KB, a cocktail of protease inhibitors consisting of 1 mM phenylmethylsulfonyl fluoride (PMSF), 0.01% aprotinin, and 10/xg/ml leupeptin should be included. After 10 min of incubation on ice, extracts are cleared by centrifugation (15 rain, 14,000 g, 4°) and kept frozen at - 7 0 ° until use. Depending on the experimental design, DTT may be omitted in the extraction buffer. In this case, oxidation of proteins should be prevented by flushing the samples with nitrogen. Extracts prepared in the same way can also be used to study the effect of other thiol compounds on NF-KB 27j. p. Dignam, R. M. Lebovitz,and R. G. Roeder, Nucleic Acids Res. 11, 1475 (1983). 28E. Schreiber,P. Matthias,M.M. MUller,and W. Schaffner,NucleicAcids Res. 17,6419(1989).

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DNA-binding activity in cell-free systems. In this case, aliquots of the extracts are treated for 30 min with serial dilutions of the thiol under test and then subjected to gel retardation assays. Procedure to Determine NF-KB Activation in Gel Retardation Assay

A DNA probe with a KB motif and flanking sequences is synthesized on a DNA synthesizer (Applied Biosystems, Warrington, UK) and purified by high-performance liquid chromatography (HPLC) or OPC cartridges (Applied Biosystems). Normally, we use a 25-mer consisting of the NF-KB binding site and flanking regions of the mouse kappa (K) light chain enhancer (5'-AGCq-'I'CAGAGGGGATTTCCGAGAGG-3', binding sites underlined). The oligonucleotide is annealed with its complementary strand and radiolabeled by the random-primed labeling procedure (25 ng DNA) using [o~-32p]NTP and Klenow enzyme (Boehringer Mannheim, Mannheim, Germany). Free nucleotides are removed on a push column (Stratagene, Heidelberg, Germany), followed by precipitation in ethanol. For the binding reaction, 2-4/xl of the total cell extract (equivalent to about 10/xg protein) is mixed in a 20-t~l volume with 4/zl of 5-fold binding buffer (20 mM HEPES, pH 7.5, 50 mM KC1, 5 mM DT]', 2.5 mM MgC12, 20% Ficoll), 2 /xg poly(dI-dC) as nonspecific competitor DNA, 2 /~g bovine serum albumin (BSA), and 5,000-10,000 counts per minute (cpm) Cerenkov of the labeled oligonucleotide. After a 30 min binding reaction at room temperature, samples are loaded on a 4% nondenaturing polyacrylamide gel (acrylamide:bisacrylamide, 29:1) and run in 0.5× TBE (TBE is 89 mM Tris, 89 mM boric acid, 2 mM EDTA), pH 8.0, at 12 V/cm for 2-3 hr. After electrophoresis gels are placed on a Whatman (Clifton, N J) 3MM paper, dried on a gel dryer, and exposed to an X-ray film at -70 °. Results are documented by autoradiography and densitometric scanning. Quantitative data can also be obtained by using a phosphoimager device (Molecular Dynamics) or by scintillation counting of the sliced gel. Because extracts obtained from various cell types may sometimes give rise to several shifted bands, the specificity of the DNA-NF-KB complex should by verified by oligonucleotide competition analysis or the use of antibodies specific to certain NF-KB protein subunits. In competition studies the shifted complex should be abolished by an excess of unlabeled wild-type oligonucleotide, whereas derivatives point-mutated in the KB site should not affect binding of NF-KB to the radioactive probe. An example of a gel retardation assay demonstrating the effect of thioredoxin on NF-KB DNA binding in intact cells is given in Fig. 1. HeLa ceils were pretreated for 2 hr with graded concentrations of recombinant thioredoxin, followed by stimulation with TPA for another 2 hr. Inhibition of NF-KB activation by thioredoxin is dose dependent.

258

TH1OREDOXIN AND GLUTAREDOXIN - TPA

TRX

I

(~.g/ml)

NF-KB

~"

0

2

10

[26]

+ TPA 20

i

0

2

10

20

f

!. ~

Fxo. 1. Gel retardation assay showing the effects of thioredoxin (TRX) on NF-KB DNAbinding activity. HeLa cells (1 × 106 cells/well) were seeded in a 6-well plate. Twelve hours later cells were treated with the indicated concentrations of recombinant thioredoxin. After 2 hr, cells were further incubated in the absence or presence of TPA (25 ng/ml). Total cellular extracts were prepared after an additional 2 hr and incubated with the 3ep-labeled NF-KBspecific oligonucleotide. The protein-DNA complex was resolved on a 4% polyacrylamide gel run in 0.5× TBE buffer. The position of the NF-xB-specific D N A complex is marked by an arrowhead.

Effect of Overexpression of Thioredoxin on NF-KB-Dependent Gene Expression A n o t h e r a p p r o a c h to a n a l y z e t h e effects o f t h i o r e d o x i n o n t r a n s c r i p t i o n f a c t o r a c t i v a t i o n is b a s e d o n t h e t r a n s i e n t t r a n s f e c t i o n o f t h i o r e d o x i n c D N A in c o m b i n a t i o n with r e p o r t e r g e n e assays. I n a t y p i c a l e x p e r i m e n t , H e L a ceils a r e c o t r a n s f e c t e d w i t h a N F - K B - c o n t r o l l e d c h l o r a m p h e n i c o l a c e t y l transferase (CAT) plasmid and increasing amounts of thioredoxin cDNA c l o n e d i n t o an e u k a r y o t i c e x p r e s s i o n vector. F o l l o w i n g 2 d a y s o f i n c u b a t i o n , cells a r e e i t h e r left u n t r e a t e d o r s t i m u l a t e d with T P A . T h e effect o f t r a n s i e n t e x p r e s s i o n o f t h i o r e d o x i n o n N F - K B a c t i v a t i o n is m o n i t o r e d b y m e a s u r i n g t h e C A T activity in e x t r a c t s o f t r a n s f e c t e d cells.

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There are a number of methods available for introducing D N A into mammalian cells. The choice of the transfection technique depends greatly on the cell line to be transfected and the design of the experiment. Useful techniques are the DEAE-dextran method, transfection with a calcium phosphate precipitate, electroporation, and liposome-mediated D N A transfer. For transient expression of foreign genes the DEAE-dextran method is a rather convenient and efficient method for a wide range of cell types. The technique is based on the binding of D N A to DEAE-dextran, which is then applied to and endocytosed by the cells. A CAT reporter gene construct directed by a KB-controlled herpes simplex thymidine kinase (tk) promoter can be obtained by insertion of four spaced NF-KB binding sites with the sequence 5'-GGGGAACTTTCC3' into the HindlII-BamHI-opened vector pBLCAT2. In addition to the CAT gene, the vector contains T-regulatory sequences from SV40 and the gene for ampicillin resistance. 29 A vector coding for human thioredoxin (pCMV-ADF) can be obtained by insertion of the full-length thioredoxin cDNA (about 3.6 kb) into the NotI-XbaI-cleaved vector pRc/CMV (Invitrogen, San Diego). Expression is directed by the strong promoter/enhancer of human cytomegalovirus (CMV). In addition, the vector contains a polyadenylation signal from bovine growth hormone, an ampicillin resistance gene, and a neomycin selection gene for stable transfectants. It should be noted that for introducing D N A into eukaryotic cells the quality of the plasmid preparation is crucial. Plasmids are therefore routinely purified by suitable column materials (i.e., Maxiprep columns, Qiagen) or equilibrium centrifugation in CsCl-ethidium bromide density gradients.

Procedure to Transfect HeLa Cells by DEAE-Dextran Method One day before transfection, HeLa cells are seeded in 60-cm2 petri dishes at a density of 3 × 106 cells/dish. On the following day, cells are washed twice with transfection medium (serum-free RPMI 1640 medium, without bicarbonate; GIBCO, Grand Island, NY). A DEAE-dextran solution (Sigma, Deisenhofen, Germany; molecular weight 500,000) is prepared from an autoclaved working stock (10 mg/ml PBS) and adjusted to 1 rag/ ml with transfection medium. Two milliliters of the solution is mixed with 2 ml of D N A dissolved in PBS to give a final D N A concentration of 1418/xg/ml. After washing the cells, the mixture is then added to the cells and incubated for i hr at room temperature with occasional gentle shaking. The amount of D N A that can be used is dependent on the cell type, the plasmid, and the D N A preparation. To enhance the uptake of DNA, cells 29 B. Luckow and G. Schiitz, Nucleic Acids Res. 15, 5490 (1987).

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are further treated with dimethyl sulfoxide (DMSO). After the DNA solution has been soaked off, 10% (v/v) DMSO in RPMI 1640 medium is added to the cells and incubated for 90 sec. As this procedure is quite toxic, the duration of the DMSO treatment should carefully be checked for each cell type. The cells can be further treated with chloroquine, which is believed to enhance the transfection efficiency by inhibiting degradation of D N A during its transit to the nucleus. Cells are washed in RPMI 1640 medium and treated with a freshly prepared chloroquine solution (10/zg/ml). The cells are returned to the incubator and incubated for 2-4 hr. After transfection, cells are washed twice with culture medium and incubated for a further 48-60 hr. In a typical experiment we usually apply a constant amount of CAT reporter gene plasmid (4-8/zg DNA) and increasing amounts of the thioredoxin-encoding plasmid ranging from 0.25 to 10/zg D N A to a 60-cm 2 petri dish. Because cell viability and transfection efficiency are influenced by the amount of D N A transfected, the D N A concentration for each sample should be normalized with irrelevant plasmid DNA, namely, the parental vector DNA. Forty-eight hours after transfection the cells are stimulated with TPA (25 ng/ml) and assayed for CAT activity after a further 15-18 hr of incubation.

Procedure to Determine NF-KB Transactivating Activity by Chloramphenicol Acetyltransferase Assay The most widely used reporter gene is the prokaryotic gene coding for chloramphenicol acetyltransferase (CAT). However, the use of other genes, such as the gene for firefly luciferase, is becoming increasingly popular. The CAT system has several advantages as a reporter gene including the absence of endogenous activity in eukaryotes, high protein stability, and high sensitivity. Several modified protocols to measure CAT activity are available. In the original protocol, 3° cells expressing the CAT gene are opened, and a low-speed extract is prepared. The extract is incubated in the presence of [14C]chloramphenicol and acetyl-coenzyme A (acetyl-CoA). If there is active CAT enzyme present, the substrate chloramphenicol becomes acetylated at the 1' and/or 3' position (Fig. 2). The acetylated derivatives are resolved from the substrate by thin-layer chromatography (TLC) and quantitated by autoradiography, scintillation counting, or direct scanning of the TLC plate. Alternate procedures to measure CAT activity involve the use of [14C]acetyl-CoA31 or immunological techniques. A CAT 30 C. M. Gorman, L. F,. Moffat, and B. H. Howard, Mol. Cell. Biol. 2, 1044 (1982). 31 j. R. Neumann, C. A. Morency, and K. O. Russian, BioTechniques 5, 444 (1987).

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261

NF-KB-C~

3-AcCAM

1-A~AM

CAM

FIG. 2. Autoradiogram of a CAT assay showing the position of the substrate chloramphenicol (CAM) and the l'-acetylated (1-AcCAM) and 3'-acetylated (3-AcCAM) reaction products. In the experiment cells were transfected with the NF-KB-CAT construct using the DEAEdextran method. Forty-eight hours after transfection, cells were either left untreated or stimulated with TPA and assayed for CAT activity after an additional 15 hr of incubation.

enzyme-linked immunosorbent assay (ELISA) can be purchased, for instance, from Boehringer Mannheim. The method of choice is the original protocol as described by Gorman et al. 3° Two days after transfection HeLa cells are harvested with a rubber policeman and washed twice in PBS. Cells from a 10-cm 2 or 60-cm z petri dish are resuspended in approximately 150 to 200/zl of 250 mM Tris-HC1, pH 7.8, and opened by three freeze-thaw cycles and vigorous vortexing. After centrifugation extracts are heated at 65° for 10 min to inactivate endogenous acetyltransferases and stored at - 7 0 °. For the enzyme assay, extracts should be normalized for protein content or activities of other cotransfected marker enzymes, such as luciferase or/3-galactosidase. Normally, we mix 20-80/zl of the enzyme extract (100-150/zg protein) with 2.22 kBq [14C]chloramphenicol (CFA 515, Amersham, Braunschweig, Germany) and 0.53 mM acetyl-CoA (Boehringer Mannheim) in 250 mM Tris-

262

THIOREDOXIN AND GLUTAREDOXlN

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HC1, pH 7.8, to give a final volume of 150/~1. Stock solutions of acetylCoA are quite unstable and should be stored at - 7 0 °. Depending on the CAT enzyme present, the mixture is incubated at 37 ° for 2-12 hr. To ensure linearity of the assay, the reaction should be stopped before significant amounts of the substrate are converted. After the reaction acetylated derivatives are extracted by the addition of 600 txl cold ethyl acetate. The samples are vortexed vigorously and centrifuged briefly to separate the phases. From the top, 500/xl of ethyl acetate is removed and evaporated in a speed vacuum concentrator. The reaction product is resuspended in a small volume of ethyl acetate and spotted on a silica TLC plate (Sigma). Thinlayer chromatography is carried out in chloroform/methanol (95:5, v/v). Finally, the reaction products are quantitated by autoradiography or direct scanning of the TLC plate. With a constant initial amount of substrate, CAT activity can be quantitated as the percentage of chloramphenicol converted to acetylated forms. A typical example of a CAT assay is given in Fig. 3. In this experiment cells have been cotransfected with a KB-controlled CAT reporter construct together with increasing amounts of the thioredoxin-encoding plasmid pCMV-ADF. The experiment illustrates that overexpression of thioredoxin dose-dependently inhibits NF-KB-mediated CAT expression. In contrast, expression of a CAT construct that is directed by the transcription factor AP-1 is enhanced by transient expression of thioredoxin. These results therefore suggest that thioredoxin may play an important role in transcriptional processes and affects gene expression controlled by distinct transcription factors in a different fashion. Concluding Remarks NF-KB is activated by posttranslational mechanisms involving the dissociation of the inhibitory protein IKB from the inactive complex. The release of IKB, a key step for NF-KB activation, is rather incompletely understood. Studies have shown that the process is promoted by prooxidants such as hydrogen peroxide or oxidized glutathione. 13-1s Several antioxidants, in contrast, efficiently inhibit NF-KB activation. 16-2° It is rather unlikely that the direct target of these redox processes is the IKB protein. Some observations suggest that the release of IKB requires the previous activation of kinases and proteases which ultimately initiate proteolytic degradation of IKB. ~2We have seen that thioredoxin can efficiently block NF-KB activation even at micromolar concentrations, suggesting that inhibition of NF-KB activation is probably not caused by a radical scavenging effect. Thioredoxin may rather counteract signaling events triggered by kinases, proteases, or other enzymes.

[261

"rHxoRzoOXIN AND ACTIVATIONOF NF-KB A

NFkB-CAT

25

_

_

_

_ _

263

B

AP-1 -CAT

_ _

-

-

70

-

60

~

15

4o

lO

<E

o

0

0

I

I

I

I

I

I

I

I

I

I

I

I

I

0.25

0.5

1

2

5

10

0

0.25

0.5

1

2

5

10

pCMV-ADF (pg)

0

pCMV-ADF (pg)

FIG. 3. Effects of transient expression of thioredoxin on NF-KB- and AP-l-dependent CAT activity. HeLa cells were seeded in 60-cm2 petri dishes and cotransfected with the indicated amounts of the thioredoxin-encoding plasmid pCMV-ADF together with 8 ~g of either a NF-KB-dependent (A) or an AP-l-dependent (B) CAT reporter gene construct. Hatched bars represent TPA-stimulated cells, and filled bars, unstimulated cells. After the enzyme reaction (6 hr), products were separated on a TLC plate and quantitated using a TLC linear analyzer. Values represent the percentage of radioactivity of acetylated chloramphenicol transferred to the added substrate.

It has been reported that NF-KB activation is also inhibited by the treatment of cells with thiols such as cysteine derivatives, although relatively high concentrations are required. 13-17 Because cyst(e)ine and glutathione serve an important role in various immunoregulatory processes, 1-3'~5 it will be of interest to investigate whether one of these redox couples influences the redox state of thioredoxin or vice versa. Moreover, the regulatory role of thioredoxin reductase is still poorly understood and remains to be investigated. A large body of evidence exists that the redox-controlled mechanism of NF-KB activation is centrally involved in immunological disorders such as human immunodeficiency virus (HIV) infection.2,3'32Because HIV repli32 W. Dr6ge, H.-P. Eck, H. N~iher, U. Pekar, and V. Daniel, Biol. Chem. Hoppe-Seyler 369, 143 (1988).

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[27]

cation is largely controlled by NF-KB, the decreased levels of thiols observed in HIV-infected persons may be directly responsible for disease progression. It will be of interest to study expression of thioredoxin in these patients. Preliminary results indicate that cells overexpressing TAT protein, a regulatory product of HIV-1, contain significantly lower levels of thioredoxin (K. Schulze-Osthoff, H. Schenk, and W. Dr6ge, unpublished data, 1995). Whereas expression of certain genes is promoted under prooxidant conditions, other genes may be induced by antioxidants. We and others have observed that activation of AP-1, another important transcription factor, is strongly potentiated by thioredoxin. 2°'33 This suggests a role for thioredoxin in the regulation of numerous genes and may explain several immunostimulatory effects of thioredoxin observed in earlier studies. 23'34 It appears worthwhile to apply the experimental procedures described here to study the regulation of other transcription factors by thioredoxin. Acknowledgments The a u t h o r s gratefully acknowledge Dr. M. Gtitlich (Munich) and Dr. P. A. Baeuerle (Freiburg) for the kind gift of plasmids.

33M. Meyer, R. Schreck, and P. A. Baeuerle, EMBO J. 12, 2005 (1993). 34y. Tagaya, H. Wakasugi, H. Masutani, H. Nakamura, S. Iwata, A. Mitsui, S. Fujii, N. Wakasugi, T. Tursz, and J. Yodoi, Mol. Immunol. 27, 1279 (1990).

[27] T h i o r e d o x i n

Genetics

B y MARJOR1E RUSSEL

Introduction Studies on Escherichia coli thioredoxin have provided a textbook example of the power of combined genetic and biochemical approaches. This chapter describes genetic approaches taken to elucidate the biological role(s) of thioredoxin in the cell and in bacteriophage replication and assembly, and to understand the basis for some of its structural and biochemical properties. Biological Roles of Thioredoxin Thioredoxin was originally purified from E. coli (as a heat-stable small protein that served as a hydrogen donor for ribonucleotide reductase) METHODS IN ENZYMOLOGY,VOL. 252

Copyright © 1995 by AcademicPress, lnc. All rightsof reproductionin any form reserved.

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in 19643 The first thioredoxin mutants were isolated 10 years later by Chamberlin,2 although they were not identified as such at the time. The mutants were isolated by virtue of their inability to support plaque formation by bacteriophage T7, and the gene was called tsnC (for T seven negative). Modrich and Richardson3'4 used one of the mutants to demonstrate that phage T7 DNA polymerase contained a host-encoded subunit; they isolated the host subunit and, based on its size and heat stability, identified it as thioredoxin. One of the mutants (tsnC or trxA 7004) completely lacked thioredoxin5 and was later shown to be a deletion mutant. 6 Thus, thioredoxin was not essential for the viability of E. coli, and ribonucleotide reductase, an essential enzyme, must have another hydrogen donor. This genetic result led Holmgren to search for an alternative hydrogen donor for ribonucleotide reductase; glutaredoxin, another small, redox-active protein, was subsequently isolated from the thioredoxin deletion mutant strain] Unlike thioredoxin-S2, which is reduced by NADPH and thioredoxin reductase, glutaredoxin-S2 is reduced nonenzymatically by glutathione.8 Advances in molecular biology and so-called reverse genetics (protein leads to cloned gene which leads to mutants) enabled Hoog et al. 9A° to use amino acid sequence information to clone and then sequence the glutaredoxin gene (grx). The cloned grx gene was disrupted (by the introduction of a kanamycin resistance gene within the grx coding region), and the disrupted gene was introduced into the E. coli chromosome in place of the functional gene. H The resulting strain, which lacked glutaredoxin (verified by radioimmunoassay and an in vitro assay that coupled it to reduction of CDP to dCDP by ribonucleotide reductase), was healthy and viable. Thus, glutaredoxin could not be the sole in vivo hydrogen donor for ribonucleotide reductase. It seemed likely that either thioredoxin or glutaredoxin could serve this function. Thus, an attempt was made to construct a double mutant lacking both proteins by introducing the disrupted grx::kan gene into a strain in which the trxA gene had been deleted. Initial attempts failed, c T. C. Laurent, E. C. Moore, and R. P. Reichard, J. Biol. Chem. 239, 3436 (1964), 2 M. Charnberlin, J. Virol. 14, 509 (1974). 3 p. Modrich and C. C. Richardson, J. Biol. Chem. 250, 5508 (1975). 4 p. Modrich and C. C. Richardson, J. Biol. Chem. 250, 5515 (1975). 5 A. Holmgren, I. Ohlsson, and M.-L. Grankvist, J. Biol. Chem. 253, 430 (1978). 6 M. Russel and P. Model, J. Biol. Chem. 261, 4997 (1986). 7 A. Holmgren, Proc. Natl. Acad. Sci. U.S.A. 73, 2275 (1976). A. Holmgren, J. Biol. Chem, 254, 3664 (1979). 9 J.-O. Hoog, H. Jornvall, A. Holmgren, M. Carlquist, and M. Persson, Eur. J. Biochem. 136, 223 (1983). m J.-O. Hoog, H. yon Bahr-Lindstrom, H. Jornvall, and A. Holmgren, Gene 43, 13 (1986). 11 M. Russel and A. Holmgren, Proc, Natl. Acad. Sci. U.S.A. 85, 990 (1988).

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suggesting that cell viability required one protein or the other. However, when the selective (kanamycin-containing) plates were incubated for several days (instead of overnight) small colonies arose. The slow-to-appear cells lacked thioredoxin and glutaredoxin, suggesting the existence of yet a third hydrogen donor for ribonucleotide reductase. Holmgren and coworkers have isolated a new glutaredoxin-like protein from cells lacking thioredoxin and glutaredoxin. 11a Perhaps reverse genetics applied to this protein will provide an answer to the 30-year-old question, "What reduces ribonucleotide reductase?" Genetic analysis has confirmed a more definitive in vivo role for thioredoxin and glutaredoxin in sulfate assimilation.12 Although bacteria lacking either thioredoxin or glutaredoxin grow in defined medium in the absence of amino acids, the strain lacking both proteins requires glutathione, methionine, or cysteine. This requirement is due to an additional mutation that occurred in the trxA grx mutant strain and enabled it to grow better. The additional mutation was mapped and shown to lie in cysA, which encodes sulfate permease. Mutation in cysC [kinases adenosine 5'-phosphosulfate (APS) to form 3'-phosphoadenosine 5'-phosphosulfate (PAPS)] or addition of excess cystine (which represents the cysteine regulon) also restored viability to the trxA grx mutants. Why should elimination of sulfate assimilation be required for viability of trxA grx mutants? It had previously been shown that, in vitro, both thioredoxin and glutaredoxin were efficient hydrogen donors for PAPS reductase, the enzyme in the sulfate assimilation pathway that reduces PAPS to s u l f i t e . 13 Furthermore, mutants in the gene that encodes PAPS reductase (cysH) accumulate secondary mutations in cys genes that act earlier in the pathway, suggesting that PAPS, the activated sulfate intermediate, is toxic to E. coli. TM Russel et al) 2 proposed that trxA grx double mutant cells are functionally cysH-, and that the resultant PAPS accumulation created a selection for cys mutations able to prevent PAPS synthesis. It was concluded that thioredoxin and glutaredoxin can replace one another in sulfate reduction reactions but that one or the other was essential. Analysis of thioredoxin mutants was facilitated by the development of a simple and specific in vivo assay for its redox activity.6 Escherichia coli contain methionine sulfoxide reductase which reduces methionine sulfoxide to methionine and uses thioredoxin as a hydrogen donor) 5 A methioninerequiring mutant forms colonies on minimal plates containing glucose and 11aF. Aslund, B. Ehn, V. A. Miranda, C. Pueyo and A. Holmgren, Proc. Nat. Acad. ScL U.S.A. 91, 9813 (1994). 12M. Russel, P. Model, and A. Holmgren, J. Bacteriol. 1"/2, 1923 (1990). 13M. L.-S. Tsang, J. Bacteriol. 146, 1059 (1981). 14 D. Gillespie, M. Demerec, and H. Itikawa, Genetics 39, 433 (1968). 15j. A. Fuchs and J. Carlson, Thioredoxins Struct. Funct. Colloq. Int. C N R S - N A S A , 111 (1983).

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10-100 tzg/ml L-methionine sulfoxide as a methionine source; however, a mutant containing a trxA deletion does not (efficiency of colony formation <10 -5 that on methionine-containing plates). T r x A substitution mutants that lacked in vitro activity, such as Cys--->Ser mutants, failed to grow in this in vivo assay, whereas mutants that were active in vitro also utilized methionine sulfoxide in place of methionine in vivo. Even when excess glutaredoxin is provided (by the grx gene on a multicopy plasmid), growth of the m e t - trxA double mutants is very slow, with visible colonies forming only after several days of incubation at 37 °. Thus, neither glutaredoxin nor any other redox-active protein in E. coli can efficiently substitute for thioredoxin as a source of reducing power for methionine sulfoxide reductase. Interestingly, although the first mutant (trxB) deficient in thioredoxin reductase was isolated in a m e t - mutant strain on the basis of its slow growth on methionine sulfoxide, 16 a m e t - trxB::kan null mutant formed colonies on methionine sulfoxide-containing plates about as efficiently as m e t - trxB + cells. 6 Thus some level of reduced thioredoxin must exist in E. coli, even in the absence of thioredoxin reductase.

met

Role of T h i o r e d o x i n in B a c t e r i o p h a g e Replication a n d A s s e m b l y The original tsnC or trxA mutants were completely blocked in phage T7 D N A synthesis, although phage transcription and protein synthesis patterns were normal. 2 Extracts of T7-infected tsnC mutant cells showed no phage D N A polymerase activity, although activity could be restored by extracts from uninfected tsnC + ceils. 3 Purified T7 D N A polymerase from infected tsnC + cells is composed of p5 and thioredoxin in a 1:1 stoichiometry. 4 Subsequently it was shown that p5 alone contains all the catalytic activities of the polymerase, and that thioredoxin confers the striking processivity to the enzyme that enables it to polymerize thousands of nucleotides without dissociationJ 7 This feature of T7 D N A polymerase led to its development (minus its potent 3'---~5'-exonuclease activity) as the reagent of choice for dideoxynucleotide-based D N A sequencing. An early question about T7 D N A polymerase function was what role the redox activity of thioredoxin played. Holmgren et al. TM purified thioredoxin from another tsnC mutant, sequenced the protein to identify its Gly92---~Asp mutation, and demonstrated that the mutant protein retained substantial redox activity although it was completely nonfunctional for 1~j. Fuchs, J. Bacteriol. 129, 967 (1977). 17S. Tabor, H. E. Huber, and C. C. Richardson, J. Biol. Chem. 262, 16212 (1987). ~ A. Holmgren, G.-B. Kallis, and B. Nordstrom, J. BioL Chem, 256, 3118 (1981).

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T7 D N A replication; this demonstrated that the two activities were not inextricably linked. Adler and Modrich 19 found that neither thioredoxin$2 nor modified thioredoxin-(SH)2 in which a single cysteine had been methylated formed a complex with p5, suggesting that the sulfhydryls were important either conformationally or as direct participants in subunit interactions. The ability of T7 to grow on trxA mutant strains in which one or both active site cysteine residues of thioredoxin had been changed to serine or alanine z° definitively demonstrated that the redox capacity of thioredoxin is not required for stimulation of D N A polymerase activity. How thioredoxin interacts with p5 to confer processivity is not yet certain. Himawan and Richardson m have used genetic analysis to begin to address this question. They have isolated T7 gene 5 mutants that have gained the ability to grow on E. coli which specify a mutant thioredoxin (Gly74--+Asp) that is defective in complex formation with p5. Reasoning by inference from the structure of the Klenow fragment of E. coli DNA polymerase I (which is homologous to p5), they suggest that thioredoxin may form the "thumblike" domain that in the Klenow fragment has been proposed to act as a clamp to hold the D N A in the binding cleft. Several of the gene 5 suppressor mutants are located in a region of p5 consistent with this hypothesis, and two of them (E319K, E319V) might function by eliminating direct charge repulsion between Asp-74 of the mutant thioredoxin and Glu-319 of p5. 21 The requirement for thioredoxin in filamentous phage assembly, like that in T7 D N A replication, was uncovered by the isolation of an E. coli mutant unable to support filamentous phage growth at 42 ° (phage growth was normal at 340).22 The mutant, originally called tip (for filamentous phage production), was identified as a thioredoxin mutant by determining the size of the cloned tip gene product, 23 by sequencing mutant and wildtype alleles of the cloned tip gene, 24 and by measuring thioredoxin activity in tip mutant extracts. 25 Thioredoxin is required for filamentous phage assembly, as phage infection, gene expression, and D N A synthesis occur normally in trxA mutant cells. The isolation of phage mutants able to grow on the original fip/trxA mutant (Pro34---)Ser in the thioredoxin active site) supports this because such phage mutants had incurred a mutation in a morphogenetic gene, gene I. 2z Whether pI and thioredoxin interact has 19 S. Adler and P. Modrich, J. BioL Chem. 258, 6956 (1983). 20 H. E. Huber, M. Russel, P. Model, and C. C. Richardson, J. Biol. Chem. 261, 5006 (1986). 21 j. S. Himawan and C. C. Richardson, Proc. Natl. Acad. Sci. U.S.A. 89, 9774 (1992). z2 M. Russel and P. Model, J. Bacteriol. 154, 1064 (1983). 23 M. Russel and P. Model, J. Bacteriol. 157, 526 (1984). 24 M. Russel and P. Model, Proc. Natl. Acad. Sci. U.S.A. 82, 29 (1985). 25 C. J. Lira, B. Hailer, and J. A. Fuehs, J. Bacteriol. 161, 799 (1985).

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never been shown directly because pI is a low abundance membranespanning protein and has not been purified. Although Russel and Model proposed that thioredoxin-mediated cycles of oxidation and reduction might drive the polymerization of coat proteins along the length of the filamentous phage genome, they showed that this was not correct by constructing active site mutants in which one or both cysteine residues were changed to serine. 6'24 The mutant proteins were inactive in both in vivo and in vitro redox assays, but cells containing the mutant proteins (and no wild-type thioredoxin) supported filamentous phage growth. 6 In a complementary approach, additional thioredoxin mutants unable to support phage growth were isolated; some of the mutant proteins (and the original Pro34---~Ser mutant) retained full redox activity both in vivo and in vitro. 6 Thus, the redox activity of thioredoxin is not necessary for T7 D N A replication or for filamentous phage assembly. Interestingly, t r x A mutants unable to support filamentous phage assembly are also defective in T7 D N A synthesis, suggesting that the same interactive surface of thioredoxin is used in both systems. Like T7 D N A polymerase, filamentous phage assembly requires thioredoxin-(SH)2. Phage assembly is reduced 10-fold in trxB mutant cells that lack thioredoxin reductase24; however, in t r x B mutant cells that contain mutant thioredoxins with serine substitutions at either of the active site cysteines, high level phage production is restored. 6 That assembly occurs at all in t r x B t r x A + cells is consistent with the methionine sulfoxide-based redox assay (see above) which indicated that some thioredoxin-(SH)2 is present in cells lacking thioredoxin reductase. These observations indicate that it is thioredoxin-(SH)2 which is required for assembly and, furthermore, that the Cys--oSer mutant thioredoxins mimic the thioredoxin-(SH)2) conformation. The similarity of wild-type and Cys---~Ser double mutant thioredoxin has been confirmed directly by nuclear magnetic resonance (NMR). 26 The amino acid substitution mutants of trxA that reduce or eliminate T7 DNA replication and filamentous phage assembly (Pro34---~Ser, Gly74--+Asp, Pro76---~Ala, Gly92-+Asp, and Gly92--~Ser) are not contiguous in the linear thioredoxin sequence. Rather, they are located at or adjacent to the tips of the three "lobes" of thioredoxin that form its active site. Thus, they are clustered at a single face of the folded protein which forms the flat and relatively hydrophobic region that has been proposed to form a surface for interaction with other proteins. 2v Several second site "pseudorevertants" of the Gly92--~Ser mutant, which have regained function in filamentous 26 H. J. Dyson, M. Jeng, P. Model, and A. Holmgren, FEBS Lett. 339, 1l (1994). 27 H. Eklund, C. Cambillau, B.-M. Sjoberg, A. Holmgren, H. Jornvall, J.-O. Hoog, and C.-I. Branden, E M B O J. 3, 1443 (1984).

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phage assembly, were isolated (Ala67--~Val, Ala67---~Pro, and Pro68-->Leu). The nature and location of the second site mutations, namely, in the "bridge" (residues 64-70) that connects the two highly structured domains of thioredoxin, suggest that a relatively minor shift in the orientation of the two domains is responsible for the restoration of function. Although second site pseudorevertants of the GlyV4---~Asp and Gly92--*Asp mutants were sought, only true revertants were obtained. Perhaps charge repulsion between these residues and acidic residues in filamentous phage pI accounts for this, as has been suggested for trxA Asp-74 and T7 p5 Glu-319. Other viruses coopt other host proteins and use them for quite different purposes than the host. One wonders why T7 and filamentous phage have chosen thioredoxin. Although this question is not really answerable, the choice might be a consequence of the abundance of thioredoxin, the availability of its interactive surface, and, for filamentous phage assembly (which takes place at the cell membrane), because of the high concentration of thioredoxin at the periphery of the cytoplasm, close to the membrane? 8'29

Thioredoxin M u t a n t s as Tools to Explore Structure and Mechanism The tsnC and tip mutants were the only trxA mutants available until the advent of molecular biology and techniques for introducing defined mutations into cloned genes. Thus, the use of mutant proteins to examine thioredoxin structure and function is a relatively new development. Filamentous phage cloning vectors that contain the trxA gene have been particularly useful for the isolation of thioredoxin mutants. The phage genome consists of single-stranded DNA, thus providing a template for site-directed mutagenesis. The ability of the resultant mutants to support filamentous phage assembly can then be assessed directly by plating the trxA mutant phage on cells that lack thioredoxin. A great deal of attention has been focused on the highly conserved residues surrounding the active site cysteines of thioredoxin (Trp-Cys-GlyPro-Cys-Lys). Using cloned genes that overproduced mutant thioredoxin, Gleason et aL 3° found that decreasing (to Cys-Ala-Cys) or increasing (to Cys-Gly-Arg-Pro-Cys) the spacing of the cysteines by one residue resulted in the loss of redox function in vivo (using the methionine sulfoxide assay) hut that, when overproduced, both mutant proteins supported T7 and filamentous phage plaque formation. In vitro, the insertion mutant served as substrate for thioredoxin reductase and reduced ribonucleotide reduc28H. Nygren,B. Rozell,A. Holmgren,and H.-A. Hansson,FEBS Lett. 133~145 (1981). 29C, A. Lunn and V. P. Pigiet,J. Biol. Chem. 257, 11424 (1982). 30F. K. Gleason, C. J. Lim, N. M. Gerami, and J. A, Fuchs,Biochemistry 29, 3701 (1990).

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tase, but with greatly reduced efficiency; the deletion mutant was inactive. Despite the high degree of conservation in all known bacterial thioredoxins, Pro-34 and Lys-36 (or Arg) have proved surprisingly tolerant to substitution. When overproduced from a plasmid, the original tip mutation (Pro34--~Ser) supported plaque formation by T7 and filamentous phage and retained full redox function in vivo and in vitro, 6'2°'3° and a Lys3S--~Glu mutant retained substantial function both in vivo and in vitro. 3° The structure of the latter mutant has been obtained; it showed good agreement with the structure of wild-type thioredoxin, although some changes in the active site region preceding the disulfide were noted. 31 Minarik et al. 32 constructed trxA mutants at positions Gly-33 and Pro34. In cells that overproduced the mutant thioredoxins, a Leu-Gln mutant supported T7 and f l plaque formation at reduced efficiency (10 -5 and 10 -8, respectively); with Trp-Pro efficiencies were approximately 10 -v, and with Lys-Phe no plaques were detected. The redox activity of these mutants was not reported. A Pro34--~His mutant that changes the thioredoxin active site to one which mimics the two proposed active sites of protein disulfide-isomerase (Cys-Gly-His-Cys) has also been constructedY This mutant protein has a higher redox potential and reduces insulin more slowly than wild-type thioredoxin; however, it is about 10-fold more efficient in disulfide formation, making it about 10% as efficient as authentic protein disulfideisomerase on a molar basis? 4 The His substitution interferes with complex formation with T7 p5, renders the mutant thioredoxin inactive in the in vivo methionine sulfoxide assay, and may not function in filamentous phage assemblyY Taken together, the analysis of several different Pro-34 mutants illustrates the plasticity of thioredoxin: despite its strict conservation, some substitutions at this position appear to retain full or enhanced redox function (i.e., Pro34---~Ser), whereas the Pro34---~His mutation essentially creates a new redox activity. The ability of T7 or filamentous phage to utilize Pro-34 mutants is unrelated to the effects of these mutants on redox function and is likely to reflect subtle changes in the conformation of the active site surface. All prokaryotic and eukaryotic thioredoxins contain a conserved tryptophan (Trp-31 in E. coli thioredoxin) that is exposed at the active site. This position was also partially tolerant to change. Krause and Holmgren 35 3~ M. Nikkola, F. K. Gleason, J. A, Fuchs, and H. Eklund, Biochemistry 32, 5093 (1993). 32 p. Minarik, M. Kollarova, and A. Brunovska, J. Basic Microbiol. 33, 213 (1993). 33 G. Krause, J. Lundstrom, J. Lopez-Barea, C. Pueyo, and A. Holrngren, J. BioL Chem. 266, 9494 (1991). 34 j. Lundstrom, G. Krause, and A. Holmgren, J. Biol. Chem. 267, 9047 (1992). 3s G. Krause and A. Holmgren, J. Biol. Chem. 266, 4056 (1991).

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constructed four replacement mutants (Tyr, Phe, His, and Ala). These mutant proteins showed good or improved (Trp31Tyr) catalytic efficiency with thioredoxin reductase, although all had decreased ability to reduce ribonucleotide reductase and insulin. The Tyr and Phe mutants supported T7 and filamentous phage growth and were functional in the in vivo methionine sulfoxide assay. A site-directed trxA mutant has been used to confirm that an obligatory isomerization of the imide peptide bond between Ile-75 and Pro-7636 is the rate-limiting step in thioredoxin folding; replacement of Pro-76 with Ala eliminated the slowest kinetic phase in thioredoxin folding? 7 By contrast, the original tip mutation (Pro34---~Ser) had no effect on folding kinetics. Richards and co-workers have used thioredoxin as a model protein and constructed site-directed mutants for more general studies of residue packing in hydrophobic protein cores. In one study, a set of single amino acid substitutions at Leu-42 and Leu-78 was analyzed. All mutants except those to Pro retained the ability to function in filamentous phage assembly, despite changes in size, hydrophobicity, or charge; nonetheless, proteins in the latter class were significantly destabilized? 8 Richards and co-workers point out that inferences about protein function based on a poorly understood in vivo biological assay must be made with caution. Hellinga and Richards have designed a computational procedure to model the introduction of new ligand-binding sites into the interior of preexisting proteins, and they have used this procedure to predict that four substitutions in the hydrophobic core of thioredoxin (LeuT----~Cys,Phe12--*His, Val16---~His, LeuSS~Met) would be sufficient to create a copper-binding site. 39'4° The mutant thioredoxin, called chelatin, bound copper; remarkably, it supported filamentous phage growth when present in sufficient.quantity, an indication that the thioredoxin structure had not been grossly perturbed. Russel and Model had previously reported that some thioredoxin mutants supported filamentous phage growth when the mutant protein was expressed at high levels, but not when it was expressed at low levels. 6 Hellinga and Richards 4° used low level expression of chelatin from a filamentous phage carrying the quadruple trxA mutant gene and selection for plaque formers to identify additional mutations in trxA (at Pro-68 or at Asp-26) that presumably stabilized a thioredoxin structure even closer to the wild type. These quintuple mutant proteins and additional derivatives were analyzed in detail. Although they could fold in the absence of metals, the mutant proteins did 36 R. F. Kelley and E. Stellwagen, Biochemistry 23, 5095 (1984). 37 R. F. Kelley and F. M. Richards, Biochemistry 26, 6765 (1987). 38 H. W. Hellinga, R. Wynn, and F. M. Richards, Biochemistry 31, 11203 (1992). 39 H. W. Hellinga and F. M. Richards, Z Mol. Biol. 222~ 763 (1991). 40 H. W. Hellinga, J. P. Caradonna, and F. M. Richards, J. Mol. Biol. 222, 787 (1991).

