Current Topics in Membranes and Transport, Volume 04

Current Topics in Membranes and Transport Vdllllls 4

Advisory Board

Robert W . Berliner Britton Chance I . S. Edelman Aharon Katchalsky (deceased) Adam Kepes Richard D. Keynes Philip Siekevitz Torsten Teorell Daniel C. Tosteson Hans H . Ussing

Contributors

Richard P. Durbin Mahendra Kumar Jain Howard E. Morgan Carolyn W . Slayman Carol F . Whitjeld

Current Topics in Membranes and Transport

VOLUME 4

Edited by Felix Bronner Department of Oral Biology University of Connecticut Health Center Farmington, Connecticut and

Arnost Kleinzeller Graduate Division of Medicine University of Pennsylvania Philadelphia, Pennsylvania

1973

Academic Press

New York and London

A Subsidiary of Harcourt Brace Jovanovich, Publishers

COPYRIGHT 0 1973, BY ACADEMIC PRESS, INC. ALL RIGHTS RESERVED. NO PART OF THIS PUBLICATION MAY BE REPRODUCED OR TRANSMllTED IN ANY FORM OR BY ANY MEANS, ELECTRONIC OR MECHANICAL, INCLUDING PHOTOCOPY, RECORDING, OR ANY INFORMATION STORAGE AND RETRIEVAL SYSTEM, WITHOUT PERMISSION IN WRITING FROM THE PUBLISHER.

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United Kingdom Edition published by ACADEMIC PRESS, INC. (LONDON) LTD. 24/28 Oval Road, London NWI

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List of Contributors, vii Preface, ix Contents of Previous Volumes, xi Aharon Katzir-Katchalsky, 1913-1972, xiii Bibliography of the Principal Publications of Aharon Katzir-Katchalsky on Membrane Phenomena, xix The Genetic Control of Membrane Transplant CAROLYN W. SLAYMAN

I. Introduction, 1 11. Isolation of Transport Mutants, 3 111. Criteria for Identifying Genes That Affect Transport Directly, 136 IV. Linkage Relationships of Transport Mutants, 140 V. Dominance and Recessiveness, 146 VI. Regulation of Transport Systems, 150 VII. Usefulness of Mutants in Understanding Transport Mechanisms, 151 VIII. Conclusion, 155 References, 155 Enzymic Hydrolysis of Various Components in Biomembranes and Related Systems MAHENDRA KUMAR JAIN

I. Introduction, 176 11. Action of Hydrolytic Enzymes on Model Systems, 184 111. Effect of Enzymic Hydrolysis on System Properties of Biomembranes, 191 IV. Effect of Enzymic Hydrolysis on Transport Systems, 217 V. Catabolism of Membrane Components by Endogenous Enzymes or Intracellular Catabolism, 233 VI. Conclusions and Epilog, 236 References, 238 Regulation of Sugar Transport in Eukaryotic Cells HOWARD E. MORGAN AND CAROL F. WHITFIELD

I. Kinetic Characterization of Passive Transport, 256 11. Nonhormonal Regulation of Sugar Transport, 261 111. Hormonal Control of Sugar Transport, 274 IV. Mechanisms of the Regulation of Transport, 287 V. Summary, 296 References, 297 V

CONTENTS

Vi

Secretory Events in Gastric Mucosa

RICHARD P. DURBIN I. Introduction, 305 11. Comparative Aspects, 305 111. Structural Aspects, 307 IV. Coupling of Secretion to Metabolism, 313 V. Conclusion, 319 References, 319 Author Index, 323 Subject Index, 340

List of Contributors Cardiovascular Research Institute and Department of Physiology, University of California, San Francisco, California Mahendra Kumar Join, Department of Chemistry, Indiana University, Bloomington, Indiana* Howard E. Morgan, Department of Physiology, The Milton S. Hershey Medical Center, The Pennsylvania State University, Hershey, Pennsylvania Carolyn w. Slayman, Departments of Human Genetics, Microbiology, and Physiology, Yale University School of Medicine, New Haven, Connecticut Carol F. Whitfleld, Department of Physiology, The Milton S. Hershey Medical Center, The Pennsylvania State University, Hershey, Pennsylvania Richard P. Durbin,

* Present address: Department of Chemistry, University of Delaware, Newark, Delaware. vii

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The fourth volume of Current Topics in Membranes and Transport extends the analysis of significant transport processes and structures. Carolyn Slayman presents a comprehensive summary of the genetics of transport, Jain reviews some aspects of the bilayer nature of the biological membrane, Morgan and Whitfield deal with sugar transport and its control in eukaryotes, and Durbin reviews some problems of gastric ion secretion. The editors hope that all interested in biological transport will find these reviews rewarding and provocative. Last year we dedicated the volume to the memory of Aharon KatairKatchalsky. Caplan’s thoughful appreciation in this volume reminds us of the breadth of Aharon’s work and of the human qualities that went into it. We hope this series will continue to reflect the scientific ideal of Aharon Katdr-Katchalsky, namely a passionate belief in the dispassionate search for scientific truth. FELIX BRONNER ARNOST KLEINZELLER

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Contents of Previous Volumes Volume 1

Some Considerations about the Structure of Cellular Membranes MAYNARD M. DEWEYAND LLOYDBARR The Transport of Sugars across Isolated Bacterial Membranes H. R. KABACK Galactoside Permease of Escherichia coli ADAMKEPES Sulfhydryl Groups in Membrane Structure and Function ASERROTHSTEIN Molecular Architecture of the Mitochondrion DAVIDH. MACLENNAN Author Index-Subject Index Volume 2

The Molecular Basis of Simple Diffusion within Biological Membranes W. R. LIEBAND W. D. STEIN The Transport of Water in Erythrocytes ROBERTE. FORSTER Ion-Translocation in Energy-Conserving Membrane Systems B. CHANCE AND M. MONTAL Structure and Biosynthesis of the Membrane Adenosine Triphosphatase of Mitochondria ALEXANDER TZAGOLOFF Mitochondria1 Compartments : A Comparison of Two Models HENRYTEDESCHI Author Index-Subject Index

xi

xii

CONTENTS OF PREVIOUS VOLUMES

Volume 3

The Na+, K+-ATPase Membrane Transport System: Importance in Cellular Function E. LINDENMAYER, AND ARNOLD SCHWARTZ, GEORGE JULIUS C. ALLEN Biochemical and Clinical Aspects of Sacroplasmic Reticulum Function ANTHONY MARTONOSI The Role of Periaxonal and Perineuronal Spaces in Modifying Ionic Flow Across Neural Membranes W. J. ADELMAN,JR.AND Y. PALTI Properties of the Isolated Nerve Endings GEORGINA RODR~GUEZ DE LORESARNAIZAND EDUARDO DE ROBERTIS Transport and Discharge of Exportable Proteins in Pancreatic Exocrine Cells : In Vitro Studies J. D. JAMIESON The Movement of Water Across Vasopressin-Sensitive Epithelia RICHARD M. HAYS Active Transport of Potassium and Other Alkali Metals by the Isolated Midgut of the Silkworm AND KARLZERAHN WILLIAMR. HARVEY Author Index-Subject Index

Aharon Katzir-Katchalrky

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Aharon Katzir-Katchalsky, 1913-1972 To write an objective biographical appraisal of Aharon Katchalsky’s contribution to the membrane field, with the recollection of his brutal and senseless death a t the hands of political assassins still raw, is scarcely possible. It is too early, and for one who knew him as a friend, and fell under his spell as a teacher, too painful. Every aspect of his work is colored by memories and associations: the elation he could evoke a t a seminar or discussion around the coffee table; the sense of excitement-sometimes bordering on euphoria-he never failed to communicate to the audience during his lectures; the intense pleasure he took (and gave) in debating a theoretical point, chalk in hand. He loved elegance and style wherever he found them, but especially in a page of mathematics; a blackboard covered with equations in his own graceful handwriting often possessed something of the richness of a medieval manuscript. Of what manner of a man he washis warmth, humanity, and largeness of spirit-others have written eloquently. For those of us who had found inspiration in his teaching, including his oldest colleagues, he always remained in the profoundest sense of the word a mentor. In a way it is an invidious task to separate Katchalsky’s studies on membranes from the totality of his work. Viewed as a whole, his work is as impressive in its unity as it is astonishing in its variety. The twin themes of mechanochemical coupling and chemodiffusional coupling dominate large parts of it, both being intimately related to the most characteristic properties of biological systems. His contributions to the understanding of such processes do not, however, stand apart from his contributions to polymer chemistry and nonequilibrium thermodynamics. His ideas on membranes encompassed many dimensions of the problem and had wide ramifications. The earliest papers, written in the late ~ O ’ S ,were directed towards the proper understanding of existing experimental techniques and the proper interpretation of permeability measurements commonly made on biological membranes. The implications were speedily realized to have importance in the technological application of membranes-especially in desalination. For Katchalsky, however, synthetic membranes remained models for biological systems, and by the mid-60’s the problem of the coupling of chemical reaction and transport had become paramount in his thoughts. In his last years he was deeply concerned with the mechanism of memory recording, which involved considerations relating to hysteretic effects in both biopolymers and biomembranes. As always the thermodynamic implications of the phenomena intrigued him, and frequently led him far into the realm of philosophical speculation. Katchalsky’s early work on membranes and transport phenomena in general was a natural outgrowth of his studies, covering more than a decade, of the chemical physics and biophysics of macromolecules. At the outset of his career he was profoundly impressed by the notion that large polymeric molecules might play an essential role in all processes occurring in living systems-as a consequence of, among other things, their conformational degrees of freedom. This was a t a time when little was known of protein structure and DNA was still to be recognized as the carrier of genetic information. However, the work of Staudinger and later Kern in the 30’s had suggested that charged synthetic polymers would be useful as models of biological macromolecules. I n the brief period between the end of World War I1 and the beginning of the Israeli War of In-

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AHARON KATZIR-KATCHALSKY, 1 9 1 3-1 972

dependence, Katchalsky developed his ideas during a seminal spell in the laboratory of the redoubtable Werner Kuhn in Basel, and returned to Palestine (as it then was) to found a school of polyelectrolyte chemistry a t the Hebrew University on Mount SCOPUS. With the conclusion of hostilities this school shifted to Rehovot, where he was called upon to establish a polymer department a t the newly-created Weizmann Institute of Science. Starting with papers in the first volumes of the Journal of Polymer Science in 1946 and 1947,Katchalsky and his colleagues produced a veritable avalanche of theoretical and experimental publications, making an explosive impact on the rapidly growing field of polyelectrolyte solutions and gels. The impact of these studies was also felt in the equally rapidly developing technology of ion-exchange resins and membranes, stimulating the concerns which led ultimately to Katchalsky’s later work on the permeation of salt through charged membranes. In the papers written during this “polyelectrolyte” period the seeds of his deep and lasting interest in mechanochemistry are to be found, and the profound influence of Gibbsian thermodynamics on his thinking is already strikingly in evidence. In the early 50’s Staverman demonstrated the utility of nonequilibrium thermodynamics in describing membrane processes, and introduced the concept of a reflection coefficient in relation to the osmometry of polymer solutions. Kirkwood showed that local force equations (written in terms of resistance coefficients), in contrast to local flow equations, may be integrated across a membrane to give global phenomenological relations. But very few workers appreciated the power of the method until 1958, when Kedem and Katchalsky, and also Spiegler, published almost simultaneously and quite independently their classical papers on the application of nonequilibrium thermodynamics to membrane transport. Kedem and Katchdsky were concerned primarily a t that time with the permeability of biological membranes to nonelectrolytes; Spiegler waa concerned with transport processes in ion-exchange membranes. Katchalsky had always understood the importance of thermodynamics as an organizing principle, often claiming that it “plays the role of scientific logics.” It was therefore characteristic of his thinking to invoke nonequilibrium thermodynamics when his attention turned to membranes and transport. Undoubtedly the contribution for which Katchalsky is chiefly known (and may well be chiefly remembered) among workers in the membrane field is precisely his collaborative study of permeability with Kedem, which led to the derivation of what they called “practical” phenomenological equations. These Kedem-Katchalsky (or K-K) equations already possess a time-honored air. As Richardson has pointed out, it is little over a decade since the most complete versions of the equations were publbhed, yet they have already taken their place alongside the Nernst-Planck and the Goldman equations as standard working models for physiologists and biophysicists. I n fact the KedemKatchalsky equations are familiar to a generation of young biophysicists who may never read the original papers. In one simple form, applicable to systems involving a single solute, the equations describe volume flow (Jy)and solute flow (J.)through a homogeneous membrane in the absence of electric current:

J.

=

c,(l

- u)JV + COAT

(2)

Here A p and AT are the hydrostatic and osmotic pressure differences respectively, and cs is an average of the solute concentrations in the solutions on either side of the membrane. The “practical” phenomenological coefficients appearing in Eqs. (1) and (2) are the filtration coefficient L,, the solute permeability U , and the reflection coefficient Q

xvii

AHARON KATZIR-KATCHALSKY, 1913-1 972

originally introduced by Staverman. Prior to the derivation of these equations there existed no self-consistent treatment of permeability, and certainly none general enough to cover the whole range of physiological phenomena. Conventional descriptions of membrane transport made use of two independent flow equations, one for volume and one for solute, with only two coefficients. Thus volume flow was represented by an expression of the type

Jv = Lp (Ap - AT) and solute flow by the Fickian form

both manifestly incomplete in the light of present-day concepts, since the possibility of coupling between flows is entirely ignored. Although experimentalists had become progressively aware of the inadequacy of expressions such as these, and although nonequilibrium thermodynamic treatments of transport were in the air and indeed osmotic pressure measurement had been characterized in terms of the reflection coefficient, it remained for the Kedem-Katchalsky equations to link the phenomena, for the first time, into a single gestalt capable of yielding an internally coherent description in physiological terms. This literally revolutionized membrane studies. Equation (2) indicates that the solute permeability w is to be determined under condi. an alternative version tions of zero volume flow, by measuring the ratio ( J s / A ~ ) ~ v 4 In of the equations a “second permeability” w‘ appears, to be determined under conditions such that the hydrostatic and osmotic pressure differences just balance, i.e., by measI. version can be written conveniently in a form uring the ratio ( J a / A ~ ) ~ 9 - ~This such that the all-important property of Onsager symmetry (identity of the cross-coefficients) is displayed:

J,

- A T ) + ca(l - U ) L ~ ( A T / C J - a ) L P ( A p- A T ) + C ~ W ’ ( A T / C J

= Lp(Ap

J a = c,(l

(3) (4)

Equations (1) and (3) are identical, and the last term in Eq. (4) is obviously just o’Aa. Clumsy though they are, these expressions bring out the nature of the thermodynamic forces conjugate to the flows J, and J., which are seen to be ( A p - AT) and ( A r / c . ) respectively. Such conjugate flux-force pairs are generated, according to the methodology of nonequilibrium thermodynamics, by first deriving the so-called “dissipation function,” i.e., the temperature times the rate of entropy production due to irreversible processes in the membrane. This takes the form

T(diS/dt) = J v ( A p

- AT)

J&a0

(5)

where the quantity Apec denotes the concentration-dependent part of the chemical potential difference of the solute across the membrane. Equation ( 5 ) is exact for dilute solutions providing that local processes within the membrane obey linear relations. Kedem and Katchalsky sought to elicit, from the global phenomenological relations corresponding to this dissipation function, a set of transport coefficients which to some extent would have the virtue of familiarity to researchers in the field (and thus be readily accessible experimentally and compatible with existing data), to some extent would be insensitive to concentration changes, but above all would be thermodynamically selfconsistent. To reconcile these requirements they were compelled to introduce the

xviii

AHARON KATZIR-KATCHALSKY, 191 3-1 972

appropriate average concentration csr such that Apse = A a j c ,

(6)

Consequently, cs is a logarithmic average. This transformation of the dissipation function has the virtue of “saving the phenomenon,” and led directly to the formulation of the practical flow equations. It is perhaps a reflection of the importance attached to the Kedem-Katchalsky equations that they did not fail to attract their share of criticism. It has been variously claimed that they are misleading on a t least two grounds: that their range of validity (the linear regime) must be vanishingly small, and that global Onsager symmetry cannot actually hold when bulk flow occurs. This is not the place to analyze such criticisms in depth, but a comment seems to be called for. It is basic to the nonequilibrium thermodynamic approach that the forces be small enough to ensure linearity of the flux equations. The transformation from ApBcto AT is itself nonlinear since it invokes the logarithmic average concentration-but it is the key to the entire representation. Certainly this limits the applicability of Eqs. (1) and (2) to very small values of J , or A T , but it does not by any means render them invalid (especially for the description of physiological membrane processes). On the contrary, it has been shown repeatedly that the KedemKatchalsky equations represent a first-order expansion of the integrals of the local frictional equations; indeed the expansion indicates that when the concentration ratio exceeds (say) 2:1, so that c8 departs by more than a few percent from the arithmetic average, use of the latter can preserve the linear formalism if J , is not too large. I t also emerges from such calculations that the reflection coefficients appearing in Eqs. (1) and ( 2 ) [or Eqs. (3) and (4)]are identical, and hence global Onsager symmetry does in fact obtain, a result which has been verified experimentally in several systems. But clearly it is in the nature of linear phenomenological equations that they represent behavior within certain limits-they are not expected to be exact under all circumstances. However small the range of the Kedem-Katchalsky relations may be, their heuristic and practical importance remains unquestionably immense. Perhaps the turning point in the realization by biologists of the significance of the new approach was the memorable symposium on membrane transport and metabolism held in Prague in 1961, when by all accounts Katchalsky electrified the audience by his presentation. At any rate, from then on the biological literature reflects a growing interest in the formalism. Hard on the heels of this meeting appeared an interpretation of the practical phenomenological coefficients in terms of friction and distribution coefficients. Here Kedem and Katchalsky turned their attention to charged membranes and demonstrated that for electrolyte permeation through such membranes, the reflection coefficient u may assume negative values, indicating “anomalous” osmosis. The mechanism of anomalous osmosis had been a controversial topic: its elucidation in phenomenological terms was thus a vivid demonstration of the scope of the method. The analysis was performed for a membrane conforming to the well-known Teorell-MeyerSievers fixedcharge model, yielding results in good agreement with the experimental data of Loeb and of Grim and Sollner. This study led naturally to the consideration of electric current flow through charged membranes. In an elegant treatment it was shown that if the potential across the system is applied by means of electrodes reversible to one of the ions present, the dissipation function requires just one more term-current times the potential difference between the electrodes. A fully developed treatment of the permeability of highly charged membranes t o electrolytes was presented in a series of three successive papers by Kedem and Katchalsky in 1963. These papers analyzed the properties of composite membranes in terms of series

AHARON KATZIR-KATCHALSKY, 1 9 1 3-1 9 7 2

xix

and parallel arrays, emphasizing the fundamental importance of polarity and circulation (respectively) in understanding the behavior of such structures. I n particular, this analysis showed that mosaic membranes may exhibit pronounced negative anomalous osmosis, i.e., high negative values of u, an effect which had been predicted qualitatively much earlier by Sollner and demonstrated by Neihof and Sollner. The substance of Katchalsky’s work on membrane and other transport processes up to this point was incorporated in the book “Nonequilibrium Thermodynamics in Biophysics” by Katchalsky and Curran, published in 1965. I n the late 60’s Katchalsky became increasingly absorbed in the problem of coupling between diffusion and chemical reaction, especially as manifested in biological membranes. An examination of the thermodynamics and kinetics of active transport in erythrocytes (with Blumenthal and Ginzburg) was followed by an analysis of facilitated diffusion (with Blumenthal) which modeled, inter a h , the allosteric transitions of a carrier protein. At this time periodicity in membranes assumed an important place in his thinking-especially periodicity related to the presence of chemical reaction. Together with Spangler he showed that if an autocatalytic system undergoes a phase transition with metastable states, oscillatory behavior can be achieved by appropriately coupling the reaction t o a membrane transport process regulating the flow of product or reactant. These considerations invoked the concept of thermodynamically metastable ateady states and suggested the possibility that hysteresis loops might exist in transitions between steady states. Such hysteresis cycles in biomembranes were linked t o similar phenomena in biopolymers and t o the phenomenon of memory. I n 1969 Katchalsky wrote the following credo*:

I believe that the ultimate goal of biological study is to “translate” the phenomena of life into meaningful physical concepts. It is rather clear that present-day physics is still unable t o deal adequately with the complex and diversified expressions of life, and many years of research and contemplation await the scientists before they will be able to fit animate and inanimate matter into a common, unified conceptual framework. The driving force of quantitative biological study is, however, our mystical conviction that “Nature” is one and that future generations will comprehend life within an integrated “Natural Philosophy of the Physical World.” The profound belief in the unity of nature expressed here led Katchalsky t o pondex deeply the chemical basis of morphogenesis and the origin of life. His ideas were influenced primarily by the early work of Turing on the ability of homogeneous chemical processes to develop structure spontaneously (as a consequence of a random disturbance) and the more recent work of Prigogine on the thermodynamic theory of structure and stability. Structures which survive only by the dissipation of an energy input were termed by Prigogine “dissipative structures.” They can appear in systems maintained far from thermodynamic equilibrium, and are characterized by an unstable transition point (a “symmetry-breaking” transition or instability). Such transitions in chemical systems require nonlinear reaction schemes such as occur in the autocatalytic process referred to above. Katchalsky considered that dissipative structures may not only play a major role in the maintenance of certain cellular patterns, but that they may also have participated in the development of the earliest structures from which life arose. Prior to his end Katchalsky was preoccupied with several complementary interests: the nature of prebiotic peptide synthesis, the consequences of chemodiffusional coupling,

* I n “Biology and the Physical Sciences” (S. Devons, ed.). Columbia Univ. Press, New York.

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AHARON KATZIR-KATCHALSKY, 1 9 1 3-1972

and the basis of the molecular memory record. Through all these there runs as a unifying thread the notion of generation, storage, and retrieval of information. This notion is strongly reflected in his later writings concerning membrane phenomena. He viewed chemodiffusional coupling as a structuring agent closely related to biological morphogenesis, and this led him to speculate that active membranes might be dissipative structures whose dynamics are governed by the very chemodiffusional processes participating in their action. Indeed he conjectured, in agreement with Prigogine, that all living organization may be based on dissipative structures, fixed to dxerent degrees by covalent bonds. His deep concern with information flow, energy flow, and the establishment of pattern and shape led him to break fresh ground in the application of thennodynamics to complex systems. A love of thermodynamic rigor expressed itself in everything he did, and his final major work-which was cut short even as it began to flourish-was an attempt to develop a “network thermodynamics” especially suited to the organizational complexity of biological systems. By generalizing network theory to include irreversible thermodynamic systems, thermodynamics was to be brought within the framework of modern dynamic systems analysis. The bond graph approach to systems analysis, still in its infancy, impressed Katchalsky with its versatility as a representation of arbitrarily complex networks. In his last paper on this subject, completed just before he died, he showed, together with Oster and Perelson, that the bond graph technique can be used to describe diffusion-reaction systems including facilitated and active transport, the rectification properties of complex membranes, and relaxation oscillations in coupled membrane systems. Many of Katchalsky’s ideas on these and other matters live on in the minds of his colleagues, and will ultimately see the light of day. But although the direction in which the broader thrust of his thinking lay may be known, we are denied the pleasure of ever witneesing its realization. His work will be carried forward, but we shall never know which path Katchalsky himself would have trodden. The particular flavor and style that was his will be missing, and so will the insight and the flair for finding connections among a bewildering variety of seemingly unrelated concepts. Katchalsky was born in Lodz, Poland, came to Israel in 1925, and received his M.Sc. and Ph.D. degrees from the Hebrew University in 1937 and 1940. He had many honors, and engaged in a host of activities. He was the first President of the Israel National Academy of Sciences and Humanities, President and then Honorary Vice-president of the International Union for Pure and Applied Biophysics, a Council Member of the International Council of Scientific Unions, a Foreign Member of the United States National Academy of Sciences, a Council Member of the European Molecular Biology Organization, and Visiting Miller Professor of the University of California a t Berkeley. He was a true Renaissance man, a citizen of the world, renowned for his charismatic charm, his encyclopedic knowledge of topics ranging from history to philosophy and metaphysics, his love of the cut and thrust of scientific dialogue. In his work he constantly emphasized historical perspective, and in his passing he symbolized the historic confrontation of his people-creativity versus hatred and destruction. The cruel snuffing out of his life deprived us all of a very precious source of enlightenment, but he left a legacy of riches to build on for a long time.

S. R. CAPLAN

Bibliography of the Principal Publications of Aharon Katzir-Katchalsky on Membrane Phenomena Kedem, O., and Katchalsky, A. (1958). Thermodynamic Analysis of the Permeability of Biological Membranes to Non-electrolytes. Biochim. Biophys. Acta 27, 229. Katchalsky, A. (1961). Membrane Permeability and the Thermodynamics of Irreversible Processes. “Membrane Transport and Metabolism” (A. Kleinzeller and A. Kotyk, eds.), p. 69. Academic Press, New York. Kedem, O., and Katchalsky, A. (1961). A Physical Interpretation of the Phenomenological Coefficients of Membrane Permeability. J . Gen. Physiol. 45, 143. Katchalsky, A,, and Kedem, 0. (1962). Thermodynamics of Flow Processes in Biological Systems. Biophys. J . 2, 53. Kedem, O., and Katchalsky, A. (1963). Permeability of Composite Membranes. Part I. Electric Current, Volume Flow and Flow of Solute Through Membranes. Trans. Faraday SOC.59, 1918. Kedem, O., and Katchalsky, A. (1963). Permeability of Composite Membranes. Part 11. Parallel Elements. Trans. Faraday SOC.59, 1931. Kedem, O., and Katchalsky, A. (1963). Permeability of Composite Membranes. Part 111. Series Array of Elements. Trans. Faraday SOC.59, 1941. Ginaburg, B. Z., and Katchalsky, A. (1963). The Frictional Coefficients of the Flows of Nonelectrolytes through Artificial Membranes. J . Gen. Physiol. 47, 403. Katchalsky, A., and Curran, P. F. (1966). “Nonequilibrium Thermodynamics in Biophysics,” Harvard Univ. Press, Cambridge, Massachusetts. Blwnenthal, R., Ginzburg, B. Z., and Katchalsky, A. (1967). Thermodynamic and Model Treatment of Active Ion Transport in Erythrocytes. “Hemorheology” (Proc. First Intern. Conf.), p. 91, Pergamon, New York. Katchalsky, A. ( 1967). Membrane Thermodynamics. “The Neurosciences, A Study Program” (G. C. Quarton, T. Melnechuk, and F. 0. Schmitt, eds.), p. 326. The Rockefeller Univ. Press, New York. Katchalsky, A., and Spangler, R. (1968). Dynamics of Membrane Processes. Quart. Rev. Biophys. 1, 127. Katchalsky, A. ( 1968). Thermodynamic Treatment of Membrane Transport. Pure Appl. Chem. 16,229. Katchalsky, A. (1968). Thermodynamic Consideration of Biological Membranes. “Membrane Models and the Formation of Biological Membranes” (L. Bolis and B. A. Pethica, eds.), p. 318. North-Holland, Amsterdam. Blumenthal, R., and Katchalsky, A. (1969). The Effect of the Carrier AssociationDissociation Rate on Membrane Permeation. Biochim. Biophys. Acta 173, 367. Katchalsky, A. (1969). Membrane Thermodynamics. “Membranes $. Permeabilite SBlective,” p. 19. Editions du Centre National de la Recherche Scientifique, Paris. Katchalsky, A. ( 1969). Non-equilibrium Thermodynamics of Bio-Membrane Processes. “Theoretical Physics and Biology” (M. Marois, ed.), p. 188. North-Holland, Amsterdam. Katchalsky, A., and Oster, G. (1969). Chemico-Diffusional Coupling in Biomembranes. xxi

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AHARON KATZIR-KATCHALSKY, 1 9 1 3-1 972

“The Molecular Basis of Membrane Function” (D. C. Tosteson, ed.), p. 1. PrenticeHall, Englewood Cliffs. Katchalsky, A. (1970). Thermodynamic Consideration of Active Transport. “Permeability and Function of Biological Membranes” (L. Bolis, A. Katchalsky, R. 1). Keynes, W. R. Loewenstein, and B. A. Pethica, eds.), p. 20. North-Holland, Amsterdam. Katchalsky, A. (1971). Thermodynamics of Flow and Biological Organization. ZYGON: J . Relig. Sci. 6, 99. Katchalsky, A. (1971). Biological Flow Structures and Their Relation to ChemicoDiffusional Coupling. Neurosci. Res. Prog. Bull. 9, 397. Oster, G., Perelson, A., and Katchalsky, A. (1971). Network Thermodynamics. Nature (London) 234, 393. Katchalsky, A., and Neumann, E. (1972). Hysteresis and Molecular Memory Record. Int. J . Neurosci. 3, 175. Oster, G. F., Perelson, A. S., and Katchalsky, A. (1973). Thermodynamics of Biological Networks. Quart. Rev. Biophys. 6, 1.

The Genetic Control of Membrane Transport CAROLYN W. SLAYMAN Departments of Human Genetics. Microbiology. and Physiology. Yale Univwsily School of Medicine. New Haven. Connecticut

. .

Introduction . . . . . . . . . . . . . . . . . . Isolation of Transport Mutants . . . . . . . . . . . . A Resistance t o Analogs . . . . . . . . . . . . . . B Selection of Nongrowing Cells . . . . . . . . . . . C Direct Screening for Uptake . . . . . . . . . . . . D . Recognition of Transport Defects in Higher Organisms . . . . E . The Strategy of Recovering Transport Mutants . . . . . . F. The Problem of Multiple Transport Systems for a Single Substrate . I11. Criteria for Identifying Genes that Affect Transport Directly . . . . IV Linkage Relationships of Transport Mutants . . . . . . . . A . Escherichia coliand Salmonella typhimurium . . . . . . . B . Neurospora crassa . . . . . . . . . . . . . . . V Dominance and Recessiveness . . . . . . . . . . . . . VI . Regulation of Transport Systems . . . . . . . . . . . . VII . Usefulness of Mutants in Understanding Transport Mechanisms . . . A . In Determining the Number of Separate Transport Systems for a Particular Substrate . . . . . . . . . . . . . . B In Identifying the Components of a Transport System . . . . C . In Determining the Functionof a Component . . . . . . . VIII . Conclusion . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . .

I I1

. . .

.

.

.

1 3 3 129 132 133 133 134 136 140 140 144 146 150 151

151 152 152 155 155

1. INTRODUCTION

The genetic approach to the study of membrane transport can be an extremely useful one. By isolating and mapping mutants defective in the transport of a particular substrate. it is possible to determine the number of systems involved and even the number of subunits in each system; by analyzing the kinetics and the biochemistry of transport in the mutants. 1

2

CAROLYN W. SLAYMAN

one can often obt,ain information about molecular mechanisms. Progress in both of these areas is discussed in this article, together with some of the problems that remain. Membrane genetics is still a relatively young field. The first realization that transport systems, like simple cytoplasmic enzymes, are under genetic control came in the 1950s with work on cystinuria in man and lactose transport in Escherichia coli. Cystinuria had long been known to be an inherited disease, and in fact was one of the “inborn errors of metabolism” (together with pentosuria, alkaptonuria, and albinism) discussed by Archibald Garrod in his famous Croonian lectures in 1908. By 1951 cystinuria had been shown to involve the excretion of abnormal amounts of arginine, lysine, and ornithinc, as well as cystine. The structural relationship among this group of amino acids led Dent and Rose (1951) to postulate that the disease was caused by a primary defect in transport across the renal tubules. Since that time cystinuria has been found to affect the intestinal mucosa as well, and the transport defect has been well characterized i n vitro in intestinal tissue obtained by biopsy (Thier et al., 1964, 1965; McCarthy et al., 1964). In addition, other inherited diseases have been shown to lead to transport defects in man (for example, Hartnup disease, iminoglycinuria, methionine malabsorption, tryptophan malabsorption, X-linked hypophosphatemia), and the idea that all mammalian transport systems are under genetic control-even though mutations may often be lethal or otherwise undetectable-is now widely accepted. The genetic analysis of transport in microorganisms also dates to the 1950s. Although the earlier results of Davis and of Doudoroff had drawn attention to the cryptic nature of certain enzyme systems in bacteria (in which intact cells were unable to metabolize a given substrate even though cell-free extracts contained the necessary enzymes), it was the work of Cohen and Rickenberg (1955) and Rickenberg et al. (1956) that first demonstrated the existence of a specific bacterial transport system. These investigators showed that the product of the lacy gene is required for the transport of p-galactosides in E . coli, and that its synthesis is regulated jointly with the synthesis of the enzyme 8-galactosidase. The kinetics of the transport system have since been examined in detail, both in wild-type E. coli and in lacy mutants, and more recently the l a c y gene product ([%I protein”) has been isolated by Fox and Kennedy (1965). In microorganisms, and particularly in E . coli, this initial work has been followed by the isolation of many different kinds of transport mutants, and the notion that transport systems are genetically determined has been amply documented. This article discusses tfhetechniques that have been developed to isolate

GENETIC CONTROL OF MEMBRANE TRANSPORT

3

and characterize transport mutants, the criteria for identifying genes that affect transport directly, the linkage relationships of transport mutants in several organisms (E. coli, Salmonella typhimurium, Neurospora crassa), the regulation of synthesis of transport systems, and the usefulness of mutants in understanding transport mechanisms. I n .order to simplify the discussion, basic information about the properties of existing transport mutants has been collected in Table I, leaving the text free to consider the general topics just listed. [For further information on microbial transport systems, the reader is referred to recent reviews by Heppel (1971), Kaback (1970a, b, 1972), Lin (1970, 1971), Oxender (1972a, b), Roseman (1972), and Simoni (1972); and for a more complete description of genetic defects affecting transport in man, to reviews by Rosenberg (1969), Rosenberg and Scriver (1969), Scriver (1969), Thier and Alpers (1969), Scriver and Hechtman (1970), and to several chapters in Stanbury et al. (1972).] II. ISOLATION OF TRANSPORT MUTANTS

As interest in the genetic analysis of transport has increased, a variety of methods havc been developed for the isolation of transport mutants, particularly in microorganisms. Some are selection methods, making use of conditions under which the transport mutant can grow but the wild-type cell cannot (or the mutant survives and the wild type is killed); others are merely screening methods, permitting the rapid identification of transport mutants among large numbcrs of wild-type cells. The particular strategy to be used in a given instance depends, as discussed below, on the function of the transport system in question and on whether or not there are alternate routes for the substrate to enter the cell. A. Resistance to Analogs

One of the most powerful methods involves selecting mutants whose growth is resistant to an appropriate analog. This approach has been particularly successful in the isolation of amino acid, purine, and pyrimidine transport mutants (Table 11), but has also been used for carbohydrate, cation, and anion transport mutants. Cclls can, of course. become resistant to analogs through several kinds of mutations-not only those reducing the uptake of the analog, but very frequently those affecting later steps in metabolism (see review by Umbarger, 1971). In studying the effects of D-cycloserine (an analog of alanine) in Streptococcus strain Challis, for example, Reitz et al. (1967) found two classes of resistant mutants. The first possessed a defective

TABLE I Mutations Affecting Membrane Transport Amino Acids and Peptides Organism

Transport System

Specificity

Escherichia coli

Aspartate

A constitutive, high-affinity transport system which is fairly specific f o r L-aspartate (K, = 3.7 x 10-6M I . Ki's for D-aspartate, L-glutamate, L-glutamine, and a series o f aspartate analogs (N-formyl-L-aspartate, N-methyl-DL-aspartate, a-methyl-DL-aspartate, p-methyl-DL-aspartate, DL-eryfhro-p-hydroxyespartate, and DL-fhreo-p-hydroxyaspartate) are substantially higher, ranging f r o m 1.8 t o 8.4 x lo4 M (Kay, 1971 ).

asf

C4 Dicarboxylic acids (aspartate, fumarate, malate, succinate).

A n inducible, low-affinity system which takes u p aspartate, fumarate, malate, maleate, and succinate w i t h Km's o f 10 t o 30 x 10-6M. (Kay and Kornberg, 1971;Loefal., 1972).

dct

Glutamate

A transport system, partially repressed in wild-type E. coli (Marcus and Halpern, 1969).which is fairly specific for L-glutamate (K, = 7.7 x 10-6M; Halpern and Even-Shoshan, 1967).Competitively inhibited by D-glutamate, L-glutamine, and several glutamate derivatives (L-glutamate-y-methyl and -y-ethyl esters, p-hydroxy-DL-glutamate, a-methyl -DL-glutamate) w i t h Ki's o f 2.10 x 10-5 t o 3.35 x 10-3M (Halpern and Even-Shoshan, 1967).Shows complex kinetics w i t h nonlinear double reciprocal plots under some

4

Gene

glfC

Amino Acids and Peptides Linkage

Near xyl (Kay and Kornberg, 1969; Lo etal., 1972). May be allelic with the FMmutant of E. coli. isolated by its inability to grow on succinate and later shown to be deficient in the uptake of dicarboxylic acids and to map near x y l (Herbert and Guest, 1970).

Method of Isolating Mutants

Transport Defect in Mutants

Resistance to DL-rhreo-phydroxyaspartaw (in E. coli K-12; Kay, 1971). An unsuccessful attempt was made to isolate ast mutants from a dct strain (which lacks the general dicarboxylic acid transport system; see below) by selecting for the inability to use aspartate as sole nitrogen source; several mutants with this phenotype were found but proved t o transport aspartate as well as the parent strain (Kay, 1971).

Lack the high-affinity aspartate transfor aspartate port system. The V,,, uptake in one ast mutant, HA3, was decreased from 39 to 25 nmolesl minute per milligram dry weight, and and a single Km (30 x 1 0 6 MI was observed, compared with two Km's (39 and 3.7 x 10-6M) in the wildtype. The defect is also seen in isolated membrane vesicles from ast mutants (Kay, 1971).

Resistance to 3-fluoromalate (Kay and Kornberg, 1969; Lo era/., 1972) or L(-)-tartrate (Kay and Kornberg, 1971). dct mutants are also unable to grow on malate, succinate, or fumarate as sole carbon source (Kay and Kornberg, 1969, 1971). Revertants, selected for the ability t o grow on any one of the C4 dicarboxylic acids, have simultaneously recovered the ability to grow on a l l the other acids (Kay and Kornberg, 1969,1971).

Lack tho low-affinity dicarboxylic acid uptake system. I n one dct for aspartate mutant, F M l , the V,, uptake was decreased to 1.5 inmolesl minute per milligram dry weight, and there was a single K, of 3.5 x 10-6M (Kay, 1971).

dct mutants must be distinguished from strains with a primary block in the tricarboxylic acid cycle; such strains often showa secondary impairment of the ability to take up one or more C4 acids (Kay and Kornberg, 1971). Between ma and pyrE (Marcus and

Halpern, 1969).

Isolated, in wild-type E. coli W, H,or K-12 (site), by the ability to grow o n glutamate as carbon source (Halpern and Umbarger, 1961; Halpern and Lupo, 1965; Marcus and Halpern, 1967).g/tcC mutants also show increased sensitivity to 2methyl-DLglutamate (Halpernand Umbarger, 1961).

gltc is believed to be the operator for gltS (see below).gltC mutants show quantitative but not qualitative alterations in glutamate transport; in a series of such strains the V,, was increased by a factor of 2 to 7, while the K,,, was unchanged (Halpern and Lupo, 1965; Marcus and Halpern, 1969). Furthermore,gltC andgltS are very closely linked, and gltC mutants

(Con tinued) 5

TABLE I Mutations Affecting Membrane Transport (Continued) Amino Acids and Peptides Oraanism

TransDort Svstem

Escherichia coli IC0n't.l

SDecificitv

Gene

conditions; interpreted in terms of an allosteric model in which succinate, aspartate, a-ketoglutarate, yaminobutyrate, or glutamate can activate transport by binding a t a second site (Halpern and EvenShoshan,

1967). A glutamate-binding protein, released from g l C mutants during spheroplast formation, is thought to be involved in glutamate transport. It has a KD of 6.7 x 1 0 6 M (close to the K, of the transport system), is inhibited competitively by L-glutamate-y-methyl ester and noncompetitively by alanine, and can restore the capacity of spheroplasts for glutamate uptake (Barash and Halpern, 1971).

g/fS

gltR

Glutamine

A highly specific transport system for glutamine (K, = 0.8 x l o 7 M ) ,competitively inhibited by y-glutamylhydrazide and y-glutamylhydroxamate but not by any naturally occurring amino acids. Transport is thought to involve a glutamine-binding protein, which is released by osmotic shock and has a K D (for glutamine) of 3 x M. Both transport and the binding protein are repressed by growth in a rich medium (Weineret a/., 1971; Weiner and Heppel,

-

1971).

-

6

Amino Acids and Peptides Linkage

Method of Isolating Mutants

Transport Defect in Mutants are derepressed (with high levels of glutamate transport activity) even in the presence of a normal g/tR gene (the postulated repressor gene; see below). The kinetics of transport in gitCC strains are discussed further in Halpern (19671. Halpern and EvenShoshan (1967). and Frank and Hopkins (1969).

Closely linked to g/tC (see above; Marcus and Halpern, 1969).

Isolated, from agltCc parent strain (see above), by loss of the ability to grow on glutamate (Marcus and Halpern, 19691. Might also be isolated from gltCc by resistance to 2methyl-D L-glutamate (Halpern and Umbarger, 1961; Halpern and Lupo, 1965).

g/tS i s thought to be the structural gene for the glutamate transport system.g/rS mutants with qualitatively altered transport systems have been isolated; in strain CS 7/50, for e x ample, the Vmax was decreased by a factor of 3,and the K,,, was increased by a factor of 20 (from 5 x 10.6 M to 1 x 1 0 4 M ) (Marcusand Halpern, 1969). No mutants with altered allosteric properties (see Specificity) have yet been reported.

Near met.4; not linked to gltc and g/tS (Marcus and Halpern, 1969).

Temperature-sensitive g/tR mutants were isolated, from wild-type E. coli, by their ability to grow on glutamate at 42' but not a t 30" (Marcus and Halpern, 1969).

g/tR i s thought to be the repressor gene for the glutamate transport operon. In one temperature-sensitive g/tR mutant (CS ZTC), the V,,, of transport a t 42' was increased by a factor of 4.5, with no change in Km. Furthermore, brief periods of heating a t 44' in the absence of growth increased the differential rate of synthesis of glutamate transport activity during subsequent growth a t 30°, suggesting that the repressor is thermolabile in this mutant (Marcus and Halpern, 1969).

Ability to grow on glutamine as sole carbon source (Weiner e t al.. 1971; Weiner and Heppel, 1971).

Increased transport of glutamine. Mutant strain GLNP 1 showed a 3-fold higher initial uptake rate (with a normal Km) and had three times more binding protein than the parent strain (Weiner and Heppel, 1971).

Resistance to y-glutamylhydrazide (Weiner and Heppel, 1971).

Decreased transport of glutamine. In mutant strain GH 20, both the initial uptake rate and the amount of binding protein were decreased by about 90% (and the small amount of uptake

7

(Continued)

TABLE I Mutations Affecting Membrane Transport (Continued) Amino Acids and Peptides Organism

Transport System

Specificity

Gene

Escherichia

coli (Con%)

Basic amino acids (arginine, lysine, ornithine)

argf Wild-type E. coli K-12 has three kinetically distinct transport systems for basic amino acids (Rosen, 1971a):

(1) a specific, high-affinity system for arginine (K,,, = 2.6 x lo-* M ) ; (2) a specific system for lysine (K, = 1 x 10-5 M ) , inhibited by thiosine; and

(3)a general system (LAO) for lysine (K, = 0.5 x 10-6 M ) , ornithine (K,,, = 1.4 x 10-6 M ) ,arginine, and canavanine. Osmotic shock causes the release of several argininebinding proteins, one of which may play a role i n the arginine-specific transport system (Wilson and Holden, 1969a, b; Rosen, 1971a). and i n addition a lysinearginine-ornithine-binding protein (LAO), which appears to be associated with the general transport system. The L A O protein has a molecular weight of 30,000 and KD's of 3.0 x 10-6 M for lysine, 1.5 x 1 0 - 6 M for arginine, and 5.0 x 10'6M for ornithine (Rosen, 1971a). The lysine-specific system i s not affected by osmotic shock under the usual conditions. I n cells in which the general system has been repressed, however, lysine transport becomes sensitive to osmotic shock and a lysine-binding (LS) protein i s released. This protein is labile at 4" and is rapidly inactivated at ionic strengths above 0.02 (Rosen, 1971b).

8

Amino Acids and Peptides Linkage

Method o f Isolating Mutants

Transport Defect i n Mutants that remained may have been mediated by another system, since it was inhibited by glutamate). The genetic relationship between strains GLNP 1 and GH 20 has not yet been established, but the quantitative correspondence b t w e e n uptake rates and amounts o f binding protein in the two strains has been used to support the idea that binding i s involved in transport (Weiner e r a / . , 1971; Weiner and Heppel, 1971 ).

Near serA (Taylor, 1970).

Resistance t o canavanine (€. coli W, Schwartz e t a l . , 1959; E. coli K - I 2, Maas, 1965; Rosen, 1971a).

Canavanine-resistant ( a r g f ) mutants are generally defective in the transport of all three basic amino acids (Scwartz e r a / . , 1959; Maas, 1965; Rosen, 1971a; Maas. cited i n Rosen, 1971a), b u t the exact function o f the argP locus i s not clear. Rosen (1971a) reported, in one such mutant (Can R22). that two transport systems were affected; arginine transport was completely missing, and ornithine and lysine transport via the L A O system were partially reduced. Both the arginine-specific binding protein and the LAO-binding protein have now been found t o be present i n Can R22, and the mutant shows normal facilitated diffusion of arginine (assayed by coupling arginine uptake to arginine decarboxylase, and measuring COP production) (Rosen. personal communication). Rosen has therefore postulated that the lesion i s in energy coupling, presumably in a factor common t o both the arginine-specific and L A O transport systems. However, Maas (personal cornmunication) found that another argP mutant, JC 182-5, produces an altered LAObinding protein with a lowered binding capacity for arginine, ornithine, and lysine; the arginine-specific binding proteins appear normal i n JC 182-5. Further work will be

9

(Continued)

TABLE I Mutations Affecting Membrane Transport Konrinuedl Amino Acids and Peptidm Organism

Transport System

Specificity

Gene

Eschsrichie coli ICon'rJ

Aromatic amino acids (tryptophan, tyrosine, phenylalanine)

Wild-type E. coli K-12 has at least f i w transport systems for the aromatic amino acids (Brown, 19701:

aroP

(1) a general aromatic system for tryptophan (Km = 4.0 x lO-7M). tyrosine (K, = 5.7 x l o 7 M1,and phenylalanine (K,,, = 4.7 x 10-7 M I, with much lower affinities for several other amino acids (histidine, leucine, methionine, alanine, cysteine, and aspertate). The general system is inhibited by p-fluorophenylalanine, @-2-thienylalanine,and 5-methyltryptophan; (2) a specific system for tyrosine (K,,, = 2.2 x 1 0 - 6 ~ ) . inhibited by p-fluorophenylalanine (and sewral other analogs) and b y high concentrations of phenylelanine;

(3)a specific system for phenylalanine (K,,,

= 2.0 x

10-6 M I , similarly inhibited by p-fluorophenylalanine (and several other analogs) and by high concentrations of tyrosine; (41 a specific system for tryptophan (K, = 3.0 x 10-6 M I , inhibited by 4methyltryptophan (and other analogs); and

(5) an inducible system for tryptophan (Boezi and DaMoss, 1961 ; Burrous and DeMoss, 1963).

10

trpf

Amino Acids and Peptides Linkage

Method of Isolating Mutants

Transport Defect in Mutants required to identify the primary effect of the argP gene on the LAO system (whether on binding, energy coupling, or both), and t o learn why arginine-specific transport i s affected in some argP mutants (Can R22) but apparently not in others (JC 182-5).

Between leu and pan (Brown, 1970).

Resistance to thiosine (Rosen, personal communication).

Lack the specific transport system for lysine but contain the lysinebinding protein (Rosen, personal communication).

Resistance to p-2-thienylalanine (E. coli K-12; Brown, 1970). The aroP mutants were also resistant to p-fluorophenylalanine and 5-rnethyltryptophan, and additional aromatic transport mutants might be isolated using these analogs.

Lack the general aromatic transport system (Brown, 1970).aroP mutants retain the specific tryptophan, tyrosine, and phenylalanine systems, with K;s similar to those of the wild-type strain but with somewhat lower V,,,'s. This latter finding, if significant, may mean that the general and specific transport systems share the component determined by the gene aroP (Brown. 19711.

Isolated in an aroP mutant of

Not discussed.

E. coli K-12 strain W31 10, which has a high level of the tryptophan-specific transport system, by resistance to 4methyltryptophan (Yanofsky, cited in Oxender, 1972a). Between trpA and ton6 (Thorne and Corwin, 1970).

Resistance to indole acrylic acid (Thorne and Corwin, 1970).

Two lines of evidence have led to the view that a gene for tryptophan transport is located between trpA and ton6 (Thorne and Corwin, 1970, 1971): (1) Deletions in this region cause a 10-fold decrease in tryptophan uptake, and ( 2 ) low uptake in point mutants, selected by resistance to indole acrylic acid, can be restored to normal by introduction of the F'trp episome. However, the uptake of other amino acids was not measured in these experiments, and the results might be explained by the observation that deletions extending into the ton6

11

(Continued)

TABLE I Mutations Affecting Membrane Transport (Continued) Amino Acids and Peptides Organism

Transport System

Specificity

Gene

Eschen'chia coli IConW

Histidine

Not characterized.