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bind copper and mercury, although alternative binding modes were utilized rather than the predicted one. This study provides an important demonstration that rational mutation can be used to gain a deeper understanding of metal binding sites in particular, and protein motifs in general.

Thioredoxin M u t a n t s from Other Organisms Only a few thioredoxin mutants have been isolated in other organisms. Muller and Buchanan 41 cloned and then disrupted the trxM gene encoding thioredoxin m in the cyanobacterium Anacystis nidulans. In a carefully controlled series of experiments, they demonstrated that the protein is essential for photosynthetic growth of this organism. Given the large body of biochemical work on the effect of plant thioredoxin rn on the activity of certain light-regulated proteins, extension of this genetic result should be very informative. Other workers have used mutants of E. coli and spinach chloroplast thioredoxin to examine activation of certain plant enzymes. Thus, de Lamotte et al. 42 changed Asp-61 to Ash (E. coli thioredoxin) and showed that whereas the ability of the mutant protein to reduce insulin and to be reduced by thioredoxin reductase remained unchanged, its ability to activate spinach fructose-l,6-bisphosphatase (FBPase) and corn NADP ÷dependent malate dehydrogenase (MDHase) in the light-activated system was increased. Brandes et al. 43 cloned the spinach gene for thioredoxin f~ constructed Cys46---~Ser (equivalent to Cys-32 in E. coli thioredoxin) and Cys49---~Ser (equivalent to Cys-35 in E. coli thioredoxin) mutants, and demonstrated that only the Cys49---~Ser mutant retained the ability to activate FBPase, thereby conclusively identifying Cys-46 as the primary nucleophile of thioredoxin f. Loferer et al. 44 isolated a Bradyrhizobium japonicum mutant defective in cytochrome-c oxidase activity and in formation of a nitrogen-fixing endosymbiotic association with soybean; surprisingly, the sequence of the cloned gene (tlpA) showed it to be a novel thioredoxin-like protein anchored in the cytoplasmic membrane with the thioredoxin domain facing the periplasm. TlpA may function as a periplasmic protein disulfide oxidoreductase. Although the targets of TlpA are not yet known, the mutant phenotypes point to hitherto unexpected roles for a thioredoxin-like protein. 4~ E. G. Muller and B. B. Buchanan, J. Biol. Chem. 264, 4008 (1989). 42 G, F. de Lamotte, M. M. Miginiac, P. Decottignies, M. Stein. P. Minard, and J. P. Jacquot, Eur. J. Biochem. 196, 287 (1991). 43 H. K. Brandes, F. W. Larimer, M. K. Geck, C. D. Stringer, P. Schurmann, and F. C. Hartman, J. Biol. Chem. 268, 18411 (1993). 44 H. Loferer, M. Bott, and H. Hennecke, E M B O J. 12, 3373 (1993).

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Muller 45 cloned two thioredoxin genes from the yeast Saccharomyces cerevisiae. Either could be deleted with no effect on cell growth or morphology; yeast with simultaneous deletion of both genes were viable, but they showed a longer generation time (and a longer S phase), an increase in cell size, and a requirement for exogenous methionine. In yeast, as in E. coli, glutaredoxin appears to be unable to substitute for thioredoxin in sulfate assimilation. Ren et aL 46 constructed site-directed mutants of a human thioredoxin also known as A D F (adult T-cell leukemia-derived factor). They used the mutant proteins to identify the noncatalytic cysteine responsible for interchain disulfide (i.e., dimer) formation. The properties of a mutant protein lacking the residue, which lies close to the active site, suggest that thioredoxin activity might be regulated by the oxidation state of this cysteine. Acknowledgments I t h a n k Peter Model and A r n e H o l m g r e n for m a n y interesting and useful discussions about thioredoxin. This work was supported by a grant from the National Science Foundation ( D M B 88 17641).

45 E. G. Muller, J. Biot Chem. 266, 9194 (1991). 46 X. Ren, M. BjOrnstedt, B. Shen, M. L. Ericson, and A. Holmgren, Biochemistry 32, 9701 (1993).

[28] F e r r e d o x i n : T h i o r e d o x i n

System

By PETER SCHURMANN

Introduction The ferredoxin: thioroxin system is a well-established regulatory system in oxygenic photosynthesis. 1'2 It regulates, by light-driven thiol-disulfide exchange, the activity of key photosynthetic enzymes. Electrons provided by the excitation of chlorophyll are transferred via ferredoxin and ferredoxin : thioredoxin reductase (FTR) to two types of enzyme-specific thiore1 B. B. B u c h a n a n , Arch. Biochem. Biophys. 288, 1 (1991). 2 B. B. B u c h a n a n , Photosynth. Res. 33, 147 (1992).

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doxins, f and m. The thioredoxins as well as FFR each contain one redoxactive disulfide bridge involved in catalysis, and FTR has in addition a 4Fe-4S cluster. Reduced thioredoxin f then activates (reduces) regulatory enzymes of the reductive pentose phosphate cycle (i.e., fructose-l,6-bisphosphatase, sedoheptulose-l,7-bisphosphatase, phosphoribulokinase), whereas reduced thioredoxin m activates chloroplast enzymes not involved in the cycle (NADP+-dependent malate dehydrogenase, coupling factor ATPase) and deactivates NADP+-dependent glucose-6-phosphate dehydrogenase. In vitro both thioredoxins can be reduced chemically by nonphysiological dithiols, like dithiothreitol (DTT), thus replacing the lightdependent reducing system. In the dark the reduced enzymes are reoxidized by oxygen. The ferredoxin : thioroxin system has been demonstrated in several organisms, and its components have been isolated and characterized. 3-8 The best known system is the one from spinach. Both types of spinach chloroplast thioredoxins have been cloned and expressed in Escherichia coil, 9-1~ which considerably simplifies their purification. The FTR has also been cloned, ~3-~5 but not overexpressed yet. In the following the purification of the recombinant thioredoxins l a n d m expressed in E. coli is described and an update of the earlier purification procedure for F T R 3 from spinach leaves is given.

3 R. A. Wolosiuk, P. Schtirmann, and B. B. Buchanan, this series, Vol. 69, p. 382. 4 N. m. Crawford, A. De La Torre, C. Lara, and B. B. Buchanan, Methods Chloroplast Mol. Biol., 735 (1982). s p. Schtirmann, K. Maeda, and A. Tsugita, Eur. J. Biochem. 116, 37 (1981). 6 M. Droux, J.-P. Jacquot, M. Miginiac-Maslow, P. Gadal, J. C. Huet, N. A. Crawford, B. C. Yee, and B. B. Buchanan, Arch. Biochern. Biophys. 252, 426 (1987). 7 A. Tsugita, K. Yano, L. Gardet-Salvi, and P. Schtirmann, Protein Sequences Data Anal, 4, 9 (1991). 8 H. Iwadate, K. Yano, M. Kamo, L. Gardet-Salvi, P. Schtirmann, and A. Tsugita, Eur. J. Biochem. 223, 465 (1994). 9 M. Kamo, A. Tsugita, Ch. Wiessner, N. Wedel, D. Bartling, R. G. Herrmann, F. Aguilar, L. Gardet-Salvi, and P. Schiirmann, Eur. J. Biochem. 182, 315 (1989). "~F. Aguilar, B. Brunner, L. Gardet-Salvi, E. Stutz, and P. Schtirmann. Plant Mol. Biol. 20, 301 (1992). 11 N. Wedel, S. Clausmeyer, R. G. Herrmann, L. Gardet-Salvi, and P. Schtirmann, Plant Mol, Biol. 18, 527 (1992). t2 M. Morris, G. DelVal, and P. Schtirmann, manuscript in preparation, 1995. 13 M. Szekeres, M. Droux, and B. B. Buchanan, J. Bacteriol. 173, 1821 (1991). 14 S. Marc-Martin, A. Spielmann, E. Stutz, and P. Schiirmann, Biochem. Biophys. Acta (Bioenergetics) 1183, 207 (1993). 1.sE. Falkenstein, A. von Schaewen, and R. Scheibe, Biochim. Biophys. Acta (Bioenergetics) 1185, 252 (1994).

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Thioredoxin f

Assay Methods Principle. The assay is based on the capacity of thioredoxin f t o activate chloroplast fructose-1,6-bisphosphatase (FBPase) when reduced either photochemically (in the presence of thylakoids, ferredoxin, and FTR 16) or, as described here, chemically by DTr. The activity is determined in a twostage assay system: FBPase is first activated during incubation with reduced thioredoxin )~ the resulting FBPase activity is measured, after dilution into the reaction mixture, by a colorimetric determination of the inorganic phosphate (Pi) released or by a coupled spectrophotometric test following NADP + reduction (in the presence of excess phosphoglucose isomerase and glucose-6-phosphate dehydrogenase). The colorimetric assay is ideally suited to look for the presence of thioredoxin f in column fractions. The spectrophotometric assay should be used for all quantitative determinations. Activation. Approximately 0.25 unit FBPase (isolated according to Schtirmann and Wolosiuk 17) is activated for 5 min (or longer if needed) at 25° in 50/zl containing 100 mM Tris-C1, pH 7.9, 5 mM DTT, and thioredoxin f to be assayed. Colorimetric Assay. After activation, 0.45 ml of a reaction mixture containing 100 mM Tris-Cl, pH 7.9, 0.1 mM sodium EDTA, 1.5 mM MgSO4, and 1.5 mM sodium fructose 1,6-bisphosphate is added and incubated at 25 °. The reaction is stopped after 5 to 15 min by the addition of 2 ml reagent for the Pi analysis. The samples are allowed to stand for 5 min before the absorbance at 730 nm is determined spectrophotometrically. One-half micromole Pi yields an absorbance of about 0.67 in a cuvette of 1-cm light path. Conical glass centrifuge tubes have proved to be ideal for this assay. Preparation of Phosphate Analysis Reagent. Combine 150 ml of water, 25 ml of 7 M H2SO4, and 25 ml of ammonium molybdate [(NH4)6MO7024"4H20, 6.6 g/100 ml water], mix well, then add 3.1 g of FeSO4, and dissolve. The reagent is stable for 1 to 2 days. Spectrophotometric Assay. An aliquot (5-10/xl) of the activation mixture is injected into a 1-ml spectrophotometer cuvette containing 1 ml of the following reaction mixture (25°): 100 mM Tris-Cl, pH 7.9, 0.1 mM sodium EGTA, 1.5 mM MgSO4, 1.0 mM sodium fructose 1,6-bisphosphate, 1.75 units phosphoglucose isomerase (EC 5.3.1.9, glucose-6-phosphate iso16 p. Schiarmann and J.-P. Jacquot, Biochim. Biophys. Acta (Protein Structure and Molecular Enzymology) 569, 309 (1979). 17p. Schttrmann and R. A. Wolosiuk, Biochim. Biophys. Acta (Protein Structure and Molecular Enzymology) 522, 130 (1978).

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rnerase), 0.7 units glucose-6-phosphate dehydrogenase (EC 1.1.1.49), 0.3 mM NADP ÷, and 14 mM 2-mereaptoethanol. The N A D P + reduction is followed at 340 nm with a recording spectrophotometer.

Production and Purification of Recombinant Thioredoxin f Principle. Recombinant thioredoxin f, strongly expressed in E. coli, is present in the cells mostly in insoluble form deposited in inclusion bodies. This presents the advantage that the inclusion bodies can be easily purified and the insoluble thioredoxin freed from the bulk of contaminating proteins. It is then solubilized in an alkaline buffer and renatured by dialysis. A final anion-exchange chromatography step removes any contaminating or incorrectly folded protein. Construction of Expression Vector. The coding part of thioredoxin f h a s been introduced into a modified pKK233-2 expression vector to yield a fusion protein z° with a slightly modified and 3-amino acid longer N terminus than the native thioredoxin f. The resulting plasmid, which is under the control of the isopropyl-/3-D-thiogalactopyranoside (IPTG)-inducible lac promoter, is used to transform E. coli strain BN103. Culture. One and one-half liters culture medium (15 g Bacto-tryptone, 7.5 g Bacto-yeast extract, 7.5 g NaC1 in 1.5 liters), supplemented with 50 t~g ampicillin/ml, are inoculated with 15 ml of an overnight preculture of transformed E. coli and incubated in a fermentor. After about 2 hr at 37°, when an OD600 of 0.6 is attained, 1 mM IPTG is added to induce the biosynthesis of thioredoxin f. The cells are harvested (4000 g for 10 rain, 4°) 4 to 5 hr after induction when the culture reaches the stationary phase. Lysis. The harvested E. coli cells (about 10 g wet weight) are resuspended in 30 ml lysis buffer containing 50 mM Tris-Cl, pH 7.9, 0.1 mM sodium EDTA, 100 mM NaC1, 100 ~M phenylmethylsulfonyl fluoride (PMSF) and incubated for 15 rain at 25 ° with 1 mg/ml of lysozyme, then frozen. After thawing, the cell suspension, which becomes rather viscous owing to released DNA, is incubated for 1 hr at 37 ° with 10/~g/ml DNase, 10 p~g/ml RNase, 10 mM MgCI2. Purification of Inclusion Bodies. The lysed cells are centrifuged for 10 rain at 48,000 g, the supernatant cell extract which contains some soluble thioredoxin fis discarded, and the insoluble cell material, composed mainly of inclusion bodies, is washed four times with 30 ml of 50 mM Tris-Cl, pH 7.3, 1 M urea, 10 mM sodium E D T A by resuspension with slow stirring during 10 rain, followed by 10 rain of centrifugation at 20,000 g, and two times with cold distilled water (centrifugation for 15 rain at 15,000 g). This procedure yields at least 1 g of inclusion bodies/10 g cells.

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Solubilization. The inclusion bodies are solubilized at 1 mg/ml in 50 mM K2HPO4, 1 mM sodium EDTA, adjusted to pH 10.5 with KOH, at room temperature with very slow stirring during 4 hr. Insoluble material is removed by centrifugation. Renaturation. The dissolved protein is renatured by two successive dialyses against 8 liters each of 20 mM triethanolamine-acetate buffer, pH 7.0, first at room temperature for 24 hr followed by 24 hr at 5 °. Precipitated material, containing some thioredoxin f, is removed by centrifugation. Purification of Thioredoxin f. After addition of dimethyl sulfoxide (DMSO) to 2% v/v, the renatured protein is collected on a 2.6 × 12 cm Q-Sepharose column equilibrated with 20 mM triethanolamine-acetate, pH 7.0, containing 2% DMSO and eluted with a 1000-ml linear gradient of 0 to 300 mM sodium acetate in column buffer. Thioredoxin f elutes as a unique sharp peak at about 150 mM sodium acetate. The thioredoxin fractions are combined, the DMSO concentration increased to 4%, and thioredoxin fconcentrated by ultrafiltration on a YM10 membrane (Amicon, Danvers, MA), with slow stirring. Excessive concentration should be avoided as thioredoxin f tends to precipitate at concentrations above 100 tzM. Higher concentrations can best be obtained by concentrating in Centricon-10 concentrators. A 1.5-liter bacterial culture yields 30 to 50 mg thioredoxin f. Properties. Recombinant thioredoxinfhas a molecular weight of 13,975, a pI of 6.1, and a molar absorbancy at 278 nm of 16,830 M -1 c m -1. It is indistinguishable from the native protein in its capacity to activate FBPase. The protein is stable when stored frozen; however, repeated freezing and thawing should be avoided.

Thioredoxin m

Assay Methods Principle. The assay is based on the capacity of thioredoxin m to activate chloroplast NADP+-dependent malate dehydrogenase (NADP+-MDH) when reduced photochemically (in the presence of thylakoids, ferredoxin, and F F R 16) or, more conveniently, chemically by DTT. As for thioredoxin f we routinely use the reduction by DTT and measure the thioredoxin activity in a two-stage assay system: N A D P + - M D H is activated with reduced thioredoxin m prior to measuring the N A D P + - M D H activity spectrophotometrically by following N A D P H oxidation in presence of oxaloacetate.

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Activation. One-half unit NADP+-MDH (isolated and partially purified from corn TM) is incubated for 5 min (or longer if needed) at 25 ° in 50/xl containing 100 mM Tris-C1, pH 7.9, 5 mM DTT, and thioredoxin m to be assayed. Spectrophotometric Assay. After activation an aliquot (5-10/xl) of the mixture is injected into a 1-ml spectrophotometer curvette containing 1 ml of reaction mixture (25 °) composed of 100 mM Tris-C1, pH 7.9, 0.5 mM oxaloacetate, and 0.2 mM NADPH. The NADPH oxidation is followed at 340 nm with a recording spectrophotometer. Production and Purification of Recombinant Thioredoxin m Principle. Thioredoxin m expressed in E. coli is present as soluble protein and released quantitatively from the bacteria by osmotic shock. 19 Nucleic acids, present in the extract, and contaminating proteins are removed by hydrophobic interaction, size-exclusion, and anion-exchange chromatography. Construction of Expression Vector. The coding part of thioredoxin m (isomer mc) 11 has been introduced into a modified pKK233-2 expression vector to yield a fusion protein 12 extending the N terminus by 6 residues including one more negative charge (Glu). The resulting plasmid, which is under the control of the IPTG-inducible lac promoter, is used to transform E. coli strain BN103. Culture. The transformed bacteria are grown at 37° in a modified LuriaBertani (LB) medium (10 g Bacto-tryptone, 5 g Bacto-yeast extract, 5 g NaC1 in 1 liter) supplemented with 50/xg/ml ampicillin to an OD600 of 0.6, when IPTG is added to a final concentration of 1 raM. The cells are havested by sedimentation at 4000 g for 10 rain 2 to 3 hr after induction while the culture is still in the log phase. Release by Osmotic Shock. The harvested bacterial cells are carefully resuspended with a glass rod in 0.1 times the original volume of cold hypertonic buffer (20% w/v sucrose, 20 mM Tris-C1, 2.5 mM sodium EDTA, pH 8.0) and left on ice for 10 min. The cells are collected again by centrifugation for 1 rain at 8000 g. The supernatant is decanted and the cells resuspended as before in an equivalent volume of cold hypotonic buffer (20 mM Tris-C1, 2.5 mM sodium EDTA, pH 8.0). After 10 min of incubation on ice, the cells are removed by centrifugation for 1 rain at 8000 g. The supernatant contains the released thioredoxin m. Purification of Thioredoxin m. Enough 1 M NaKPi, pH 7.0, and solid (NH4)2SO4 are added to the supernatant containing the thioredoxin m to L8j..p. Jacquot, B. B. Buchanan, F. Martin, and J. Vidal, Plant Physiol. 68, 300 (1981). ~9N. G. Nossal and L. A. Heppel, J. Biol. Chem. 241, 3055 (1966).

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increase the buffer concentration to 50 rnM and the (NH4)2SO4 concentration to 0.5 raM. After centrifugation for 30 rain at 48,000 g, the supernatant is loaded on a 2.5 × 26-cm Phenyl-Sepharose (Pharmacia) column equilibrated with 50 mM NaKPi, pH 7.0, and 0.5 M (NH4)2SO4. The column is washed with 300 ml of equilibration buffer and eluted with a 600 ml linear gradient of 0.5 to 0 M (NH4)2SO4 in 50 mM NaKPi, pH 7.0, followed by 300 ml buffer. This hydrophobic interaction chromatography removes quantitatively contaminating nucleic acids. The active fractions are concentrated by ultrafiltration on a YM10 membrane (Amicon), centrifuged 30 rain at 48,000 g, and separated on a 2.6 × 95-cm Sephadex G-50F column equilibrated in 20 mM Tris-Cl, pH 8.3, 50 mM NaC1. Thioredoxin m elutes as the major peak, well separated from higher molecular weight proteins and essentially pure. For a final polishing, the thioredoxin m fractions are combined, concentrated, and diafiltered with 20 mM Tris-C1, pH 8.3, on a YM10 membrane (Amicon) and subjected to a separation on a 6-ml Resource-Q column (Pharmacia) equilibrated in diafiltration buffer. Thioredoxin m is eluted with a 150-ml linear gradient of 0 to 200 mM NaC1 in column buffer. The thioredoxin fractions are combined, concentrated, and diafiltered with 20 mM Tris-C1, pH 8.3. Yields of more than 20 mg thioredoxin m per 1 liter bacterial culture can be obtained. Properties. The recombinant thioredoxin m has a molecular weight of 13,384, a calculated pI of 4.5, and a molar absorbancy at 278 nm of 20,500 M -1 cm<. x2 It is indistinguishable from the native protein in its activation behavior. It is very soluble, can be easily concentrated, and is stable when stored frozen. Ferredoxin : Thioredoxin Reductase

Assay Method Principle. The FTR is assayed by measuring light-dependent activation of FBPase in a two-stage assay system. The F F R fraction to be tested is incubated in the light in the presence of thylakoids, ferredoxin, thioredoxin f, and FBPasea6; the resulting FBPase activity is measured, after dilution into the reaction mixture for the coupled spectrophotornetric test. Procedure. Activation is carried out in conical glass centrifuge tubes containing 100 ~1 of the following: 100 mM Tris-C1, pH 7.9, 10 mM sodium ascorbate, 0.1 mM dichlorophenol indophenol, 14 mM 2-mercaptoethanol, 10 /~M ferredoxin, 1 t~M thioredoxin f, 0.5 units FBPase, 17 thylakoids 2° 20 j._p. Jacquot, M. Droux, M. Miginiac-Maslow, C. Joly, and P. Gadal, PlantSci. 35, 181 (1984).

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equivalent to 10 tzg chlorophyll, and the FTR fraction to be assayed. After 5 min of incubation at 25 ° in the dark under argon, the tubes are illuminated for 5 to 15 min. An aliquot (5-10/zl) of the activation mixture is tested for FBPase activity by the spectrophotometric test as described above for thioredoxin f.

Purification of Ferredoxin : Thioredoxin Reductase from Spinach Step 1: Leaf Extract. Six kilograms of spinach leaves (1.2-kg lots) are blended for 3 min in a gallon size Waring blendor containing 1200 ml of 20 mM K2HPO4 and 14 mM 2-mercaptoethanol and filtered through four layers of cheesecloth. To keep a low temperature during blending, onethird frozen leaves are mixed with two-thirds fresh leaves. Step 2: Acid Precipitation. The green filtrate, divided into batches small enough to be centrifuged in one run, is acidified to pH 4.9 with 2 M formic acid and immediately centrifuged at 13,000 g for 5 rain. The green pellets are discarded, and the pale yellow supernatant is adjusted to pH 7 with 2 M NH4OH and maintained at pH 7 throughout the following step. Step 3: Ammonium Sulfate Fractionation. Solid ammonium sulfate is added to give 50% saturation. The precipitate is removed by centrifugation (8 min at 13,000 g) and the ammonium sulfate concentration in the supernatant increased to 80% saturation. The precipitate is collected by centrifugation (10 rain at 13,000 g) and dissolved in 30 mM Tris-Cl, pH 7.9, 14 mM 2-mercaptoethanol (25 ml/kg starting material). Step 4: Acetone Precipitation. Three volumes of cold aetone ( - 2 0 °) are added with constant stirring, and the precipitate is immediately collected by centrifugation at - 1 5 ° (10 min at 9000 g). The pellets are resuspended in 30 mM Tris-Cl, pH 7.9, 14 mM 2-mercaptoethanol (20 ml/kg starting material) and dialyzed overnight against 4 liters of 10 mM Tris-Cl, pH 7.9, 14 mM 2-mercaptoethanol to remove traces of acetone. Polyethylene glycol 20,000 is added to 2% w/v to the dialysis buffer to avoid excessive swelling of the dialysis bags. Dialysis tubing with a molecular weight cutoff of 10,000 is used. Step 5: Buffer Exchange and Concentration. The dialyzed protein solution is clarified by centrifugation (30 min at 48,000 g) and passed at 250 ml/hr over a 5 x 27 cm Sephadex G-25 column in 30 mM Tris-Cl, pH 7.9, 14 mM 2-mercaptoethanol, 400 mM NaC1. The excluded protein fractions are concentrated by ultrafiltration on a YMI0 membrane (Amicon) to below 50 ml and clarified by centrifugation as before.

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Step 6." Gel Filtration. The supernatant fraction from Step 5 is sizefractionated at 90 ml/hr on a 5 x 92 cm Sephacryl S-100HR column in 30 mM Tris-Cl, pH 7.9, 14 mM 2-mercaptoethanol, 400 mM NaC1. The fractions with FTR activity are concentrated and diafiltered on a YM10 membrane with 20 mM Tris-C1, pH 7.9, 14 mM 2-mercaptoethanol, 100 mM NaCI. Steps 1 through 6 are repeated once, and the concentrated protein solutions, from 12 kg starting material, are combined for the following chromatographic separations. Step 7: Anion-Exchange Chromatography. The diafiltered and clarified fraction from Step 6 is loaded on a 5 x 25 cm Q-Sepharose FastFlow (Pharmacia) column equilibrated with 20 mM Tris-C1, pH 7.9, 14 mM 2-mercaptoethanol, 100 mM NaC1. The column is eluted with a 4000-ml linear gradient of 100 to 500 mM NaC1 in the same buffer. The fractions with FTR activity are pooled. Step 8a: Hydroxyapatite Chromatography. The pooled fractions from Step 7 are applied without further treatment to a 2.5 x 19-cm MacroPrep (40-~m particle size) ceramic hydroxyapatite column (Bio-Rad) equilibrated with 10 mM NaKPi buffer, pH 6.8, 14 mM MET. The column is washed with 120 ml equilibration buffer and eluted with a 990-ml linear gradient of 10 to 400 mM NaKPi, pH 6.8, containing 14 mM MET followed by 60 ml 400 mM NaKPi, pH 6.8, 14 mM MET. Step 8b: Affinity Chromatography. Alternatively, the last step can be accomplished by affinity chromatography on ferredoxin-Sepharose. The fractions from Step 7 are concentrated and diafiltered on a YM10 membrane with 25 mM NaKPi, pH 6.8, and loaded on a 2.6 x 6 cm ferredoxin-Sepharose column22 equilibrated with the same buffer. The FTR is eluted with a 500-ml gradient of 0 to 750 mM NaC1 in equilibration buffer. Step 9: Concentration and Storage. The active fractions from the last step are combined, concentrated, diafiltered on a YM10 membrane with 25 mM NaKPi, pH 6.8, and stored at liquid nitrogen temperature. Yields of 0.3 to 0.5 mg pure FTR per kilogram spinach leaves are obtained. Properties. Ferredoxin:thioredoxin reductase is a heterodimer composed of a 12,669-Da noncatalytic8 and a 12,959-Da catalytic subunit. It shows a visible absorption peak at 408 nm with a molar absorbancy of 17,400 M -1 cm -1 (Ref. 6) owing to the presence of the 4Fe-4S cluster. The best preparations have an absorption ratio at 408/278 nm of 0.37. The

21S. Ida, K. Kobayakawa,and Y. Morita,FEBSLett. 65, 305 (1976).

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protein is stable over months when stored in the above buffer at liquid nitrogen temperature.

Acknowledgment Work done in the authors lab has been supported by the Schweizedscher Nationalfonds (31-28811.90 and 31-37725.93).

[29] G l u t a r e d o x i n o

By ARNE HOLMGRENand FREDm~ ASLUND Introduction Glutaredoxin (Grx) was discovered as a glutathione (GSH)-dependent hydrogen donor for ribonucleotide reductase (RR) in a mutant of Escherichia coli lacking thioredoxin (Trx), the classic dithiol hydrogen donor) Purification and characterization of E. coli Grx 2'3 showed a small protein, which also exhibited a general GSH-disulfide oxidoreductase activity with clear similarities to previously described eukaryotic GSH-disulfide oxidoreductases (or transhydrogenases),48 later renamed thioltransferases.6 The thioltransferase has no relation to the GSH transferases, which were later discovered, and the reactions catalyzed are oxidoreductions rather than transferase reactions. Because the structure of glutaredoxin has revealed a GSH binding site, we propose the name glutaredoxin rather than thioltransferase for this group of enzymes (see below). The amino acid sequence of E. coli B Grx was determined 7 and shown to contain 85 amino acid residues including a redox active disulfide within an active site sequence of -Cys-Pro-Tyr-Cys- located in positions 11 and 14 of the molecule (Mr 10,000). A clear homology to a protein originally J A. Holmgren, Proc. Natl. Acad. Sci. U.S.A. 73, 2275 (1976). 2 A. Holmgren, J. Biol. Chem. 254, 3664 (1979). 3 A. Holmgren, J. Biol. Chem. 254, 3672 (1979). 4 E. Racker, J. Biol. Chem. 217, 867 (1955). 5 S. Nagai and S. Black, J. BioL Chem. 243, 1942 (1968). 6 p. Askel6f, K. Axelsson, S. A. Ericksson, and B. Mannervik, F E B S Lett. 38, 263 (1974). 7 J.-O. H66g, H. J6rnvall, A. Holmgren, M. Carlquist, and M. Persson, E l m J. Biochem. 136, 223 (1982).

METHODS IN ENZYMOLOGY, VOL. 252

Copyright © 1995 by Academic Press, Inc. All rights of reproduction in any form reserved.

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known as phage T4 thioredoxin, now renamed T4 Grx, was noted. 8,9Cloning of the gene for E. coli glutaredoxin (grx) 1° and construction of high level expression systems resulted in the isolation of two forms of Grx. I°'1~ Grx85 is the major product, whereas a longer form called Grx-90 was also present as 5-10% of the expressed material. 1°,1a This form is also expressed in wild-type E. coli cells as shown by Western blot analysisJ ~ The longer form could be removed from Grx-85 preparations for structural studies by nuclear magnetic resonance (NMR); it is not yet known if Grx-90 has any additional functions in the cell, as the activity in enzyme assays and with ribonucleotide reduetase is identical to Grx-85.1~,~2 The three-dimensional structure of E. coli Grx has been determined in solution by high-resolution NMR spectroscopy in both oxidized (Grx-S2) and reduced from [Grx(SH)2]. 13-1~ The solution structure by NMR of the mixed disulfide of Grx and GSH has been determined for a mutant form of glutaredoxin, GrxC 1 4 S . 16-1s The three-dimensional structure of Grx is similar to that of Trx; however, the sequence identity is very low, and Grx contains a folding that includes a binding site for GSH, which is also present in other GSHdependent enzymes like GSH peroxidaseJ 6-18 The first mammalian Grx was isolated from calf thymus as a hydrogen donor for the corresponding ribonucleotide reductaseJ 9,2° Determination of the amino acid sequence 2~,z2 demonstrated the same active site, -CysPro-Tyr-Cys-, and a protein with elongations at both the N- and C-terminal ends. Glutaredoxin or thioltransferase has also been isolated originally 8 H. Eklund, C. Cambillau, B.-M. SjOberg, A. Holmgren, H. J6rnvall, J.-O. HO6g, and C.-I. Br~ind6n, E M B O J. 3, 1443 (1984). 9 H. Eklund, M. Ingelman, B.-O. Stiderberg, T. Uhlin, P. Nordlund, M. Nikkola, U. Sonnerstam, T. Joelson, and K. Peteratos, J. Mol. Biol. 228, 596 (1992). 10J.-O. H0/Sg, H. yon Bahr-Lindstr6m, H. Jtirnvall, and A. Holmgren, Gene 43, 13 (1986). 11 O. Bj6rnberg and A. Holmgren, Protein Expression Purif 2, 287 (1991). 12 V. Sandberg, B. Kren, J. A. Fuchs, and C. Woodward, Biochemistry 30, 5475 (1991). 13 p. Sodano, K. Chary, O. BjOrnberg, A. Holmgren, B. Kren, J. Fuchs, and K. Wtitrich, Eur. J. Biochem. 200, 369 (1991). 14 p. Sodano, T. Xia, J. H. Bushweller, O. BjOrnberg, A. Holmgren, M. Billeter, and K. Wiithrich, J. Mol. Biol. 221, 1311 (1991). 15 T. Xia, J. H. Bushweller, P. Sodano, M. Billeter, O. BjOrnberg, A. Holmgren, and K. Wtithrich, Protein Sci. 1, 310 (1992). 16j. H. Bushweller, F. A.slund, K. Wiithrich, and A. Holmgren, Biochemistry 31, 9288 (1992). 17j. H. Bushweller, M. Billeter, A. Holmgren, and K. Wtithrich, J. Mol. Biol. 235, 1585 (1994). a8 j. H. Bushweller, A. Holrngren, and K. Wtithrich, Eur. J. Biochem. 218, 327 (1994). 19 M. Luthman, S. Ericksson, A. Holmgren, and L. Thelander, Proc. Natl. Acad. Sci. U.S.A. 76, 2158 (1979). 20 M. Luthman and A. Holmgren, J. Biol. Chem. 257, 6686 (1982). 2a I.-M. Klintrot, J.-O. H66g, H. J6rnvall, A. Hohngren, and M. Luthman, Eur. J. Biochem. 144, 417 (1984). 22 I. A. Papayannopoulos, Z.-R. Gan, W. W. Well, and K. Biemann, Biochem. Biophys. Res. Commun. 159, 1448 (1989).

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from bovine liver4 or kidney, 23 yeast, 5 and pig liver24-26; the latter protein has also been cloned z7 and expressed 28 by Wells and co-workers. They initially showed that the pig liver enzyme contained 8% carbohydrate as first reported for the rat enzyme. 29'3° Later results showed that the protein lacked carbohydrate, and Grx is therefore identical to thioltransferase. 2°-22 Reaction mechanisms have been proposed based on work on Grx from pig liver31 and erythrocytes. 32The amino acid sequences of 10 different glutaredoxins are aligned in Fig. 1. This includes the finding that E. coli contains a total of three glutaredoxins, called Grxl, Grx2, and Grx3. Grx2 and Grx3 were discovered in double mutants of E. coli Agrx, AtrxA. 33 Grx3 (9 kDa) is an inefficient hydrogen donor for RR, which is induced more than 20-fold in the double mutant. 34'35 The structure and function of Grx2 (27 kDa) remain unknown. 34 Of particular interest is the finding that 350 copies of a virus-coded Grx exist in the particle of vaccinia virus. 36'37 This supports a role of Grx in establishing the virus-host relation. A chapter by Dyson elsewhere in this volume 38 describes structural studies of thioredoxin and glutaredoxin in solution by NMR. Previously, a chapter in this series has summarized results up to 1984, including basic methods for measuring glutaredoxin. 39 Extensive reviews on glutaredoxin have also been published. 4°-42

23 K. Larson, V. Eriksson, and B. Mannervik, this series, Vol. 113, p. 520. 24 Z.-R. Gan and W. W. Wells, J. Biol. Chem. 261, 996 (1986). 25 Z.-R. Gan and W. W. Wells, Anal. Biochem. 162, 265 (1987). 26 Z.-R. Gan and W. W. Wells, J. Biol. Chem. 262, 6699 (1987). 27 y. Yang, Z.-R. Gan, and W. W. Wells, Gene 83, 339 (1989). 28 y . Yang and W. W. Wells, J. Biol. Chem. 265, 589 (1990). 29 B. Mannervik, K. Axelsson, and K. Larson, Biochem. J. 149, 785 (1978). 30 K. Axelsson, S. Erickson, and B. Mannervik, Biochemistry 17, 2978 (1978). 31 Z.-R. Gan and W. W. Wells, J. Biol. Chem. 262, 6704 (1987). 32 S. A. Gravina and J. J. Mieyal, Biochemistry 32, 3368 (1993). 33 M. Russel and A. Holmgren, Proc. Natl. Acad. Sci. U.S.A, 85, 990 (1988). 34 F. Aslund, B. Ehn, A. Miranda-Vizuete, C. Pueyo, and A. Holmgren, Proc. Natl. Acad. Sci. U.S.A. 91, 9813 (1994). ~5 A. Miranda-Vizuete, E. Martinez-Galisteo, F. Aslund, J. Lopez-Barea, C. Pueyo, and A. Holmgren, J. Biol. Chem. 269, 16631 (1994). 36 G. P. Johnson, S. J. Goebel, M. E. Perkus, S. W. Davis. J. P. Winslow, and E. Paoletti, Virology 181, 378 (1991). 37 B.-Y. Ahn and B. Moss, Proc. Natl. Acad. Sci. U.S.A. 89, 7060 (1992). .~8H. J. Dyson, this volume [30]. 39 A. Holmgren, this series, Vol. 113, p. 525. 40 A. Holmgren, in "Handbook of Glutathione" (J. Vina, ed.), p. 145. CRC Press, Cleveland, Ohio, 1990. 41 j. A. Fuchs, in "Glutathione: Chemical, Biochemical and Medical Aspects" (D. Dolphin, R. Poulson, and O. Avramovic, eds.), p. 551. Wiley, New York, 1989. 42 W. W. Wells, Y. Yang, and T. L. Deits, Adv. Enzymol. 66, 149 (1993).

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A s s a y s of G l u t a r e d o x i n Escherichia coli R i b o n u c l e o t i d e Reductase A s s a y ( C D P R e d u c t i o n A s s a y )

The principle of the assay has b e e n described in detail by H o l m g r e n 239 and Thelander and c o - w o r k e r s J 3 The Grx activity is measured as the conversion of [3H}CDP to [3H]dCDP catalyzed by R R (10/xg of subunits R1 and R2 in equal proportions) in the presence of 4 m M G S H , 2 m M N A D P H , 7 tzg/ml yeast glutathione reductase, 1,6 m M ATP, 0.7 m M [3H]CDP [500050,000 counts/min (cpm)/nmol], 12 m M MgCle, and 40 m M H E P E S , p H 7.6, with bovine serum albumin (BSA, 0.5 mg/rnl) in a final volume of 120/~1. Without Grx, 4 m M G S H gives no activity. T o ensure that R R is fully active, it is advisable to p r e t r e a t the enzyme with 10 m M dithiothreitol ( D T I ' ) (30 min at 37 °) and r e m o v e the D T T by gel filtration before the assay (the enzyme m a y be stored in this f o r m in liquid nitrogen), At low concentrations (1-5 m M ) , D T T m a y be used instead of G S H , N A D P H , and g l u t a t h i o n e reductase as the ultimate hydrogen donor. Phosphate buffers should be avoided, because phosphate ion will inhibit activity by complexing the Mg 2~. The presence of phosphate may be tolerated if MgCI2 is exchanged for 0.45 M sodium acetate. 44 One unit of activity corresponds to the formation of l nmol of d C D P per minute at 37 °. The Km value for E. coli G r x l is 0.13 ~M, and the Vm~,x is almost equal to the Vr,~ with Trx (K~ of 1.5 /xM). The thioredoxin system may be used as a positive control in assays. 2,45 43L. Thelander, B.-M. Sj6berg, and S. Eriksson, this series, Vol. 51, p. 227. 44N. C. Brown, Z. N. Canellakis, B. Lundin, P. Reichard, and L. Thelander, Eur. J. Biochem. 9, 561 (1969). 45A. Aberg, S. Hahne, M. Karlsson, A. Larsson, M. Orm0, A. Ahgren, and B.-M. Sj6berg, J. Biol. Chem. 164, 12249 (1989).