Glycine. alanine, wine

Wild-type E. coli K-12 has t w o (or three) kinetically distinct transport systems for glycine. atanine, and serine (Wargel etal., 1970; Qxender, 1972a):

-

cyc

(1) a system for L-alanine (Km = 5.7 x 10-5 M ) and probably L-serine. inhibited by L-cycloserine and 0-carbamyl-Dserine; and

(2) and (3?)a system for glycine, D- and L-alanine. and D-serine. with nonlinear double reciprocal plots which have been resolved into t w o sets of Km's and VmaX's (Km's = 2.5 and 9.1 x 10-5 M for glycine, and 2.5 and 8.2 x 1 0 %M for D-alanine). This system is inhibited by D-cycloserine.

Leucine, isoleucine, valine

There appear t o be multiple transport systems for the branched-chain amino acids i n E, coli, but the kinetic picture i s not yet clear. Guardiola and laccarino (1971) reported a single set of Km's for laucine, isoleucine, and valine (7 x 10-6 M, 4.8 x l o 6 M and 12 x l o 6 M, respectively); but Piperno and Oxender (19681observed two K,'s for valine (0.7 and 8 x 1 0 6 MI, Furlong and Weiner (1970) observed t w o Km'S for leucine (0.2 and 2 x 10-6 M I, and more recently 12

brnP brnQ

Amino Acids and Poptades Linkage

Method of Isolating Mutants

Transport Defect i n Mutants region lead t o decreased transport of a variety of amino acids, as i f tun8 influences general membrane permeability (Yanofsky, cited in Oxender, 1972a).

c y c r l (cycA) maps near purA (Russell. 1972); and cyc'2 and cycr3. which are cotransducible with one another, map at least 0.5 minute away (Curtiss et a/., 1965).

Penicillin treatment of a histidine-requiring strain i n low-histidine medium I€.coli W; Lubin eta/., 1960).

Defective i n the uptake of histidine (Lubin e t a / . , 1960).

Resistance t o D-cycloserine; three successive steps in resistance (cycrl, cycr2. and cycr3) have been identified in €. coli K-12 by Curtiss ot a/. (1965). Other investigators have selected by means of resistance to D-cycloserine (E. colt W, Kessel and Lubin, 1965; Wargel et al., 1971) or Dw i n e (E. coli W, Davis and Maas. 1949; Kessel and Lubin, 1965: E. coli K-12, Cosloy and McFall. 1971; Oxender, 1972a), or by penicillin treatment of glycine-requiring strains I€.culi W and B) growing in lowglycine medium (Kessel and Lubin. 1965). The relationship of these mutants t o the cyc loci has not been renorted

Defective in the transport of glycine. D- and L-alanine, and Dderine (Schwartz et a/., 1959; Levine and Simmonds, 1960. 1962; Lubin era!., 1960; Kessel and Lubin. 1965; Kaback and Kostellow. 1968; Kaback and Stadtman, 1968; Wargel et at.. 1971; Oxender, 1972a). Thecycrl strains lack the high-affinity component of transport, and the cycr2 and cycr3 strains lack the lowaffinity component (Wargel er &., 1971).

Isolated, i n a Dserine-resistant mutant that lacked the glycine. D- and L-alanine. D-serine systemls), by penicillin salection on L-alanine (Oxender, 1972a).

brnP maps near leu, and bmQ near phoA (Guardiola and laccarino, 1971).

Resistance t o valine (Guardiola and laccarino, 19711. The growth of wild-type E. coli K-12 i s inhibited by valine, which blocks isoleucine biosynthesis through feedback inhibition o f acetolactate synthetase (Leavitt and Umbarger. 1962). I n addition t o the

13

The double mutant has lost about

95%of the L-alanine transport capacity. and is assumed t o lack the

L-alanine. L-serina system as well as as the glycine. D- and L-alanine, D-serine system (Oxender, 1972al. brnP mutant M I 183a showsd abnormal Km's for isoleucine ( 3 6 x 106M.comparedwith4.8 x 1 0 - 6 M in the parent strain) and for leucine (0.4 and 7 x 1 0 6 M, respectively), although not for valine (12 and 12.5 x 1 0 8 M ) ; Guardiola and laccarino 11971) concluded that it might h a w a qualitatively altered transport sys-

(Continued)

TABLE I Mutations Affecting Membrane Transport (Continued) Amino Acids and Peptides Organism

Escherichia coli (Con't.)

Transport System

Specificity

Gene

Guardiola, De Felice, and laccarino (oersonal communication) detected four systems for the branchedchain amino acids, a shared system with K,'s of about 2 x 10.6 M for leucine, isoleucine, and valine, and specific systems for each of the three amino M. acids with Km's of about 50 x At least two binding proteins have been isolated. One has comparable affinities for leucine, isoleucine, and valine, and presumably is associated with the shared LIV transport system. It has a molecular weight of about 36,000, and both i t and LIV transport are repressed when leucine is present in the growth medium (Piperno and Oxender, 1968; Anraku, 1968a, b, c; Nakane eta/., 1968; Penrose eta/., 1968, 19701. The second protein binds only leucine. It is remarkably similar to the first in molecular weight, amino acid composition. and K D for leucine; it, like the first, is repressed by leucine; and the two proteins cross-react immunologically, suggesting a common developmental origin (Furlong and Weiner, 1970; Furlong, personal communication I .

dlu

14

A m i n o Acids and Peptides Linkage

Method of Isolating Mutants valine-resistant mutants defective in transport, others have been described that possess either a valine-resistant acetolactate synthetase or an increased rate o f isoleucine biosynthesis (Glover, 1962; Ramakrishnan and Adelberg, 1964,1965).

Transport Defect in Mutants t e m for the branched-chain amino acids, and thus that brnP might be the structural gene f o r this transport system. By contrast,brnO m u t a n t M I 1 7 4 b f o r isoshowed decreased V,,,'s leucine, valine, and leucine (by a factor of 5 t o 10). and it was suggested that brnO might be a regulatory gene (Guardiola and laccarino, 1971). With the discovery o f multiple transport systems f o r leucine, isoleucine, and valine (see Specificity), however, the kinetic analysis o f b r n P a n d brnO mutants w i l l have to be carried o u t in greater detail before one can conclude which system o r systems are altered b y these mutations and which are unaffected. Recently Guardiola, De Felice, and laccarino (personal communication) measured the amounts and the dissociation constants of b o t h the L I V and the leucine-binding proteins in 6rnP and b r n 0 mutants, and have found the proteins to be normal. They conclude tentatively that brnP and brn0 d o n o t code f o r either binding protein.

Isolated, in a leucine-requiring strain, b y the ability t o use D-leucine as a source o f Lleucine (Rahmanian and Oxender, 1972a.b).

Increased transport o f D- and Lleucine, isoleucine, and valine via the shared system, and increased amounts o f the LIV-binding protein. dlu is concluded t o be a regulatory gene f o r the L I V system (Rahmanian and Oxender, 1972b).

Isolated, in a dlu parent strain (see above), by resistance t o azaleucine (Rahmanian and Oxender, 1972b).

One class o f azaleucine-resistant mutants lacks L I V transport activity b u t retains the LIV-binding protein; another class shows reduced transport activity f o r branched+hain amino acids and also f o r other, unrelated amino acids (Rahmanian and Oxender, 1972b).

15

(Continued)

TABLE I Mutations Affecting Membrane Transport (Continued Amino Acids and Peptides Organism

Transport System

Escherichia

Cystine ,

co/i (Con%) diaminopimelic acid

Specificity Wild-type E. coli W has two transport systems for

Gene

-

cystine: (1) a general system (K,,, = 3 x lo-' M ) , inhibited by diaminopimelic acid (DAP), and (2) a specific system (K, = 2 x 1 0 8 M ) ,not inhibited by DAP. The activity of the general system is reduced by osmotic shock, and a cystine- and DAP-binding protein (molecular weight 28,000; K D for cystine = 2 x l o 7 M ) has been partially purified from the shock fluid (BergereraL, 1971).

Proline

Wild-type E. coli takes up proline with a K,,, of 6.4 x 10-7 M. Uptake is inhibited by azetidine-2-carboxylic acid and 3.4-dehydroproline (with Ki's of 2.4 x 10-5 M, and 2.6 x 1 0 6 M, respectively) and by several other proline analogs (Tristram and Neale, 1968). A prolinebinding activity has recently been partially purified from membrane vesicles (Gordon era/., 1972).

-

Dipeptides

A transport system for dipeptides containing two L-amino acids or glycine plus one L-amino acid (Kessel and Lubin, 1963;Sussman and Gilvarg, 1971).

-

Oligopeptides

A transport system for oligopeptides containing lysine, omithine, glycine, tyrosine, and perhaps other amino acids (Payne, 1968;Sussman & Gilvarg, 1971).

-

16

Amino Acids and Peptidm Linkage

Method of Isolating Mutants

Transport Defect in Mutants

Diaminopimelic acid-requiring mutants of E. coli W normally grow slowli with DAP as sole supplement, and require lysine, i n addition, for normal growth; "D" mutants were isolated which had lost the partial requirement for lysine (Leive and Davis. 19651.

Lack the specific cystine transport system (Berger et a/., 1971).

Penicillin treatment of a proline-requiring strain of €. coli W or B in low-proline medium (Lubin etal., 1960; Kessel and Lubin, 1962).

Defective in proline transport, both in intact cells (Kessel and Lubin, 1962) and in isolated membrane vesicles (Kaback and Stadtman, 1966; Kaback and Deuel, 1969). Normal, however, in the passive diffusion of proline across the membrane (Kessel and Lubin, 1962; Kaback and Stadtman, 1966).

Resistance t o 3.4-dehydroproline or L-azetidine-2-carboxylic acid ( E . coli strain C4; Tristram and Neale, 1968). Al I dehydroprol ine-resistant strains tested showed crossresistance t o azetidine, b u t by contrast the azetidine-resistant strains (14 tested) were sensitive t o dehydroproline.

Some of the dehydroproline-resistant mutants were defective i n proline uptake; others showed normal uptake b u t excreted large quantities of proline (Tristram and Neale, 1968). Most azetidine-resistant mutants were reduced i n proline uptake. One mutant was of particular interest because i t s transport system had normal affinities for proline and dehydroproline but a 10-fold reduced affinity for azetidine. In a few azetidine-resistant mutants, proline uptake was normal, proline was not excreted in large quantities, and the mechanism o f resistance is unknown (Tristram and Neale, 19681. The genetic relationship among the various mutants has not yet been determined.

Penicillin treatment of a glyauxotroph of E. col; W o n glycylglycine medium (Kessel and Lubin, 19631.

Defective uptake of glycylglycine and other dipeptides (Kessel and Lubin, 1963).

Resistance t o triornithine

Indirect evidence (from growth experiments) for a defect i n the transport of oligopeptides (Payne and Gilvarg, 1968; Payne, 1968).

(E. coli W; Payne and Gilvarg, 1968; Payne, 1968).

17

(Continued)

TABLE I Mutations Affecting Membrane Transport (Continued) Amino Acids and Peptides Gene

Organism

Transport System

Specificity

Salmonella typhimurium

Aromatic amino Wild-type S. ryphimurium has at least six transport aroP acids (tryptosystems for the aromatic amino acids (Ames, 1964): phan, tyrosine, ( 1 ) a nonspecific aromatic system which transports phenyla'anine'tryptophan (K, = 5 x 10-7 M),tyrosine (Km = 2 x 10-6 histidine) M),phenylalanine (Km = 5.9 x l o 7 M),and histidine (K, = 1.1 x lo4 M).Inhibited by 2-methylhistidine, 3-pyrazolealanine, 2-thiazolealanine, p-fluorophenylalanine,5-methyltryptophan, p-2-thienylalanine, p-3-furylalanine, azaserine, and the phosphonate derivatives of phenylalanine and tyrosine (Ames, 1964; Ames and Roth, 1968); (2) and (3) at least two specific histidine systems, designated J-P and K-P. with K,'s of M and l o 7 M ,respectively (Arnes and Lever, 1970);

(4)a specific tryptophan system (Ames, 1964); (5)a specific tyrosine system, inhibited b y p-fluorophenylalanine and by high concentrations of phenylalanine (Ames, 1964);and

(6)a specific phenylalanine system ( K , = 2 x which is also inhibited by p-fluorophenylalanine (Ames, 1964).

M),

Recently reviewed by Ames (1974a.b)

hisP

18

~~

Amino Acids and Peptides Linkage Near p r o A (Ames and Roth, 1968).

Method o f Isolating Mutants Resistance t o azaserine (Ames, 1964) or t o tyrosine or phenyl. alanine phosphonate derivatives (Ames and Roth, 1968). Mutants resistant to p-fluorophenylalanine and 5-methyltryptophan were also isolated, but turned out to be phenylalanine excretors rather than transport mutants (Ames,

1964).

Transport Defect i n Mutants Lack the general aromatic transport system. aroP mutants are not very defective in the uptake o f tryptophan, tyrosine, or phenylalanine (since these amino acids are also transported b y the specific systems), but do show greatly reduced uptake of p-fluorophenylalanine. Control experiments, i n which aroP culture supernatant was found t o have no effect on uptake by wild-type cells, ruled out the possibility that the primary defect in aroP was the excretion of some compound that inhibited FPA uptake (Ames, 1964).

An attempt t o isolate specific phenylalanine transport mutants by resistance t o p-fluorophenylalanine (FPA) in the presence of tyrosine (to inhibit the general aromatic transport system) was n o t successful; the mutants that were isolated had normal rates of FPA uptake (Ames,

1964). Near p u r F (Ames and Roth, 1968). Closely linked t o hisJ and dhuA (Ames and Lever, 1970).

Resistance t o 2-hydrazino-3(4-imidazolyl) propionic acid (HIPA) (Shifrin eta/., 1966; Ames and Roth, 1968).

Lack the two specific histidine transport systems, J-P and K-P. Only a small amount of residual histidine transport remains i n hisP mutants, with a K, of about 10-6 M; this residual transport occurs via the general aromatic system and at least two additional low-affinity systems; (Ames and Lever, 1970). Evidence t o be described below indicates that the hisP gene product, known to be a protein because o f the existence of amber mutants (Ames and Roth, 1968). interacts with two binding proteins-J and K-to constitute the specific histidine transport systems. The amount o f the binding proteins is not altered i n hisP mutants (Ames and Lever, 1970).

19

(Contimed)

TABLE I Mutations Affecting Membrane Transport (Continuedl Amino Acids and Peptides Organism

Transport System

Specificity

Gene hisJ

Salmonella typhimorium (Con%I

dhuA

20

Amino Acids and Peptides Linkage

Method of Isolating Mutants

Transport Defect i n Mutants

See hisP

Isolated, from a dhuA hisparent strain (see below), b y loss of the ability t o grow on D-histidine while s t i l l retaining sensitivity t o HlPA (Ames and Lever, 1970).

Lack the J-binding protein, which binds D- and L-histidine and HI PA, and interacts with the P protein t o constitute one mode of specific histidine transport (Ames and Lever, 1970). hisJ mutants retain the K-P system, and can transport histidine with a K,, of about 2 x 10-7 M. Evidence that hisJ is i n fact the structural gene for the J-binding protein has come from the study of a strain that contains two mutations in hisJ, both induced by the frameshift mutagen ICR191; the initial mutation caused loss of both the J-P mode of transport and the J protein, and the second mutation caused partial restoration of both. In this strain the J protein is abnormal-it is temperature-sensitive and has altered chromatographic and electrophoretic properties, Concomitantly, histidine transport via the J P system has become temperature-sensitive, reinforcing the idea that the J protein is an obligatory component of the transport system, as well as indicating that hisJ is i t s structural gene (Ames and Lever, 1972). A detailed biochemical characterization of the J protein has recently been reported (Lever, 1972).

See hisP.

Isolated, from a his- auxotroph, by the ability t o use D-histidine as a source of L-histidine (KrajewskaGrynkiewicz eta/., 19711.

Shows a 2- to 3-fold increase i n the transport o f L-histidine, a significant transport o f D-histidine (not seen in the parent strain), and an increased sensitivity t o HlPA (KrajewskaGrynkiewicz eta/., 1971; Ames and Lever, 1970). In addition,dhuA mutants contain a 4- to 5-fold increased level of J-binding protein (which has the same chromatographic properties and binding constant as the protein from wild type) (Ames and Lever, 1970). All these results are consistent with the idea that dhuA i s a regulatory gene for hisJ.

21

(Continued)

TABLE I Mutations Affecting Membrane Transport (Continued) Amino Acids and Peptides Oraanism

TransDort Svstem

Salmonella typhimurium (Con 't.I

Tryptophan

Pseudomona aeruginosa

Specificity

Gene -

Leucine

Not characterized (but note the complexity of the branched-chain amino acid transport systems in E. coli).

Methionine

Wild-type S. typhimurium has two transport systems for methionine, one with an affinity 50 times that of the other. Methionine transport is inhibited at least partially b y the analogs a-methylmethionine, ethionine, norleucine, and methionine sulfoxime, but it has not yet been reported whether these compounds block the high-affinity transport system, the lowaffinity system, or both (Ayling and Bridgeland, cited in Smith, 19711.

Proline

There is kinetic evidence for two proline transport systems in wild-type P. aeruginosa, one saturating below 1 x 10-6 M, and the other above 20 x M. The first, high-affinity system is quite specific for L-proline and is inhibited only by close analogs (thioproline, dehydroproline, L-azetidine-2-carboxylic acid); it is induced by growth in the presence o f proline (Kay and Gronlund, 1969b).

Aromatic amino acids (tryptophan, tyrosine, phenylalaninel

Preliminary evidence suggests that wild-type P. aeruginosa has at least two transport systems for the aromatic amino acids, differing in their relative affinities for tryptophan, tyrosine, and phenylalanine, and also in their sensitivity t o inhibitors (D-phenylalanine,p-fluorophenylalanine, 5- and 6-fluorotryptophan) (Kay and Gronlund, 19711.

22

-

metP

-

Amino Acids and Peptides Linkage Between trp and chr

Method of Isolating Mutants -

This region is thought t o include a gene that affects tryptophan transport (system not specified), since deletion mutants take u p greatly reduced amounts of tryptophan (Thorne and Corwin, 1970; but see discussion of similar mutants in E. cold.

(Thorne and Corwin, 1970).

Near chr, on the side distal t o trp (Thorne and Corwin, 1972).

Maps i n the leu-jwrE region (Ayling and Bridgeland, cited i n Smith, 1971).

Transport Defect in Mutants

Similarly, the trp-chr region is believed t o contain a gene affecting leucine transport, since deletions in this region lead t o a decrease in leucine uptake (Thorne and Corwin, 1972); from a comparison of several deletion mutants, it was concluded that the postulated leucine gene i s not the same as the postulated tryptophan gene. Resistance t o a-methylmethionine sulfoxime (Ayling and Bridgeland, cited in Smith, 1971).

Lack the high-affinity methionine transport system (Ayling and Bridgeland, cited in Smith, 1971).

Slow growth on proline as carbon source (Kay and Gronlund, 1969a).

Defective uptake of proline via the high-affinity system (Kay and Gronlund, 1969a.b).

Slow growth on tryptophan as carbon source (mutant strains TClO and TA3). or resistance t o 5-fluorotryptophan (mutant strain 5FT3). The genetic relationship among these three strains is not certain. 3 0 p fluorophenylalanine-resistant mutants were also isolated, but none was defective in aromatic amino acid transport (Kay and Gronlund, 1971).

Strain TClO was primarily defective in the uptake of tyrosine, strain 5FT3 in the uptake of tryptophan, and strain T A 3 in the uptake of both. I t was suggested that the mutations may have affected, respectively, the postulated tyrosine-preferring system, the postulated tryptophan-preferring system, and a common element of both systems (Kay and Gronlund, 1971). Since kinetic constants have not yet been reported f o r any of the mutants (or for the wild type),

23

(Continuedl

TABLE I Mutations Affecting Membrane Transport (Continued) Amino Acids and Peptides Organism

Transport System

Specificity

Gene

Aeudomonas aeruginosa /Con?.)

Pseudomonas fluorescens

Streptococcus faecalis

Arginine

Not characterized.

lsoleucine

Not characterized.

L-Alanine, P-alanine, L-proline

Wild-type P. fluorescens has a common transport system for L-alanine (K, = 1.4 x 1 0 4 M),palanine (Km '6-8 x 1 0 5 M ) , and L p r o l i n e (Km = 2.6 x 1 0 5 M),and in addition two specific transport systems for L-alanine (Km's = 2 and 8 x 10-6 M )end one for Lproline (Km = 5 x 10-6 M ) (Hechtmen and Scriver, 1970a.b).

-

S. faecalis is thought t o take up L-lysine and hydroxyL-lvsine via a specific transport system, and L-lysine, hydroxy-L-lysine, and L-arginine via a general system (Friede etal., 1972). The kinetic constants of the t w o systems have not yet been reported.

-

Wild-type S faecalis has two transport systems for acidic amino acids (Reid etal., 1970):

-

Lysine, arginine

Aspartate, glutamate

( 1 ) a high-affinity (HA) system which transports aspartate (K, = 1 x 10-5 MI, glutamate (K, = 3 x 10-5 MI, 2-amino-3phosphonopropionicacid, and a-methylglutamic acid; and (2)a low-affinity ( L A ) system which transports aspartate (K, = 8 x lo4 M )and glutamate (K, = 1.2 x M )and is inhibited by glutamine. 24

Amino Acids and Paptides Linkage

Method of Isolating Mutants

Transport Defect in Mutants however, this conclusion must remain tentative-particularly in view of the large number of aromatic amino acid transport systems known t o be present in other bacterial species (see E. coli. S. typhimurium).

Slow growth on arginine as carbon source (Kay and Gronlund, 1969~).

Defective uptake of arginine (Kay and Gronlund. 1969aJ.

Slow growth on isoleucine as carbon source (Kay and Gronlund, 1 9 6 9 ~ ) .

Defective uptake of isoleucine (Kay end Gronlund, 1969e).

Resistance t o 4-mathyltryptophan [Hechtman and Scriver, 1970a.b).

Greatly reduced uptake of p-alanina and partially reduced uptake of Lalanine and proline; attributed t o e defect in the common transport system for these three amino acids (Hechtman and Scriver, 1970a). A t low temperatures the mutant did show facilitated diffusion o f p-alanine. suggesting that the block may be in energy coupling rather than In the carrier (Hechtman and Scriver, 1970b).

Resistance t o h y d r o x y - l lysine (Friede etel., 1972).

The hydroxylysine-resistant straln was found t o take up lysine more slowly than the wild type, end is believed t o be defective in the specific lysine transport system. The results are complicated by the fact that the double reciprocal p l o t of lysine uptake is still curvilinear i n the mutant, however. and a detailed kinetic analysis has not yet bean made (Friede e t a/., 19721.

Inability t o use arginine as an energy source (Barter and Strsughn, 1971).

Defective in the uptake o f arginine and citrulline (measured indirectly) (Baxter and Straughn. 1971).

Penicillin treatment in lowglutamete medium (Utech etal., 1970).

Mutant strain R4 lacks the H A system for aspartate end glutamate. The L A system remains, with Km's of 8 x lo4 M for aspartate and 9.6 x 10-3 M for glutamate (Utech eta!., 1970).

(Continued)

TABLE I Mutations Affecting Membrane Transport (Continued) Amino Acids and Peptides Organism

Transport System

Specificity

Streptococcus strain Challis

Alanine, glycine

Wild-type S. faecah (Mora and Snell, 1963) and wildtype Streptococcus strain Challis (Reitz et a/., 1967) contain a transport system for glycine and D- and L-alanine, inhibited by D-cycloserine.

-

Neurospora crassa

Neutral L-amino acids

Transport system I (Pall, 1969). present i n rapidly growing cells, takes up most neutral L-amino acids (DeEusk and DeEusk, 1965; Stadler, 1966; Lester, 1966; Wiley and Matchett, 1966; Pall, 1969; Wolfinbarger and DeEusk, 1971a). Km's have been reported for leucine (1 .2 x 104 M I ,valine (4.7 x 1 0 4 M I ,tryptophan (5-6 x 10-5 M ) ,phenylalanine (5-6 x 10-5 M ) , and histidine (6.5-8 x 1 0 4 M ) (Wiley and Matchett, 1966,1968; Pall, 1969; Pall and Kelly, 1971; Magill eta/., 1972). System I also takes up acidicamino acids (en.. L-aspartate) a t low p H (Wolfinbarger eta/.,

mtr

1971). Recently, an amino acid-binding protein has been isolated from the osmotic shock fluid of early exponentialphase Neurospora hyphae, and has been postulated t o play a role in transport system I (Wiley, 1970). The protein binds tryptophan with a K D of 8x M, very similar t o the K, for tryptophan transport; tryptophan binding is inhibited by other neutral amino acids (phenylalanine, leucine) but not b y arginine or lysine; and both the binding protein and the activity of the transport system are repressed b y growth in the presence of tryptophan, and are decreased in at least onemtr mutant (Wiley, 1970).

A glycoprotein, extracted from Neurospora conidia b y KCI, has also been postulated t o be a component of system I; it is absent in pmn mutants (but also in pmb mutants; see below) (Stuart and DeBusk, 1971) The relationship between this glycoprotein and the neutral amino acid-binding protein has not been discussed.

26

Gene

A m i n o Acids and Peptides Linkage

Linkage group I V (Stadler, 1966). The mtr locus has been extensively studied, since techniques are available t o select for b o t h forward mutations ( b y means o f analog resistance) and reverse mutations ( b y the ability of an amino acid auxotroph to grow at l o w amino acid concentrations) (Stadler, 1967; Brink eta/., 1969). Finestruc ture mapping has revealed three clusters o f mutational sites; no intragenic complementation was observed (Stadler and Kariya, 1969). mfr- is recessive t o the wild-type allele (Stadler, 1966).

Method o f Isolating Mutants

Transport Defect in Mutants

Resistance to D-cycloserine. In addition t o transport m u tants, other D-cycloserineresistant mutants were f o u n d that contained increased amounts o f D-alanine: Dalanine ligase and alanine racemase, enzymes k n o w n t o be inhibited b y D-cyclow i n e (Reitz e f a/., 1967).

Defective in the uptake o f D- and Lalanine (glycine uptake n o t measured) (Reitzef a/., 1967).

Resistance t o 4-methyltryptophan (Lester, 1966; Stadler, 1966) orp-fluorophenylalanine (Stadler, 1966).Sixty independently isolated mutants, resistant t o b o t h analogs, have all been shown t o map i n t h e same region o f linkage group I V (Stadler, 1966; Stadler and Kariya, 1969);onlyone mutant has been isolated w i t h this pattern o f resistance and found t o be unlinked t o mfr (Stadler and Kariya, 1969).

Defective in system I . m f r mutants were originally f o u n d t o have decreased ( b u t s t i l l measurable) transport rates f o r neutral amino acids, including tryptophan, phenylalanine, tyrosine, methionine, valine, leucine, and histidine (Lester, 1966; Stadler, 1966). Subsequently, it was shown that the remaining uptake o f neutral amino acids (tryptophan, leucine) i n at least one mfr mutant was c o m pletely inhibited b y arginine, indicating that it occurred via system II (see below); b y contrast, neutral amino acid uptake in wild-type Neurospora is only partially inhibited b y arginine and involves b o t h systems I and II (Pall, 1969). Several rnfr mutants have been described that may have qualitative alterations in system I. These include tryp-auxotrophs which have become resistant to inhibit i o n b y phenylalanine (Stadler, 1966); a temperaturesensitive mfr mutant (Stadler and Kariya, 1969);a mutant w i t h decreased affinities f o r neutral amino acids (Pall, personal communication); and n e d mutants (see below). None o f these strains has been studied in detail, b u t qualitatively altered mtr mutants should prove useful i n establishing whether mtr is a structural gene for transport system I, and also in testing the hvpothesis that the neutral amino acidbinding protein (Wiley, 1970) and the KClextractable glycoprotein (Stuart and DeBusk, 1971) are involved in transport.

27

(Continued)

TABLE I Mutations Affecting Membrane Transport (Continued) ~

~~

~~

Amino Acids and Peptides arganism

Transport System

Specificity

Gene

Neurospora crassa IC0n~t.1

Most amino acids

Transport systam II (Pall, 1969) i s active in 3day hyphae. and takes up a wide variety of amino acids: D and L, 0: and fl, neutral, basic, and acidic, Km's have been measured fo[ L-tryptophan (45x 1 0 6 Mi, L-phenylalanine (2 x 10-6M ) , 0-phenylalanine (2-3 x 106 M I . glycine (5 x 10-6MI. L-aspartate (12 x 104 M ) . and L-asparagine (8 x 10-6 M ) ;and Ki's have been measured for several additional amino acids (Pall. 1969, 1970al.The activiry of system I I increases when cells are starved for carbon or nitrogen (Pall, 1969; SanchezataL. 1972).

su-mtr

'The symbols for Neurospora genes have been modified, where necessary, to follow the recommendations of Barratt and Perkins (1965) on Neurospora genetic nomenclature.

28

Amino Acids and Peptides Linkage

Method of Isolating Mutants

Transport Defect in Mutants

Linkage group I V ; probably allelic w i t h mtr (Wolfinbarger and DeBusk, 1971a).

Resistance t o p-fluorophenylalanine (Wolfinbarger and DeBusk, 1971a).

Defective in the transport of phenylbut not argialanine (via system I?) nine (Wolfinbarger and DeBusk, 1971a).

Has not been mapped;

Inability of a his- auxotroph t o grow o n histidine in the presence o f arginine, which blocks systems II and Ill (the other routes by which histidine could be taken up; see below) (Woodward e t a/., 1967; Magill era/., 1972).

Defective in system I. Residual uptake of histidine b y t h e neua mutant was completely blocked b y arginine. Systems I I and I l l were shown t o be present, with normal Km's and Vmax's; and histidine.once taken up, was shown t o be incorporated normally into protein (Magill etal., 1972).

Ability of a his-auxotroph t o grow on histidine in the presence of methionine and arginine, which block systems I , Il,and Ill,thereby inhibiting the growth of the parent strain (Magill e t a/., 1972).

Believed to be altered in system I , Histidine uptake b y system I i n the neur mutant required unusually high concentrations of methionine for inhibition (20 m M instead of 2 mM). suggesting that the relative affinities of system I for the various neutral amino acids may have been altered; this idea has not yet been checked directly.

it will be important t o

establish whether neua is allelic t o mtr (and neur; see below),

Linkage group IV (Magill eta/., 1972); may be allelic t o mtr.

Systems II and III were shown t o be present in neur; and histidine, once taken up, was incorporated normally into protein (Magill eta/., 1972). Linkage group I (Stadler, 1967; Brink etal., 1969).

Ability of a t r y p - m t r parent strain to grow on low concentrations of tryptophan (Stadler, 19671,or ability of a his-mtr strain t o grow on histidine in the presence of arginine (which would normally block the uptake of histidine via systems 1 1 and Ill;see below) (Brinketa/., 1969). Of 109 mutants isolated b y the latter method.49 proved to b e mtr+ revertants and 60 t o b e s u m t r (Brink eta/., 1969). Allsu-mtr isolates mapped in the same region of linkage group I; furthermore, the suppressors were not ailelespecific, and any su-mtr could suppress any mtr (Stadler. 1967; Brinketal., 1969).

29

Believed t o be regulatory mutants with increased levels of system I I (Pall, 1968). This explanation is consistent with t w o observed properties of su-mtr strains (Stadler, 1967): (1) they have recovered the ability t o take up neutral amino acids (tyrosine, leucine, isoleucine. valine, methionine, cysteine), but uptake has become sensitive t o inhibition by arginine and lysine; and (2) they have regained sensitivity t o p-fluorophenylalanine but are s t i l l resistant t o 4-methyltryptophen. This latter finding, unexplained at the t i m e s u m t r strains were originally described, is consistent with the known properties of system II, which has a 20-fold lower affinity for tryptophan (and possibly for tryptophan analogs) than for phenylalanine (Pall, 1969).

(Continued)

TABLE I Mutations Affecting Membrane Transport (Continued) A m i n o Acids and Peptides Organism

Transport System

Specificity

Gene

Neurospora crassa (Con%)

Basic L-amino acids

Transport system Ill (Pall, 1970a) i s present in rapidly growing cells, and takes u p basic amino acids (Bauerle and Garner, 1964; Roess and DeBusk, 1968; Pall, 1970a; Wolfinbarger and DeBusk, 1971a). Km's have been reported for L-lysine (K, = 5 x 1 0 - 6 M ) . L-arginine (K, = 2.3 x 1 0 - 6 M ) , a n d L-histidine (K, = i.ex10-3~).

baf

bm-1

30

A m i n o Acids and Peptides Linkage

Method o f Isolating Mutants

Transport Defect in Mutants su-mtr mutants may also have an i n creased level o f transport system I V (see below),and the possibility must be considered that this gene affects transport in an indirect way, b y altering carbon and/or nitrogen metabolism (Pall, personal communication).

Maps o n linkage group IV, about 2 5 recombination units distal t o cot (Thwaites, personal communication).

Isolated b y an indirect p r o cedure, making use o f t h e fact that the pyr-3arg-12S parent strain i s inhibited b y arginine; bat mutants are selected b y their resistance to arginine (Thwaites, 1 9 6 7 ) .

Defective i n system Ill, w i t h essentially normal activities o f systems I and II. The result is that arginine and lysine can still be taken u p b y bat, via system I I , b u t their uptake is i n hibited b y a wide range o f amino acids (Thwaites and Pendyala, 1969; Pall, 1970a).

Linkage group V (Wolf inbarger and DeBusk, 1971a).

Resistance to canavanine (Roess and DeBusk, 1968; Wolfinbarger and DeBusk, 1971a).

Defective in arginine and lysine u p take and (partially) in histidine uptake (via system I l l ? ) (Roess and DeBusk, 1968; Wolfinbarger and DeBusk, 1971a).

Linkage group II (Magill eta/., 1972).

Inability o f a his-auxotroph to grow on histidine in the presence o f methionine, which blocks systems I and II (Magill eta!., 1972).

Defective in system Ill?Uptake o f histidine b y the basa mutant was completely inhibited b y methionine Systems I and II were shown to be present, w i t h normal Km's and Vmax's, and histidine, once taken up, was shown t o be incorporated normally i n t o protein (Magill era/., 1972).

Resistance t o canavanine and thialysine (Sanchez et a/., 1972).

Defective in system I l l ? Uptake o f basic amino acids is reduced in bm-1 mutants and is completely inhibited b y neutral amino acids (Sanchez eta/., 1972).

Linkage group V (Woodward eta/., 1967).

Inability o f a his-auxotroph t o grow o n histidine in the presence o f a neutral amino acid (Woodward eta/., 1967).

Defective in system Ill?Reported t o have abnormally low amounts o f "basic amino acid-inhibited" histidine uptake (Woodward eta/., 1967).

Linkage group V I I (Choke, 1969).

A b i l i t y o f a his-auxotroph t o grow o n histidinol (Choke,

Possibly defective in basic amino acid transport (via system Ill?). Very indirect evidence based o n growth experiments alone: t h e growth o f hishlp-1 double mutants on histidine

1969).

31

(Continued)

TABLE I Mutations Affecting Membrane Transport Kontinoedl Amino Acids and Peptides Organism

Transport System

Specificity

Gene

Neurospora crassa (Con't.1

Acidic D-and L-amino acids (aspartate, glutamate)

Transport system IV (Pall, 1970b) is present in nitrogen-or sulfur-starved hyphae, and takes up L-aspartate (K,,, = 1.2 x lo6 M I , D-aspartate (K,,, = 5.4 x 10-6M),L-glutamate (K,,, = 1.6 x 106 M I , D-glutamate (K,,, = 9.0 x 10-5 M ) ,and L-cysteic acid (K, = 7 x 10sM).

The following two mutants may have defects in amino acid transport, but have not been well characterized.

-

hlp-2

32

Amino Acids and Peptides Linkage

Method of Isolating Mutants

Transport Defect in Mutants was completely inhibited by neutral amino acids, while the growth o f the his-parent strain required both a neutral and a basic amino acid for complete inhibition (Choke, 1969). Choke has suggested that the basic amino acid transport system may have been altered in h/p-1 to acquire an affinity for histidinol. Note: The relationship among the various genes affecting basic amino acid transport is not yet clear, particularly since transport experiments b y different investigators have been done under very different conditions. A careful kinetic study i s needed t o check on the possibility that there may be more than one basic amino acid transport system in Neurospora, and qualitatively altered mutants are needed to establish which of the genes are structural genes.

N o clear-cut system-lV mutants have been isolated.

Unlinked t o m t r (D. Boone, cited in Stadler and Kariya, 1969).

Spontaneous mtr-like mutant in h p A m t r 119 stock, with partial resistance t o 4 m e t h y l tryptophan and p-fluorophenylalanine. I n a his-background prevents growth on histidine in the presence of arginine (D. Boone, cited in Stadler and Kariya, 1969).

Not characterized.

Linkage group V I I (Choke. 1969).

Ability of a his-auxotroph to grow on histidinol (Choke, 1969).

N o t characterized directly. The growth of a his-hlp-2 double mutant on histidine was stimulated b y aromatic amino acids and inhibited b y methionine, isoleucine, valine, or asparagine. Choke 11969) suggested that hlp-2 might have an altered permease which had acquired the ability t o take u p histidinol, but no direct uptake measurements have been made.

33

(Continued)

TABLE I Mutations Affecting Membrane Transport (Continued) Amino Acids and Peptides Organism

Transport System

Neorospofa crassa

The remaining mutants show reduced uptake of one or more groups of amino acids, but are difficult to interpret in terms of known transport systems. They may represent regulatory genes or other genes that affect transport indirectly.

/Con’C.I

Specificity

Gene fpr- 1

mod-5

nap

on(55701t)

34

A m i n o Acids and Peptides Linkage Linkage group V (Kinsey and Stadler. 1969).

Linkage group V I (St. Lawrence era/., 1964).

Method of Isolating Mutants

Transport Defect in Mutants

Resistance o f an mtrsu-mfr parent strain (see above) t o p-fluorophenylalanine (Stadler, 1966; Kinsey, 1967; Kinsey and Stadler. 1969). fpr-1 i s also resistant t o 4 m e t h y l t r y p t o p h a n (Kinsey and Stadler, 1969).

N o t characterized in detail. Partial defect in the uptake o f phenylalanine, p-fluorophenylalanine, tryptophan, and leucine; normal uptake o f lysine (Kinsey and Stadler, 1969). Possibly a regulatory gene, since Pall (personal communication) f o u n d fpr-1 t o have reduced levels o f b o t h systems I and II.

Reverses the inhibition o f the

N o t characterized in detail. m o d 4 shows a decreased lag i n the uptake o f aromatic amino acids and dipeptides (St. Lawrence etal., 1964).

f r y p - 3 parent strain b y leucine, yeast extract, or peptone (St. Lawrence etal., 1964).

Linkage group V (Jacobson and Metzenberg, 1968).

Resistance t o ethionine and p - f luorophenylalanine. Later found t o be resistant t o aminopterin, glycine, and 4 m e t h y l tryptophan (Jacobson and Metzenberg, 1968).

Primary defect not clear. Very slow uptake of neutral and acidic amino acids (methionine, ethionine, phenyla1anine.p-fluorophenylalanine, a-aminoisobutyrate, aspartate, and glutamate); partially reduced uptake o f proline and lysine; normal uptake o f arginine, glucose, and sulfate. Normal oxygen consumption and growth rate (Jacobson and Metzenberg, 1968).

Linkage group V I (Davis and Zimmerman, 1965).

In an arg-parent strain, prevented the utilization o f arginine f r o m the medium (Davis and Zimmerman, 1965).

Primary defect n o t understood. Reduced uptake o f arginine, lysine, other unrelated amino acids, and uridine (but at least in the case o f arginine, uptake is abnormal only in NHq+-containing medium). Resistant t o p - f l u o rophenylalanine (Davis and Zimmerman. 1965). Primary defect n o t clear. Jacobson and Metzenberg (1968) reported that, like nap.55701 showed decreased uptake o f neutral and acidic amino acids (methionine, ethionine, phenylalanine,p-fluorophenylalanine, aspartate, glutamate, and possibly proline) b u t normal uptake o f basic amino acids (arginine, Iysine), glucose, and sulfate, and normal oxygen consumption and growth rate, Protoplasts o f 55701 appeared t o show increased osmotic fragility, and Kappy and Metzenberg (1967) suggested that the primary defect might be in some structural element o f t h e plasma membrane.

Linkage group I (Kappv and Metzenberg, 1967; Jacobson and Metzenberg, 1968) b u t see below.

35

(Continued)

TABLE I Mutations Affecting Membrane Transport (Continued) Amino Acids and Peptides Organism

Transport System

Specificity

Gene

Neurospora crassa (Con?.)

Saccharomyces cerevisiae

Basic amino acids (arginine, lysine)

There are two transport systems for the basic amino acids in S. cerevisiae: (1) one specific for L-lysine (K, = 2.5 x 1 0 6 M),not inhibited significantly by ornithine or arginine; and

36

lys-pl

Amino Acids and Peptides Linkage

Method o f Isolating Mutants

Transport Defect in Mutants

Resistance t o citrulline, in a pyr-3arg-125 parent strain sensitive t o arginine and citrulline b y virtue o f feedback control of the accumulation of argininespecific carbamyl phosphate (Thwaites et a/., 1970). These mutants appear t o be allelic w i t h 55701 ,and a detailed complementation map of the locus-involving 80 independently isolated mutants-has been worked out (Thwaites e t a / . , 1970). More recently, a question has arisen concerning the link age relationships of the original im(55701t) strain Although Metzenberg (personal communication) found that the transport defect and the temperature sensitivity segregated together through 12 crosses, and that both are closely linked to mating type on Iinkage group I, Tisdale and DeBusk (1970) have reported that a "transport regulatory" gene segregates from the temperature4ens.itive gene, and is located about 20 map units from rryp-1 on linkage group I l l (DeBusk, personal communication) These differences remain t o be worked out Resistance to thiosine (Grenson, 19661.

Defective i n the specific lysine transport system (the high-affinity component of lysine transport), with the Vmax reduced in mutant strain R A 309 by a factor o f 50 (Grenson, 1966).

37

(Con tinued)

TABLE I Mutations Affecting Membrane Transport (Conrinued) Amino Acids and Peptides Organism

Transport System

Saccharornyces cerevisiae (Con %)

Specificity (2) one that transports L-arginine (Krn = 10-5 M )and,

Gene arg-pl

a t higher concentrations, L-lysine (Krn = 2.5 x 104 M )

and L a n i t h i n e (Km = 3 x lO-3M). Competitlvely inhibited by L-canavanine ( K ; = 6 x 10-5 M )and D-arginine (K; = 7.5 x l o 4 M ) (Grenson eta/., 1966).

Dicarboxylic amino acids (aspartate, glutamate, a-aminoadipate)

Not characterized in detail

Histidine

High-affinity transport system for L-histidine. Not inhibited by other naturally occurring L-amino acids, but weakly inhibited by 1-methylhistidine (Crabeel and Grenson. 1970).

Methionine

High-affinity transport system for L-methionine (K, = 1.2 x 10-6M); competitively inhibited by L-ethionine, DLselenomethionine, D-methionine (Gits and Grenson, 1967).

Most neutral and basic amino acids

A general transport system for a wide range of amino acids. K,'s have been reported for L-arginine (7.6 x 10-6 M),L-lysine (3.1 x MI, L-citrulline (7.4-8 x 10-5 M),and L-tryptophan (0.9-1.3 x MI. The general transport system also takes up L-histidine, Lserine, L-alanine, L-methionine. L-valine, and possibly L-glutamate, but not proline. Inhibited by ammonium ions (Grenson eta/., 1970).

(60615)

38

Amino Acids and Peptides Linkage Unlinked t o lys-pl (Grenson, 1966).

Method o f lsolatina Mutants

TransDort Defect in Mutants

Resistance to canavanine (Grenson eta/., 1966; BBchet ef a/., 1970).

Defective in the arginine transport system (which is also responsible for the l o w a f f i n i t y component of lysine transport), w i t h the Vmax reduced in mutant strain MG 168 b y a factor o f 2 6 (Grensonetal., 1966).

Inability of a lys-auxotroph to use ol-aminoadipate as a source of lysine (Joiris and Grenson, 1969).

Defective in the transport of aspartate, glutamate, and olaminoadipate, w i t h the Vmax reduced by a factor o f 100 in mutant strain MG 9 5 6 (Joiris and Grenson, 1969).

Slow growth of a his-auxotroph at low histidine concentrations (Crabeel and Grenson, 1970).

Defective in the transport o f histidine (Crabeel and Grenson, 1970).

Unlinked t o lys-pi or arg-pl (Gits and Grenson, 1967).

Resistance to low concentrations of L-ethionine (Gitsand Grenson, 1967).

Not linked t o any of the genes determining specific amino acid transport systems (see above; Grenson eral., 1970).

Resistance to canavanine i n an arg-pi parent strain, growing in the absence o f ammonium ions (so that the general transport system is functional; see Specificity) (Grenson era/., 1970). gap alone does not confer resistance to amino acid analogs, which can also be taken up by the appropriate specific amino acid transport systems; gap arg-pi double mutants are resistant to canavanine because they lack both the general and the argininespecific systems. gap mutants can also be recognized by their slow growth on citrulline, which appears t o be taken u p only by the general amino acid transport system (Grenson etal., 1970).

Defective in the general amino acid transport system. gap mutants can take u p most amino acids via the various specific transport systems, but they d o n o t exhibit the usual increase in uptake rate (signifying the appearance of the general system) when ammonium ions are removed from the medium (Grenson etal., 1970).

A wild-type strain derived from six species of Saccharornyces (Bussey and Umbarger, 1970b).

Strain 60615 has a decreased affinity for leucine transport compared with 1.05 x wild-type strain L P (K,'sof 10-3M and 3.0 x M, respectively).

39

Defective in the transport of methio nine (Gits and Grenson, 1967).

(Continued)

TABLE I Mutations Affecting Membrane Transport (Continuedl Amino Acids and Peptides Organism

Transport System

Specificity

Gene

Saccharo myces cerevisiae

1Con't.l

60615/f 12

a@ (apf)

Aspergillus nidulans

7

N o t characterized.

fpa D

Amino Acids and Peptides Linkage

Method of Isolating Mutants

Transport Defect in Mutants Leucine-binding proteins have been isolated from both strains, and because their affinities differ by a similar factor ( K 's of 1.68 x 1 0 5 M and 2.47 x 10- M),Bussey and Umbarger (1970a)

9

have suggested that the binding protein may be involved i n transport. This appears t o be a transport system for a rather wide range of amino acids (leucine, isoleucine, valine, threonine, tryptophan, tyrosine; Eussev and Umbargar. 1970a.b). b u t its specificity has not been investigated in detail, and its relationship to the systems described by Grenson and coworkers has not been established. Comparisons are made even more difficult by the complex origin o f strain 60615 (see Method of isolating mutants).

Unlinked t o (Grenson er el., 1970).

-

Isolated from strain 60615 (see above) by resistance t o trifluoroleucine (Bussey and Umbarger, 1970b).

Derepressed for leucine (and valine and tyrosine! transport, w i t h no change in Km (Bussey and Umbarger. 1970a.b).

Resistan- t o ethionine (Sorsoli eta/., 1964; Surdin et at., 1965; Cherest and de RobichonSzu lmajster. 1966; de Robichon-Szulmajster and Chersst. 19661 or top-fluorophenylalanine (Grens.cn and Hennaut, 1971). aap mutants are also resistant t o canavanine, @-2-thienylalanine,thiosine, norleucine, aretidine-2carboxylic acid, cyclolaucine, and cycloserine (Grenson and Hennaut, 19711. They can be selected most effciently b y resistance to a pair of amino acid analogs (e.g., canavanine plus 0-thienylalanine) or by the ability t o use glycine as sole carbon source (Grenson and Hennaut, 1971). Resistance t o p-fluorophenylalanine. @aD mutants are also resistant t o ethionine and t o

Reduced activity of several amino acid transport systems (arginine. lysina. leucine, proline, general). In each case the Vmax is depressed but transport is not eliminated completely. Granson and Hennaut (1971) suggest t h a t aap (apf) may determine a factor common to the various amino acid transport systems.

41

The uptake of purines and pyrimidines is unaffected (Grenson and Hennaut, 1971).

Defective i n the uptake o f phenylalanine, tyrasine, tryptophan, methionine. leucine, and aspartic acid, and partially

(Conrinued)

TABLE I Mutations Affecting Membrane Transport (Continued) Amino Acids and Peptides Organism

Transport System

Gene

Specificity

~~

Aspergi/lus nidulans (Con'r.) Ochromonas danica

Methionine. wine

Not characterized.