Fie;. 1. Sequence alignments of glutaredoxins. The alignments are based on the NMR structures of E. coli Grxl. 14-17 Residues identical in all sequences are printed on a black background. The primary structures were retrieved from the following references: vaccinia virus [G. P. Johnson, S. J. Goebel, M. E. Perkus, S. W. Davis, J. P. Winslow, and E. Paoletti, Virology 181, 378 (1991)], calf thymus,21,~2pig liver~6,28(thioltransferase), rabbit bone marrow IS. Hopper, R. S. Johnson, J. E. Vath, and K. Biemann, J. Biol. Chem. 264, 20438 (1989)I, yeast 1 [Z.-R. Gan, M. A. Polokoff, J. W. Jacobs, and M. K. Sardana, Biochem. Biophys. Res. Commun. 168, 944 (1990)], yeast 2 [C. P. Hollenberg, U. Kleinhans, K. Luetzenkirchen, M. R. Rad, and G. Xu, EMBL/GenBank/DDBJ databanks (1992)] (hypothetical Grx-like protein), phage T4 [B.-M. Sj6berg and A. Holmgren, J. Biol. Chem. 247, 8063 (1972)], E. coli Grxl,7 E. coli Grx3 [F. Aslund, M. Nikkola, T. Bergman, K, D. Berndt, H. J6rnvall, and A. Holmgren, Z Biol. Chem. submitted (1995)1, and Methanobacterium thermoautotrophicum IS. C. McFarlan, C. A. Terrell, and H. P. C. Hogenkamp, J. Biol. Chem. 167, 10561 (1992)].

288

TmOREDOXlN AND GLUTAREDOXIN

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Low Molecular Weight Disulfide Reduction ([3-Hydroxyethylene Disulfide Assay) Glutaredoxin has a G S H binding site, defined by N M R 17 and sitedirected mutagenesis. 46 This G S H binding site is the rationale behind the activity of Grx to reduce glutathionyl mixed disulfides. This activity has been shown to be dependent on only the presence of the N-terminal of the active site half-cystines 47 (see Fig. 1). However, an important exception to this reaction mechanism is the activity of Grx with RR, which has a redox-active disulfide in the active site. Only dithiol Grx [Grx-(SH)2] is able to react with a mechanism similar to Trx-(SH)2.16 The substrate in this case is thus a protein disulfide and not a GSH-mixed disulfide. A n y type of disulfide may be used as a low molecular weight substrate provided that it readily forms a mixed disulfide with GSH. The most frequently used are/3-hydroxyethylene disulfide ( H E D ) and S-sulfocysteine, since the use of these two substrates is not complicated by a high spontaneous background reaction which complicates e.g. the use of L-cystine. The H E D assay is based on the following reactions (where XSSX is H E D and GSSG is oxidized glutathione): G S H + XSSX--9 GSSX + XSH G S H + GSSX GSSG + N A D P H + H + Net:

g~utaredoxin~ GSSH + XSH glutathi. . . . .

ductase > 2 G S H + N A D P +

(1) (2) (3)

N A D P H + H + + XSSX --~ N A D P + + 2XSH

Activity is measured as the decrease of N A D P H absorbance at 340 nm. The reagents in the H E D assay are as follows. A cuvette contains 0.50 ml of 1 m M GSH, 0.4 m M N A D P H , 2 m M E D T A , 0.1 mg/ml BSA, 6/xg/ml glutathione reductase in 0.1 M Tris-C1, p H 8.0. The disulfide substrate H E D is added to each curvette (including the reference curvette) to a final concentration of 0.7 mM. A mixed disulfide between G S H and H E D is formed within 2 min, whereafter Grx is added to the sample cuvettes and buffer to the reference cuvette. Addition of 5 pmol of E. coli G r x l (5/xl of 1.0/zM G r x l ) gives a decrease of absorption at 340 nm of 0.140 min -1. The reaction is linear up to a rate of A340 of -0.200 min -a. The spontaneous background reaction is -0.020 min -1. One unit of activity is defined as the consumption of 1/xmol of N A D P H per minute calculated from the expression (&A340 × V)/(min x 6.2), where Vis the cuvette volume 46 M. Nikkola, F. K. Gleason, M. Saarinen, T. Joelson, O. Bj6rnberg, and H. Eklund, J. BioL Chem. 266, 16105 (1991). 47 y. Yang and W. W. Wells, J. Biol. Chem. 266, 12759 (1991).

1291

GLUTAREDOXIN

289

in milliliters and 6.2 is the millimolar extinction coefficient for NADPH. Pure Grxl, Grx2, and Grx3 from E. coli have the specific activities 220] ~ 287, 34 and 35034 units/rag. Reduction of Dehydroascorbic Acid Reduction of dehydroascorbic acid (DHA) is performed either indirectly, by calculation of the oxidation of N A D P H as described above, or directly as the increase of absorbance at 265 nm (the millimolar absorption coefficient of ascorbic acid is 12.59) by the formation of ascorbic acid. The ingredients in the assay may be as listed above (with the addition of D H A instead of H E D ) or as described by Wells and co-workers48:137 mM sodium phosphate, pH 6.8, i mM EDTA, 2 mM GSH, and 1 mM D H A (without N A D P H and glutathione reductase). Radioirnmunoassay and ELISA of Escherichia coli Grxl The first E. coli strains constructed lacking Grxl were confirmed to lack Grxl protein by a radioimmunoassay, using iodinated purified Grxl. 33 In a subsequent characterization of an analogous set of mutants, MirandaVizuete and co-workers developed a highly sensitive enzyme-linked immunosorbent assay (ELISA) for Grxl determinationsY Purification of Escherichia coli Glutaredoxins Escherichia coli Glutaredoxin 1 The gene coding for the Grxl protein has been cloned 1° and overexpressed in a hpL plasmid system, it The method described below was used for preparation of material used for the determination of the three-dimensional structure of Grxl by NMR. 14'15,17 Escherichia coli N4830/pAHOB1 cells are grown at 30° and induced by heat treatment for 5 hr at 40 °, resulting in expression of Grxl as 20% of the soluble E. coli protein, u Bacterial cells are disrupted in 50 mM Tris, pH 7.5, 1 mM EDTA, and 1 mM phenylmethylsulfonyl fluoride (PMSF). A crude extract is obtained by centrifugation (10,000 g for 15 min). Ammonium sulfate is added to 85% solution, and the precipitated protein is dialyzed against 20 mM potassium phosphate, pH 7.3, 2 mM EDTA. v After dialysis, the material is applied to a column of DEAE-cellulose equilibrated with 20 mM potassium phosphate, pH 7.3, 2 mM E D T A and eluted with 0.2 M potassium phosphate, pH 7.3, 2 mM EDTA. The active fractions are then further purified to homogeneity on 48 W. W. Wells. D. P. Xu, Y. Yang, and P. A. Rocque, J. BioL Chem. 265, 15361 (1990).

290

THIOREDOXIN AND GLUTAREDOXIN

[291

a column of Sephadex G-50. Grx-90 (10% of the material) can be isolated on a small scale by separation on a Mono Q column (Pharmacia, Piscataway, NJ) equilibrated with 50 mM Tris-C1, p H 8.0, 1 mM EDTA, eluting the material with a gradient of 0-350 mM NaC1 in the same buffer. For largescale isolation of Grxl-85, DEAE-cellulose chromatography at pH 6.0 with a gradient of 0.10 to 0.40 M ammonium acetate is employed. 13 To avoid aggregation, the solution is raised to pH 7.5 before ultrafiltration concentration (lyophilization should be avoided), as the solubility of Grxl is very low below pH 5.0. Methods for the preparation of 15N,13C-labeled Grxl and a mixed disulfide of the 15N,13C-labeled Grxl C14S mutant with GSH have been developed) s Escherichia coli Grxl is available from IMCO (Stockholm, Sweden). Escherichia coli Glutaredoxins 2 and 3 The Grx2 and Grx3 forms have been purified from an E. coli AtrxA, grx :: kan strain. 34 A crude extract of the bacteria is prepared as above, whereafter nucleic acids are precipitated with 0.6-0.8% final concentration of slowly added streptomycin solution (10%). The supernatant is acidified to pH 5, and precipitated proteins are discarded by centrifugation. After neutralization, protein is collected with a 40-95% ammonium sulfate precipitation by centrifugation. The precipitate is extensively dialyzed against a buffer of 20 mM Tris, pH 9.5, 1 mM E D T A and then applied to a column of DEAE-cellulose equilibrated with this buffer. Grx2 and Grx3 coelute from the column on addition of 50 mM Tris-Cl, pH 8.0. After concentration and incubation with DTT (10 mM), the 27-kDa E. coli Grx2 separates from the 9-kDa Grx3 on Sephadex G-50 chromatography; alternatively, Grx2 may be removed by heat treatment 75 ° for 5 rain. Activated Thiol-Sepharose Chromatography Activated thiol-Sepharose chromatography is a recently developed technique for affinity chromotography of G r x . 34 This step is for practical reasons usually applied after a gel-filtration step, since it requires the reduced form without DTT. Presumably Grx-(SH)2 binds to the GSH matrix in the column, which is in the form of a GSH-2-pyridyl disulfide, as a mixed disulfide. To stabilize the binding, a buffer of pH 6 is used. For both E. coli Grx148a and pig liver glutaredoxin 47 it has been shown that the Nterminal of the active site cysteine residues has a pKa value that is unusually low, whereas the C-terminal active site residue shows a higher apparent pK~ value (around 9). 49 Thus, at pH 6.0 the SH group of the N-terminal 4sa O. Bj6rnberg and A. Holmgren, to be published. a9 T. E. Creighton, Prog. Biophys. Mol. Biol. 33, 231 (1978).

[291

GLUTAREDOXIN

291

active site cysteine would be in the thiolate form ready to initiate a nucleophilic attack on the GSH-2-pyridyl disulfide in the thiol-Sepharose (Pharmacia). Purification of Mammalian Glutaredoxin The first purification of mammalian glutaredoxin to homogeneity was performed by Luthman and Holmgren,2° which enabled the subsequent determination of its primary structure. 22'22 Partial purifications of presumably the same enzyme had been attempted since 1955 by others. 4'5'23 A breakthrough in the purification of mammalian glutaredoxin came with the pI shift method developed by Gan and Wells,2s which is described in detail in a chapter by Wells et al. elsewhere in this volume.5° Human placenta Grx has been purified to homogeneity, and the amino acid sequence was determined,51 based on the method by Luthman and Holmgren2° and the aforementioned pl shift method. The gene for human Grx was cloned and characterized. 5~ Concluding Remarks Glutaredoxin has been found in all organisms containing GSH, and even in one glutathione-negative species, namely, the archaebacterium Methanobacterium thermoautotrophicum. 52 Its presence in the genome of the fast replicating phage T4 demonstrates its importance for DNA synthesis as a hydrogen donor for RR. 53'54 Except as hydrogen donor for RR, the only well-established function of Grxl in vivo is that for sulfate (or PAPS) reduction. 55'56 Either Grxl or Trx has to be present in order to permit growth in liquid minimal media. 33 Neither Grxl nor Trx is essential for DNA synthesis, owing to the presence in E. coli of the Grx isoenzymes Grx2 and Grx3. 34 However, our data strongly suggest that in wild-type cells Trx and Grxl are both important hydrogen donors for RR, as the levels of ribonucleotide reductase activity in trx A, grx (the gene for Grxl) double mutants are increased 19- to 23-fold and only slightly elevated in single mutantsY In an attempt to investigate if a mutator phenotype was associs0 W. W. Wells, D. P. Xu, and M. P. Washburn, this volume [4]. 5t C. A. Padilla, E. M. Martinez-Galisteo, J. A. B~ircena, G. Spyrou, and A. Holmgren. Eur. J. Biochem. 227, 27 (1995). 52 S. C. McFarlan, C. A. Terrell, and H. P. C. Hogenkamp, J, Biol. Chem. 267, 10561 (1992). 53 O. Berglund, J. Biol. Chem. 244, 6303 (1969). 54 D. M. LeMaster, J. ViroL 59, 759 (1986). 55 M. Russel, P. Model, and A. Holmgren, J, Bacteriol. 172, 1923 (1990). 56 M. Russel, this volume [27].

292

THIOREDOXIN AND GLUTAREDOXIN

[29]

ated with the lack of Trx and/or Grxl, these mutants were investigated for sensitivity to different mutagenic agents. No major differences in sensitivity was recorded between the different mutants, suggesting well-balanced deoxynucleoside-triphosphate (dNTP) pools despite the lack of efficient hydrogen donor systemsY The interconversion of the reducing equivalents between GSH and ascorbic acid is likely to be of great physiological importance 57 and to be mediated by Grx. 48 The general activity of Grx with low molecular weight disulfides or GSHmixed disulfides (see above) has led to its characterization by a number of assays using different disulfide substrates. As it has not been apparent in the earlier studies that the actual substrate in the reaction is a mixed disulfide with GSH, Grx has been named after its activity with its supposed disulfide substrate. Examples of names referring to Grx in the literature include GSH-homocystine reductase 4 and thiol-disulfide transhydrogenaseJ Based on a model which did not include a GSH-mixed disulfide intermediate, Grx was named thioltransferase. 6 To avoid further confusion, we advocate the glutaredoxin name, which is easy to associate with the name of the functionally related disulfide reducing protein thioredoxin. Thus, Grx-(SH)2 acts either as a dithiol reductant with protein disulfides or as a specific GSH-mixed disulfide reductase because of its GSH binding site. The substrate specificity of Grx is often different from that of thioredoxin, which has only low activity (high Km value) with low molecular weight disulfides. For proteins it appears that there is a clear difference in substrate specificity owing to steric and charge factors around the disulfide. This forms the basis for unknown redox regulation effects on proteins with synergistic or opposing effects of Trx and GSH and Grx, Acknowledgments This research was supported by grants from the Swedish Cancer Society (961), the Swedish Medical Research Council (Project 13X-3529), the Inga-Bfitt and Arne Lundbergs Stiftelse, the Knut and Alice Wallenbergs Stiftelse, and the Karolinska Institute. The excellent secretarial assistance of Mrs. Lena Ringd~n is gratefully acknowledged.

57 A. Meister, Biochem. Pharmacol. 44, 1905 (1992).

[30]

NMR

OF THIOREDOXIN

AND GLUTAREDOXIN

[30] N u c l e a r M a g n e t i c R e s o n a n c e and Glutaredoxin

293

of Thioredoxin

By H. JANE DYSON Introduction Thioredoxins and glutaredoxins from a number of sources, with molecular weights of the order of 9000-12,000, are sufficiently small to be amenable to nuclear magnetic resonance (NMR) spectroscopy. NMR spectra of thioredoxin were reported as early as 1976,1 but extensive NMR studies on these proteins were not made until the advent of high-field spectrometers in the 1980s. One of the major advances has been the development of methods to utilize NMR information to derive three-dimensional structures of proteins and other macromolecules (reviewed in Clore and Gronenborn2). This application has provided a major impetus in the study of thioredoxins and glutaredoxins by NMR, as X-ray crystallography cannot be used to derive structures in the majority of cases because crystals are not formed. An exception to this is the well-known X-ray crystal structure of the oxidized form of Escherichia coli thioredoxin,3-5 which was crystallized in the presence of Cu(II).6 Reduced E. coli thioredoxin could not be crystallized under the same conditions owing to the redox reaction between the copper ions and the protein dithiol, and its three-dimensional structure has therefore been solved by NMR spectroscopy;7 high-resolution structures of both forms of the protein under identical conditions have been determined. ~ The structure of reduced human thioredoxin cloned from T lymphocytes and overexpressed in E. coli has also been solved by NMR, 9 and a comparison of the solution structures of the oxidized and reduced forms of a A. Holmgren and G. Roberts, FEBS Letr 71, 261 (1976). 2 G. M. Clore and A. M. Gronenborn, Annu. Rev. Biophys. Biophys. Chem. 20, 29 (1991). 3 B.-O. SOderberg, A. Holmgren, and C.-I. Br~indfn, J. Mol. Biol. 90, 143 (1974). 4 A. Holmgren, B.-O. S6derberg, H. Eklund, and C.-I. Br~indfn, Proc. NatL Acad. Sci. U.S.A. 72, 2305 (1975). S. K. Katti, D. M. LeMaster, and H. Eklund, J. Mol. Biol. 212, 167 (1990). A. Holrngren and B.-O. SOderberg, J. Mol. Biol. 54, 387 (1970). 7 H. J. Dyson, G. P. Gippert, D, A. Case, A. Holmgren, and P. E. Wright, Biochemistry 29, 4129 (1990). 8 M.-F. Jeng, A. P. Campbell, T. Begley, A. Holmgren, D. A. Case, P. E. Wright, and H. J. Dyson, Structure 2, 853 (1994). 9 j. D. Forman-Kay, G. M. Clore, P. T. Wingfield, and A. M. Gronenborn, Biochemistry 30, 2685 (1991).

METHODS IN ENZYMOLOGY, VOL. 252

Copyright © 1995 by Academic Press. Inc. All rights of reproduction in any form reserved,

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THIOREDOXIN AND GLUTAREDOXIN

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structurally similar mutant human protein has been published. 1° The Xray structure of T4 glutaredoxin (thioredoxin) has been determined, ~1 but E. coli glutaredoxin has not been crystallized. The NMR solution structures of both oxidized and reduced glutaredoxin have been determined, m3 As well as providing structural information, the NMR spectrum of a protein can also be used to probe enzyme mechanism and function and the binding of ligands. A number of such studies have been performed for thioredoxins and glutaredoxins and are summarized in this chapter.

Nuclear Magnetic Resonance of Thioredoxin The high thermal stability and small size of thioredoxin (108 amino acids for E. coli and 105 for human) make it a suitable candidate for NMR spectroscopy. Other desirable attributes in a protein to be studied by NMR are a high solubility without aggregation (for maximum sensitivity of measurement), stability at relatively low pH values (for lowered solvent exchange of amide protons), and a high yield of recombinant protein from the bacterial expression system used (for maximum efficiency of incorporation of isotope labels, if these are to be used). Thioredoxins from E. coli and human sources meet many of these criteria. Solution Conditions for Nuclear Magnetic Resonance Measurement Thioredoxin is prepared in the oxidized form using E. coli strain SK3981 containing the thioredoxin gene (trxA) cloned on a derivative of the plasmid pBR325. TM Preparation of labeled thioredoxin utilized strain Cla, which grows well on minimal medium, t5 Purification of protein by column chromatography TM follows a modification of the method of Holmgren and ReichardJ 6 Reduced thioredoxin for NMR is prepared from the oxidized protein by reduction with dithiothreitol (DTT). Interference from the 1H signals of the DTT is avoided either by use of fully deuterated DTT or by passage of the solution containing reduced thioredoxin and unlabeled DTT through 10j. Qin, G. M. Clore, and A. M. Gronenborn, Structure 2, 503 (1994). 11 B.-O. SSderberg, B.-M. Sit)berg, U. Sonnerstam, and C.-I. Brtind6n, Proc. Natl. Acad. Sci. U.S.A. 75, 5827 (1978). i2 p. Sodano, T.-H. Xia, J. H. Bushweller, O. Bjt~rnborg, A. Holmgren, M. Billeter, and K. Wtithrich, J. Mol. Biol. 221, 1311 (1991). 13T.-H. Xia, J. H. Bushweller, P. Sodano, M. Billeter, O. BjSrnberg, A. Holrngren, and K, Wtithrich, Protein Sci. 1, 310 (1992). 14 H. J. Dyson, A. Holmgren, and P. E. Wright, F E B S Lett. 228, 254 (1988). 15 K. Chandrasekhar, G. Krause, A. Holmgren, and H. J. Dyson, F E B S Lett. 284, 178 (1991). 16 A. Holmgren and P. Reichard, Eur. J. Biochem. 2, 187 (1967).

[30]

NMR

OF THIOREDOXIN AND GLUTAREDOXIN

295

i

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1H Chemical Shift (ppm) FIG. 1. The 500 MHz 1H NMR spectrum of oxidized Escherichia coli thioredoxin in 90% 1H20/10% 2H20, pH 5.7, 30°. The resonance of the 1H20 protons at approximately 4.6 ppm has been deleted for clarity,

a final Sephadex G-25 column under argon to remove the D T T . ~7 Both the oxidized and reduced forms of E. coli thioredoxin are soluble to at least 4 mM at pH 5.7 in 100 mM phosphate buffer1,14 and to 5-8 mM in 150 mM NaCI, 20 mM phosphate, pH 5.7. is This is the lowest pH that can be used before broadening of resonances in the NMR spectrum is observed, indicating the onset of aggregation, t Under these conditions, the 1H NMR spectrum is well-dispersed with narrow resonance lines, typical of a wellfolded, monomeric protein of this size (Fig. 1). The related protein cloned from human T lymphocytes is approximately 25% homologous with the E. coli protein, mainly in the active site region. 19 Only the reduced form of unmodified human thioredoxin has so far been studied by NMR, because of the presence of three cysteine residues in addition to the two in the active site. The additional thiols cause problems with aggregation of the protein on oxidation.2° Reduced human thioredoxin ~7 H. J. Dyson, A. Holmgren, and P. E. Wright, Biochemistry 28, 7074 (1989). is D. M. LeMaster and F. M. Richards, Biochemistry 27, 142 (1988). 19 H. Eklund, F. K. Gleason, and A. Holmgren, Proteins 11, 13 (1991). ~0 j. D. Forman-Kay, G. M. Clore, P. C. Driscoll, P. Wingfield, F. M. Riehards, and A. M. Gronenborn, Biochemistry 28, 7088 (1989).

296

THIOREDOXIN AND GLUTAREDOXIN

[30]

is soluble to at least 2 mM, and solutions prepared in 100-150 mM phosphate buffer containing 0.2 mM DTT under argon appear to be stable at pH 7.0 or 5.5 for at least 2 months, over a temperature range of 40-500. 20 Solubility is reduced below pH 5.6. 21 The recombinant human thioredoxin isolated from an E. coli expression system is heterogeneous at the N terminus, with about 30% containing an additional methionine residue, 2° although the N-terminal methionine is processed more efficiently during bacterial growth in minimal media. 22

Resonance Assignment It is obvious from Fig. 1 that the assignment of all of the proton resonances of thioredoxin to individual protons in the protein molecule is not possible from the one-dimensional (1D) spectrum alone. The development of two-dimensional (2D) and higher dimensional spectroscopy has been an important breakthrough in the resonance assignment problem for macromolecules, e3Higher dimensions are obtained by frequency and phase modulation in a series of 1D spectra, giving spectra with chemical shift (frequency) scales in two or more dimensions that contain cross-peaks off the planar or body diagonal representing the 1D spectrum. Depending on the sequence of the radiofrequency pulses used to obtain the spectrum, different information is conveyed by the presence of cross-peaks at particular positions. For example, in a 2D correlated (COSY) spectrum, a cross-peak between proton resonances at two frequencies indicates that they are coupled, usually by being attached to adjacent carbon or nitrogen atoms, but in a 2D nuclear Overhauser effect (NOESY) spectrum, a cross-peak indicates that the protons are close together, usually within 5 ~ , but not necessarily coupled or close together in the amino acid sequence. It is in fact the information obtained from long-range NOE cross-peaks obtained from NOESY spectra that allows three-dimensional structures to be calculated from NMR data. The two-dimensional spectra of thioredoxin are well-dispersed, as seen, for example, in Fig. 2, which shows a portion of the COSY spectrum of oxidized E. coli thioredoxin. This particular part of the 2D IH spectrum, containing the cross-peaks between the amide protons and C"H, is known as the fingerprint region, since there is only one cross-peak for each amino acid residue (2 for glycine) in this region of the spectrum. Complete 1H resonance assignments have been obtained for the reduced and oxidized 21 j. D. Forman-Kay, G. M. Clore, and A. M. Gronenborn, Biochemistry 31, 3442 (1992). 2z j. D. Forman-Kay, A. M. Gronenborn, L. E. Kay, P. T. Wingfield, and G. M. Clore, Biochemistry 29, 1566 (1990). 23 K. Wiithrich, "NMR of Proteins and Nucleic Acids." Wiley, New York, 1986.

[30}

N M R OF THIOREDOXIN AND GLUTAREDOXIN

297

-3.0 ~Val 86

~' Ala 29

3.5

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915

I

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[

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FIG. 2. Portion of a 500 MHz 2QF COSY spectrum of oxidized thioredoxin in 90% IH20/ 10% 2H20, pH 5.7, 30°. This part of the spectrum, known as the fingerprint region, shows the cross-peaks that arise from the coupled N H and C~H protons of the amino acid residues, and each four-lobed cross-peak shows one such connectivity. The inset shows schematically the position of this region in the full 2D spectrum of the protein.

forms of E. coli thioredoxin 17 and for the reduced form of human thioredoxin 2° using IH two-dimensional methods alone.

Structure Determination from 1H Data Once resonance assignments have been obtained, it is possible to calculate a three-dimensional structure of the molecule. This is done by extracting interproton distances from the NMR spectrum via the nuclear Overhauser effect (NOE). An NOE is typically observed between two protons that lie in the folded protein within about 5 A, and the intensity of the NOE is proportional to the inverse sixth power of the internuclear distance. Observation and quantitation of the NOEs between protons, especially those between residues far distant in the sequence, give a set of distance restraints that can be used together with other information such as dihedral angle restraints provided by spin-spin coupling constants, distance geometry, or similar algorithms to give the three-dimensional structure. Such a structure has been obtained for reduced E. coli thioredoxin from 2D 1H

298

THIOREDOXIN AND GLUTAREDOXIN

[30]

FIG. 3. Stereo view showing the best-fit superpositions of the backbone (N, C ", and C) atoms of 12 NMR solution structures of reduced E. coli thioredoxin (residues 3-108) obtained using the distance geometry program DISGEO followed by molecular dynamics refinement with AMBER. The active site (residues 32-35) is at the bottom right of the structure. [Adapted with permission from H. J. Dyson, G, P. Gippert, D. A. Case, A. Holmgren, and P. E. Wright, Biochemistry 29, 4129 (1990). Copyright 1990 American Chemical Society.]

structure has been obtained for reduced E. coli thioredoxin from 2D ~H data, 7 and a superposition of 12 of these structures is shown in Fig. 3. N M R solution structures are usually presented as families. The slight differences in the calculated structures arise because the distances and dihedral angles obtained from N M R data are not known accurately. The data are therefore presented as a set of boundary conditions (upper and lower bounds) rather than as discrete distances or angles. Individual structures calculated using these ranges may have slightly different starting and end points, hence the variability in the structures. The correspondence between the independently calculated structures [usually expressed as a root-mean-square difference (rmsd) between the structures or from a mean structure] gives a measure of how well the distance and dihedral angle restraints determine the structure.

Multidimensional Heteronuclear Nuclear Magnetic Resonance and Determination of High-Resolution Structures Although the homonuclear proton spectrum in this case allowed us to obtain a recognizable and even quite high-resolution structure by some criteria, 24 the problem of resonance overlap in two-dimensional spectra of thioredoxin is still substantial. The introduction of the stable NMR-active 24 M. W. MacArthur and J. M. Thornton, Proteins 17, 232 (1993).

[301

NMR

OF THIOREDOXIN AND GLUTAREDOXIN

299

nuclei 13C and 15N into the protein gives much greater resolution in multidimensional spectra and allows the assignment of a much greater proportion of the available NOEs as distance restraints,2 as well as a more accurate determination of dihedral angle constraints from a number of homonuclear and heteronuclear coupling constants. The additional resolution obtainable from three-dimensional heteronuclear NMR spectra is illustrated in Fig. 4 for oxidized E. coli thioredoxin: the dispersal of resonances into a third dimension according to the z3C chemical shift greatly reduces resonance overlap and allows unambiguous assignment of many more NOE crosspeaks. Assignments of the 15N and ~3C spectra have been made for both oxidized and reduced E. coli thioredoxin,~5"25and high-resolution structures for both forms of the protein using the greater volume of data available from the three-dimensional heteronuclear spectra have been calculated, s Extensive 15N assignments and coupling constant measurements have been made for reduced human thioredoxin,22 and a high-resolution structure has been calculated. 9 The 13C, 1H, and 15N resonance assignments have also been made for the reduced form of a triple mutant of human thioredoxin, in which the three cysteine residues other than those in the active site have been mutated to alanines.26 The mutant has been used in structural studies for the comparison of the oxidized and reduced forms of human thioredoxin. ~° The three-dimensional structures of the E. coli and human thioredoxins are quite similar. The E. coli protein in both the X-ray crystal structure 5 and the NMR solution structuresv,8 has a central five-stranded twisted t3 sheet with two long, regular ot helices and two shorter helices, one irregular helix and one apparently 310 helix. The solution structure of reduced human thioredoxin9,~° shows a similar overall topology, and the central 13 sheet is retained, including the presence of a 13 bulge between the fourth and fifth 13 strands. There are also four helices in human thioredoxin, but they are in general longer and more regular? The active site is similar among all of the thioredoxin structures studied, consistent with the high degree of sequence similarity in this region.

Deuterium Labeling of Thioredoxin Oxidized Escherichia coli thioredoxin has also been extensively used as a paradigm for a number of methodological advances in the labeling of proteins for NMR. A number of papers from the laboratories of LeMaster 25 K. Chandrasekhar, A. P. Campbell, M.-F. Jeng, A. Holmgren, and H. J. Dyson, J. Biomol. N M R 4, 411 (1994). 26 j. D. Forman-Kay, G. M. Clore, S. J. Stahl, and A. M. Gronenborn, J. Biomol. N M R 2~ 431 (1992).

38: <:::+ I

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[30]

N M R OF THIOREDOXIN AND GLUTAREDOXIN

301

and Richards have explored the possibilities of deuteration as a means of resonance assignment, especially of prochiral protons such as C~H~ and leucine and valine methyl groups. 18'27-29 Oxidized E. coli thioredoxin was also used to show the utility of selective enrichment with 2H, 13C, and 15N,3°'3~ and a number of resonance assignments were made for oxidized thioredoxin by utilizing these methods. 18'3° The advantages of deuteration for the proton resonance assignment of larger proteins have been pointed out, 32 and selective deuteration is now being applied as a method for identification of protein folding intermediates of thioredoxin. 33

Additional Information from Nuclear Magnetic Resonance Spectrum A number of measurements and observations can be made in the NMR spectrum that give a wide variety of information in addition to the determination of the three-dimensional structure. 15N and 13C relaxation measurements in solution can give an estimate of the backbone and side chain dynamics of the polypeptide chain at each residue. The backbone and tryptophan side chain dynamics of oxidized and reduced thioredoxin have been estimated using uniformly aSN-labeled protein 34 and show a number of regions, especially in turns and loops, where backbone flexibility is high compared to that of the polypeptide present in ordered secondary structure. 27 D. M. LeMaster, FEBS Lett. 223, 191 (1987). ~ D. M. LeMaster, FEBS Lett. 233, 326 (1988). 29 D. M. LeMaster, this series, Vol. 177, p. 23. 30 D. M. LeMaster and F. M. Richards, Biochemistry 24, 7263 (1985). 3~ D. M. LeMaster and F, M. Richards, in "Thioredoxin and Glutaredoxin Systems: Structure and Function" (A. Holmgren, C.-I. Br~ind6n, H. J6rnvaU, and B.-M. Sj0berg, eds.), p. 67. Raven, New York, 1986. 3~ D. M. LeMaster, Q. Rev. Biophys. 23, 133 (1990). 33 D. S. Wishart, B. D, Sykes, and F. M. Richards, Biochim. Biophys. Acta (Protein Structure and Molecular Enzymology) 1164~ 36 (1993). 34 M. J. Stone, K. Chandrasekhar, A, Holmgren, P. E. Wright, and H. J. Dyson, Biochemistry 32, 426 (1993).

F1G. 4. (A) Portion of a 500 MHz IH NOESY spectrum of oxidized thioredoxin in 99% 2H20, pH 5.7, 30°, showing the NOE cross-peaks between protons attached to aliphatic carbon atoms. Cross-peaks to the C~H resonance of Pro-76 are circled, but it is apparent that resonance overlap even in this relatively clear region of the 2D spectrum makes the cross-peaks ambiguous. (B) The same portion of a single plane (13C = 56.1 ppm) of a three-dimensional 13C N O E S Y - H M Q C (heteronuclear multiple quantum correlation) spectrum of oxidized thioredoxin acquired under the same conditions. The NOE connectivities to the Pro-76 C~H are now unambiguous. [Adapted with permission from H. J. Dyson and P. E. Wright, in "TwoDimensional NMR Spectroscopy--Applications for Chemists and Biochemists" (W. R. Croasmun and R. M. K. Carlson, eds.), VCH Publ., New York, 1994.]

302

THIOREDOXIN AND GLUTAREDOXIN

[30]

The rather low proportion of flexible regions in thioredoxin is consistent with the exceptionally high thermal stability of this protein in either oxidation state. 35 The active site loop is also apparently flexible, but measurements are hampered because the sequence contains a proline, for which the lSN relaxation parameters are not measurable, and a glycine, for which the amide proton has an extremely rapid exchange with solvent. 17,36Differences in the flexibility of loops adjacent to the active site occur between the oxidized and reduced forms of the protein (see later section). Insights into the mechanism of thioredoxin can be gained by a study of the pH dependence of the NMR spectrum. The thiol-disulfide exchange reaction of thioredoxin, apparently two hydride transfers, probably proceeds under aqueous conditions via concerted electron-proton transfer. Control of the pH in the active site region is therefore likely to be crucial to the reaction. The pKa for deprotonation of one of the active site cysteines in reduced E. coli thioredoxin, Cys-32, was shown in chemical modification studies to be considerably lowered from normal, to a value of about 6.7, 37 consistent with its role as a thiolate primary nucleophile in the thioldisulfide exchange reaction. An NMR study of reduced and oxidized thioredoxin as a function of pH between pH 5.7 and pH 1038 indicated that the mechanism is probably considerably more complex, pKa values of 7.1 and 8.4 were obtained for two groups titrating in the active site region of reduced thioredoxin, consistent with the values previously obtained for the two active site thiols. 37 However, a third group also titrates in the active site region, giving a pKa of 7.5 in oxidized thioredoxin and approximately 7.1 in reduced thioredoxin. Mutant studies by N M R 39 and other methods 4°'41 indicate that this titrating group is most likely the buried carboxyl group of the highly conserved aspartic acid at position 26, which is thus revealed as an important factor in the pH control of the mechanism of E. coli thioredoxin. The behavior of human thioredoxin is significantly different? 1 This protein is apparently not subject to the same aggregation problems encountered with the E. coli protein at pH values lower than about pH 5.5, and a set of NMR data between pH 2 and pH 10 has been obtained. No obvious effect of the buried aspartate carboxyl group is seen in these studies, possibly owing to the absence of the salt-bridging side chain of Lys-57 present in the E. coli protein. The pKa of the Cys-32 thiol is about 35 T. Hiraoki, S. B. Brown, K. J. Stevenson, and H. J. Vogel, Biochemistry 27, 5000 (1988). 36 M.-F. Jeng and H. J. Dyson, Biochemistry 34, 611 (1995). 37 G. B. Kallis and A. Holragren, J. Biol. Chem. 255, 10261 (1980). 38 H. J. Dyson, L. L. Tennant, and A. Holmgren, Biochemistry 30, 4262 (1991). 39 H. J. Dyson, M.-F. Jeng, P. Model, and A. Holmgren, FEBS Lett. 339, 11 (1994). 40 K. Langsetmo, J. A. Fuchs, and C. Woodward, Biochemistry 30, 7603 (1991). 41 K. Langsetmo, J. A. Fuchs, C. Woodward, and K. Sharp, Biochemistry 30, 7609 (1991).

[30]

NMR OF THIOREDOXINAND GLUTAREDOXIN

303

6.5; this low value is suggested zl to be due to the presence of a salt bridge between the Cys-32 thiolate and the Cys-35 amide proton. 9 Although such an interaction is present in both the crystal and solution structures of oxidized E. coli thioredoxin, it is absent in the reduced form of the protein owing to the van der Waals contacts between the two sulfur atoms. 8 Additional structural information is available from N M R studies of the water molecules bound to proteins. 42 Determination of the positions of bound water molecules in the solution structure of reduced human thioredoxin 43 revealed the presence of four tightly bound water molecules, one of which appears to be in close proximity to the active site, between the side chains of Asp-26 and Cys-35. Several of the positions found for reduced human thioredoxin are similar to those seen in the highly refined X-ray structure of oxidized E. coli thioredoxin) Differences between Oxidized and Reduced Thioredoxin The oxidized and reduced forms of E. coli thioredoxin display some functional differences not entirely caused by the chemical difference between the disulfide and the dithiols. 44 A comparison of the structures of oxidized and reduced thioredoxin 7'8 shows only very small differences between the two forms. The N M R spectra of oxidized and reduced thioredoxin are very similar, except in the region of the active site, where significant differences in the chemical shifts of a number of resonances is observed. 15,~7'25 Backbone coupling constants appear to be very similar between the two forms, but a number of N O E s in the active site region are different. 25 In summary, structural differences are very small between oxidized and reduced E. coli thioredoxin. Thus, the functional differences must be explained in another way. A clue to the reason for the functional difference comes from the study of the backbone and tryptophan sidechain dynamics of thioredoxin. 34 A significant difference in the behavior of several loops, especially that between residues 73 and 76 in contact with the active site, suggest that the two forms of the protein may have significant differences in the flexibility of the active site region, allowing the reduced form of the protein to take up functionally significant conformations of slightly higher energy that are accessible because of the greater flexibility. This interpretation is borne out by a comparison of the rates of amide proton hydrogen exchange in the two forms of the protein, 36 which indicate 42G. Otting, E. Liepinsh, and K. Wtithrich, Science 254, 974 (1991), 43j. D. Forman-Kay, A. M. Gronenborn, P. T. Wingfield,and G. M. Clore, J. Mol. Biol. 220, 209 (1991). 44M. Russell, this volume [27].

304

THIOREDOXIN AND GLUTAREDOXIN

[30]

a significantly greater protection in the active site region in oxidized thioredoxin, consistent with lower flexibility of the polypeptide chain. Nuclear Magnetic Resonance of Glutaredoxin Nuclear magnetic resonance measurements and solution structures have been made for the glutaredoxin from E. coli 12,~3,45 using mainly ~H 2D methods, with the addition of three-dimensional 15N NOESY spectra for the resolution of ambiguities in NOE assignments and for measurement of coupling constants.

Solution Conditions for Nuclear Magnetic Resonance Measurement The solution conditions required for the glutaredoxin NMR measurements were less than optimal for structure determination, 12'4s owing to aggregation of the protein below pH 7.0. The NMR spectra were therefore acquired using a 2.5 mM solution at pH 7.0 in 50 mM phosphate buffer. Glutaredoxin is apparently susceptible to proteolysis, but this problem was overcome by the use of protease inhibitors. 45 Heterogeneity in the NMR spectra of oxidized glutaredoxin was also apparent because of the presence of about 10% of a form elongated at the N terminus or conformational isomerism.45 No conformational heterogeneity was reported for reduced glutaredoxin.12 A more detailed description of the conditions used to obtain glutaredoxin in quantities sufficient for NMR is given by •slund and Holmgren. 46

Resonance Assignments and Structure Determination The 1H resonance assignments were made for oxidized45 and reduced lz glutaredoxin, and three-dimensional structures were calculated. 12,13 The structure of glutaredoxin consists of a four-stranded /3 sheet and three helices, and it is very similar in both oxidized and reduced forms of the protein. 13 A superposition of 20 structures for each form of the protein is shown in Fig. 5. A comparison of the hydrogen exchange behavior of the two proteins by NMR reveals a number of differences in local dynamics near the active site and the C-terminal a helix) 3 Interestingly, despite a very low sequence homology even in the vicinity of the active site, a9 the topology of glutaredoxin resembles that of thioredoxin, lacking only the N-terminal/3 strand and the first helix. 45 p. Sodano, K. V. R. Chary, O. Bj/Srnberg, A. Holmgren, B. Kren, J. A. Fuchs, and K. Wtithrich, Eur, J, Biochem. 200, 369 (1991). 46 A. Holmgren and F./~slund, this volume [29].