Organism

Transport System

Man

Cystine, lysine, Kidney: In vivo clearance studies and in vitro transornithine arginine port experiments with kidney slices have pointed (kidney, intestine) t o the existence o f at least t w o (and probably three) transport systems for cystine and the dibasic amino acids in the kidney:

Specificity

Disease Cystinuria

( 1 ) a specific system for cystine, n o t inhibited by lysine, ornithine, or arginine (Fox eta/., 19641; ( 2 ) a system for lysine, ornithine, and arginine, not inhibited by cystine (Foxeta/., 1964);and

( 3 ) (less conclusively) a shared system for all four amino acids, postulated on the basis o f combined genetic and clearance data (Dent and Rose, 1951). However, attempts t o demonstrate in vitro a mutual competitive inhibition among the four amino acidsas would be expected for a shared system-have been unsuccessful (Fox e t a/., 1964). Rosenberget a/. (1967) were able t o detect t w o kinetically distinct lysine transport systems in both rat and human kidney slices, b u t which ( i f either) might correspond to the shared cystine-lysine-ornithine-arginine system was not established. Intestine: In the intestine the shared system has been demonstrated by oral loading experiments (Milne e t a/., 1961; Asatoor e t a/., 1962; Rosenberg e t a/., 1965) and by direct measurements o f transport in jejunal mucosa obtained by biopsy (Thier era/., 1964, 1965; McCarthy eta/., 1964). The specific cystine and lysine-ornithinearginine systems appear not t o be present in the intestine.

42

Amino Acids and Paptides Linkage

-

Method o f Isolating Mutants L-3-aminotyrosine and phenylanthranilic acid (Sinha, 1969).

defective in the uptake of histidine, glycine, and glutamic acid (Sinha, 1969).

Resistance to ethionine (Hochberg et al., 1972).

Decreased uptake o f methionine, ethionine, serine (Hochbergetal., 1972).

Mode of Inheritance Autosomal recessive (Harris et al., 1955a.b).

Phenotype

Urinary excretion of dibasic amino acids and cystine

Control Type 1 Type II Type I I I

Normal Markedly increased Markedly increased Markedly increased

I , l l , a n d I I I havebeen shown t o be multiple alleles o f the same gene, since I I I , II Ill, and I I II double heterozygotes all have cystinuric phenotypes (Rosenberg, 1966. 1967; Rosenberg etal., 1966b).

Transport Defect

Increased renal clearance of cystine, lysine, ornithine, and arginine (Dent and Rose, 1951; Dent e t al., 1954; Arrow and Westall, 1958; Robson and Rose, 1957; Doolan et al., 1957; Frimpter e t a/., 1962; Crawhall etal., 1969; Rosenberg and Scriver, 1969).

More recently, cystinuria has been subdivided into three types on the basis of combined studies of urinary excretion and intestinal transport (Rosenberg e t al., 1966a):

Homozygotes

Transport Defect in Mutants

Heterozygotes -

Normal Increased Increased

Postulated to be a primary defect in the transport of cystine, lysine, orni thine, and arginine via the shared system (Dent and Rose, 1951). The defect has been demonstrated clearly in the intestine b y oral loading experiments (Milne etal., 1961; Asatoor e t al., 1962; Rosenberg et al., 1965) and by transport measurements on jejunal rnucosa obtained by biopsy (Thier etal., 1964, 1965; McCarthy etal., 1964).

In the kidney, in vivo clearance studies are consistent with such a defect (see Phenotype), but in vitro transport experiments have given a Intestinal transport o f dibasic amino acids and cystine somewhat confusing picture. Homozygotes Heterozygotes The postulated shared transPresent port system for Absent cystine, lysine ornithine. and Cystine-present arginine has not been detected Lysine-absen t Present in normal kidney slices (see Specificit v ) ; no clearcut abnormality in cystine transport was found in kidney slices from cystinuric patients (Fox e t al.. 1964); and both o f the lysine transport systems seen in normal subjects were shown t o be present in vitro in cystinuric patients, although the data were n o t sufficient t o conclude whether the kinetic parameters o f either

(Continued) 43

TABLE I Mutations Affecting Membrane Transport (Continuedl Amino Acids and Peptides Organism

Transport System

Specificity

Disease

Man (Con ‘t.I

“Isolated” cystinuria

Dibasic aminoaciduria

Neutral amino acids (kidney. intestine)

There appear t o be multiple transport systems for new tral amino acids in the human kidney and intestine:

(1) A common system for neutral a-amino acids w i t h aliphatic or aromatic side chains. Such a system seems likely on the basis of the urinary excretion pattern i n Hartnup disease (see Transport defect), b u t direct evidence for i t s existence has n o t yet been reported.

44

Hartnup disease

A m i n o Acids and Peptides Mode o f Inheritance

Phenotype

Transport Defect system had been altered (Rosenberg eta/., 1967). A likely explanation f o r the discrepancy between the in vivo clearance data and t h e transport results is that a shared reabsorptive system does exist in the kidney (and isdefective in cystinuria), b u t that its kinetics are obscured b y simultaneous secretion o f cystine and the dibasic amino acids.

Probably recessive (data f r o m a single family) (Erodehl eta/., 1967).

Increased clearance o f cystine (Brodehl era/., 1967).

Postulated t o be defective in t h e renal transport of cystine via the specific system.

Recessive (reported in Finland; Perheentupa and Visakorpi, 1965; Kekoma'ki eta/., 1967).

Severe protein intolerance. Increased renal clearance o f lysine and arginine (ornithine not measured) (Perheentupa and Visakorpi, 1965; Kekoma'ki etal., 1967).

Dominant (reported in Canada; Whelan and Scriver, 1968).

Increased clearance o f dibasic amino acids (Whelan and Scriver, 1968).

Postulated t o be defective in the renal transport o f lysine, ornithine, and arginine (and possibly, in three o f the four cases, in intestinal transport as well). The genetic relationship among the three forms o f dibasic aminoaciduria is n o t y e t clear (Scriver and Hechtman, 1970).

Recessive (reported in Japan; Oyanagi etal., 1970).

Severe mental retardation, physical retardation, and increased renal clearance o f dibasic amino acids (Oyanagi etal., 1970).

Autosomal recessive (reviewed in Rosenberg and Scriver. 1969).

Increased excretion o f many neutral amino acids (see Transport defect); pellagralike skin rash and temporary cerebellar ataxia (Baron etal., 1956). thought t o reflect niacin deficiency resulting f r o m the malabsorption o f tryptophan (Milne, 1963).

Kidney: indirect evidence ( f r o m clearance data) f o r a defect in the renal transport o f many neutral amino acids (alanine, serine. threonine, asparagine, glutamine, valine, leucine, isoleucine, phenylalanine, tyrosine, tryptophan, hlstidine, and citrulline) (Baron etal., 1956; Jepson, 1972). Intestine: indirect evidence f o r a defect in the intestinal absorption o f tryptophan (oral loading experiments; Milneetal., 1960) and other neutral amino acids (Scriver and Shaw, 1962; Scriver, 1965). In virro studies are needed to c o n f i r m that there is a primary defect in the

45

(Continued)

TABLE I Mutations Affecting Membrane Transport (Continued) Amino Acids and Peptides Organism

Transport System

Specificity

Disease

Man 1Con't.l

Methion i ne (intestine)

(2) A specific system for methionine, postulated on the basis of methionine malabsorption syndrome but not characterized directly.

Methionine malabsorption

Tryptophan (intestine)

(3)a specific system for tryptophan, postulated on the basis of tryptophan malabsorption syndrome; not characterized directly.

Tryptophan malabsorption

Glycine, proline, hydroxyproline (kidney, intestine)

Three transport systems have been postulated for glycine, proline, and hydroxyproline in the human kidney (Scriver, 1967, 1968; Scriver and Wilson, 1967): (1) a common system for the three amino acids, responsible for the reabsorption of about 40% of the filtered glycine and more than 90% of the filtered proline and hydroxyproline a t normal plasma concentrations (Rosenberg and Scriver, 1969). This system was first suggested by the finding that patients with elevated plasma proline concentrations (due to an inherited disorder of proline catabolism; Scriver e t a/., 1961; Schafer et a/., 19621 and also normal subjects, after proline infusion (Scriver et a/., 1964). excreted increased quantities of glycine and hydroxyproline. Direct measurements on several mammalian tissues in vitro (rat kidney slices, Wilson and Scriver, 1967; hamster intestine, Lin e t a / . , 1962; fetal rat bone, Finerman and Rosenberg, 1966; rabbit renal tubules, Hillman et a/., 1968; Hillman and Rosenberg, 1969, 1970) have revealed inhibition among the three amino acids, as expected for a common transport system, but have indicated that there are also

(2)a specific high-affinity system or perhaps two systems for glycine (and alanine), and 46

Iminoglycinuria

Amino Acids and Peptides Mode of Inheritance

Phenotype

Transport Defect transport of this group of amino acids, leading to the observed abnormalities in absorption b y the kidney and intestine.

Probably recessive (data from a single family; Hooft etal., 1968).

White hair; convulsions; mental retardation; attacks of hyperpnea; large amounts of methionine and a-hydroxybutyric acid (thought t o be a bacterial degradation product of methionine) in the feces (Smith and Strang, 1958; Hooft etal., 1965).

Postulated t o be a primary defect i n the intestinal absorption of methionine. Consistent with this view, oral loading of methionine produced diarrhea and an increase in fecal and urinary a-hydroxybutyric acid, while oral loading of six other amino acids failed t o produce these effects (Hooft etal., 1965). In vitro transport studies have not been performed.

Probably recessive (data from a single family; Drummond e t a/., 1964).

Recurrent febrile episodes; growth retardation; irritability; constipation; hypercalcemia; increased excretion of indoles (bacterial degradation products of tryptophan), resulting in a "blue diaper syndrome" (Drummond e t a/., 1964).

Postulated t o be a primary defect in the intestinal absorption of tryptophan. Oral loading of tryptophan caused an increase in fecal tryptophan and in the urinary excretion of indoles (Drummond e t a/., 1964). but in vitro transport experiments have not been done.

Autosomal recessive. There appear t o be at least three phenotypic types of iminoglycinuria: Intestinal absorption of glycine, proline, and hydroxyproline

Urinary excretion of glycine, proline, and hydroxyproline

(1) Described by

Homozygotes

Heterozygotes

Homozygotes

Heterozygotes

Increased

Normal

Defective

-

Defective

-

Normal

-

Tadaer a/. (1965); Morikawa et a/.(1966) ( 2 ) Described by

Increased

(3) Described by Scriver (1968); Rosenberg et a/. (1968)

Increas-d (glycine) Normal (proline, hydroxyproline)

Goodman ef a/. (19C7)

Increased

Increased (glycine) Normal (proline, hydroxyprol ine)

These types of iminoglycinuria may represent different mutant alleles at the same locus, since Scriver (1968) has described a patient with iminoglycinuria who had one hyperglycinuric pagent and one normal parent.

47

Thought t o be a primary defect in the transport of glycine, proline, and hydroxyproline via the common system.

(Continued)

TABLE I Mutations Affecting Membrane Transport (Conrinued) Amino Acids and Peptides Organism

Man (Con ‘t.I

Transport System

Specificity

(3) a specific high-affinity system f o r proline and hydroxyproline (and alanine). Recently, i t has been shown that the various systems are easily distinguishable in the developing rat kidney, the common system being present at b i r t h and the high-affinity proline and glycine systems appearing at 1 and 3 weeks after birth, respectively (Baerlocher e t a / . , 1970, 1971a.b).

48

Disease

Amino Acids and Peptides Mode of Inheritance

Phenotype

Transport Defect

More recently Greene et a/. 11973) have reported a new type of hyperglycinuria in two brothers who appear to have a qualitatively altered glycine-proline-hydroxyproline transport system (see Transport defect). Detailed study of the parents was not possible, so it was not clear whether the two patients were homzygous for a new type of mutation affecting the common transport system, or whether they were doubly heterozygous for two d i f ferent mutations. The genetic relationship of this new type of hyperglycinuria t o the previously described types of iminoglycinuria has not been established.

Kidney: The most frequent cases of iminoglycinuria appear t o involve quantitative reductions i n the common transport system without any obvious qualitative change. Renal clearance measurements have indicated that at least some homozygotes lack the common system entirely (Scriver, 1968; Rosenberg ef a/., 1968). They reabsorb about 60% of the filtered glycine at normal plasma glycine concentrations, but show no inhibition of glycine reabsorption when proline is infused intravenously; by contrast, normal subjects reabsorb more than 95% of the filtered glycine, and show a decrease t o 50% during proline infusion. (The proline-insensitive glycine reabsorption in both cases is thought t o represent the activity of other glycine transport systems.) Some (but not all) heterozygotes appear t o have intermediate levels of the common glycine-proline-hydroxyproline system, since they excrete increased quantities of glycine but not proline or hydroxyproline (Scriver, 1968; Rosenberg et a/., 1968). (This pattern is consistent with the measured affinities of the common system for the three amino acids in normal subjects: lower for glycine than for proline and hydroxyproline.) I n addition, when the plasma proline concentration is increased b y infusion in such heterozygotes, the maximum tubular reabsorption of proline can be shown t o be reduced t o about one-half the normal value (Scriver, 1968). By contrast with these apparent quantitative alterations, the new form of hyperglycinuria described by Greene e t a / . (1973) appears t o represent a qualitative change in t h e glycineproline-hydroxyproline transport system. Glycine reabsorption is reduced ( t o 73% of the filtered load) and n o t further inhibited by proline. suggesting that no glycine i s being transported

(Continued) 49

TABLE I Mutations Affecting Membrane Transport (Continuedl Amino Acids and Peptides Organism

Transport System

Specificity

Disease

Man (Con ‘t.)

Carbohydrates Organism Escherichia coli

Transport System L-Arabinose

Specificity

Gene

There are two inducible transport systems for L-arabinose in E. coli, both under the control ofaraC, the regulatory gene for the L-arabinose operon (Schleif, 1969; Brown and Hogg, 1971,1972):

( 1 ) a l o w a f f i n i t y system (K,

=

araE

1.O x 1 0 4 M ) and

(2) a high-affinitysystem (K, = 8 . 3 x 10-6M). Both are competitively inhibited b y D-fucose, Dxylose, and p-methyl-L-arabinoside; in addition the high-affinity system is inhibited by D-galactose (Brown and Hogg, 1972). A protein that binds L-arabinose has been isolated from E. c o l i b y Hogg and Englesberg (1969) and b y Schleif (1969),and appears to be involved in the highaffinity transport system (Brown and Hogg. 1972). The protein is released from the cells b y osmotic shock, suggesting that it i s located in the periplasmic space between the cell wall and the cell membrane (Neu and Heppel, 1965). I t has a molecular weight of 32,000 t o

50

araF

A m i n o Acids and Peptides Mode o f -

Inheritance

Phenotype

Transport Defect b y the common system. A t the same time proline is reabsorbed w i t h a shift in concentration dependence (maximal reabsorption being reached o n l y at higher filtered loads), suggesting a change in the a f f i n i t y o f the transport system f o r proline. Intestine: I t seems clear that t h e comm o n glycine-proline-hydroxyproline system also exists in t h e intestine since, in oral loading experiments, some patients w i t h iminoglycinuria exhibit an intestinal defect in t h e transport of these three amino acids (Morikawa eta/., 1966; Goodman eta/., 1967). Other patients d o not, however (Scriver, 1968; Rosenberg eta/., 1968).and it is n o t k n o w n whether they possess a different mutational alteration in the common system or whether other intestinal transport systems obscure the defect.

Carbohydrates Linkage

Method o f Isolating Mutants

Transport Defect in Mutants

Near thy; unlinked t o araDABC (Isaacson and Englesberg, 1964; Englesberg era/., 1965).

Lack the low-affinity L-arabinose The growth o f araA and araD strains (which lack L-arabinose transport system (Brown and Hogg, isomerase and L-ribuloseS1971,1972);contain normal amounts phosphate 4epimerase. reo f the L-arabinose-binding protein spectively) is inhibited b y L(Schleif, 1969; Hogg and Englesberg, arabinose, and in at least some 1969). araA andaraD strains,araE mutants can be selected b y resistance t o arabinose (Isaacson and Englesberg, 1964; Hogg and Englesberg, 1969; Schleif, 1969; Hogg, 1971 ) . (araC-mutants are also selected b y this procedure but can be recognized b y their pleiotropic negative phenotype; see below.)

Map location n o t known. Unlinked t o araE and araDABC

Lack the high-affinity L-arabinose Identified in an araE parent strain b y the inability t o metab- transport system; in addition, t h e L-arabinose-binding protein is either olize L-arabinose (on tetrazo51

(Contimed)

TABLE I Mutations Affecting Membrane Transport (Continued) Carbohydrates Organism

Transport System

Specificity

Gene

35,000,consists o f a single polypeptide chain,and binds 0.8-1 .O moles o f L-arabinose per mole w i t h a K x o f 2-5.7 x 10-6 M (Schleif, 1969; Hogg and Englesberg, 1969). Binding, like transport via the high-affinity system, is strongly inhibited b y D-galactose (Brown and Hogg, 1972).

Escherichia coli (Con?.)

araC

A t least four d i f ferent transport systems exist in E. coli for galactose and galactosides: Lactose (TMG I )

An inducible system which transports w a n d p-D-galactosides (including lactose), p-D-thiogalactosides, and galactose. First described b y Cohen and Rickenberg (1955). Rickenberg e t a / . (1956),and Pardee (1957). K,'s for commonly used substrates are: lactose, 6-9 x 1 0 4 M; melibiose,2 x 1 0 4 M;o-nitrophenylQ-Dgalactoside (ONPG), 3-10 x l o 4 M; methyl-1-thio-p-Dgalactoside (TMG), 5 x l o 4 M; p-Dgalactosyl-1 -thiop-Dgalactoside (TDG),2-5 x 1 0 6 M; phenyl-1 -thiop-D-galactoside (TPG), 2.5 x 1 0 4 M (Kepes, 1960; Kepes and Cohen, 1962; Winkler and Wilson, 1966; Carter e t a / . , 19681.

52

lacy

~

~

~~

Carbohydrates Linkage

Method o f Isolating Mutants

Transport Defect in Mutants

(Hogg and Englesberg, 1969;Schleif, 1969; Brown and Hogg, 1972).

liumarabinose agar medium) (Brown and Hogg, 1972). Earlier Hogg and Englesberg (1969) and Schleif (1969). b y means o f direct testing, had found t w o mutants w i t h decreased L-arabinose-binding activity. In addition, Hogg (1971) has described a method to screen systematically for binding mutants b y plating o n medium containing antiserum to t h e binding protein; mutants lacking the protein d o n o t produce a visible precipitin reaction.

altered (detectable as cross-reacting material) o r missing altogether (Brown and Hogg. 1972).

Between thr and leu. closely linked t o araD, araA, and araB tn t h e order DABC (Isaacson and Englesberg, 1964; Englesberg eta/., 1965).

araC-mutants can be isolated (along w i t h araA, araB, and araD mutants) b y their i n ability t o grow o n Larabtnose as sole carbon source (Englesberg, 1961 1. Alternatively, they can be selected in an araA o r a r a D genetic background b y resistance t o arabinose (see above; Hogg and Englesberg, 1969; Schleif, 1969).

A regulatory gene which exerts positive control over the operon araD araA JraB (coding f o r L-ribulose4phosphate 4epimerase, Larabinose isomerase, and L-ribulokinase, respectively) and also over t h e unlinked genesaraE and araF (thought t o code for components o f t h e low-affinity and h i g h a f f i n i t y arabinose transport systems; see above). araC- mutants are unable t o synthesize the three arabinose enzymes o r either o f the transport systems, while araCc m u tants synthesize these proteins constitutively (Englesberg eta/., 1964; Novotny and Englesberg, 1966; Englesberg eta/., 1965; Sheppard and Englesberg, 1967; Englesberg eta/., 1969; Brown and Hogg, 1 9 7 2 ) .

araCC mutants can be selected b y resistance t o D-fucose (Englesberg et a/., 1964; Doyle eta/., 1972). which normally inhibits induction o f t h e arabinose operon (Beverin eta/., 1971). Closely linked t o lac/ (the regulatory gene for the lactose operon), /acZ (the structural gene f o r pBalactosidase), and /acA (the structural gene f o r thiogalactoside transacetylase) in the order / Z Y A (Jacob and Monod, 1961 1 .

Inability t o ferment lactose (Rickenberg era/., 1956)./acY mutants are then distinguished f r o m l a c 2 (p-galactostdaseless) mutants b y t h e fact that they are cryptic; when treated w i t h toluene t o disrupt the cell membrane, they can hydrolyze ONPG. Resistance to O-nitrophenyl1-thio-p-Dgalactoside, which

53

l a c y is well documented to be t h e structural gene for t h e M protein (see Specificity), which in t u r n i s a c o m ponent o f the lactose transport system: M protein is not detectable in uninduced cells, indicating that i t s synthesis is under the regulation of the lac/ gene (see below), nor is it detectable in l a c y (amber) mutants (Foxeta/., 1967). One particular Y mutant (8204-ts3), studied in some detail, was shown t o have b o t h a tempera-

(Continued)

TABLE I Mutations Affecting Membrane Transport (Continued) Carbohydrates Organism

Transport System

Specificity

Gene

The kinetics of transport have been worked out in detail (reviewed b y Kepes, 197la,b),and have led t o t h e notion o f a saturable carrier which mediates either facilitated diffusion or, when coupled t o metabolic energy, active transport, Energy coupling in the latter case is viewed as having n o effect o n the entry o f substrate b u t instead as preventing exit, b y greatly reducing the affinity o f the carrier for substrate at the inner surface o f the membrane (Winkler and Wilson, 1966).

Escherichia (Con't.)

COIl

A membrane protein ( M protein) has been isolated f r o m cells that possess the P-galactoside transport system, b y taking advantage o f the fact that sulfhydryl groups o n the protein can be specifically protected f r o m reaction w i t h Nethylmaleimide if a tightly bound substrate (such as TDG; see above) is present (Fox and Kennedy, 1965; Fox eta/., 1967; Carter eta/., 1968). The protein has been solubilized f r o m t h e membrane b y means o f detergents; it i s a single polypeptide chain w i t h a molecular weight of 30,000 (Jones and Kennedy, 1969).

lac1

Melibiose ( T M G II)

A n inducible transport system f o r galactose and certain and pgalactosides, including melibiose (6-0-a-Dgalactosyl-Dglucose) , galactinol (6-O-a-D+alactosylD-glucitol), and TMG, b u t n o t lactose o r ONPG (Prestidgeand Pardee, 1965; Leder and Perry, 19671. Coordinately regulated w i t h the enzyme olgalactosidase (Buttin, 1968; Schmitt, 1968).

01-

54

melt3

Carbohydrates Linkage

Method of Isolating Mutants inhibits the growth o f cells that have a functional lactose transport system (Muller-Hill eta/., 1968; Wong er a/., 1970; T. H. Wilson eta/., 1970). Decreased ability t o accumulate TMG-14C as screened directly b y autoradiography o f colonies (Zwaig and Lin, 1966; Wong et a/., 1970; T . H. Wilson era/., 1970)

Transport Defect in Mutants ture-sensitive lactose transport system and a temperature-sensitive M protein ( F o x et a/., 1967). More recently,another class of rnutants ha: been isolated which appear t o possess qualitatively altered lactose transport systems (Wong eta/., 1970; T. H. Wilson eta/., 1970; Wilson and Kusch, 1 9 7 2 ) . These mutants contain a normal (or perhaps greater than normal) number o f lactose carriers, judged b y four assays that measure carrier activity independent o f coupling t o metabolic energy: (1) t h e rate of entry o f ONPG (facilitated diffusion; Winkler and Wilson, 1966); (2) the initial rate o f entry o f T M G ; (3) counterflow o f T M G ; and (4) direct assay o f the M protein. Active transport ( o f T M G ) is reduced drastically in the mutants, however, and the defect has been traced t o an abnormally high exit rate (which is not a generalized leak, since it is specific f o r substrates of t h e lactose transport system). One mutation o f this t y p e has been mapped in (or near) the lactose operon, consistent w i t h t h e idea that it is a mutation in the Y gene, causing a qualitative change in the M protein such t h a t transport is n o longer energy-coupled.

See above.

Variety o f methods reviewed b y Gilbert and Muller-Hill (1970).

Secondary changes in the level o f lactose transport and also in the levels o f P-galactosidase and thiogalactoside transacetylase; lac/ i s t h e regulatory gene of the lactose operon (Jacob and Monod, 1961).

Closely linked t o metA (Schmitt, 1968).

Inability t o grow o n melibiose; mutants lacking the transport system must then be distinguished ( b y direct assay) f r o m those lacking olgalactosidase and f r o m regulatory mutants deficient in both (Buttin, 1968;Schmitt. 1968).

Defective in the uptake o f melibiose and other galactosides (Schmitt, 1968; Buttin, 1968); n o t characterized in detail.

Considerable differences exist among the various wild-type strains of E. coli w i t h respect 55

(Continued)

TABLE I Mutations Affecting Membrane Transport (Continued) Carbohydrates

Organism

Transport System

Specificity

Gene

Escherichia coli (Con't.1

p-Methylgalactoside

A n inducible transport system for galactose and pgalactosides, with K,'s of 5 x 10-7 M for galactose (Rotman and Radojkovic. 1964); 2.8 x 10-6 M for 1 -09-D-galactosyl-glycerol (Boos, 1969); and 2 x 1 0 3 M for methyl-1 9-D-galactoside (MG; Rotman er a/., 1968). Also studied b y Rotman (1959). Horecker eta/. (1960a,b), Osborn etal. (19611, Buttin (1963a,b), Ganesan and Rotman 11966). Booset a/. (1967). Boos and Wallenfels (19681,and Singer and Englesberg (19711,This system, rather than the one described below, plays the key physiological role in growth on low concentrations of galactose and i n the induction of thegal operon (Wu and Kalckar, 1966; Wu, 1967; Wu eta/., 1969; Kalckar, 1971 1. A galactose-binding protein, which appears t o be involved i n transport via the 0-methylgalactoside system, was first described by Anraku (1968a,b,c), and has been characterized i n detail by Boos (1969), Boos and Gordon (1971),and Boosetal. (1972). Like the transport system, the binding protein has the highest affinities for galactose, P-galactosyl-glycerol, and glucose; and i n addition, the protein has been shown t o undergo a conformational change in the presence of substrate, detected as an increase in electrophoretic mobility and in fluorescence. Genetic evidence suggests that the binding protein also plays a role in galactose chemotaxis (Hazelbauer and Adler, 1971; Kalckar, 1971; Boos, 1971) (see Transport Defect i n Mutants).

56

mglP

Carbohydrates Linkage

Method o f Isolating Mutants

Transport Defect in Mutants

t o the melibiose operon; i n duction o f the permease and a-galactosidase is temperatureSensitive in E. cot; K-12 and does n o t occur during growth at 37' or higher (Buttin. 1968); E. coli B is inducible at all temperatures; and E. c o l i ML lacks the permease and a-galactosidase altogether (Pardee, 1957; Prestidge and Pardee, 1 9 6 5 ) . One mutant (W4345) which lacks the p-methylgalactoside permease was shown t o be closely linked t o his (Ganesan and Rotman, 1966); t h e remaining mutants have not yet been mapped.

W3092i was isolated as an "inducible" mutant f r o m a galactokinaseless parent strain that is normally "constitutive" f o r the other t w o enzymes o f the gal operon (galactose-1 -phosphate uridyltransferase and UDPGal 4-epimerase); the p-methylgalactoside transp o r t system, when functional, maintains a high intracellular concentration of galactose in the absence o f the kinase, and the galactose in t u r n serves as an endogenous inducer o f the gal operon (Wu and Kalckar, 1 9 6 6 ) . More recently, p-methylgalactoside transport mutants have been isolated o n the basis o f lowered accumulation o f galactose-l4C b y an autoradiographic technique (Boos and Sarvas, 1970),or b y t h e inability t o show chemotaxis toward galactose (Hazelbauer and Adler. 1971).

57

Defective in the p-methylgalactoside transport system, w i t h little o r no u p take o f galactose (at l o w concentrations), p-methylgalactoside, o r galactosyl-glycerol (Wu, 1967; Wu eral., 1969; Boos and Wallenfels, 1968; Boos, 1969). It has not yet been established that this phenotype corresponds to a single gene, since o n l y one o f t h e mutants has been mapped (see Linkage). More is k n o w n about the biochemistry o f the mutants. Some (but not all) o f t h e m contain reduced levels o f galactosehinding protein (Boos and Sarvas, 19701,and recently t w o mutants have been identified w i t h qualitatively altered galactosehinding proteins: ( 1 1 Strain AW550 (isolated b y i t s failure t o show chemotaxis at l o w galactose concentrations and also found t o be defective in galactose transport; Hazelbauer and Adler, 1971 ). A p r o tein has been isolated f r o m this strain which cross-reacts w i t h antibody t o the wild-type galactose-binding protein b u t does not b i n d galactose at low concentrations o r show the substrate-induced conformational change characteristic of t h e wild-type protein (Boos, 1971). (2) Strain EH3039 (defective i n t h e p-methylgalactoside transport system). Protein f r o m this mutant does n o t b i n d galactose at l o w concentrations o r show a substrate-induced conformational change detectable b y acrylamide gel electrophoresis, b u t does-at high concen-

(Continued)

TABLE I Mutations Affecting Membrane Transport (Conrinued) Carbohydrates Organism

Transport System

Specificity

Gene

Escherichia coli (Con't.)

mglR

Galactose

A n inducible system which is specific f o r galactose (K, = 1 0 4 M ) (Rotman etal., 1968).

(galP)

Hexose phosphates

A n inducible system which transports a variety o f hexose phosphates, including glucose G-phosphate, mannose Gphosphate, fructose G-phosphate, 2 d e o x y glucose Gphosphate. glucose 1 -phosphate, and fructose 1-phosphate (Fraenkel etal., 1964; Pogell etal., 1966; Winkler, 1966; Dietz and Heppel, 1971a.c; Ferenci etal., 1971). The K, for glucose Gphosphate is 2 . 7 5 x 1 0 4 M,and Ki's for mannose 6-phosphate and fructose 6-phosphate are 5 x 1 0 4 M and 4 x l o 4 M, respectively (Pogell etal., 1966; Winkler. 1 9 6 6 ) . Interestingly, the system is induced b y exogenously added, b u t not b y endogenously formed, glucose Gphosphate (Heppel. 1969; Winkler, 1970,1971b; Dietz and Heppel, 1971b).

uhp

Carbohydrates Linkage

Method of Isolating Mutants

Transport Defect in Mutants t r a t i o n s 4 x h i b i t a substrate-induced increase in fluorescence. A t r y p t i c digest o f the mutant protein contained one altered peptide. A revertant-selected for recovery o f transport ability-contained a normal galactose-binding protein, as judged b y binding affinity, electrophoresis, fluorescence, and peptide mapping (Silhaw and Boos, 1972; Boos, 1972). These results indicate that strain EH3039 land probably strain AW550 as well) carry mutations i n the structural gene for the galactose-binding protein (although this gene has not yet been shown t o be mglP), and furthermore that the binding protein plays an obligatory role in b o t h the p-methylgalactoside transport system and galactose chemotaxis.

Maps between 5 6 a n d 74 minutes (Lengeler e t a/., 1971).

A b i l i t y o f carbonstarved cells t o grow o n p-methylgalactoside (Lengeleretal., 1971) .

mg/R mutant L104 is constitutive for p-methylgalactoside transport b u t s t i l l f u l l y inducible f o r the gal operon; mg/R i s concluded t o be a regulatory gene f o r m g l P (Lengeler eta/., 1971 1.

Selected b y penicillin treatment o f cells on a glucose G-phosphate medium (Winkler, 1966) or, starting w i t h a PEPcarboxylase-negative parent strain growing o n acetate as carbon source, b y resistance t o growth inhibition b y glucose 6-phosphate. (Kornberg and Smith, 1969). This latter metho d is based on the fact that d u r ing growth of PEP-carboxylasenegative cells o n acetate, C4 acids are synthesized b y t h e glyoxylate cycle; and isocttrate lyase, the key enzyme o f the glyoxylate cycle, i s inhibited b y C3 acids (PEP, pyruvate) derived f r o m glucose 6-phosphate.

Defective in the transport of hexose phosphates (Winkler, 1966; Kornberg and Smith, 1969).

N o t linked t o his (Ganesan and Rotman. 1966). Maps near pyrE (Kornberg and Smith, 1969).

59

(Continued)

TABLE I Mutations Affecting Membrane Transport (Continued) Carbohydrates Organism

Transport System

Specificity

Gene uhpc

Escherichia coli (Con't.)

L-aGlycerophosphate

A transport system, inducible b y L-a-glycerophosphate, conveniently studied in a mutant lacking alkaline phosphatase and LIU-GP dehydrogenase (so that L-a-GP can neither be hydrolyzed extracellularly nor metabolized intracellularly). Under these conditions LIU-GP IS accumulated, unchanged, in an energy-de, o f 1.2 x 1 0 6 M. Uptake pendent process w i t h a K is competitively inhibited b y high concentrations o f ~ DL-glyinorganic phosphate (Ki = 7.5 x 1 0 - 3 or ceraldehyde 3-phosphate (Ki = 5 x 1 0 4 M ) (Hayashi eta/., 1964).

g/P T

Glycerol

A system for the facilitated diffusion o f glycerol, induced by glycerol (in cells possessing glycerol kinase) or b y L-a-GP (Sanno eta/., 1 9 6 8 ) .

glpf

60

~

~~

Carbohydrates Linkage

Method o f Isolating Mutants

Transport Defect in Mutants

Closely linked t o ohp (Ferenci and Kornberg, 1971).

Selected, in a wild-type strain, b y the ability t o grow on fructose 1-phosphate (which is a substrate f o r , b u t cannot induce,the hexose phosphate transport system) (Ferenci e t a / . , 1971) or, in an enzymeI mutant (see below), b y the ability t o grow o n glucose 1 -phosphate (Dietz and Heppel, 1 9 7 1 ~ )(In . the absence o f enzyme I o f the phosphotransferase system, glucose 1-phosphate can support growth o n l y if it can be taken u p b y the hexose phosphate transport system.)

F o r m the hexose phosphate transport system constitutive1y;ohp may be an operon, w i t h uhpc mutations in the control gene (Ferenci and Kornberg, 1971; D i e t z a n d Heppel, 1 9 7 1 ~ ) .

Maps at 42 minutes, between t y r and his (Cozzarelli era/., 1968) and close o r adjacent t o g/pA, the gene f o r the anaerobic L a - G P dehydrogenase (Kistler era/., 1969; Kistler and Lin, cited in Berman-Kurtz era/., 1971).

Isolated b y the inability t o ferment L a G P (Hayashi eta/., 1964). b y slow growth o n a dual carbon source consisting o f a large amount o f L a - G P and a trace of casein hydrolyzate (Cozzarelli e t a / . , 1968). or b y resistance to phosphonomycin, an antibiotic which is taken up b y the L a G P transport syst e m (Hendlin eta/., 1969).

Defective in L a G P transport. g / p T is believed t o be the structural gene for the transport system (rather than a regulatory gene) because all L a - G P transport mutants map in the same small region, well separated f r o m the k n o w n regulatory geneglpR (see below), and because residual transport in g / p T mutants is still inducible in the normal way (Cozzarelli etal., 1968). Defective in t h e facilitated diffusion of glycerol (Sanno, cited in EermanKurtzeral.. 1971).

Maps at 7 6 minutes, close o r adjacent t o g/pK, t h e gene f o r gly cerol kinase (Cozzarelli and Lin, 1966; Sanno, cited in Eerman-Kurtz et a/., 1971). Maps a t 66 minutes, close o r adjacent t o g/pD (the structural gene for the aerobic L a - G P dehydrogenase) b u t not near the other genes it regulates: g/pT, A,

Constitutive, noninducible. and temperaturesensitive control mutants have been isolated b y several methods, described in detail b y Cozzarelli e t a / . (1968).

f , K (Cozzarelli e r a / . . 1968; Berman-Kurtz etal., 1971).

Secondary alterations in the transport of L a - G P and the facilitated d i f f u sion of glycerol.g/pR is thought t o be a regulatory gene, exerting control over at least three operons: g l p T a n d g/pA ( L a - G P transport and anaerobic L a - G P dehydrogenase; Cozzarelli eta/., 1968; Kistler and Lin, cited in Berman-Kurtz et a/., 1971 );g/pF and g / p K (facilitated diffusion o f glycerol and glycerol kinase; Sanno, cited i n Berman-Kurtz eta/., 1971; Cozzarelli and Lin,

-

61

(Continued)

TABLE I Mutations Affecting Membrane Transport (Continued) Carbohydrates Organism

Transport System

Specificity

Gene

Escherichia coli (Con't.)

Ma1t ose

First described b y Wiesmeyer and Cohn (1960); kinetics have not been analyzed in detail.

ma16

malT

Phosphotransferase system

A system that carries o u t the phosphorylation of a variety o f sugars and their "group translocation" i n t o bacterial cells according t o the following scheme: enz I

Phosphoenolp y r uvat e

t

HPr

7Phospho-H Pr + pyruvate

Phospho-HPr

+

62

enz II sugar

Sugar-P + HPr

ptsl (ctr)

Carbohydrates Linkage

Method o f Isolating Mutants

Transport Defect in Mutants 1966); and glpD (anaerobic L a - G P dehydrogenase; Cozzarelli eta/., 1968).

The m a l B region, located between m e t A and u v r A (Schwartz, 1966). appears t o include more than one gene involved in maltose transport (cited i n Thirion and Hofnung, 1972). I n addition, it includes a gene (/amB) required for the synthesis o f A bacteriophage receptors (Thirion and Hofnung, 1972). and polar mutants in malB are often b o t h defective in maltose transport and resistant t o A (Lederberg, 1955; Schwartz, 196713; Thirion and Hofnung, 1972).

Isolated, in E. coli strain K-12, b y the inability t o grow on maltose as sole carbon source; must be distinguished f r o m other rnaltose-nonutilizing mutants in t h e m a l A and m a l B gene clusters (Schwartz, 1966, 1967a,b; Hatfield etal., 1969). E. coli strain B,which is u n able t o grow on maltose and is resistant t o bacteriophage A , i s considered t o be ma/B(Chung and Greenberg, 1968; Ronen and Raanen-Ashkenazi, 1971).

Inability t o grow o n maltose as Located in the ma/A sole carbon source (f.coli region. together w i t h malP (thought t o be the K-12; see above). structural gene for maltodextrin phosphorylase) and malQ (thought t o be the structural gene for amylomaltase) (Hatfield etal., 1969; Hofnung and Schwartz, 1971 ; H o f nungetal., 1971 1. Map between purC and supN (Epstein etal., 1970); this location is consistent w i t h the data o f Wang e t a/. (1969) and Bourd eta/. (1968).

Inability t o grow o n glucose (strain MM-6; Monod, cited in Asensio eta/., 1963; Fraenkel etal., 1964; the ctr mutants of Wang and Morse, 1966,1968; Wangetal., 1969); inability t o ferment fructose + rnannose ( F o x a n d Wilson, 1968); inability t o ferment mannitol + sorbitol (Epstein etal., 1970).

63

Believed t o have a primary defect in the transport o f maltose, b u t n o t characterized in detail (Schwartz, 1966,1967abl.

Defective in maltose transport, a m y lomaltase, and maltodextrin phosphorylase, and resistant t o bacteriophage A.malT is thought t o be a regulatory gene exerting positive control over b o t h t h e A and B operons (Schwartz, 1967b; Hatfield etal.,

1969).

Defective in the uptake o f a variety o f sugars and in enzyme I. The m u tants can be divided i n t o t w o classes o n the basis o f their growth characteristics and the amount o f remaining enzyme I : ( 1 ) "Leaky" mutants, which have significant residual enzyme-I activity (Fox and Wilson, 1968; Saier eta/., 1970; Epstein etal., 1970). These strains d o n o t grow, o r grow slowly, o n sugars k n o w n t o be substrates for the phosphotransferase system in E. coli, including glucose, fructose,

(Continued)

TABLE I Mutations Affecting Membrane Transport (Continued) Carbohydrates Organism Escherichia c o l i (Con't.)

Transport System

Specificity

Gene

First described b y Kundigetal. (1964) and reviewed b y Roseman (1969). Enzyme I and the low-molecularweight protein, HPr, are common t o all sugars phosphorylated b y the system and are found primarily in the cytoplasm; enzyme I has been extensively purified f r o m E. c o l i IM. Saier, cited in Kundig and Roseman, 1971a). and HPr has been obtained in homogeneous f o r m f r o m E. coli (Anderson e t a / . , 1968) and S. typhimurium ( A . Nakazawa, cited in Kundig and Roseman, 1971a). By contrast there are a family of "enzymes 11,"

which

c a n be distinguished f r o m one another on the basis o f their specificities toward different carbohydrates. Each "enzyme 1 1 " i s a complex, membrane-bound fraction; recently,the constitutive enzymes II (for glucose, mannose,and fructose) of E. c o l i have been shown t o contain at least t w o proteins (11-A and Il-B),and t o require phosphatidylglycerol and a divalent cation (Ca2+ o r Mg2+) for activity (Kundig and Roseman, 1971b).

In E. coli and S. typhimuriurn, relatively few sugars are substrates for t h e phosphotransferase system, while In in S. aureus, all sugars that have been examined are substrates for the system (see below).

64

Carbohydrates Linkage

Method of Isolating Mutants

Transport Defect in Mutants mannose, mannitol, and sorbitol [Tanaka eta/., 1967; F o x and Wilson, 1968; Epstein eta/., 1970 (except that the latter strains d o grow o n glucosell.

(2) "Tight" mutants, which have very l o w levels o f enzyme I ( F o x and Wilson, 1968; Wangetal., 1969; Saier e t a/., 1970; Epstein eta/., 1970; Morseetal., 1971l.These strains grow poorly o n t h e above sugars and in addition o n other compounds (maltose, lactose, melibiose, glycerol, succinate) which are not substrates f o r the phosphotransferase system (PTS) in E. coli (Wang and Morse, 1966,1968; Fox and Wilson, 1968; Epstein eta/., 1970). It has been suggested that the failure of this second group of mutants t o grow o n non-PTS sugars stems f r o m an i n creased sensitivity t o catabolite repression, such that the enzymes required t o metabolize t h e sugars are n o t induced (Pastan and Perlman, 1969; Berman eta/., 1970; Epstein eta/., 1970). Consistent w i t h this view is the fact that cyclic AMP, added t o the medium, can restore b o t h inducibility and growth (Pastan and Perlman, 1969; de Crombrugghe era/., 1969; Berman eta/., 1970; Epstein eta/., 1970; Dahl etal., 1971). In addition,growth o n i n d i vidual non-PTS sugars can be restored b y suppressor mutations which i n crease the synthesis o r t h e activity o f the corresponding enzymes w i t h o u t affecting the level o f enzyme I (Wang and Morse, 1967; Morse e t a/., 1969; Wangetai., 1970; Bermaneta/., 1970; Berman and Lin, 1971: BermanKurtzetal., 1971; Dahl eta/., 19711. Recently, a temperature-sensitive pts/ mutant has been isolated and shown t o possess a heat-labile enzyme I; this result indicates t h a t p t s l i s the structural gene f o r enzyme I (Epstetn etal., 1970).

65

(Continued)

TABLE I Mutations Affecting Membrane Transport (Continued) Carbohydrates Organism

Transport System

Specificity

Escherichia coli (Con't.)

Gene ptsH

Glucose (via the phosphotransferase system)

Originally characterized as a transport system f o r glucose and alkyl @ - a n dP-glucosides (Englesberg etal., 1961; Hoffee and Englesberg, 1962; Hoffee et a/., 1964; Rogers and Yu, 1962; Hagihara etal., 1963; Winkler, 1971a). N o w k n o w n t o involve a constitutive enzyme II o f the phosphotransferase system.

Fructose (via the phosphotransferase system)

p-Glucosides (via the phosphotransferase system)

umg

ptsF

A l k y l 0 - a n d aryl Pglucosides (Schaefler, 1967; Schaefler and Maas, 1967).

Mannitol, via the phosphotransferase system

bgl6

mtlA

66

Carbohydrates Linkage

Method of Isolating Mutants

Very closely linked t o

Inability t o ferment fructose

ptsl in the orderpurC p tsl ptsH supN ( Ep s t ei n

+ mannose (Fox and Wilson, 19681 or mannitol + sorbitol

eta/., 19701, consistent

(Epstein eta/., 1970).

w i t h the data of Bourd etal. (19681 and Wang etal. (1970). Earlier evidence for close linkage came from mutant strain P34, isolated b y Gershanovitch e t a / . (1967a.b), which maps in the same region and lacks both enzyme I and HPr activities (Bourd e t a / . , 19681; P34 does not revert, so it may be a deletion covering both t h e p t s l and ptsH genes.

Transport Defect in Mutants Defective in the uptake of a variety of sugarsand in HPr (Foxand Wilson, 19681. In a recent study all theptsH mutants isolated were phenotypically leaky,and it was suggested that HPr-which presumably has a very stable tertiary structure, in view of i t s resistance t o heat denaturationmay retain at least partial activity following most amino acid substitutions (Epstein eta/., 1970).

Maps at 23.5 minutes, cotransducible w i t h purB (Kornberg and Smith, 1972).

The growth of E. coli on fructose is normally inhibited b y glucose and, in many strains, by noncatabolizable glucose analogs such as 2sleoxyglucose and amethylglucoside. umg mutants were selected b y the ability t o grow on fructose in the presence of 2deoxyglucose (Kornberg and Reeves, 1972apl.

Defective in the uptake o f glucose and amethylglucoside, and in enzyme II for glucose (Kornberg and Reeves, 1972a,b,cl. Similar mutants have been described b y Schaefler (1967) and Foxand Wilson (19681.

Located between thy and his at about 42 minutes (Ferenci and Kornberg, 19711.

Inability t o grow on fructose as sole carbon source (Ferenci and Kornberg, 1971I.

Defective in fructose uptake and in enzyme II for fructose (Ferenci and Kornberg, 1971I.

Closely linked t o bg/A (the structural gene for aryl P-glucosidesplitting enzyme) and bglC (regulatory gene), between pyrE and ile (Schaefler and Maas, 19671.

Isolated from a P-glucosidefermenting parent strain (bg/B+l b y the inability t o ferment arbutin @-hydroquinonyl~-Dglucoside) (Schaefler, 1967; Fox and Wilson, 19681.

Defective in the uptake o f P-glucosides and in enzyme II for pglucosides (Foxand Wilson, 1968).

71 minutes; cotransducible w i t h lct (Solomon and Lin, 1972).

Inability t o utilize mannitol as carbon source (Solomon and Lin, 1972).

Lack enzyme I I for mannitol (Solomon and Lin, 19721.

(Continued) 67

TABLE I Mutations Affecting Membrane Transport (Continued) Carbohydrates Organism

Transport System

Specificity

fscherichia

Gene

mt/C

coli (Con't.)

Sa/monel/a typhimurium

Phosphotransferase system

Very similar to the phosphotransferase system of (see above; Kundig and Roseman, 1 9 7 1 a p ) .

f.coli

carA btsl)

Carbohydrates Method of Isolating Mutants

Linkage

Transport Defect in Mutants

Closely linked t o m t l A (see above) and t o mtlD (thought t o be the structural gene f o r mann i t o l dehydrogenase) (Solomon and Lin, 1972).

Constitutive mutants were isolated b y the ability t o grow on mannitol as sole carbon source,at a concentration near or below the threshold of induction o f the necessary enzymes (Solomon and Lin, 1 9 7 2 ) .

Regulatory gene affecting mtlA (enzyme II f o r mannitol) and mt/D (mann i t o l dehydrogenase) (Solomon and Lin, 1972).

Originally reported t o map nearpro (Levinthal and Sirnoni, 1969); n o w thought t o map near cysA and trzA (Eerkowitz, 1971; Cordaro and Roseman, 1972) The latter position is consistent w i t h the location o f p t s l mutants in E. coli (see above).

Inability t o ferment melibiose (Simoni eta/., 1967; Levinthal and Simoni, 1969; Levinthal, 1971) o r sorbitol (Eerkowitz, 1971 1.

Defective i n the uptake of a variety of sugars and in enzyme I o f the phosphotransferase system (Simoni eta/., 1967; Levinthal and Simoni, 1969; Berkowitz, 1971). As in E. coli, there are t w o classes o f enzyme- I mutants (Saier e r a / . , 1970): (1 ) "Leaky" mutants, containing

0.55% of the wild-type level of enzyme I; these strains grow poorly on glucose, fructose, mannose, rnannitol, sorbitol, and N-acetylglucosamine). (2) "Tight" mutants, containing no detectable enzyme I (less than 0.1% o f the wild-type level); these strains grow poorly on the above sugars and in addition on maltose, lactose, melibiose, and glycerol. They are also reported t o be poorly motile and t o show altered activities o f some membranehound enzymes, suggesting a structural defect in the cell membrane (Saier and Roseman, 19726.b). The sensitivity o f pfs mutants t o carbohydrate repression is discussed b y Saier and Roseman (1972b) and Saier e t a / . (1971).

Adjacent t o carA. in the order cysA rrzA cart? carA (Cordaro and Roseman, 1972).

Inability t o ferment melibiose (Levinthal and Simoni, 1969).

Defective in the uptake o f a variety of sugars and in HPr o f t h e phosphotransferase system (Levinthal and Simoni, 1969; Saier eta/., 1970).

-

(Continued) 69

TABLE I Mutations Affecting Membrane Transport (Continuedl Carbohydrates Organism

Transport System

Salmonella typhimurium (Con 't.I

Mannirol (via the phosphotransferase system)

Aerobacter aerogenes

Specificity

Gene m tl

Deoxyribose

Not characterized.

Phosphotransferase system

See description under E. coli.

deo P

Mannitol (via the phosphotransferase system) Fructose (via the phosphotransferase system)

"Enzyme 11" for fructose consists of a high-molecularweight protein (probably constitutive) and a smaller, inducible protein (Km factor), which increases the affinity of the system for fructose, from a K,,, of 20-80 mM t o a K,,, of less than 1 m M (Hanson and Anderson, 1968).