130]

NMR

OF THIOREDOXIN AND GLUTAREDOXIN

305

k

C

C

FIG. 5. Stereo views of the 20 conformers used to represent the solution structure of (A) reduced and (B) oxidized E. coli glutaredoxin. The complete polypeptide backbone from residues 1 to 85 is shown. Conformers 2 to 20 were superimposed with conformer 1 (which has the smallest residual violations) for pairwise minimal rmsd of the backbone atoms N, C", and C' of residues 1 to 8, 13 to 43, and 60 to 83. The active site (residues 11-14) is at the top right of the structure. ]Reproduced with permission from (A) P. Sodano, T.-H. Xia, J. H. Bushweller, O. Bj6rnberg, A. Holmgren, M. Billeter, and W. Wtithrich, J. Mol. Biol, 221, 1311 (1991). Copyright 1991 Academic Press; and (B) T.-H. Xia, J. H. Bushweller, P. Sodano, M. Billeter, O. Bj6rnberg, A. Holmgren, and K. Wiithrich, Protein ScL 1, 310 (1992). Copyright 1992 Cambridge University Press.]

306

THIOREDOXIN AND GLUTAREDOXIN

[301

Structure of Mixed Disulfide Intermediate by Nuclear Magnetic Resonance The mechanism of thiol-disulfide exchange of glutaredoxin was thought to be similar to that of thioredoxin, proceeding via a mixed disulfide intermediate between the Cys-ll thiol and a molecule of glutathione. 47 To investigate the mixed disulfide intermediate, a mutant glutaredoxin was prepared with Cys-14 substituted by serine. Glutathione could then be bound to the remaining active site cysteine, Cys-ll, forming a mixed disulfide which is presumed to have the same structure as that of the complex with the wild-type protein, but which is stable because reaction of the second thiol (now a serine) with a second molecule of glutathione and release of the oxidized glutathione dimer are not possible. The C14---~S glutaredoxin mutant and the mixed disulfide complex with glutathione have been prepared and characterized by 15N NMR47; the studies confirm that the glutathione molecule is attached via a disulfide bond to Cys-ll. The reported solution structure of the complex shows that the covalently attached glutathione molecule lies along a shallow cleft in the protein that includes a number of hydrophobic residues. 4s Conclusions For small, soluble, monomeric proteins such as thioredoxin and glutaredoxin, NMR spectroscopy can provide a great deal of site-specific information, including, but not limited to, distance and dihedral angle data that can be used to obtain high-resolution three-dimensional structures. In addition, questions of dynamics and mechanism can be addressed, giving insights into the mode of action of these ubiquitous proteins at the level of individual atoms and functional groups. Acknowledgments I thank Professor Arne Holmgren for helpful discussions and Dr. Peter Wright for a critical reading of the manuscript, This work was supported by Grant GM43238 from the National Institutes of Health.

47 j. H. Bushweller, F./~slund, K. Wiithrich, and A. Holmgren, Biochemistry 31, 9288 (1992). 48 j. H. Bushweller, M. Billeter, A. Holmgren, and K. Wfithrich, Z Mol. Biol. 235, 1585 (1994).

[31 ]

SELENOPHOSPHATE

309

[31] Selenophosphate By RICHARD S. GLASS and THRESSA C. STADTMAN Introduction M o n o s e l e n o p h o s p h a t e has b e e n identified l as the biological selenium donor required in p r o k a r y o t e s for the synthesis of selenocysteine in selenium-dependent enzymes 2'3 and for 2-selenouridine residues in tRNAs. 4-6 This reactive, oxygen-labile c o m p o u n d is synthesized f r o m A T P and selenide by selenophosphate synthetase, the product of the s e l D gene. 7-9 Using 31p nuclear magnetic resonance ( N M R ) spectroscopy 6 the enzyme product was detected as a new resonance at 23.3 p p m c o m p a r e d to orthophosphoric acid (0 ppm). W h e n selenide containing 77Se (spin ½) was substituted for normal selenium isotope abundance selenide, the resonance at 23.3 p p m was split, and the magnitude of the coupling constant (561 Hz) showed that selenium is b o n d e d directly to phosphorus in the new compound. Chemical synthesis of authentic m o n o s e l e n o p h o s p h a t e t provided a reference c o m p o u n d that was used to establish identity of the selenophosphate c o m p o u n d f o r m e d by the enzyme. 6'9 In this chapter a procedure for the chemical synthesis of O,O,Otrimethylsilylselenophosphate and its hydrolysis under anaerobic conditions in aqueous buffers is described. Selenophosphate, either 75Se-labeled or nonradioactive, is synthesized under strictly anaerobic conditions f r o m A T P and selenide by selenophosphate synthetase, isolated from an overproducing E x c h e r i c h i a c o l i strain. 1,2'6,8'9

I R. S. Glass, W. P. Singh, W. Jung, Z. Veres, T. D. Scholz, and T. C. Stadtman, Biochemistry 32, 12555 (1993). 2 W. Leinfelder, K. Forchhammer, B. Veprek, E. Zehelein, and A. B0ck, Proc. Natl. Acad. Sci. U.S.A. 87, 543 (1990). 3 K. Forchhammer and A. Bock, J. Biol. Chem. 266, 6324 (1991). 4 A. J. Wittwer, L. Tsai, W.-M. Ching, and T. C. Stadtman, Biochemistry 23, 4650 (1984). 5 A. J. Wittwer and T. C. Stadtman, Arch. Biochem. Biophys. 248, 540 (1986). 6 Z. Veres, L. Tsai, T. D. Scholz, M. Politino, R. S. Balaban, and T. C. Stadtman, Proc. Natl. Acad. Sci. U.S.A, 89, 2975 (1992). 7 A. Ehrenreich, K. Forchhammer, P. Tormay, B. Veprek, and A. B6ck, Eur. J. Biochem. 206, 767 (1992). I. Y. Kim, Z. Veres, and T. C, Stadtman, J. Biol. Chem. 267, 19650 (1992). 9 Z. Veres, I. Y. Kim, T. D. Scholz, and T. C. Stadtman, J. Biol. Chem. 269, 10597 (1994).

METHODS IN ENZYMOLOGY, VOL. 252

Copyright © 1995 by Academic Press. Inc. All rights of reproduction in any form reserved.

310

MIMICS OF BIOLOGICAL THIOLS AND 1NHIBITORS

[3 1]

Chemical Synthesis of Selenophosphate

Synthesis of (CH~O)~PSe Materials and Reagents Trimethyl phosphite (Aldrich, Milwaukee, WI). Selenium powder (Aldrich) Benzene Procedure. Following the procedure of Bhardwaj and Davidson, 1° 6.2 g (50 mmol) of trimethyl phosphite, 5 g (65 mmol) of selenium powder, and 12 ml of benzene are stirred at room temperature for 24 hr. The mixture is then filtered. The benzene is distilled off at atmospheric pressure and the residue distilled under vacuum to afford 6 g (60% yield) of O,O,Otrimethyl selenophosphate, (CH30)3PSe, bp 72 ° (11 mm), literature value 1° bp 730-74 ° (11 mm).

Synthesis of (TMSO)~PSe O,O,O-Trimethylsilyl selenophosphate, (TMSO)3PSe, can be conveniently prepared by the reaction of iodotrimethylsilane (TMSI) with (CH30)3PSe.

Materials and Reagents TMSI (Aldrich, Milwaukee, WI)

Procedure. A 1.4-g (6.9-mmol) sample of (CH30)3PSe and 5 g (21 retool) of TMSI are heated at 95 ° for 20 hr. The solution is then fractionally distilled to afford 2.59 g (97% yield) of (TMSO)3PSe, bp 80 ° (0.6 mm).

Hydrolysis of (TMSO)3PSe Hydrolysis of the O-trimethylsilyl groups of (TMSO)3PSe can be accomplished easily on a small scale. 1 However, owing to its insolubility in water, it is better to first convert (TMSO)3PSe to water-soluble (DIPEAH) + [(TMSO)2P(Se)(O)]- and then hydrolyze as described below, if the reaction is done on a large scale.

Materials and Reagents (TMSO)3PSe in CHC13 (60 mM) Tricine (0.1 M ) - K O H buffer, containing 20 mM dithiothreitol (DTT), 60 mM MgCI2, pH 7.2 10 R. K. Bhardwaj and R. S. Davidson, Tetrahedron 43, 4473 (1987).

[311

SELZNOeHOSPnA'rE

311

KOH, 6 M HC1, 6 M Procedure. (TMSO)3PSe dissolved in chloroform to a final concentration of 60 mM is mixed with an equal volume of 0.1 M Tricine-KOH buffer, pH 7.2, containing 20 mM DTT and 60 mM MgC12. The separation of phases is enforced by centrifugation. The pH of the water phase is adjusted by adding 6 M KOH or HCI to the desired value, All experiments are carried out using argon to protect the selenium compounds against oxidation.

Synthesis of (DIPEAH)+[(TMSO)2P(Se)(O)]In the presence of the sterically hindered base diisopropylethylamine selectively reacts with one of the TMS groups of (TMSO)3PSe in dichloromethane (DCM) to generate the water-soluble salt (DIPEAH)+[(TMSO)2P(Se)(O)] --. Because all reactants are soluble in the organic solvent, this reaction is conveniently accomplished. 1

(DIPEA), 2-propanol

Materials and Reagents DIPEA (Aldrich) 2-Propanol DCM (TMSO)3PSe Hexane Procedure. A degassed solution of 390 mg (3.0 mmol) of DIPEA and 180 mg (3.0 mmol) of 2-propanol dissolved in 2 ml of anhydrous DCM is cannulated into a degassed solution of 188 mg (0.50 mmol) of (TMSO)3PSe dissolved in 2 ml of anhydrous DCM under an argon atmosphere and cooled in a dry ice-acetone bath. After being stirred for 5 rain, the reaction mixture is warmed to room temperature. After 15 min at room temperature, the mixture is concentrated under vacuum to about 0.5 ml and cooled to 0°, and hexane is added dropwise until 150 mg (70% yield) of (DIPEAH) + [(TMSO)eP(Se)(O)]- precipitates as a colorless solid.

Hydrolysis of (DIPEAH) +[(TMSO)2P(Se)(O)]Owing to the water solubility of the salt, it is more convenient to hydrolyze it than (TMSO)3PSeJ 1 The hydrolysis of the TMS groups occurs on dissolution in water. 11R. Kamifiski,A. Skowrofiska,J. Michalski,and R. S. Glass, unpublished.

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Materials and Reagents Tricine (0.9 M ) - K O H buffer KOH, 6 M HC1, 6 M Procedure. A 22-mg (1.0-mmol) sample of (DIPEAH)+[(TMSO)2 P(Se)(O)]- is dissolved in 0.2 ml of doubly distilled water. The sample is then diluted to a total volume of 0.5 ml with 0.9 M Tricine-KOH buffer adjusted to the desired pH value by adding 6 M KOH or HC1. The solutions are analyzed by 31p NMR spectroscopy.

Enzymatic Synthesis of Selenophosphate

Preparation of Enzyme Escherichia coli MC410012 containing two plasmids, pGP1-2 (carrying the kanamycin resistance gene and the heat-inducible T7 polymerase gene 13 and pMN340 (carrying the ampicillin resistance gene and selD behind the T7 promoter 2, is cultured under overproducing conditions and used as the source of enzyme.9 Harvested cells are frozen in liquid nitrogen and stored at - 8 0 ° prior to use. Sonic extracts are prepared from one part (wet weight) of E. coli cells suspended in 2 volumes of 50 mM Tricine-KOH buffer, pH 7.2, containing 2 mM DT-I', 0.1 mM MgTitriplex (Merck, Darmstadt, Germany), 10 tzg/ml DNase I, and 0.5 mM phenylmethylsulfonyl fluoride. Soluble proteins in the extract are precipitated by addition of ammonium sulfate (385 mg/ml) and redissolved in buffer A (50 mM Tricine-KOH, pH 7.2, containing 2 mM DTT and 0.1 mM Titriplex). One method of purification that gives essentially homogeneous enzyme9 consists of two successive DEAE-Sepharose chromatographic steps (each preceded by gel filtration on Sephadex G-25 to remove salt), precipitation of enzyme from dilute solutions by addition of ammonium sulfate, and finally molecular sieve chromatography on Sephacryl S-200. By this procedure an average of 100 mg of highly purified enzyme is obtained from 15 g of cell paste. 9 Although such preparations appear homogeneous by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis, the 37-kDa protein usually is contaminated with trace amounts of adenylate kinase. By taking advantage of the hydrophobic property of selenophosphate synthetase, about half of the enzyme could be completely 12 M. J. Casadaban, J. Mol. Biol. 1@4, 541 (1976). 13 S. T a b o r and C. C. Richardson, Proc. Natl. Acad. Sci. U.S.A. 82, 1074 (1985).

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resolved from the more hydrophilic adenylate kinase contaminant by a single passage over a phenyl-Sepharose column. As complete elution of selenophosphate synthetase from phenylSepharose requires inclusion of 20-40% ethylene glycol in the buffer, a modified enzyme purification procedure employing chromatography on phenyl-Sepharose as a first step seems feasible. In preliminary trials 14 the redissolved protein precipitate from the crude sonic extract is adjusted to 0.5 M ammonium sulfate and applied directly to a phenyl-Sepharose column equilibrated with buffer A containing 0.5 M ammonium sulfate. Stepwise elution of the column with buffer containing 0.5, 0.25, and 0.1 M ammonium sulfate removes adsorbed protein, but no selenophosphate synthetase emerges until buffer only is applied to the column. Many other proteins are present in the first fractions containing residual ammonium sulfate, but successive fractions eluted with buffer alone and with buffer containing 10 and 40% v/v ethylene glycol contain the enzyme as the major protein species as judged by S D S - P A G E analysis. With this simple one-step procedure, about half of the selenophosphate synthetase is recovered in a highly enriched form suitable for use as a reagent for preparation of selenophosphate. Use of a shallow linear gradient of 0.1 M ammonium sulfate to buffer followed by another gradient of buffer to 40% ethylene glycol is indicated as a procedure for the recovery of a larger amount of highly purified enzyme.

Assay of Enzyme Activity In the reaction catalyzed by selenosphosphate synthetase, ATP and selenide are converted to equimolar amounts of selenophosphate, AMP, and orthophosphate (Pi) [Eq. (1)]. As ATP + NaSeH--+ SeP + Pi + AMP

(1)

an assay of enzyme activity the selenide-dependent formation of [14C]AMP from [14C]ATP is measured after separation of labeled nucleotides by thinlayer chromatography. Reaction mixtures (100 /xl) containing 100 mM Tricine-KOH, pH 7.2, 2 mM DTI', 1.5 mM [854C]ATP (0.25 /zCi), 3 mM MgCI2, 20 mM KC1, 0.5-5 /zM selenophosphate synthetase, and 1.5 mM NaSeH (added last) are incubated under argon in sealed tubes for 5-20 min at 37 °. A stock solution of the selenide is prepared by reduction of selenite (Na2SeO3; 30 raM) with 250 mM DTT at pH 8.5 under argon. Each 5-/,1 aliquot of this solution is calculated to contribute excess DTT equivalent to about 8 mM in the final reaction mixture. Reactions are terminated by addition of HC104, and after neutralization with K O H the ~4Z. Veres, I. Y. Kim, and T. C. Stadtman, unpublished.

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nucleotides in suitable aliquots of the supernatant separated by chromatography on cellulose PEI-F Baker, Phillipsburg, NJ) developed in 1.0 or 1.6 M of the nucleotide spots, detected by UV quenching, scintillation spectroscopy.

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solutions (5-15/zl) are thin-layer sheets (J. T. LiCI. The radioactivity is determined by liquid

Preparation of [75Se]Selenophosphate For convenience of subsequent isolation of the radioactive product, x,9 a 2-ml reaction mixture containing 100 mM Tricine-KOH, pH 7.2, 20 mM KC1, 2 mM DTT, 1.5 mM ATP, 3 mM MgC12, 1.5 mM Na75SeH (0.4/zCi), and 40/zM selenophosphate synthetase is incubated for 2.5 hr under argon at 37 °. The mixture also contains an additional 8 mM DTT added with the selenide solution. The reaction mixture, after adjustment to 20 mM DTT and l mM MgTitriplex, is applied to a Sephadex G-10 column (1 × 50 cm) equilibrated with 20 mM DTT and 2 mM MgTitriplex in 100 mM Tricine-KOH, pH 7.2. The column is eluted with the same buffer (18 ml/ hr) and 0.45-ml fractions are collected. The radioactive selenophosphate is eluted in fractions 23 to 27 and is completely separated from enzyme (fractions 15-20) and residual [75Se]selenide (fractions 29-35). The 75Se in the fractions is measured using a Beckman Model 5500 3, counter. The pooled selenophosphate peak fractions also contain AMP and ATP from the incubation mixture. To obtain selenophosphate free of nucleotides an ion pairing high-performance liquid chromatography (HPLC) procedure, essentially as described by Lazzarino et al.,15is used. For this purpose an Apex octadecyl 15 cm × 4.6 mm, 5-/zm particle size column supplied by Jones Chromatography (Lakewood, CO) is recommended, and the pairing ion is tetrabutylammonium obtained from Janssen Chimica (New Brunswick, NJ). Buffers used contain 10 mM tetrabutylammonium hydroxide, 10 mM K2HPO4, 1% (v/v) methanol, and 2 mM DT-F, pH 7.0 (buffer A), or 2.8 mM tetrabutylammonium hydroxide, 100 mM KHzPO4, 30% (v/v) methanol, and 2 mM DTT, pH 5.5 (buffer B). The column is eluted isocratically with buffer A for 3 min, followed by linear gradients of 0-40% buffer B for 3 to 5 rain and 40-44% buffer B for 5 to 16 rain. At 18 rain buffer B reaches 100%, and the column is washed for an additional 15 min with buffer B before returning to the original condition. The column is eluted at room temperature at a flow rate of 0.8 ml/min, and 0.4-ml fractions are collected. The 75Se in the fractions is measured by y counting, and the presence of [75Se]selenophosphate is verified by 31p NMR. In this chromatoi5 G. Lazzarino, D. DiPierro, B. Tavazzi, L. Cerroni, and B. Giardina, A n a l Biochem. 197, 191 (1991).

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graphic system selenophosphate is eluted at 10.7 min, AMP at 13.1 rain, and ATP at 23 min. Recovery of selenophosphate is 95%.

Selenophosphate Properties Selenophosphoric acid, H3PO3Se, is a tribasic acid with pK2 and pK3 values of approximately 4.6 and 8.8, respectively. ~Salts of the acid are very sensitive to air oxidation but only slowly hydrolyze in aqueous solution in the range pH 2-9 at room temperature provided that air is rigorously excluded. To lessen the chance of oxidative decomposition of the salts, DTT is added. The most convenient method for analyzing solutions of the salts is 31p NMR spectroscopy. The 3lp chemical shift depends on pH, l and in samples of sufficiently high concentration the 31p side bands, owing to coupling with natural isotopic abundance 77Se (7.58%), are detectable.

Selenophosphate Synthetase Selenophosphate synthetase (seID gene product) is a 37-kDa protein that contains 7 cysteine residues, at least one of which is essential for catalytic activity.:,8 The enzyme does not use sulfide as substrate, and other common nucleoside triphosphates do not replace ATP. 6'16 Mg 2+ is required and cannot be replaced by other divalent cations such as Mn 2+, Co 2+, or Zn 2+. In addition, a monovalent cation is required for enzyme activity. K ÷, Rb +, or NH4 +, but not Na + or Li +, activate the enzyme.9 Although the selenophosphate product formed by the enzyme is extremely oxygen labile, selenophosphate synthetase is not inactivated by exposure to oxygen. No loss of activity was detected after passage of a stream of oxygen through a solution of the enzyme in pH 7.2 buffer for 10 min in the absence of added reducing agent. Although sensitive to treatment with H202, a relatively high concentration (3 raM) was required to destroy 50% of the activity during 30 min. 9 The enzyme retains full activity during prolonged storage at - 8 0 °. It withstands heating at 60° for 5 rain without loss of activity, and fully active enzyme was recovered after precipitation with ammonium sulfate at pH 3.5 or acidification to pH 5 with HC1.

16 I. Y. Kim, Z. Veres, and T. C. Stadtman, J. BioL Chem. 268, 27020 (1993).

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[32] A n e t h o l e D i t h i o l e t h i o n e : Biochemical Considerations

By MARIE-ODILE CHRISTEN Introduction Certain sulfur-containing compounds likely play vital roles in the physiology of the organism, and others are among the effective drugs currently employed in human therapy. The cyclic sulfur-containing compounds, termed 1,2-dithiol-3-thiones, are representatives of a specific class of sulfurcontaining compounds, primarily of synthetic origin but also found in trace amounts in certain cruciferous vegetables. ~ These agents possess antioxidant, chemoprotective, and chemotherapeutic activities. 2-6 One substituted member of this class is prescribed as choleretic and sialagogue,~-~° another was used as antischistosomal agent. One molecule of special interest is 5-(p-methoxyphenyl)-3H-1,2-dithiol3-thione, also termed anethole dithiolethione (see Fig. 1) (ADT, the abbreviation to be used herein; trade names: Sulfarlem $25, Sialor). ADT has been used for many years as a choleretic, and it is also employed as a sialagogue in treating xerostomia ("dry mouth"). In addition, ADT exerts chemoprotective effects. Another 1,2-dithiol-3-thione termed oltipraz was previously used as an antischistosomal agent. Studies performed with anethole dithiolethione are briefly reviewed here, with particular emphasis on biochemical mechanisms. Early experiments with dogs bearing fixed biliary fistulae revealed that ADT induces an intense, enduring choleresis after ingestion, which can be 1 L. Jirousek and J. Starka, Nature (London) 45, 386 (1958). 2 S. S. Ansher, P. Dolan, and E. Bueding, Hepatology (Baltimore) 3, 932 (1983). 3 G. M. Feldman, J. M. Naples, J. L. Seed, and E. Bueding, Parasite Immunol. 7, 567 (1985). 4 D. Mansuy, A. Sassi, P. M. Dansette, and M. Plat, Biochem. Biophys. Res. Commun. 135, 1015 (1986). 5 S. J. Stohs, T. A. Lawson, L. Anderson, and E. Bueding, Mech. Ageing Dev. 37, 137 (1986). 6 p. Dansette, A. Sassi, M. Plat, M. O. Christen, and D. Manusy, in "Drug Metabolism-From Molecules of Man" (D. J. Benford, J. W. Bridges, and G. G. Gibson, eds.), p. 755. Taylor & Francis, London, 1987. 7 R. Hausler, Schweiz. Rundsch. Med/Prax. 68, 1063 (1979). 8 N. Lozac'h and M. Stavaux, Adv. Heterocycl. Chem. 27~ 151 (1980). 9 E. Bueding, P. Dolan, and J. P. Leroy, Res. Commun. Chem. Pathol. Pharmacol. 37, 293 (1982). lo S. Archer, Annu. Rev. Pharmacol. Toxicol. 25, 485 (1985).

METHODS IN ENZYMOLOGY, VOL. 252

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H3CO

S FIG. 1. 5-(p-Methoxyphenyl)-3H-1,2-dithiol-3-thione or anethole dithiolethione (ADT).

demonstrated by monitoring the secretion of biliary cholesterol, u ADT acts by increasing the total output of bile, and the bile that is secreted has a markedly altered chemical composition. Hence, ADT acts like a total choleretic; that is, it increases not only the volume of bile but also its content of bile acids, bile salts, and cholesterol. H Such findings have led to the use of ADT in treating symptoms associated with hepatobiliary dysfunctions. Xerostomia is the most prevalent type of drug-induced salivary gland dysfunction. Systemic sialagogues (e.g., pilocarpine), which possess parasympathomimetic activity and therefore stimulate the smooth muscle and glands of the gastrointestinal tract, are commonly used to treat this condition (see Ref. 12). ADT has also been used to treat the xerostomia that occurs in patients receiving psychotropic drugs ~3,14 and that which occurs during chemotherapy, 15 irradiation therapy, 16 and in autoimmune diseases. 17 By monitoring changes in muscarinic acetylcholine (ACh) receptor binding with [3H]quinuclidinyl benzilate, TM it has been shown that treatment of rats with A D T (300 mg/kg/day) for 7 days increased the density (maximal binding capacity; Bmax) of muscarinic ACh receptors on acinar cells of the salivary glands while not affecting the apparent dissociation constant (KD) of binding (i.e., no effect on binding affinity). These results, taken together with the widely held view that hypersensitivity is due to an increased density 11 R. Kourilsky, B. Halpern, and O. Gaudin, Journdes ThOrapeutiques de Paris 381 (1955). 12 p. C. Fox, J. Dent. Res. 66(Special issue), 689 (1987), J3 G. Lelord, C. Mercat, and Fuseilier, Gaz. Med. (Fr.) 76, 2257 (i969). 14R. de Buck, R. Titeca, and I. Pelc, Acta Psychiatr. Belg. 73, 510 (1973). 15 H. C. Falkson, S. Afr. Med. J. 49, 690 (1975). 16 p. Pazat, Vie Med. 52, 2751 (1971). 17 M. Schiodt, P. Oxholm, and A. Jacobsen, Scand. J. RheurnatoL SuppL 61, 250 (1986). Is y. Ukai, N. Taniguchi, K. Takeshita, K. Kimura, and H. Enomoto, Arch. Int. Pharmacodyn. Ther. 271, 206 (1984).

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of receptors, 19indicate that ADT augments salivary secretion by increasing the density of muscarinic A C h receptors, an effect resembling that following denervation or chronic exposure to antagonists or blocking agentsJ s The actions of ADT and other antioxidants have also been examined using schistosome granuloma formation in mice.3The developing egg granuloma, considered to be a manifestation of delayed-type hypersensitivity,2° secretes inflammatory mediators (e.g., prostaglandins, leukotrienes) and superoxide anion radical (O2 ~) (see Ref. 21). As reactive oxygen appears to be crucially involved in the synthesis of inflammatory mediators, it was considered that antioxidants might reduce the hypersensitivity response to schistosome eggs. In support of this hypothesis, administration of ADT and 2(3)-tert-butyl-4-hydroxyanisole (BHA) to female CD-1 mice, commencing 4 weeks after infection with cercariae of Schistosoma mansoni, decreased the size of the inner fibrotic region of the hepatic granuloma. Administration of the specific O2~ scavenger copper diisopropylsalicylate (an artificial superoxide dismutase; SOD) caused a similar decrease in granuloma size, indicating an involvement of 02 ~ in the granulomatous reaction. As both ADT and BHA increase hepatic glutathione levels and the activities of glutathione reductase, glutathione S-transferases, and NAD(P)H:quinone reductase (e.g., Refs. 2, 22, and 23; see below), actions which have been linked to inhibition of the formation of reactive oxygen species, their inhibitory effects are consistent with an oxidant-dependent mechanism for granuloma development. 3 Chemoproteetive Effects of Anethole Dithiolethione Ansher et al. 2 determined the chemoprotective effects of anethole dithiolethione (ADT) and BHA against the toxicity of CCI4 and acetaminophen (AAP). Female mice were given a high dose (500 mg/kg) of the dithiolethione by gastric intubation 4 days and 2 days before the hepatotoxic agent, or were fed a diet containing 0.75% BHA for 12 days. Acetaminophen was administered intraperitoneally (i.p.) in various doses (300, 500, 600, or 700 mg/kg), and CC14 (i0 g/kg or 30 mg/kg) was given by gastric intubation. Mortality (an estimate of acute toxicity), determined 24 hr after administration of acetaminophen or CC14, was reduced by pretreatments with ADT a9 D. R. Burt, I. Creese, and S. H. Snyder, Science 196, 326 (1977). 20 K. S. Warren, E. O. Domingo, and R. B. T. Cowan, Am. J. PathoL 51, 735 (1967). 21 S. W. Chensue, S. L. Kunkel, G. I. Higashi, P. A. Ward, and D. L. Boros, Infect. lmmun. 42, 1116 (1983). z2 R. P. Batzinger, S.-Y L. Ou, and E. Bueding, Cancer Res. 3B, 4478 (1978), z3 A. M. Benson, Y. N. Cha, E. Bueding, H. S. Heine, and P. Talalay, Cancer Res. 39, 2971 (1979).

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and BHA. For the 10 g/kg dose of CC14, mortality approached 50%, but in pretreated animals it was reduced to 18% with the BHA-containing diet and to 22% with 500 mg/kg of ADT. Pretreatment with A D T or B H A also reduced or prevented the depletion of hepatic glutathione produced by the two hepatotoxic agents. Liver damage that followed administration of CC14 o r acetaminophen, as determined by serum transaminase and sorbitol dehydrogenase activities, was lower after treatment with the two agents. Oral administration of ADT (at 500 mg/kg, p.o.) 4 days and 2 days before analysis caused a 4- to over 6-fold increase in the activities of hepatic glutathione S-transferases. It was proposed that the increases in hepatic glutathione concentration promoted by A D T could account for its chemoprotective and radioprotective effects, as the reduced form of glutathione (GSH) interacts with, and inactivates, toxic metabolites of CC14 and acetaminophen. Further experiments e4-27have revealed that administration of a low oral dose of ADT 1 hr before hepatotoxins can also elicit pronounced protective effects against CC14 in rats and AAP in mice. When female mice were given A D T (10 mg/kg, p.o.) 1 hr before acetaminophen (450 mg/kg, i.p.), then anesthetized with sodium pentobarbital (360 mg/kg, i.p.) and sacrificed 4 hr or 18 hr after injection of acetaminophen, analyses of blood and liver showed that the intensity of acetaminophen-induced cytolysis was minimized, as evidenced by the decreased serum aminotransferase activities at the two times selected for measurementY Treatment with AAP induced marked decreases in hepatic glutathione content and in glutathione-related enzyme activities that were evident 4 hr after administration of acetaminophen. The depletion in glutathione (GS) occurred mainly in the reduced form (GSH), leading to a lower GSH/total GS ratio (71% in ADT-treated animals versus 96% in controls). However, this decline was transitory; 18 hr after acetaminophen administration the total GS content was increased more than 2-fold with respect to controls, presumably owing to a compensatory "rebound" in glutathione synthesis. 28 This protective effect 24 j. M, Warnet, M. O. Christen, M. Thevenin, D. Biard, A. Jacqueson, and J. R. Claude, in "Local Immunity International Symposium Series" (J. P. Revillard and N. Wierzbicki, eds.), Vol. 3, p. 249. Fondation Franco-Allemande, Suresnes, France 1987. 25 j. M. Warnet, M. O. Christen, M. Thevenin, D. Biard, A. Jacqueson, and J. R. Claude, Arch. ToxicoL 13(Suppl.), 322 (1989). 26 j. M. Warnet, M. O. Christen, M. Thevenin, D. Biard, A. Jacqueson, and J. R. Claude, Pharmacol. ToxicoL 65, 63 (1989). 27 j. M. Warnet, M. O. Christen, M. Thevenin, C. Martin, J. Cesbrom and J. P.Claude, SFT/ SIT Joint Meeting on Mechanisms o f Toxicity and Repair ll: Clinical Aspects, Venice, Nov. 5-6, 1990, Abstract. 28 A. Meister, Fed. Proc. 43, 3031 (1986).

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of ADT might be related to its properties of increasing tissue GSH concentration and enhancing hepatic glutathione-related enzyme activities, as has been observed at higher dosage (see above). Buthionine sulfoximine (BSO; 2-cyclohexen-l-one) is apparently a selective inhibitor of y-glutamylcysteine synthetase, which catalyzes the first step in GSH synthesis,29'3° and therefore it is used in attempts to elucidate the biological significance of GSH. In an effort to determine whether the protective effect of A D T can be attributed to a stimulation of GSH synthesis, female mice were given ADT (10 mg/kg, p.o.) 1 hr before acetaminophen (450 mg/kg, i.p.), with or without BSO (0.01 mmol/kg, i.p.), and were sacrificed 5 hr after ADT ingestion. 27 ADT significantly increased survival of the mice given a lethal dose of acetaminophen plus BSO, an effect that coincided with a preventive effect of ADT on the inhibition of GSH synthesis. Such results indicate that the hepatoprotective effect of ADT against acetaminophen toxicity may be explained by its stimulation of GSH synthesis via the y-glutamylcysteine synthetase pathway. Ethane production rate provides a measure of in vivo lipid peroxidation which has toxicological significance.31 Mice that are fasted for 72 hr (to deplete hepatic GSH contents) become much more sensitive to acetaminophen toxicity and exhale large amounts of ethane. 3x Mansuy et aL 4 showed that ADT is a good inhibitor of ethane exhalation in animals intoxicated with either CCI4 o r acetaminophen (see also Ref. 6). Low doses of ADT (IDs0 of 0.7 mg/kg) inhibited ethane exhalation by mice that had been intoxicated with 500 mg/kg acetaminophen and prevented mortality due to acetaminophen overdose. Furthermore, ADT inhibited lipid peroxidation that had been induced in rat liver microsomes either chemically by FeSO4 and reducing agents (cysteine or ascorbate) or enzymatically by NADPH and CC14. ADT (IC~0 of - 2 / z M ) was about as potent as propylgallate and much more potent than vitamin E or levamisole. The antioxidant effect of ADT, by decreasing the formation of lipid hydroperoxides, could have protected against CC14- and acetaminophen-induced hepatotoxicity, and the dithiolethione function might be important for this inhibitory action. 4 Several types of organic sulfur derivatives, including isothiocyanates, thiocarbamates, and carbon disulfide, inhibit chemical carcinogenesis in laboratory animals, and some of these are powerful inducers of detoxification enzymes, such as glutathione S-transferase and NAD(P)H:quinone 29 O. W. Griffith and A. Meister, J. BioL Chem. 25,1, 7588 (1979). 30 G. Seelig and A. Meister, this series, Vol. 113, p. 379, 3~ A. Wendel, S. Feuerstein, and K. H. Konz, Biochem. Pharmacol. 13, 2051 (1979).

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reductase. 32 This latter mechanism appears to be involved in the actions of dithiolethiones.

Biochemical Mechanisms Underlying Beneficial Effects of Anethole Dithiolethione What is the connection between the biochemical actions and protective effects of the 1,2-dithiol-3-thiones and their therapeutic, or postulated therapeutic, uses? It seems necessary to consider several mechanisms, all related to antioxidant potential, in answering this question. Lipid peroxidation may be significantly implicated in acetaminophen-induced toxicity (e.g., Ref. 31), indicating that an interaction might occur between dithiolethiones and lipid peroxidation. Decreases in lipid peroxidation and D N A damage caused by antioxidants such as A D T may be mediated via decreased interactions of free radicals with complex lipids and DNA. Antioxidants may act directly to prevent such injurious reactions or indirectly by enhancing the formation of GSH and glutathione-metabolizing enzymes, which would then provide protection. 5 The glutathione redox cycle can be altered at several points, including depletion of GSH pools, inactivation of glutathione reductase, or inhibition of glutathione peroxidase or glutathione Stransferase. 33 The effects of dithiolethiones are most notably associated with increases in tissue GSH content and in the activities of enzymes catalyzing the inactivation of toxic compounds. 34 Protective actions of dithiolethiones appear to result from the effect of increasing GSH which directly interacts with toxic electrophilic compounds. Dithiolethiones promote the synthesis of GSH by enhancing the activities of oxidized glutatione (GSSG) reductase, glucose-6-phosphate dehydrogenase (G6PD), and 6phosphogluconic acid dehydrogenase (PGAD), the dehydrogenases generating N A D P H which is vital for GSSG reductase activity (see Ref. 34). The glutathione S-transferases, whose activities are increased by dithiolethiones, catalyze the conjugation of GSH with many carcinogens and their metabolites, the conjugates being metabolized and finally excreted. 34 Some GSH transferases bind directly to electrophiles, thereby promoting their inactivation (see Ref. 34). Protective effects of dithiolethiones may also be due to their action of increasing the activity of NAD(P)H:quinone reductase,

32M. De Long, P. Dolan, A. B. Santamaria, and E. Bueding, Carcinogenesis (London) 7, 977 (1986). 33B. Mannervik and U. Danielson, Crit. Rev. Biochem. 23~283 (1988). 34S. S. Ansher, P. Dolan, and E. Bueding, Food Chem. Toxicol. 24, 405 (1986).

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which catalyzes the reduction of the toxic quinones to the less noxious hydroquinones for subsequent conjugation and excretion. 35 What is the connection between the biochemical actions and protective effects of anethole dithiolethione (ADT) that have been discussed above and its therapeutic, or postulated therapeutic, uses? In attempting to answer this question, it seems necessary to deal with several mechanisms, all related in some way to antioxidant potential. A relationship appears to exist between glutathione content and lipid peroxidation. These two mechanisms, lipid peroxidation and glutathione depletion, are related to one another, and their inhibition may well underlie the protective actions of ADT. Lipid peroxidation processes may be significantly ,implicated in acetaminophen-induced toxicity,31 indicating that an interaction exists between dithiolethiones such as ADT and lipid peroxidation (see above). Thus, antioxidants like ADT may protect against lipid peroxidation and DNA damage by decreasing the interaction of free radicals and reactive oxygen species with complex lipids and DNA. Antioxidants may act directly to prevent these tissue-damaging reactions or indirectly by enhancing the formation of reduced glutathione and glutathionemetabolizing enzymes, which would then provide cellular protection (see Ref. 5; see above). These contentions provide a setting for considering further the free radical-scavenging (antioxidant) properties of ADT. Radiolysis experiments have been conducted to define the radicalscavenging activity of A D T . 36'37 Ethanol was used as the solvent because it generates radicals of the type R. [i.e., H3C-C"H-OH or H3C-CH(OH) OO "] which represent models for the free radicals that are implicated in lipid peroxidation. Results indicated that ADT does not seem to react appreciably with peroxyl radicals (ROe ") or with superoxide anion radicals (027) in this system but that it efficiently scavenges nonoxygenated radicals (R.). Hence, in contrast to the mechanism of action of vitamin E, ADT exerts its antioxidant action by scavenging the nonoxygenated precursors (R .) of RO2 ". These results were supported by other data which showed that the presence of ADT in the medium limits the degradation (peroxidation) of linoleic acid [tested as aerated linoleic acid (L) micelles exposed to irradiation], as well as the formation of conjugated dienes, ADT being consumed proportionally to the concentration absorbed. 37 Because ADT efficiently scavenges free radicals of the type R., it may act indirectly to suppress the generation of lipid peroxides (LO2) (see [33] in this volume). 35 E. Horning, J. P. Thernot, and E. HeRon, J. Toxicol. Environ. Health 4, 341 (1979). 36 p. Ruimy-Ifrah, D. Jore, M. O. Chrisen, P. Dansette, D. Mansuy, and C. Ferradini, J. Chim. Phys. 88, 907 (1991). 37 p. Ruimy-Ifrah, D. Jore, M. O. Christen, and C. Ferradini, J. Chim. Phys. 90, 1067 (1993).