-

Streptococcus lactis strain C2F

Lactose (via the phosphotransfer ase system)

Two wild-type strains of S. lactis appear t o transport lactose b y different mechanisms-strain C2F via the phosphotransferase system, and strain 7962 by some other route (McKayetal., 1970).

-

Streptococcus lactis strain 7962

Lactose

Staphylo. coccus aureus

Phosphotransferase system

Similar t o the phosphotransferase system of E. coli, except for the involvement of factor Ill (see below) (Kennedy and Scarborough, 1967; Simoni etal., 1968; Laue and MacDonald, 1968a.b; Hengstenberg et a/., 1968b. 1969).

70

car

Carbohydrates Linkage

Method o f Isolating Mutants

Transport Defect in Mutants

Maps at about 115 minutes o n the Salmonella chromosome, close t o the gene for mannitol 1 -phosphate dehydrogenase (Berkowitz, 1971).

Inability t o ferment mannitol (Berkowitz. 1971 ).

Defective in the uptake o f mannitol and in mannitol phosphotransferase (Berkowttz. 1971).

Maps at about 34 minutes (Hoffee, cited in Sanderson, 1970).

Inability t o ferment deoxyribose (Hoffee, 1968).

Defective in the uptake of deoxy ribose (Hoffee, 1968).

Inability t o ferment mannitol (Tanaka and L i n , 1967).

Lack enzyme 1;defecttve in the uptake of D-mannttol (and presumably, on the basis o f growth characteristics, D-sorbitol , D-g lucose , D-mannose, and D-fructose) (Tanaka and L i n , 1967).

Same as above

Lack HPr; same transport defect as enzyme4 mutants (Tanaka and Lin, 1967).

Same as above.

Lack enzyme 11 for D-mannitol (Tanaka and Lin. 1967).

Inability t o ferment fructose (Sapico etal., 1968).

Lack t h e K, factor b u t s t i l l contain t h e l o w a f f i n i t y enzyme II for fructose. Grow slowly on fructose b u t normally on glucose, mannose, mannit o l , and glycerol (Hanson and Anderson, 1968).

Inability t o ferment lactose (McKav e t al., 1970).

Defective in enzyme I1 and factor I l l for lactose; contain normal amounts o f enzyme I and HPr (McKayetal., 1970).

Same as above

Defective in lactose uptake (McKay eta/., 1970).

Inability t o ferment sucrose, mannitol, or maltose (Egan and Morse, 1965a) or sucrose t fructose, or lactose t mannose (Hengstenberg eta/., 1969).

Defective in the uptake o f a variety o f sugars (lactose, sucrose, maltose, galactose, mannitol, mannose, fructose, trehalose, glycerol), all believed to be substrates o f the phosphotransferase system in S. aureus (Egan and Morse,

71

(Continued)

TABLE I Mutations Affecting Membrane Transport (Continued) Carbohydrates Organism

Transport System

Specificity

Gene

Staph ylo coccus aureus ICon't.)

Lactose (via the phosphotransferase system)

S. aureus differs from E. coli in phosphorylating a wider variety o f sugars (including lactose) by means of i t s phosphotransferase system, and also in requiring-in addition t o the sugarspecific, membranebound enzyme Il-a sugar-specific cytoplasmic factor Ill (Simoni er a/., 1968; Hengstenberg eta/., 1969). Factor Illlachas now been purified and characterized (Nakazawa eta/., 1971; Schrecker and Hengstenberg, 1971; Hays and Simoni, 1971).

Pseudo rnonas aeruginosa

Glycerol

Wild-type P. aeruginosa appears to have two inducible systems for the uptake o f glycerol. w i t h Km's of 7.8 x 10-6 M and 4.8 x 1 0 4 M. It is not clear whether uptake occurs by facilitated diffusion (as in E. coli) or b y active transport; an energy requirement has not been demonstrated (Tsay eta/., 1971).

Neurospora crassa

Glucose

Dglucose i s taken up by t w o systems in Neurospora: (1 A high-affinity system. repressed during growth on glucose, with a K,,, of 1-7 x 10-5 M for glucose and 4 x 10-3 M for sorbose. This system is clearly energydependent (inhibited b y azide and dinitrophenol), and can accumulate 3-0-methylglucose as the free sugar against a considerable concentration gradient (KlingmOller. 1967b.c; KlingmOller and Huh, 1972; Scarborough, 1970ap; Schneider and Wiley, 1971a.b.c; Nevilleetal., 1971); (2) A low-affinity system, present constitutively, with a Krn of 8-25 x 10-3M for glucose. Scarborough (1970b) reported very little uptake of sorbose via this system at an external concentration o f 10 mM, but it seems likely that sorbose is a substrate at much higher concentrations (Crocken and Tatum, 1967). The mechanism of uptake by the low-affinity system has not yet been settled. Scarborough (1970a) reported that 3-0methylglucose could not be accumulated against a con-

72

cnc

-

-

sor

~

~

_

_

_

Carbohydrates Linkage

Method o f Isolating Mutants

Transport Defect in Mutants 1965a.b; Hengstenberg e f a/., 1968b). and defective i n enzyme I (Hengstenberg e t a/., 1968a. 1969; Simoni e t a/., 1968).

Inability t o ferment lactose

+ mannitol (Hengstenberg eta/., 1969). Like /acY in E. coli, closely linked t o /acZ (the structural gene for pgalactosidase) and lac/ (the regulatory gene).

Inability t o ferment lactose (McClatchy and Rosenblum, 1963; Morseetal., 1968a.b) or lactose + galactose (Hengstenberg eta/., 19691.

Defective in t h e uptake o f lactose and in enzyme Illac( M o m e t a / . , 1968; Hengstenberg eta/., 1968, 1 9 6 9 ) .

Inability t o ferment lactose (Hengstenberg eta/., 1968b, 1969).

Defective in lactose uptake and in factor Illlac(Hengstenberg e t a/., 1968b. 1969).

Method n o t given,

Defective in glycerol uptake (a complete kinetic analysis was n o t reported, so it is n o t clear whether the mutant lacks one of the t w o uptake systems o r whether it lacks b o t h ) . Very l o w levels o f glycerol4inding protein (Tsayetal., 1971).

Resistance t o sorbose, which in hibits the growth o f wild-type Neurospora ( KI ingmll Iler and Kaudewitz , 1966; KI ingmul ler , 1967a).

T w o o f the classes o f sorbose-resistant mutants ( A and B; mapping o n l i n k age groups V I and VII, respectively) were found t o be partially defective in sorbose uptake (Klingmuller, 1967d). Fructosegrown cells (in which the h i g h a f f i n i t y glucosesorbose system should have been derepressed) showed essentially normal K,'s f o r sorbose, b u t a 3 0 4 3 % decrease i n Vmax; glucose uptake appeared normal, however. In view o f the overlapping substrate specificities o f the t w o hexose transport systems in Neurospora, detailed kinetic studies are needed of b o t h repressed (glucosegrown) and derepressed (fructosegrown) mutant cells t o determine which o f t h e t w o systems i s altered and t o what extent.

+ galactose

Six sor genes have been reported, located o n linkage groups I, Ill, V, Vl,and V I I (and one not yet located, b u t not closely linked t o any of the others) (Klingmilller, 1967a).

Defective in HPr (Hengstenberg eta/., 1969). and presumably in the uptake o f the above sugars.

73

(Con tinued)

TABLE I Mutations Affecting Membrane Transport (Continued) Carbohydrates Organism

Transport System

IC0n't.l

Saccharomyces cere visiae

Gene

centration gradient,and concluded that the low-affinity system involved facilitated diffusion. However, Schneider and Wiley (1971a) did observe accumulation of unaltered 3-0-methylglucose against a concentration gradient, inhibited b y azide and dinitrophenol, and concluded that the l o w a f f i n i t y system was capable of active transport.

Neurospora crassa

Aspergillus nidulans

Specificity

Hexoses

Kinetic evidence indicates that there are at least four constitutive transport systems for hexoses in A. nidulans : (1) glucose (K, = 4-6 x 10-5 M ) , competitively inhibited by glucose analogs (2-deoxy-D-glucose, 3-0-methyl-D-glucose, G-deoxy-D+lucose) and at higher concentrations, by D-galactose (Ki = 1.1 x M ) and D-mannose (Ki = 1.3 x M ) (Brown and Romano, 1969; Markand Romano, 1971). MI, competitively (2) D-galactose (K, = 3 x inhibited by D-fucose (Ki = 2.9 x l o 4 M I , D-glucose (Ki = 5.0 x l o 4 MI, and D-mannose (Ki = 5.9 x l o 4 M (Mark and Romano, 1971). (3) D-fructose ( K , = 2 x l o 4 M ) ,not inhibited by any sugar tested (Mark and Romano, 1971). (4) L-sorbose, competitively inhibited by D-glucose and D-mannose; kinetic constants not reported (Elorza and Arst, 1971).

Lactose

Not characterized

lac-1

Galactose

An inducible system for Dgalactose and its nonmetabolizable analogs D-fucose and L-arabinose. The mechanism of uptake is not clear; various investigators have postulated active transport (de RobichonSzulmajster, 1961 1, phosphorylative transfer (Van Steveninck and Rothstein, 1965; Van Steveninck and Dawson, 1968; Van Steveninck, 1972). and facilitated diffusion (Cirillo. 1968; Kuo e t a/., 1970; Kuo and Cirillo, 1970).

gal2

sorA

i-, C,

~ 1 3 . gal4

74

Carbohydrates Linkage

Method of Isolating Mutants

Transport Defect in Mutants

Maps on linkage group I (Elorza and Arst, 1971).

Resistance to sorbose; must be distinguished f r o m s o r B mutants, in which sorbose resistance results f r o m loss o f phosphoglucomutase (Elorza and Arst, 1971).

Greatly reduced uptake o f Lsorbose, w i t h a slight reduction in the uptake o f D-glucose (Elorza and Arst, 1971). Presumably,sorA mutants are defecfive i n the sorbose transport system described under Specificity, b u t this interpretation is complicated b y the fact that they are also resistant t o 2deoxy-D-glucose (a substrate f o r the glucose transport system; Mark and Romano, 1971).

Maps on linkage group VI (Gajewski etal., 1972).

Inability t o utilize lactose as carbon source (Gajewski e r a / . , 1972).

Greatly reduced uptake o f lactose (Gajewski e t a / . , 19721.

Maps o n linkage group X I I . N o t linked to the structural genes f o r the galactose pathway enzymes, which map in a cluster o n linkage group I I (Douglas and Hawthorne, 1964; Mortimer and Hawthorne, 1966; Bassel and Mortimer. 1 9 7 1 ) .

Can metabolize galactose only at high external concentrations (Douglas and Condie, 1954).

Defective in the uptake o f D-galactose (de RobichonSzulmajster. 1961) and D-fucose and L-arabinose (Cirillo, 1968; Kuo etal., 1970).

Regulatory mutants w i t h altered inducibility o f b o t h t h e galactose transport system and the galactose pathway enzymes: i - a n d C mutants are constitutive, and ga13 and ga14 mutants are noninducible (Douglas and Hawthorne, 1966; Cirillo, 1968).

75

(Continued)

TABLE I Mutations Affecting Membrane Transport (Continued) Carbohydrates Organism

Transport System

Man

Glucose (kidney, intestine)

Specificity Intestine: a Naclependent system for D-glucose, D-galactose, and structurally related hexoses (reviewed by Crane, 1960,1968). K, for D-glucose (in the presence of saturating amounts of Na+) is 1.5 m M in the hamster intestine (Lyon and Crane, 1966) and 4.2 m M in man (Elsasetal., 1970).

Disease Glucosegalactose malabsorption

Kidney: There appear to be two transport systems for glucose in the proximal renal tubule,on the basis of in vivo studies in dogs (Silverman er al., 1970) and man (Elsasetal., 1970). and uptake measurements on isolated brush border preparations from the rabbit (Chesnev, 1971; Busseetal., 19721. System A (Busse eta/., 1972) resembles the intestinal glucose transport system in having a relatively low affinity for glucose (K, = 3 m M in the rabbit) and i n being inhibited b y D-galactose; system B has a higher affinity for glucose (K, = 0.07 mM) and is strongly inhibited b y D-mannose but not b y D-galactose.

Renal glyco. suria

76

Carbohydrates Mode o f Inheritance Autosomal recess1ve (Elsasefal., 1 9 7 0 ) . Intestinal glucose transport is absent in h o m o zygotes and shows a somewhat reduced Vmax ( t o 75% o f the normal value) in heterozygotes. Renal glucose transport is partially impaired in homozygotes and slightly (but probably significantly) lowered in heterozygotes.

Abnormal Phenotype Profuse diarrhea following the ingestion o f glucose, galactose, o r disaccharides containing one or b o t h o f these hexoses. Oral loading with glucose, galactose, or 3-O-methylglucose results in little or n o increase in b l o o d hexose and in the prompt appearance o f large quantities of free hexose in the feces (reviewed in Rosenberg. 1969).

Transport Defect Intestine: Homozygotes appear t o lack the intestinal transport system for glucose and galactose. Biopsy specimens f r o m t w o such patients failed t o accumulate glucose in excess o f the medium concentration (Eggermont and Loeb, 1966; Elms eta/., 1970),and an isolated brush border preparation f r o m another patient did n o t bind Dgalactose-14C o r p h l o r i z i n 3 H (a competitive inhibitor of transport), as shown b y autoradiography (Schneider eta/., 1966). The defect appears t o b e limited t o the glucosegalactose transport system and does n o t extend t o Nadependent transport generally; Eggermont, Meeuwisse, and their colleagues found t h a t 22Na absorption in vivo, and Na,K&imulated ATPase activity and Nadependent amino acid accumulation in vifro, were normal in patients w i t h glucosegalactose malabsorption (Eggerrnont and Loeb. 1966; Meeuwisse and Dahlqvist, 1968). Kidney: The single homozygote studied in detail had a n abnormal renal titration curve f o r glucose, w i t h a reduced minimal threshold. (The maximal reabsorptive capacity could not be determined accurately because the filtered load o f glucose was inadequate). The results are consistent w i t h the view that t h e patient lacked one o f the t w o renal transport systems for glucose (presumably system A ; see Specificity) b u t retained t h e other, h i g h e r a f f i n i t y system (B) (Elsasetal., 1970).

Probably several d i f ferent autosomal recessive mutations. Three pedigrees have been studied in detail (Elsas and Rosenberg, 1963; Elsasefa/., 1971). I n pedigree Holm, the m u -

Appearance o f abnormally large amounts o f glucose in the urine, b u t w i t h no clinical symptoms (reviewed in Rosenberg, 1969)

77

Kidney: T w o types o f renal glycosuria have been distinguished, based o n the shape o f the in vivo glucose titration curves. I n one (type A ) , the curve has a normal shape b u t reaches an abnormally l o w TmG (maximal tubular absorptive capacity f o r glucose); in t h e other ( t y p e 6).glyco-

(Continued]

TABLE I Mutations Affecting Membrane Transport (Continuedl Carbohydrates Organism

Transport System

Specificity

Disease

Man (Con 't.)

Purines and Pyrimidines Organism

Transport System

Specificity

Escherichia coli

Uracil

Not characterized.

Salmonella typhimurium

Guanine, h y p o xanthine, xanthine

Purine transport in E. coli and presumably in Salmonella is mediated b y membrane-bound phosphoribosyltransferases, and results in the intracellular accumulation o f nucleoside monophosphates. The transport o f guanine and hypoxanthine involves a phosphoribosyltransferase (or possibly more than one) specific f o r the 6-OH purines (Hochstadt-Ozer, 1972a.b). while adenine is transported b y a separate enzyme (Hochstadt-Ozer and Stadtman, 1971a.b.c). Extracellular purine nucleosides are first cleaved to the free bases b y nucleoside phosphorylases and then taken u p b y the same route (Hochstadt-Ozer, 1972a).

78

Gene uraP

-

Carbohydrates Mode of Inheritance

Abnormal Phenotype

tation is clearly i n herited as an autosomal recessive, leading t o mild type-A glycosuria (see Transport defect) in the heterozygotes and severe type-A glycosuria in the homozygote. Pedigrees Cov and Hol can also b e interpreted i n terms of auto. soma1 recessive mutations (one as in pedigree Holm; another producing m i l d type-A glycosuria in the h o m o zygote; and a t h i r d producing type-B glycosuria i n the heterozygote), b u t the data are less complete and the possibility o f autosoma1 dominants w i t h variable expression cannot be ruled o u t

Transport Defect suria begins at an abnormally l o w filtration rate, b u t t h e TmG-when reached-is normal The t w o kinds o f titration curves have been interpreted kinetically t o mean that type A i s the result o f a reduced VmaX of glucose transport, while type B i s the result of an increased K, (Woolf e t 4.. 1966). Intestine: Glucose transport I S normal (Elsas and Rosenberg, 1969). Because renal glycosuria affects the kidney and not the intestine, it is reasonable t o suggest that the defect is in the higher-affinity glucose transport system (system B). In vitro measurements, perhaps w i t h the k i n d o f brush border preparation described b y Busse et a/. (1972). would be required t o test this idea, and t o conf i r m the presence o f a normal system A in patients w i t h renal glycosuria.

Purines and Pyrimidines Linkage Maps at 50 minutes (see Taylor, 1 9 7 0 ) .

-

Method of Isolating Mutants

Transport Defect in Mutants

Resistance t o 6-azauraciI (see Taylor, 1 9 7 0 ) .

Defective in uracil uptake (see Taylor, 1970).

Resistance to 6 m e r c a p t o p u rine or 8azaguanine (Kalle and Gots, 1961).

Defective in uptake o f guanine and hypoxanthine (Zimmerman and Magasanik, 1964). Guanine-hypoxanthine phosphoribosyltransferase activity was present in cell-free extracts f r o m the mutant (Zimmerman and Magasanik. 1964). b u t appeared t o b e an altered enzyme w i t h an abnormal elution pattern f r o m DEAE-cellulose and an abnormal substrate specificity (Adye and Gots, 1966).

Resistance t o 8azaguanine (Thakar and Kalle, 1 9 6 8 ) .

Defective i n the uptake o f guanine and xanthine; purine phosphoribosyltransferases appeared normal (Thakar and Kalle, 1 9 6 8 ) .

(Continued) 79

TABLE I Mutations Affecting Membrane Transport (Continued) Purines and Pyrimidines Organism

Transport System

Specificity

Salmonella typhimurium 1Con't.l

Gene gxu

Streptococcus faecalis

Purines

Not characterized

Aspergillus nidulans

Adenine, guanine, hypoxanthine

Not characterized.

Xanthine, uric acid

Not characterized.

uap

ua Y

Saccharornyces cere visiae

Cytosine

Not characterized.

cyt-p, FCY-2

Uracil

There appear t o be t w o uptake systems for uracil in wild-type S. cerevisiae, w i t h Km's o f 5 x 10-6 M and 2.5 x 104 M. (Grenson, 19691.

ura-p, FUR4

80

Purines and Pyrimidines Linkage NearproAB (Benson 8t a/., 1972).

Method of Isolating Mutants 2 3 o f 5 3 p r o A B deletion mutants were found t o be also deleted in gxu, an adjacent gene. The mutants were resistant t o Bazaguanine, and when carrying an additional purE mutation were unable t o use guanine or xanthine as a purine source (Benson 8f a/.. 1972).

Transport Defect in Mutants Defective in uptake and phosphoribosyltransferase activities for guanine and xanthine (Benson 8t a/., 19721. The genetic relationship among these various purine transport mutants remains t o be established.

Resistance t o 8-azaguanine or 6-mercaptopurine (Brockmaneral., 1961).

Lack guanine-hypoxanthine phosphoribosyltransferase; transport not studied (Brockman etal., 1961 1.

Resistance to Bazaxanthine (Brockman etal., 1961 1.

Lack xanthine phosphoribosyltransferase (Brockman eta/., 1961).

Resistance t o Bazsadenine (Brockman etal., 1961 1.

Lack adenine phosphoribosyltransferase (Brockman era/., 19611.

Resistance t o Bazaguanine or t o purine (Darlington and Schazzochio, 1967).

Thought t o be defective in the uptake of adenine,guanine, and hypoxanthine (an indirect argument based on growth data) (Darlington and Schazzochio, 1967).

Resistance t o 2-thiouric acid or Z-thioxanthine (Darlington and Schazzochio, 1967).

Thought t o be defective in the uptake of xanthine and uric acid (an indirect argument based on growth data) (Darlington and Schazzochio, 1967).

Same as above.

Thought t o be a regulatory gene controlling the xanthine-uric acid transport system and also xanthine dehydrogenase and urate oxidase (Darlington and Schazzochio, 1967).

Not yet established whether cyr-p and F C Y Z are allelic.

Inability t o use cytosine as sole source of nitrogen (Grenson, 1969); resistance t o 5-fluorocytosine (Jund and Lacroute. 1970).

Both cyr-p and FCY-2 are defective in cytosine uptake (and have a normal level o f cytosine deaminase, which converts cytosine t o uracil) (Grenson, 1969; Jund and Lacroute, 1970).

ura-p and F U R 4 are allelic (Grenson, 1969).

Resistance t o 5-fluorouracil or, in a cpa strain (which lacks the argininespecific carbarnil phosphate syn-

Defective i n uracil uptake (by both systems) (Grenson, 1969; Jund and Lacroute. 1970).

81

(Continuedl

TABLE I Mutations Affecting Membrane Transport (Continued) Purines and Pyrimidines Organism

Transport System

Specificity

Gene

Saccharornyces cerevisiae (Con’t.)

Uridine

N o t characterized.

Adenine, hypoxanthine

Wild-type S. cerevisiae appears t o take u p purines b y t w o systems: an adenine transport system (K, = 1.2 x M I , which also accepts hypoxanthine. pyrimidine (4-APP). and 4aminopyrazolo [3,4-d] some other analogs;and a guanine transport system which accepts hypoxanthine and E-azaguanine (Pickering and Woods, 1972b).

82

wid-p, FUI-1

app-1

Purines and Pyrimidines Linkage

Method o f Isolating Mutants

Transport Defect in Mutants

thetase and is sensitive t o inhibition b y uracil, uridine. or cytosine), resistance t o uracil (Grenson, 1969; Jund and Lacroute. 1970). ups and F U R - I are allelic (Grenson, 1969).

Resistance t o uracil o r cytosine (Grenson, 1969); resistance t o 5-fluorouracil (Jund and Lacroute, 1970).

Lack uracil phosphoribosyltransferase activity and show reduced rates o f uracil uptake (Grenson, 1969; Jund and Lacroute, 1 9 7 0 ) . Grenson (1969) has concluded that the effect on uptake is an indirect one-that uracil is accumulated intracellularly b y ups mutants and blocks the transport system b y feedback inhibition. Consistent w i t h this idea, she observed that p y r i midine starvation o f a ups strain resulted in a partial restoration of transport. Hochstadt-Ozer and Stadtman (1971b) have pointed out, however, that t h e amount o f transport was small in these experiments (about 3%o f that seen in other starved strains), that ups strains might have l o w activities of uracil phosphoribosyltransferase (which was not assayed directly), and that an obligatory role o f t h e phosphoribosyltransferase in transport cannot be ruled o u t . In this case the identity of t h e ura-pgene product and i t s role in transport, relative to the phosphoribosyltransferase, w o u l d be interesting t o invest igate.

N o t yet k n o w n whether wid-p and FUI-1 are allelic.

Resistance o f a cpa strain t o uridine (Grenson, 1969); r e sistance t o 5-fluorouridine (Jund and Lacroute, 1970).

Defective in uridine uptake (Grenson, 1969; Jund and Lacroute, 1 9 7 0 ) .

Resistance t o 4-APP (see Specificity; Pickering and Woods, 1972a.b).

Defective in the uptake o f adenine and hypoxanthine. Purine phosphoribosyltransferase activities are higher than normal in app-1 mutants, h o w ever. Either the mechanism of purine uptake is different f r o m that in bacteria (see above) (Pickering and Woods, 1972a.b). or the mutation

-

83

(Continued)

TABLE I Mutations Affecting Membrane Transport (Continued) Purines and Pyrimidines Organism

Transport System

Specificity

Gene

Sacchsromyces cerevisiae (Con’t.)

Chinese hamster cells

Thymidine

Not characterized.

TP-

Miscellaneous Compounds Organism

Transport System

Specificity

Escherichia Vitamin 812

Uptake of vitamin 612 by E. coli consists of two

coli

phases: (1) an initial rapid phase which is energyindependent and is thought to represent the binding of 812 to a membrane carrier (Km = 5 x 10-9 M ) ;and (2) a slower, energydependent phase (Di Girolamo and Bradbeer, 1971; Taylor et a/., 1972).

Biotin

Not yet clear whether there is a specific biotin transport system in E. coli, or whether biotin simply diffuses into the cell and is covalently bound (Campbell eta/., 1972).

Thiamine

An energydependent transport system with a Km of 8.3 x l o 7 M, inhibited by the analogs pyrithiamine and oxythiamine (Miyata et a/., 1967; Kawasaki etal., 1969a). Thiamine is normally accumulated as the pyrophosphate, and because the kinases are membranebound, there has been speculation that uptake occurs by group translocation, as in the bacterial phosphoenolpyruvate phosphotransferaseand purine phosphoribosyltransferase systems. Recently, however, mutants lacking thiamine kinase have been shown to take up free thiamine against a concentration gradient, a result that argues against an obligatory role of phosphorylation in transport; likewise, mutants lacking thiamine monophosphokinase can accumulate thiamine mono84

Gene

-

bir

-

Purines and Pyrimidines Linkage

Method of Isolating Mutants

Transport Defect in Mutants has altered the phosphoribosyltransferase, increasing its activity in cellfree extracts while at t h e same time making it unable t o function in transport.

-

Direct autorad lographic screening method (Breslow and Goldsby, 19691.

Defective in thymidine uptake (Breslow and Goldsby, 1969).

Miscellaneous Compounds Linkage

Between argC and thiA, close t o rif (Campbell eta/., 1972).

Method of Isolating Mutants

Transport Defect in Mutants

Isolated, in a parent strain which requires either vitamin 812 or methionine, by t h e inability t o grow at low 612 concentrations in the absence of methionine (Di Girolamo eta/.. 1971).

Defective in the initial rapid binding of 612 (Di Girolamoetal., 1971).

Same as above.

Defective in the slow, energydependent phase of 812 uptake and i n the conversion of 6 1 2 t o other cobalamins (Di Girolamo e t a/., 19711.

Inability of a biotin-requiring strain (bio-) t o grow at low biotin concentrations (Campbell eta/., 1972).

The primary effect of the b i r mutation i s not yet certain. bio-bir- strains are deficient in biotin uptake (or binding); and biot bir- strains are derepressed for at least one biosynthetic Qene (bioD),so that they overproduce and excrete biotin (Campbell eta/., 1972).

Isolated, in a thiamine-requiring strain of E. coli, by the inability t o grow at low thiamine concentrations (Kawasaki eta/., 1969b).

Defective in thiamine uptake ( b u t with elevated amounts of thiamine kinase) (Kawasaki eta/., 1969b).

(Continuedl 85

TABLE I Mutations Affecting Membrane Transport (Continued) Miscellaneous Compounds Organism

Transport System

Escherichia coli (Con’t.)

Specificity phosphate (Kawasaki and Yamada, 1972). A thiaminebinding protein ( K D = 10-7 M ) has been isolated f r o m E. c o l i by osmotic shock; both it and thiamine transport are repressed by growth in the presence o f thiamine (Iwashimaetal., 1971; Kawasaki and Esaki, 1971).

Shikimic acid

Not characterized.

Glycolate

Not characterized.

Bacillus subtilis

Citrate

An inducible transport system for citrate (K, = 2.3 x l o 3 M ) . conveniently studied i n an aconitaseless mutant that cannot metabolize citrate (Willecke and Pardee. 1971a).

Saccharomyces cefevisiae

Ureidosuccinic acid

Ureidosuccinic acid (USA), the first specific intermediate o f the pyrimidine pathway, is transported by a system which i s derepressed in a low-nitrogen medium (e.g., proline) and repressed in a high-nitrogen medium (e.g., ammonium sulfate, glutamate) (Drillien and LaCroute, 1972).

Aspergillus nidulans

Gene

S-Adenosylmethionine

A transport system for S-adenosylmethionine (K, = 3.3 x l o 6 M ) and S-adenosylhomocysteine, competitively inhibited by S-adenosylethionine (Murphy and Spence, 1972).

Acetate

Not characterized.

shiA

-

weP-1

ure-1, 2.3.4

Sam-pl

-

Miscellaneous Compounds Linkage

Closely linked to his andaroD (Pittard and Wallace, 1966).

Method of Isolating Mutants

Transport Defect in Mutants

Isolated, in an aroD parent strain (unable t o convert dehydroquinate t o dehydroshikimate) by the inability t o grow on shikimic acid. Two mutants were obtained: A82879, which required all the aromatic amino acids and vitamins for growth, and AB2880, which required only shikimic acid and tyrosine (Pittard and Wallace, 1966).

Defective in the uptake of shikimic acid and probably dehydroshikimic acid. There was no measureable uptake in AB 2879, and uptake in A82880 was reduced b y approximately 40% (Pittard and Wallace, 1966).

Ability t o use glyoxylate but not glycolate as sole source of carbon and energy (Ornston and Ornston, 19701.

Defective in the uptake o f glycolate (Ornston and Ornston, 1970).

Two mutants have been isolated with partial defects in citrate transport (McKillen eta/., 1972).

-

Resistance to USA, which inhibits the growth of the wildtype strain o n proline medium (Drillien and Lacroute, 1972).

Defective in USA uptake (Drillien and Lacroute, 19721.

Ability of a pyrimidine-requiring parent strain t o grow on USA in a high-nitrogen mediu m (Lacroute, 1971; Drillien and Lacroute, 1972).

Constitutive for the USA transport system and the general amino acid transport system, both of which are normally repressed in high-nitrogen medium. I t has been postulated that the ure strains are not true regulatory mutants but instead are altered in nitrogen metabolism (Drillien and Lacroute, 1972).

Isolated, in a methioninerequiring parent strain (rnet2a). by the inability t o grow on S-adenosylrnethionine (Spence and Shapiro, 1967).

Defective in the uptake o f S-adenosylmethionine and S-adenosylhomocysteine (Spence, 1971; Murphy and Spence, 1972).

Inability t o grow on acetate (Lanier, 1971).

Defective in the uptake of acetate and probably pyruvate (Lanier, 1971).

87

(Con tin uedl

TABLE I Mutations Affecting Membrane Transport (Continued) Miscellaneous Compounds Organism

Transport System

Man

Vitamin 8 1 2

Spec if ic it y The absorptlon of vitamin 8 1 2 f r o m the intestine requires an ”intrinsic factor” elaborated b y the gastric mucosa.

Disease Heredltary malabsorption o f vitamin B12

Inorganic Cations Organism

Transport System

Escherichia coli

K

Specificity Wild-type E. coli K-12 takes up K b y at least t w o systems:

Gene kdpA, B

C. D

( 1 ) A high-affinity system, which allows cells t o grow rapidly in media containing l o w concentrations o f K (as l o w as 10-5 M ) b u t i s repressed when cells are grown in high-K media (Epstein and Waters, cited in Epstein and Kim, 1971).

(2) A lower-affinity system. thoroughly characterized kinetically. which takes u p K in exchange for Na and H (Schultz e t a / . , 1963). Recently, Bhattacharya e t a / . (19711 showed that membrane vesicles f r o m E. coli B and K-12 can accumulate K (and R b ) in a process that is stimulated by the cyclic depsipeptide antibiotic valinomvcin.

88

(kac)

Miscellaneous Compounds Mode of Inheritance Probably an autosomal recessive (Imerslund, 1960; Spurlinget al., 1964; Mohamed et a/., 1966). In two families parents of affected children have shown normal absorption of vitamin 612 (Gr%beck etal., 1960; Spurling etal., 1964). while in a third family, the parents showed a partial defect (Mohamed eral., 1966).

Abnormal Phenotype Megaloblastic anemia which responds t o parenteral therapy with vitamin 812 but not t o intrinsic factor; persistent proteinuria (not understood) (Imerslund, 1960; Grassbeck eral., 1960; Spurling et al., 1964; Mohamed e t al., 1966). Must be distinguished from classic pernicious anemia in the adult, caused by a deficiency of intrinsic factor.

Transport Defect Presumably a defect in the intestinal transport o f vitamin 612.

Inorganic Cations Linkage Four cistrons, mapping in a cluster in the order DCBA near the gal region, and possibly constituting an operon (Epstein and Davies, 1970).

Method of Isolating Mutants Inability t o grow at very low K concentrations (2 x M) (Epstein and Davies, 1970).

Inability t o grow at low K Three independently concentrations (E. coli 6; isolated kac mutants map near gal (Burmeister, Damadian, 1966, 1968). 1969) and may be allelic with kdp (see above).

trkA and Bare closely linked t o strA; trkC t o pdxA; rrkD t o ilv; and trkE t o trp (Epstein and Kim, 1971). Nocom-

Isolated, from a kdp parent strain (see above), by the inability t o grow at intermediate K concentrations (10-4 t o 5 x 10-3 M ) (Epstein and Kim, 1971).

Transport Defect in Mutants Lack the high-affinity K transport system (Epstein and Waters, cited in Epstein and Kim, 1971).

Defective in K uptake at low concentrations (below lo4 M ) but normal at higher concentrations (Damadian, 1968). May lack the high-affinity K transport system described for E coli K-12 by Epstein and Waters (see above). Also defective in the uptake o f inorganic phosphate (Damadian. 1967). possibly indicating a link between K and PO4 uptake (Weiden e t a l . , 1967). Defective in the lower-affinity system (or systems). trkA, D, and E have reduced rates of K uptake, and t r k 6 and Care defective in the retention o f K (Epstein and Waters, cited i n

(Continued) 09

TABLE I Mutations Affecting Membrane Transport (Continued Inorganic Cations Organism

Transport System

Specificity

Gene

Escherichia coli (Con't.)

Mg

Kinetic studies of Mg transport in wild-type E. coli cor have not yet led t o a clear picture of the system or systems involved. Km's of 3 x lo4 M t o 5 x lW4 M have been reported for cells grown under various conditions (broth, synthetic medium), and C o and Mn have shown t o inhibit uptake (Silver, 1969;Silver and Clark, 1971; Lusk and Kennedy, 1969;Nelson and Kennedy, 1971). mng Recently, on the basis of studies with Co-resistant mutants (see Transport defect), Nelson and Kennedy (1972)have postulated that wild-type E. coli has two transport systems for Mg:

( 1 ) a constitutive system, competitively inhibited by Co; and (2)a system which is repressed during growth at high Mg concentrations (and has l i t t l e affinity for Co). The kinetic constants of the two systems (K, for Mg. Ki for Co and Mn, VmaXI have not yet been reported.

90

Inorganic Cations Linkage

Method of Isolating Mutants

plementation occurred among 19 trkA mutations or among 6 frkB mutations, indicating that these are probably single cistrons. Not enough trk C, D, and E mutations were available to permit significant comDlementation studies.

Transport Defect in Mutants Epstein and Kim, 1971).Consistent with the whole-cell results, membrane vesicles from a trkA mutant were shown t o have abnormally low rates of valinomycinstimulated K uptake (see Specificity), and vesicles from a trkB mutant were (at least in some experiments) defective in K retention (Bhattacharyaet a/., 1971). t r k D appears t o control a minor transport system (possibly a third, previously undetected system) which is normally overshadowed by the major transport system associated with the rrkA locus; r r k D mutants are isolated only in trkA- strains (Epstein and Kim, 1971 ).

Maps near leu (Lubin Inability t o grow at low K conmay and Kessel, 1960); centrations (€. co/i B; Lubin be allelic with trkC and Kessel, 1960). (Epstein and Kim, 1971).

Defective in the retention of K (Lubin and Ennis, 1964;Lubochinsky eta/., 1964,1966;Gunther and Dorn, 1966;Zimmermann and Pilwat, 1971; Pilwat and Zimmermann, 1972).

Ability to grow in the presence M Co (Nelson and of 5 x Kennedy, 1971,1972).

cor mutants are thought t o lack the constitutive Mg-Co transport system, since they take up very l i t t l e Co and, when grown at high Mg concentrations, very little Mg (Nelson and Kennedy,

1972). Maps between aroD and his (Silver eta/., 1972).

Ability to grow in the presence of M Mn (Silver eta/.,

1972).

mng mutants take up Mg with a normal Km (studied over the range 3 x 10-5t o 1 x lo4 M )but are slightly less sensitive t o Mn inhibition (Silver e t a/., 1972);these experiments were performed with broth-grown cells, which would normally possess both of the Mg transport systems postulated by Nelson and Kennedy

11972). rnng mutants are not resistant t o Co, nor are cor mutants resistant t o Mn (Silver ef a/., 1972).suggesting that they may be defective in different transport systems. This notion could be tested b y a kinetic comparison (over a wide range of Mg, Mn. and Co concentrations) o f the two mutants grown on low and high Mg.

(Continued) 91

TABLE I Mutations Affecting Membrane Transport (Continued) Inorganic Cations Organism

Transport System

Escherichia Fe coli (Con't.)

Specificity E. coli takes up Fe by at least two routes: (1) i n the form of a chelate with enterobactin (also called enterochelin), a cyclic trimer of 2.3-dihydroxyN-benzoylserine (O'Brien and Gibson, 1970). synthesized from chorismic acid via 2.3-dihydroxybenzoate (Young etal., 1971); and (2) as a chelate with citrate (Young et a/., 1967).

Gene entA, 6,

" D'

The former system is induced in Fe-deficient medium (Young et a/., 1967; Brot and Goodwin, 1968; Young and Gibson, 1969b; Bryce and Brot, 1971) and the latter in citrate-containing medium (Cox ef a/., 1970).

Salmonella typhimurium

Fe

Fe transport i n Salmonella, as in E. coli, involves chelatin with enterobactin (enterochelin) (Pollack and Neilands, 1970) or with a variety of other catechols or hydroxamates (Luckey etal., 1972).

en b class I and I I

sidA-H, J-M

Aerobacter aerogenes

Mg

An energy-dependent transport system for Mg, inhibited by Ni, Co, and (less effectively) Zn (Webb. 1970a).

92

-

~ _ _ _ _

~

Inorganic Cations Linkage

Method of Isolating Mutants

Transport Defect in Mutants

entA, B, C, D, €, F, and fep (see below) are closely linked t o one another, and are located between purE and lip (Cox etal., 1970; Youngetal., 1971; Luke and Gibson, 19711, This gene cluster has been postulated to be an operon, repressed in the presence of Fe; the order of genes within the cluster is not known.

Inability t o grow on Fe-deficient medium unles citrate i s present t o induce the alternative system for Fe uptake (Young et al., 1971).

See above.

See above.

fep mutants can synthesize enterobactin, but are defective i n the uptake of the Fe-enterobactin complex (Luke and Gibson, 1971).

enb class I and class I I

enb mutants were originally

enb class-I I mutants d o not accumu-

mutants are closely linked, and located at about 20 minutes on the Salmonella chromosome (Pollack etal., 1970). It is not yet known how many genes are included in the enb region but, as in €. co/i, this is thought to be an operon repressed i n the Dresence of Fe.

isolated by their inability t o grow on minimal medium, by their failure t o respond t o any of the usual growth factors, and by the fact that an unusually large halo of bacteria grew up around each colony of revertants t o wild type (now known t o be due t o the excretion of enterobactin by the revertants) (Pollack etal., 1970).

late any detectable intermediates and are thought t o be blocked in the synthesis of 2.3-dihydroxybenzoic acid; enb class-I mutants accumulate 2.3-dihydroxybenzoic acid and are thought t o be blocked in i t s conversion t o enterobactin (Pollack et al., 1970).

sidA-G and sidM are cotransducible with panC at 9 minutes; sidJ with enb; and sidK and L are unlinked t o either panC or enb (Luckey etal., 1972).

Isolated, in an enb parent strain, b y resistance t o albomycin, a hydroxamate antibiotic (Luckey et al., 1972). The mutants were subdivided into 12 phenotypic classes (sidA-H, J-M) on the basis of their growth responses t o other hydroxamates and catechols.

Appear t o lack various transport systems for hydroxamates and catechols (Luckey etal., 1972).

Ability t o grow in the presence of 10-3 M Co (Webb, 1970b).

Defective in the uptake o f Mg, Co, and Ni (Webb. 1970b).

Defective i n Fe uptake via the enterobactin system. entC mutants are blocked in the first step of enterobactin biosynthesis (the conversion of chorismic acid to isochorismic acid); ent6 mutants in the second step (the conversion of isochorismic acid t o

2.3-dihydro-2.3-dihydroxybenzoic acid); entA mutants in the third step (the oxidation of 2.3-dihydro-2.3dihydroxybenzoic acid t o 2.3-dihydroxybenzoic acid); and entD, €, and F mutants in the last step (the conversion of 2.3-dihydroxybenzoic acid t o enterobactin) (Young e t al., 1971; Luke and Gibson, 1971).

93

(Continued)

TABLE I Mutations Affecting Membrane Transport (Continued) Inorganic Cations Organism

Transport System

Hydrogenomonas eutropha

NH4

Streptococcus faecalis

K, Na, H

Specificity Not characterized.

Wild-type S. faecalis, like many other microorganisms, takes up K in exchange for Na and H (Zarlengo and Schultz, 1966). The overall process appears t o be closely related t o a membrane-bound ATPase: both the ATPase and K transport are inhibited by N,N'dicyclohexylcarbodiimide, Dio 9, and chlorhexidine (Harold e t a/., 1969a.b); both are stimulated during growth on low-K medium (Abrams and Smith, 1971 1; and in DCCD-resistant mutants, both have become insensitive t o DCCD (Abrams e t a/., 1972; Harold and Papineau, 1972b). The ATPase has been purified and extensively characterized by Abrams and his colleagues (Abrams eta/., 1960; 1972; Abrams, 1965; Abrams and Baron, 1967, 1968, 1970; Schnebli and Abrams, 1970;Schnebli e t a/., 1970; Baron and Abrams, 1971). I t has a molecular weight of 385,000, and consists o f six 01 and six (3 subunits, each with a molecular weight of 33,000; and it is attached t o the bacterial membrane by another protein, nectin, of molecular weight 37.000. The precise relationship between the ATPase and cation transport remains unclear. Recently, Harold proposed a mechanism based on the Mitchell model for cation transport in mitochondria (Harold and Papineau, 1972a. b; Harold, 1972):

(1) The primary process is postulated t o be the electrogenic extrusion of H ions from the cell, coupled t o the membrane-bound ATPase and creating a membrane potenti 150-200 m V (inside negative). (2) K is vu.tulated to enter the cell along i t s electrochemical gradient 4n a passive, carrier-mediated process.

(3) Na (moving outward) is exchanged for H (moving inward along i t s electrochemical gradient) in a second passive, carrier-mediated process. The main evidence for this scheme comes from indirect measurements of membrane potential (by means of lipid-soluble cations; Harold and Papineau, 1972a.b) and intracellular pH (by means of the distribution of dimethyloxazolidinedione; Harold e t a/., 1970a); and from the effects of ion-conducting reagents on cation fluxes (Harold and Baarda, 1967a.b; 1968a.b; Harold e t a/., 1970a; Harold and Papineau, 1972a.bl. 94

Gene -

CnK6

TrK8

-

dcc

Inorganic Cations Linkage

Mapping techniques are not available for S. faecalis, so the genetic relationship of CnK6 to the other cation transport mutants (see below) has not been established.

Method of Isolating Mutants

Transport Defect in Mutants

Slow growth on NH4 as sole nitrogen source at low p H (Strenkoski and DeCicco, 197 1a).

Defective in the transport o f NH4, and dependent on the diffusion of NH3 (an indirect argument based on growth experiments) (Strenkoski and DeCicco, 1971b).

Ability to survive P32 suicide in low-K medium (see isolation of PO4 transport mutants of S. faecalis) (Harold e t al., 1967). C n ~ mutants 6 require abnormally high K concentrations for growth at pH 6, and might also be isolated in this way.

C n ~ mutants 6 are deficient in the net uptake of K or the lipid-soluble cation DDAt in exchange for H at p H 6 (but, when preloaded with Na, can exchange K for Na at nearly normal rates) (Harold et a/., 1970a; Harold and Papineau, 1972b).

Same as above (Harold and Baarda, 19676). T r ~ mutants 8 require abnormally high K concentrations for growth at p H 8, and might be isolated in this way.

is Uptake of K in T r ~ mutants 8 abnormally sensitive t o inhibition by Na (Harold and Baarda, 1967b).

Inability to grow at low K concentrations (Harold e t a/., 1970a).

Strain 7683 is defective in the uptake of K, tris, or H in exchange for Na (Harold e t a/., 1970a; Harold and Papineau, 1972b).

Resistance t o DCCD (Abrams e t a l . , 1972).

dcc mutants possess an altered membrane ATPase which is not inhibited

b y DCCD. Reconstitution experiments have shown that the purified ATPase and nectin (the protein required t o attach the enzyme t o the membrane) are normal in the mutants, and indicate that the defect must be in a third component (a "carbodiimide-sensitizing factor") of the overall ATPase complex (Abrams et a/., 1972). K uptake and H release b y dcc mutants are resistant t o DCCD. consistent with the postulated role of the ATPase i r i cation transport (Abrams et a/., 1972; Harold and Papineau, 1972b). According t o Harold's hypothesis (see Specificity), C n ~ and 6 dcc mutants are said t o have primary defects in the electrogenic extrusion of H ions;

(Continued) 95

TABLE I Mutations Affecting Membrane Transport (Continued) Inorganic Cations Organism

Transport System

Specificity

Gene

Alternatively, one might visualize a direct coupling o f K, Na, and H movements, mediated by a single carrier and driven either by ATP directly (as in mammalian cells) or b y the electrochemical gradient for H ions. As long as the coupling is not obligatory-i.e., as long as the fluxes of K, Na, and H can be dissociated from one another under some experimental conditionssuch a model could account for most of Harold's results with lipid-soluble cations and ion-conducting reagents.

Strepfococcus faecalis (Con't.I

6acillus subtilis

K

Not characterized.

6acillus megaterium

Fe

Wild-type 6. megaterium secretes schizokinen (a secondary hydroxamic acid; structure determined b y Mullis ef a/., 19711, and takes up Fe as the schizokinen-Fe chelate.

-

(SK11)

AAD-1

Sfaphylococcus aureus

Fe

Neurospora crassa

K

Wild-type Neurospora takes up K in exchange for Na and H, in an energy-dependent process (Slayman and Slayman, 1968). A t l o w p H the system obeys standard Michaelis kinetics as a function of the extracellular concentration, with a K,,, of 1.1 x l o 3 M; at high pH, however, the kinetics become sigmoid. The results have been interpreted in terms of two alternative multisite models. (1 The transport system contains a second site which must be filled with a cation (H at low pH, K at high pH) in order for transport t o occur. (21 The transport system i s an allosteric protein consisting o f multiple subunits, each with a binding site for K; cooperative interactions among the subunits give rise to a sigmoid curve at high pH, but H serves as an allosteric activator of the system, causing a shift toward a standard Michaelis curve at low p H (Slayman and Slayman, 1970).

96

frk-1

Inorganic Cations Linkage

Method of Isolating Mutants

Transport Defect in Mutants strain 7683 in the passive exchange of H for Na; and T r ~ in 8 the passive uptake of K. According t o the singlepump hypothesis, strains C n ~ and 6 7683 are primarily defective in efflux sites, and T r ~ in 8 influx sites.

Inability t o grow at low K concentrations (Lubin, 1964).

Oefective in the retention o f K (Willis and Ennis, 1968).

Requirement for schizokinen for growth (Arceneaux and Lankford, 1966).

Blocked in the biosynthesis of schizokinen and in the uptake of Fe b y this route (Arceneaux and Lankford, 1966; Davis etal., 1971; Davis and Eyers, 1971).

Isolated, in strain SKI1 (see above), b y resistance t o the ferric hydroxamate antibiotic A22765 (Davis and Byers, 1971).

Defective in the uptake of some Fe chelates (A22765, Desferal) but n o t in the uptake of others (schizokinen) (Davis and Eyers, 1971).

-

Resistance t o the ferric hydroxamate antibiotic A22765 (Kniisel etal., 1969).

Defective i n the uptake of Fe chelates such as A22765 (Zimmermann and Kniisel, 1969).

Linkage group I l l (Slayman and Tatum, 1965).

Inability t o grow at low K concentrations; selected by inositolless death or filtration enrichment (Slayman and Tatum, 1965; Slayman and Kopsack, unpublished results).

Oefective in K influx (Slayman and Tatum, 1965; Slayman, 1970, and unpublished results). The best-studied trk-1 mutant, strain R2449, appears t o have a qualitatively altered transport system, with an abnormally low affinity for K and also an abnormal p H dependence of transport. According t o both the allosteric and the two-site models (see Specificity), the higher K requirement o f the mutant would reflect an increase in the K,,, of the transport site(s). The abnormal p H dependence would come from an altered equilibrium between the t w o conformations of the carrier, in the allosteric model, or from altered affinities of the modifier site for H and K, in the two-site model (Slayman, 1970).

97

(Continued)

TABLE I Mutations Affecting Membrane Transport (Continued) Inorganic Cations Organism

Transport Svstem

Specificity

Neuro-

Gene

trk-2

spora crassa (Con 't.)

Organism

Transport System

Sheep

Na, K

Specificity Sheep red b l o o d cells, like most other animal cells, transport K inward and Na outward in an ATPdependent process catalyzed b y an (Na + K)-activated membrane ATPase. The system is specifically inhibited b y cardiac glycosides such as ouabain (reviewed b y Glynn, 1968; Skou, 1971).