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Use of new fluorometric methods readily revealed the cytoprotective effects and free radical-scavenging properties of A D T and showed that A D T acts on the injury that appears to be the initial intracellular target of toxic lipoperoxidative mechanisms. Acetaminophen was added to the culture medium of L 929 or Hep G2 cell lines (human liver) in the absence or presence of N-acetylcysteine or ADT, and mitochondrial alterations were assessed with rhodamine 123 (Rh 123) using the fluorometric microtitration method. 38'39 After 72 hr of contact with acetaminophen, cell viability was 49.9% versus control in cells exposed to acetaminophen (150/zg/ml) alone, and 81% versus control in cells exposed to acetaminophen plus A D T (12 /xg/ml). A D T was superior to N-acetylcysteine in increasing cell viability and in protecting cells against both acetaminophen- and CC14-induced toxicity (see [34] in this volume). Concluding Remarks Among the cyclic sulfur-containing compounds, termed 1,2-dithiol-3thiones, a molecule of particular interest is ADT, which is marketed as a choleretic and sialagogue, and which also exerts chemoprotective effects. The chemoprotective effects of this 1,2-dithiol-3-thione may be related mainly to the increase in the activity of detoxification enzymes such as glutathione S-transferases and NAD(P)H:quinone reductase, which promote the elimination of xenobiotics and their activated metabolites via conjugation reactions, particularly with glutathione. A D T may protect against lipid peroxidation and D N A damage by decreasing the interactions of free radicals with these macromolecules. How safe is ADT? Current evidence also based on a wide clinical experience indicates that the compound has low mammalian toxicity. Indeed, its advantage as a therapeutic agent is strengthened by its low toxicity and by its lack of mutagenicity. A D T has already been administered to large numbers of people, as a choleretic and sialagogue] 3,4°without apparent adverse side effects] Because of its potency and low toxicity, anethole dithiolethione is of high therapeutic benefit.

38p. Rat, M.O. Christen, M. Thevenin, J. M. Warnet, and M. Adolphe, Proceedings of the VI International Congress of Toxicology, Rome, June 28-July 3,1992 [Toxicol. Letr (Suppl.), 214 (1992)]. 39p. Rat, M. O. Christen, M. Thevenin,J. M. Warnet, and M. Adolphe, Seventh International Workshop on In Vitro Toxicology INVITOX, De Harm, Belgium October 5-9, 1992. 40j. Breunsbach and V. Dvorak, Strahlentherapie 148, 298 (1974).

324

MIMICS OF BIOLOGICAL THIOLS AND INHIBITORS

[33]

[33] U s e o f L i n o l e i c A c i d R ~ a d i o l y s i s f o r A s s a y o f Antioxidant Action of Anethole Dithiolethione B y M A R I E ODILE CHRISTEN, L. FACKIR a n d DANIEL JORE

Introduction Sulfarlem (p-anisyldithiolthione) (ADT, Fig. 1) is a sulfur-containing heterocyclic compound which exhibits free radical scavenging (antioxidant) propertiesJ -3 Radiolysis may be used in the characterization of the radical mechanisms involved in free radical scavenging. The data obtained in homogeneous ethanolic solution 4 have shown that Sulfarlem appears to be a better scavenger of carbon-centered radicals (i.e., H 3 C - - C H - - O H ) than of oxygenated ones [i.e., HaC--CH(OH)OO]. A study performed in aerated linoleic acid micelles presented the first evidence that Sulfarlem 5was capable of limiting peroxidation of polyunsaturated fatty acids. The study described in this chapter shows how radiolysis experiments performed in oxygen-saturated micellar media can afford quantitative results concerning the prevention of linoleic acid peroxidation by ADT. y-Radiolysis can therefore be an efficient method of testing the capability of a supposed antioxidant compound to play such a role toward peroxidation chains. Oxygen-saturated aqueous linoleic acid (10 -2 M) micelles are submitted to y-irradiation (6°C0) in the absence and in the presence of variable amounts of A D T (ranging from 5 × 10 -6 to 9.9 × 10 -5 M). Materials and Methods Linoleic acid (LH) is from Merck (Darmstadt, Germany) and Sulfarlem (ADT) is a L.T.M.-Solvay Pharma product. The water used for y-irradiations is triply distilled, and all vessels used are heated at 400 ° for 4 hr after washing, y-Irradiation has been performed in a 6°Co irradiator with a dose 1 D. Mansuy, A. Sari, P. Dansette, and M. Plat, Biochim. Biophys. Res. Commun. 135, 1015 (1986). 2 p. Dansette, in "Local Immunity, Tissue Fibrosis, Immune Cells and Mediators" (J. P. Revillard and N. Wierzbicki, eds.), p. 203. Fondation Franco-Allemande, Paris, 1987. 3 p. Dansette, A. Sari, M. Plat, M. O. Christen, and D. Mansuy, in "Drug Metabolism-From Molecules to Man" (B. J. Bonford, J. W. Bridges, and G. G. Gibson, eds.), p. 755. Taylor & Francis, London, 1987. 4 p. Ruimy-Ifrah, D. Jore, M. O. Christen, P. Dansette, D. Mansuy, and C. Ferradini, J. Chim. Phys. 88, 907 (1991). 5 p. Ruimy-Ifrah, D. Jore, M. O. Christen, and C. Ferradini, Z Chim. Phys. 90, 1067 (1993).

METHODS IN ENZYMOLOGY, VOL. 252

Copyright © 1995 by Academic Press, Inc. All rights of reproduction in any form reserved.

[33]

ASSAY OF ANETHOLE DITHIOLETHIONE ACTION

325

H3CO

~s F16. 1. Structure of sulfarlem (anethole dithiolethione, ADT).

rate of 85 Gy/hr. The dosimetry is done according to Fricke's method: radiooxidation of an aerated Mohr salt solution (10 -3 M) in the presence of H2SO4 (0.4 M), taking A,~x (Fe 3+) = 304 nm, ~304 = 2204 M -1 c m -1 at 25 °, and the yield of Fe 3+ formation G(Fe 3+) = 16.2 x 10 -7 mol/J. The micelles are prepared with linoleic acid (10 -2 M) in water, adjusted to p H 10 before irradiation with introduction of the necessary quantity of N a O H solution (0.1 M). All titrations are made with a Beckman D U 70 spectrophotometer with an optical pathway of 1 or 0.2 cm. The absorption spectra of the micelles present two maxima at 270 and 234 nm (see Fig. 2, spectrum a). When A D T is added, A D T absorbs at 428 nm with a molar extinction coefficient equal to 4.9 × 103 M -1 cm -1.

Radiolysis of Aqueous Linoleic Acid Micelles Within the nanosecond time scale, -/-irradiation of water leads to the formation of three radical moieties, .OH, H., and e-aq. The yields of formation of these species, G values, expressed in moles per joule, are as follows6'7: G.oH = 2.8 × 10 -7 mol/J, Ge aq 2.8 x 10 -7 mol/J, and GH. = 0.6 X 10 -7 mol/J. When the irradiation is performed in the presence of oxygen, the following reactions occur: =

e-aq + 0 2 "--+ 0 2 7

H" +

0 2 ---+

HO2"

where HO2- is the acidic form of 027 (pKa HO2"/O2 v = 4.8). When irradiating at p H 10 in the presence of 02, only two species are obtained: -OH 6 C. Ferradini and J. Pucheault, "Biologie de l'Action des Rayonnements Ionisants," Masson. Paris, 1983. 7 I. G, Draganic and Z. D. Draganic, in "The Radiation Chemistry of Water," p. 91. Academic Press, New York, 1971.

326

[33]

MIMICS OF BIOLOGICAL THIOLS AND INHIBITORS _~

i

,

i

i

i

a

E5 2.69

,

i

0.9

2.07 ,"'-~'',

o.3

[~;"#/' f--. ".', 1.14

/~,~./.L\.":"

0. a3

\~'C. - F

i

i

? IGAE234p115x10-3

0.6 ,i,,

I

/

o~

<:~ 2 . 3 8

,.76

i

/

/GAE/7"

2.7d= -72,~10-3,

I

,

o

25 . . . . .

,

DOSE

soGy

;--, .,,5,,:,,

0.52

-0. i

2oo

2i5

2~o

2,i~

2~o

2~5

260

3c;-~

a~o

a:~

asonm

X FIG. 2. Absorption spectra of irradiated oxygen-saturated LH micelles (10 -2 M) for (a) initial solution and doses of (b) 7 Gy, (c) 14 Gy, (d) 21 Gy, (e) 28 Gy, and (f) 42 Gy. Dose rate, 85 Gy/hr; optical pathway; 0.2 cm. Inset: Plots of AOD versus (dose) at A = 270 nm (+) and A = 234 nm (×).

and 02 ~ with the yields G.oH = 2.8 × 10 -7 m o l / J and G ( 0 2 ~ ) = Ge-aq + Gn. = 3.4 × 10 -7 mol/J. In an aqueous micellar m e d i u m of linoleic acid ( L H ) at p H 10, where 02- is relatively poorly reactive, it can be admitted that L H reacts with •O H according to reaction (1). L H + - O H ~ L" + H z O

(1)

The obtained L. free radical, in the presence of oxygen, m a y lead to the formation of a peroxyl radical LO2" according to reaction (2). L- + 02 --* LO2"

(2)

The values of the rate constants 8 of reactions (1) and (2) are as follows: kl = 109 M -1 sec -1 and k2 = 3 × 108 M -1 sec -1. LO2" m a y then react with L H [reaction (3)], affording the L O O H hydroperoxide and a new Lfree radical. 8 j. R. Bucher, M. Tien, and S. D. Aust, Biochem. Biophys. Res. Commun. 111, 780 (1983).

[33]

ASSAY OF ANETHOLEDITHIOLETHIONEACTION

327

LO2" + L H ~ L O O H + L-

(3)

Reactions (2) and (3) are responsible for the peroxidation chain. LO2" may also react with another peroxyl radical according to reaction (4), which, as in all biradical reactions, leads to chain termination. LO2" + LO2" --+ L O O L + 02

(4)

The value of the rate constant 9 of reaction (4) is equal to 10v M -~ sec -l. Evaluation of L H peroxidation may be obtained by spectrophotometric measurements of L H degradation observed at 270 nm and, for instance, by measurement of the formation of conjugated dienes 1° which absorb at 234 nm. Quantitatively, these effects are expressed in terms of a parameter GAs, which is directly obtained from the absorption spectra according to GAs, = AOD,/dose, with AOD, = ODa (irradiated solution) - O D , (initial solution) Dose is expressed in Gy. Therefore, 5 O D , = Ae, × concentration of product = G [product] × dose

Experimental Results

y-Irradiation of Linoleic Acid Micelles in Absence of Anethiole Dithiolethione T o determine the ability of A D T to prevent linoleic acid from peroxidation, it is necessary to examine the evolution of L H subjected to y-irradiation in the absence of ADT. For this purpose, aqueous L H micelles [LH] = 10 2 M) have been irradiated in the presence of oxygen with doses between 7 and 43 Gy. It can be observed from Fig. 2 that the absorption at 270 nm decreases according to the absorbed dose, whereas the absorption increases at 234 nm, this maximum being characteristic of the formation of conjugated dienes. The inset (Fig. 2) indicates that the differential optical densities AOD234and AOD270vary linearly as a function of the dose. The slopes of such A O D J d o s e curves are proportional to the yield of conjugated dienes and to the yield of degradation of linoleic acid. 9K. Hazegawa and L. K. Patterson, Photochem. Photobiol. 28, 817 (1978). t0 B. Halliwell and J, M. C. Gutteridge, "Free Radicals in Biology and Medicine." Oxford Univ. Press (Clarendon), Oxford, 1985.

328

[33]

MIMICS OF BIOLOGICAL THIOLS AND INHIBITORS

y-Irradiation of Linoleic Acid Micelles in Presence of Anethiole Dithiolethione To examine the possibility that A D T prevents LH from peroxidation, y-irradiation of O2-saturated LH aqueous micelles was performed in the presence of various quantities of ADT. Fig. 3 shows the differential absorption spectra obtained when irradiating O2-saturated L H micelles (8.7 × 10 -3 M) in the presence of A D T (2.7 x 10 -3 M) as a function of the dose. It can be observed that the absorption maximum at 270 nm decreases, whereas the maximum at 234 nm increases. Fig. 3 (inset) shows that the change in ODa is linear as a function of the dose. Fig. 4 shows that the absorption at 428 nm due to A D T decreases, indicating A D T consumption. Figure 4 (inset) indicates that this consumption is proportional to the irradiation dose. The slopes of the curves of AOD~ as a function of dose allow for the quantification of the phenomena. The slopes AODzy0/dose and AOD234/dose (45 X 10 -3 and 83 x 10 -3) are smaller than the values obtained in the absence of A D T (72 x 10 -3 and 115 x 10 -3, respectively), indicating

0.7 ~

~

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at / ~ ~

o. .1 r~

/

~

~

',

/

048

~

x


~\

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/

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0.34

I/i,~:~'-<~ \ ~

0.25

%,,

0.22

/

+

/

'k\

0.~.

.

_

oosE

~

_1o

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\

-0. t4

-0,26

\ x .// e

-0.38 -0.5 ~_00

2i5

230

245

260

275

290

305

320

335

350 N m

X FIG. 3. Differential absorption spectra of irradiated oxygen-saturated LH micelles (8.7 × 10 -3 M ) in the presence of A D T (2.7 x 10 -5 M ) (referenced to initial solution) at doses of (a) 7 Gy, (b) 14 Gy, (e) 21 Gy, (d) 28 Gy, and (e) 35 Gy. Dose rate, 85 Gy/hr; optical pathway, 0.2 crn. Inset: Plots of 2~OD versus (dose) at )t = 270 nm ( + ) and A = 234 nm (x).

[33]

ASSAY

OF ANETHOLE

DITHIOLETHIONE

329

ACTION

0.2 CO

0.16

r~ cJ

c$

~t

~

g

c~ 0.05

0.12

I

~DOSE 0 10 20 30c_,y

0.08 0.04 -0

-0.04 \

~.._-~ ~_~ ",... j-

~,?-~_ c ,~.. . ,., ~.,.'~...~-,~ i I

-0.08 -0.12 -0. ~.6 -0.2

300

325

350

375

400

425

450

475

500

525

550 r[ m

X FIG. 4. Differential absorption spectra of irradiated oxygen-saturated LH micelles (8.7 × in the presence of ADT (2.7 x 10-5 M) (referenced to initial solution) at doses of (a) 7 Gy, (b) 14 Gy, (c) 22 Gy, (d) 28 Gy, and (e) 35 Gy. Dose rate, 85 Gy/hr; optical pathway, 1 cm. Inset: Plots of AOD versus (dose) at A = 428 nm. 10 _3 M)

that the peroxidation of L H is less important when A D T is present in the medium. Such experiments have been p e r f o r m e d for eight different concentrations of A D T ranging f r o m 5 × 10 -6 to 9.9 × 10 -2 M. In all cases, the results obtained are analogous to those described above. The slopes of AOD270/dose, AOD234/dose, and AOD428/dose have been measured and are plotted versus A D T concentration in Fig. 5. It can be observed that, according to the initial concentration of A D T , the degradation of L H (measured at 270 nm) decreases, the formation of conjugated dienes (measured at 234 nm) decreases, and the consumption of A D T (measured at 428 nm) increases and reaches a roughly constant value for concentrations of A D T above 6 × 10 -5 M. Discussion The fact that the degradation of L H and the formation of dienes decrease in the presence of A D T indicates that A D T is capable of preventing peroxi-

330

[33]

MIMICS OF BIOLOGICAL THIOLS AND INHIBITORS 140

•

I

'

I'

'

i

•

~

,

o

120

,,< 100 uU

.,=3

80

~ 0 0

~ -

60

0 40 20

i

0 0

i 2

i 4

,, 6

8

10

[ADT] (10"s M) FIG. 5. Plots of GAa~ versus [ADTi.iti.d for h = 270 nm (fq) GAez70 X 103), 234 nm (©) (GAe2~ X 103), and 428 nm (©) (GAe42s x 104).

dation of LH. At the same time, ADT is consumed. The possible radical mechanisms involved in such an antioxidant action may be of two types. In one, ADT may be an efficient scavenger of LO2" peroxyl radicals and may react with LO2" according to reaction (5), where LO2" + ADT--0 L O O H + A.

(5)

A. is a monoelectronic oxidized species derived from ADT. Reaction (5) competes with reaction (3), thus limiting the peroxidation chain. In the other mechanism, ADT may be an efficient scavenger of the nonoxygenated radicals L- and may react with L. free radicals according to reaction (6), where L. + ADT --o L + + ADT ~

(6)

ADT: is a monoelectronic reduced species derived from ADT. Reaction (6) competes with reaction (2), thus limiting the formation of LO2" and, therefore, the peroxidation chain. The previously described results, 4 obtained in homogeneous ethanolic solution, seem to indicate that ADT is more reactive toward nonoxygenated radicals than toward oxygenated ones, suggesting that the second mechanism is correct. When comparing the data obtained in aerated systems~

[34]

COLD L I G H T F L U O R I M E T R Y TO E V A L U A T E BIOTHIOLS

331

and those of the above reactions performed in oxygenated systems, which are almost identical, the concentration of oxygen being multiplied by a factor of 5, it remains difficult to choose between the two hypotheses. Nevertheless, whether ADT scavenges L. or LO2", its action results in a very efficient prevention of the peroxidation of linoleic acid. In conclusion, the use of 7-radiolysis of oxygen-saturated linoleic acid-aqueous micelle systems affords an efficient method for evaluating the ability of antioxidant molecules such as ADT to prevent peroxidation chain phenomena. Acknowledgment W e are g r a t e f u l to A. R o u s c i l l e s for e x c e l l e n t t e c h n i c a l assistance.

[34] C o l d L i g h t F l u o r i m e t r y : A M i c r o t i t r a t i o n T e c h n o l o g y for Cell Culture to Evaluate Anethole Dithiolethione and Other Biothiols By

PATRICE

RAT,

MARIE-ODILE

CHRISTEN,

MARC

THEVENIN,

JEAN-MICHEL WARNET, and MONIQUE ADOLPHE Introduction Biothiols are compounds of growing biological interest. This chapter describes screening these drugs using a new microtitration technology, cold light fluorimetry (CLF), and comparing it to classic fluorimetric methods. Until now, in vitro screening of such molecules was done using photometric or fluorimetric microtitration methods which are not sufficiently sensitive and/or specific. The only fluorimetric methodology available for cell culture is flow cytometry, which is not adapted to screening because the equipment is too heavy and expensive. The classic microtitration fluorimetric methods are reviewed and the cold light concept is discussed. The three main microplate fluorescence readers have been tested. These fluorimeters read correctly in ultraviolet (UV) or visible spectra but rarely in both, and they have low sensitivity. Therefore a new fluorimetric technology has been developed which proved to be sensitive and could be adapted to direct screening in 96-weU microplates. This new fluorimeter includes cold light technology with chemical and physical modifications of the illumination system, and it allows detection of fluorescence in the UV, visible, and near-infrared regions by increasing photonic energy, by reducing the calorific part of light, and by decreasing METHODS IN ENZYMOLOGY,VOL. 252

Copyright © 1995 by AcademicPress, Inc All rights of reproductionin any form reserved.

332

MIMICS OF BIOLOGICAL THIOLS AND INHIBITORS

[34]

background. The detection limit of the probes is improved (by 500- to 1000fold) in UV, visible, and near-infrared spectra for cell proliferation (DNA/ Hoechst 33258), viability (cell integrity/fluorescent neutral red), and metabolic evaluation (mitochondria/rhodamine 123). The cytoprotective effect of biothiols can be detected with this new technology with high sensitivity. Drug-induced cell injury was evaluated on the L929 or Hep G2 (human liver) cell line, with or without biothiols such as N-acetylcysteine or anethole dithiolethione. Materials and Methods Fluorescence Readers

We have tested several prototypes in order to obtain a final reader based on the cold light concept (Fluorolite 1000/3, Dynatech) with high sensitivity and specificity, well-adapted for cell culture. It has been compared to the three main fluorescence readers now available: Microfluor from Dynatech (using a mercury vapor lamp), Cytofluor from Millipore (Bedford, MA) (using a tungsten lamp), and Fluoroskan II from Labsystem (using xenon lamp). Our new reader illuminating system includes a very high energy krypton-xenon lamp. Only 1% of total photonic energy is used with cell culture. Fluorescent Probes

For comparison of the different readers, we use several fluorescent probes covering different parts of spectrum. For the UV spectrum (excitation, 360 nm; emission, 450 nm), Hoechst 33258 (Molecular Probes, Eugene, OR) is used at a final concentration of 1-5/z/ml, as in the flow cytometry method (FCM). 1'2 For the visible spectrum (excitation, 490 nm; emission, 535 nm), rhodamine 123 (Molecular Probes) is used on cells at a final concentration of 10/zg/ml as in FCM. 3-5 For the near-infrared spectrum (excitation, 540 nm; emission, 600 nm), a neutral red probe (Fluka, Ronkonkoma, NY) is dispensed on different cells in microplates at a standard concentration (50 tzg/ml) following the Borenfreund protocol. 6'7 1 R. P, Haugland, in "Handbook of Fluorescent Probes and Research Chemicals" (K. D. Larrison, ed.), p. 175. Molecular Probes, Eugene, Oregon, 1992. 2 R. Rago, J. Mitchen, and G. Wilding, A n a l Biochem. 191, 31 (1990). 3 L. V. Johnson, M. L. Walsh, and L. B. Chen, Proc. Natl. Acad. Sci. U.S.A. 77, 990 (1980). 4 L. B. Chen, I. C. Summerhayes, L. V. Johnson, M. L. Walsh, S. D. Bernal, and T. J. Lampidis, Cold Spring Harbor Syrnp. Quant. Biol. 46, 141 (1981). 5 X. Ronot, L. Benel, M. Adolphe, and J.-C. Mounolou, Biol. Cell t~7, 1 (1986). 6 F. C. Kull and P. Cuatrecasas, Appl. Biochem. Biotechnol. 8, 97 (1983). 7 E. Borenfreund and J. A. Puerner, J. Tissue Cult. Methods 9, 7 (1984).

[34]

COLD LIGHT FLUORIMETRY TO EVALUATE BIOTHIOLS

333

Cell Lines Two cell types are used, a fibroblast cell line (L929) and a hepatic cell line (Hep G2). The growth medium used ]minimum essential medium (MEM), Ham's] depends on the cell line and is mixed with 10% fetal calf serum (GIBCO, Grand Island, NY). Before the assay, cells are maintained in an incubator [5% (v/v) COz at 37°]. In all cases, fluorescent probes are added when the cells are in the growth phase.

Drugs Acetaminophen (AAP) is purchased from Upsa Laboratories (France). This hepatotoxin is tested on cells at a concentration corresponding to the blood level observed in human intoxication (250/zg/ml). Two biothiols are evaluated as A A P antidotes on microplates at a concentration of 1 /xM and after a 48 hr-pretreatment periodS: N-acetylcysteine (Sigma, St. Louis, MO), the reference AAP antidote used in human intoxication, and anethole dithiolethione (Sulfarlem, Sialor, Solvay-Pharma LTM), previously successfully tested against AAP intoxication. 9

Results and Discussion

Standard Fluorimetric Technology Initially, standard microplate fluorescence readers were tested. These fluorimeters read correctly in the UV or visible spectrum but rarely in both, and generally with a low sensitivity. The illuminating system influences the fluorimetry reader sensitivity (Table I). The fluorimeter with a xenon lamp is the most energy-producing system in standard fluorimetric technology. Even in this case, however, sensitivity (ng/ml) has to be improved in order to be adapted to cellular level. Heat energy associated with the xenon lamp is very high (75% of the total energy) and reduces the photonic energy percentage. Any increase in this photonic energy induces a high increase in heat which generates noise in optical systems and induces a very high background. The solution to these problems was to dissociate the photonic energy from the calorific energy in order to obtain a cold light emission.

R. J. Riddell, D. S. Panacer, S. M. Wilde, R. H. Clothier, and M. Balls, A T L A , Altern. Lab. Anita. 14, 86 (1986). 9 J.-M. Warnet, M. O. Christen, M. Thevenin, B. Diard. A. Jacqueson, and J. R. Claude, Pharmacol. ToxicoL 65~ 63 (1989).

334

MIMICS OF BIOLOGICAL THIOLS AND INHIBITORS

[34]

TABLE I CLASSIC ILLUMINATING SYSTEMS: INFLUENCE ON FLUORIMETRY READER SENSITIVITYa

Detection limita Visible Illumination Excitation/emission (nm) Lamp type of reader Mercury vapor Tungsten Xenon

UV, Hoechst 3 3 2 5 8

Rhodamine 123

Neutral red

350/450

490/535

540/600

+ + + to + + + + 0 + + to + + +

++ +++ +++

0 + to + + + + to + + +

a Detection limit zones of different fluorescent probes: + and + +,/zg/ml; + + +, ng/ml; + + + +, pg/ml; 0, background/signal ratio <2 for controls.

Cold Light Fluorimetry Development The new cold light fluorimetric technology was developed with modifications relative to the standard technology: a krypton-xenon mixture was used inside the lamp, the size and axis of the lamp were altered, filter quality and position were changed, an infrared blocking system was added, and a light beam drive, optical trap, and spectral repartition were included. These modifications led to an increase in the light flow from 1000 to 2300 lumens for the same power of 75 W (Table II), with a corresponding decrease in calorific energy (from 75 to 25% of total energy). The detection limits of fluorescent probes were improved in all tested parts of the spectrum (UV, visible, and near-infrared) and were as low as femtograms per milliliter (Table III). Energetic Equation in Cold Light Fluorimetry. The relationship between the lamp voltage (X) and fluorescent units (FSU) of probes (Y) is defined by the formula Y = 9.6303 x

10 °'64714x

TABLE II LIGHT ENERGY REPARTIT1ON IN CLASSICAND COLD LIGHT FLUORIMETRY

Parameter

Classic fluorimetry

Cold light fluorimetry (CLF)

Calorific energy (% of light Cow) UV-visible photonic energy (% of light flow) Light flow (lumens) Electric power (W)

65-70 35-30 1000 75

25-30 75-70 2300 75

[34]

COLD LIGHTFLUORIMETRYTO EVALUATEBIOTHIOLS

335

TABLE 1II DETECTIONLIMITOF FLUORESCENTPROBESWITHCOLDLIGHTFLUORIMETR'~' Detection limit Visible Illumination Excitation/emission (nm) Lamp type of reader Krypton-xenon lamp (with cold light concept)

UV, Hoechst 33258

Rhodamine 123

Neutral red

350/450

490/535

540/600

<500 fg/ml

<30 pg/ml

<200 pg/ml

By varying the voltage of the light source f r o m 0 to 12 V, it is possible to increase considerably the fluorescence signal (Fig. 1) and to measure very low concentrations of fluorescent probes. For example, a signal of 100 FSU at 2 V can be increased by 64-fold at 4 V. In all cases, the fluorescent signal/background ratio must be higher than 5. Reproducibility. Intrarun and interrun variability were low, as assessed by the coefficient of variation, which ranged from 0.9 to 1.9% for intrarun comparisons and f r o m 0.8 to 1.6% for interrun. This high reproducibility

120000

100000

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60000-

40000"

20000-

0

~" 0

,

~

2

,

4

-

,

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Voltage

FIG. 1. Energy equation: FSU = f(voltage). Y = 9.6303 × I0064714X,r

=

0.973.

336

1341

MIMICS OF BIOLOGICAL THIOLS AND INHIBITORS 800

600

P

~

400

200

I

20

I

40

!

60

I

80

I

100

120

Concentration pg/mi (Hoe¢hst 33258) Fro. 2. Hoechst 33258 detection limit with cold light fluorimetry. Y = 34.65 + 6.5573X, r = 0.989.

is an essential criterion for fluorimetric microtitration assays used in pharmacological and toxicological studies. Sensitivity. The detection limit of probes was evaluated in the UV spectrum with Hoechst 33258 (H33258) and in the visible spectrum with neutral red. H33258 is a useful probe for D N A evaluation, x°'n but it cannot be used in microtitration methods using standard fluorimeters, because of their weak sensitivity in U V spectrum. 12 In contrast, the cold light fluorimeter can easily detect the probe with very high sensitivity (near fg/ml) (Fig. 2). Neutral red probe, commonly used for cytotoxicity studies in photometric microtitration assays, 13-16 was proposed as a fluorescent probe. ~7 Its use 10 W. L. Richards, Exp. Cell Res. 159, 235 (1985). 11 H. K. Plummer, In Vitro Cell Dev. Biol. 28, 4 (1992). 12j. Rao and W. R. Otto, Anal. Biochem. 207, 435 (1992). 13 E. Combrier, P. Metezeau, X. Ronot, H. Kiefergachelin, and M. Adolphe, Toxicol. in Vitro 4, 751 (1990). 14 O. Blein, M. Adolphe, B. Lakhdar, J. Cambar, G. Gubanski, D. Castelli, C. Contie, F. Hubert, F. Latrille, P. Masson, J. Clouzeau, J. F. Le Bigot, O. Da Silva, and K. G. Dossou, Toxicol in Vitro 5, 555 (1991). 15 A. Fautrel, C. Chesne, A. Guillouzo, G. Desousa, M. Placidi, R. Rahmani, F. Braut, J. Pichon, H. Hoellinger, P. Vintezou, I. Diarte, C. Melcion, A. Cordier, G. Lorenzon, M. Benicourt, B. Vannier, R. Fournex, A. F. Peloux, N. Bichet, D. Gouy, J. P. Cano, and R. Lounes, ToxicoL in Vitro 5, 543 (1991).

[34]

COLD LIGHT FLUORIMETRY TO EVALUATE BIOTHIOLS

337

TABLE IV NEUTRAL RED DETECTIONLIMITIN PHOTOMETRYAND FLUORIMETRY Type of Detection

Lamp type

Neutral red detection limit (/~g/ml)

Photometric (540 nm) Fluorimetric (excitation, 540 nm; emission, 600 nm)

Tungsten lamp Tungsten lamp Xenon lamp Krypton-xenon lamp (cold light fluorimetry)

0.05 75 0.01 0.00019 (->190 pg/ml)

was limited, however, because of the lack of an adequate fluorescence reader, requiring a 20-fold higher concentration (1 mg/ml) than in photometric methods. The application of the cold light concept to the neutral red detection led to an improvement of detection limits by 500- to 1000fold (Table IV). Importance of Microplate Plastic in Microtitration Fluorimetry. To improve the fluorescence signal, the choice of plastic microplates is decisive. Black microplates used for fluorimetric biochemistry assays are not adapted for cell biology, because they are neither treated for cell culture nor sterilized. Currently, we use standard cell culture translucid microplates for microtitration fluorimetric assays. To prevent fluorescence contamination between two wells observed with some probes (cross-talk fluorescence), a special plate template is necessary (Fig. 3). Absorption of UV light by plasIics is well-known. Nevertheless, in some readers, light flow illuminates the cells through a plastic well, leading to a background increase and a photonic energy decrease. An epifluorescence reading (as in fluorescence microscopy) is used in CLF to eliminate this phenomenon. In addition, autofluorescence of plastic microplates in the visible range could induce a background signal. The high-energy system in CLF reduces this problem and maintains a good signal/background ratio. In the future, the system will be improved by using a special cell culture microplate for fluorimetric assays. Three important features of such microplates have to be emphasized: cell culture treatment and sterilization, black sides for each well to eliminate cross-talk fluorescence, and a translucid bottom to control cells by fluorescence microscopy before reading and to allow direct photographs and image analysis. 16A. F. Peloux, C. Federici, N. Bichet, D. Gouy, and J. P. Cano, A T L A , Altern. Lab, Anirn. 20, 8 (1992). 17 j. S. Essig-Marcello and R. G. Van Buskirk, In Vitro Toxicol. 3, 219 (1990).

338

[34]

MIMICS OF BIOLOGICAL THIOLS AND INHIBITORS

1

2

3

4

5

6

Cla, Clb Clc C1(

6

7

8

9

10

11

12

Sla 511~ Slc" Sld"Ii

I

Well without cells

c

=: :=

D

SZa S 2 b l S 2 C I s 2 ,

S3aiS3bi

s3c

S3. ,,: ........... [

[~

i

[

Vehicle Control {cells + Fluorescent probe}

F

S4a S4blS4Cl S4c

SSa SSb S5c

SS~= ............. : ['S]Sample --{C¢IIs

G H

+ trcatmcat

+ Fluorescentprobe}

: ....... ........

FIG. 3. Plate template for cold light fluorimetry with clear, cell culture-treated microplate.

Application of Cold Light Fluorimetry to Biothiol Evaluation The eytoprotective effect of two biothiols, N-acetylcysteine (NAC) and anethole dithiolethione (ADT), against acetaminophen cytotoxicity has been evaluated. Acetaminophen Cytotoxicity Evaluation. In multicentric studies, AAP cytotoxicity on hepatocytes has been extensively evaluated using the photo120 -

100.

*

• 80-

L929 - NR assay

[ ] HepG2-NR assay

•

L929 - Rh123 assay *

[ ] HepG2-Rh123 assay *

~" 60~

4020

Control without AAP

24 H treatment with AAP (250 p.g/ml)

FIG. 4. A c e t a m i n o p h e n ( A A P ) cytotoxicity evaluation on the L929 and H e p G2 cell lines. • , P < 0.05. C o m p a r i s o n of neutral red and r h o d a m i n e 123 assay. Rhodamine123 with hepatic cell line H e p G2 is the most sensitive system for detecting A A P toxicity.

[34]

COLD LIGHT FLUORIMETRY TO EVALUATE BIOTHIOLS

339

200

1Control [~]AAP []AAP+NAC ['/"AAAP+ADT 100 '7.

=~

__ ~~ _ 0

m

Control

AAP (200 txg/ml)

AAP NAI or ADT (1~ M)

FIG. 5. Fluorescent neutral red assay to detect the cytoprotective effect of A D T or N A C against acetaminophen toxicity [48 hr of pretreatment with antidotes (1 /zmol) followed by treatment with A A P (200/xg/ml, 48 hr)].

metric neutral red assay (IC50 at 24 hr of 975 to over 2000 /xg/ml). 1~-16 Because A A P toxicity is targeted to mitochondria, 18 it was convenient to use a human Hep G2 cell line which has a large number of mitochondria and a growth completely adapted to microtitration assays. 19 Therefore, preliminary experiments were performed to evaluate whether AAPinduced cell injury can be easily detected with the CLF technology in two cell types (L929 and Hep G2). Cell viability alterations induced by A A P were detected as soon as 24 hr after incubation in rhodamine 123 assays (Fig. 4), whereas they are observed only after 48 hr of incubation in neutral red assays at a concentration (250/~g/ml) similar to that observed in the first steps of human A A P intoxication. Fluorescent Neutral Red Assay. A significant cytoprotective effect of ADT, superior to that of NAC, can be observed against A A P injury (Fig. 5). This result, observed with the human Hep G2 cell line, after a 48-hr 18 p. C. Burcham and A. W. Harman, Toxicol. Lett. 50, 37 (1990). 19 E. Bornefreund, H. Babich, and N. Martinalguacil, In Vitro Cell Dev. Biol. 26, 1030 (1990).

340

MIMICS O F B I O L O G I C A L T H I O L S A N D I N H I B I T O R S

[34]

pretreatment with each biothiol (1/xM), confirms the protective effect of A D T observed in animals. 9 This marked cytoprotective effect of A D T was confirmed with rhodamine 123 cell viability assay. R h o d a m i n e 123 A s s a y . The relationships between the mitochondrial transmembrane potential, ATP concentration, and cytotoxicity are well known. 3,2° The rhodamine 123 assay is relevant for evaluating cytotoxicity,21-23 and has shown an increase in mitochondrial activity potential (marker of cellular energy) when A D T and N A C were used as A A P antidotes. 24 Conclusion A new fluorimetric method, cold light fluorimetry, exhibiting high specificity and sensitivity, has been developed. It could represent a major improvement in cytotoxicity screening of drugs. The fluorimeter that we developed incorporating the cold light concept reads UV, visible, and near-infrared fuorescence by increasing the photonic energy (1000 to 2300 lumens) and reducing the calorific part of the light. The detection limit of probes for evaluation of cell proliferation (DNA/H33258), viability (cell integrity/fluorescent neutral red), or metabolism (mitochondria/rhodamine 123) is improved (by 500- to 1000-fold) in the UV, visible, and near-infrared spectra, and many other probes can also be used. The cytoprotective effect of biothiols like N-acetylcysteine or anethole dithiolethione can be assayed with high sensitivity and specificity using this new technology. Cold light fluorimetry allows the detection of very low concentrations of probes (picograms to femtograms per milliliter), directly in 6, 12, or 96well microplates or petri dishesY Thus, it is a promising alternative to flow cytometry for providing a cell viability index in pharmacological and toxicological studies.

20E. Y. Wu, M. T. Smith, G. Bellomo, and D. Di Monte, Arch. Biochem. Biophys. 282, 358 (1990). 21E. G. Adams, K. Barden, R. Badiner, R. Ulrich, and B. K. Bhuyan, Cell Tissue Kinet. 17, 670 (1984). z2C. A. Rahn, D. W. Bombick, and D. J. Doolittle, Fundam. Appl. Toxicol. 16, 435 (1991). 23p. Rat, M. O. Christen, M. Thevenin, J.-M. Warnet, and M. Adolphe, Toxicol. Lett. $1, 214 (1992). 24p. Rat, Nouv. Dermatol. 12, 471 (1993). 25p. Rat, J. P. Simoulin,J. C. Hildeilfinger,J.-M. Warnet, and M. Adolphe, Toxicol. Letr S1, 238 (1992).

[351

EBSELEN

341

[351 Ebselen B y HELMUT SIES

Introduction Ebselen, 2-phenyl-1, 2-benzisoselenazol-3(2H)-one, has attracted interest since it was found that it exhibits activity as an enzyme mimic. The reaction catalyzed is that of a glutathione (GSH) peroxidase, namely, the reduction of a hydroperoxide at the expense of thiol. In 1984, the glutathione peroxidase-like activity of ebselen (then called P Z 51) was describedJ "2 Since then, extended research on this interesting molecule has covered areas from pulse radiolytic studies on radical reactivity through its biological properties in cells and organs, all the way to clinical settings (see Ref. 3; also, this series4). Basically, this compound is considered capable of contributing to the antioxidant defense in tissues, so that a potential pharmacological application becomes of interest. Clinical studies are underway. The reader is referred to other reviews 3'4 for detailed information on the properties of the molecule.

Range of H y d r o p e r o x i d e S u b s t r a t e s a n d Thiols Maiorino et aL 5 found that ebselen also mimics the phospholipid hydroperoxide G S H peroxidase reaction. Indeed, ebselen plus glutathione was shown to protect low-density lipoprotein ( L D L ) against peroxidation 6 and to be capable of reducing high-density lipoprotein ( H D L ) - and LDLassociated cholesteryl ester and phospholipid hydroperoxides. 7 The nature of the hydroperoxide substrate, therefore, covers a wide range, from hydrogen peroxide to smaller organic hydroperoxides up to membrane-bound phospholipid and cholesteryl ester hydroperoxides. Likewise, the thiol utilized is not restricted to GSH. Unlike the enzymes catalyzing the G S H peroxidase reaction, which contain specific binding I A. Mailer, E. Cadenas, P. Graf, and H. Sies, Biochem, Pharmacol. 33, 3235 (1984). 2 A. Wendel, M. Fausel, H. Safaghi, G. Tiegs, and R. Otter, Biochem. Pharmacol. 33, 324l (1984). 3 H. Sies, Free Radical Biol. Med. 14, 333 (1993). 4 H. Sies, this series, VD1.234, p. 476. 5M. Maiorino, A. Roveri, and F. Ursini, Arch. Biochem. Biophys. 295, 404 (1992). 6 C. E. Thomas and R. L. Jackson, J. Pharmacol. Exp. Ther. 256, 1182 (1991). 7W. Sattler, M. Maiorino, and R. Stocker, Arch. Biochem. Biophys. 309, 214 (1994).

METHODS IN ENZYMOLOGY, VOL. 252

Copyright © 1995 by Academic Press, lnc. All rights of reproduction in any form reserved.