98

Gene

L/ h (fTIL/M)

Inorganic Cations Linkage Linkage group V (Slayman and Kopsack, unpublished results).

Mode o f Inheritance The L / h gene controls the K concentration o f sheep red cells, w i t h L (low-po tassi u m ) almost completely dominant over h (high-potassium) (Evans and King, 1955; Evans ef a/., 1956; Kidwell ef a/., 1958; Rasmusen and Hall, 1966; Tucker and Ellory, 1970; Eagleton ef a/., 1970). I n addition, a correlation exists between potassium types and the M f L blood group system such that H K sheep (hh) are homozygous for the gene controlling the M antigen (MM), homozygous L K sheep ( L L ) are homozygous f o r the gene controlling the L antigen (mLmL1, and heterozygous L K sheep are M m L (Rasmusen and Hall, 1966, 1967;Tucker, 1968; Lauf and Tosteson. 1969; Ellory and Tucker, 1969a; Rasmusen, 1969). I t is n o t yet clear that L / h and m L l M are identical, and i n fact several lines o f evidence suggest that they may be closely linked b u t separate genes: (1) In reticulocytes f r o m genetically L K sheep, mL is expressed (that is, L antigen is present) b u t L is n o t (the cells contain a high potassium

Method o f Isolating Mutants Same as above.

Transport Defect in Mutants Defective in t h e retention o f K. w i t h an abnormally rapid K - K exchange (Slayman, unpublished results).

Abnormal Phenotype Red cells f r o m H K sheep contain about 80 mmoles K/liter and those f r o m L K sheep, about 1 3 mmolesfliter. The Na concentrations are complementary t o the K concentrations, so that the sum o f Na t K is the same in the t w o cell types; CI concentrations, water content, and cell volumes are also the same, as are plasma K and Na concentrations (Tosteson and Hoffman, 1960). The difference between H K and L K is conspicuous o n l y in mature red cells f r o m adult animals. In genetically L K lambs, and in adult L K animals that have undergone massive hemorrhage so that large quantities o f new cells are entering t h e circulation, the red cells contain high K concentrations (Lee eral., 1966; reviewed in Tucker, 1971).

99

Transport Defect Mature H K and L K cells differ in two main respects w i t h regard t o cation movements across their membranes: ( 1 ) B o t h cation transport and the (Na t K)-stimulated ATPase are less active in L K cells-4 t o 8-fold (Tosteson and Hoffman, 1960; Tosteson, 1963; Dunham and Hoffman, 1971a; Hoffman and Tosteson, 1971) and 4- t o 13-fold (Tosteson e t a / . , 1960; Tosteson, 1963; Whittington and Blostein, 1971 ), respectively. (2) Conversely, the passive permeability t o K ( K leak) is 2 t o 5 times greater in L K cells (Tosteson and Hoffman, 1960; Dunham and Hoffman, 1971a). Perhaps the most interesting feature o f this system is that treatment o f L K cells w i t h anti-L serum (formed in an H K sheep against L K red cells) causes a dramatic stimulation o f cation transport, w i t h a 2 t o 5-fold increase in active potassium influx (Ellory and Tucker, 1969a. 1970a; Lauf e f a/., 1970; Ellory eral., 1972) and in (Na + K)-stimulated ATPase activity (Ellory and Tucker, 1969a). Part o f the difference between H K and L K cells, and also part o f t h e stimulating effect o f anti-L, can be accounted f o r b y a conversion o f p u m p sites t o leaks during the maturation o f the L K cell, and a reconversion t o pumps in the presence o f anti-L. Using ouabain-3 binding as an assay, Dunham and Hoffman (1971a.b) f o u n d 4 2 p u m p sites per H K cell and 7.6 per L K cell la ratio o f about 6 : l . consistent w i t h the ratios o f cation transport and ATPase activity), and an intermediate

(Continued)

TABLE I Mutations Affecting Membrane Transport (Continued) Inorganic Cations Organism

Transport System

Specificity

Sheep (Con‘t.I

100

Gene

Inorganic Cations Mode of Inheritance

Abnormal Phenotype

concentration) (Ellory and Tucker, 1 9 7 0 ~ ) . (2) I n adult L K cells, where both are expressed, it is possible to distinguish two responses t o anti-L antiserum-a marked stimulation of K transport (see Transport defect) as well as complement-mediated immune hemolysis. The first response is abolished i f the cells are pretreated with trypsin but the second i s not, a result most easily interpreted i n terms of separate L and m L gene products (Lauf etal., 1971). (3)Finally, goat red cells also have the H K I L K transport system and M I L blood groups, but in this case the two are clearly controlled by unlinked genes; sheep anti-L serum stimulates transport in L K goat cells, pointing to a close relationship between the L genes of the two species, but does not hemolyze goat cells, suggesting that the mL genes may be different (Ellory and Tucker, 1970b).

Transport Defect number of pump sites in cells from L K lambs. I n addition, when L K cells were treated with anti-L, there was a 2-fold increase in ouabain-binding sites (Lauf et a/,, 1970; Ellory etal., 1972) and, at least in one case, a concomitant decrease in the K leak (Ellory etal., 1972; but see Lauf etal., 1970). These quantitative changes can be interpreted in terms of t w o general hypotheses concerning the nature o f HK and L K transport systems. On the one hand, one might imagine that the L gene controls a labile element required for transport, and that the element loses activity during maturation of rhe L K cell but can be reactivated by antiserum. (In what may be a parallel case, several mutant forms of the enzyme p-galactosidase in E. coli are known to be reactivated by antibody; Rotman and Celada, 1968; Celada et sl., 1970; Messer and Melchers, 1970.) The dominance o f L over h, according to this hypothesis, could result from preferential incorporation of the defective L element into the membrane, or from interactions among defective and normal subunits in an oligomeric protein (see discussion of dominance and recessiveness in the text). Alternatively, the L K phenotype might reflect the delayed synthesis of an inhibitor of transport, with antibody somehow masking or inactivating t h e inhibitor. In this case the dominance of L could b e explained if h were a mutant allele, coding for a defective inhibitor. Either hypothesis would have t o account for the recent demonstration that there are qualitative as well as quantitative differences between HK, LK, and anti-L treated L K cells: in their relative affinities for K and Na (Hoffman and Tosteson, 1971; Ellory e t a/., 1972) and ouabain (Dunham and Hoffman, 1971a1, and in some of the partial reactions of the ATPase (Whittington and Blostein,

(Continued) 101

TABLE I Mutations Affecting Membrane Transport (Continuedl Inorganic Cations Organism

Transport System

Specificity

Disease

Sheep (Con 't.)

Man

Hereditary spherocytosis

Na (passive per meabili ty 1

Deer mouse IPeromyscus maniculatus)

Man

Hereditary spherocytosis

Na. K

ATPasedeficient hemolytic anemia

H (passive permeabi Iity )

Renal tubular acidosis

102

Inorganic Cations Mode of Inheritance

Abnormal Phenotype

Transport Defect 1971; Blostein eta/., 1971; Blostein and Whittington, 1972). It would be interesting to know whether these kinetic differences are present at the reticulocyte stage or whether they appear-along w i t h the change in number of pump sites-as the red cell matures.

Autosomal dominant (reviewed b y Jandl and Cooper, 1972).

Hemolytic anemia with jaundice and splenomegaly. The circulating red cells tend to assume a spherical shape, and are more readily sequestered in in the spleen and hemolyzed. The red cells also show an abnormal osmotic fragility, and undergo premature autohemolysis when incubated in vitro (reviewed b y Jandl and Cooper, 1972). A similar but less severe condition involving elliptical red cells has been described b y Honig e t a/., (1971).

Increased permeability t o Na, leading to a compensatory increase in (Na + KI-activated ATPase and-in the absence of glucose-to swelling and hemolysis (Jacob and Jandl, 1964). Believed t o be a primary defect in t h e microfilaments of the red cell membrane. Proteins extracted from the membranes of hereditary spherocytes are abnormal in solubility and in aggregation as a function of ionic strength (Jacob e t a / . , 1971I. In addition, brief exposure of normal red cells t o vinblastine, colchicine, or strychnine-compounds known t o precipitate microfilament proteinsgenerates cells very similar t o hereditary spherocytes in morphology, osmotic fragility, and permeability t o Na (Jacob e t a / . , 1972).

Autosomal recessive (Huestis and Motulsky, 1956).

Hemolytic anemia with jaundice and splenomegaly. Spherical red cells (Anderson e t a/., 1960).

Believed t o be analogous t o hereditary spherocytosis of man.

Autosomal dominant with variable expression (data from two families; Harvald e t a/., 1964).

Hemolytic anemia, with red cells of normal morphology (Harvald e t a / . , 1964).

Postulated t o be a defect in the (Na + K)-activated ATPase and in the transport of Na and K. The data are incomplete, however, since only total red cell ATPase [(Na + KI-activated and Mg-ATPase] was assayed, and since cation fluxes were not studied (Harvald eta/., 1964).

Can be inherited as an autosomal dominant, with variable expression in some families and full expression in others. Most cases of renal tubu-

Sustained metabolic acidosis, with a low concentration of bicarbonate and an elevated concentration of chloride in the plasma. In many patients there are additional distur-

Thought t o be a primary defect in the ability t o generate steep H ion gradients between blood and urine, possibly because the distal tubular cells are abnormally permeable t o H ions. Bicarbonate reabsorption and ammonia

(Continued) 103

TABLE I Mutations Affecting Membrane Transport (Continued) Inorganic Cations Organism

Transport System

Specificity

Disease

Man (Con 't.I

Inorganic Anions Organism ~~~

Transport System

Specificity

Gene

~

Escherichia coli

PO4

There are a t least two transport systems for phosphate in E. coli (Bennett and Malamy, 1970a,b, 1971; Medveczky and Rosenberg, 19711: (1) A high-affinity system with a Km of 7 x 10-7 M, inhibited by arsenate. This system appears to involve a phosphatebinding protein,Kg = 8 x 10-7 M . which has a molecular weight of 42,000 and binds one phosphate per molecule of protein (Medwczky and Rosenberg, 1989, 1970). Uptake requires K (Weiden e t a / . , 1967; Medveczky and Rosenberg. 19711 and is regulated by the intracellular pool of inorganic PO4:

(2) A lower-affinity system with a K, of 9.2 x 10-6 M, inhibited by Ni2+ but not by AsO4. Bennett and Malamy (1971) have discussed the conditions under which the two systems are formed, and have reported that during growth in the presence of gluwsed-phosphate, an additional POq-As04 transport system is induced.

104

pitA

Inorganic Cations Mode of Inheritance ~~

Abnormal Phenotype ~~

Transport Defect

~

lar acidosis have negative family histories, however, and the syndrome may arise secondary to pyelonephritis, acquired hyperglobulinemia, or drug therapy (reviewed b y Seldin and Wilson, 1972).

bances of electrolyte metabolism, including an excessive urinary loss of potassium (leading t o hypokalemia, with weakness or paraiyiis) and calcium and phosphate (leading to osteomalacia, nephrocalcinosis, and renal stones) (reviewed by Seldin and Wilson, 1972).

excretion are normal (reviewed by Seldin and Wilson, 1972).

Inorganic Anions Linkage Maps between x y l and malA (Bennett and Malamy, 1970a).

Method of Isolating Mutants Resistance t o As04 (Bennett and Malamy, 1970a.b); slow growth at low PO4 concentrations (Medveczky and Rosenberg, 1970).

Transport Defect in Mutants Defective in the high-affinity phos. phatearsenate transport system. The original pitA mutant (UR13). isolated and mapped b y Bennett and Malamy (1970a,b), was unable t o accumulate A S 0 4 and took UP PO4 at a reduced rate. Mutants 10-1 and 20-2 (Medveczky and Rosenberg, 1970,1971) have been characterized in greater detail; they lack both the high-affinity phosphate transport system and the phosphate-binding protein. and retain only the low-affinity system. Further experiments are needed t o see whether mutants 10-1 and 20-2 map at the pitA locus.

Resistance to A s 0 4 in the presence of La-glycerophosphate as phosphate source (Bennett and Malamy, 1970a,b); resistance to AS04 (Medveczky and Rosenberg, 1970. 1971).

Possible defects in the low-affinity phosphate transport system: Strain U R I (Bennett and Malamy, 1970a.b) appears t o contain at least two mutations-in pitA and in one or more unlinked genes-and transports phosphate at about 10% of the normal rate; it may be defective in both phosphate transport systems. Strain 20-1 (Medveczky and Rosenberg, 1971) lacks the low-affinity

(Continued) 105

TABLE 1 Mutations Affecting Membrane Transport (Continuedl Inorganic Anions ~

~~

Organism

Transport System

Specificity

Gene

Escherichia coli (Con?.]

so4

N o t characterized,

cysP

cysB

cys€

Salmonella typhimurium

SO4

A transport system f o r inorganic sulfate (K, = 3.6 x cysA 10-5 M I , inhibited b y sulfite, thiosulfate, and chromate. Repressed during growth on cysteine; derepressed during growth on Ldjenkolate (Dreyfuss, 19641. A sulfatebinding protein which may be involved in transport has been crystallized and extensively characterized (Pardee, 1967; Langridgeet a/., 1970).The protein is located in the periplasmic space (between the cell wall and the cell membrane) (Pardee and Watanabe, 1968) and i s released during protoplast formation (Dreyfuss and Pardee, 1965) or osmotic shock (Pardee, 1966). I t has a molecular weight o f 32,000 and binds sulfate w i t h a dissociation constant on the order o f 10-7 M, depending on the ionicstrength (Pardee, 1966). Binding is inhibited b y sulfite, thiosulfate, and chromate, and synthesis o f the binding protein is repressed during growth on cysteine (Pardee er a/., 1966).

106

Inorganic Anions Linkage

Method of Isolating Mutants

Transport Defect in Mutants phosphate transport system and i s derepressed for the high-affinity system; it has not been mapped. Defective in SO4 transport and sulfite reductase (Jones-Mortimer, 1968).

About 5 3 minutes (Jones-Mortimer, 1968; Taylor, 19701

Require cysteine for growth (Jones-Mortimer , 1968).

25 minutes (Yanofsky and Lennox, 1959; Signer et a/., 1965; Taylor, 1970).

Same as above.

72 minutes (JonesMortimer. 1968; Taylor, 1970).

Same as above

A second group of pleiotropic mutations affecting SO4 transport and cysteine biosynthesis (JonesMortimer, 1968).

76 minutes (Mizobuchi e t a/., 1962; Demerec etal., 1963; Sanderson,

Require cysteine for growth; must be distinguished from other cys- mutants that are defective in cysteine biosynthesis (Mizobuchi eta/., 1962; Dreyfussand Monty, 1963).

Defective in sulfate uptake (Dreyfuss, 1964; Ohtaetal., 19711.cysA is not the structural gene for the sulfatebinding protein, however: (1) Although many chromate-resistant cysA a, b, or c mutants produce abnormally low amounts of binding protein, the protein appears to be identical to that of the parent strain as tested by acrylamide gel electrophoresis, immunodiffusion, and heat stability. (2) Two mutants with deletions covering the entire cysA region produce the binding protein in normal amounts. (3)Nonsense mutants in each of the three cysA cistrons produce the binding protein in normal amounts. Ohta et a/. (19711 concluded, on the basis of this evidence, that cysA determines some other component of the transport system, and in addition exerts a regulatory effect (seen in some cysA mutants but not others) on the production of binding protein. The binding protein itself may be necessary for transport, but seems to saturate the system in low amounts (i.e., well before it is produced in maximal quantities, as in derepressed cells). The structural gene for the binding protein has not been identified,

1970).

Pleiotropic mutations affecting

SO4 transport and cysteine biosynthesis (Jones-Mort imer, 1968).

Resistance to chromate (Pardee 8t a/., 1966; Ohta etal., 1971). Most chromateresistant mutants are cysA, but some are in other cys regions. Ability to use Lcysteine sulfinic acid but inability to use either sulfate or thiosutfate as sulfur source; 52 strains isolated in this way were all cysA mutants (Ohta etal., 1971).

107

(Con timed)

TABLE 1 Mutations Affecting Membrane Transport (Continued) Inorganic Anions Organism

Transport System

Specificity

Gene cysB

Salmonella typhimurium (Con 't.I

Salmonella pullorum

SO4

Not characterized.

Pseudomonas pseudomallei

As03 (PO41

Not characterized

Streptococcus faecalis

PO4

Wild-type S. faecalis has two transport systems for inorganic PO4: one (the 01 system) with a pH optimum of 8-9 and a K, of approximately 10-5 M PO4 or AsO4; and the other (the p system) with a pH optimum of 5.5 and a K, that rises rapidly with increasing pH (Harold etal., 1965; Harold and Eaarda, 1966).

-

Bacillus cereus

PO4

A system that transports phosphate (K, = 3.5 x 10-5 M ) ,pyrophosphate, phosphite (Km = 4.8 x l o 4 M ) , and arsenate (K, = 2.4 x 10-5 M ) (Rosenberg and LaNauze, 1968; Rosenberg et al., 1969).

-

Neurospora crassa

SO4

Wild-type Neurospora has two transport systems for inorganic sulfate (Marzluf, 1970a.b; Roberts and Marzluf, 1971): System I, which predominates in conidia, has Km's of 2 x 104 M for sulfate and 4 x 1 0 6 M f o r chromate; and

108

cys-13

cys-14

Inorganic Anions Linkage 5 2 minutes (Mizobuchi eta/., 1962; Demerec e t a / . , 1963; Sanderson, 1970).

Method o f Isolating Mutants

Transport Defect in Mutants

Require cysteine for growth (Mizobuchi et a/., 1962). Resistant t o chromate (Ohta eta/., 1 9 7 1 ) .

A regulatory gene; mutants have reduced levels o f the enzymes o f the cysteine pathway, including the binding protein. A temperaturesensitive c y s 6 mutant was shown t o produce binding protein o n l y a t the lower temperature, b u t once p r o duced, the protein had normal heat stability (Ohtaetal., 1971).

Strain MS35 was one o f 45 isolates o f S. pullorurn that required cysteine for growth (Kline and Schoenhard, 1970).

Strain MS35 was f o u n d t o contain t w o mutations, one leading t o a defect in SO4 transport and the other leading t o temperature sensitivity o f sulfite reduction (Kline and Schoenhard, 1970).

Resistance t o arsenite (Arima and Beppu, 1964).

Decrexed permeability t o arsenite (an indirect argument) (Arima and Beppu, 1964).

32P suicide. After treatment w i t h a mutagen, cells were grown briefly in medium containing 32PO4 of high specific activity,and then stored at -BOO for 30 days; at that time the majority o f cells had been inactivated b y decay o f the 32P incorporated into nucleic acids, b u t transport mutantsunable t o grow on l o w concentrations o f phosphatesurvived selectively (Harold e t a / . , 1965).

Defective in PO4 and A s 0 4 uptake at high pH; thought t o lack t h e a system (see Specificity) (Harold era/., 1965; Harold and Baarda, 1966).

Resistance t o PO3 in the presence o f aminoethylphosphonate as a source of P (Rosenberg and LaNauze, 1968).

Defective in the uptake o f phosphate, phosphite, and arsenate (Rosenberg and LaNauze, 1968).

Maps o n linkage group I, t w o units t o the right o f his-3 (Marzluf, 1970a).

Resistance t o chromate i n the presence o f methionine as S source (Marzluf, 1970a).

Lack transport system I for sulfate and chromate (Marzluf, 1970a.b; Robertsand Marzluf, 1971).

Maps on linkage group IV, approximately 21 units f r o m cot-1 (Marzluf, 1970al.

Originally isolated as chromate-cesistant, b u t when retested, f o u n d t o be sensitive (Marzluf, 1970a).

Lack transport system II for sulfate (Marzluf, 1970a.b).

109

(Continued)

TABLE 1 Mutations Affecting Membrane Transport (Continued) Inorganic Anions Organism

Transport System

Specificity

Gene

System II, which predominates in growing hyphae, has a K,,, of 8 x 106 M for sulfate.

Neurospora crassa (Con 't.I

Both transport systems, along with a group of related enzymes (aryl sulfatase, choline sulfatase, and cholineOsulfate permease1,are repressed during growth on methionine (Metzenberg and Parson, 1966);and both systems, together with the same group of enzymes, are under the control of the cys-3 locus (Marzluf and Metzenberg, 1968)and thescon locus (Burton and Metzenberg. 1972).

cys-3

scon

There appear to be two distinct transport systems for PO4 in Neurospora (Lowendorf & Slayman, 1970 and manuscripts in preparation; Lowendorf,

nuc-I

19721: (1) A low-affinity system is present in cells grown on the standard minimal medium (Vogel's; 37 mM PO4) and possesses rather complex kinetics. The K,,, of this system increases from less than 2 x 10-5M a t pH 4 to 9 x 104 M a t pH 7.2,while the Vmax remains nearly constant at 1.5-1.6mmoles/liter cell water per minute. The results can be accounted for by assuming that H (or OH) serves as a modifier of transport.

nuc-2; pcon

(2)In additi0n.a high-affinity system is derepressed in cells that have been grown on limiting phosphate.This system has simpler kinetics, with a K,,, of about 2 x 10-6M and a VmaXof 4 to 5 mmoles/liter cell water per minute over the entire pH range tested. I t s formation is blocked by cycloheximide.

Aspergillus nidulans

SO4

A transport system for SO4 (K,,, = 7.5 x los MI, S203(Km=7.5x105MI.Se04(Krn=7.7x105M).

s -3

and MoO4, repressed during growth on methionine (Tweedie and Segel, 1970;Bradfield eta/., 1970).

A. C

110

Inorganic A n io n s Linkage

M e t h o d o f Isolating Mutants

Transport Defect in Mutants

Maps o n t h e left a r m of linkage group II (Metzenberg and Ahlgren, 1970).

Require cysteine f o r g r o wt h .

A regulatory gene w h i ch exerts positive control over the synthesis o f b o t h sulfate permeases, a r yl sulfatase, choline sulfatase, and c h o l i n e a sulfate permease; cys-3- strains d o n o t make any o f these enzymes (Marzluf and Metzenberg, 1968; Metzenberg and Ahlgren, 1971) ,

Maps on t h e right a r m of linkage group V, b e yo nd his-6 (B urton and Metzenberg, 1972)

Hydrolysis o f in d o x y l sulfate (producing a blue color) during g r o wt h in the presence o f m e thionine and sulfate ( B u r t o n and Metzenberg, 1972).

A second regulatory gene f o r th e proteins controlled b y cys-3 (see above); scone mutants are derepressed f o r all these proteins ( Bu r to n and Metzenberg, 1972).

Linkage group I (Ishikawaetal., 1969).

I n a b ilit y o f wild-type Neurospora t o grow o n R N A , o r i n a b ilit y of an adenine-requiring strain t o use R N A as a source o f adenine (Ishikawa eta/.,

Postulated t o be a regulatory gene fo r phosphorus metabolism, analogous t o cys-3 in th e sulfur system (see above) (Lehman e t a/.. 1973). nuc-l mutants cannot be derepressed f o r PO4 transp o r t at high p H o r fo r acid and alkaline phosphatase (Lehman eta/., 1973; T o h e and Ishikawa, 1971 ) .

1969).

Linkage group I I (Ishikawa era/., 1969).

nuc-2/pcon is postulated t o b e a

Same as above

second regulatory gene f o r phosphorus metabolism, analogous t o scon (see above) (Lehman etal., 1973).pconc mutants are derepressed fo r PO4 at high p H and f o r alkaline phosphatase (although not f o r acid phosphatase), while nuc-2 mutants are repressed f o r these activities (Lehman eta/., 1973; Toh-e and Ishikawa, 1971). See, however, Hasunuma and lshikawa (1972) fo r a discussion o f an alternative hypothesis-that nuc-1 and nuc-2 are structural genes f o r a nuclease.

Maps o n linkage group V I (Dorn, 1967).

I n a b ilit y t o g r o w on SO4 as sole source o f sulfur ( Do r n ,

1967). I n a b ilit y t o g r o w on SO4 as sole source of sulfur (Hussey etal., 1965).

Defective in SO4 transport (indirect argument based o n g r o w th experiments) ( Ar st. 19681. Defective in SO4 transport (Spencer eta/., 1968).

(Continued) 111

TABLE 1 Mutations Affecting Membrane Transport

(Continued)

Inorganic Anions Organism Aspergillus nidulans (Con%) Penicillium notaturn

Transport System

Specificity

Gene Gamma

SO4

Organism

Transport System

Man

PO4

Similar t o that of A. nidulans (see above; Tweedie and Segel, 1970;Bradfield etal., 1970).

Specificity Kidney: Evidence has been presented f o r t w o PO4 transport systems in the human kidney (Glorieux and Scriver, 1972):one accounting f o r about t w o thirds o f the total net reabsorptive capacity, regulated b y parathyroid hormone (PTH),and the other,accounting f o r the remaining reabsorptive capacity, regulated b y Ca.

38632M

Gene Familial h y pophosphatemic rickets

Intestine: Likewise, there appear t o be t w o PO4 transport systems in the jejunal mucosa, w i t h apparent Km's o f 6 x 1 0 - 6 a n d 6 x lo4 M (Short etal., 1973).

CI

N o t characterized

112

Congenital chloridorrhea

Inorganic Anions Linkage

Method of Isolating Mutants

-

-

-

-

Transport Defect in Mutants Defective in SO4 transport (Bradfield eta/., 1970). Defective i n t h e transport o f SO4,

S2O3,SeO4, and M o o 4 (Tweedie and Segel, 1970).

Linkage

Abnormal Phenotype

X-Linked dominant (Wintersetal., 1957, 1958; Graham et a/., 1959; Burnett eta/., 1964).

L o w concentration o f i n organic phosphate in the serum, associated w i t h increased urinary excretion o f phosphate; in some cases reduced gastrointestinal absorption of Ca and high incidence o f rickets, not responsive t o physiological amounts of vitamin D (reviewed b y Rosenberg, 1969; Williams and Winters, 1972).

Transport Defect Defective in the PTH-regulated PO4 transport system in the kidney (Glorieux and Scriver, 1972) and in the h i g h a f f i n i t y PO4 transport syst e m in the jejunal mucosa (Short e t a/., 1972).The latter defect, b y leading t o the formation o f insoluble calcium phosphate complexes i n t h e intestinal lumen, could account f o r the reduced absorption of Ca in t h e intestine (Rosenberg, 1969).

A n alternative theory o f familial hypophosphatemic rickets-that the primary lesion is i n the conversion o f vitamin D t o i t s active metabolite(s) (DeLucaetab, 1967; Avioli era/., 19671, leading indirectly t o decreased intestinal Ca transport, secondary hyperparathyroidism, renal loss of PO4, hywphosphatemia, and bone disease-now seems unlikely t o be correct. Hypophosphatemia frequently occurs w i t h o u t any detectable impairment o f Ca absorption, and in addition, it has recently been demonstrated that in patients w i t h untreated hypophosphatemia, serum levels of P T H are normal (Arnaud eta/., 1971). Probably autosomal recessive (Perheentupa eta/., 1 9 6 5 ) .

Diarrhea; high fecal CI concentrations (and almost complete absence of CI in the urine); metabolic alkalosis (Darrow, 1945; Gamble eta/., 1945).

113

Thought t o be a defect in t h e absorpt i o n o f CI i n t h e colon and terminal ileum (Evanson and Stanbury, 1965). Direct f l u x measurements have n o t been performed.

(Continued)

TABLE 1 Mutations Affecting Membrane Transport (Continued) Multiple Transport Systems or General Membrane Permeability Organism Escherichia

coli

Membrane Property

Gene

Several mutants o f €. coli have been described w i t h multiple transport defects. In some cases the energy supply t o a large number o f transport systems has been blocked [e.g., mutants lacking Ca,Mg-ATPase (Eutlin etal., 1971 ;Simoni and Shallenberger, 1972; Schairer and Haddock, 1972) o r mutants lacking enzyme I o r HPr of the phosphoenolpyruvate phosphotransferase system] . Others may be cases i n which the transport systems cannot be formed or are.not fully functional, because of an alteration in membrane structure [the kmt strains o f von Meyenburg (1971) or the mutants of Crandall and Koch ( 1 9 7 1 ) l .

kmt

In addition, a large number o f mutants are k n o w n w i t h abnormal permeability, usually t o a variety o f chemically unrelated compounds. These are almost certainly cellsurface mutants, b u t because o f the complexity o f the surface layers o f €. coli [which include an outer membrane w i t h attached lipopolysaccharides, a rigid peptidoglycan layer, and a plasma membrane (see, for exarnple,Schnaitman, 1971a,b)l, it is n o t always possible t o say which portion o f the surface has been altered.

acrA

envA

114

Multiple Transport Systems or General Membrane Permeability Linkage Between s t r A and metB (von Meyenburg, 1971).

Method o f Isolating Mutants

Transport Defect

Delayed growth at l o w glucose Appear t o transport a variety o f concentrations (von Meyenburg, amino acids, carbohydrates, PO4, and SO4 w i t h 20-500-fold increased Km's 1971). (measured indirectly). Postulated t o have an altered cell wall o r plasma membrane, such that the attachment o f the various binding proteins i s weakened (von Meyenburg, 1971). Inability t o grow at l o w lactose concentrations at 42" (Crandall and Koch, 1971).

Cannot f o r m functional transport systems f o r lactose o r uracil when growing at 42' (but glucose and a-methylglucoside transport are normal) (Crandall and Koch, 1971 ) . I t would be interesting t o have information about additional transport systems in these mutants,and also information about the presence o r absence o f k n o w n transport proteins (e.g., the M protein o f the lactose system).

Near lac (Nakamura, 1968). The relationship of acrA t o mrc (see below) has n o t been established

Sensitivity t o acriflavine (Nakamura, 1965).

General increase in binding (uptake?) o f basic dyes (acriflavine, toluidine blue, crystal violet, m e t h y l green, pyronine B ) and in sensitivity t o lipophilic substances (phenethyl alcohol, sodium dodecyl sulfate) (Nakamura, 1965,1966,1967,1968). Could result f r o m an alteration in the plasma membrane (Nakamura and Suganuma, 1972) or in the outer layers of the cell surface.

Near leu (Yura and Wada, 1968; Taylor. 19701.

Resistance t o azide o r phenethyl alcohol (Yura and Wada. 1968); formation o f filaments at 42O (Van de Putte eta/., 1964).

Transport properties have n o t been studied directly, b u t azi strains are thought t o have an altered plasma membrane (Vura and Wada, 1968).

Maps between leu and

Isolated as a mutation f r o m rough t o smooth colony mor. phology (Normark et a/., 1969).

Increased sensitivity to ampicillin, chloramphenicol, kanamycin, n o w biocin, actinomycin D, rifampicin, and gentian violet (Normark et al.. 1969; Normark, 1970) which,at least in the case o f gentian violet, can be correlated w i t h increased uptake (Normark and Westling, 1971 ). Believed t o be defective in the outer layer (LPS membrane) of the cell surface (Normark. 1970,1971).

ari, at 1.5 minutes (Normark, 1970).

~~

..

r

(Continuedl

TABLE 1 Mutations Affecting Membrane Transport (Continued) Multiple Transport Systems or General Membrane Permeability Organism

Membrane Property

Gene

Escherichia coli (Con‘t.I

IpcA. B

rn tcA

mtc8

tolAB

116

M u l t i p l e Transport Systems o r General Membrane Permeability Linkaqe

Method of lsolatinq Mutants

TransDort Defect

Near p y r F (Wijsman, 1972).

Isolated as temperaturesensitive mutants, able t o grow normally at 28" b u t not at 42"; 0.5% NaCl restores growth at the high ternperature (Wijsman, 1972).

Fail t o plasmolyze at 42", possibly because of a defective plasma m e m brane (Wijsman, 1972).

Between ara and lac (Tamaki eta/., 1971 1 .

Sensitivity t o novobiocin (Tamakietal., 1971).

Defect in t h e outer layer o f the cell surface (the LPS membrane), leading t o increased sensitivity t o novobiocin. spiramycin, and actinomycin D . and t o resistance t o some phages (Tamaki etal., 1971 1.

Between T6 and pur (Sugino, 1966); near lac (Otsuji, 1968); relationship t o acrA (see above) has not been established.

Sensitivity t o methylene blue (Sugino, 1966) o r t o mitomycin C (Otsuji, 1968; Imae. 1968.

Increased sensitivity t o a wide variety ot substances (mitomycin C, basic dyes, sodium dodecyl sulfate, sodium deoxycholate, colicins E l , E2, E3, K) (Sugino, 1966; Otsuji, 1968; Imae, 1968; Otsuji eta/., 1972).

Near metC; possibly identical w i t h to/C (see below) (Otsuji eta/., 1972).

Sensitivity t o mitomycin C (Otsuji e t a / . , 1972).

Resembles mtcA except that it is resistant t o colicin E l (Otsuji etal., 1972).

Near gal (Nomura and Witten, 1967; Nagel de Zwaig and Luria, 1967; H i l l and Holland 1967). Includes three complementat ion groups (Bernstein eta/., 1972).

Tolerance t o colicins E l , E2, E3, K, and A (Nomura and Witten, 1967; Nagel de Zwaig and Luria, 1967; Hill and Holland, 1967; Bernstein era/., 1971).

Between s t r a n d his (Nagel de Zwaig and Luria, 1967; Hill and Holland, 1967); near m e t C (Whitney, 1971).

Tolerance t o colicin E l (Nagel de Zwaig and Luria, 1967; Hill and Holland, 1967).

to/ mutants show increased sensitivity t o a variety of compounds: t o l A B t o deo xycholate, E D T A , vancomyci n, and bacitracin (Nagel de Zwaig and Luria, 1967; Bernstein eta/., 1971, 1972); tolC t o deoxycholate, methylene blue, and acridines (Nagel d e Zwaig and Luria, 1967); and t o l D t o various antibiotics and detergents (Rolfeetal., 1971). More recently, purified membranes f r o m t o l A 6 and to/C mutants have been demonstrated t o lack particular proteins found in wild-type membranes (Onodera eta/., 1970; R o l f e a n d Onodera. 1971).

Between s t r A and malQPT (Rolfe etal., 1971).

Tolerance t o colicins E2 and E3 at 40" (Nomura and Witten, 1967; Rolfe eta/., 1971). Sensitivity t o actinomycin D (Sekiguchi and lida, 1967).

117

Primary defect n o t known; the mutants show increased sensitivity t o lysozyme, as well as t o actinomycin D (Sekiguchi and lida. 1967).

(Continued)

TABLE 1 Mutations Affecting Membrane Transport (Continued) Multiple Transport Systems or General Membrane Permeability Organism

Escherichia coli (Con%)

Membrane Property

Gene

-

M u l t i p l e Transport Systems or General Membrane Permeability Linkage -

Maps at 2 5 minutes (Taylor. 1 9 7 0 ) .

Method of Isolating Mutants

Transport Defect

Sensitivity t o synergistin A (Ennis, 1971).

Increased sensitivity t o a variety of drugs (strain H l O l : actinomycin D, bacitracin, carbomycin, clindamycin, erythromycin, spiramycin Ill, vernam y c i n A, and also t o detergents; strain H I S : acridine orange, amicetin, carbomycin, clindamycin, e r y t h romycin, fusidic acid, puromycin, spiromycin Ill,vernamycin). In addit i o n , HI35 is resistant t o phage T4. Primary defect n o t known.

A b i l i t y o f a tryptophan-requiring amber mutant t o grow in minimal medium supplemented w i t h t R N A f r o m an sul-carrying strain (Yamamoto eta/., 1971).

Increased uptake o f R N A (Yamamoto eta/., 1971).

Resistance t o phage TI; sensit i v i t y t o Cr3+ (Wang and Newton, 1969a).

May be a general defect in the plasma membrane, involving b o t h transport and the attachment o f phages and colicins. Wang and Newton (1969a.b) showed that t o n 8 mutants require very high concentrations o f Fe for growth, are defective in t h e 2 . 3 d i h y droxybenzoylserinedependent transp o r t o f Fe, and are abnormally sensitive t o Cr (which presumably inhibits the uptake of free Fe b y an alternative route; see discussion of Fe transp o r t in E. coli). More recently, Yanofsky (cited in Oxender. 1972a) has observed that deletions extending i n t o the t o n 6 region result i n decreased transport o f several amino acids. t o n 8 mutants are also resistant to phages TI and $80 and t o colicins €3. I,and V (Taylor, 1970).

Isolated, in a parent strain w i t h deletions covering mal8 (maltose permease) and l a c y (lactose permease), b y the ability t o grow on maltose and lactose. Alternatively, isolated in a wild-type parent strain b y increased sensitivity t o deoxycholate (Ricard era/., 1 9 7 0 ) .

Increased passive permeability t o sugars and increased sensitivity t o deoxycholate (Ricard e t a / . , 1970)

119

(Continued)

TABLE 1 Mutations Affecting Membrane Transport (Continuedl Multiple Transport Systems or General Membrane Permeability Organism

Membrane Property

Gene

Escherichia coli (Can't.)

Numerous mutants of E. coli have been isolated with primary defects in lipid synthesis, and some of them have been shown to possess secondary defects in transport or permeability.

-

plsA

fabA

fabB

120

Multiple Transport Systems o r General Membrane Permeability Linkage

Method of Isolating Mutants

Transport Defect

Isolated as a temperature-sensifive mutant; able t o grow at 30" b u t n o t at 41" (Hirota e r a / . . 1969).

Several membrane defects, including an abnormally large passive permeability t o o-nitrophenyl-p-D-ghlactoside (ONPG) (other compounds n o t tested), increased sensitivity t o deoxycholate, and intense fluorescence w i t h the d y e 1-anilino-8-naphthalenesulfonic acid (ANSI (Hirota e t a / . , 1969).

Requirement f o r glycerol (Hsu and Fox. 1970).

Primary defect in biosynthetic glycerol-3-phosphate dehydrogenase, which supplies L-glycerol 3-phosphate f o r lipid synthesis. When lipid synthesis is blocked b y withholding glycerol f r o m the growth medium, the p-galactoside transport system cannot be induced (Hsu and Fox, 1970).

Stimulation o f growth b y glycerol-3-phosphate ( K i t o e r a / . , 1969). Resistance t o radiation suicide b y glycerol 3-phosphate-H3 in a parent strain which incorporates exogenous glycerol 3-phosphate efficiently i n t o lipids; the parent strain IS defective in alkaline phosphatase and catabolic glycerol-3-phosphate dehydrogenase, and also constitutive for glycerol-3-phosphate transport (Cronan e t a / . , 1970; Godson, 1973).

Several mutants have been isolated w i t h defects in glycerol-3-phosphate acyltransferase, which is involved in the conversion o f glycerol 3-phosphate t o phosphatidic acid. One strain has an enzyme w i t h a 10-fold lower a f f i n i t y f o r glycerol 3phosphate ( K i t o er a/., 1969). and other strains have heatlabile enzymes (Cronan er a/., 1970; Hechemy and Goldfine, 1971; Godson, 1973).

Between p y r D and pyrC (Cronan e t a / . , 1 9 7 2 ) .

Growth requirement f o r an unsaturated f a t t y acid (Silbert and Vagelos. 1967; Cronan e t a / . , 1969; Henning et a / . , 1969; Esfahani et a/., 1969).

Primary defect in p-hydroxydecanoyl thioester dehydrase, the first enzyme in unsaturated fatty acid biosynthesis (Silbert and Vagelos, 1967; Esfahani e t a / . . 1969).

Between aroC and purF (Epstein and Fox, 1970; Schairer and Overath, 1969).

Same as above (Cronan e t a / . , 1969; Schairer and Overath, 1969; G. Wilson e r a / . , 1970).

Unable t o synthesize unsaturated f a t t y acids; enzymic defect n o t k n o w n (Cronan e t a / . , 1969; Birge and Vagelos, 1972). fabA and B mutants are useful for transport studies because their membrane composition can be varied according to the unsaturated f a t t y acid provided in the growth medium (Silbert and Vagelos, 1967; Silbert

Maps between purE and proC (Cronan and

Godson, 1972).

121

(Con tinued)

TABLE 1 Mutations Affecting Membrane Transport (Continued) Multiple Transport Systems or General Membrane Permeability Organism

Membrane Property

Gene

Escherichia coli (Con %I

Salmonella typhimurium

The car ( p t s ) mutations i n Salmonella, like the corresponding mutations in E. coli, are pleiotropic because they block the energy supply t o multiple carbohydrate transport systems (discussed under Mutations affecting the transport of carbohydrates).

Bacillus subtilis

Several mutants of B. subfilis are known in which lipid synthesis is defective; in some cases, corresponding defects in transport have been described.

122

-

Multiple Transport Systems or General Membrane Permeability Linkage

Method of Isolating Mutants

Transport Defect eta/., 1968; Silbert, 1970; Schairer and Overath, 1969; Overath e t a / . , 1970; Fox, 1969; Foxetal., 1970; G. Wilson eta/., 1970; Wilson and Fox, 1971; Esfahani et a/., 1969, 1971).

Requirement for high Mg concentrations for growth (Lusk era/., 1968). Now known to be inhibited b y Na; Mg overcomes the inhibition (Lusk and Kennedy, 1972).

Altered phospholipid metabolism, with Na stimulating the synthesis of cardiolipin and inhibiting t h e synthesis of phosphatidylethanolamine. Following the addition of Na, the P-galactoside transport system is also inhibited, suggesting a general change in the functioning of the membrane. Primary defect unknown (Lusk and Kennedy, 1972).

Primary defect in lipid synthesis, leading t o a deficiency in phosphotidylethanolamine (Beebe, 19711. Abnormally low rates of uptake of several amino acids (lysine, tryptophan, phenylalanine, serine, threonine, proline, methionine, glycine), pyruvic acid, and purines and pyrimidines (uracil, uridine, thymine, adenosine) (Beebe, 1972).

-

Requirement for glycerol (Mindich, 1970a).

Defective i n the synthesis of glycerol for lipids (probably via a glycerol-3phosphate dehydrogenase) (Mindich, 1970a). Used t o demonstrate that membrane proteins continue t o be made and that the citrate transport system can be induced in the absence of phospholipid synthesis (Mindich, 1970a.b; Willecke and Mindich, 1971).

Requirement for short branched-chain fatty acids (Willecke and Pardee, 1971b).

Defective i n branched-chain a-keto acid dehydrogenase (Willecke and Pardee, 1971b). ~ ~ _ _ _ _

123

(Continued)

TABLE 1 Mutations Affecting Membrane Transport (Continued) M u l t i p l e Transport Systems or General Membrane Permeability Organism

Membrane Property

Staph y-

Gene -

lococcus aureus

Neurospora crassa

A variety of Neurospora mutants has been isolated w i t h m u l t i p l e transport defects [see fpr-1, mod-5, nap, un(UMSOO),on (55701 t ) under Mutations affecting the transport of amino acids andpeptides1 ,as well as a series o f "osmotic" mutants that m a y have generalized defects in t h e cell membrane and/or cell wall.

In addition, t w o mutants of Neurospora are k n o w n t h a t are defective in f a t t y acid synthesis, but these strains have not been used f o r transport studies.

124

0s-1, 2 , 3, 4,5, cut, flm-2

cel (01)

Multiple Transport Systems or General Membrane Permeability Method of Isolating Mutants

Linkage

Transport Defect

Requirement for glycerol (Mindich, 1971).

Defective in the synthesis of glycerol for lipids. Used to demonstrate that the lactose transport system, induced in the absence of phospholipid synthesis, is only partly functional; lactose uptake by intact cellsdeclined to 30-50% of the control value, while phosphotransferase activity for pgalactosides in isolated membrane vesicles remained normal (Mindich, 1971).

os-l,3, 4, 5, cut, and f/m-2 all map on linkage group I, although not adjacent to one another (Mays, 1969). 0s-2 is on linkage group I V (Schroeder, cited in Mays, 1969).

Abnormal morphology (Kuwana, 1953; Perkins, 1959; Emerson, 1963). Inhibition of growth by the addition of 4% NaCl to the medium (Perkins, 1959; Emerson, 1963; Mays, 1969).

Primary defect unknown. These strains are inhibited in growth media of high osmolality (Perkins, 1959; Slayman and Slayman, 1965; Mays, 1969). are altered in the composition of their cell walls (Emerson and Emerson, 1958; Hamilton and Calvet, 1964; Trevethick and Metzenberg, 1966; Livingston, 1969). and have an abnormally high passive permeability to K and probably to other ions and small molecules (Slayman and Slayman, 1965). Both Mays (1969) and Slayman and Slayman (1965)have suggested that the primary change may be in some structural element of the cell membrane, such that the membrane becomes leaky and cell wall synthesis (which may be mediated by membranebound enzvmes) is abnormal.

Linkage group I V (Perkinseta/., 1962)

A segregant from a cross involving a morphological mutant isolated by S. R . Gross (Perkins eta/., 1962). Originally reported to grow on oleic acid, other higher fatty acids, or Tween, and named 01. More recently found to require saturated fatty acids and not t o grow on highly purified unsaturated fatty acids; renamed cel (chain elongation) (Henry and Keith, 1971).

Primary defect in the biosynthesis of long-chain saturated fatty acids.

Requirement for unsaturated fatty acids (Lein and Lein, 1949).

Primary defect in the synthesis of unsaturated fatty acids (Lein and Lein, 1949).

-

125

(Continued)

TABLE 1 Mutations Affecting Membrane Transport (Continued) Multiple Transport Systems or General Membrane Permeability Organism

Membrane Property

Gene fas

Saccharomyces cerevisiae

01-1, 2, 3. 4

nys-1, 3, pol- 1 , 2 3. 5

Organism Man

Membrane Property The following five syndromes involve multiple abnormalities of renal tubular reabsorption. They may result from generalized damage t o the proximal tubule, as has been suggested in the case of Fanconi syndrome (Leaf, 1966). or from a metabolic disturbance in the supply of energy for transport (Rosenberg, 1969).

Disease Fanconi syndrome

Lowe's

syndrome

Busby syndrome

126

M u l t i p l e Transport Systems o r General Membrane Permeability Linkage

Method o f Isolating Mutants

Transport Defect

Nine complementation groups (Schweizer eta/., 1971) involving at least three unlinked genetic regions (Henry and Fogel. 1971).

Requirement for long-chain f a t t y acids (Schweizer and Bolling, 1970; Schweizer e t a/., 1971; Henry and Fogel, 1971).

Defective in fatty acid synthetase, a muttienzyme complex o f molecular weight 2.3 x 106 containing seven enzymic activities and a carrier protein (Lynen, 1967; Schweizer and Bolling, 1970; Schweizer eta/., 1971; Henry and Fogel, 19711.

Four genes (Keith e t a / . , 1969). w i t h o/-1 mapping distal t o tr-5 o n chromosome VI I (Resnick and Mortimer,

Requirement for unsaturated f a t t y acids (Resnick and Mortimer, 1966).

Defective in the A9-desaturation o f palmitate and stearate ( K e i t h eta/., 1969; Wisnieski eta/., 19701.

Resistance t o polyene antibiotics, which b i n d t o sterols in the cell membrane (Ahmed and Woods, 1967; Woods, 1971; Molzahn and Woods,

Altered sterol composition (Woods, 1971; Thompson eta/., 1971; Molzahn and Woods, 1972). The relationship between nys mutants and 01 mutants (see above) has been discussed b y Bard

1972).

(1972).

1966). pol-1 and pol-3 are allelic t o nys-I and nys-3, respectively (Molzahn & Woods,

19721.

Mode of Inheritance

Abnormal Phenotype

Transport Defect

Can be inherited as an autosomal recessive (Dent and Harris, 1951). Can also be acquired f r o m poisoning w i t h heavy metals, Lysol, or outdated tetracycline, or as a secondary result o f Wilson’s disease, galactosemia, von Gierke’s disease, o r cystinosis (Leaf, 1966).

Increased excretion o f glucose and all amino acids, in spite o f normal or reduced plasma concentrations of these compounds; chronic acidosis; osteomalacia w i t h hypophosphaternia (reviewed in Leaf,

X-Linked recessive (Lowe eta/.. 1952)

Increased excretion o f glucose, amino acids, organic acids; decreased production o f ammonia in t h e kidney; glaucoma; cataracts; bone disease; mental retardation ( L o w e e t a/., 1952).

Primary defect unknown.

N o t established; the syndrome was observed in three of six children o f asymptomatic parents (Rowley er a/.,

Increased excretion o f all amino acids; growth retardation; poor muscular development; right ventricular hypertrophy (Rowley e t a/., 1961; Rosenbergetal., 19611.

Primary defect unknown.

1961).

Primary defect unknown.

19661.