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sites for that thiol, ebselen can utilize the reducing equivalents from, for example, dithioerythritol,8 N-acetylcysteine,9 or dihydrolipoate.1° Metabolism a n d Disposition

Ebselen metabolism has been studied in isolated perfused liver11 and intact rats, pigs, and human volunteers. 12,13The detected metabolites share the characteristics that the isoselenazole ring is opened by cleavage of the Se-N bond. It is important to note that no unchanged ebselen was detectable in urine, plasma, or bile. a2 The facile ring opening is probably a basis for this. Whether ebselen is attacked by sulfhydryl compounds already in the stomach or intestine before absorption or while transported through the mucosa is not yet known. In model studies using bovine serum albumin, rapid binding of ebselen was observed, TM probably to the reactive thiol group at cysteine-34, the location identified for albumin-bound cysteine or glutathione, a5 In model experiments, the binding of ebselen to glutathione transferases was studied, using the ebselen-albumin complex as a s o u r c e . 16 The physiological transport of ebselen across cell membranes and into the cytosol and further subcellular compartments is likely to involve ebselen in its protein-bound form. Wendel et al. 2 observed that the selenium contained in ebselen was not bioavailable; it did not enter the body pool of selenium. This is considered as a basis for the lack of toxicity observed in experimental studies. Elimination occurs via the ebselen metabolites, so that selenium toxicity is not a problem in this context. However, ebselen in the selenol form can be a substrate for pig liver flavin-containing monooxygenase, yielding a thiol oxidant. 17 Thus, in tissues expressing such activities, potentially harmful bioactivation could occur. ~8 s A. MOiler, H. Gabriel, and H. Sies, Biochem. Pharmacol. 34, 1185 (1985). 9 I. A. Cotgreave, M. S. Sandy, M. Berggren, P. M. Moldeus, and M. T. Smith, Biochem. PharmacoL 36, 2899 (1987), 10 I. A. Cotgreave, P. Moldeus, R. Brattsand, A. Hallberg, C. M. Andersson, and L. Engman, Biochem. Pharmacol. 43, 793 (1992). 11 A. MiJller, H. Gabriel, H. Sies, R. Terlinden, H. Fischer, and A. R0mer, Biochem. Pharmacol. 37, 1103 (1988). 12 H. Fischer, R. Terlinden, J. P. L6hr, and A. ROmer, Xenobiotica 18, 1347 (1988). 13 n . Sies, in "Selenium in Biology and Medicine" (A. Wendel, ed.), p. 153, Springer-Verlag, Heidelberg, 1989. 14H. Nomura, H. Hakusui, and T. Takegoshi, in "Selenium in Biology and Medicine" (A. Wendel, ed.), p. 189. Springer-Verlag, Heidelberg, 1989. 15 T. Peters, Jr., Adv. Protein Chem. 37, 161 (1985). 16T. Nikawa, G. Schuch, G. Wagner, and H. Sies, Biochem. Pharmacol. 47, 1007 (1994). 17 D. M. Ziegler, P. Graf, L. L. Poulsen, W. Stahl, and H. Sies, Chem. Res. Toxicol. 5,163 (1992). 18 T. P. M. Akerboom, H. Sies, and D. M. Ziegler, Arch. Biochem. Biophys. 316, 220 (1995).

[36]

THIOLCOMPOUNDSAND HTLV-I

[36] T h i o l C o m p o u n d s and Adult T-Cell Leukemia Infection: A Potential Therapeutic Approach

343

Virus

B y NORIHITO SATO, SATOSHI IWATA, A K I R A YAMAUCHI, TOSHIYUKI HORI, a n d JUNJI Y o D o l

Introduction Adult T-cell leukemia (ATL) is a peculiar form of acute leukemia of mature T lymphocytes which is caused by the human lymphotropic retrovirus HTLV-I. 1-3 HTLV-I is also related to HTLV-I-associated myelopathy (HAM), 4 or tropical spastic paraparesis (TSP), 5 although the pathogenic role of HTLV-I is still unclear in these diseases. T cells infected with HTLV-I generally overexpress interleukin 2 (IL2) receptor oe chain (IL-2Ra, Tac antigen), which is one of the markers of activated T lymphocytes. In 1985, our laboratory found IL-2Ra-inducing activity in culture supernatants of HTLV-I-transformed T cell lines, 6 Subsequent purification revealed that the IL-2Ro~-inducing activity resided in a 13-kDa protein. 7 cDNA cloning of the molecule, which we designated as ATL-derived factor (ADF), led to an unexpected finding that ADF is a human homolog of bacterial thioredoxin, 8 a dithiol-reducing enzyme. described in detail in other chapters of this volume. In the studies on ADF/ human thioredoxin (hTRX), we and collaborators have found that the expression of ADF/hTRX is highly upregulated in HTLV-I-transformed as well as in Epstein-Barr virus (EBV)-transformed lymphoid cells and that extracellularly applied recombinant A D F has growth-promoting activity on the virus-transformed cells under certain conditions, indicating that A D F / hTRX may act as an autocrine growth factor in HTLV-I- or EBVJ. Yodoi and T. Uchiyama, lmmunol. Rev. 92, 136 (1986). 2 T. Uchiyama, J. Yodoi, K. Sagawa, K. Takatsuki, and H. Uchino, Blood 50, 481 (1977). 3 j. Yodoi, K. Takatsuki, and T. Masuda, N. EngL J. Med. 290, 572 (1974). 4 M. Osame, K. Usuki, and S. Izumo, Lancet 1, 1031 (1985). 5 A. Gessain, F. Barim, and J. C. Vernant, Lancet 2, 407 (1985). K. Teshigawara, M. Maeda, K. Nishino, T. Nikaido, T. Uchiyama, M. Tsudo, M. Wano, and J. Yodoi, J. Mol. Cell. Imrnunol. 2, 17 (1985). 7 y. Tagaya, M. Okada, K. Sugie, T. Kasahara, N. Kondo, J. Hamuro, K. Matsushima, C. A. Dinarello, and J. Yodoi, J. IrnrnunoL 140, 2614 (1988). Y. Tagaya, Y. Maeda, A. Mitsui, N. Kondo, H. Matsui, J. Hamuro, N. Brown, K.-I. Arai, T. Yokota, H. Wakasugi, and J. Yodoi, EMBO J. 8, 757 (1989).

METHODS IN ENZYMOLOGY, VOL. 252

Copyright © 1995 by Academic Press. Inc. All rights of reproduction in any form reserved.

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MIMICS OF BIOLOGICAL THIOLS AND INHIBITORS

[36]

transformed cells. 9,x° These findings also prompted us to study the pathogenic roles of ADF/hTRX and other thiol compounds in the virus transformation process. The results of our studies imply that modulation or modification of thiol-related processes may have some therapeutic potential in ATL and HTLV-I related diseases. Materials and Methods

Media and Cells The cell lines ATL-2 and HUT 102 are established from HTLV-I infected peripheral T lymphocytes from ATL patients. Jurkat is a HTLV-I negative T-cell line. These cells are maintained in RPMI 1640 medium (GIBCO-BRL, Grand Island, NY) supplemented with 10% fetal calf serum (FCS) (Filtron PTY Ltd., Brooklyn, Australia) in 5% (v/v) CO2 in air at 37 °. Human peripheral blood lymphocytes (PBL) are isolated from healthy donors, cultured in RPMI 1640 medium supplemented with 10% FCS in the presence of 0.1/zg/ml PHA-P (Difco, Detroit, MI) for 3 days, then in the presence of 1 nM human recombinant IL-2 (Takeda Chemical) for 4 days, and used as PBL blasts. Standard RPMI 1640 medium contains I00/zM cystine and 1 /zg/ml glutathione. Cystine-free, GSH-free RPMI 1640 medium (thiol-free medium) is obtained from Nikken Bio Medical Laboratory Inc. (Kyoto, Japan).

Viability Assay Cells were washed once with phosphate-buffered saline (PBS) and twice with thiol-free medium, then cultured in thiol-free medium supplemented with 10% FCS for 48 hr in 24-well plates (Nunc, Roskilde, Denmark). Cellular viability is determined by trypan blue dye exclusion. An aliquot of cells is mixed with an equal amount of 0.16% (w/v) trypan blue in PBS. The stained and unstained cells are then counted manually.

Flow Cytometric Analysis of DNA Content Nuclear DNA content is analyzed by flow cytometry (CYTORON; Ortho Diagnostic Systems K. K., Tokyo, Japan) after staining with propidium iodide (PI; Calbiochem-Behring, La Jolla, CA) according to the method 9 N. Wakasugi, Y. Tagaya, H. Wakasugi, A. Mitsui, M. Maeda, J. Yodoi, and T. Tursz, Proc. Natl. Acad. Sci. U.S.A. 87, 8282 (1990). 10A. Yamauchi, H. Masutani, Y. Tagaya, N. Wakasugi, A. Mitsui, H. Nakamura, T. Inamoto, K. Ozawa, and J, Yodoi, Mol. lmrnunol. 29, 263 (1992).

[36]

THIOLCOMPOUNDSAND HTLV-I

345

described by Nicoletti et al. n Cells are pelleted, resuspended in hypotonic fluorochrome solution (50/xg/ml PI in 0.1% (w/v) sodium citrate and 0.1% (w/v) Triton X-100), and placed at 4 ° in the dark overnight before the analysis. An alternate protocol for flow cytometric analysis of apoptotic cell death is provided by Darzynkiewicz et al. aa

L-Cystine Transport A s s a y

L-Cystine transport is measured by the method described by Bannai et al. with some modificationsJ 3 [14C]-Cystine (NEN, Boston, MA) is added to 37 kBq per 10 6 cells (final concentration of 6 ~M) in thiol-free medium. After incubation at 37 °, the transport is terminated by washing with icecold PBS three times. The pellet is lysed by the addition of 0.5 N NaOH, then subjected to radioactivity measurement and protein determination.

Results a n d D i s c u s s i o n Thiol compounds in the extracellular environment, blood plasma in vivo and culture medium in vitro, all modulate various aspects of lymphocyte functions. Cysteine, a sulfur amino acid, has been shown to increase the D N A synthesis of mitogenically stimulated lymphocytes and cytotoxic Tlymphocyte activity. TM 2-Mercaptoethanol or reduced glutathione have also been reported to potentiate the cytotoxic activity or proliferation of routine lymphocytes induced by IL-2. Though the disulfide cystine does not have free thiols itself, it can be incorporated into the cells, reduced to cysteine, used as the precursor of thiol compounds, 15 and thus can affect lymphocyte function via the modulation of thiol-related intracellular processes. As shown in Fig. 1, thiol deprivation from the culture medium resulted in the loss of viability in HTLV-I infected cell lines, whereas virus-negative cell lines remained viable 48 hr after thiol deprivation. The relative sensitivity of HTLV-I-infected cell lines to cystine deprivation suggests that HTLV-I infection causes the derangement of cellular thiol metabolism and that environmental thiols could be a potential therapeutic target. Apoptosis, the active process of cell death, occurs under many physiolog11I. Nicoletti,G. Migliorati,M. C. Pagliacci,F. Grignani,and C. Riccardi,J. ImmunoL Methods 139, 271 (1991). 12Z. Darzynkiewicz,S. Bruno, G. Del Bino, W. Gorczyca,M. A. Hotz, P. Lassota, and F. Traganos, Cytometry 13, 795 (1992). 13S. Bannai, H. Sato, T. Ishii, and Y. Sugita, J. Biol. Chem. 264, 18480 (1989). 14H. Griinder,H.-P. Eck, B. Bennighoff,S. Roth, and W. Dr6ge, Cell Immunol. 129,32 (1990). 158. Bannai, Biochim. Biophys. Acta 779, 289 (1984).

346

[36]

MIMICS OF BIOLOGICAL THIOLS AND INHIBITORS 100 -T.,

A

n-

I

,.T... 80-

! i

70"

i i

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I

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i

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!

30"

l

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o

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i MT-2

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K562

FIo. 1. Effects of cystine deprivation on the viability of various cell lines. Cells were cultured for 48 hr in glntathione-free RPMI 1640 supplemented with 10% FCS in the absence (solid bars) or presence of 200 /xM cystine (open bars). Cell viability was determined by trypan blue dye exclusion. [Reprinted from Molecular Immunology, Volume 29, A. Yamauchi, H. Masutani, Y. Tagaya, N. Wakasugi, A. Mitsui, H. Nakamura, T. Inamoto, K. Ozawa, and J. Yodoi, Lymphocyte transformation and thiol compounds; the role of ADF/thioredoxin as an endogenous reducing agent, page 267, Copyright (1992), with kind permission from Elsevier Science Ltd., The Boulevard, Langford Lane, Kidlington OX5 1GB, UK.]

ical and pathological conditions, such as embryonic development, growth factor withdrawal, and cytolysis caused by cytotoxic lymphocytes, cytokines, or cancer chemotherapeutic drugs. N-Acetylcysteine, which acts as both a precursor of glutathione and an antioxidant itself, has been demonstrated to block the apoptosis induced by various stimuli, including human immunodeficiency virus (HIV) infection and growth factor withdrawal} 6'17 These findings suggest that intracellular redox status, especially that of thiol compounds, may play a central role in apoptotic cell death. D N A content measurement after the extraction of fragmented D N A suggested that cell death induced by thiol deprivation in ATL-2 cells was apoptotic rather than simply necrotic (Fig. 2). The finding that cystine deprivation can induce apoptosis in HTLV-I-infected cell lines supports the importance of thiolrelated processes in apoptosis. The induction of apoptosis is either desirable or undesirable depending on the circumstances. In the case of HTLV-I infection, which is clinically manifested as neoplastic growth of lymphocytes, 16W. Malorni, R. Rivabene, M. T. Santini, and G. Donelli, FEBS Lett. 327, 75 (1993). 17 D. M. Hockenberry, Z. N. Oltvai, X.-M. Yin, C. L. Milliman, and S. J. Korsmeyer, Cell (Cambridge, Mass.) 75, 241 (1993).

[361

TmOL COMPOUNDSArqO H T L V - I

A

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B

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DNA lluorescence

Fie. 2. Flow cytometric analysis of the DNA content of ATL-2 cells. ATL-2 cells were cultured for 24 hr in the absence (B) or presence (A) of 200/zM cystine, and then the DNA content was measured by propidium iodide staining. The percentages of nuclei with decreased DNA content (apoptotic nuclei) are indicated.

the induction of apoptosis may be beneficial or effective in therapy. The finding raises the possibility that HTLV-I-infected tumor cells may be selectively induced to undergo apoptosis by modifying thiol compounds in the extracellular environment. 1500

1000

12.

o

500

i

i

i

2

1

0

conc. of rADF (txM)

F1G. 3. Effect of recombinant ADF (rADF) on L-cystine incorporation of PBL blasts. The PBL blasts were incubated in Dulbecco's PBS containing 0.5 mM MgCI2, 0.9 mM CaCI2, and 5.6 mM o-glucose supplemented with approximately 6/xM of L-[14C] cystine (2/xCi/ml) in the presence or absence of rADF for 1 hr. Radioactivity per microgram protein of the lysate is shown.

348

MIMICS OF BIOLOGICAL THIOLS AND INHIBITORS

[361

One of the thiol compounds which represents a potential therapeutic target is ADF/hTRX. We have previously shown that HTLV-I- or EBVinfected cell lines tend to produce a larger amount of ADF/hTRX than virus-negative cell lines. 1° Extracellular secreted A D F may act as an autocrine growth factor in the virus-transformed cell lines because supplementation of recombinant A D F can promote the growth of cells or support their survival in low serum cultures. 9 This extracellular action of ADF/hTRX can in part be explained by the enhancement of cellular cystine uptake. Micromolar concentrations of recombinant A D F augmented the uptake of cystine by PBL blasts about 6- to 8-fold (Fig. 3). The increase of intracellular thiol pools by ADF/hTRX may promote this proliferation. In addition to these extracellular actions, intracellular ADF/hTRX has been shown to be important for the growth of HTLV-I infected cells: in some HTLV-Iinfected cell lines, the expression of ADF/hTRX has been shown to change during cell cycle transitions, indicating that ADF/hTRX may be required for cell cycle progression in these cells) s Furthermore, 13-cis-retinoic acid, which has been reported to inhibit thioredoxin reductase, ~9showed growth inhibitory effects in HTLV-I-infected cell lines. 2° Collectively, these findings suggest that ADF/hTRX plays critical roles in the continuous growth or survival of the HTLV-I-infected cells and that the inhibition of the A D F / hTRX system may be a new therapeutic approach for HTLV-I-related disorders. Certainly, glutathione, the most abundant intracellular thiol, can be another potential therapeutic target. Although it is not clear whether dysregulation of glutathione is involved in the pathogenesis of HTLV-I infection, it would seem to be worth studying the effect of modifying glutathione metabolism on HTLV-I-infected cells. We hope that further studies to dissect the role of various thiol compounds, including ADF/hTRX and glutathione, in the HTLV-I transformation process will aid the establishment of new therapies for ATL and HTLV-I-associated diseases.

lS y. Ueda and J. Yodoi, unpublished observation, 1993. 19 K. U. Schallreuter and J. M. Wood, Biochem. Biophys. Res. Commun. 160, 573 (1989). 20 H. Masutani, H. Nakamura, Y. Ueda, Y. Kitaoka, T. Kawabe, S. Iwata, A. Mitsui, and J. Yodoi, in "Microbial Infections: Role of Biological Response Modifiers" (H. Fridman, ed.), p. 265. Plenum, New York, 1992.

AUTHOR

INDEX

349

Author Index

Numbers in parentheses are footnote reference numbers and indicate that an author's work is referred to although the name is not cited in the text.

A Abate, C., 175 Abeles, R. H., 100 o Aberg, A., 287 Adams, E. G., 340 Adams, J. A., 9 Adamson, S. R., 187, 193(9) Adler, S., 268 Adolphe, M., 323, 331432, 336, 340 Affara, N., 39, 42(11) Agostino, A., 241 Aguilar, F., 222, 275, 277(10) Ahgren, A., 287 Ahmad, H., 54, 73 Ahmad, M., 253 Ahmed, S., 133, 155, 161 Ahn, B.-Y., 33, 36(20), 285 Aillet, F., 53 Akasaka, M., 41, 42(28, 34) Akerboom, T. P., 181 Akerboom, T. P. M., 73,152, 153(60, 61), 342 Alary, R., 232 Albermann, K., 254, 262(14), 263(14) Albers, J. W., 151 Albers, R. C., 129 Alexander, R. W., 253 Alin, P., 65 Allen, L., 68 Alien, R. H., 95, 98(13) Aim, D. M., 220 Ames, B. N., 86 Amman, E., 245 Amstad, P., 181,253 Andalibi, A., 177 Anders, M. W., 18 Anderson, E. I., 32

Anderson, L., 316, 321(5), 322(5) Anderson, M. E., 26-27, 27(3), 28(6), 29, 66, 67(1), 103 Anderson, M. T., 168 Anderson, W. B., 132, 133(7, 8), 135(7), 137, 138(26), 139(7), 140(8), 152 Andersson, C. M., 342 Ando, S., 90 Andreesen, J. R., 39 Ansher, S. S., 316, 318(2), 321 Aoyama, E., 211 Aperia, A., 149 Apontoweil, P., 84-85, 85(6) Arai, K.-I., 200, 343 Archer, E. G., 104 Archer, S., 316 Areseni, M., 94, 97(8) Arizmendi, C., 189 Arnold, H., 91 Arscott, D., 194 Arscott, L. D., 191, 193, 202 Arvidson, K., 9 Asada, K., 31 Ase, K., 133, 136(18) Ashton, F. M., 229 Askeli3f, P., 283, 292(6) o Aslund, F., 283-285, 287, 287(16), 288(16), 289(34, 35), 290(34), 291(34, 35), 292(35), 304, 306 Augereau, J. M., 132, 136(14), 143(14) Aumann, K.-D., 38, 40, 41(21), 42(21), 47, 48(63), 49(63), 52(21) Aust, S. D., 326 Austin, D. F., 3 Avissar, N., 40 Aw, T. Y., %4, 9, 9(11), 12, 13(28), 17.72 Awasthi, Y. C., 53-54, 73.75

350

AUTnOR~NDEX

Axelsson, K., 33, 37(14), 283, 285, 292(6) Aykaqc, G., 150

Benen, J., 194 Ben-Hayyim, G., 41, 42(33), 51(33) Benicourt, M., 336, 339(15) Benninghoff, B., 253, 263(1), 345 I3 Benson, A. M., 318 Berends, W., 84-85, 85(6) Babich, H., 339 Berget, P. B., 84 Babson, J. R., 5, 95, 109 Berggren, M., 342 Badiner, R., 340 Berglund, O., 291 Baeuerle, P., 168, 169(7), 177 Baeuerle, P. A., 254, 262(12-14), 263, Bergman, T., 287 Bergmann, D., 240 263(13, 14) Berkel, W. V., 194 Bai, C., 9, 10(26), 11(26), 17 Bernal, S. D., 332 Baker, D. H., 53, 96, 97(17) Berry, M. J., 40, 43 Balaban, R. S., 309, 315(6) Berthe-Corti, L., 91 Ballard, D. W., 175, 177(2) Berties, P. J., 149, 151(27) Balls, M., 333 Balogh, A., 229-230, 230(11), 231(11, 16), Bertoglio, J., 254 Bertorello, A. M., 149 236(11), 238(11, 16), 240(16) Besse, I., 230 Baltimore, D., 168-169 Betzel, C., 194 Bannai, S., 104, 345 Beutler, E., 68, 72-73, 73(3), 74(3, 4, 11, 12), Banu, L., 40, 43 75, 75(4, 11), 76(12), 77(6), 79-80, 82, 91 Barbado, C., 88 Beyaert, R., 254, 262(18) B~ircena, J. A., 291 Bhardwaj, R. K., 310 Barden, K., 340 Bardes, E. S. G., 131,133, 153, 156, 157(17- Bhuyan, B. K., 340 19), 158(17), 159, 159(17), 160(18), Biaglow, J. E., 105, 111 Biard, D., 319, 320(27) 164(17-19), 166(18, 19) Bichet, N., 336, 336(16), 337, 339(15, 16) Barim, F., 343 Bieber, P., 168, 169(7) Baron, C., 43 Biemann, K., 33, 284, 285(22), 287, 287(22), Battling, D., 275 291(22) Bartosz, G., 73 Billeter, M., 284, 287(14, 15, 17), 288(17), Batzinger, R. P., 318 289(14, 15, 17), 294, 304(12, 13), 305-306 Baxter, M. L., 41, 42(41) Binkley, F., 92 Bayes, J. B., 150, 151(44) Birkmann, A., 39 Beatrice, M. C., 14 Bishop, W. R., 124, 125(3), 133, 154 Beatty, P. W., 5, 95, 109 Bjelland, S., 36 Beaune, P. H., 59 Bj6rnberg, O., 284, 287(14, 15), 288, 289(11, Bechtel, D. B., 229 14, 15), 290, 290(13), 294, 304, 304(12, Becket, T., 91 13), 305 Beckman, J. S., 145 Bj6rnstedt, M., 199-200, 201(11), 205, Beckman, T. W., 145 205(10), 209-210, 210(10), 211(10), Begley, T., 293, 299(8), 303(8) 212(10, 12), 213(10, 12), 214(10, 12), Beinert, H., 192 215(10), 216(10, 12), 217(10, 12, 13), Beiswanger, C. M., 147-148 218(13), 219(10, 12), 274 Bell, R. M,, 131,133, 137(23), 152-153, 153(5, 6), 154-156,156(9), 157,157(17-20), 158, Blaauboer, B., 59, 65(12) 158(17), 159, 159(17), 160(18, 20), 161(9), Black, S., 283, 285(5), 291(5), 292(5) Blackmore, P. F., 149 164(17-19), 166(18, 19), 167, 167(20) Blanco, A., 88 Bellomo, G., 78, 340 Blein, O., 336 Benel, L., 332

AUTHORINDEX Block, G., 4, 8(13) Bl6cker, H., 40-41, 41(21), 42(21, 23), 52(21) Bloomenthal, J., 131, 133, 156-157, 157(17), 158(17), 159, 159(17), 164(17) Bloomquist, D. R., 128 Blot, W. J., 3 Blumberg, P. M.. 134 Blume, K. G., 9l Board, P. G., 53-54, 72, 76(5) B~Sck, A., 39, 43, 309, 312(2), 315(2) Bock, R. M., 191 Bodenstein-Lang, J., 230 Boettcber, B., 66, 103 Bogaards, J. J. P., 59, 65(12) Bolt, H. M., 63 Bolton, A. E., 207 Bombick, D. W., 340 Borchardt, R. A., 153, 159 Borenfreund, E., 332 Bornefreund, E., 339 Boros, D. L., 318 Borresen, H. C., 91 Borsook, H., 31 Bosch-Morell, F., 148, 150 Bott, M., 201,273 Bourier, S., 105 Bowman, B. B., 4, 9(11), 10, 11(27), 12(27) Boxer, L., 253 Bradford, M. M., 206, 207(44) Brand6n, C.-I., 269, 284, 293-294 Brandes, H. K., 273 Branton, D., 79 Brattsand, R., 342 Braut, F., 336, 339(15) Breunsbach, J., 323 Brigelius, R., I52, 153(60) B rigelius-Floh6, R., 38, 40, 41(21), 42(21), 47, 48(63), 49(63), 52(20, 21) Brimijoin, S., 150 Brodie, A. E., 5, 92, 95, 109 Bromberg, Y., 214 Brosius, J., 245 Brot, N., 202 Brown, L. A., 66 Brown, N., 200, 343 Brown, N. C., 287 Brown, S. B., 302 Brown-Hovanec, J., 102 Brownie, A. C., 31 Brunner. B., 222, 275, 277(10)

351

Bruno, S., 345 Brunovska, A., 271 Buch, A., 230 Buchanan, B. B., 199, 220-222, 224(6), 228230, 230(11), 231, 231(11, 16, 18, 19), 233(25), 236(11, 20), 238(11, 12, 16, 19, 20, 22), 239-240, 240(16), 244, 273275, 279 Bucher, J. R., 326 Bueding, E., 316, 318, 318(2, 3), 321, 321(5), 322(5) Burcham, P. C.. 339 Burk, R. F., 39-40, 46 Burkitt, M. J., 53 Burns, D., 133, 137(23) Burns, D. J., 153(6), 154, 156(9), 157, 161(9) Burns, R. F., 141 Burt, D. R., 318 Busam, K. J., 181 Bushuk, W., 229, 235 Bushweller, J. H., 284, 287(14-17), 288(16. 17), 289(14, 15, 17), 290(18), 294, 304(12. 13), 305-3O6 Butler, J. A., 12 Butterworth, M,. 103 Byers, T. E., 3

C Cadenas, E , 341 Calligaro, A., 52 Cambar, J., 336 Cambillau, C., 269, 284 Cameron, N. E., 150 Camoin, L., 41, 42(33), 51(33) Campbell, A. P., 293,299, 299(8), 303(8, 25) Campbell, K., 41, 42(39) Canada, A. T., 86 Canellakis, Z. N., 287 Cano, J. P., 336, 336(16), 337, 339(15, 16) Cantley, L., 149 Capron, A., 40, 41(22), 42(22) Caradonna, J. P., 272 Carey, J., 177 Carlquist, M., 265, 283 Carlson, D. E., 244 Carlson, J, 266 Carothers, D., 188 Carothers, D. J., 186, 194, 194(3) Carpenter, C. L., 149

352

AUTHORINDEX

Carroll, M. D., 4 Carroll, P. B., 148, 151 Carver, D. K., 47 Casadaban, M. J., 312 Casasco, A., 52 Case, D. A., 293, 298, 299(7, 8), 303(7, 8) Casini, A. F., 38 Castelli, D., 336 Cavanagh, J. B., 147 Cerny, T., 11l Cerroni, L., 314 Cerutti, P., 132, 134(10), 181 Cerutti, P. A., 253 Cesbron, J., 319 Cha, Y. N., 318 Chamberlain, S. H., 230 Chamberlin, M., 265, 267(2) Chambers, I., 39, 42(11) Chandrasekhar, K., 202, 294, 299, 299(15), 301,303(15, 25, 34) Chang, K., 150, 15l(46) Chang, L.-S., 26, 27(3) Chang, M., 71 Chary, K., 284, 290(13) Chary, K. V. R., 304 Chen, E., 133 Chert, J., 145 Chen, L. B., 332, 340(3) Chen, S., 132, 140(11) Chen, Z., 133, 145 Chen, Z.-H., 132-133, 135(15), 137, 138(26), 152 Chensue, S, W., 318 Cherian, M. G., 102 Chesne, C., 336, 339(15) Chibata, I., 87 Chiesa, R., 223 Chifflet, S., 223 Chikuma, M., 211 Ching, W.-M., 309 Cho, E. S, 102 Chomczynski, P., 182 Christen, M.-O., 316, 319, 322-324, 329(27), 330(4, 5), 331,333, 340, 340(9) Christensen, H. N., 104 Christine, L., 31 Christodoulides, L. G., 53 Chu, C., 230 Chu, F.-F., 40-41, 42(17, 31), 44, 50(55), 52(17)

Chung, J. S., 33, 36(19) Chvapil, M., 148 Cidlowski, J. A., 175 Civan, A. M., 149 Clancey, C. J., 226 Clark, J. D., 154 Clarke, R., 9 Claude, J. R., 319, 320(27), 333, 340(9) Clausmeyer, S., 275, 279(11) Cleland, W. W., 225, 228(10) Clore, G. M., 200, 293-296, 296(20), 297(20), 299, 299(2, 9, i0, 22), 302(21), 303, 303(9, 21) Clothier, R. H., 333 Clouzeau, J., 336 Coassin, M., 52 Coates, R. J., 3-4, 4(6), 8(6, 13) Coca-Prados, M., 41, 42(35) Cohen, C. M., 79 Cohen, G., 39, 51(14) Cohen, G. M., 103 Cohen, H., 40 Cohen, H. G., 143 Colburn, N. H., 132, 140(5) Coleman, J. E., 155 Coles, B., 54, 63(6), 65 Combrier, E., 336 Committee on Medical and BiologicalEffects of Environmental Pollutants, 38 Comporti, M., 38 Conley, R. R., 202 Contie, C., 336 Cookson, E., 40-41, 41(22), 42(22, 41) Cooper, M. R., 91 Cordier, A., 336, 339(15) Cotgreave, I. A., 342 Cotter, M. A., 150 Coussens, L., 133 Cowan, R. J., 91 Cowan, R. B. T., 318 Cramer, S. P., 124-125, 125(3), 126, 133, 154 Crapo, J, D., 41, 42(25) Crawford, D., 181,253 Crawford, N. A., 221,231,236(20), 238(20), 244, 275 Creese, I., 318 Creighton, T. E., 290 Cremers, T. L., 128 Cresswell, C. F., 88 Cretin, C., 245, 247, 249, 251

AUTHOR INDEX Criqui, M. C., 41, 42(32) Crofts, A. R., 220 Cronshaw, A. D., 54, 64(8) Crook, E. M., 3l Crosby, G. A., 128 Crothers, D. M., 169, 177 Crout, B. O., 147 Cuatrecasas, P., 332 Cuervo, A. M., 149 Curan, G. L., 150, 15l(45) Curran, T., 175

D Daglish, C., 33 Dahl, R. H., 85 Dahm, L. J., 3, 4(6), 8(6), 9, 11(22) Dalan, P., 52 Dale, G. L., 72, 74(4), 75(4), 77(6) Daniel, V., 263 Danielson, U., 321 Danner, R., 147 Dansette, P., 316, 322, 324, 330(4) Dansette, P. M., 316, 320(4) Danson, M. J., 187,189(8), 190,190(8), 193(8) Darlison, M. G., 194 Darzynkiewicz, Z., 345 Da Silva, O., 336 Davenport, H. W., 31 Davidson, P. C., 12 Davidson, R. S., 310 Davis, N. K., 87, 88(24) Davis, S. W, 285, 287 Davison, M., 187, 189(8), 190(8), 193(8) Daws, T., 84 Dear, T. N., 4l, 42(39) &Beer, F. C., 177 de Buck, R., 317 Decottignies, P., 231,240, 244-245, 247,249, 251,273 DeForge, L. E, 253 Deits, T. L., 37-38, 285 DeKok, A., 194 de Lamotte, F., 244 de Lamotte, G. F., 273 de la Motte-Guery, F., 231 De La Torre, A., 275 Del Bello, B., 38 Del Bino, G., 345 Delcos, K. B., 134

353

Delgado-Escueta, A. V., 149 De Long, M., 321 DelVal, G., 275,279(12), 280(12) DeMatteis, M. C., 78 Demerec, M., 266 Demple, B., 85 Deneke, S. M., 66 Desousa, G., 336, 339(15) DeVeaux, L. C., 41, 42(40), 43(40), 51(40) Diard, B., 333,340(9) Diarte, I., 336, 339(15) Dieteren, N., 194 Dignam, J. P., 256 Di Ilio, C., 53 Di Monte, D., 340 Dinarello, C. A., 343 Ding, J. L., 67, 69(12) DiPierro. D., 314 d'Ischia, M., 115 Distlerath, L. M., 59 Dixon, N. E., 88 Dolan, P., 316, 318(2), 321 Domingo, E. O., 318 Donaudy, F., 115 Donelli, G., 346 Doolittle, D. J., 340 Doroshow, J. H., 40, 42(17), 44, 50(55), 52(17) Dossou, K. G., 336 Dothey, C., 33, 36(19) Doty, D. M., 79 Draganic, I. G., 325 Draganic, Z. D., 325 Dresser, C. M., 4 Driscoll, P. C., 295, 296(20), 297(20) Dr6ge, W., 104, 168, 253-254, 262(15, 16, 20), 263, 263(1-3, 15, 16), 264, 264(20), 345 Droux, M., 231,236(20), 238(20), 244,275,280 Dubendorff, J. W., 245, 246(17) Dubrow, R., 180 Duckworth, H, W., 229 Duddy, S. K., 132, 134(9), 152 Dudman, N. P, 98 Dufaure, J.-P., 41, 42(38), 51(38) Duhe, R. J., 115,117(7) Dunn, J. J., 245,246(17) Duran, N., 141,142(28) Durr, A, 41, 42(32) Dutka, S., 251 Dutton, P. L., 227 du Vigneaud, V., 92

354

AUTHORINDEX

Dvorak, V., 323 Dyer, J. A., 41, 42(29, 30) Dypbukt, J. M., 53 Dyson, H. J., 199, 200(6), 202, 202(6), 269, 285, 293-295, 295(14), 297(17), 298-299, 299(7, 8, 15), 301-302, 302(17), 303(7, 8, 15, 17, 25, 34, 36)

Esworthy, S., 40, 42(17, 31) Esworthy, S. R., 41 Evenson, J. K., 41, 42(29, 30) Evered, D. F., 3, 9(1) Exton, J. H., 149 Eyer, P., 110 F

E

Fackir, L., 324 Falin, Z., 41, 42(33), 51(33) Eakardt, F., 90 Falkenstein, E., 275 Eberle, D., 9 Falkson, H. C., 317 Ebling, J. G., 158 Fanburg, 13. L., 66 Eccles, T. K., 129 Farber, C. M., 31, 32(11) Eck, H,-P., 104, 253, 263, 263(1-3), 345 Fariss, M. W., 95 Eckardt-Schupp, F., 91 Fariss, M. J. W., 22 Eckert, K. G., 104-108, 110 Fausel, M., 341,342(2) Edlund, T., 170 Fautrel, A., 336, 339(15) Ehn, B., 285, 289(34), 290(34), 291(34) Favilli, F., 3 Ehrenreich, A., 309 Federici, C., 336(16), 337, 339(16) Eichele, E., 44, 47(46), 48(46), 51(46) Eklund, H., 201-202, 240, 269, 271,284, 288, Feeley, R. M., 3 Feldman, G. M., 316, 318(3) 293, 295, 303(5), 304(19) Felipo, V., 148, 150-151, 152(63) Eklung, H., 199, 200(4), 201(4), 202(4) Fernandez, H. L., 147 Eldjarn, L., 91 Fernandez-Checa, J. C., 15 Eley, J. W., 3-4, 4(6), 8(6, 13) Ferradini, C., 322, 324-325, 330(4, 5) Ellis, W. W., 5, 18, 95, 109 Ferri, L., 40, 51(15) Ellman, G. L., 225 Feuerstein, S., 320, 321(3l), 322(31) Elvin, C. M., 88 Fickenseher, K., 241 Engel, R., 53 Fiering, S. N., 174 English, L., 149 Fiers, W., 254, 262(18) Engrnan, L., 342 Finkelstein, J. D., 92 EngstrOm, N.-E., 203 Finn, K. A., 65 Ennen, J., 168, 254, 262(16), 263(16) Finn, O. J., 157 Enomoto, H., 317, 318(18) Fischbein, J. W., 66 Epp, O., 44, 46(51), 47(51), 49(51), 50(51) Fischer, H., 342 Epstein, L., 11l Flagg, E. W., 3-4, 4(6), 8(6, 13) Epstein, W., 86 Flammang, T. J., 59 Erdbrfigger, W,, 254, 262(20), 264(20) Fleck, J., 41, 42(32) Ericksson, S., 33, 37(14), 284-285, 287 Floh6, L., 38-41, 41(21), 42(21, 26), 44, 44(26), Ericksson, S. A., 283, 292(6) 46(8, 48), 47, 47(8, 46, 47, 48), 48(8, 46Ericksson, V., 33 48, 62), 49(63), 50(47, 48), 51, 51(8, 46, Ericson, M.-L., 200, 205(10), 210, 217(13), 47), 52, 52(20, 21) 218(13), 254, 274 Florencio, F. J., 231,233(25) Eriksson, V., 285, 291(23) Fluegge, U. I., 159 Eshdat, Y., 41, 42(33), 51(33) Fogelman, A. M., 177 Essig-Marcello, J. S., 336(17), 337 Folch, J., 5 Estrela, J. M., 94, 97(8) Follmann, H., 230 Esworthy, R. S., 44, 50(55), 52(17)

AUTHOR INDEX Foltmann, B., 36 Foltz, C. M., 39, 209 Fontecave, M., 244 Forchhammer, K., 43, 309, 312(2), 315(2) Forman, H. J., 66-67, 71 Forrnan-Kay, J. D., 200, 293, 295-296, 296(20), 297(20), 299,299(9, 22), 302(21), 303, 303(9, 21) Fournex, R., 336, 339(15) Fox, P. C., 317 Foyer, C. H., 88 Fradelizi, D., 254 Frampton, J., 39, 42(11) Frangos, M., 150, 151(46) Fraser, G. M., 54, 63(6) Fraumeni, J. F., 3 Frayer, W., 13, 71 Frederick, J. R., 220 Freedman, R. B., 36-37, 240 Freeman, B. A., 145 Fridovich, I., 86 Fried, M., 177 Fried, M. G., 169 Friedrich, M. J., 41, 42(40), 43(40), 51(40) Fritsch, E. F., 157, 247 Fuchs, J., 267,284, 290(13) Fuchs, J. A., 83-85, 87, 87(5), 88(23), 89(3), 201-202, 266, 268, 270-271, 271(30), 284-285,302, 304 Fuji, T., 133, 136(18) Fujii, H., 78 Fujii, S., 263 Fujii, T., 124, 127(2), 130(2), 131(2), 154 Fuji-Kuriyama, Y., 41, 42(24) Fujita, Y., 95 Fuseilier, 317, 323(13)

G Gabriel, H., 342 Gadal, P., 244-245, 249, 251,275, 280 Gaines, R. L., 229 Gallego, J., 148-150 Galter, D., 254, 262(15), 263(15) Gan, Z.-R., 33, 37, 37(15), 38, 284-285, 285(22), 287, 287(22, 26), 291(22, 25) Ganapathi, M. K., 194 Ganong, B. R., 158 Ganther, H. E., 39, 46, 209, 212