(Continued] 127

128

CAROLYN W. SLAYMAN

TABLE 1 Mutations Affecting Membrane Transport (Continued) Multiple Transport Systems or General Membrane Permeability Orqanism Man (Con 't.I

Membrane Property

Disease G~UCOglycinuria

Glucoaminoaciduria

alanine transport system and presumably owed its resistance to the decreased uptake of D-cycloserine; the second had a normal transport system but increased activities of alanine racemase and D-alanine :D-alanine ligase, two enzymrs that are sensitive to inhibition by D-cycloserine. Similarly, BQchet et al. (1970) found that Saccharomyces cerevisiae could become resistant to canavanine (an arginine analog) in either of two ways: through mutations inactivating the arginine transport system, or through mutations (in three genetic loci) reducing the repressibility of ornithine transcarbamylase by arginine. For this reason analog-resistant mutants must always be tested directly for transport defects, as discussed below. In a variation of the analog resistance method, it is sometimes possible to find conditions under which naturally occurring compounds inhibit growth, so that the corresponding transport mutants can be selected by their failure to be inhibited. For example, the growth of wild-type E. coli I<-12 on minimal medium is sensitive to valine, which blocks isoleucine biosynthesis through feedback inhibition of acetolactate synthetase (Leavitt and Umbarger, 1962). By selecting for resistance to valine, mutants have been obtained that are defective in the transport of branchrdchain amino acids (valine, isoleucine, lrucine) (Guardiola and Iaccarino, 1971), as well as mutants that possrss either a valinc-resistant acetolactate synthetase or an othrrwise increased rate of isolrucine biosynthesis (Glover, 1962; Ramakrishnan and Adclberg, 1964, 1965). Similarly, double mutants of Neurospora carrying a suppressible pyr-3 mutation and the suppressor, arg-12n, are highly sensitive to inhibition by arginine. pyr-3 blocks the synthesis of carbamyl phosphate in the pyrimidine pathway (CAP,,,) ; a~-g-12~, which leads to an altered ornithine transcarbamylase, restores growth by permitting the use of carbamyl phosphate that is normally

129

GENETIC CONTROL OF MEMBRANE TRANSPORT

Multiple Transport Systems or General Membrane Permeability Mode of Inheritance

Abnormal Phenotype

Transport Defect

Autosomal dominant (data from a single family) (Kaser er a/., 1962).

Increased excretioii of glucose and glycine (Klser era/., 1 9 6 2 ) .

Primary defect unknown.

Autosomal dominant (data from a single family) (Luder and Sheldon, 1955).

Increased excretion of glucose and amino acids; growth retardation (Luder and Sheldon, 1955).

Primary defect unknown.

restricted to the arginine pathway (CAP,,,); and arginine in turn inhibits growth by fcrdback control of the enzyme producing CAP,,, (Davis, ~ for resistance to arginine, 1967). By selrcting in a pvr-3 a ~ g - 1 2strain Thmaites (1967) has obtained mutants defrctive in the transport of basic amino acids. Arabinose and glucose 6-phosphate transport mutants of E. coli have been isolated in an analogous way, in strains sensitive to arabinose (brcausc they lack L-arabinose isomerase or ~-ribulosedphosphatr 4-epimerase ; Isaacson and Englesberg, 1964; Hogg and Englesberg, 1969; Hogg, 1971) or glucose 6-phosphate (because they lack phosphoenolpyruvate carboxylase ; Kornberg and Smith, 1969). B. Selection of Nongrowing Cells

In the analog resistance method, conditions are chosen under which the transport mutant can grow while the wild-typr cell cannot. If the converse conditions can be arranged-that is, if a medium can be designed that permits the growth of the wild type but not of the desired mutant-mutant sclection is also possible. In E. coli, this is usually accomplished by means of the penicillin method, originated by Davis (1948) and Lcderberg and Zindcr (1948), and modified for the sclection of transport mutants by Lubin et al. (1960) and Lubin (1962). In the presence of penicillin, growing cells are unable to synthesize new cell walls and therefore lyse. If, for example, cells of a prolinc-requiring strain are treated with a mutagen and suspended in a medium containing penicillin and a low concentration of proline, mutants tjhatl have lost the ability to transport proline will not grow and will therefore survive. By this mrthod mutants of E. coli have been obtained that are defective in the transport of proline (Lubin et al.,

TABLE II Transport Mutants Isolated by Analog Resistance Substrate Amino acids Alanine, glycine, w i n e

Organism

Analog

E. coli (Curtiss e t a / . , 1965; Kessel and

D-C ycloserine

Lubin, 1965; Wargel e t a / . , 1971) Streptococcus strain Challis (Reitz eta/., D-Serine

Aromatic amino acids (tryptophan, tyrosine, phenylalanine)

0-2-Thienylalanine 4-Methyltryptophan

5-fluorotryptophan p-fluorophenylalnine

Aspartate Basic amino acids (arginine, lysine, ornithine)

Azaserine Tyrosine or phenylalanine phosphonate derivatives lndole acrylic acid DL-threo-0-h ydroxyaspartate Canavanine

Th iosi ne

Glutamine

Isoleucine, valine, leucine Methionine

Hydroxy-L-lysine ?-GI utamylhydrazine 2-Hydrazino-344imidazolyl) propionic acid Azaleucirie

1967) E. coli (Davis and Maas, 1949; Kessel and Lubin, 1965; Cosloy and McFall, 1971; Oxender, 1972a) E. coli (Brown, 1970) E. co/i (Yanofsky, cited in Oxender, 1972a) N. crassa (neutral amino acid transport system; Lester, 1966; Stadler, 1966) P. aeruginosa (Kay and Gronlund, 1971) N. crassa (neutral amino acid transport system; Stadler, 1966; Wolfinbarger and DeBusk, 1971a; see also Kinsey, 1967; Kinsey and Stadler, 1969; Jacobson and Metzenberg, 1968) S. cerevisiae (Grenson and Hennaut, 1971) A. nidulans (Sinha, 1969) S. typhimurium (Ames. 1964) S.typhimurium (Ames,and Roth, 1968)

E. coli (Thorne and Corwin, 1970)

E. coli (Kay, 1971) E. coli (Schwartz eta/., 1959; Maas, 1965; Rosen, 1971a) N. crassa (Roess and DeBusk, 1968; Wolfinbarger and DeBusk, 1971a) S. cerevisiae (Grenson eta/., 1966. 1970; Bdchet eta/., 1970) E. coli (Rosen, personal communication) S. cerevisiae (Grenson, 1966) S. faecalis (Friede eta/., 1972) E. coli (Weiner and Heppel, 1971) S. typhimurium (Shifrin eta/., 1966; Ames and Roth, 1968)

E. coli (Rahmanian and Oxender, 1972a.b)

N. crassa (Jacobson and Metzenberg, 1968) S. cerevisiae (Gits and Grenson, 1967;

Eth ionine

Surdin et a/., 1965; Cherest and de Robichon-Szulmajster, 1966; de Robichon-Szulmajsterand Cherest, 1966) 0. danica (Hochberg eta/., 1972)

130

TABLE I I (Continued) Transport Mutants Isolated by Analog Resistance Substrate

Analog

Organism

Methionine (Con’t.1

a-Methylmethionine

Proline

3.4-Dehydroprol ine L-Azetidine-2-carboxylic acid Triornithine

Oligopeptides

S. ryphimurium (Ayling and Bridgeland, cited in Smith, 1971) E. coli (Tristram and Neale, 19681 E. coli (Tristram and Neale, 1968)

E. c o l i (Payne and Gilvarg, 1968; Payne, 19681

Carbohydrates GI ucose

N. crassa (Klingmliller and Kaudewitz,

Sorbose

1966; Klingmuller, 1967a)

A. nidulans (Elorza and Arst, 1971) L-a-Glycerophosphate lactose Purines and pyrimidines Uracil

Uridine Cytosine, cytidine Guanine, hypoxanthine Guanine, xanthine Adenine, hypoxanthine Adenine, hypoxanthine, guanine Xanthine, uric acid

Phosphonomycin o-Nitrophenyl-1 -thio0-D-galactoside

E. coli (Hendlin eta/., 1969) f. coli (Moiler-Hill eta/., 1968; Wong

6-Azauracil 5-F luorouraci I

E. coli (Lavalle, cited in Taylor, 1970) S. cerevisiae (Grenson, 1969; Jund and LaCroute, 1970) S. cerevisiae (Jund and Lacroute, 19701 S. cerevisiae (Jund and Lacroute, 1970) S. typhimurium (Kalle and Gots, 1961)

5-Fluorouridine 5-f luorocytosine 6-Mercaptopurine, 8-Azaguanine 8-Azaguanine 4-Aminopyrazole [3,4dl pyrimidine 8-Azaguanine, purine 2-Thiouric acid 2-Thioxanthine

Cations Fe

etal., 1970;T. H. Wilson eta/., 1970)

S. typhimurium (Thakar and Kalle, 1968)

S.cerevisiae (Pickering and Woods, 1972a.b) A. nidulans (Darlington and Schazzochio, 1967) A. nidulans (Darlington and Schazzochio, 1967) A. nidulans (Darlington and Schazzochio, 1967)

S. aureus (Knusel etal., 1969)

A22765

6.megaferium (Davis and Eyers, 1971) S. typhimurium (Luckey e t a/., 1972) E. coli (Nelson and Kennedy, 1971, 1972) A. aerogenes (Webb, 1970a) E. coli (Silver etal., 1972)

Albomycin co

Mn Anions so4

CrO4

S. ryphimurium (Pardee et a/., 1966; Ohta eta/., 1971) N. crassa (Marzluf, 1970al 8. cereus (Rosenberg and LaNauze, 1968) P. pseudomallei (Arirna and Beppu, 1964) E. coli (Bennett and Malamy, 1970a.b)

As04

131

132

CAROLYN W. SLAYMAN

1960; Kessel and Lubin, 1962), histidine (Lubin et al., 1960), glycine (Kessel and Lubin, 1965), glycylglycine (Kessel and Lubin, 1963) , and glucose 6-phosphate (Winkler, 1966), among others, and a mutant of Streptococcus faecalis defective in the transport of glutamine (Utech et al., 1970). In Neurospora, nongrowing cells can be selected in two somewhat different ways: by inositolless death (taking advantage of the fact that spores of an inositol-requiring strain, germinating in an inositol-free medium, die unless the presence of a second mutation prevents germination; Lester and Gross, 1959) ; and by filtration enrichment (in which growing cells are removed by filtration, while nongrowing spores are left behind; Woodward et al., 1954). Both methods have been used to obtain potassium transport mutants of Neurospora (Slayman & Tatum, 1965; Slayman, unpublished results). C. Direct Screening for Uptake

The selection methods that have just been described are indirect and frequently yield a mixture of mutants, some defective in transport and others with various metabolic blocks; they have the advantage, however, of producing a small number of isolates to be tested while eliminating a very large number of wild-type cells. When selection methods are not available, it is important to have a technique for the rapid screening of many isolates. One such technique-based on autoradiography-was originally designed by Zwaig and Lin (1966) for the detection of mutants of E. coli resistant to catabolite repression, and later modified by T. H. Wilson et al. (1970) and Wilson and Kusch (1972) to obtain mutants that were energy-uncoupled for lactose transport. In this method clones are spot,ted onto an agar plate containing thiomethylgala~toside-1~C (TMG, a substrate for the lactose transport system) ; sterile filter paper is pressed onto the surface of the agar, a portion of each clone is removed onto the paper, and the paper is exposed to x-ray film for several days. Wild-type clones that have accumulated TMG-1% appear black on the developed film, while transport-negative clones appear as very faint gray spots, and energy-uncoupled mutants have an intermediate color. Autoradiographic techniques have also been used by Boos and Sarvas (1970) to identify galactose transport mutants of E. coli, and by Breslow and Goldsby (1969) to isolate thymidine transport mutants of Chinese hamster cells. A somewhat analogous immunological procedure has been designed to screen for mutants of E. coli that lack arabinose-binding protein, believed to be a component of the arabinose transport system (Hogg, 1971). Cells

GENETIC CONTROL OF MEMBRANE TRANSPORT

133

are inoculated onto plates containing antiserum to purified arabinosebinding protein, incubated until visible colonies have been formed, and then partially lysed in situ by exposure to toluene-chloroform vapor. When the colonies are washed from the plate, clear precipitin spots can be seen at the sites of the wild-type colonies, while the absence of a precipitin reaction indicates a mutant colony unable t o produce binding protein. D. Recognition of Transport Defects in Higher Organisms

Although the various selection and screening procedures can readily be applied to cells in culture, the detection of transport defects in whole organisms is more difficult. I n some cases there are pronounced secondary symptoms which can eventually be traced back to the primary transport defect. For example, Hartnup disease is characterized by a pellagra-like rash now thought to be caused by a deficiency of nicotinamide, resulting in turn from the reduced absorption of tryptophan across the intestinal mucosa (Rlilne, 1963); and in hereditary vitamin BlZmalabsorption, the predominant symptom is megaloblastic anemia, reflecting the deficiency of vitamin B1z (Imerslund, 1960; Grasbeck et al., 1960; Spurling et al., 1964; Mohamed et al., 1966). In other cases the symptoms are more closely linked t o the primary transport defect, as in cystinuria, in which the decreased reabsorption of cystine in the kidneys leads to the formation of cystine stones in the urine (reviewed by Rosenberg and Scriver, 1969). All the inherited transport defects now known in man involve one of three tissues: kidney, intestinal mucosa (often both), or erythrocytes. I n part this presumably reflects the relative ease with which transport can be studied (and mutations detected) in these tissues compared with less accessible ones, but it may also mean that tissues specialized for transport are more likely to have multiple transport systems for any given substrate, so that mutations affecting any one system are less likely to be lethal. E. The Strategy of Recovering Transport Mutants

An essential part of any procedure for isolating transport mutants is to find conditions under which the mutants arc viable. An analog-resistant strain, lacking a particular transport system, must be able to survive without it; a nongrowing strain selected by the penicillin method, inositolless death, or filtration enrichment must be able to grow on some other medium in order to be recovered; and a strain isolated by the autoradio-

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CAROLYN W. SLAYMAN

graphic or immunological technique has to form colonies before these techniques can be applied. Although mutations affecting general membrane structure are often lethal (and can be studied only under special circumstances; for example, in temperature-sensitive strains; Table I), most mutations inactivating particular transport systems seem not to lead to general disorganization of the membrane. The cell survives as long as it has some other way to obtain (or does not need) the substrate in question. In many cases this is achieved relatively easily. Strains defective in the transport of a sugar or group of sugars can always be grown on another carbon source, and strains defective in amino acid transport can (in the microorganisms commonly used for genetic studies) synthesize their own amino acids. When the substrate is an essential one, the problem becomes more difficult. For inorganic cations and anions, one approach has been to add high concentrations to the medium, in the hope that the ion will enter the cell by diffusion (see, for example, the isolation of potassium transport mutants of E. coli and Neurospora; Lubin and Kessel, 1960; Epstein and Kim, 1971; Slayman and Tatum, 1965). Microorganisms have highly impermeable cell membranes, however, and this procedure is often inefficient, yielding mutants with qualitatively altered transport systems but not mutants lacking transport ability completely. A better approach is to supply an organic derivative of the ion that can be taken up by some other route: for example, cysteine as a replacement for SO4in S. typhimurium and Aspergillus nidulans (Dreyfuss and Monty, 1963; Hussey et al., 1965), and L-a-glycerophosphate and aminoethylphosphonate as sources of POa in E. coli and Bacillus subtilis (Bennett and Malamy, 1970a, b; Rosenberg and LaNauze, 1968). Alternatively, antibiotics are now available that increase membrane permeability to one or more ions in a highly specific way, and it seems likely that mutants lacking ion transport systems might be viable in the presence of such antibiotics. Antibiotic-dependent strains of this sort have not yet been reported, but Harold and Papineau (197213) recently observed that cation fluxes in a sodium transport mutant of S. faecalis are greatly stimulated by the antibiotic monensin, which is known to increase membrane permeability to sodium and hydrogen ions. F. The Problem of Multiple Transport Systems for a Single Substrate

Very often, both in microorganisms and in higher organisms, multiple transport systems exist for a given substrate or group of related substrates (Table I). Although this usually means that a mutation inactivating one

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of the transport systems is not lethal, since the defect can be compensated for by the other system(s), at the same time it creates a clear problem in the detection of such mutants. Occasionally one can take advantage of either qualitative or quantitative differences among the multiple systems. Salmonella typhimurium, for example, takes up histidine by a t least three routes (Ames, 1964; Ames and Lever, 1970). Among them, the “J” system is capable of transporting D-histidine in addition to L-histidine. It is normally present in small amounts, but regulatory (dhuA) mutants with increased levels of the J system can be selected by the ability to grow in D-histidine (KrajewskaGrynkiewicz et al., 1971: Ames and Lever, 1970; Ames, 1974a). Similarly, by selecting for the ability t,o use D-leucine, Rahmanian and Oxender (1972a, b) obtained mutants of E. coli with increased levels of one of the lrucine-isoleucine-valine transport systems (see also Kuhn and Somerville, 1971). I n S. cerevisiae, which has two transport systems for methionine, Gits and Grenson (1967) used low concentrations of ethionine (a methionine analog) to isolate mutants lacking the system with greater affinity. An alternative approach is t,o inactivate all but one of the multiple transport systems, and then to select directly for mutants defective in the remaining one. This can be accomplished in several ways: 1. By competitive inhibition. Neurospora has four major amino acid transport, systems, three of which (I, 11, and 111) can take up histidine (Pall, 1969, 1970a, b). To isolate mutants lacking system I, Woodward et al. (1967) and Magill et al. (1972) selected for the inability of a histidinerequiring strain to use exogenous histidine in the presence of high concentrations of arginine, a competitive inhibitor of systems I1 and 111. 2. By feedback inhibition or repression. Saccharomyces cerevisiae transports arginine by two systems, one specific for arginine and the other functioning for most neutral and basic amino acids. The latter is feedbackinhibited in the presence of high NH, concentrations (Grenson et al., 1970); Grenson et al. (1966) and BBchet et al. (1970) were able to select mutants lacking the specific system by resistance to canavanine (an arginine analog) in the presence of NH,. 3. By mutation. In E. coli, the original attempts to isolate mutants lacking the major potassium transport system (Schultz et al., 1963) were unsuccessful, because a minor, high-affinity system is induced a t low extracellular potassium concentrations and is able to support growth. In k d p mutants lacking the inducible system, however, trk mutants defective in the major system have recently been obtained (Epstein and Kim, 1971).

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111. CRITERIA FOR IDENTIFYING GENES THAT AFFECT TRANSPORT DIRECTLY

The selection and screening methods that have just been discussed allow the isolation of mutants with decreased rates of uptake of a particular substrate or class of substrates. It is important to realize that such mutations do not necessarily affect transport directly, but instead may act indirectly in any one of several ways: 1. The least interesting, for the present, purposes, can occur when the amount of uptake depends on subsequent metabolism of the substrate ; in this situation a mutational defect in one of the later steps may lead to an apparent reduction in “uptake.” For example, Cq acids (succinate, fumarate, malate, aspartate) enter E. coli by an inducible uptake system (Kay and Kornberg, 1971); they show mutual competitive inhibit,ion, indicating a common carrier, but the intxacellular concentration of free acid remains low and most of the isotope is rapidly incorporated into material precipitable by trichloroacetic acid (TCA). Several types of mutants have been isolated with reduced uptake of Ca acids. Some (dct mutants) have been found to contain normal amounts of the enzymes of the tricarboxylic acid cycle, and are thought to be defective in the transport system itself. However, other mutants, known to lack succinic dehydrogenase activity, also show a drastically reduced ability to take up labeled succinate (although they are virtually normal in their ability to take up labeled fumarate or malate). In these strains the uptake of radioactivity is decreased because the succinate cannot be further metabolized. Presumably, a more sensitive assay, carried out over shorter time intervals and measuring acid-soluble rather than acid-insoluble radioactivity, would reveal that the transport system is present in such mutants. Likewise, assays of amino acid uptake in bacteria frequently involve the measurement of TCA-precipitable radioactivity that has been incorporated into protein. Ames (1964, 1974b), who introduced this method, has pointed out that it gives a valid estimate of the initial rate of uptake only when the concentration of radioactive amino acid in the medium is low, so that transport is rate-limiting for the overall process. Ames has further noted that, in such an assay, a mutant defective in the incorporation of the amino acid into protein (for example, a mutant with an altered tRNA synthetase) might incorrectly be classified as a transport mutant; the error would be revealed by an uptake measurement that does not depend on protein synthesis (for example, an assay in the presence of chloramphenicol or in glucose-starved cells).

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2. A second kind of indirect effect can occur if the mutant produces an inhibitor of transport. Ames (1964) found that a class of p-fluorophenylalanine-resistant mutants of Salmonella, first thought to be defective in transport, in fact excreted large amounts of phenylalanine into the medium; supernatant fluid from a suspension of mutant cells, when added t o a wild-type suspension, competed with pheny1alanine-l4C or p-fluorophenylalanine-"C and thereby decreased the measured rate of transport. 3. Mutations in energy metabolism might also be expected to alter transport indirectly. I n this case the abnormalities would tend to be general ones, affecting transport through multiple systems and perhaps other energy-requiring processes as well. Mutations altering D-lactic dehydrogenase of E. coli, glycerol-3-phosphate dehydrogenase of Staphylococcus aureus, enzyme I and HPr of the bacterial phosphoenolpyruvate phosphotransferase system, or the membrane ATPase of Streptococcus faecalis (Table I) might be placed in this category although, at least in the last-mentioned two cases, the enzymes can be considered to be actual components of the transport systems. Diffuse transport diseases in man, such as the Fanconi syndrome, might also be explained by a primary disturbance in energy metabolism (Rosenberg, 1969). 4. A final class of mutations affecting transport indirectly includes those altering the fundamental structure-either lipid or protein-of the cell membrane. Recent experiments have indicated, for example, that transport rates can be highly dependent on the lipid composition of the membrane. In these experiments unsaturated fatty acid-requiring mutants of E. coli wcrr grown on a wide range of fatty acid supplements, with the result that they incorporated a corresponding range of unsaturated fatty acids into their membrane phospholipids (Schairer and Overath, 1969; G. Wilson el al., 1970; Wilson and Fox, 1971). Under each set of conditions, the Arrhenius plot of transport rate as a function of temperature appeared biphasic, the slopes extrapolating to an intersection a t a clear transition temperature. The transition temperatures varied from 7" for cells grown on linolcate to 30" for cells grown on elaidate, and were interpreted to mean that transport depends on the liquidity or mobility of the lipid phase of the membrane. [For further discussion of transition temperatures, see Kaback (1972) and Hochstadt-Ozer (1972a)l. It is clear, however, that these are not specific effects on individual transport systems. I n the experiments of Wilson and Fox (1971), cells containing any given fatty acid showed the same transition temperature for two independent transport systems, the p-galactoside system and the p-glucoside system; and furthermore, Overath et al. (1970) observed similar transitions for cell rcspiration and growth. Therefore mutational changes in the gross lipid composition of the membranc-although interesting in themselves-are not likely to

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be mistaken for mutations affecting transport specifically. [By contrast, if a minor lipid species were a cofactor for a particular transport system, its loss or alteration might be classified as a transport mutation. The only lipid cofactor presently known is phosphatidylglycerol, which stimulates enzyme I1 of the phosphoenolpyruvate phosphotransferase system (Kundig and Roseman, 1971b; Kaback and Milner, 1970). Phosphatidylglycerol comprises 5-15y0 of the cellular phospholipid in E. coli, however (Cronan and Vagelos, 1972), and is presumably involved in multiple membrane functions.] Less is known about the organization of proteins in cell membranes and about the possible secondary effects on a transport system that might result from mutational changes in neighboring proteins. One can imagine that such effects might be appreciable. Even in relatively simple subcellular structures such as multienzyme complexes, a missense mutation leading to an amino acid substitution in one subunit can affect the state of aggregation of the complex and thereby produce gross changes in the activity of the other subunits [see, for example, the work of Yanofsky and Ito (1966) and Bauerle and Margolin (1966) on the anthranilate synthetase-phosphoribosyl transferase complex of E. coli and Salmonella, and the work of Giles and his colleagues on the arom aggregate in Neurospora (Case and Giles, 1971; Jacobson et al., 1972)l. I n a membrane, such interactions almost certainly take place in transport systems that have multiple subunits, but they could also occur between a transport system and neighboring membrane proteins. It might then become difficult, both in theory and in practice, to distinguish between those proteins (and their corresponding genes) involved in transport directly and those involved only indirectly. Mutations affecting transport indirectly are clearly worth investigation, since they can provide useful information about the metabolic control of transport and about the synthesis and general organization of the cell membrane. By far the most attention has been paid to mutants having well-defined, presumably primary defects in transport, however, partly because such strains are easier to identify and partly because they offer a way to dissect transport mechanisms a t the molecular level. These mutants (see Table I), whenever they have been sufficiently well characterized, correspond to one of three kinds of genes: 1. Structural genes for transport proteins. The ultimate test of a structural gene-that mutations in it should lead to corresponding changes in the amino acid sequence of the protein product-has not yet been met for any transport gene, because no transport protein has yet been sequenced. I n several cases preliminary evidence is quite convincing, however. Two mutant strains of E. coli, defective in transport via the

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P-methylgalactoside system, have been found to possess qualitatively altered galactose-binding proteins (Boos, 1971, 1972; Silhavy and BOOS, 1972). I n one case the protein does not bind galactose a t low concentrations and does not exhibit the normal substrate-induced conformational change ; in the second casc the protein shows similar defects, and in addition a tryptic digest has revealed an altered peptide fragment. Several other bacterial transport mutants that are temperature-sensitive for transport have been shown to possess correspondingly heat-labile proteins. These include a l a c y mutant of E. coli with an altered M protein (Fox et al., 1967), a hisJ mutant of Salmonella with a n altered histidine-binding protein (Ames and Lever, 1970, 1972; Lever, 1972), and a ptsI mutant of E. coli with an altrrrd enzyme I of the phosphoenolpyruvatc phosphotransfcrasc system (Epstcin et al., 1970). In addition, a dicyclohexylcarbodiimide (DCCD)-resistant mutant of S. faecalis has bren shown to have an ab-

FIG.1. Partial linkage map of E . coli, adapted from Taylor (1970), including genes affecting transport and permeability and also related genes mapping in the same operons. Genes in parentheses have been only approximately mapped a t the positions shown. Symbols are described in Table 111.

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CAROLYN W. SLAYMAN

normal “carbodiimide-sensitizing factor,” one of the components of the membrane ATPase complex thought to play a role in cation transport in this organism (Abrams et al., 1972). With considerable effort being made in many laboratories to characterize transport mutants biochemically, it is likely that the list of confirmed structural genes will continue t o grow. 2. Genes that, code for enzymes involved in the synthesis of transport cofactors. Whenever a transport system requires a particular cofactor-for example, a low-molecular-weight, ion-chelating group or a specific lipid-it should be possible to isolate corresponding mutants. Only one clear-cut case is known at the present time: the entA, B, C , D,El and F mutants of E. coli, which lack the six enzymes involved in the biosynthesis of enterobactin and are unable to transport iron by chelation with this compound (Young et al., 1971; Luke and Gibson, 1971). Again it seems likely that further examples will be discovered in the next few years. 3. Regulatory genes that control the expression of the above kinds of structural genes. Regulatory genes are well known for many transport systems [lacl in E. coli (Jacob and Monod, 1961); araC in E. coli (Englesberg et al., 1964, 1965, 1969; Novotny and Englesberg, 1966; Sheppard and Englesberg, 1967); gltR in E. coli (Marcus and Halpern, 1969); dhuA in Salmon.ella (Ames and Lever, 1970); cys-3 and scan in Neurospora (Marzluf and Metzenberg, 1968; Metzenberg and Ahlgren, 1971; Burton and Metzenberg, 1972)], and may explain numerous cases in which transport activity is changed quantitatively but not qualitatively by mutation. More is said about the regulation of synthesis of transport systems in Section VI, p. 150.

IV. LINKAGE RELATIONSHIPS OF TRANSPORT MUTANTS

A large number of transport mutants, as well as mutants with general defects in membrane structurc and permeability, have been mapped in E. coli and t,o a lesser extent in Salmonella and Neurospora (Figs. 1-3; Tables III-V). Several clear-cut patterns can be seen. A. Escherichia coli and Salmonella iyphimurium

I n the bacteria, in which genes of related function are often clustered, the genes that code for sugar transport systems tend to map next to the genes that code for the corresponding enzymes and to be regulated together. The best-known example is the lactose operon of E. coli, containing the genes for 0-galactoside transport ( l a c y ), fl-galactosidase (la&), and

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(deo P)

FIG.2. Partial linkage map of S. typhimurium, adapted from Sanderson (1970), including genes affecting transport and permeability. As in the map of E . coli, genes in parentheses have been only approximately mapped a t the positions shown. Symbols are described in Table IV.

thiogalactoside transacetylase (lacA), all regulated by the lacI gene (,Jacob and Monod, 1961; Table I). A similar situation exists in the p-glucoside oprron (including bylB for p-glucoside transport and bglA for aryl p-glucosidase) and the glycerol phosphate operons (with glpF for the facilitated diffusion of glyccrol mapping next to gZpK for glycerol kinase, gZpT for L-a-glycerol-phosphate transport mapping next to glpA for L-a-glycerol-phosphate dehydrogenase, all regulated by gZpR) (Table I). The advantage of this kind of coordinate regulation is clear, since the transport system for a given sugar and the enzymes required to metabolize that sugar can be induced or repressed togcthrr, as conditions dictate. I n addition, there are several regions of the chromosome of E. coli that

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CAROLYN W. SLAYMAN

TABLE Ill Escherichia coli Symbol acrA [araAl IaraBl araC [araDl araE argP aroP azi [bglAl bglB bglC b ir brnP brnQ cycA CYSB

cysE CYSP

dc t entA-F envA fabA fabB fep [gIpAl [glp D1

Map Location (11) 1 1 1 1

56 57 2 2 73 73 73 77 1 10

83 25 72 153) 69 14 1.5

22 44 14 43

IglpKI SIP R

66 76 76 66

sip T

43

gltC glrR g/ ts

73 79 73 26 16 (70)

sip F

gts kdpA-D kmt [IacAl lac1 lac Y [IacZI IpcA IPCB ma16 [malPl [malQl

10 10 10 10

(5) 1571 79 66 66

Phenotypic Trait Affected Sensitivity t o acriflavine, phenethyl alcohol, sodium dodecyl sulfate [ L-Arabinose isomerase] [ L-Ribulokinasel Regulatory gene f o r araA, araB, araD, araE [ L-Ribulose-5-phosphate 4-epimerase] L-Arabinose transport Arginine transport Aromatic amino acid transport Sensitivity t o sodium azide, phenethyl alcohol [ A r y l 0-glucosidasel 0-Glucoside transport (enzyme II ) Regulatory gene for bglA, b g l B Biotin transport Leucine, isoleucine, valine transport Leucine, isoleucine, valine transport Glycine, alanine, serine transport Regulatory gene Regulatory gene SO4 transport C4 Dicarboxylic acid transport Fe transport (synthesis o f enterobactin) Sensitivity t o ampicillin, chloramphenicol, kanamycin, novobiocin, actinomycin D,rifampicin, gentian violet p-Hydroxydecanoyl thioester dehydrase Unsaturated fatty acid biosynthesis Fe transport [ L-a-Glycerophosphate dehydrogenase] [ La-Glycerophosphate dehydrogenase] Facilitated diffusion o f glycerol [Glycerol kinasel Regulatory gene f o r gIpA, D, F, K, T La-Glycerophosphate transport Operator (glrS) Regulatory gene for glrS, Glutamate transport Inability t o plasmolyze K transport (inducible system) Multiple transport systems (amino acids, carbohydrates. P04, SO41 [Thiogalactoside transacetylase] Regulatory gene for IacA, Y, Z b-Galactoside transport [p-Galactosidase] Sensitivity t o novobiocin, spiramycin, actinomycin D Sensitivity t o novobiocin, spiramycin, actinomycin D Maltose transport [Maltodextrin phosphorylasel [Amylomaltase]

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OF MEMBRANE TRANSPORT

TABLE I I I (Continued) Escherichia coli Symbol ma1 T I melA I melB mglP mgl R

Map Location

mtcA mtcB mtlA mtlC (mtlD1 pitA plsA ptsF ptsH ptsl rbsP shiA rolA to16 tole tolD ton8

66 81 81 (40) (17 ) (36) 12 58 71 71 71 (68) 13 (42) 47 47 74 38 17 17 58 (20) 25

trkA trkB trkC trkD trkE uhp umg uraP

63 63 1 73 25.5 72 23.5 50

mng

Phenotypic Trait Affected Regulatory gene Ia-Galactosidasel Melibiose transport P-Methylgalactoside transport Regulatory gene for mglP M g transport Sensitivity t o mitomycin C, acridines, methylene blue tolC7, sensitivity t o mitornyctn C, acridines, rnethylene blue Mannitol transport (enzyme I I ) Regulatory gene f o r mtlA, D Mannitol-1 -phosphate dehydrogenase PO4 transport Glycerol-3-phosphate acyltransferase Fructose transport (enzyme I I ) HPr of the phosphotransferase system ctr, enzyme I o f the phosphotransferase system D-Ribose transport Shikimate and dehydroshikimate transport Tolerance t o colicins E2. E3, A , K Tolerance t o colicins E l , E2, E3, A, K Tolerance t o colicin E l Tolerance t o colicins E2, E3 Resistance t o phages T1, @80,and colicins B, I, V , altered Fe and amino acid transport K transport K transport K transport K transport K transport Hexose phosphate transport Glucose transport (enzyme Ill Uracil transport

contain multiple genes affecting the cell surface. The first, from 1 to 3 minutes on the map of Taylor (1970), was originally pointed out by Yura and Wada (1968) and is now known to include: envA and azi, two genes associated with sensitivity to antibiotics, sodium azide, and phenethyl alcohol, and presumably somehow influencing general membrane permeability; brnP and aroP, controlling the transport of leucine-isoleucine-valine and the aromatic amino acids, respectively; and also (not shown in Fig. 1) aceE and F , coding for pyruvate dehydrogenase, a membrane-bound enzyme complex.

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CAROLYN W. SLAYMAN

TABLE IV Salmonella typhimurium Symbol aroP cysA cys6 deoP dhuA enb gxu hisJ hisP

metP mtlA. B ptsH PtSl sidA-H, M sidJ

Map Location

28 76

52 (34) 73

20 (12) 73 73 5 115 77 77

9 20

Phenotypic Trait Affected Aromatic amino acid transport SO4 transport Regulatory gene Deoxyribose transport Regulatory gene for hisJ Fe transport (Enterobactin synthesis) Guanine, xanthine transport Histidine transport Histidine transport Methionine transport Mannitol transport car6; HPr o f the phosphotransferase system carA; enzyme I of the phosphotransferase system Fe transport Fe transport

The second region is between 11 and 12 minutes (Taylor, 1970) and includes genes influencing sensitivity to acridines, mitomycin C, methylene blue, phenethyl alcohol, and sodium dodecyl sulfate (acrA, mtc), and also (not shown in Fig. 1) genes affecting septum formation and radiation sensitivity (Zon,, min, ras) (Taylor, 1970). Ot,her portions of the map containing multiple membrane-related genes are the regions from 16 to 17 minutes (with kdpA, B , C, D and tolA and B ) , from 25 to 26 minutes (with tonB, trkE, and gts), and from 69 to 70 minutes (with pitA, dct, and kmt). Whether these regions have any significance at the level of regulation of gene expression is not yet clear. B. 'Neurosporacrassa

I n the fungi, unlike the bacteria, genes of related function are not generally clustered. The only exceptions are regions such as arom in Neurospora and his-4 in yeast, which code for multienzyme aggregates (reviewed by Calvo and Fink, 1971). There has been speculation that such regions, like bacterial operons, may be transcribed into polycistronic mRNA, and that the protein products may be synthesized as a single polypeptide chain which is then cleaved by proteolytic enzymes, either before or after formation of the aggregate (Jacobson et al., 1972). If this is a special mechanism that has evolved for the assembly of protein aggregates, it could conceivably hold true for some portions of the cell membrane as well. Not enough membrane-related genes are known in

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the fungi to provide a clear test of this idea. The few that have been mapped show no particular tendency to be located together; of the seven Neurospora loci which control sensitivity to growth media of high osmolality (0s-1, 2 ,3 , 4 , 5 ; cut;$m-2) and which may determine elements of the cell wall (Livingston, 1969) or cell membrane (Slayman and Slayman, 1965), six are located on linkage group I but not adjacent to one another (Mays, 1969). Likewise,

I Ir)

I ul r,

0

II'

I

*

I

x L L

m L

I

c

In

E

0

e

0

n

P

H

10 map u n i t s

FIG.3. Partial linkage map of N . massa, adapted from Radford (1972), including genes affecting transport and permeability. Genes in parentheses to the right of each linkage group have been mapped on that linkage group, but their precise location is not known. Symbols are described in Table V.

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CAROLYN W. SLAYMAN

TABLE V Neurospora crassa Symbol bas-a bat cut cys-3 cys-13 cys-14 flm-2 fpr-1 hip-1 hlp-2 mod-5 mtr

Map Location

II IV I

nap neu-r

II I IV I V VI I VI I VI IV V IV

nuc-1 nuc-2 0s-1 0s-2 0s-3 0s-4 0s-5 pcon

I II I IV I I I I1

pmb pmn scon sor su-mtr

V IV V I, Ill. v, VI, V I I I

trk-1 trk-2 4 5 5 7 0 1t) un ( UM300)

Ill V I VI

Phenotypic Trait Affected Basic amino acid transport via system I l l Basic amino acid transport via system I l l Sensitivity t o high osmolarity Regulatory gene SO4 transport SO4 transport Sensitivity t o high osmolarity A m i n o acid transport A m i n o acid transport A m i n o acid transport A m i n o acid transport Neutral amino acid transport via system I A m i n o acid transport Possibly allelic w i t h mtr; neutral amino acid transport via system I Regulatory gene affecting PO4 transport Regulatory gene affecting PO4 transport Sensitivity t o high osmolarity Sensitivity t o high osmolarity Sensitivity t o high osmolarity Sensitivity t o high osmolarity Sensitivity t o high osmolarity Possibly allelic w i t h nuc-2; regulatory gene affecting PO4 transport Basic amino acid transport Probably allelic w i t h mtr; neutral amino acid transport Regulatory gene affecting SO4 transport Sorbose (glucose) transport Regulatory gene affecting general amino acid transport via system II K transport K transport A m i n o acid transport A m i n o acid transport

two genes affccting potassium transport in Neurospora (trk-1, trk-2) are not linked (Slayman & Tatum, 1965; Slayman, unpublished results). V. DOMINANCE AND RECESSIVENESS

Most transport mutations are recessive when studied a t the gross phenotypic level. Where homozygotes are analog-resistant or require ab-

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normally high substrate concentrations for growth or display pronounced clinical symptoms, the corresponding heterozygotes are sensitive to analogs (hisP in Salmonella, Ames and Roth, 1968; mtr in Neurospora, Stadler, 1966), are able to grow at low substrate concentrations (trk-1 in Neurospora, Slayman and Tatum, 1965) and appear clinically normal (cystinuria, Hartnup disease, methionine and tryptophan malabsorption, iminoglycinuria, glucose-galactose malabsorption, renal glycosuria, vitamin BIZ malabsorption ; see Table I). Such phenotypic recessiveness is readily understood if one assumes that the wild-type allele is capable of supplying at least some transport function. However, a few dominant transport mutations have been described, including X-linked hypophosphatcmic rickets in man (Winters et al., 1957, 1958), LK sheep red cells (Evans and King, 1955; Evans et al., 1956), f p a D (resistance to p-fluorophenylalanine) in Aspergillus (Sinha, 1969), and at least one kdpC mutant in E. coli (Epstein and Kim, 1971). In addition, when careful transport measurements are made, even mutations that are recessive a t the gross phenotypic level are often found to be expressed to an intermediate degree. Both in cystinuria (Rosenberg e2 al., 1966a) and in iminoglycinuria (Goodman et al., 1967; Scriver, 1968; Rosenberg et al., 1968), some heterozygotes excrete significantly larger amounts of amino acids than do normal subjects, and therefore have a partial drfect in renal tubular reabsorption. Likewise, transport measurements in yeast diploids have revealed that arg-p, lys-p, met-p, and Sam-p heterozygotrs take up the corresponding substrates (arginine, lysine, methionine, and S-adenosylmethionine) at rates halfway between those measured for homozygous mutant and wild-type cells (Grenson et al., 1966; Grenson, 1966; Gits and Grenson, 1967; Murphy and Spence, 1972). It is useful to consider three levcls at which dominance or codominance can arise : during regulation of gem expression, during assembly of the transport system into the membrane, and as a result of intcractions among multiple components of the transport system.

1. I n the lactose operon of E. coli, dominant mutations of lacI (the regulatory gene) havr been described that prevent the synthesis of the lacZ, l a c y , and lacA gene products (P-galactosidase, the p-galactoside transport system, and thiogalactoside transacetylase). These mutations+-can be understood as causing a change in the structure of the repressor protein such that it can no longer be removed from the operator by inducer, with the result that transcription of the operon is blocked (reviewed by Gilbert and Mullrr-Hill, 1970). The situation is complicated by the fact

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CAROLYN W. SLAYMAN

that the repressor protein is a tetramer, and that interactions can occur among its subunits. However, if the heterozygous cell contains a mixture of repressors-normal i+ tetramers, je tetramers, and hybrid tetramers with a mixture of subunits-then the i8 tetramers (and also, apparently, proteins containing three i8subunits) will bind to the operators whether or not any inducer is present. As a result, je is dominant over i+,and the products of the lactose operon are not synthesized. 2. Even when transport proteins are made, they must be integrated into the membrane, and there are several aspects of this process that can lead to the partial expression (or even the complete dominance) of a mutant allele. If, for example, the membrane has an unlimited capacity for transport protein, one would expect to see pure gene dosage effects, in which a heterozygous diploid cell (with one normal copy of the transport gene) contains only half as many transport sites as a homozygous wild-type cell (with two normal copies). If the membrane has a limited capacity for transport protein, and if a mutant protein competes effectively for integration into the membrane without being able to function in transport, one might again find decreased transport activity in the heterozygous cell. (In the extreme case of a mutant protein that is integrated more efficiently into the membrane than the wild-type protein, dominance could conceivably be complete.) Experimental evidence for a limited number of transport sites has recently been presented in E. coli (Fox, 1969) and in yeast (Hennaut et al., 1970). I n E. coli, Fox compared the expression of lacZ, l a c y , and lacA in a normal strain possessing a single copy of the lactose operon and a partial diploid strain with two copies of the operon. As predicted, the diploid had twice the normal amount of p-galactosidase and thiogalactoside transacetylase (the soluble products of lac2 and ZacA), and also twice the normal amount of M protein (the membrane-bound product of lacZ, assayed by substrate binding; see also Kennedy, 1970), consistent with the idea that protein synthesis proceeded in proportion to gene dosage. Significantly, however, 0-galactoside transport activity had incrcased only slightly in the diploid, by a factor of 1.1 to 1.4. It appeared that although M protein was incorporated into the membrane, only part of it had become associated with functional transport sites. Further experiments have suggested that the limiting factor, in the presence of multiple copies of the l a c y gene, is the rate of synthesis of membrane lipids, since p-galactoside transport activity cannot be induced a t all in E. coli in the absence of phospholipid synthesis (Fox, 1969; Hsu and Fox, 1970; Wilson and Fox, 1971; but see Mindich, 1970 b ; Willecke and Mindich, 1971, for somewhat different results in other organisms). If, for whatever reason,

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there is a limited rate at which transport protein can be integrated into active membrane sites, one would expect-in partial diploids containing some lacy alleles-to observe a reduction in transport resulting from competition between mutant and wild-type protein Experiments of this kind have not been reported. I n a second, possibly analogous example, three types of cystinuria have been distinguished on the basis of quantitative differences in the transport of cystine and the basic amino acids (Rosenberg, 1966). Normally these amino acids are transported by a single, shared system in the intestine and by both shared and specific systems in the kidney. The mutation leading to type I cystinuria appears to inactivate the shared system completely, since type I homozygotes show no detectable transport of cystine and the basic amino acids in the intestine. By the same argument the mutations leading to type I1 and I11 cystinuria-known to be allelic to type I (Rosenberg, 1966)-appear to cause qualitative changes in the shared system, since type I1 and I11 homozygotes have only partial defects in intestinal transport. Interestingly, however, type I1 and 111 heterozyyotes show a significant defect in renal transport, while type I heterozygotes appear normal. These results could be interpreted to mean that altered transport proteins, produced by the type I1 and I11 alleles, compete with the normal protein for assembly into the membrane, while the type I protein is either more severely altered such that it does not compete, or else is missing altogether. 3. A third way in which dominance can arise, in the special case of a protein with several identical subunits, is by interactions between mutant and wild-type subunits such that the activity of the protein is decreased. If the number of subunits is large, the majority of wild-type subunits will be found in hybrid molecules containing at least some mutant ones; and if thr negative interaction is strong, the mutant alleles will appear dominant. The possibility of this kind of dominance was first pointed out by Garen and Garen (1963) and Bernstein et al. (1965), and several cases have since been described, although none involves a transport system. Glutamic dehydrogenase of Neurospora, for example, is a n oligomeric protein coded for by the am locus; and in heterokaryons between certain am and am+ strains, the enzyme formed is heat-labile, electrophoretically abnormal, and only partially active (reviewed in Fincham and Day, 1972). Similarly, in the lactose operon of E. coli, dominant i- mutations can arise, producing inactive repressor subunits which mix with normal subunits to form inactive hybrid tetramers (Gilbert and Muller-Hill, 1970). Whether or not this kind of mechanism can account for any of the dominant transport mutations remains to be established.

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VI. REGULATION OF TRANSPORT SYSTEMS

Transport systems are not, in many cases, permanent constituents of plasma membranes. In microorganisms their presence is often determined by the growth medium; carbohydrate transport systems tend to be inducible by their substrates, as do amino acid transport systems when the amino acid in question can be used as a carbon or nitrogen source (e.g., glutamate and tryptophan in E. coli, aromatic amino acids in Salmonella; Table I). Other microbial transport systems are present only during certain stages of the life cycle. SO4uptake in Neurospora is mediated by two systems, for example, one in conidia and the other in growing hyphae (Marzluf, 1970a, 1972; Roberts and Marzluf, 1971). Although both systems are completely repressed during growth on methionine, and are known to be regulated in this respect by two genes (cys-3, scon) that control a group of related enzymes (aryl sulfatase, choline sulfatase, and choline-0-sulfate permease; Marzluf and Metzenberg, 1968; Burton and Metzenberg, 1972), the mechanism of the first type of regulation-during the life cycle-remains unclear. The only genetic modification that has yet been achieved is in the cys-14 mutant, which lacks the hyphal SO4 transport system and in which the conidial system appears to persist. I n Neurospora, the four major amino acid transport systems are also subject to regulation during the life cycle. Systems I (for neutral amino acids) and I11 (for amino acids) are present in rapidly growing cells, while systems I1 (for most D- and L-, neutral, basic, and acidic amino acids) and IV (for acidic amino acids) are present in older hyphae and tend to increase during carbon or nitrogen starvation (Pall, 1969, 1970a, b). It is probable that a t least some of the mutations affecting multiple amino acid transport systems in Neurospora [fpr-1, mod-5, nap, un(UM300), un(55701t)l are in regulatory genes, perhaps controlling carbon or nitrogen metabolism, but the exact mechanism of regulation is likely to prove very complex. I n multicellular organisms there is the added complication that a given transport system may be present in one tissue but not in others. The pattern of distribution can best be studied for genetically defective transport systems which are easily distinguished from their normal counterparts. I n man, mutations frequently affect transport across both the intestinal epithelium and the renal tubular epithelium-for example, in cystinuria, Hartnup disease, iminoglycinuria, and glucose-galactose malabsorption (Table I). However, the same mutations appear not to alter transport in other tissues. Cystine and the basic amino acids are transported normally in leukocytes and cultured fibroblasts from patients with cystinuria, for example (Becker and Green, 1958; Rosenberg and

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Downing, 196.5; Groth and Rosenberg, 1972), and one can conclude that separatr systems, coded for by separate genes, are responsible for transport in these cell types. Presumably, many of the medium-dependent, time-dependent, and tissue-dependent changes in transport activity can be accounted for by standard regulatory mechanisms affecting the rate of synthesis of transport proteins. Othrrs may reflect changes in the integration of transport proteins into the membrane or in the transport process itself (for example, by changes in thc intracellular concentration of activators or inhibitors of transport). The study of thesc phenomena is made difficult by the fact that only a few transport proteins can be assayed directly, independently of thrir transport function. Only when dirrct assays are available is it possible to distinguish between regulation of synthesis, of integration into the membrane, and of activity.

VII. USEFULNESS OF MUTANTS IN UNDERSTANDING TRANSPORT MECHANISMS A. In Determining the Number of Separate Transport Systems for a Particular Substrate

The kinctics of transport can be quite complex when multiple systems are present, particularly when the systems have similar affinities for substrate. I n this circumstance mutants have often hclped to simplify the analysis. Of the two systems for aspartate uptake in E. coli, for example, ast mutants lack the specific one, and dct mutants the general one for C4 dicarboxylic acids. The biphasic curve of uptake versus concentration scen in wild-type cells is resolved clearly into single saturation curves in the two mutants (Kay, 1971). Similarly, hisJ mutants lack one of the histidine-specific transport systems in Salmonella, permitting the study of the remaining hisK system(s) (Ames and Lrver, 1970); and mtr and bat mutants of Neurospora and gap, lys-p, arg-p, and met-p mutants of yeast have helprd to decipher even more complex sets of general and specific amino acid transport systems in these organisms (Pall, 1969, 1970a; Grenson et al., 1966, 1970; Gits and Grcnson, 1967; Crabeel and Grenson, 1970). In man the study of iminoglycinuria has contributed to the notion of multiple renal transport systems for glycine, prolinr, and hydroxyproline. Homozygotes for iminoglycinuria reabsorb only 60% of the filtered glycine and, unlike normal subjects, do not show inhibition of glycine reabsorption when proline is infused. They are thought to lack a common renal system

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for both amino acids (and hydroxyproline) ,while retaining specific systems (Scriver, 1968; Rosenberg et al., 1968). B. In Identifying the Components of a Transport System

Mutants can also make it possible to decide whether or not a protein is part of a particular transport systcm. Numerous binding proteins have been isolated from bacteria during the past few years, and the argument that they are involved in transport has often been an indirect one, based on correlations between the affinities of the binding protein and the transport system for substrates and inhibitors, and on the amounts of binding protein and transport measured in cells grown under various conditions (for example, in the presence and absence of inducer). I n several cases, however, the isolation of qualitatively altered mutants has provided good evidence for a primary role of binding protein in transport. Ames and Lever (1972) have recently described a hisJ mutant of Salmonella in which the J binding protein is heat-labile and has abnormal chromatographic and electrophoretic properties; a t the same time histidine transport via the J system is temperature-sensitive. Other examples-including mglP, lacy, and ptsI mutants of E. coli, and a dcc mutant of S. faecalis-have already been discussed (Section 111). A further complication arises when a subunit is shared among several transport systems. Again mutants can be helpful. The idea that the bacterial phosphoenolpyruvate phosphotransfrrase system consists of both substrate-specific components (cnzymcs IJ) and common components (enzyme I and HPr) was greatly strengthened by the isolation of the corresponding mutants, those defective in the transport of a single carbohydrate found to lack an enzyme 11,and those unable to transport a whole series of carbohydrates found to lack cneyme I or HPr (Table I). Other mutants that have helped to establish a relationship among scveral transport systems are: hisP in Salmonella, which appears to lack a subunit required by two histidine-specific transport systems (Ames and Lever, 1970) and probably by histidinol and arginine transport as well (Ames et al., 1972); and aap mutants in yeast, with greatly decreased activities of the specific arginine, lysine, leucinc, and proline transport systems and also thc gcneral amino acid transport system (Grenson and Hennaut, 1971). I n neither of the latter two cases has the corresponding subunit been purified and characterized, however. C. In Determining the Function of a Component

Once a gene product is known to participate in a particular transport system (or systems) , more sophisticated questions can be asked regarding

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its function. Careful measurements of transport in qualitatively altered mutants can in theory establish which portions of the transport process are defective, and can lead to the assignment of a precise function to the gene product. I n the p-galactoside transport system of E. coli, it appears that the lacy gene product-the M protein-plays a role both in substrate binding and in the coupling of metabolic energy to transport. Binding is the basis for its assay, through the ability of substrate to protect it from reacting with N-ethylmaleimide (Fox and Kennedy, 196Fj). In addition Wong et al. (19701, T. H. Wilson et al. (1970), and Wilson and Kusch (1972) have described mutants, cotransducible with the lactose operon and presumably mapping in lacy, that arc LLenergy-uncoupled’lfor P-galactoside transport. These strains are grossly deficient in the ability to accumulate P-galactosides against a concentration gradient; when cells are exposed to a wide range of extracellular thiomethylgalactoside concentrations, they reach steady-state intracellular concentrations only 10-2075 of those achieved by wild-type E. coli. At the same time, however, the mutants can be shown to contain a normal (or possibly greater than normal) number of p-galactoside carriers in their cell membranes. They are capable of facilitated diffusion [energy-independent entry of substrate, measured by onitrophenyl-0-D-galactoside (ONPG) hydrolysis] a t 130-1800/0 of the normal rate; and when preloaded with TMG and then resuspended in TMG-14C they show rapid counterflow. Direct assay has indicated an excess of M protein (163% of the normal amount) in a t least one of the mutants. A reasonable model for p-galactoside transport in E. coli postulates a cycle of transformations of the carrier in which the energy-dependent step is a conversion of hl protein, at the inner surface of the membrane, to an altered form with greatly reduced affinity for galactosides. At the rxternal surface of the membrane the high-affinity form is regenerated, and it is the asymmetry of binding on the two sides of the membrane that results in the intracellular accumulation of galactoside (Fox and Kennedy, 196Fi; Winkler and Wilson, 1966). The precise nature of the energydependent step is not yet known. It has been suggested by various investigators to involve reaction of the transport system with ATP (Scarborough et al., 1968), response of the system to either a potential difference or a gradient of hydrogen ions across the membrane (Pavlasova and Harold, 1969) or, most recently, a direct coupling of transport to electron flow through D-lactic dehydrogenase, possibly involving a cycle of oxidation and reduction of the M protein (Barnes and Kaback, 1971; Kaback and Barnes, 1971). According to any of these models, the energy-uncoupled mutants would be viewed as having an abnormal R I protein that can bind substrate and carry it across the membrane, but cannot, interact with the energy source in the step that results in a lowered intracellular

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affinity. One intriguing point is that, the defect is not a complrtr one; the mutants arr uncoupled at low pH but are capablr of relatively normal active transport at high pH (Wilson and Kusch, 1972), and this proprrty mill have to be included in any attempt to interpret the mutation in molccular terms (whether by an alterrd rrsponsr to a hydrogrn ion or potential gradirnt, or by altered reaction with ATP or D-lactic dehydrogrnase). I n addition, it will bc of interest to lrarn whrthrr the mutant protein is altered in any other resprrts. Preliminary indications are that it binds substrates with essentially normal Km)s,at lrast in those casrs that have beep tested (Wong et al., 1970; T . H. Wilson et al., 1970), but that it is abnormally sensitive to sulfhydryl reagrnts (Wilson and I h c h , 1972). The incrrased carrier activity rcflected in the measurements of ONPG hydrolysis and TMG counterflow presumably also stems from an alteration in the M protein, but it is not yet clear whether there is an incrrased amount of M protein in the membranc (as suggestcd by the preliminary direct assay) or increased turnover. The 0-galactosidr transport systrm of E. coli is rrlatively simplr in that it appears to be codrd for by a single cistron, lacy, and to involve a single specific protein subunit, the h l protein. I t is vrry likely that othrr transport systems will be found which havc multiple subunits. I n these instances the subunits may play relatively specialized (although probably not completely independent) roles-some may be responsible primarily for substrate binding, while others may mrdiatc thr reaction with the energy source. One possible example is thr sulfate transport system of Salmonella. All mutants so far reported which arc defrctivc specifically in sulfate transport map in the cysA region, composed of thrcc cistrons. These cistrons are known to codr for polypeptide products since amber mutations (nonsense mutations suppressible a t the level of translation of mRNA into protein) have been isolated in each of them (Ohta et al., 1971), but the proteins themselves have not been identified and their function in transport is unknown. At the same time a sulfate-binding protein has been extensively characterizcd in Xalmonella and is believcd, on indirect grounds, to play a role in transport. It is located a t or near the cell surface (Pardee and Watanabe, 1968), and both binding and transport are affected in parallel by osmotic shock, inhibition by a series of anions, repression during growth on cysteine, and derepression during growth on djenkolic acid (Pardee et al., 1966). The structural gene for the binding protein has not been identified but is known not to be cysAa, b, or c; two mutants with long deletions covering the entire cysA region produced t hr binding protein in normal amounts, as did strains with nonsense mutations in each of the thrre cistrons (Ohta et al., 1971). This, then, is an example of a transport system that may contain as many as

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four different, subunits, only one of which is directly involved in substrate binding. VIII. CONCLUSION

It seems usclful, in concluding t8his article, to point to three general areas wherc :wtive work is just beginning. (1) Although great progress has been made in cataloging transport mutants of microorganisms, comparatively few mutants arc known in higher organisms. The next few years should see additions to the list of transport diseases in man and also-of prrhaps greater use expcrimcntally-the isolation of transport mutants in tissue culture cells. ( 2 ) Where mut8antfshave been identified with primary defects in transport, it is now possible to dissect transport mechanisms at the molecular level. Altered proteins can be examined in terms of their ability to bind substrate, to undergo substrate-induced conformational changes, to be integrated into the cell membrane, and to interact with the energy source for transport; ultimately it should be possible to rclatc changes in these properties to particular amino acid replacements in the protcins. (3) Finally, the development of improved techniques for the isolation of pleiotropic membrane mutants should lead, in the next frw years, to increased information about the interrelationships among transport systems and thc overall organization of the cell membrane. ACKNOWLEDGMENTS The author is grateful to the following people for supplying preprints and unpublished information: Drs. G. F. Ames, A. G. UeBusk, C. E. Furlong, J. Guardiola, J. Hochstadt-Ozcr, H. It. Kaback, W. K. Maas, C. W. Magill, It. L. Metzenberg, D. L. Oxender, M. L. Pall, B. P. Rosen, L. E. Itosenberg, S. Silver, D. R. Stadler, A. L. Taylor and W. M. Thwaites; and t o Dr. J. F. Hoffman and Dr. J. R. Sachs for helpful discussions about HK/LK sheep erythrocytes. Research in the author’s laboratory has been supported by grants from the National Institute of General Medical Sciences (GM15761), the National Genetics Foundation, and by a Research Career 1)evelopment Award (GM 20163). REFERENCES Abrams, Abrams, Abrams, Abrams, Abrams, Abrams, Abrams,

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Enzymic Hydrolysis of Various Components in Biomembranes and Related Systems MAHENDRA K U M A R J A I N Department of Chemistry. Indiana Universil y . Bloomington. Indiana*

I . Introduction

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

A . Scope of This Article . . . . . . . . . . . . . . . B. Types of Hydrolytic Enzymes . . . . . . . . . . . . I1. Action of HydrolyticEnzymes on Model Systems . . . . . . . . A. Action of Lipolytic Enzymes on Lipid Aggregates . . . . . . B Action of Proteolytic Enzymes on Lipid-Protein Films . . . . . 111. Effect of Enzymic Hydrolysis on System Properties of Biomembranes . . A . Perturbation of Cell Wall Components . . . . . . . . . . B Perturbation of Plasma Membranes . . . . . . . . . . C . Asymmetry Across Biomembranes . . . . . . . . . . . D Membrane Hyperstructure . . . . . . . . . . . . . E Unmasking and Release of Membrane-Bound Proteins . . . . . IV Effect of Enzymic Hydrolysis on Transport Systems . . . . . . . A Excitable Membranes . . . . . . . . . . . . . . B . Respiratory Chain . . . . . . . . . . . . . . . . C Calcium Transport System . . . . . . . . . . . . . D . Sodium plus Potassium Transport System . . . . . . . . . E . Hormone-Sensitive Transport Systems . . . . . . . . . . V Catabolism of Membrane Components by Endogenous Enzymes or Intracellular Catabolism . . . . . . . . . . . . . . . . . VI Conclusions and Epilog . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . .

.

.

. .

. . . . .

176 177 178 184 184 189 191 191 202 207 209 210 217 217 220 224 226 229 233 236 238

* Present address: Department of Chemistry. University of Delaware. Newark. Delaware . 175

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MAHENDRA KUMAR JAlN

1. INTRODUCTION*

A substantial body of experimental information has accumulated concerning the nature of the surface components of living cells. Histological, immunological, electrophysiological, and physicochemical methods have led to the general conclusion that the cell surface contains a variety of complex lipids, proteins, and carbohydrates organized in a lamellar array. Structural and functional specialization in surface membranes is manifested in their peculiar chemical composition and their organization in such a way that various components are integrated into a functional whole which transcends the sum of the properties of its parts (Jain, 1972), [The point of view involved here is that the functional interface between the intracellular and extracellular compartments is a domain that we shall call the surface membrane, or simply biomembrane. It consists of a thin (ca. 100 thick) inner zone which is hereafter referred to as the plasma membrane; the outer zone is referred to as a coat.] The system properties of surface membranes include, among others, barrier characteristics, spatiotemporal anisotropy of interfacial processes, and the integrative contribution of functional units. Thus, for example, carbohydrates may be present in biomembranes in the form of glycoproteins and glycolipids and be involved in such diverse macroscopic membrane functions as adhesion or electrophoretic mobility, or act as antigens. Similarly, on the microscopic or molecular level, virtually all the multienzyme systems of cells contain lipids as an integral part of their structure, which are required for their activity. The importance of individual components in membrane function is a reminder that the chemical behavior of each component in a membrane system may be dependent on direct and/or indirect interactions with other components of that system. Methods employed to study membrane phenomena frequently involve dissociation of the system into its constituents and/or component subunits. Thus purely analytical studies yield limited insight into membrane phenomenology, since membrane functions are largely attributable to system properties. Among methods of study of membranes applicable to

* The following abbreviations are used in this article: AChase, acetylcholinesterase; ANS, anilinenaphthalenesulfonicacid; BLM, bimolecular lipid membrane or black lipid membrane; CCFP, carbonyl cyanide-ptrifluoromethoxyphenylhydrazone; CD, circular dichroism; ESR, electron spin resonance; G-6-Pase, glucose-6-phosphatase; LAPase, leucine aminopeptidase; NANA, N-acetylneuraminic acid; PC, phosphatidylchloine; PE, phosphatidylethanolamine;PEP, phosphoenolpyruvate; PI, phosphatidylinositol; PMR, proton magnetic resonance; PNPase, p-nitrophenylphosphatase; PPase, pyrophosphatase; PS, phosphatidylserine; SDS, sodium dodecyl sulfate; SMP, submitochondrial particles with inside-out geometry; SR, sarcoplasmic reticulum.

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intact or nearly intact systems, enzymic hydrolysis of membrane-bound components deserves special consideration. I n view of their specificity of action and the generally mild conditions under which they work, hydrolytic enzymes are excellent tools for the selective modification of membrane structure by elimination or alteration of a specific component or group of components. A membrane thus modified is then subject to study by other methods. A. Scope of This Article

To a first approximation, the biomembrane may be visualized as an array of lipoprotein complexes held together in a matrix formed from a lamellar sheet of phospholipid molecules. The intermolecular interactions that determine the organizational variables characterizing the biomembrane as a system are probably of prime importance for understanding the interaction of macromolecules with the membrane as a whole and with specific components of it. As implied in the title of this article, biomembranes may be perturbed by a variety of target-specific enzymes; from resultant changes measured by other techniques, significant information is obtained regarding the nature of the target molecules, the relationship of target molecules to their neighbors, and chemical changes in target molecules as they relate to membrane structure and function. It is this line of reasoning that is detailed in the following sections of this article. Early sections are devoted to a discussion of the action of hydrolytic enzymes on model systems of aggregated lipids. Later sections focus more directly on biomembranes themselves. Although the literature covered is extensive, this article is not meant to be comprehensive. Some of the information obtained and conclusions reached through use of hydrolytic enzymes stand out as unique; but in general most of the information is supplcmentary or complementary to that already secured by independent methods. In the published literature little attention has been directed to the methodology of chemical modification of membrane components. Therefore the organization of this article is based solely on morphological features of biomembranes. The discussion follows (1) the modification of gross morphological features of membranes, (2) the alteration of features relating to the general system properties, and finally (3) the perturbation of specific molecular features which give rise to specialized membrane functions. However, of necessity the following discussion has a somewhat biased perspective, especially for the reader looking for a specialized experimental technique which can give definite answers to unsolved problems. The merits and pitfalls of the present technique can be appreciated only in an overview.

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B. Types of Hydrolytic Enzymes

Many lipases, saccharidases, and proteases have been described in the literature (Barman, 1969; Boyer, 1971, 1972). However, only a few of these, in varying degrees of purity, have been studied with regard to their action on biomembranes. 4 suitable choice from among these enzymes may be important not only from the point of view of substrate specificity but also from considerations of their size and topography, which determine the accessibility of their active sites to potential substrates. A short discussion of some of these hydrolytic enzymes follows. 1. LIPOLYTIC ENZYMES (Ansell and Hawthorne, 1964; Van Deenen and De Haas, 1966; Lennarz, 1970; Thompson, 1970)

Generally speaking, lipolytic enzymes do not act on their substrates in

true solution. These enzymes are unusual in the sense that they have substrates that are not soluble in water. However, lipids do form a variety of dispersed aggregated phases in aqueous as well as organic media, reflecting their amphipathic character (Bangham, 1963; Jain, 1972). These phases include emulsions, monolayers, lamellar lipid phases in various forms, and liquid crystals. Characteristically, all these phases have extended interfaces separating apolar chains from the aqueous medium. The polar groups of the lipid molecules are located a t these interfaces, and thus special problems arise with respect to the accessibility of the substrate to enzymes and the release of products. Moreover, lipolytic enzymes are subject to the action of the asymmetric surface forces of the ionic double layer and must penetrate the shield of organized water that surrounds the polar groups of the substrate. Such aspects of specific interfacial affinity of the enzymes suggest unexploitcd variation of the method of protein purification by “affinity” chromatography. Various reactions catalyzed by lipolytic enzymes are shown in Fig. 1. Lipase or glycerol-ester hydrolase (EC 3.1.1.3) catalyzes hydrolysis of triglyceridcs to diglycerides and fatty acid ions (Benzonana and Desnuelle, 1965; Slotboom et aE., 1970a, b ; Wills, 1965). Several lipases are activated by additives such as proteins, amphipaths, bile salts, acidic phospholipids, and hormones (see Table I). Stimulation of in vivo lipolytic activity by hormones may occur by either of two mechanisms (Illiano and Cuatrecasas, 1971a, b; Scow and Chernick, 1970) : fast-acting lipolytic hormones act through cyclic AMP, whereas slow-acting hormones act primarily through a mechanism involving RNA and protein synthesis. However, this distinction is somewhat arbitrary. Other agents enhance the rate of lipolysis by modifying the structure of the oil-water interface rather than by modifying the structure of the en-

179

ENZYMIC HYDROLYSIS IN BIOMEMBRANES

$H,OH 0 HOFH CH2-0- P-X

0-

'R2C-O-$H

c!

CH,-O-C-R

FIG.1. Reactions catalyzed by various lipolytic enzymes. (1) Phospholipase A:, (2) phospholipase AZ,(3) phospholipase B, (4) and (5) lysophospholipases, (6) phospholipase D, (7) phospholipase C, (8) phosphatidic acid phosphatase, (9) lipase.

zyme. It is believed that the enzyme is adsorbed by its emulsified substrate, and that the initial rate of reaction is a function of the number of enzyme molecules adsorbed a t the interface. However, lipophilic bonding unfolds lipase and destroys its activity, as it does for many other enzymes (Brockerhoff, 1971). Phospholipase A or phosphatide acyl hydrolase (EC 3.1.1.4) hydrolyzes only one of the fatty acyl ester linkages of 1,2-diacyl-3-n-glyceryl phosphatides. The enzyme phospholipase A1 attacks a t the 1 position, whereas phospholipase Az cleaves the fatty acid a t position 2. Although both of these enzymes show a distinct substrate specificity in the hydrolysis of purified phospholipids, there is evidence that this specificity is reduced in the presence of lipid mixtures (Van Deenen, 1969). Enzymes from different 3ources also display different substrate specificities (Bird et al., 1965; Van Deenen and De Haas, 1963; Woelk and Debuch, 1971) and cofactor requirements (Dawson, 1966; Kirschmann et al., 1964; Klibansky et al., 1962; Ibrahim et al., 1964; Salach et al., 1971; Uthe and Magee, 1971a, b). Thus the snake venom enzyme (mostly At) preferentially hydrolyzes lecithin and/or plasmalogen and is significantly more active on lipoprotein than on pure lipid substrates, but it does not hydrolyze sphingomyelin. Pancreatic phospholipase Az rapidly attacks very acidic lipids such as phosphatidylglycerol and cardiolipin and is inhibited by Na and K ions. Phospholipase A is also inhibited by various analogs of glycerophosphatides

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TABLE I EFFECTOF VARIOUSADDITIVES ON Lipolytic enzymes

THE

RATE OF LIPOLYSISBY LIPOLYTICENZYMES Effect

Lipase

Activated by proteins (Brockerhoff, 1971), amphipaths (Muller and Alaupovic, 1970), bile salts (Benzonana and Desnuelle, 1965), acidic phospholipids (Kariya and Kaplan, 1971), hormones (Appleman and Sevilla, 1970; Chariot et al., 1970), and detergents (Egelrud and Olivecrona, 1973). Phospholipase A Activated by cationic amphipaths (Shah and Schulman, 1967), organic solvents (Marinetti, 1965; Dawson, 1963), n-alkanols (Jain and Cordes, 1973), deoxycholate (Uthe and Magee, 1971a,b; Ibrahim et al., 1964), and bile salts (Boucrot and Clement, 1971). Inactivated by anionic amphipaths (Shah and Schulman, 1967; Quarles and Dawson, 1969a,b; however, see Dawson, 1966) Phospholipase B Activated by anionic amphipaths (Bangham and Dawson, 1960, 1962; Dawson, 1968; Dawson and Hauser, 1967; Kates et al., 1965). Phospholipase C Activated by anionic amphipaths (Kates, 1957; Dawson, 1968) and n-alkanols (Jain and Cordes, 1973) Phospholipase D Activated by anionic amphipaths (Quarles and Dawson, 1969a,b; Dawson and Hemington, 1967; Dawson, 1963) and ethyl ether (Condrea et al., 1964). Inhibited by cationic amphipaths (Quarles and Dawson, 1969a,b) 'Iriphosphoinositide Activated by cationic amphipaths and inhibited by phosphomonoesterase and anionic amphipaths (Dawson, 1968) -phosphodiesterase. Activated by chloroform (Ryan and Mavrides, 1960) Glucuronidase Sphingomyelinase Activated by methylesters of long-chain fatty acids, phosphatidic acid, dicetylphosphate, octadecylamine, various detergents (Gatt et al., 1973 and references therein) Ceramide trihexoside-aActivated by taurocholate Triton X-100 (Ho, 1973) galactosidase

+

(Rosenthal and Han, 1970). The term phospholipase A as used in this article generally refers to impure preparations. Lysophospholipase or lysolecithin acyl hydrolase (EC 3.1.1.5) catalyzes deacylation of lysolecithin. No specificity is indicated with respect to the position that can be attacked. Although the Enzyme Commission lists phospholipase B as a synonym for lysolecithin acyl hydrolase, the former term originally signified an enzyme capable of hydrolyzing fatty acids from both the 1- and 2-positions of phospholipids, and this is still considered to

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be the reaction catalyzed by a crude preparation from Penicillium notatum (Dawson and Hauser, 1967). I n the text the term phospholipase B is used in the latter sense. Several other enzymes catalyzing acylation and deacylation of various complex lipids and cholesterol esters have been described (Van Deenen and De Haas, 1966; Thompson, 1970). Phospholipase C or phosphatidylcholine cholinephosphohydrolase (EC 3.1.4.3) catalyzes cleavage of phosphorylcholine from phosphatidylcholinc (PC). Under proper conditions and in the presence of Ca ion, the enzyme from Clostridium welchii has the following substrate specificity P C > SM > PS > PI > LysoPC. PE is hydrolyzed only in the presence of PC (Stahl, 1973; also see McIlwain and Rapport, 1971). It is inhibited by phosphonate analogs of glycerophosphatides (Rosenthal and Pousada, 1968). A highly purified enzyme preparation from Bacillus cereus completely hydrolyzes P C and PE, and to a lesser extent PS; however, this enzyme does not attack sphingomyelin (Roelofsen et al., 1971; Zwaal et al., 1971). The distinct behavior of the Clostridium and Bacillus preparations probably reflects the presence of two phospholipases C in C. welchii (Pastan et al., 1968); one enzyme preferentially hydrolyzes spingomyelin, and the other preferentially hydrolyzes PC. Both of these enzymes are activated by organic solvents, but Ca ion has a differential action (see also Bangham and Dawson, 1962; Kates, 1957) (Table I). Triphosphoinositide phosphomonocsterase and phosphodiesterase hydrolyze triphosphoinositides; the phosphomonoesterase successively dephosphorylates the phospholipid, yielding diphosphoinositide and monophosphoinositide, while the phosphodiesterase cleaves it into diglyceride and inositol triphosphate. Sodium phospoinositide in aqueous “solution” forms highly negatively charged micelles which are only partially hydrolyzed by the enzymes. After addition of long-chain cations, however, the reaction proceeds more nearly to complction ; anionic amphipaths are potent inhibitors (Table I). Phospholipase D or phosphatidylcholine phosphohydrolase (EC 3.1.4.4) catalyzes hydrolysis of phospholipids such as PC, PE, PS to diglyceride phosphatc and a base. Cardiolipin-specific (On0 and White, 1970a, b) and sphingomyelin-specific (Soucek et al., 1971) enzymes have also been characterized. Several other catabolic enzymes have also bcen described. Since they bear little relevance to this article, no attempt is made to elaborate on their chemistry (see Gatt, 1970; Roscman, 1970; Thompson, 1970) 2. GLYCOLYTIC ENZYMES

Various glycosphingolipids (gangliosides) and glycoproteins (Fig. 2) play an important role in membrane phenomena. These cell wall components can be selectively cleaved by the variety of enzymes listed below.

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cH,cohNp "

OH

B gal NAc(l-c4)gol(l~4)glc-cer B B Gal(1-3) NANA

FIQ.2. (A) Proposed structure of monosialoganglioside. (B) Abbreviated structure of di- and trisialogangliosides. (C) Left: Glycoproteins from erythrocyte membrane. X --t = NAN-galactose; few polysaccharide chains (branched); OH- stable. Right: glycoproteins from sheep submaxillary mucin. X + = NAN-acetylgalactosamine; many disaccharide units; OH- labile. All these complex glycolipids and glycoproteins appear to be intergral parts of plasma membranes. The galactose-NANA bond is cleaved by neuraminidase.

1. a-Amylase or a-l,4-glucan 4-glucanohydrolase (EC 3.2.1.1) hydrolyzes in a random fashion the a-1,4-glucan link in polysaccharides of three or more a-l,4-linked D-glucose units. Ca ion is required for activity. This enzyme also shows proteolytic activity toward tropocollagen (Steven et al., 1971). 2. 0-Amylase or a-1 ,4-glucan maltohydrolase (EC 3.2.1.2) hydrolyzes a a-1,4-glucan link in polysaccharides, removing successive maltose units from the nonreducing ends of the chains. 3. Glucose amylase or a-l,4-glucan glucohydrolase (EC 3.2.1.3) hydrolyzes a-1 ,4-glucan links in polysaccharides, removing successive glucose units from the nonreducing ends of the chain. 4. Cellulase or /3-1,4-glucan glucanohydrolase (EC 3.2.1.4) hydrolyzes 0-1,Cglucan links in cellulose. 5. Chitinase or poly-0-1 ,4-(2-acetamido-2-deoxy)-~-glucoside glucano-

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hydrolase (EC 3.2.1.14) is specific for linear polymers of N-acetyl-Dglucosamine. 6. Lysozyme or N-acetylmuramide glucanohydrolase (EC 3.2.1.17) hydrolyzes @-1,4-links between N-acetylmuramic acid and other sugar residues in mucopolysaccharides and mucopeptides. Neuraminidase or mucopolysaccharide N-acetylneuraminylhydrolase (EC 3.2.1.18) hydrolyzes terminal a-2,6-links between N-acetylneuramic acid (NANA) or sialic acids and amino sugar residues of various mucopolysaccharides (Gottschalk, 1958). The rate of hydrolysis of a particular substrate is influenced by the position of the sialic acid residue relative to the penultimate sugar of the carbohydrate chain. The optimal p H for hydrolysis by purified enzyme is low (4 to 5), and detergent is required for maximal activity. Sialyl groups of gangliosides are hydrolyzed by silidase from Vibrio, whether the substrate is in a dispersed or a micellar form (Lipovac et al., 1971). It is, however, possible that when a particulate enzyme is removed from its membrane environment during purification it loses part of its specificity and much of its activity; thus a membrane-bound preparation from calf brain has been found to be specific for gangliosides and does not hydrolyze sialosyl lactose or sialoglycoproteins (Leibovitz and Gatt, 1968). Hyaluronidase or hyaluronate glucanohydrolase (EC 3.2.1.35) catalyzes the hydrolysis of links between 2-acctamido-2-deoxy-~-glucose and D-glucuronate residues in hyaluronate. It also attacks chondroitin and mucoitin sulfates. For a description of other glycolytic enzymes involved in catabolism, see Svennerholm (1970).

3. PROTEOLYTIC ENZYMES The enzymes that cleave peptide bonds are well known and have regulatory functions in the catabolism of proteins and, consequently, in all enzyme-catalyzed 1 zactions (Rechcigl, 1971). 1. Carboxypeptidase A or peptidyl-L-amino acid hydrolase (EC 3.4.2.1) attacks the terminal N-residue of a polypeptide. Carboxypeptidase B or peptidyl-L-lysine hydrolase (EC 3.4.2.2) hydrolyzes N-terminal L-lysine, L-arginine, or L-ornithine in polypeptide chains. 2. Pepsin (EC 3.4.4.1), pronase and thermolysin are endonucleases which cleave peptide bonds with little specificity. Trypsin (EC 3.4.4.4) hydrolyzes peptides, amides, and esters a t bonds involving the carboxyl group of basic amino acid residues such as L-arginine and L-lysine. Its activity shows a salt dependence, especially a t low p H (Ness el al., 1971; Yon, 1958). 3. Chymotrypsin (EC 3.4.4.5) hydrolyzes peptides, amides, and esters a t

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bonds involving the carboxyl groups of aromatic amino acids. Its activity shows a salt dependence (Ness et al., 1971). 4. Papain (EC 3.4.4.10) and ficin (EC 3.4.4.12) hydrolyze peptides, amides, and esters a t bonds involving basic amino acids. Other peptidases of interest are muraminyl-alanine amidase (Csuzi, 1970), cathepsin, elastase, collagenase, nagarase, subtilisin, and arylamidase, which show a rather high degree of selectivity. The use of hydrolytic enzymes, particularly lipolytic ones, for determination of fatty acid distribution in complex lipids, recognition of molecular species, structural identification of glycerides in general, determination of stereochemical configurations, and preparative applications is well documented (Van Deenen and De Haas, 1966; Van Deenen, 1969, 1971). Although quite a few of these enzymes have not been highly purified, one need not lack confidence in the major conclusions.

II. ACTION OF HYDROLYTIC ENZYMES O N MODEL SYSTEMS

Biomembranes consist of two semi-infinite interfaces which are the site of a variety of biochemical processes. As emphasized in the preceding section, the nature of the interfaces is particularly important to considerations of the activity of adsorbed enzymes. At the interface an enzyme is subject to the action of asymmetric surface forces when it is absorbed or when it acts on substrate present at the interface. Thus before elaborating on the action of hydrolytic enzymes on biomembranes, it seems pertinent to consider the mode of action of these enzymes on some model systems. None of the substrates of phospholipases form ideal aqueous solutions. Enzymic hydrolysis is limited therefore by the size and shape of the substrate micelles, and by the arrangement and packing of the substrate and enzyme molecules a t the micelle surface. Furthermore, the products of lipolysis may modify the structural organization of the substrate. Such considerations are developed in the following discussion, with a view to identifying the relative position and orientation of the hydrophilic groups of various membrane constituents, so that models of the molecular organization and surface topography of biomembranes can be built and tested. A. Action of Lipolylic Enzymes on Lipid Aggregates

A bimolecular lamellar lipid membrane may be considered to consist of two monomolecular layers of lipid molecules oriented anisotropically ; that

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is, the polar groups are directed toward the more polar of the two phases, and the apolar chains are directed toward the other phase. Therefore the following discussion is divided into two parts; the first part summarizes the action of lipolytic enzymes on lipid monolayers, and the second develops their action on bimolecular lipid membranes. 1. ACTION ON INSOLUBLE LIPID MONOLAYERS AT INTERFACE

THE

AIR-WATER

The local concentration of a substrate in a monolayer is usually very high, while the enzyme concentration in the bulk phase and the stationary concentration of the enzyme interacting with the substrate a t the surface is usually low. Although the number of substrate molecules per unit area of the interface can be increased by compressing a monolayer, complication;, arise due to decreased penetrability and other steric factors regulatiug access of hydrolytic enzyme to the substrate. Similarly, monolayers of very low surface pressure tend to inactivate enzymes, presumably because of increased penetrability of proteins in the lipid monolayer (cf. Miller and Ruysschaert, 1971). The hydrolysis of lipid molecules in monolayers by lipolytic enzymes can be followed by observing changes in surface pressure or surface potential or the composition of the film, depending on the solubility of the products in the bulk phase. Thus i t is possible to study how lipolytic activity is affected by varying the packing density of the lipid molecules in the monolayer, or by changing the interfacial potential, or by altering the structure of the substrates, or by introducing additives into the monolayer or counterions into the bulk phase (Dawson, 1969). The kinetics of lipase-catalyzed reactions a t the lipid monolayer interface have been studied rather extensively. As shown in Fig. 3, various lipases seem to respond differentially to alteration of surface pressure. Furthermore, extent and magnitude of the effect (initial rate of substrate hydrolysis) depend both on the nature of the substrate and the enzyme. Thus, for example, pancreatic lipase reacts with its substrates in a monolayer. The reaction is first order with respect to enzyme and substrate and is independent of surface pressure in the range 0.5-10 dynes.cm-'. Lagocki et al. (1973) also observed base-catalyzed hydrolysis of glycerides in monolayer (Alexander and Rideal, 1937). However, above 12 dynes. cm-1 significant activation of the rate of lipolysis has been observed (Esposito et al., 1973). The rate of lecithin hydrolysis by various phospholipases is significantly affected by the surface pressure of the monolayer. Thus hydrolysis of lecithin catalyzed by phospholipase A (Colacicco and Rapport, 1966; Dawson, 1969; Hughes, 1935; Shah and Schulman, 1967), phospholipase B from P. notatum (Bangham and Dawson, 1960; Dawson and Hauser, 1967),

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I

I

I

I

I

B

SURFACE PRESSURE (dynes cm-11

FIG.3. A Rates of hydrolysis of corresponding substrates by lipases as a function of surface pressure in lipid monolayers. Curve 1, di- and trioleoyl glycerol by pancreatic lipase (Lagocki et al., 1970); curve 2, egg lecithin by phospholipase A from N . naju (Colacicco and Rapport, 1966); curve 3, yeast lecithin by phospholipase B from P . nolaturn (Bangham and Dawson, 1960); curve 4, dipalmitoyl lecithin by phospholipase C from C. welchii (Miller and Ruysschaert, 1971); curve 5, yeast lecithin by phospholipase D from cabbage. The curves are only approximate and are adapted to illustrate the trends rather than the exact magnitude of the effects. The exact position of the plateau may be a function of either the nature of the phospholipase or the substrate. Available data do not permit resolution of this ambiguity. B. The effect of surface ) measured by pressure on the initial rate of hydrolysis by phospholipase A ( N . ~ a j a as the initial decrease in surface potential in the first 2 minutes for various lecithin monolayers. Curve ( l ) , dipalmitoyl; curve (2) egg; curve (3) soybean; curve (4)dioleoyl. (From Shah and Schulman, 1967.)

phospholipase C (Bangham and Dawson, 1962; Miller and Ruysschaert, 1971), or phospholipase D (Quarles and Dawson, 1969b) is drastically reduced below 10 dynes.cm-' and above 30 dynes.cm-' (Fig. 3). The exact position of the rate maximum appears to be a function of the nature of the

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acyl chains in the lecithin and the nature and concentration of divalent ions and other surfactants in the medium. (Dawson, 1966; Shah and Schulman, 1967; Colacicco, 1971). It may be noted that surface pressure for maximum rate increases with increasing unsaturation of acyl chains (Fig. 3B) or decreasing intermolecular spacings in monolayers. Thus the steric factors involved in the formation of enzyme-substrate complexes a t the monolayer interface are reflected in the kinetics of lipolysis of these various lecithins. Similar factors may be responsible for the difference in the rate of hydrolysis by various phospholipases as plotted in Fig. 3A. However, the possibility of differential penetrability for various phospholipases has not yet been ruled out. Generally speaking, all phospholipases cease to attack phospholipid in monolayers a t film pressures above 28 dynes.cm-l. A t these high pressures the spacing between the phospholipid molecules is probably insufficient to allow the penetration of the active center of the enzyme to sites to be hydrolyzed. The limits of penetrability of the protein presumably depend on such factors as the nature of the lipid and the pH and temperature. It may be noted that various amphipathic substances, including phosphatidic acid, activate phospholipases (Table I). Available data suggest that the electrostatic zeta potential or the surface potential or dispersion or surface dilution factors may account for such activation. Some of these aspects are developed in the following discussion. It may be noted that both Crotalus atrox and Naja naja venoms can distinguish between phosphatidalcholine and PC in monolayers (Colacicco and Rapport, 1966) and in bulk/water emulsions (Gottfried and Rapport, 1962). The nature of the linkages of the hydrophobic chains in glycerophosphatides may therefore affect their interaction with phospholipases. Replacement of an acyl ester linkage of lecithin by an a,b-unsaturated ether in phosphatidalcholine may effect a change not only in steric configuration but also in dielectric properties (cf. Shah and Schulman, 1965). Also, as expected, the rate of enzymic reactions a t the interface often depends on the purity of the enzyme, since foreign proteins can be adsorbed onto the interface (cf. Khaiat and Miller, 1969) and sterically block the formation of enzymesubstrate complexes. The hydrolysis of lecithin films by N . naja venom is observed only when the concentration of venom is reduced sufficiently to minimize the effect of interfering proteins (Hughes, 1935). Similarly, addition of ovalbumin can greatly inliibit the hydrolysis. Phospholipase D-catalyzed hydrolysis of lecit’iin i s markedly inhibited when basic proteins, such as protamine, are added to the enzyme preparation (Dawson and Hemington, 1967). The relative adsorption of foreign proteins and enzymes onto the monolayer can be altered differentially by changes in the bulk p H which may affect the net charge on each protein.

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2. ACTIONON BIMOLECULAR LIPID MEMBRANES Phospholipase A hydrolyzes phospholipids present in bimolecular lipid membranes (BLMs). Since the products do not form BLMs, BLMs are lysed by the action of this enzyme. Thus BLMs cannot be prepared from lecithin in the presence of phospholipase A or prephospholipase A (Lesslauer e2 al., 1968). BLMs prepared from a synthetic analog of lecithin, which contairs a carbon or ether bond a t the C-2 position, are stable in the preF:nce of phospholipase Az, cven though the BLM resistance is lowered by 5 fzctor of 100 to 1000. The capacitance of the membrane did not change on the addition of protein. On the basis of this observation, it can be argued that the enzyme is adsorbed onto the polar head groups of the phospholipids on the membrane surface, thereby causing some alteration in the observed ionic permeability. The instability of BLMs prepared from lecithin in the presence of phospholipase A is thought to be due to incorporation of lysolecithin into the membrane. In fact, progressive transformation of liposomal bilayers into micellcs is seen in electron micrographs of aqueous lecithin mixed with lysolecithin (Bangham and Horne, 1964; Saunders, 1965) and in lecithin treated with phospholipase A (Henrikso and Henrikso, 1970). Phospholipase C (Clostridiumwelchii) has shown to interact with BLM PS in the presence of Ca. The resulting membrane is prepared from P C stable a t low enzyme concentrations; however, it has lower resistance and higher electrical capacitance (Hendrickson and Scattergood, 1972). The BLM treated with phospholipase C on only one side may be asymmetric. Phospholipase B , triphosphoinositidc phosphomonoesterase, and phosphodiesterase react with their substrates only when a small amount of anionic amphipath is present (cf. Dawson, 1968). The results have been interpreted to suggest that the formation of enzyme-substrate complexes requires a certain well-defined density of negative charges on the surface of substrate aggregates. In general, the rate of lipolysis by phospholipases seems to depend on a variety of physicochcmical factors. Thus large P C particles are not appreciably hydrolyzed by phospholipase D, whereas ultrasonically treated particles (Dawson and Hemington, 1967) and lecithin in lipoprotein combination (Condrea et al., 1964) are rapidly broken down. The reaction rate is greatly accelerated by the inclusion in the incubation media of certain organic solvents and anionic amphipaths (Dawson and Hemington, 1967; Kates, 1957), both of which are likely to influence the nature of the substrate-water interface rather than the enzyme itself. Similar results have bccn obtained for the dcacylating enzyme of P. notatum (Bangham and Dawson, 1960). Phospholipase A and C from a variety of sources do not hydrolyze

+

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ovolecithin swollen in aqueous solution. Following sonication, however, hydrolysis proceeds at a substantial rate (Jain and Cordes, 1973). This does not appear to be due to an increase in the surface area of the substrate, since the accessible area of sonicated liposomes was only three to six times more than that of unsonicated liposomes. However, rates of hydrolysis of unsonicated and sonicated lecithin become equal in the presence of 5 mole % ’ n-hexanol. Other alkyl alcohols also have a n activating influence at prelytic concentrations, whereas a t higher concentrations they have an inhibitory effect. It seems more likely that a loosening of the lamellar bilayer structure occurs on sonication. Such differences have been noted in proton magnetic resonance (PMR) (Sheard, 1969; Finer et al., 1972) and 13Cnuclear magnetic resonance (NMR) (Sears, 1971; Sheetz and Chan, 1972) spectra. Unsonicated lecithin shows broad peaks which are sharpened as the suspension is sonicated. Although broadening could be due to “freezing” or immobilization of chains (also see below), the difference has been attributed to dipole-dipole interactions which are not completely averaged out by anisotropic segmental motions (Finer et al., 1972; however, see Sheetz and Chan, 1972). The effect of the nature of the molecular species on packing and surface characteristics has been demonstrated by a variety of experiments. Upon sonication (1,2-dibutyryl)-, (1,2-dioleoyl)-, and (l-oleoyl-2-butyry1)lecithin give asymmetrical liposomes (Attwood et al., 1965). These compounds also differ significantly in their molecular orientation in monolayers spread a t an oil-water interface, and in their rates of hydrolysis by phospholipase A. Lecithin and cholesterol are known to form molecular complexes of well-defined stoichiometry : one to one. The association of cholesterol with lecithin is reflected in the rate of hydrolysis of sonicated lecithin mixed with varying amounts of cholesterol. The rate of hydrolysis of lecithin-cholesterol mixtures decreases as the cholesterol concentration increases. The total change in the Michaelis-Menten constant is by a factor of 3, and a sharp change is observed a t 1:1lecithin/cholesterol molar ratio (Jain and Cordes, 1973). Thus intermolecular spacing and packing as altered by additives, such as cholesterol and alkanols, are manifested in the kinetics of hydrolysis of phospholipid in liposomes by phospholipases.

B.

Action of Proteolytic Enzymes on Lipid-Protein Films

Considerable information pertaining to the behavior of protein films a t interfaces has been collected (Camejo et al., 1968; James and Augenstein, 1966; Miller, 1971; Dawson and Quinn, 1971). A fundamental feature of

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lipoprotein monolayers is the association of lipid with protein by polar and/or apolar noncovalent bonding, the precise nature of which is not yet understood. Penetration of protein into lipid monolayers is influenced by the chemical nature of the lipid. The extent of the increase in surface pressure following adsorption of protein reveals the following order : cholesterol > dihydroceramide lactoside > PE > P C > phosphatidylinositol (PI) > sphingomyelin > ganglioside. Furthermore, protein is incorporated into monolayers only a t low (2-5 dynes.cm-’) initial surface pressures, and the “cutoff” point is a t about 20 dynes.cm-1. As discussed in the preceding section, hydrolysis of the lipid alters the surface pressure and surface potential of monolayers. Similar changes are expected following hydrolysis of peptide bonds in proteins adsorbed to monolayers. A decrease in surface potential has been observed following pronase treatment of lecithin mo,lolayers modified with RNase, rabbit serum albumin, globulin, or lysozyme (Colacicco, 1969, 1970). Such a decrease could also occur if proteolysis occurs in the bulk phase. This may account for the observation that the rate of trypsinization of bovine serum albumin remains unchanged even when it is sonicated with up to 50 times its weight of egg lecithin (M. K. Jain, F. P. White, and E. H. Cordes, unpublished observations). Most of these studies are preliminary and inconclusive. The available data suggest that most proteins do not penetrate into monolayers having surface pressures greater than 10 dynes.cm-‘. I n all probability those protein molecules that do penetrate into monolayers are denatured and modify the interfacial charge profile (double layer). This may further modify the adsorption behavior of proteins, as do various other additives (cf. Miller, 1971). The response of lipid monolayers modified with protein to phospholipase A treatment is comparable to that observed in the case of pure lecithin films. Many lipoprotein-bound phospholipids are excellent substrates for phospholipases (Condrea et al., 1962, 1963, 1964; Marinetti, 1965). Enhanced rates have been observed for phospholipase A-catalyzed hydrolysis of lecithin in sonicated mixtures with albumin (M. K. Jain, F. P. White, and E. H. Cordes, unpublished observations) of beef serum, egg yolk suspensions, and rat brain homogenates (Uthe and Magee, 1971a, b). Such differences are, however, less apparent with enzymes prepared from pancreas (Condrea et al., 1963; Ibrahim et al., 1964). It is obvious from the discussion in this section that the stereospecific and position-specific action of phospholipases appears to involve multiple binding sites of an electrostatic and hydrophobic nature. Increasing the degree of dispersion of substrate stimulates hydrolysis, but the shape, size, and the molecular packing in the aggregate particle may also influence the

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rate of lipolysis. The reason for such behavior is not yet understood; however, a plausible explanation must take into account the state of aggregation of the substrate, the surface potential and charge profile, and the magnitude of the surface pressure. Packing factors may also influence the effect of the ionic composition of the medium and the activating effect of various additives. The nature of ions in the medium has considerable influence on micellar complexes, especially those involving lipophilic-hydrophilic equilibria (Wolman and Wiener, 1965). Such induced changes, a shift in equilibria regulating composition and structure of lipoproteins, and the presence or absence of various additives which modify packing in the lamellar structure greatly complicate the interpretation of reactions of various hydrolytic enzymes on biomembranes. 111. EFFECT OF ENZYMIC HYDROLYSIS ON SYSTEM PROPERTIES OF BIOMEMBRANES

A. Perturbation of Cell Wall Components

Electron microscope studies on bacteria have established the relationships between cell wall and plasma membrane present as surface components (Salton, 1967; Tomasz, 1971). The innermost limiting structure adhering to the cytoplasm of the bacterial cell possesses the typical triple-layered profile of plasma membranes. It contains most of the cellular lipids, as well as proteins and carbohydrates, and it forms the main permeability barrier of the cell. In some rare instances, in bacteria living in unusual habitats, the plasma membrane is the only surface layer of the cell. I n the great majority of organisms, however, there are several additional surface-layers exterior to the plasma membrane (Martincz-Palomo, 1970; Pease, 1966; Rambourg et al., 1966). In tissues cells are separated by noncellular “cement,” connective fibers, and/or a basal membrane (Rinaldini, 1958). I n spite of some species-to-species variation in chemical composition, these outer layers are made up of a n extensive network of glycopeptide units (Heath, 1971; Rambourg, 1971 ; Winzler, 1970). The glycoprotein surface components and the intercellular ccment appear to maintain the shape and mechanical stability of the cells. Thus various cell types are more easily deformed following treatment with ncuraminidase (Weiss, 1965) and proteascs (Weiss, 1966). In gram-positive bacteria onc finds attached to the peptidoglycan, and probably on the outer surface of it, a wall gcnerally seen as a thick (200800 8) rather amorphous structure. It is made up of a variety of complex polysaccharides, polyphosphates or “teichoic acids” (cf. Heptinstall et al.,

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1970), which are the major antigenic components and may form receptors for several bactcriophagcs. The structure external to the peptidoglycan layer in gram-negative bacteria has a “double-track” profile indistinguishable in dimcnsions and form from the plasma membranc. This is the “outer membranc” and, in Escherichia coli a t least, it is covalently linked to the peptidoglycan layer. Trypsin cleaves the pcptide bond of a lysyl residue which links a lipoprotein molecule to approximately every tenth unit of the peptidoglycan (Braun and Rehn, 1969). Pronase treatmcnt also cleaves the lipoprotein-peptidoglycan complcx, yielding peptidoglycan containing a lysine and an arginine residue in the lipoprotein (Braun and Sieglin, 1970; Braun and Wolff, 1970). Electron microscopc examination of trypsin-treated cell wall preparations reveals separate independent structures which, prior to trypsin treatment, appear closcly associated with one another. In addition to contributing significantly to the structural integrity of the cell envelope, the peripheral structures of polysaccharides, lipid, and protein may act as insulation against entry of certain ions into cells, dctermine the O-antigenic specificity, act as specific intercellular cement, act as receptor sites for various bacteriophages, and bc responsible for thc endotoxin activity of the cell. The cell cnvelopc may also be implicated in processes involved in anchoring membrane bodies, chemotaxis and transport, immunological recognition, and so on (Heath, 1971 ;Marched and Andrews, 1971; Strominger and Ghuysen, 1967; Wright and Kanegasaki, 1971; Betz and Sakmann, 1971). Which molecular elements of the surface mosaic are responsible for these various biological functions is not yet established. Electron microscopy of tissues and cclls before and after enzymic digestion has givcn significant information about the morphological features characteristic of various functions (Monneron and Bernhard, 1966; Rambourg, 1971; Wolff and Schrcincr, 1971). Localization of biological activities in the various surface layers and biochemical characterization of functional units has not been possible. Howcver, intercellular material can be degraded by a variety of nonspecific enzymes or mixtures of enzymes. Cells or plasma membrane-bound vesicles denuded in this manner have been studied extensively (Weibull, 1958). These studies strongly suggest that exchange of matter between the interior of the cell and the exterior is mediated and regulated by the plasma membrane. Cells denuded of their walls persist and exhibit many of the characteristics of whole cells, provided they are maintained in solutions of appropriate osmolarity. However, some significant exceptions are often noted (Warren, 1970). Thus, for example, pepsin digestion of the cell envelope of an extreme halophile leaves a residue that no longer requires salt for stability (Kushner and Onishi, 1966). The biochemical r .turc of the ccll wall has been clarified by the use of specific enzymes (Ghuysen, 1968; Rambourg, 1971). Available data suggest

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that sialoglycoproteins generally constitute the outermost region of mammalian cells. A large proportion of the glycoprotein-bound sialic acid residues can be liberated from cells by treatment with purified neuraminidase, which has little or no effect on the general structural organization of the cell surface or the viability of the treated cells. Treatment of Novikoff ascites cells (Walborg et al., 1969), mouse TA, ascites cells (Codington et al., 1972), and reticulocytes (Harris and Johnson, 1969) with neuraminidase and various proteases releases more than 70% of the total cellular sialic acid. In contrast, sialic acid-containing lipids of the surface membranes of mouse fibroblasts are insensitive to neuraminidase treatment (Weinstein et al., 1970). Several changes result in the behavior of fibroblasts and other cells following neuraminidase treatment (cf. Ray and Simmons, 1971). These include a reduction in surface negative charge, enhanced phagocytosis, inhibition of virus- or mycoplasma-induced hemagglutination, inhibition of cell aggregation, increased cell deformability and ability to metastasize, alterations in patterns of cellular migration and recognition, interference with amino acid and cation transport, apparent increase in immunogenicity, increased susceptibility to lysis by antibody and complement, and increased availability of weak or masked antigens. A short discussion of some of these cnzyme-induccd alterations and structural modifications of cell surfaces is given in the remainder of this section. The effect of hydrolytic enzymes on intercellular or cell wall material is rather specific. Plant cells are denuded by cellulase (Cocking, 1960) ; yeast (Eddy and Williamson, 1957) , and Neurospora crassa (Bachmann and Bonner, 1959) protoplasts can be obtained by treatment with a cellulase, but Avena coleoptile protoplasts are obtained by RNase treatment (Ruesink and Thimann, 1965; Ruesink, 1971). Grasshopper embryos have been disaggregated only with hyaluronidase (St. Amand and Tipton, 1954). Proteolytic enzymes appear to be more effective in disaggregating cells from higher organisms such as kidney (Poste, 1971), liver (Tacconi de Alaniz et al., 1970), and brain (Seeds and Vatter, 1971). In fact, trypsin (Moscona, 1952), pronase (Gwatkin and Thomson, 1964), collagenase (Cavanaugh et al., 1963), and other proteolytic enzymes (Curtis, 1967, pp. 129-131) have been used rathcr extensively for disaggregation of such cells. I n general, tissues rich in intercellular contacts, such as liver, are more susceptible to damage than embryonic and cancer tissues (Chernyak et al., 1971). The enteric coat surface of cat intestinal microvilli could not be degraded by any of a variety of hydrolytic enzymes (Ito, 1965). It may be pointed out here that disaggregation of cells does not necessarily yield protoplasts; individual cells may retain most of their surface structure intact. Furthermore, it has been shown that univalent antibody fragments directed against certain membrane antigens also dissociate multicellular

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bodies of the cellular slime mold Dictyostelium discoideum into single cells (Beug et al., 1971). This demonstrates that a noncatalytic protein can also disrupt cellular aggregates by binding to specific surface sites. Chick embryo cells, which are disaggregated by trypsin (Moscona, 1952), are not so affected if this enzyme is treated with the trypsin inhibitor from soybeans. Trypsin inactivated by diisopropylfluorophosphonate separates cells agglutinated with protamine, but it does not disaggregate normal tissue (Easty and Mutolo, 1960). It may be noted that growth conditions (Johnson and Campbell, 1972), dehydration (Mattman and Webb, 1971), or pretreatment with hydrolytic enzymes significantly modify the susceptibility of cell walls to the action of (other) hydrolytic enzymes. For example, addition of trypsin prior to or simultaneously with Lactobacillus fermentii renders the latter susceptible to the action of lysosyme (Berlin and Neujahr, 1968) ; these cells are not affected by lysozyme directly, nor after pretreatment with heat, butanol, EDTA, or urea. The lysed cells after trypsin plus lysozyme treatment yield membranelike fragments which exhibit ATPase activity. The presence of cofactors or substrates can modify the extent of membrane disaggregation. When Mg is omitted from medium during lysis of L. fermentii, most of the ATPase is recovered in the soluble fraction, presumably reflecting extensive degradation of the surface membrane (Neujahr, 1970). Similarly, disintegration of tubules from axonomes of the sperm of the sea urchin is significantly influenced by the presence of ATP (Summers and Gibbons, 1971). Collagenase degrades basement membranes (Kefalides, 1968, 1969; Kefalides and Denduchis, 1969). Digestion of bovine glomerular basement membranes with purified collagenase results in the solubilization of over 90% of the carbohydrate and peptides. Characterization of the glycoproteins produced by these hydrolytic procedures indicates that two distinct types of carbohydrate units are present in the basement membranes. One type is a disaccharide unit containing glucose and galactose, and the other is a heteropolysaccharide containing galactose, mannose, hexosamine, sialic acid, and fucose and has an average molecular weight of 3500. There are approximately 10 disaccharide units for every heteropolysaccharide unit in the basement membrane (Spiro, 1967). Glycopeptides from bacterial membranes appear t o be similar (Ghuysen, 1968; Heath, 1971), and these surface components of the cells appear to be intimately associated with the general behavior of the organism. Thus the phenomenology of cell aggregation or intercellular adhesion as implied in the observations just described bears on several aspects of cellular biology; the cell surface is thought to be either a direct or indirect mediator for growth control and is thus primarily involved in phenomena such as invasive growth, loss of contact inhibition, and the metastasizing properties of the neoplastic cells (cf. Curtis, 1967).