355

Garcia, G. E, 43 Garcia-Ruiz, C., 15 Gardet-Salvi, L., 275,277(10), 279(11), 282(8) Gardetsalvi, L., 222 Gardner, P. R, 86 Garner, M. M., 169, 177 Garrick, M. D., 91 Gasa, S., 73, 78, 82 Gasco, E., 94 Gasdaska, P. Y., 203, 205(43) Gaudin, O, 317 Geck, M. K., 273 Geiger, P., 44, 50(55) Gelbart, T., 68, 91 George, G. N., 126 George, K., 149 George, S. J., 124, 125(3), 133, 154 Gerami, N. M., 270, 271(30) Gerweck, L. E., 105, 111 Gessain, A., 343 Ghosh, S., 167-168 Ghyselinck, N. B., 41, 42(38), 51(38) Giardina, B., 314 Gibson, Q. H., 187, 191(6), 193(6) Gilbert, H. F., 144 Gilbert, H. R., 226 Gilchrist, S. M., 14 Gillespie, D, 266 Gilmore, K. S., 54, 63(6), 65 Gimenez, A., 94 Gippert, G. P., 293, 298, 299(7), 303(7) Girotti, A. W., 44, 50(55) Glass, R. S., 309, 310(1), 311, 311(1), 314(1), 315(1) Gleason, F. K., 199, 200(3, 4), 201,201(3, 4), 202(4), 240, 270-271,271(30), 288, 295, 304(19) Gleye, j., 132, 136(14), 143(14) Gm(inder, H., 104, 253, 263(1), 345 Goebel, S. J., 285, 287 Goldberg, V., 149 Goldfarb, P., 39, 42(11) Goodwin, G. H., 177 Gopalakrishna, R., 132-133,133(7, 8), 135(7, 15), 136-137, 138(26), 139(7), 140(8), 145, 152 Gorczyca, W., 345 Gorman, C. M., 260, 261(30) Gosh, S., 41, 42(35)

356

AUTHORINDEX

Goto, S., 78, 82 Gougerot-Pocilado, M. A., 53 Gouy, D., 336, 336(16), 337, 339(15, 16) Graf, G., 41, 42(23) Graf, P., 341-342 Grafstrom, R. C., 9 Grankvist, M.-L., 265 Gravina, S. A., 33, 36(19), 285 Gray, L., 9 Greenberg, D. A., 149 Greenberg, J. T., 85 Greenberg, M. E., 180 Greenberg, R. S., 3-4, 8(13) Greene, D. A., 148, 151 Greene, W. C., 175, 177(2) Greengard, P., 149 Greer, S., 87-88, 88(24) Gregolin, C., 40, 44, 46, 50(59), 51(15) Gridley, G., 3 Griffith, O. W., 13, 15, 21, 29, 66, 69, 97, 320 Griffith Humphreys, W., 63 Grigat, G., 149 Grignani, F., 345 Grisar, T., 149 Grisolfa, S., 148, 150-151, 152(63) Groffen, J., 67 Gronenborn, A. M., 200, 293-296, 296(20), 297(20), 299, 299(2, 9, 10, 22), 302(21), 303, 303(9, 21) Gross, G., 40, 41(21), 42(21), 52(21) Gross, J. A., 147 Grossmann, A., 41, 42(26), 44(26) Gubanski, G., 336 Guengerich, F. P., 59, 63 Guenther, B., 201 Guerri, C., 150 Guest, J. R., 194 Guillaume, D., 149 Guillouzo, A., 336, 339(15) Gundimeda, U., 132-133, 135(15), 137, 138(26), 145, 152 Gunsalus0 I. C., 190 Gunter, E. W., 4, 8(13) Gfinzler, W. A., 39, 41, 42(26), 44, 44(26), 46(48), 47(46, 47, 48), 48(46-48), 50(47, 48), 51(46, 47) Gupta, S., 73 Gushima, H., 87 Guthenberg, C., 54 Gutteridge, J. M. C., 327

H Haber, M., 3 Habig, W. H., 55, 62(11), 65(11) Haegeman, G., 254, 262(18) Hafeman, D. G., 39 Hagen, T. M., 3-4, 9, 9(4, 11), 10, 10(4, 26), 11(4, 26, 27), 12, 12(4, 27), 66 Hager, L. J., 115, 117(4), 118(4), 121(6), 123(6) Hager, L. P., 190 Hahne, S., 287 Hakusui, H., 342 Hall, C., 161 Hall, L., 41, 42(37) Hallberg, A., 342 Hailer, B., 202, 268 Hailer, B. L., 85 Hallier, E., 63 Halliwell, B., 327 Halpern, B., 317 Hamada, M., 192 Hamlin, J. L., 180 Hammarstrom, S., 66 Hamuro, J., 200, 343 Handler, 179 Hanker, J. S., 66 Hannun, Y. A., 152 Hansson, H.-A., 270 Harden, N., 133, 155 Harman, A. W., 339 Harman, L. S., 47 Harnex, J. W., 43 Harris, J. M., 65 Harrison, D. J., 54, 64(8) Harrison, P. P., 39, 42(11) Harrop, H. A., 85 Harrsch, P. B., 221 H~irtling, S., 53 Hartman, A. M., 4 Hartman, F. C., 252, 273 Hartman, P. E., 83 Hasan, K. S., 150, 151(46) Hasse, W., 149 Hatch, M. D., 241 Hatefi, Y., 14(9), 15 Hatta, H., 41, 42(36) Haugland, R. P., 332 Haurand, M., 52 Hausler, R., 316, 323(7)

AUTHOR INDEX Hay, R. T., 175, 177(4) Hayakawa, T., 192-193 Hayashi, N., 15 Hayashi, T., 53, 175 Hayashi, Y., 90 Hayes, J. D., 53-54, 63(7), 64(8) Hazegawa, K., 327 Heath, W. F., 141 Heider, J.. 43 Heine, H. S., 318 Heisterkamp, N., 67 Hejgaard, J,, 229 Held, K. D., 85 Hellinga, H. W., 272 Helton, E., 322 Hendrickson, W. A., 124, 125(3), 133, 154 Henkel, T., 254, 262(12) Henneeke, H., 201, 273 Heppel, L. A., 279 Herbert, J. M., 132, 136(14), 143(14) Hermenegildo, C., 148, 150-151, 152(63) Herrmann, R. G., 275, 279(11) Herzenberg, L. A., 168, 172(4), 173(4), 174, 174(4), 176, 254, 262(17), 263(17) Hesketh, J. D., 220 Hess, C. E., 91 Hewick, R. M., 155 Hibberd, K. A., 84 Higashi, G. I., 318 Higashi, T., 94, 97(5) Higgs, D. J., 214 Higueras, V., 148 Hildeilfinger, J. C., 340 Hill, K. E., 39-40 Hill, R. D., 229 Hillson, D. A,, 36 Himawan, J. S., 268 Hiraoki, T., 302 Hirashima, M., 192 Hirata, M., 69 Hiyoshi, Y., 78 Ho, L., 189 Ho, Y., 41, 42(25) Hobbs, M., 103 Hochstein, P., 39, 51(14) Hockenberry, D. M., 346 Hodgson, K. O., 125 Hoekstra, W. G., 39 HoeUinger, H., 336, 339(15) Hogenkamp, H. P. C., 287, 291

357

Hokin-Neaverson, M., 149, 151(27) Hol, W.G.J., 194 Holland, D., 41, 42(33), 51(33) Hollenberg, C. P., 287 Holler, J., 53 Holler, T. P., 115, 119, 121-122, 122(10), 123(13, 14) Holmgren, A., 31,33, 37, 83, 89(2, 4), 199-200, 200(1-4), 201,201(2-4, 11), 202, 202(1, 4, 7), 203,204(29, 42), 205,205(7, 10, 42), 209-210, 210(10), 211(10), 212, 212(10, 12), 213(10,12), 214(10,12),215,215(10), 216(10, 12), 217,217(10, 12, 13), 218(13), 219(10, 12, 23), 230-231, 240, 254-255. 265-267,269-271,274, 283-285,285(20, 21), 287, 287(2, 14-17, 21, 39), 288(16, 17), 289(10, 11, 14, 15, 17, 33-35), 290, 290(13, 18, 34), 291, 291(20, 33-35), 292(35), 293-295, 295(1, 14), 297(17), 298-299, 299(7, 8, 15), 301-302, 302(17), 303(7, 8, 15, 17, 25, 34), 304, 304(12, 13. 19), 305-306 Hommel, U., 130 H60g, J.-O., 265, 269, 283-284, 285(21), 287(21), 289(10) Hopkins, P. B., 115, 118(2), 119, 121-122, 122(10), 123(13, 14) Hopper, S., 33,287 Hori, T., 343 H0rling, J., 200, 254 Homing, E., 322 Hossain, M. A., 31 Hotz, M. A., 345 Hough, D. W., 187, 189(8), 190(8), 193(8) Houghton, C. B., 102 Housey, G. M., 133 Houwerzijl, J., 91 Howard, A. J., 41, 42(25) Howard, B. H, 260, 261(30) Howbert, J. J., 141 Hu, C.-W.C., 189 Huang, C.-S., 26-27, 27(3), 28(6), 69 Hubbard, S. R., 123-124, 125(3), 133, 154 Huber, H. E., 202, 267-268, 271(20) Hubert, F., 336 Huet, J. C., 245,275 Hughes, R. E.~ 31 Hulsch, R., 91 Huneeus-Cox, F., 147 Hunjan, M. K., 3, 9(1)

358

AUTr~ORI N D E X

Hunter, W. M., 207 Hussey, A. J., 54, 63(7), 64(8) Hutcheson, S. W., 221 Hutchison, R. S., 220, 225, 226(13), 227(13) Hwang, P., 229

I Iantomasi, T., 3 Ibbetson, A. L., 37 Ida, S., 282 Ide, S., 192-193 Ido, Y., 150, 151(46) Igarashi, K., 124, 127(2), 130(2), 131(2), 133, 136(18), 154 Iggo, B., 31 Inamoto, T., 344, 346, 348(10) Ingelman, M., 284 Ishida, K., 41, 42(27) Ishii, T., 104, 345 Israel, N., 53 Issakidis, E., 240, 247, 249, 251 Issels, R. D., 103-108, 111 Itikawa, H., 266 Iwadate, H., 275, 282(8) Iwamoto, T., 66 Iwata, S., 263, 343, 348 Izumo, S., 343 Izuta, S., 100 d Jackson, B., 4, 8(13) Jackson, R. L., 341 Jackson, R. M., 41, 42(37) Jacobs, J. W., 287 Jacobsen, A., 317 Jacobson, B., 111 Jacobson, B. S., 79 Jacqueson, A., 319, 320(27), 333, 340(9) Jacquot, J.-P., 231, 240, 244, 247, 249, 251, 273, 275-276, 278(16), 279-280, 280(16) Jain, A., 13, 71 Jakobs, D., 53 Jakoby, W. B., 55, 62(11), 65(ll) Jamet, E., 41, 42(32) Janakidevi, K., 253 Javed, A. A., 194 Jeffrey, P., 241

Jeffreys, C. E. P., 31 Jellum, E., 91 Jeng, M., 269 Jeng, M.-F., 293, 299, 299(8), 302, 303(8, 25, 36) Jensen, G. L., 71 Jesse, B. W., 194 Jiao, J., 230, 231(18, 19), 238(19) Jirousek, L., 316 Jocelyn, P. C., 122, 123(12) Joelson, T., 284, 288 Johnson, D., 16 Johnson, G. P., 285, 287 Johnson, J. A., 65 Johnson, L. V., 332, 340(3) Johnson, M. D., 133 Johnson, P., 59 Johnson, R. S., 33, 287 Johnson, T. C., 230-231,233(25), 240 Joly, C., 245, 280 Jones, D. P., 3-4, 4(6), 8(6, 13), 9, 9(4, 11), 10, 10(4, 26), 11(4, 22, 26, 27), 12, 12(4, 27), 13(28), 17-19, 66, 72 Jones, G. P., 147 Jones, R., 41, 42(37) Jones, W. A., 147 Jones-Mortimer, M. C., 87, 88(24) Jore, D., 324, 330(4, 5) J6rnvall, H., 265, 269, 283-284, 285(21), 287, 287(21), 289(10) Jung, G., 44, 48(48), 50(48) Jung, W., 309, 310(1), 311(1), 314(1), 315(1) Junker, E., 111

K Kadlubar, F. F., 59 Kadner, R. J., 41, 42(40), 43(40), 51(40) Kageyama, H., 253 Kalebic, T., 168 Kalishi, D. I., 79 Kallis, G.-B., 267, 302 Kalse, J. F., 192 Kaltschmidt, B., 177 Kaltschmidt, C., 177 Kamada, T., 15 Kamel, A. H., 254 Kamifiski, R., 311 Kamo, M., 275, 282(8) Kant, J. A., 76

AUTHOR1NDEX Kaplowitz, N., 9, 15 Karlsson, M., 287 Kasahara, T., 343 Kass, G. E., 53 Kass, G. E. N., 132, 134(9), 152 Kaszubska, W., 175 Kato, J., 87 Katti, S. K., 202, 293, 303(5) Kawabe, T., 53, 175, 348 Kawakami, Y., 73, 78, 80, 82 Kawamura, T., 150, 151(46) Kawase, M., 95 Kay, L. E., 296, 299(22) Kelley, R. F., 272 Kenney, J., 253 Kensler, T. W., 65 Kern, J. M., 148 Kerr, L. A., 54, 64(8) Keryer, E., 247 Ketterer, B., 53-54, 59, 63, 63(6), 65 Kiefergachelin, H., 336 Kiess, M., 40, 41(21), 42(21), 52(21) Kihara, M., 150, 151(45) Kikkawa, U., 124, 127(2), 130(2), 131(2), 133, 136(18), 154 Kikuchi, K., 253 Kilo, C., 150, 151(46) Kim, H., 66, 189-190, 193(15), 194(15) Kim, I. Y., 309, 312(9), 313, 314(9), 315,

315(8, 9) Kim, S.-M,A., 41, 42(26), 44(26) Kimura, A., 87 Kimura, K., 317, 318(18) Kirschmeier, P., 124, 125(3), 133, 154 Kispert, A., 65 Kiss, A., 149 Kiss, F., 229-230, 230(11), 231(11, 16), 236(11), 238(11., 16), 240(16) Kistler, M., 90 Kitano, A., 189 Kitaoka, Y., 348 Kivirikko, K. I., 30 Klann, E., 132, 140(11) Klein, M., 254, 262(20), 264(20) Kleinhans, U., 287 Klein-Szanto, A.J.P., 132 Kletzien, R. F., 180 Klevitt, R., 115, 117(4), 118(2, 4) Klevitt, R. E., 115 Klintrot, I.-M., 284, 285(21), 287(21)

359

Kl6ppel, K. D., 40, 41(21), 42(21), 52(21) Kluge, T., 91 Knopf, J. L., 155 Kobayakawa, K., 282 Kobayashi, C., 41, 42(34) Kobayashi, E., 132, 136(12, 13), 141, 141(12) Kobayashi, N., 211 Kobrehel, K., 199, 228-230, 230(11), 231, 231(11, 16, 18, 19), 232,236(11), 238(ll, 12, 16, 19, 22), 239-240, 240(16) Kodama, H., 95-96 Koike, M., 190, 192-193 Kollarova, M., 247,271 Kondo, M., 87 Kondo, N., 200, 343 Kondo, T., 72-73, 77, 77(6), 78, 80, 82 Konrad, P. N., 91 Konz, K. H., 320, 321(31), 322(31) Korsmeyer, S. J., 346 Kosower, E. M., 146, 226, 231 Kosower. N. S., 146, 226, 231, 236(20), 238(20), 244 Kourilsky, R., 317 Kowalski, D. P., 3 Koyama, J., 41, 42(34) Kozma, R., 133, 155, 161 Kramer, D. M., 220 Kramer, G. F., 86 Kraus, R. J., 46 Krause, G., 200, 202, 271, 294, 299(15), 303(15) Kren, B., 83, 89(3), 284, 290(130), 304 Krishna, T. S. R., 201 Kriz, R. W., 154-155 Kruger, J. E., 229, 232(10) Kuhl, W., 79 Kuhn, L. J., 158 Kull, F. C., 332 Kumar, 8., 200, 201(11), 209-210, 210(10), 21l(10), 212(10, 12), 213(10, 12), 214(10, 12), 215(10), 216(10, 12), 217(10, 12), 219(10, 12) Kunert, K. J., 88 Kunkel, S. L., 318 Kunkel, T. A., 249 Kuno, T., 124, 127(2), 130(2), 131(2), 154 Kunseh, C., 253 Kuo, I. F., 149 Kttpfer, A., 111 Kuriyan, J., 201

360

AUTHORINDEX

Kuroki, T., 181, 253 Kurosawa, K., 15 Kurth, R., 168, 254, 262(16), 263(16) Kutalp, G,, 150

L LaBelle, E. F., 73 Ladenstein, R., 44, 46(51), 47(51), 48(51), 49(51), 50(51) Laemmli, U. K., 159, 234 Lai, L., 253 Lakhdar, B., 336 Lalor, E., 59, 65 Lambert, N., 36 Lampidis, T. J., 332 Lands, W. E., 52 Lanetti, G., 202 Langsetmo, K., 302 Lara, C., 275 Lardy, H., 16 Latimer, F. W., 273 Larsen, P. R., 40, 43 Larson, E. M., 223 Larson, K., 33, 285, 291(23) Larsson, A., 91,203, 287 Larsson, R., 132, 134(10) Lash, L. H., 9, 14-17, 17(17), 18, 18(17), 19, 21(15), 22(15) Lasovsky, J., 143 Lassota, P., 345 Latrille, F., 336 Laurent, T. C., 265 Lauterburg, B. H., 9, 111 Lawrence, R. A., 46 Lawson, T. A., 316, 321(5), 322(5) Layrisse, M., 9 Lazzarino, G., 314 Leach, F. R., 190 Le Bigot, J. F., 336 Lebovitz, R. M., 256 Lee, J., 133, 155 Lee, M.-H., 154-155, 157 Lee, S., 149 Lees, M., 5 Lees, W. J., 228 Leets, I., 9 Lehnberg, W., 41, 42(23) Leinfelder, W., 309, 312(2), 315(2) Lelord, G., 317, 323(13)

Lemaire, M., 240, 247 LeMar6chal, P., 247 LeMaster, D. M., 202, 291, 293, 295, 301, 301(18), 303(5) Lenfeld, J., 143 Leonard, W. J., 175, 177(1) Lepiniec, L., 245 LeQuesne, P. M., 149 L&Qu6c, D., 14, 14(7), 15 L6-Qu6c, K., 14, 14(7), 15 Leroy, J. P., 316 Lestas, A. N., 68 Leuner, U., 41, 42(23) Levander, O. A., 39, 214 Levitch, M. E., 190 Levy, E. J., 103 Lewis, H. M., 194 Liang, M., 104 Liao, F., 177 Liburn, J. E., 228 Liebes, L. F., 31, 32(ll) Liepinsh, E., 303 Lira, C.-J., 84, 202, 268, 270, 271(30) Lim, H. H., 161 Lim, L., 133, 155, 161 Lim, L. L., 154 Lin, A. Y., 154 Lincoln, D., 41, 42(23) Lindenbaum, J., 95, 98(13) Ling, L., 149 Lisosky, T., 90 Listowsky, I., 53 Little, C., 46 Liu, R.-M., 66 Liu, T.-C., 186, 189 Liu, Y., 9 Ljungdahl, L. G., 39 Llopis, J., 151 Llopis, J. E., 149 Lloyd, R. S., 40 Lo, S. K., 253 Loferer, H., 201,273 Logan, T. M., 86 Logerlund, T. D., 150, 151(41) LiShr, G. W., 91 L6hr, J. P., 342 Loomis, C. R., 152, 155 Loos, J. A., 91 Lopez-Barea, J. 88, 200, 271, 285, 289(35), 291(35), 292(35)

AUTHOR INDEX Lorenzon, G., 336, 339(15) Loschen, G., 44, 47(46), 48(46), 51(46) Lounes, R., 336, 339(15) Low, P. A., 150-151,151(41, 45) Lowndes, H. E., 147-148 Lowndes, J. M., 149, 151(27) Lozac'h, N., 316 Luchetta, P., 245 Luckow, B., 259 Luetzenkirchen, K., 287 Lundberg, P., 98 Lundin, B., 287 Lundstr6m, J., 200-201,271 Lunn, C. A., 270 Lunn, G., 72, 74(4), 75(4) Lusis, A. J., 177 Lusty, C. J., 192 Luthman, M., 33, 203, 204(42), 205(42), 212, 284, 285(20, 21), 287(21), 291(20) Luyten, M., 130 Lyckeborg, C., 201 Lynch, C. J., 149 Lynch, J. F., 98 Lynn, D. G.. 86

M Mabrouk, G. M., 167 MacArthur, M. W., 298 Macey, D., 98 MacGill, R. S., 141,142(29) MacGregor, A. W., 229 Maci~in, F., 149 M~idefessel, K., 149 Maeda, K., 244, 275 Maeda, M., 200, 343-344, 348(9) Maeda, Y., 200, 343 Maellaro, E., 38 Maffrand, J. P., 132, 136(14), 143(14) Mahe, Y., 254 Maiorino, M., 40, 41(21), 42(21), 44, 46, 50, 50(59), 51(15), 52, 52(20, 21), 341 Majerus, P. W.. 91 Majorino, M., 38 Makowske, M., 104 Malik, A. B., 253 Malorni, W., 346 Mandel, L. J., 14 Maniatis, T., 157, 247

361

Mannervik, B., 33, 37(14), 53-54, 65,283,285, 291(23), 292(6), 321 Manning, J. M., 28 Mansuy, D., 316, 320(4), 322, 324, 330(4) Marbach, J., 41, 42(32) Marchylo, B. A., 229, 232(10) Marc-Martin, S., 275 Marcus, D. J., 149 Marcus, F., 221,230 Marinho, P., 247 Marquis, J. K., 147 Marshall, P, A., 145 Mltrtensson, J., 13, 15, 30, 71 Martin, C., 319 Martin, F., 279 Martin, J. B., 79 Martin, J. L., 209 Martinalguacil, N., 339 Martin-Alonso, J. M., 41, 42(35) Martinez-Galisteo, E., 285. 289(35), 291(35), 292(35) Martinez-Galisteo, E. M., 291 Martinez-Torres, C., 9 Marui, N., 253 Masiarz, F. R., 230 Mason, R. P., 47 Massey, V., 187, 191(6), 193(6, 7) Masson, P., 336 Masuda, T., 343 Masutani, H., 263, 344, 346, 348, 348(10) Mathews, R. G., 193 Matignon, B., 232 Matsui, H., 200, 343 Matsushima, K., 343 Mattevi, A.. 194 Matthews, J. R., 175, 177(4) Matthias, P., 170, 176, 256 Mautner, H. G., 147 Mayer, A. M., 229 Mayhew, S. G., 245 McAllister, J. K., 202 McBain, W., 39, 42(11) McCully, V., 190 McDonald, T. F., 149 McFarlan, S. C., 287, 291 McKernan, T. B., 15, 21(15), 22(15) McLaggan, D., 86 Mclaughlin, J. K.. 3 McLaughlin, S., 154 McManis, P. G., 150, 151(41)

362

AUTHORINDEX

Meck, R., 68 Meck, R. A., 18 Medford, R. M., 253 Medh, R. D., 73 Medina, D., 209 Meier, T., 103, 106, 111 Meister, A., 13, 15, 26-27, 27(3), 28, 28(6), 29, 29(8, 9), 30, 30(1, 14), 66-67, 67(1), 68(10), 69, 69(10), 71, 103, 109, 292, 319-320 Melcion, C., 336, 339(15) Mercat, C., 317, 323(13) Meredith, M. J., 14, 94 Meriman, R. L., 141 Messing, R. O., 149 Metezeau, P., 336 Meyer, D. J., 53-54, 59, 63, 63(6), 65 Meyer, M., 263 Meyer, Y., 247 Miao, G., 175 Michael, B. D., 85 Michalski, J., 311 Mieyal, J. J., 33, 36(19), 285 Miflin, B. J., 229 Miginiac, M. M., 273 Miginiac-Maslow, M., 240, 244, 247, 249, 251, 275, 280 Migliorati, G., 345 Mihm, S., 168, 253-254, 262(15, 16), 263(2, 3, 15, 16) Mijiulac-Maslow, M., 231 Milanez,, 8., 252 Miller, F. D., 141 Miller, G. C., 141, 142(29) Milliman, C. L., 346 Mills, G. C., 39 Milner, J. A., 209, 214 Milona, L., 154 Mifiana, M.-D., 148, 150-151, 152(63) Minard, P., 231,273 Minarik, P., 271 Minnich, V., 91 Mirabelli, F., 78 Miranda-Vizuete, A., 285, 289(34, 35), 290(34), 291(34, 35), 292(35) Misiorowski, R., 148 Misuraca, G., 115 Mitchell, J. B., 111 Mitchell, J. R., 9 Mitchen, J., 332

Mitsui, A., 53, 175, 200, 263, 343-344, 346, 348, 348(9, 10) Miura, K., 104 Miwa, S., 78 Miya, T., 87 Miyamoto, K., 73 Mizoguchi, J., 41, 42(28, 34) Mizuno, M., 180 Model, P., 83, 89(4), 201-202, 231, 265-266, 266(6), 267(6), 268-269, 269(6, 24), 271(6, 20), 272(6), 291, 302 Modrich, P., 265, 267(3, 4), 268 Moffat, L. F., 260, 261(30) Mohler, D. N., 91 Moldeus, P. M., 342 Molitor, J. A., 175, 177(2) Monfries, C., 133, 155, 161 Monsalve, E., 148 Montgomery, G. A., 191 Moore, E. C., 265 Moore, M., 78 Moore, S., 28 Moore, W., 27, 30 Moore, W. R., 69 Moran, T., 41, 42(29, 30) Morency, C. A., 260 Morgenstern, R., 53 Morimoto, M., 132, 136(12), 141(12) Morir~o, T., 41, 42(27) Morino, Y., 101 Morita, Y., 282 Moriuchi, T., 41, 42(36) Morris, M., 275, 279(12), 280(12) Morris, V. C., 214 Morrissey, P., 45 Morse, M. L., 85 Mosior, M., 154 Moss, B., 33, 36(20), 285 Moss, K. M., 14(8), 15 Mounolou, J.-C., 332 Muckel, C., 152, 153(60) Mudd, S. H., 92, 97 Mueller, M. M., 170, 176 M011er, A., 341-342 M~iller, E., 111 Muller, E. G., 273-274 Mtiller, I., 44, 47(47), 48(47), 50(47), 51(47) M~iller, M. M., 256 Mullineaux, P. M., 88 Multhoff, G., 111

AUTHOR1NDEX Mundy, J., 229 Mural, R. J., 252 Muramatsu, M., 53 Murao, M., 78 Murata, K., 87 Mustre de Leon, J., 129 Mutoh, N., 90 Myllyl~, R,, 30

N Nagai, S., 283, 285(5), 291(5), 292(5) Nagamastu, M., 151 Nagarajan, B., 136 Nagele, A., 104-108, 111 N~iher, H., 263 Nairn, A. C., 149 Nakagaawa, C. W., 90 Nakagawa, T., 2ll Nakamura, H., 263, 344, 346, 348, 348(10) Nakanishi, S., 132, 136(13) Nakano, H., 132, 136(12, 13), 141,141(12) Nakashima, K., 94, 97(5) Nakata, K., 95 Nakayama, M., 211 Nakayasu, M., 103 Naples, J. M., 316, 318(3) Naruse, A., 94, 97(5) Nehring, R. E. J., 109 Neumann, D., 229 Neumann, J. R., 260 Nevries, U., 91 Niang, L. S., 41, 42(37) Nickander, K. A., 151 Nickander, K. K., 150, 151(45) Nickauder, K., 151 Nicolas, J. F., 174 Nicoletti, I., 345 Nicotera, P., 53, 78 Nies, E., 148 Nikaido, T., 343 Nikawa, T., 342 Nikkola, M., 201,271,284, 287-288 Nilsson, L., 9 Nimbona, C., 230, 231(16), 238(16), 240, 240(16) Nishihara, K., 95 Nishino, K., 343 Nishiuch, Y., 103

363

Nishizawa, A., 222, 224(6) Nishizuka, V., 124, 127(2), 130(2), 131(2), 132-133, 136(18), 149, 153-154, 154(3), 156(3) Nolan, G. P., 168, 174 Nomura, H., 342 Nordlund, P., 284 Nordstrom, B., 267 Nose, K., 181,253 Nossal, N. G., 279 Novogrodsky, A., 109 Nyengaard, J. R., 150, 151(46) Nygren, H., 270

O Oblong, J. E., 203, 205(43) Obmolova, G., 194 Oborn, C. J., 209 O'Brian, C. A., 133 O'Brian, P. J., 45-46 O'Brien, T. G., 149 Offermann, M. K., 253 Ogita, K., 133, 136(18) Ogivara, H., 53 Ogiwara, H., 175 Ohlsson, H., 170 Ohlsson, I., 265 Ohmori, S., 95 Ohno, K., 69 Ohta, J., 96 Ohtake, Y., 90 Ohtsuka, Y., 78 Oikawa, A., 103 Oishi, H., 149 Okabe, K., 192-193 Okada, M., 343 Okarnoto, T., 53, 175 Oler, A., 149 Olinescu, R, 46 OlWai, Z. N., 346 O'Malley, B. W., 175 Ono, Y., 124, 127(2), 130(2), 131(2), 133, 136(18), 154 Ookhtens, M., 15 Oort, M., 91 Orlowski, M., 29 Orm/), M., 287 Orrnstad, K., 9 Orning, L., 66

364

AUTHOR INDEX

Orrenius, S., 53, 78, 92, 132, 134(9), 152 Ort, D. R., 220 Osame, M., 343 Osborne, T. B., 232 Otter, R., 341,342(2) Otting, F., 41, 42(26), 44(26) Otting, G., 303 Otto, W. R., 336 Otulakowski, G., 194 Ou, S.-Y. L., 318 Owens, R. A., 83 Oxholm, P., 317 Ozawa, K., 344, 346, 348(10) Ozdemirler, G., 150

P Packer, L., 175, 180-181,254, 262(19) Packman, S., 189 Padilla, C. A., 291 Paglia, D. E., 91 Pagliacci, M. C., 345 Pahler, A., 201 Painter, E. P., 209 Pallardo, F. V., 94, 97(8) Palumbo, A., 115 Palumbo, E. J., 132, 140(11) Pan, Y.-C.E., 175 Panacer, D. S., 333 Paoletti, E., 285, 287 Papayannopoulos, I. A., 284, 285(22), 287(22), 291(22) Pardee, A. B., 180 Pardini, R. S., 141, 142(29) Parker, P. L, 124, 132-133 Parmentier, Y., 41, 42(32) Parsell, D., 83, 89(3) Passara, U., 168 Patel, L., 175 Patel, M. S., 186, 189, 192, 193(15), 194, 194(3, 15) Patterson, L. K., 327 Pazat, P., 317 Pearce, L. A., 91 Pearson, W. R., 53 Pegelow, C. J., 91 Peinado, J., 181 Pekar, U., 263 Pelc, I., 317 Peleg, S., 175

Peloux, A. F., 336, 336(16), 337, 339(15, 16) Pemble, S. E., 54, 63, 63(6) Penry, J. K., 147 Pepin, R, A., 194 Perham, R. N., 87-88, 88(24), 190 Perkus, M. E., 285, 287 Permark, M., 63 Pernollet, J. C., 245 Perry, A. C., 41, 42(37) Persons, D. A., 157 Persson, M., 265, 283 Pessara, U., 254, 262(16), 263(16) Peteratos, K., 284 Peters, T. J., 67, 69(12) Peters, T., Jr., 342 Peters, W. H. M., 59, 65(12) Peterson-Yantorno, K., 149 Pfeifer, M. A., 151 Pfeiffer, D. R., 14 Philbert, M. A., 147-148 Pichon, J., 336, 339(15) Pick, E., 214 Pickett, C. B., 53 Pierce, R. J., 40, 41(22), 42(22) Pigiet, V. P., 202, 270 Pincus, J. H., 149 Pisoni, R. L., 104 Placidi, M., 336, 339(15) Plat, M., 316, 320(4), 324 Plummet, H. K., 336 Poduslo, J. F., 150, 151(45) Politino, M., 309, 315(6) Poljakoff-Mayber, A., 229 Polokoff, M. A., 287 Pomeranz, Y., 229 Pons, G., 186, 194, 194(3) Poole, L R., 97 Pope, A. L., 39 Potter, D. W., 5, 95, 109 Potter, J., 92 Poulsen, L. L., 342 Powis, G., 203, 205(43) Preininger, V., 143 Preston, A. M., 253 Preston-Martin, S., 3 Prins, H. K., 91 Prohaska, J. R., 46 Prota, G., 115 Pucheault, J., 325 Puerner, J. A., 332

AUTHORINDEX Puertas. F. J., 148, 150 Puertes, I. R., 94 Pueyo, C., 88, 271,285,289(34, 35), 290(34), 291(34, 35), 292(35) Pueyo de La Cuesta, C., 200

365

Riccardi, C., 345 Richards, F. H., 91 Richards, F. M., 272, 295, 296(20), 297(20), 301,301(18) Richards, W. L, 336 Richardson, C. C., 202, 265, 267, 267(3, 4), 268, 271(20), 312 Q Richman, P., 26 Qin, J., 294, 299(10) Richman, P. G., 29 Quandt, F. N., 149 Riddell, R..I., 333 Quest, A.F.G., 131, 133, 153-154, 156, Rieber, P., 254, 262(13), 263(13) 157(17-20), 158(17), 159, 159(17), Rimsky, L., 254 160(18, 20), 164(17-19), 166(18, 19), Rivabene, R., 346 167, 167(20) Rivera-Madrid, R., 247 Roberts, A. B., 181 Roberts, C. M., 190 R Roberts, G., 293, 295(1) Rabbitts, T. H., 41, 42(39) Roberts, J. D, 249 Racker, E., 283, 285(4), 291(4), 292(4) Robins, P., 85 Rad, M. R., 287 Robinson, B. H., 194 Raefsky-Estrin, C., 194 Robinson, M. F., 12 Rago, R., 332 Robison, T. W., 66 Rahmani, R,, 336, 339(15) Roche, T. E., 186 Rahn, C. A., 340 Rocque, P. A., 31, 33(10), 37(10), 38, 289. Rajpert-De Meyts, E., 67 292(48) Raju, P. A., 174 Rodrigo, F., 94, 97(8) Ramesha, C. S., 154 Roeder, R. O., 253, 256 Ramirez, J., 9 Roederer, M., 168,172(4), 173(4), 174,174(4). Ramirez, M., 88 176, 254, 262(17), 263(17) Rao, J., 336 Rom~i, J., 146, 148-151 Rat, P., 323,331,340 R6mer, A., 342 Rauscher, F. J. III, 175 Romero, F. J., 14, 146, 148-151, •52(63) Rawson, M. D., 149 Romero, M, J., 86 Raya, A., 148-151 Ronot, X, 332, 336 Read, R., 40 Ronzio, R., 28 Redinbaugh, M. G., 79 Roos, H. D,, 91 Reed, D. J., 5, 14, 18, 22, 92, 94-95, 109 R()salki, S. B., 67 Reed, L. J., 190 Rosano. C. L., 71 Reed, R, L., 12 Ros6n, A., 200, 254 Rehr, J. J., 129 Rosen, C. A., 253 Reichard, P., 200, 202, 217, 287, 294 Rosenberg, A. H., 245, 246(17) Reid, K. G., 46 Rosenberg, H., 88 Reilly, P. E. B., 59 Roth, S., 253, 263(1), 345 Remick, D. R., 253 Rothwarf, D. M., 228 Rein X., 200, 205(10), 210, 217(13), 218(13), Rotruck, J. T., 39 274 Roveri, A., 38, 40, 41(21), 42(21), 44, 52. Reuhl, K. R., 147-148 52(20, 21), 341 Reumkens, J., 40, 52(20) Rowe, W. B., 28 Revzin, A., 169, 177 RozelL B., 270 Rhee, L., 133 Ruan, F.. 119

366

AUTHORINDEX

R~diger, H. W., 91 Ruimy-Ifrah, P., 322, 324, 330(4, 5) Rusakow, L. S., 99 Russel, M., 83, 89(2, 4), 199, 201-202, 231, 264-266, 266(6), 267(6), 268, 269(6, 24), 271(6, 20), 272(6), 285, 289(33), 291, 291(33), 303 Russian, K. O., 260 Russo, A., 111 Rutenburg, A. M., 66 Ryan, C. A., 229 S Saarinen, M., 251, 288 Sacchi, N., 182 Safaghi, H., 341,342(2) Sagara, J., 104 Sagawa, K., 343 Sakamoto, Y., 3, 94, 97(5) Sakiyama, S., 253 Sakurai, H., 211 Sall, J. M., 16, 17(17), 18(17) Sallantin, M., 245 Sambrook, J., 157, 247 Samiec, P. S., 3, 4(6), 8(6) Samols, D., 194 Samuelsson, B., 66 Sandberg, V., 284 Sandy, M. S., 342 Santamaria, A. B., 32l Santini, M. T., 346 Santucci, A., 38 Sapirstein, H. D., 235 Sardana, M. K., 287 Sari, A., 324 Sarkar, S. N., 143 Sasaki, M., 103 Sassi, A., 316, 320(4) Sastre, J., 94, 97(8) Sato, H., 104, 345 Sato, K., 53 Sato, N., 15, 343 Sattler, W., 341 Savage, D. G., 95, 98(13) Saxena, M., 54 Schade, F. U., 53 Schaewen, A. v., 275 Schaffner, W., 159, 170, 176, 256 Schaich, E., 44, 48(48), 50(48)

Schallreuter, K. U., 348 Scheibe, R., 241, 249, 252, 275 Schein, C., 161 Schenk, H., 253-254, 262(20), 264, 264(20) Scheraga, H. A., 228 Schierbeek, A. J., 194 Schiodt, M., 317 Schmalzing, G., 149 Schmelzer, J. D., 150-151, 151(45) Schmidt, A., 88 Schmitter, J. M., 247, 251 Schneider, F., 44, 48(48), 50(48) Schnellmann, R. G., 14-15 Schock, H. H., 39, 44 Schoenbeg, J. B., 3 Scholz, T. D., 309, 310(1), 311(1), 312(9), 314(1, 9), 315(1, 6, 9) Schomburg, D., 38, 47, 48(63), 49(63) Schrader, W. T., 175 Schreck, R., 168, 169(7), 254, 262(13, 14), 263, 263(13, 14) Schreiber, E., 170, 176, 256 Schreiber, J., 47 Schroeder, K. R., 63 Schuch, G., 342 Schuckelt, R., 40, 41(21), 42(21), 52(20, 21) Schulze-OsthofL K., 253-254, 262(18, 20), 264, 264(20) Schtirmann, P., 222, 244, 273-276, 277(10), 278(16), 279(11, 12), 280(12, 16, 17), 282(8) Sch~tz, G., 259 Schwartz, K., 209 Schwarz, J. R., 149 Schwarz, K., 39 Schwarz, W., 149 Sedgwick, B., 85 Seed, J. L., 316, 318(3) Seelig, G., 320 Seelig, G. F., 67, 68(10), 69(10) Segura-Aguilar, J., 148 Seifen, E. E., 143 Sekiguchi, K., 133, 136(18), 149 Selig, G. F., 26, 30(1) Seligman, A. M., 66 Selkirk, M. E., 41, 42(41) Selman, B. R., 115, 117(7) Selman-Reirner, S., 115, 117(7) Sen, R., 168-169 Sevanian, A., 52

AUTHOR INDEX Shamberger, R. J., 209 Shan, X., 72 Shapiro, B. M., 115, 117(4), 118(2, 4), 121, 121(6), 123(6) Sharma, R., 73 Sharma, V. K., 29 Sharp, K., 302 Sharp, P. A., 169 Shaw, D. C., 54 Shearman, M. S., 149 Shearman, Y. M. S., 133, 136(18) Shen, B., 200, 205(10), 210, 217(13), 218(13), 274 Sherrill, K., 203, 205(43) Shewry, P. R., 229 Shi, M., 71 Shi, M. M., 66 Shibanuma, M., 181,253 Shibata, Y., 100 Shimada, M., 78 Shin, S., 230, 238(12) Shinozuka, S., 101 Shiozaki, H., 94, 97(5) Shrager, P,, 147 Siegel, F. L., 65 Siegel, J. M., 191 Sies, H., 14, 14(8), 15, 73, 81,152, 153(60, 61), 181,341-342 Silber, R., 31, 32(11) Sillau, A. H., 10, 11(27), 12(27) Silva, C. M., 175 Sima, A. A. F., 151 Simanek, V., 143 Simondsen, R. P., 30 Simons, P. C., 54, 159, 161(29) Simoulin, J. P., 340 Singh, H., 169 Singh, S. V., 73 Singh, W. P., 309, 310(1), 311(1), 314(1),

315(1) Singhal, S. S., 54 Sj/3berg, B.-M.. 202, 269, 284, 287, 294 Skelton, D. C., 71 Skowrofiska, A., 311 Slaga, T. J., 132 Slife, C. W., 17 Sloane Stanley, G. H., 5 Smith, B. H., 147 Smith, G. D., 67, 69(12) Smith, H. M., 146

367

Smith, M. T., 340, 342 Smith, P., 161 Snyder, S. H., 318 So, E. L., 147 Sodano, P., 284, 287(14, 15), 289(14, 15), 290(13), 294, 304, 304(12, 13), 305 S6derberg, B.-O., 284, 293-294 S6derhNl, S., 203 Soeda, E., 87 Sokatch, J. R., 190, 194 Somers, C., 115 Song, P.-S., 141,142(28) Sonnerstam, U., 284, 294 Spallholz. J. E., 209 Spaltenstein, A., 115, 119 Spector, A., 32 Spencer, S. R., 63 Spielmann, A., 275 Sporn, M. B., 181 Spurr, C. L., 91 Spyrou, G., 291 Srinivas, L., 132, 140(5) Srivastava, S. K., 72-73, 73(3), 74(3, 12), 75, 76(12) Staal, F. J. T., 168, 172(4), 173(4), 174, 174(4), 176, 254, 262(17), 263(17) Stabel, E., 133 Stabel, S., 124, 132 Stabler, S. P., 95, 98(13), 99 Stadtman, T. C., 39, 43, 209, 309, 310(1), 311(l), 312(9), 313, 314(1, 9), 315,315(l, 6,8,9) Stahl, R. L., 31, 32(11) Stahl, S. J., 299 Stahl, W,, 342 Starka, J., 316 Starke, D. W., 33, 36(19) Stavaux, M., 316 Steck, T. L., 76 Steffens, G. J., 41, 42(26), 44(26) Stegink, L. D., 102 Stein, M., 231,273 Steinherz, R., 30 Stellwagen, E., 272 Stephens, P. E., 194 Stermitz, F. R., 143 Sternberg, P., 12 Stevenson, K. J., 187, 189(8), 190(8), 193(8, 9), 194, 302 Stewart, C. P., 31

368

AUTHORINDEX

Steyn-Parve, E. P., 192 Stiers, D. L., 14 Stocker, R., 34l Stockman, B. J., 115, 117(7) Stofer-Vogel, B., 111 Stohs, S. J., 316, 321(5), 322(5) Stokke, O., 91 Stone, M. J., 301,303(34) Strassburger, W., 40, 47, 48(63), 49(63), 52(20) Straub, K. D., 143 Stringer, C. D., 273 Strosberg, A. D., 41, 42(33), 51(33) Strum, J. C., 167 Studier, F. W., 245, 246(17) Stutz, E., 222, 275, 277(10) Sugahara, K., 95-96 Sugherini, L., 38 Sugie, K., 343 Sugimoto, M., 41, 42(29) Sugita, Y., 345 Sukenaga, Y., 41, 42(27) Sultzman, L. A., 154-155 Summer, K.-H., 90 Summerhayes, I. C., 332 Sunde, R. A., 41, 42(29, 30) Sundqvist, K., 9 Suthanthiran, M., 29 Suziki, K., 132, 136(13) Suzuki, T., 54 Suzuki, Y. J., 175, 177, 181,254, 262(19) Svendsen, I. B., 229 Swanson, A. B., 39 Sweatt, J. D., 132, 140(11) Swerlick, R., 253 Swift, T. R., 147, 149 Sykes, B. D., 301 Szajewski, R. P., 228 Szekeres, M., 275 Szent-Gy6rgyi, A., 31

T Tabor, S., 202, 267, 312 Tagawa, K., 15 Tagaya, Y., 200, 263,343-344, 346, 348(9, 10) Takagi, K., 41, 42(27) Takahashi, I., 132, 136(13), 141 Takahashi, K., 41, 42(34) Takahashi, K. T., 40 Takatsuki, K., 343 Takegoshi, T., 342

Takekoshi, S., 41, 42(24) Takemura, M., 96 Takeshita, K., 317, 318(18) Takeuehi, E., 253 Takeuchi, F., 100 Takeuehi, M., 150 Talalay, P., 318 Tamaoki, T., 132, 136(12, 13), 141, 141(12) Tanabe, K., 90 Tanaka, C., 124, 127(2), 130(2), 131(2), 154 Tanaka, H., 211 Tanase, S., 101 Tani, K., 87 Taniguehi, N., 73, 78, 80, 82, 317, 318(18) Tappel, A. L., 209 Tateishi, N., 3 Tateshi, N., 94, 97(5) Tavazzi, B., 314 Taylor, J. B., 63 Taylor, P., 9 Tenth, O., 126 Tennant, L. L., 302 Terlinden, R., 342 Terrell, C. A., 287, 291 Teshigawara, K., 343 Thelander, L., 33, 217, 284, 287 Thernot, J. P., 322 Thevenin, M., 319, 320(27), 323, 331, 333, 340, 340(9) Thier, R., 63 Thoene, J. G., 104 Thomas, C., 141,142(29) Thomas, C. E., 341 Thompson, G. A., 66-67 Thomson, C. D., 12 Thomson, G., 31 Thornton, 13. M., 148, 151 Thornton, J. M., 298 Thorpe, C., 193 Tiefenback, C., 143 Tiegs, G., 341, 342(2) Tien, M., 326 Tilton, R. G., 150, 151(46) Titeca, R., 317 Toftgard, R., 9 Toledano, M. B., 175, 177(1) Tolosa, S., 223 Tomanek, 102 Tormay, P., 309 Torriglia, A., 223 Totty, N., 133

AUTHOR INDEX Traganos, F., 345 Trilschler, H., 151 Triplett, L. L., 132 Trosko, J. E., 132 Tsai, L., 309, 315(6) Tsan, M. F., 71 Tsang, M. L.-S., 202, 266 Tsen, C. C., 209 Tsuboi, S., 95 Tsubouchi, R., 100 Tsuchiya, M., 181 Tsudo, M., 343 Tsugita, A., 244, 275, 282(8) Tsuji, Y., 78 Tu, C.-P. D., 53 Tuck, R. R., 150 Tuggle, C. K., 87, 88(23) Turcatti, G., 175 Turley, R. B., 79 Turner, D. C., 39 Turner, E., 115, 117(4), 118(2, 4), 121, 121(6), 123(6) Tursz, T., 200, 254, 263, 344, 348(9)

U Uchino, H., 343 Uchiyama, T., 254, 264(23), 343 Ueda, Y., 348 Ueno, Y., 175 Uhlin, T., 284 Ukai, Y., 317, 318(18) Ullrich, A., 133 Ulrich, R., 340 Ulrichova, J., 143 Upshall, D. G., 103 Urata, Y., 78, 82 Uribe, L., 67 Ursini, F., 38, 40, 41(21), 42(21), 44, 46-47, 48(63), 49(63), 50(59), 51(15), 52, 52(20, 21), 341 Usuki, K., 343 Uysal, M., 150

V Vajda, F., 149 Valente, M., 40, 51(15) Valentine, W. N., 91

369

van Bladeren, P. J., 59, 65(12) Van Buskirk, R. G., 336(17), 337 Vandenbark, G. R., 158 van der Enden, M., 150, 151(46) Vander Jagt, D. L., 54, 159, 16l(29) Vandevoorde, V., 254, 262(18) Vannier, B., 336, 339(15) van Ommen, B., 59, 65(12) Varnes, M. E., 111 Vasilets, L. A., 149 Vath, J. E., 33, 287 Veeger, C., 187, 191(6), 192, 193(6, 7), 194 Veprek, B., 309, 312(2), 315(2) Veres, Z., 309, 310(1), 311(1), 312(9), 313, 314(1, 9), 315, 315(1, 6, 8, 9) Vergin, H., 44, 47(47), 48(47), 50(47), 51(47) Vernant, J. C., 343 V6ron, M., 244 Vettakkorumakankav, N. N., 186-187, 189(8), 190(8), 192, 193(8), 194 Vettore, L., 78 Vian, J. R., 94 Vidal, J., 279 Vina, J., 94, 97(8) Vincenzini, M. T., 3 Vintezou, P., 336, 339(15) Virelizier, J.-L., 53, 175, 177(4) Visser, J., 192 Vogel, H. J., 302 von Bahr-Lindstr6m, H., 265, 284, 289(10) Voorhees, C. G., 232

W Wada, K,, 230, 240 Wagner, G., 342 Wahllander, A., 81 Wakasugi, H., 200, 254, 263, 343-344, 348(9) Wakasugi, N., 175,177(4), 344,346, 348(9, 10) Waksman, G., 201 Walaas, S. I., 149 Wallace, R., 150 Wallevik, K., 36 Walsh, C. T., 100 Walsh, M. L., 332, 340(3) Walterova, D., 143 Wang, F., 175 Wang, J., 98 Wano, M., 343 Ward, K., 150

370

AUTHOR INDEX

Ward, L. C., 147 Ward, P. A., 318 Warner, H. R., 84, 87(5) Warner, R. C., 31 Warnet, J.-M., 319, 320(27), 323, 331, 333, 340, 340(9) Warren, K. S., 318 Warso, M. A., 52 Washburn, M. P., 30, 291 Washtien, W., 100 Wasserkrub, H. I., 66 Watanabe, H., 95, 104 Watanabe, K., 41, 42(24, 28) Watefield, M., 133 Waters, D. K., 147-148 Weber, E., 229 Wedel, N., 275, 279(11) Weening, R., 91 Weinfeld, H. M., 149 Weinstein, I. B., 133 Weinstein, P. R., 148 Weissbach, H., 202 Weissbach, L., 202 Weissmann, C., 159 Weitzel, F., 52, 53(70) Wellner, D., 30 Wells, T. N. C., 175 Wells, W. W., 30-31, 33, 33(10), 35, 36(21), 37, 37(10, 15), 38, 284-285, 285(22), 287(22, 26, 28), 288-289, 290(47), 291, 291(22, 25), 292(48) Wendel, A., 41, 42(26), 44, 44(26), 46(51), 47(51), 49(51), 50(51), 52, 53(70), 320, 321(31), 322(31), 341,342(2) Wendel-Hansen, V., 200, 254 Werth, J., 202 Weselake, R. J., 229 Wessel, D., 159 West, I. C., 73 Westphal, A. H., 194 Wetterauer, B., 244 Whanger, P. D., 12 White, C. W., 99 White, J. E., 71 White, J. M., 68 Whitesides, G. M., 228 Whittrock, D. A., 151 Widersten, M., 53 Wiener, H. L., 30 Wierzbicka, G., 12, 13(28)

Wierzbicka, G. T., 4, 9(11), 10, 11(27), 12(27) Wiessner, Ch., 275 Wileken, D. E., 98 Wilde, S. M., 333 Wilding, G., 332 Wilkison, W. O., 157 Willet, R. D., 128 Williams, C. H., Jr., 186, 187(1, 2), 189, 190(1 ), 191, 191(13), 192(13), 193, 193(1, 13), 194, 194(1), 201-202 Williams, D. L,, 40, 41(22), 42(22) Williams, M. W., 3, 9 Williams, S., 4 Williamson, J. M., 66, 103 Williamson, J. R., 150, 151(46) Willott, E., 153 Wilmanns, W., 104-108, 111 Wilson, J. C., 137, 138(26) Wilson, P. B., 149 Wingender, E., 40, 41(21), 42(21), 52(21) Wingfield, P., 295, 296(20), 297(20) Wingfield, P. T., 200, 293, 296, 299(9, 22), 303, 303(9) Winkler, B. S., 3 Winslow, J. P., 285,287 Wise, R. R., 220 Wishart, D. S., 30l Within, J., 40 Wittwer, A. J., 309 Wolf, B., 40, 52(20) Wolf, C. R., 53 Wollman, E., 244 Wolosiuk, R. A., 275-276, 280(17) Wong, J. H., 199, 228-230, 230(11), 231(11, 16, 19), 236(11), 238(11, 12, 16, 19), 239240, 240(16) Wong, L., 201 Wood, J. M., 348 Wood, R. J., 53 Woodbury, D. M., 149 Woods, E. B., 15, 21(15), 22(15) Woodward, C., 284, 302 Wright, P. E., 293-295,295(14), 297(17), 298, 299(7, 8), 301,302(17), 303(7, 8, 17, 34) Wu, E. Y., 340 Wtithrieh, K., 284, 287(14-17), 288(16, 17), 289(14, 15, 17), 290(13, 18), 294, 296, 303-304, 304(12, 13), 305-306 Wynn, R., 272

AUTHORINDEX

X Xanthoudakis, S., 175 Xia, T., 284, 287(14, 15), 289(14, 15) Xia, T.-H., 294, 304(12, 13), 305 Xie, W. Q., 153, 167 Xu, D. P., 30-31, 33(10), 37(10), 38, 289, 291, 292(48) Xu, G., 287

Y Yabuuchi, S., 90 Yagi, T., 14(9), 15 Yamamoto, S., 95 Yamamoto, Y., 41, 42(34) Yamauchi, A., 343-344, 346, 348(10) Yamazoe, Y., 59 Yan, N., 27 Yang, J.-G., 40 Yang, Y., 31, 33(10), 35, 36(21), 37, 37(10), 38, 285, 287(28), 288-289, 290(47), 292(48) Yano, K., 275, 282(8) Yasda, S., 87 Yeaman, S. J., 186 Yee, B. C., 221-222, 224(6), 229-230, 230(11), 231,231(11, 16, 18, 19), 233(25), 236(11), 238(11, 16, 19, 22), 240, 240(16), 244, 275 Yeh, L. A., 149 Yin, X.-M., 346

371

Yishimura, S., 41, 42(28) Yocum, M., 126 Yodoi, J., 53, 175, 177(4), 200, 254, 263, 264(23), 343-344, 346, 348, 348(9, 10) Yokoa, M., 87 Yokota, T., 200, 343 Yoolee, K., 133 Yoshida, K., 78, 82 Yoshimura, S., 41, 42(24) Yotti, P., 132 Young, J. A., 187, 189(8), 190(8), 193(8) Young, S., 133 Yunis, A., 68

Z Zabinsky, S. I., 129 Zakour, R. A., 249 Zampini, P., 78 Zbinden, I., 181 Zbinden, J., 253 Zehelein, E., 309, 312(2), 315(2) Zheng, B., 149 Ziegler, D. M., 342 Ziff, E. B., 180 Zillman, R. R., 235 Zimmerman, R., 51 Zinoni, F., 39, 43 Ztircheer, C., 91 Zurini, M., 130

SUBJECT INDEX

373

Subject Index

A Acetaminophen, hepatotoxicity inhibitors N-acetylcysteine, 333, 338-340 anethole dithiolethione, 318-321,323, 333, 338-340 N-Acetylcysteine acetaminophen, hepatotoxicity inhibition, 333, 338-340 cystine uptake, increase in CHO cells, 105 NF-KB inhibition, 168, 174 supplementation and glutathione synthesis, 105-107 Acivicin, 3,-glutamyltransferase inhibition, 10, 18-19, 21 Aconitase, activity in Escherichia coli glutathione mutants, 86-87 ADT, see Anethole dithiolethione Adult T-cell leukemia cell culture, 344 t.-cystine transport assay, 345, 348 DNA content, flow cytometry analysis, 344-346 thiol deprivation and viability, 345, 348 thiol status and apoptosis, 345-347 thioredoxin expression, 343-344 viability assay, 344 Anethole dithiolethione chemoprotective effect against hepatotoxins, 318-321, 323, 333, 338-340 choleretic activity, 316-317 glutathione metabolism effects, 318, 320321 lipid peroxidation, inhibition, 320-324, 329-331 muscarinic ACh receptors, effect on density, 317-318 radical scavenging assay ethanol radiolysis, 322-324, 330 linoleic acid radiolysis, 324, 328-330 structure, 317, 325

therapeutic applications, 316-317 toxicity, 323 AP-1, redox regulation of activation, 175, 264 Apoptosis, thiol status and regulation, 345347 ATL, see Adult T-cell leukemia Atomic absorption spectroscopy, protein kinase C, 131, 159 ATPase Na÷,K *inactivation and neuropathy, 150-151 phosphorylation effect on activity, 149, 151 glutathione disulfide-stimulated, red blood cells assay S-(2,4-dinitrophenyl)glutathione conjugate, 80-81 membrane/polycationic bead suspension, 79-80 solubilized membranes, 80 purification. 81-82

B BSO, see Buthionine sulfoximine Buthionine sulfoximine 3,-glutamylcysteine synthetase inhibition, 10, 12-13, 29-30, 320 structure, 28 transport, 29-30

C Calphostin C inhibition of protein kinase C, 141-142 photoactivation, 142-143 c-fos

induction by PMA, 180-181, 185 reactive oxygen species, 180-181

374

SUBJECT INDEX

RNA isolation cell culture, 181-182 RNase denaturation, 181 total RNA extraction, 182-183 Northern blot analysis, 183-184 suppression by dihydrolipoic acid, 181, 185-186 Chelerythrine, inhibition of protein kinase C, 143-145 Chloramphenicol acetyltransferase, reporter gene in transcriptional transactivation assay, 258-262 Cold light fluorimetry acetaminophen hepatotoxicity assay, 332, 339-340 apparatus, 334 energetic equation, 334-335 microplate design, 337, 340 reproducibility in microtitration assay, 335-336 sensitivity, 336-337 Cystamine, y-glutamylcysteine synthetase inhibition, 27, 30 y-Cystathionase, inactivation by propargylglycine, 100-102 Cystathionine assay high-performance liquid chromatography, 95 liquid chromatography/mass spectrometry, 96 sample preparation, 94 metabolism in rat development, 97-98 pathway and cysteine synthesis, 92-93, 101, 103 serum levels, effects of cobalamin deficiency, 98 folate deficiency, 98 oxidative stress, 98-100 Cysteamine cystine uptake, increase in CHO cells, 105 supplementation and glutathione synthesis, 105-107 Cysteine biosynthetic pathway, 92-94, 96, 101 cell transport systems, 104 dietary requirement, 96 intestinal absorption, 9 oxidation in solution, 104

redox potential, 179 toxicity, 97, 103 Cystine cell uptake assay adherent cells, 107-109 adult T-cell leukemia, 345, 348 nonadherent cells, 109 effect of exogenous thiols, 105-107, 111-112 systems, 104 glutathione biosynthesis, 105-107 measurement of incorporation, 110 Cytochrome c, reduction by thioredoxin reductase and selenite, 214

D Dehydroascorbate assay of glutaredoxin, 289 enzymatic reduction, s e e Dehydroascorbate reductase preparation, 32 reduction by glutathione, 31 synthesis, 30-31 Dehydroascorbate reductase, s e e a l s o Glutaredoxin; Protein disulfide-isomerase assay incubation, 32-33 principle, 32 reagent preparation, 32 glutathione dependence, 31 kinetic parameters, 36-38 reaction mechanism, 37-38 site-directed mutagenesis, 38 species distribution, 31 DHA, s e e Dehydroascorbate DHLA, s e e Dihydrolipoic acid Diethyl pyrocarbonate, RNase denaturation, 181, 183 Dihydrolipoamide, chemical synthesis, 189190 Dihydrolipoamide dehydrogenase absorption spectrum, 193 assay acetylpyridine adenine dinucleotide as substrate, 191 diaphorase activity, 192 NADH production, 187, 190-191 reverse reaction, 191-192

SUBJECT INDEX crude extract preparation bacteria, 187-188 cultured skin fibroblasts, 189 eukaryotic cells, 188 FAD cofactor, 186, 191 kinetic mechanism, 187, 193 kinetic parameters, 193-194 metal sensitivity, 193 pH optimum, 193 primary structure, 193-194 purification, 189 stability, 193 substrates, 186 Dihydrolipoic acid, c-los suppression, 181, 185-186 Dithiothreitol, redox potential, 179 DPH, see Phenytoin D T T , see Dithiothreitol

E Ebselen glutathione peroxidase activity, 341 lipid peroxidation inhibition, 341 metabolism, 342 toxicity, 342 Electrophoretic mobility shift assay NF-KB analysis, 173-174, 179-180 cell growth, 169-170, 178 DNA binding reaction, 171, 178 probe labeling, 172-173 gel running conditions, 172 nuclear protein extraction, 170-171, 176 principle, 169, 177 EMSA, see Electrophoretic mobility shift assay Ethanol effect on glutathione levels in sciatic nerve, 150 radiolysis, anethole dithiolethione assay, 322-324, 330 N-Ethylmaleimide, inhibition of protein kinase C, 144, 146

F Ferredoxin-thioredoxin reductase absorption spectra, 282-283

375

active site thiols, 275 assay, 280-281 purification from spinach acetone precipitation, 281 ammonium sulfate fractionation, 281 chromatography, 282 gel filtration, 281-282 leaf extraction, 281 storage, 282 Fluorescence microplate readers commercial availability, 332 microplate design, 337 sensitivity and illumination sources, 333 Fructose-l,6-bisphosphatase, chloroplast enzyme assay, 223-224 light-dependent activation, 220 purification, 222-223 redox titration curve fitting of data, 227 determination of Eh, 228 establishing of equilibrium, 225-226 monobromobimane labeling of thiols, 226-227 stability of ambient redox potential, 225 subunit structure, 221

G Glutamine synthetase, inhibition by methionine sulfoxamine, 28 y-Glutamylcysteine synthetase assay, 68-71 defects in humans, 91 Escherichia coli enzyme, characteristics, 27-28 feedback inhibition, 26-27 inhibition buthionine sulfoximine, 10, 12-13, 2930, 320 methionine sulfoxamine, 28-29 ophthalmic acid, 27 rat kidney enzyme cystamine inhibition, 27 purification of recombinant protein, 27 subunit structure, 26-27 reaction catalyzed, 67 y-Glutamyltransferase acivicin inhibition, 10, 18-19, 21 assay, 67-70

376

SUBJECT INDEX

reaction catalyzed, 66 substrate specificity, 66-67 tissue distribution, 14 y-Glutamyl transpeptidase, see y-Glutamyltransferase Glutaredoxin assay dehydroascorbic acid reduction, 31, 36-38, 289 /3-hydroxyethylene disulfide assay, 288-289 immunoassay, 289 ribonucleotide reductase assay, 287 enzyme nomenclature, 292 Escherichia coli mutants assay, 266-267 cell growth, 83 locus, 89 viability, 265-266, 291-292 isoforms in Escherichia coli, 284 purification, Escherichia coli enzymes activated thiol-Sepharose chromatography, 290-291 glutaredoxin 1,289-290 glutaredoxin 2, 290 glutaredoxin 3, 290 purification, human enzyme, 35-36, 291 purification, pig liver enzyme ammonium sulfate fractionation, 34 gel filtration, 34 heat step, 34 homogenization, 33-34 ion-exchange chromatography, 34-35 recombinant enzyme, 35 sequence homology between species, 285-286 site-directed mutagenesis, 38 structure determination by nuclear magnetic resonance, 284, 293-294 mixed disulfide intermediate structure, 306 resonance assignment, 304 solution conditions, 304 substrate specificity, 292 Glutathione antioxidant defense roles, 66, 72, 103 assay cell culture, 109-110 foods high-performance liquid chromatography, 5-6

homogenization, 5 sample preparation, 5 sciatic nerve detection, 153 sample preparation, 152-153 cancer prevention, 3 cytostatic agents, effect on levels, 111 food content availability, 4 food group differences, 7-9 preparation effects, 7-8 intestinal absorption assay in rat, 9-10 hormonal regulation, 11 systemic availability, 11-13 transport, 3, 9 intracellular pools tissue distribution, 14, 94 turnover rates, 14-15, 94 metabolism in rat development, 97-98 mitochondrial transport assay glutathione oxidation, prevention, 188 glutathione reductase, 20-22 high-performance liquid chromatography, 18, 22 incubation, 21 intact renal cells, 16-17, 25 isolated mitochondria, 16-17, 25 rat renal mitochondria, 22-24 divalent anions, effect on transport system, 24 energetics, 23-24 systems, 15 osmoregulation in Escherichia coli, 86 peripheral nerve, histochemical localization, 147 red blood cells, glutathione concentration, 72 Glutathione disulfide, red blood cell transport adduct transport, 75-76 ATPase assay, 79-81 purification, 81-82 stimulation by glutathione disulfide, 78-79 energy dependence, 72 inside-out vesicles, 72, 76-78 native cells, 74-75

s u ~ j z c r INDEx

oxidatively stressed cells, 72-74 system types, 72-73 Glutathione peroxidase biological roles, 51-53 dendrogram of superfamily, 42 hydroperoxide decomposition, 51-52 kinetic mechanism, 44-46 molecular evolution, 40-43 rate-limiting step, 45 reaction mechanism, 46-48 selenium cofactor, 39-40, 46, 209 selenocysteine TGA codon, 39, 43 three-dimensional structure, 48-51 types, 40 Glutathione reductase defects in humans, 91 Escherichia coli mutants, 87-88 glutathione transport assay in mitochondria, 20-22 seed protein reduction, 230, 238 Glutathione S-transferase affinity chromatography conjugates, 54 assay, 65 gene families, 53 protein kinase C fusion protein purification, 157-158, 161-164, 166-167 purification, human enzymes kidney PI-1 enzyme, 60 liver enzymes AI-1, A1-2, A2-2, and M1 equipment, 55 hydroxylapatite chromatography, 57-58 ion-exchange chromatography, 58-59 reagents, 55-56 tissue extraction, 56-57 skeletal muscle M2-2 enzyme, 61-63 testis M3-3 enzyme, 60-61 O class enzyme expressed in Escherichia coli

affinity chromatography, 65 equipment, 63 purity analysis, 65 reagents, 64 subunit structure, 53 Glutathione synthase defects in humans, 91 Escherichia coli mutants B strains, 87 K strains cell growth, 83

377 gshA, 85-86 gshB, 84-86

yeast mutants, 90-91 GPX, see Glutathione peroxidase GSH, see Glutathione gshB gene Escherichia coli mutants, 84-86 locus. 84 GSSG, see Glutathione disulfide GST, see Glutathione S-transferase

H High-performance liquid chromatography cystathionine assay, 95 cystine incorporation into glutathione. measurement, 110 -),-glutamylcysteine synthetase assay. 7071 glutathione assay cell culture, 109-110 foods, 5-6 mitochondrial transport, 18, 22 ovothiol assay, 117 HIV, see Human immunodeficiency virus Hoechst 33258, acetaminophen hepatotoxicity assay, 332, 340 Homocysteine, effect on lipid hydroperoxide levels, 98-99 HPLC, see High-performance liquid chromatography HTLV-I, see Human T-cell lymphotropic virus type I Human immunodeficiency virus, NF-KB control of replication, 263-264 Human T-cell lymphotropic virus type I disease pathogenesis, 343 thiol deprivation and viability of infected cells, 345 thioredoxin expression in lymphoid cells. 343-344 Hydrogen peroxide NF-KB activation, 168-169 protein kinase C oxidation, 139-140 /3-Hydroxyethylene, assay of glutaredoxin. 288-289 Hypericin inhibition of protein kinase C, 141-142 photoactivation, 142-143

378

SUBJECT INDEX

L Linoleic acid, micelle radiolysis absorbance spectroscopy, 327 anethole dithiolethione assay, 327-329 micelle preparation, 325 radical reactions, 325-326 rate constants, 326-327 Lipid peroxidation chain termination, 327 inhibitors anethole dithiolethione, 320-324, 329331 ebselen, 341 glutathione, 322, 341 a-Lipoic acid, redox potential, 179

isolated mitochondria, 16-17, 25 rat renal mitochondria, 22-24 divalent anions, effect on transport system, 24 energetics, 23-24 systems, 15 isolation, renal cortical mitochondria, 16 Monobromobimane fluorescence photography of labeled proteins, 235-236 germinated wheat seed labeling, 233-234, 238-239 quantitation of labeled proteins, 236-237 redox titration of protein thiols, 226-227, 231-233

N M Malate dehydrogenase, see NADP+-malate dehydrogenase mBBr, s e e Monobromobimane 2-Mercaptoethane sulfonate, s e e Mesna 4-Mercaptohistidine, s e e Ovothiol C Mesna, glutathione recovery after cytostatic drug therapy, 111 Metaperiodate, protein kinase C oxidation, 140-141 Methionine dietary requirement, 96-97 surgical stress effect on half-life, 100 toxicity, 97 Methionine sulfoxamine inhibition glutamine synthetase, 28 y-glutamylcysteine synthetase, 28-29 phosphorylation, 28 Mitochondria digitonin fractionation, 16-17, 19-20 functional integrity assessment, 17-18 glutathione transport assay glutathione oxidation, prevention, 188 glutathione reductase, 20-22 high-performance liquid chromatography, 18, 22 incubation, 21 intact renal cells, 16-17, 25

NAC, see N-Acetylcysteine NADP+-malate dehydrogenase activation by thioredoxin, 240-241 dithiothreitol system, 244 NADP + inhibition, 249 photochemical activation, 244-245 assay, 243 CA05A/C417A mutant protein activation kinetics, 252 purification, 251 site-directed mutagenesis, 249-250 storage, 252 thioredoxin reductase assay, 250-251 cloning by polymerase chain reaction, 245-247 purification, C4 plant enzyme affinity chromatography, 242 ammonium sulfate fractionation, 242 extraction from leaves, 241-242 hydrophobic interaction chromatography, 243 storage, 243 purification from E s c h e r i c h i a coli expression system affinity chromatography, 248 ammonium sulfate fractionation, 248 cell growth, 247 N-terminal sequencing, 248-249 Neutral red, acetaminophen hepatotoxicity assay, 332, 339-340 NF-KB, s e e Transcription factor NF-KB

SUBJECT INDEX Nitric oxide, inhibition of protein kinase C, 145 NMR, s e e Nuclear magnetic resonance Northern blot analysis, c - l o s RNA, 183-184 Nuclear magnetic resonance glutaredoxin, structure determination, 284, 293-294 mixed disulfide intermediate structure, 306 resonance assignment, 304 solution conditions, 304 proton spectra of ovothiols, 120 selenophosphate, 309, 315 thioredoxin, structure determination, 293-294 deuterium labeling of protein, 299, 301 flexibility of protein, 301-302 high-resolution structure determination, 298-299 mechanistic studies, 302-303 nuclear Overhauser effect, 297-298 oxidized and reduced protein differences, 303-304 resonance assignment, 296-297 solution conditions, 294-296 species differences, 299 water molecule binding, 303

O Ophthalmic acid, inhibition of ~/-glutamylcysteine synthetase, 27-28 Ovothiol A chemical synthesis, 119-120 detection in tissue, 117 invertebrate development, role, 115, 123 isolation, 117-118 proton NMR spectra, 120 redox potential, 122-123 structure, 116 thiol pKa, 121 ultraviolet spectra, 120-121 Ovothiol B chemical synthesis, 119-120 detection in tissue, 117 invertebrate development, role, 115, 123 isolation, 117-118 proton NMR spectra, 120 structure, 116

379

thiol pKa, 121 ultraviolet spectra, 120-121 Ovothiol C chemical synthesis, 119-120 detection in tissue, 117 invertebrate development, role, 115, 123 isolation, 117-118 ovoperoxidase cofactor, 115 proton NMR spectra, 120 redox potential, 121-122 structure, 116 thiol pKa, 121 ultraviolet spectra, 118, 120-121

P PAPS reductase, s e e 3'-Phosphoadenosine 5'-phosphosulfate reductase PDI, s e e Protein disulfide-isomerase Phenytoin neuropathy induction, 147, 149 protein kinase C activation, 150 Phorbol 12-myristate 13-acetate, c - f o s induction, 180-181, 185 3'-Phosphoadenosine 5'-phosphosulfate reductase, hydrogen donors, 266, 291 PKC, s e e Protein kinase C PMA, s e e Phorbol 12-myristate 13-acetate Propargylglycine effect on levels cysteine, 102 glutathione, 102 inactivation of ~/-cystathionase, 100-102 Protein disulfide-isomerase dehydroascorbate reductase activity, 31, 36-38 purification from bovine liver, 36-37 redox potential, 201 Protein kinase C activation, 124, 132, 150, 156 assay, 137 atomic absorption spectroscopy, 131,159 concentration determination, 127-128, 159 extended X-ray absorption fine structure data processing, 128-129 spectra, 129-130 family classification, 154 inhibitors, 132-133 peripheral nerve activity, 148

380

SUBJECT INDEX

purification calpain-free enzyme, 134-135 glutathione S-transferase fusion protein affinity chromatography, 158 cell growth optimization, 157-158, 161-164, 166-167 plasmid construction, 157 oxidatively modified enzyme, 138-139 protein kinase Cy from Sf9 cells, 157 regulatory domain conserved sequence, 124, 133, 154-156 oxidation, 133 phorbol ester binding assay, 137-138, 158 zinc binding, 126-131, 156 storage, 135 thiol modification catalytic domain, 133, 137, 143 chelerythrine, 143-145 enzyme preparation, 135-136 N-ethylmaleimide, 144, 146 hydrogen peroxide, 139-140 inhibitors of modification, 136, 141-143 metaperiodate, 140-141 nitric oxide, 145 removal of excess reagent, 136-137 sanguinarine, 143-145 X-ray fluorescence data processing, 125-126 sample preparation, 125 spectra, 126-128 zinc role, 131 stoichiometry, 160, 166

R Red blood cell, glutathione disulfide transport adduct transport, 75-76 ATPase assay, 79-81 purification, 81-82 stimulation by glutathione disulfide, 78-79 energy dependence, 72 inside-out vesicles, 72, 76-78 native cells, 74-75 oxidatively stressed cells, 72-74 system types, 72-73

Rhodamine 123, acetaminophen hepatotoxicity assay, 332, 340 Ribonucleotide reductase assay of glutaredoxin, 287 hydrogen donors, 264-266, 283, 291 RNA, c-jun RNA isolation cell culture, 181-182 RNase denaturation, 181 total RNA extraction, 182-183

S Sanguinarine, inhibition of protein kinase C, 143-145 Seed protein isolation from wheat, 231-232 metabolism, 228-229 monobromobimane labeling, 232-234 polyacrylamide gel electrophoresis, 234235 redox status food processing effects, 239-240 germination, 231,238-239 maturation, 239 thiol reduction, 229-230 Selenide, metal binding, 209 Selenite cytochrome c reduction with thioredoxin reductase, 214 reduction by thiols, 209 thioredoxin, oxidation, 210, 216-217 thioredoxin reductase specificity, 215 Selenium glutathione peroxidase cofactor, 39, 46, 209 toxicity, 38-39 Selenodiglutathione chemical synthesis, 210-211 formation reaction, 209 purification, 211-212 stability, 212 thioredoxin inactivation, 217-218 kinetics of oxidation, 215-217, 219 thioredoxin reductase anaerobic reaction, 213-214 enzymatic reduction, 210 kinetics of reaction, 212-213 mechanism of inactivation, 219

SUBJECT INDEX Selenophosphate nuclear magnetic resonance, 309, 315 oxidation, 315 pK values, 315 synthesis chemical synthesis, 310-312 enzymatic synthesis enzyme preparation, 312-313 75Se-labeled compound, 314-315 Selenophosphate synthetase assay, 313-314 gene, 309 metal dependence, 315 purification of recombinant Escherichia coli enzyme, 312-313 stability, 315 substrate specificity, 315 Site-directed mutagenesis NADP+-malate dehydrogenase, 249-250 thioltransferase, 38 thioredoxin, 270-273 thioredoxin-controlled disulfide bridges, 252 Spectroscopy, see specific type

T Thioltransferase, see Glutaredoxin Thioredoxin assay of crude extracts radioimmunoassay, 206-207 spectrophotometric assay, 207-208 bacteriophage role assembly, 268-270 replication, 267-268 control of activation AP-1,264 NF-KB, 254-259, 264 lymphocyte secretion, 200-201 mutants Anacystis nidulans, 273 Bradyrhizobium japonicurn, 273 Escherichia coli assay, 266-267 cell growth, 83 viability, 265-266, 291-292 Saccharomyces cerevisiae, 274 purification Escherichia coli enzyme, 202

381

human enzyme expressed in Escherichia coli, 255 mammalian enzyme gel filtration, 203 ion-exchange chromatography, 203204 tissue homogenization, 203 redox potential, 199-200 selenodiglutathione reactions inactivation, 217-218 kinetics of oxidation, 215-217, 219 sequence homology between species, 200 site-directed mutagenesis, 270-273 structure determination by nuclear magnetic resonance, 293-294 deuterium labeling of protein, 299, 301 flexibility of protein, 301-302 high-resolution structure determination, 298-299 mechanistic studies, 302-303 nuclear Overhauser effect, 297-298 oxidized and reduced protein differences, 303-304 resonance assignment, 296-297 solution conditions, 294-296 species differences, 299 water molecule binding, 303 substrate specificity, 202 thiol redox control system, 200 thiols and dimerization, 218 Thioredoxin f assay, 276-277 light-dependent activation of chloroplast enzymes, 220, 226, 244-245,275 purification recombinant protein from Escherichia coli cell growth, 277 chromatography, 278 expression vector construction, 277 inclusion body purification, 277 renaturation, 278 storage, 278 spinach chloroplast enzyme gel filtration, 222 hydroxyapatite chromatography, 221-222 leaf homogenization, 221 Thioredoxin h C405A/C417A mutant malate dehydrogenase assay, 250-251

382

SUBJECT INDEX

distribution in plant cells, 230 seed protein reduction, 229-230, 238 Thioredoxin m assay, 278-279 light-dependent activation of chloroplast enzymes, 275 purification, recombinant protein from Escherichia coli

cell growth, 279 chromatography, 280 expression vector construction, 279 storage, 280 Thioredoxin reductase assay C405A/C417A mutant malate dehydrogenase, 250-251 crude extracts, 205-206 spectrophotometric assay, 204-205 purification Escherichia coli enzyme, 202 mammalian enzyme affinity chromatography, 204 tissue homogenization, 203 reaction catalyzed, 199 selenodiglutathione reactions anaerobic reaction, 213-214 enzymatic reduction, 210 kinetics of reaction, 212-213 mechanism of inactivation, 219 structure, 201 substrate specificity, 201-202, 205, 208 T7 phage, thioredoxin role assembly, 268-270 replication, 267-268 Transcription factor NF-KB cytoplasmic retention molecule, 168, 254, 262 electrophoretic mobility shift assay analysis, 173-174, 179-180, 257 cell growth, 169-170, 178 DNA binding reaction, 171, 178, 257 probe labeling, 172-173 gel running conditions, 172, 257

nuclear protein extraction, 170-171, 176 principle, 169, 177, 255-256 human immunodeficiency virus, control of replication, 263-264 redox regulation of activation, 175, 177, 179-180, 254, 262-263 N-acetylcysteine, 168, 174 hydrogen peroxide, 168-169, 254, 262 thioredoxin, 254-259, 264 role in disease, 177 subunit components, 168 transactivation assays chloramphenicol acetyltransferase assay, 258-262 transfection of HeLa cells, 259-260

O,(9, O-Trimethylsilylselenophosphate chemical synthesis, 310 diisopropylethylamine salt hydrolysis, 311-312 synthesis, 311 hydrolysis, 310-311

V Vitamin B6, deficiency and glutathione metabolism, 100

X XAS, see X-ray absorption spectroscopy X-ray absorption spectroscopy extended X-ray absorption fine structure principle, 125 protein kinase C data processing, 128-129 spectra, 129-130 fluorescence detection principle, 124-125 protein kinase C data processing, 125-126 sample preparation, 125 spectra, 126-128