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Moreover, the ability of a variety of cells to aggregate, to adhere selectively to each other, and to form groupings structured in a manner characteristic of the tissues of their origin, has been amply demonstrated (Lilien, 1969; Seeds and Vattcr, 1971; Anonymous, 1971). All vertebrate cells so far examined carry a net negative surface charge. Contact between cells or their processes and cellular or noncellular substrata (such as glass or Perspex) has been considered analogous to contact between negatively charged particles in suspension (Weiss, 1961a, b, 1963, 1970a, b). A variety of physicochemical perturbations inducing changes in surface charge and dielectric properties of the medium can be correlated with changes in ccll contact. However, there are some significant objections to this view. EDTA treatment reduces cell-to-cell adhesion of rabbit VZ carcinoma cells but does not affect cell-to-glass adhesion (Berwick and Coman, 1962). Cells cultured on glass appear to be in actual contact with the glass over only a small proportion of the total area in apparent contact. Observations of this type suggest that specific areas of the cell periphery are involved in contact phenomena. Thus existence of selective sites for cellular contact may also account for differences in the effects of trypsin on disaggregation of chick embryo cells (Moscona, 1961), washed mouse ascitcs cells (Easty et al., 1960), and rat Walker ascites cells (Easty and Mutolo, 1960). Freshly dissociated cells “comingled” in suspension sort out preferentially according to tissue specificity or space specificity, depending on the cell type (Lilien, 1969) ; this suggests specificity of the cellular contact process. However, the onset of such specificity is preceded by an initial period (which may last up to 24 hours) of nonspecificity, during which adhesion between heterotypic cells is as likely as between homotypic cells. A variety of biochemical studies suggests that intercellular aggregation may involve certain protein factors and metabolic processes (Moscona, 1961; Curtis, 1967), and macromolecular synthesis (Lilien, 1969). In fact, proteolytic enzymes absorbed on disaggregated cells are found to prevent the formation of glycoprotein cell coat material and so to interfere with the attachment, spreading, and growth of cells on glass (Poste, 1971). In an attempt to relate specific cell surface components to physiological functions, it was found Chat L-glutamine is required for adhesion of mouse tetratoma cclls but can be replaced by glucosamine and mannosarnine (Oppenheimer el al., 1969; Oppenheimer and Humphreys, 1971). These compounds are actively metabolized during the conversion of nonadhesive to adhesive cells. These results suggest that cell adhesion may depend upon the introduction of hexosamine or sialic acid residues, or both, into specific complex components of the cell surface. While these components have not yet been identified, this apparent relationship between the bio-

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synthesis of specific cell surface components and cell adhesion may be significant (Roth and White, 1972). It has been proposed (Roseman, 1970) that specific intercellular adhesion may be related to the interaction of cell surface-bound glycosyl transferases with their substrates-complex carbohydrates on the surface of the adjacent cell. In platelets there is evidence of bond formation between collagen and glucosyl transferase, and the process of new bond formation can be inhibited by collagenase treatment (Jamieson et al., 1971). Normal fibroblasts and other cells in tissue culture stop growing when they reach the cell density of a confluent monolayer. Under identical nutritional conditions, virally as well as chemically transformed cells grow past the confluent monolayer stage and, by piling up, eventually reach five- to ten-fold higher saturation densities. Many of the changes that occur in cells transformed to a neoplastic state by viruses may be attributed to alterations in the cell surfaces (Burger, 1970a, b). Such transformations also lead- to a change in their capacity to interact with specific proteins. A glycoprotein, isolated from wheat germ, specifically agglutinates cells transformed by various tumor viruses, while under identical conditions the parent, nontransformed cells are unreactive. After proteolysis by trypsin, pronase, or ficin, nontransformed cells are also agglutinated, apparently as a result of exposure of the same or similar receptor sites on the cell surface (Burger, 1969). Lipases, glycosidases, or mucopolysaccharidases have no effect. Similarly, proteolysis of leukemic cells results in their agglutination by concanavalin A (Inbar and Sachs, 1969), which can be explained by three types of changes in the structural organization of sites on the surface membrane. There can be an exposure of hidden sites, a concentration of exposed sites as a result of decreased cell size, and a rearrangement of exposed sites without a decrease in cell size, resulting in a clustering of sites (Ben-Bassat el al., 1971). Furthermore, malignant transformation of normal cells also results in a change in the location of amino acid and carbohydrate transport sites on the surface membrane in relation to the binding sites for concanavalin A (Inbar et al., 1971). Studies with normal cultured cells suggest that they release a factor which may be responsible for the reduced adhesiveness of transformed cells (Temin, 1967). This “overgrowth-stimulating factor” in cultured chick cells appears to be a protease of restricted substrate specificity (Rubin, 1970). Thus a change in surface structure may be involved in the conversion of the nonagglutinable state of virally transformed cells. Such changes may also account for other phenomena related to tissue growth (Burger, 1970b). However, the factors contributing to the lack of normal contact inhibition in transformed cells appear to be much more complex. Changes in the glycolipid pattern (Hakamori and Murakami,

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1968) and their extraction profile (Weinstein et al., 1970) have been noted before and after trypsin treatment. A hematoside containing NANA is the main glucolipid of the normal fibroblasts, while its precursor, ceramide lactoside, is a minor component; the transformed cells have little hematoside and greatly increased ceramide lactoside. The increase in ceramide lactoside is of special interest, as this glycolipid is a specific serological determinant present in many human tumors. Similarly, i n vivo synthesis of acidic surface components has been suggested to account for the protective influence of exogenous nucleotides against phospholipase C treatment (Montagnier, 1971). Whether such changes relate to some basic regulatory mechanism or simply to a change in the distribution profile of metabolites is not clear (Burger, 1971; Ginsburg and Neufeld, 1969; Stoffel, 1971). Some of the most important functional constituents of the red cell surface are the blood group substances that determine the immunological specificity of the cells (Boorman and Dodd, 1961). These substances are mucopolysaccharide complexes. The nature of these surface groups has been investigated by the digestion of red cells with neuraminidase (Cook et al., 1961) and pronase (Uhlenbruck et al., 1968). Concomitant with the action of neuraminidase is a reduction in anionic charge on the cell surface, hence the mobility of the cells in an electric field is altered. This difference is not due to unmasking of positive groups but to removal of negative charges. Calculations show that the amount of sialic acid released from the cells is about twice as much as required to account for the loss of surface charge. I t seems likely therefore that some of the groups released by ncuraminidase treatment were in fact not originally in a position to contribute to the surface charge of the cells. Comparable results have been obtained from chick muscle cells (Kemp, 1970), liver cells (Chaudhuri and Liberman, 1965), and red blood cells of various animal species (Eylar et al., 1962). All the species of erythrocytes examined so far have exhibited a residual negative surface charge following treatment with neuraminidase (Tenforde, 1970; Weiss, 1961a, b, 1968). Ncuraminidase is active a t the surface of human erythrocytes following acetaldehyde or glutaraldehyde fixation (Tenforde, 1970). The fixed cells release only 64% of the sialic acid released from unfixed cells. Thus a substantial fraction of these residues may reside relatively deep within the peripheral zone. Trypsinization of erythrocytes usually lowers the surface charge density, although chick red cells have their charge density raised (Seaman and Uhlenbruck, 1962). Although trypsin is strongly positively charged a t p H 7, there is no evidence that its absorption onto the cells is rcsponsible for lowering the charge dcnsity (Seaman and Heard, 1960; Heard and Seaman, 1960). Trypsin treated with diisopropyl fluoro-

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phosphate (DFP) does not affect surface charges. An effect similar to that obtained with trypsin has also been elicited by mild treatment with pronase (Cook and Eylar, 1965) and a-amylase (Glaeser and Mel, 1966). These and other studies (Cook el al., 1961; Eylar et al., 1962; Haydon and Seaman, 1967) suggest that most of the sialic acid and blood group substances are bound to the outermost surface of the cell. Not all the negative surface charge on mammalian cells is susceptible to neuraminidase or is due to sialic acid residues. For example, RNase reduces the electrophoretic mobility of two types of cultured human cells (Weiss and Mayhew, 1966). This reduction is found to be independent of and additive to the known effects of neuraminidase on these cells. Analysis of the cell supernatant showed that RNase does not release any neuraminic acids from the cells. These results cannot be attributed to absorption of RNase on the cell surface (Mayhew and Weiss, 1970), but the effect of RNase appears to be on RNA present in the peripheral zone of the cellular electrokinetic surface. It is also interesting to note that a fraction (about 18%) of calcium bound to lobster nerve homogenate is made dialyzable by RNase (Rudenburg and Tobias, 1960). I n fact, it appears that the cell surface density of sialic acid carboxyl groups and RNase-susceptible anionic groups may vary considerably in any cell or any group of cells, depending on their proliferative and mitotic status (cf. Weiss, 1970a). Changes in surface charge density can be correlated with various cellular phenomena. For example, the rate of agglutination of neuraminidasetreated red cells with polylysine can be correlated with their electrophoretic mobility (Marikovsky el al., 1966; Danon et al., 1969). Similarly, the reduction in surface charge of senescent cells and extruded erythroid nuclei is a major recognition signal which helps the macrophage to sort out undesirable altered or deteriorated cells (Danon, 1970). Surface charge density and its perturbation have also been correlated with the phenomena of intercellular contact (Bangham and Pethica, 1960) and cellular deformability (Weiss, 1965 and 1966). The stimulated growth of confluent cells following treatment with low concentrations of trypsin (Sefton and Rubin, 1970) may also be due to an altered surface charge density which may eventually result in altered permeability characteristics or in alteration of surface recognition sites. Quite a few of the surface receptor components of biomembranes have been isolated. The enterovirus receptors from HeLa cells, whether on intact cells, plasma membranes, or a Triton-X-100-solubilized receptor are inactivated by treatment with a variety of organic solvents, and by trypsin and chymotrypsin (McLaren et al., 1968). Phospholipase C, RNase, and DNase have no effect. Similarly, neuraminidase treatment

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destroys surface receptors for Newcastle disease virus, myxovirus, enterovirus (Gottschalk, 1960; Marcus and Schwartz, 1968), and influenza virus (Cook et al., 1961; Eylar et al., 1962). Phospholipase C (Friedman and Pastan, 1968, 1969; Mizutani and Mizutani, 1964), lysolecithinase (Barbanti-Brodano el al., 1971), and protease (Hoyle and Hlmeida, 1971) treatment of host cells has been shown to inhibit infectivity and growth of several viruses. Several sialoglycopeptides which possess weak M- and N-antihemagglutinating activity are liberated from the peripheral zone of intact human erythrocytes or stroma by the action of trypsin (Jackson and Seaman, 1972; Seaman and Jackson, 1971, and references therein). Some of the glycopeptides have been isolated and partially characterized. They differ from one another in molecular weight, net charge, and amino acid and carbohydrate composition. These results support the concept of glycoprotein “subunits” in the peripheral zone of human erythrocyte membrane, which are exposed and accessible to tryptic attack. The neuraminylcontaining glycopeptide fragments are somewhat heterogeneous and probably represent portions of the original viral, M, and N active glycoproteins. The thrombocyte-specific antigen shows related behavior (Barber and Jamieson, 1971). Glycolipids of the surface membrane are resistant to neuraminidase and appear to be buried within the membrane matrix or are otherwise sterically inaccessible to this enzyme (Weinstein et al., 1970). Fibroblasts treated with trypsin show greater reactivity toward an antihematoside antiserum than do untreated cells (Hakamori et al., 1968). Similarly, antibody prepared against human red cell ceramide tetrahexoside fails to agglutinate intact erythrocytes but strongly agglutinates trypsinized erythrocytes (Koscielak et al., 1968). This implies that glycolipids are masked by trypsin-sensitive protein or glycoprotein a t the cell surface, or that they are sterically inaccessible to the enzymes a t both the inner and outer surfaces (cf. Bretscher, 1971). Such steric factors may be responsible for the differential activity of lysozyme on gram-positive and gram-negativc bacteria. Gram-positive bacteria, with their high content of murein and low content of lipoprotein and lipopolysaccharide in the cell envelope, are usually attacked to some degree by lysozyme. Gramnegative bacteria are as a rule resistant to lysozyme attack. This resistance is attributed t o the more complex structure of their cell envelope which consists of a double membrane with the murein component embedded in it. In certain instances an increase in sensitivity to lysozyme is achieved by pretreatment of the cells, for example, by moderate heat, lyophilization, slight denaturation, or treatment with hot formamide, phenol, or trichloroacetic acid, or treatment with trypsin.

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Neuraminidase treatment causes alteration in three immunogenic characteristics (Ray and Simmons, 1971, Simmons et al., 1971): increased immunogenicity, increased susceptibility to complement lysis, and increased availability of weak or masked antigens on the cell surface. To some extent these properties may correlate with one another. However, it is apparent that an increase in immunogenicity and susceptibility to lysis is not always due to an increase in the number of antibody binding sites. The various components of the cell surface involved in immunogenicity may be inactivated by trypsinixation. When sensitive lymphocyte cells are incubated with trypsin, they are no longer inhibited by antigens (David et al., 1964). Furthermore, upon contact with specific antigens, the sensitized lymphocytes release a soluble factor which is inactivated by trypsin (David, 1968). Similarly, the role of membrane-bound components was demonstrated in the development of cellular immunity of cultured cancer cells (Dwyer and Mackay, 1971; Watkins et nl., 1971) and human lymphocytes (Grothaus et al., 1971). Pretreatment of cultured cells with neuraminidase activates immune recognition of antigens associated with tumor cells. In some cases a t least, these antigenic components have been isolated. The glycoproteins on the surface of red cells carry the "-antigens, and the receptors for phytoagglutinin, myxovirus, and the cold agglutinin. Ficin treatment permits the nondializable material to be fractionated into various glycoproteins containing specific immunological receptors (Ebert et al., 1971). Lauf et al. (1971) showed that an isoimmune antiserum (anti-L serum) prepared by immunization of high-K-type (HK) blood group M-positive sheep with blood from low-K-type (LK) blood group Lpositive sheep contains one or more antibodies which stimulate K influx four- to sixfold in LK red cells. This antiserum also stimulates the transport of ATPase in ghosts derived from LK cells. This stimulation is due both to an alteration in the number of pump sites and to changes in kinetic properties of the ATPase. These experiments suggest that the L antigen is intimately involved with the molecules responsible for cation pump activity. Neuraminidase treatrncnt of L-positive LK and M-positive HK sheep red cells did not affect the interaction of these cells with their homologous antibodies as measured by K influx, complement-mediated immune lysis, and adsorption of antibody. Trypsin treatment of LK and HK cells did not interfere with hemolytic action of anti-L and anti-M antibodies, respectively. However, pretreatment of LK cells with trypsin rendered these cells insensitive to the K-pump-stimulating antibody present in the anti-L serum. Such obscrvations prove not only the surface localization of the pump but also suggest that the pump is a multicom-

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ponent system and that specific modification with trypsin leads to disruption of the antigen-antibody interaction. Although it is difficult to arrive a t general conclusions from the studies described in this scction, the surface coat should be considered part of a functional entity generally termed the biomembranc. A challenging aspect of the study of the outer coat of the biomcmbrane is the possibility that cellular specificity may find its expression in this region. The main characteristics of glycoproteins and glycolipids located in the surface region are : they are macromolecular, heterogencous, and vary in size and composition ; they are biphasic-the polar portion consisting of strong cation exchange properties-whereas gangliosides havc ceramide residues buried in the lipid bilayer; their surface components appear to be the site of local synthesis and interchange of carbohydrate rcsidues. The transition between the plasma membrane and the environment of the cell is not necessarily a sharp discontinuity. There are a host of demonstrable phenomena and functions ascribed to the surface coat for which definite biochemical features have yet to be found. Removal of surface groups has profound effects on the behavior of various types of cells. For example, removal of sialic acid from the zona pellucida of rabbit ova strongly inhibits the penetration of sperm (Soupart and Clewe, 1965). This suggests that sialic acid plays a role in the initial attachment of sperm to the ovum, as it does in the attachment of viruses and bacteriophages to their host cells. Treatment of leukemia cells with neuraminidasc inhibits their normal secretion of proteins (Glick et al., 1966). Sialic acid may therefore play a role in secretion. The normal circulation of lymphocytes in the rat is altered drastically by treatment with neuraminidase (Woodruff and Gesner, 1967), with a mixture of glycosidases (Gesner and Ginsburg, 1964), or with trypsin (Woodruff and Gesner, 1968). Therefore the unique behavior of these cells may depend on their surface characteristics. Similarly, ingestion of proteolytic enzymes results in a marked hypertrophy of the submaxillary glands of rats (Ershoff and Baj wa, 1963). Also, murine leukemia cells treated with neuraminidase fail to grow in normal mice, although they grow in irradiated mice (Bagshawe and Currie, 1968). Active uptake of sheep and chick erythrocytes into the surfaces of cockroach phagocytic hemocytes is abolished by pretreatment of hemocytes with trypsin (Scott, 1971). Papain treatment of rabbit lungs causes loss of the tethering properties of alveolar walls to airways (Caldwell, 1971). Such observations suggest that the modification of surface coats of various cell types may have far-reaching but rather specific effects. The experimental data presented thus lead to the hypothesis that, on the cell surface, both glycolipids and glycoproteins play a n

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important role in determining specificities of intercellular and antibody interactions by acting as “recognition sites.” Malignant transformations may also result from changes in such recognition sites (Sanford, 1967; Sanford and Codington, 1971). Thus the molecular processes underlying such changes may be generally traced to surface components. However, the role of the plasma membrane and its lipid components must not be underestimated when considering membrane processes in general (Poole et al., 1970; Lucy, 1970). Such aspects are discussed in the following section. B. Perturbation of Plasma Membranes

Some of the functional and structural changes that occur when intact cells are treated with various hydrolytic enzymes emphasize the system properties of the cell membrane. Thus, following phospholipase C treatment of erythrocytes, there is shrinkage of the cells, sphering, and increased susceptibility to osmotic stress. Progressive hemolysis ensues, leaving ghosts which are characterized by a focal electron-dense area intimately associated with each membrane (Bowman et al., 1971). Correlative experiments on phospholipase C treatment suggest that diglyceride separates from muscle microsomal membrane as dense droplets (Finean and Martonoshi, 1965). The total area of the membrane is reduced in proportion to the loss of lipid during hydrolysis. Maximum treatment of erythrocytes (Casu et al., 1968), erythrocyte ghosts (Coleman et al., 1970; Glaeser and Mel, 1966; Leonard and Singer, 1968; Simpkins et al., 1971a; Zwaal et al., 1971), lobster axons (Simpkins et al., 1971b), bovine brain myelin fragments (McIlwain and Rapport, 1971), submitochondrial particles with inside-out geometry (SMP) (Simpkins et al., 1971a)) isolated liver cells (Gallai-Hachard and Gray, 1968), brain microsomes (Stahl, 1973), and muscle microsomes (Finean and Martonosi, 1965) with phospholipase C liberates approximately 70% of the membrane phospholipid phosphorus. Similar results have been obtained following treatment of erythrocytes with sphingomyelinase (Colley et al., 1973). It is however interesting to note that significant species difference is observed in the susceptibility of erythrocytes to these enaymes.Electron microscope studies of phospholipase C-treated rat kidney mitochondria (Ottolenghi and Bowman, 1970), erythrocyte ghosts (Ottolenghi and Bowman, 1970; Bowman et al., 1971), muscle mitochondria (Grossman et al., 1967), rat liver microsomes (Trump et al., 1970), muscle microsomes (Finean and Martonoshi, 1965), skeletal muscle fibers (Strunk et al., 1967), and Bacillus subtilis proplast membranes (Nanninga et al., 1973), have revealed that there are discrete areas of digestion on the membrane survace. These areas are distributed over the surface

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of the membrane but do not coalesce. Similar droplets were not detected in phospholipase C-treated myelin (McIlwain and Rapport, 1971). In erythrocyte ghosts lipolysis by phospholipase C causes a 4 5 5 5 % reduction in surface area. From these data, it has been computed that in red cell ghosts about 70% of the area is occupied by.lipids, whereas for muscle microsomes the corresponding figure is 90% (Finean et al., 1971). A molecular interpretation of this observation is difficult. It cannot, for example, be concluded explicitly that (cf. Glaser et al., 1970) “2530% of the phospholipids are in a physical state different from the remainder of the lipids, perhaps involved in a more tightly coupled interaction with the membrane proteins.” As emphasized earlier in this article and elsewhere (Zwaal et al., 1971), the extent of lipolysis in biomembranes significantly depends upon the chemical nature of the phospholipids involved. It is noteworthy that in electron micrographs phospholipase-C-treated lasma membrane appears to have many ringlike structures rat liver 70 wide with diameters of more than 350 A (Benedetti and Emmeabout lot, 1965; 1966). The rings consist of globular subunits. This difference in behavior, as compared to that of erythrocytes, may be attributed to the differences in their content of sphingomyelin (absent in liver microsomes) and cholesterol. These observations give morphological and chemical evidence that there are specific areas on the plasma membrane where phospholipase C produces gross structural changes, presumably in the lipid region of the membrane. Furthermore, increased mobility is observed in the lipid region after phospholipase C treatment of axonal membranes (Simpkins et al., 1971b), erythrocyte ghosts, and SMP (Simpkins et al., 1971a). The mobility of the fatty acid chains increases with increasing distance from the charged head group. However, resonance spectra of protein labels do not change a t all following phospholipase C treatment. The presence or absence of cholesterol does not alter these conclusions. Electron spin resonance (ESR) and electron microscope data also suggest that, even after removal of about 70% of the lipid phosphorus and cleavage of the ionic head groups, a significant porportion of the bilayer character remains. Thus stability of an ordered membrane structure relies very little on ionic interactions of charged head groups. These conclusions are in accord with the observations that BLMs can be prepared from such lipids as glycerol distearate and sorbitan tristearate (Sessa et al., 1968; Tien and Diana, 1967). Differential activity of intact and ghost erythrocytes has been noted toward trypsinization. In intact erythrocytes or ghosts, a concentrationdependent trypsin-induced inactivation of membrane acetylcholinesterase (AChase) activity and release of sialic acid-containing glycopeptides

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occurs (Carraway and Triplett, 1971). In intact cells there is little difference in the protein pattern before and after trypsin treatment. However, treatment of intact ghosts results in virtually complete destruction of the protein pattern. This indicates that all proteins in the ghost are accessible to attack by trypsin. In fact, trypsin releases up to 50% of the total peptide from ghosts. The main effect as seen by electron microscopy is disaggregation of the membrane continuum into smaller segments which still have a trilamellar structure whose thickness is reduced about 4070. This reflects removal of material from the outer surface of the membrane. Such preparations were found to have lost 80% of their (Na I()-ATPase activity, but only 15% of this activity was lost when ATP was present during the tryptic digestion (Marchesi and Palade, 1967). Study of the K)-ATPase activity effects of trypsin on intact red cells showed (Na to be unaffected and cholinesterase activity to be lost (Herz et al., 1963). As noted earlier, the action of phospholipase A on lipid monolayers and BLMs produces significant disorganization, presumably because of the formation of lysolecithin and consequent bilayer-micelle transformation (cf. Haydon and Taylor, 1963). Similar changes have been observed in electron micrographs of biomembranes treated with lysolecithin (Agostini and Hasselbach, 1971). Following phospholipase A2 treatment, both lobster nerve and erythrocyte ghost membranes show extensive (40-50% in nerve, and 5 5 7 0 % in erythrocyte) cleavage of phospholipid (Simpkins et al., 1971~).ESR studies show that in the resulting membrane the fatty acid chains become less mobile all along their lengths and that there is increased accessibility of some thiol groups. Qualitatively, similar perturbation of protein structurc can be produced by lysolecithin treatment of nerve and erythrocyte ghosts. The perturbations induced by phospholipase A treatment can be picked up by a variety of ESR probes (Hsia et al., 1971; Hubbell and McConnell, 1969), PMR (Kamat and Chapman, 1968; Sheard, 1969), anilinenaphthalenesulfonic acid (ANS) fluorescence depolarization (Azzi and Vainio, 1971; Weidekamm et al., 1971), and circular dichroism (CD). Treatment of erythrocyte ghosts with phospholipase A (Simpkins et al., 1971c) or C (Leonard and Singer, 1968) hydrolyzes about 70% of the membrane phospholipid, but the CD spectra of the treated and untreated membranes are essentially identical. It may, however, be noted that small changes in CD spectra observed by several investigators (Gordon et al., 1969; Wallach, 1969) could possibly arise as a result of increased light scattering from particulate membrane fragments (Glaser et al., 1970; Singer and Glaser, 1971). Changes in CD spectra (Gordon et al., 1969) and signals from ESR probes for proteins (Simpkins et al., 1971a, b) in membranes perturbed by phospholipase A appear to be identical to those produced by binding of lysolecithin to

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erythrocyte ghosts. These results imply that the structure of a substantial fraction of the phospholipids in the plasma membrane can be changed without affecting the properties of membrane-bound protein (however, see Section 111, E). Significant differences have been noted in the behavior of phospholipases on intact cell membranes as compared to their action on model lipid systems or microsomes or membrane fragments (Balantine and Parpart, 1940). For example, phospholipase A from snake venom is unable to hemolyze fresh erythrocytes from sheep (Casu et al., 1968), beef (Uthe and Magee, 1971a, b), human (Roelofsen et al., 1971), or rabbit (Habermann and Krusche, 1962), or platelets (Bradlow and Marcus, 1966; Kirschmann et al., 1964). Comparable results have been obtained for the bee venom enzyme (Habermann, 1971). Similarly, phospholipase C does not hemolyze intact erythrocytes (Roelofsen et al., 1971; Laster et al., 1972). However, phospholipase A causes hemolysis of washed erythrocytes (Lankisch and Vogt, 1971a, b), of butanol-treated cells (Burt and Green, 1971), and of ghosts obtained either by osmotic or immune lysis (Casu et al., 1968; Condrea et aZ., 1970; Roelofsen et al., 1971; Uthe and Magee, 1971b). Phospholipase A can also hemolyze intact red cells in combination with plasma or a basic lytic factor* (Condrea et al., 1970; Habermann, 1971; Lankisch and Vogt, 1971a, b ; Uthe and Magee, 1971a, b ; Vogt et al., 1970), or with added lecithin (W. B. Campbell, quoted in Simpkins et al., 1971c) or egg yolk (Wahlstrom, 1971), or following treatment with sulfhydrylblocking agents (Lankisch and Vogt, 1971a, b) or deoxycholate (Heemslterk and Van Deenen, 1964). Similarly, red cells treated with sublytic conccntrations of deoxycholate or Triton X-100 can be hemolyzed by phospholipase C (Roelofsen et al., 1971). Intact erythrocytes are hemolyzed by crude phospholipases of Leptospira (Kasarov, 1970). These observations are in accord with a membrane structure that does not allow phospholipascs to reach thc substrates. It is, however, confusing to note that erythrocytes pretreated' with trypsin, pronase E, or neuraminidase are not lysed by cither phospholipase A2 or C. In contrast, several enzymes such as N-acetylmuraminidase (Katz et al., 1971), proteases, hyaluronidase, lysozyme, and neuraminidase (Bender et al., 1971; Simpkins, 1971) have been reported to be lytic. This difference

* The toxicity of the venoms is a combined effect of various proteinaceous components (Sarkar and Devi, 1968). Even in the lytic action of whole venoms on erythrocytes two components are involved: a basic peptide and phospholipase A. The basic protein factors that bind to membrane can cause nonosmotic hemolysis which can be intensified by adding phospholipase A. Phospholipase A alone causes hemolysis only indirectly by splitting extracellular phospholipids. The role of other hydrolytic enzymes present in venoms is still a matter of conjecture.

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could be due either to a species difference or to the difference in the concentration of enzyme used. To account for the lytic effect of proteases and glycosidases, it has been suggested that the site of lytic action may be membrane protein (Simpkins, 1971). In fact, protein has been postulated even as the site for the lytic action of surfactants (Tomizawa and Kondo, 1971). The mechanisms by which lipolysis produces changes leading to osmotic or secondary lysis have been characterized. Changes in membrane permeability as noted for red cells (Balantine and Parpart, 1940), isolated liver cells (Gallai-Hachard and Gray, 1968), and other cells (Habermann, 1971) following lipolysis may be the first step in the overall process (see Jain, 1972, Ch. VI). [Although permeability changes can account for changes in the shape of a cell which may ultimately lead to lysis (Braasch, 1966), several other alternatives have been suggested. According to Roy (1945), lysolecithin may solubilize cholesterol in the red cell membrane with consequent loosening of the membrane structure. I n fact, transfer of red cell cholesterol to plasma has been noted after phospholipase A treatment (Klibansky et al., 1962).] Thus if the barrier properties of the membrane are altered, the osmotic balance across the membrane is also altered; consequently, a unidirectional flow of water is expected. In fact, an early decrease in mean corpuscular volume suggests that the action of phospholipase C (Bowman et al., 1971) and phospholipase A (Klibansky et al., 1962) secondarily results in a shrinkage of cell size. Thus a variety of agents that interact with membrane lipids and cause expansion of membrane area are known to protect red cells from “lysolecithin-induced” lysis (Roth and Seeman, 1971). Similar protection is induced by hydrocortisone sodium succinate added to sheep red blood cells during treatment with phospholipase C (Eaglstein et al., 1969). This phenomenon may also account for inhibition of phospholipase-A-induced swelling of mitochondria (see Section IV, B) by local anesthetics (Seppala et al., 1971) and uncouplers of oxidative phosphorylation (Nils-Erik and Seppala, 1970; Weinbach and Garbus, 1968a). The molecular architecture of the cell membrane determines the shape of the cell. Theoretically, a liquid membrane should give rise to a spherical shape. However, quite a few cells are known to exist in more complex shapes. Little is known about the biochemical basis of the maintenance of the biconcave shape of erythrocytes, for example. It could arise from changes in the surface area of the membrane (Braasch, 1971) or from the (ATP Ca)-dependent sol-gel transformation of certain cell proteins (Szasz, 1970; Szasz et al., 1967; Weed et al., 1969). Sphering of red cells from dogs, rabbits, or mice is induced by the action of trypsin (Szasz et al., 1967) and snake venom (Avi-Dor et al., 1960; Balozet, 1962; Danon

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et al., 1961; Essex and Markowitz, 1930; Gitter et al., 1959). Following enzymatic treatment the hematocrit of the blood samples increases. This indicates that the sphered cells resist centrifugal packing more than normal cells. The viscosity of the blood rises more than threefold, and the cell is rendered more rigid. Plasma from immunized animals neutralized phospholipase A and prevented the sphering effect (Balozet, 1962). Similarly, normal and filamentous whole cells and isolated envelopes of E. coli B were exposed to amylase, DNAse, lipase, lysozyme, pepsin, phospholipase A and C, and trypsin (Weinbaum et al., 1967). Modification of cell rigidity was determined by sphere formation in both the whole cells and in isolated envelopes. Enzymes capable of converting trypsinized normal and untreated filamentous whole cells and untreated envelopes to spheres included lysozyme plus EDTA, and phospholipases C and D. The phospholipase C-sensitive component is protected more completely in normal than in filamentous whole cells by a protein layer which is easily attacked by trypsin. Thus sphering of cells may involve changes in membrane permeability and water flux (see Section IV, A). C. Asymmetry across Biomembranes

Yet another aspect of the topography of membranes arises from the asymmetry of biomembranes a t the two interfaces. Membranes have two sides: an outside and an inside. It appears that asymmetry of organization plays a significant role in the function of the membrane, and that each side of the membrane has properties determined not only by the intrinsic character of its constitutents but also by the distinct content of the compartment it faces. As early as 1926, it was noted that intracellular microinjection of protease destroyed the cellular integrity of amebas (Northrop, 1926), whereas externally applied protease had little or no effect. Similar effects have been observed in sea urchin eggs (Hagstrom and Hagstrom, 1954). A direct study of the asymmetry of the erythrocyte membrane has been made on right-side-out (RO) and inside-out (10) erythrocyte ghost vesicles (Steck et al., 1971). By digestion with proteolytic enzymes (trypsin, chymotrypsin, and pronase), the accessibility of substrate protein on each of the erythrocyte membrane’s two faces was found to be distinctly different. Quantitatively, the titration studies demonstrated that the RO vesicles were rapidly and extensively hydrolyzed, while the I0 vesicles were attacked to only a limited extent. Gel electrophoresis of total membrane protein revealed a different digestion pattern for the two vesicle species. Disruption of the membrane with small amounts of sodium dodecyl sulfate (SDS) led to extensive, in-

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discriminate proteolysis. Thus the data provide evidence for membrane asymmetry in accord with data collected by immunological methods (Nanni et al., 1969; Wintzer and Uhlenbruek, 1967), topooptical staining (Romhanyi and Deak, 1969), and several other techniques (Phillips and Morrison, 1971; Wallach, 1972). It is, however, interesting to note that the inner mitochondria1 membrane, which is known to be asymmetric (Racker et al., 1970), is hydrolyzed by pancreatic phospholipase A a t the same rate as SMP (Scarpa and Scherphof, 1971). The asymmetry of biomembranes is probably best reflected in the differential affinity of the outer and inner interfaces for fixing and staining agents. The folding and tilting of the isolated mernbrane sheets with respect to the plane of section hampers establishment of the asymmetry of the two dense layers. Moreover, the asymmetry of the membrane element appears to depend on the type of fixation. Fixation with osmium tetroxide and staining with uranyl acetate yield a rather symmetrical membrane element; if this procedure is preceded by fixation with glutaraldehyde, the inner leaflet is found to be somewhat thicker (Farquhar and Palade, 1963). Although such observations make the interpretation of data difficult, significant information can still be obtained. The plasma membrane of nervous elements fixed in lanthanum permanganate shows a characteristic unit membrane structure with differential staining of the external dense stratum in contrast to the internal one (Doggenweiler and Frenk, 1965). Pretreatment with trypsin, pronase-EDTA, DNase, a-amylase, and neuraminidase did not remove the lanthanum-staining material, which could, however, be removed by phospholipase C (Lesseps, 1967). Similarly, asymmetry has been noted in the distribution of sialic acid. Colloidal Fe hydroxide (CIH) or periodic acid-silver methenamine selectively stain carbohydrates. The stained membranes have been found to be dotted by electron-dense granules confined only to the outer side of the membrane element. However, pretreatment of the membranes with neuraminidase prevented the subsequent staining almost completely (Patterson and Touster, 1962; Rambourg and LeBlond, 1967). Similarly, differential staining of myelin before and after digestion with phospholipase A and trypsin suggests asymmetry with respect to the distribution of lipids and proteins (Adams and Bayliss, 1968). It may be noted here that asymmetry in biomembranes is not restricted to a differential affinity of the outer and inner membrane leaflets for k i n g and staining agents. In fact, as noted later, considerable evidence has accumulated suggesting the asymmetrical location of various specific enzymes, ionogenic groups (Fleischer et al., 1967), immunologically active groups (Benedetti and Emmelot, 1968), and other functional macromolecules of biomembranes (Bretscher, 1972; Heidrich, 1971; Phillips and Morrison, 1971).

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D. Membrane Hyperstructure

As noted earlier, trypsinization of various membranes removes 1 5 5 0 % of the protein. Phospholipase A and C treatment hydrolyzes 6 5 7 5 % of the membrane lipid. After incubation of rat liver plasma membranes with trypsin and fixation with glutaraldehyde and osmium tetroxide, the :Vera11 width of the membrane element is reduced from about 80 to 60 A ; the reduction involves both the two dense strata and the central light gap (Benedetti and Emmelot, 1968). Similarly, trypsin releases up to 50% of the total protein from erythrocyte ghosts with a 40% decrease in overall membrane thickness (Finean and Coleman, 1970). The main effect seen on membrane morphology is a break-up of the membrane sacs to give large independent segments of membrane. X-ray diffraction studies further suggest that in trypsinized red cell ghosts, after drying, lipid-protein interactions are modified so that all the lipid is crystallized out and forms an independent phase. Phospholipase C digests the part of the membrane phospholipid that normally separates from the membrane when it is dried. This lipid contains mainly the less highly charged choline- and ethanolamine-containing phospholipids. The phospholipids that remain part of the residual lipoprotein framework and which resist the action of phospholipase C include the more highly charged molecules such as PS and the inositol phosphatides. These results are in general accord with solvent extraction studies made on mitochondria (Fleischer et al., 1967; Benedetti and Emmelot, 1968) in which 90% of the total lipid has been removed. This implies that a protein matrix is involved in membrane stabilization. Recently, the concept of “subunits” in biomembranes has become fashionable, although not justifiably so. It is generally postulated that plasma membranes consist of an array of structurally equivalent lipoprotein oligomers, or simply arrays of lipoprotein microspheres embedded in a protein matrix. The term (‘membrane subunit” has been applied to a wide class of membrane entities, including those visualized by electron microscopy, and has been invoked to explain the functional organization of membranes and the process of membrane reaggregation after solubilization. If the existence of membrane subunits is accepted, a crucial question is whether these subunits form the membrane element as a bidimensional lattice of repeating protomers, or whether they are randomly associated with the matrix of a lamellar lipid layer. The relevance of globular repeating units, as observed in electron micrographs, to membrane function arises from the fact that such structures contain the enzymatic activities. Multienzyme particles have been released from plasma membranes by treatment with low concentrations of papain

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(Eichholz, 1968; Emmelot et al., 1968; Oda and Seki, 1966). However, some doubt is cast upon the idcntity of papain-rcleased particles by the observation that papain treatment does not release the subunits found in freeze-etched preparations. After negative staining with phosphotungstic acid, some membrane surfaces show a very fine granular structure with smooth edges. The edges of other membranes are covered by an orderly array of small, globular units with an average diameter of 5&60 A (Benedetti and Emmelot, 1965). These knobs have been observed in, on, or away (up to 20 A) from the membranes. Papain treatmrnt of rat liver membranes releases particles 50-100 in diameter, which contain most of the leucine aminopeptidase (LAPase) activity. Papain-treated membranes lack the globular knobs. In contrast to papain trypsin does not release sedimentable (or soluble) LAPase activity from the same membrancs. However, trypsintreated membranes show various aspects of membrane structure indicative of “subunit” structure. Papain trcatment of the brush border of the intestinal epithelium also yields particlcs measuring 40-60 A in diameter, and the membranes contain high invertase and LAPase activity. The microvillus sheets are devoid of particles after papain treatment (Eichholz, 1968; Emmelot et al., 1968). A fair percentage of rat liver plasma membranes, which do not contain globular knobs, exhibit an array of subunits arranged in hexagons and an occasional pentagon after negative staining a t 37°C (Benedetti and Emmelot, 1965; Emmelot and Benedetti, 1967). The aggregated repeating unit frequently contains a small hydrophilic hole in the middle; the centerto-center distance of the subunit is 80-90 A. This repeat pattern docs not arise when the negative staining of these membranes is carried out a t low tcmperature. One possible interpretation of the temperature-dependent emergence of the hexagonal (plus pentagonal) repeat pattern in the rat liver plasma membranc is based on the phase transitions of phospholipids. Digestion of rat liver membranes with neuraminidase or trypsin does not affect the emergence of the pattern; however, after trypsin or papain treatment, the ave:age center-to-center distance of the hexagonal subunits is reduced to 70 A, and evidence for the presence of a hydrophilic hole in the center of the repeat unit is less distinct or absent (Bcnedetti and Emmelot, 1968). This suggests that the amount of protein present in the membranes may influence the size of the lipid micelles in equilibrium with the bilayer (cf. Glauert and Lucy, 1968). E. Unmasking and Release of Membrane-Bound Proteins

The nature of lipid-protein interactions and organization in biomembranes is still a matter of conjecture. Among other methods labeling

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(cf. Bretscher, 1971) and freeze-etching (Muhlethaler, 1971) have rather convincingly demonstrated that approximately 20y0 of the volume of a plasma membrane is constituted by proteins. Trypsin generates new polar groups a t its site of action, and freeze-etching of proteolyzed membranes (Fig. 4) reveals a significant decrease in the amount of membrane-bound protein (however, see Benedetti and Delbauffe, 1971) without affecting the plane of fracture. Treatment of erythrocyte membranes with phospholipase A and low levels of saponin induces alteration in the fracture plane of freeze-etched preparations. Increasing levels of saponin, lysolecithin, and SDS lower the number of membranes fracturing tangentially, presumably because an altered balance between polar and apolar forces in the membranes allows the fracture plane, normally guided by hydrophobic regions in the membrane, to follow directions other than those along the membrane plane (Branton, 1971; Speth et al., 1972). It is an interesting fact that the action of proteases on membranes is often limited. For example, tryptic digestion of rabbit liver microsomes stopped when about 22-25y0 of the membrane-bound protein was released (Ito and Sato, 1969). Further addition of enzyme had no effect; the same result was obtained with other proteolytic enzymes. The amino acid composition of peptides obtained through stepwise addition attack by pronase on the membrane of ddycoplasma laidlawii, including the residual protein, failed t o show any increase in thc proportion of nonpolar residues (Morowitz and Terry, 1969). Thus there is no indication of increased hydrophobic interaction of lipid with the least accessible protein. This implies that the hydrophobic character of lipid-protein interactions is mainly due to tertiary and quaternary structures. These results are, however, a t variance with the data on amino acid analysis of membrane proteins (Hatch and Bruce, 1968; Wallach, 1972) and related observations. Moreover, a glycoprotcin of molecular weight near 31,000 in human red blood cell mcmbranes is cleaved by trypsin into soluble glycopeptides of molecular weight near 10,000, although the remaining protein is quite hydrophobic; this again suggests the existence of a single membrane protein differentiated into exterior and interior regions (Winzler, 1969). Perturbation of the membrane environment leads to the modification of catalytic propertics not only of membranc-bound proteins but also of enzymes in solution. I n fact, proteolysis of various cnzyme systems in an isotropic environment is known to modify their catalytic properties sclectively, and this effect depends significantly upon the presence of substrates, cofactors, and modifiers (Geller et al., 1971; Krebs et ul., 1968; Lccocq, 1971; Menon, 1971; Pontrcmoli et al., 1971; Selmeci and Posch, 1971; Srtlow et al., 1972; Spitnik-Elson and Breiman, 1972; Taketa and Pogell, 1965; Kohno and Yourno, 1971). Conventional methods for the release of loosely bound components

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from aggregates such as biomembranes depend upon mechanical breakage, osmotic rupture, physicochemical and physiological disruption, or some kind of autolytic breakdown. However, treatment of biomembranes with hydrolytic enzymes may disaggregate the membrane so that the resulting fragments or subunits have little tendency to reaggregate. For example, crude snake venom or purified phospholipase A has been shown to solubilize choline dehydrogenase (Rendina and Singer, 1958), NADH-ferricyanide oxidoreductase (King and Howard, 1960), NADH dehydrogenase (Briggs et al., 1970; Minakami et al., 1960; Ringler et al., 1960), D-lactate-cytochrome reductase (Gregolin and Singer, 1963), n-glycerophosphate dehydrogenase (Ringler and Singer, 1958; Ringler, 196l), P-hydroxybutyrate dehydrogenase (Fleischer et al., 1966), and various segments of the electron transfer chain (Bachmann et al., 1966; Fleischer and Fleischer, 1967) from mitochondria. It may be noted that release of NADH dehydrogenase by phospholipase A is correlated with hydrolysis of cardiolipin (Awasthi et al., 1970), and that the solubilized preparation is insensitive to inhibition by amytal or antimycin A (Minakami et al., 1960). From a review of the published information, it seems obvious that no general rule can be given as to the ability of phospholipases to solubilize enzymic activities (see Biggs et al., 1970). Thus NADH dehydrogenase from cardiac mitochondria (King et al., 1966), choline dehydrogenase from rat liver mitochondria (Rendina and Singer, 1958), and succinate dehydrogenase from beef heart mitochondria (Cerletti et al., 1969) are not solubilized by phospholipase A treatment. This difference may reflect a difference in topological distribution or orientation, or a difference in intramembrane binding of these various enzymes in the electron transfer chain. Correlative experiments with phospholipase C indicate that the phosphoryl group of phospholipids plays an important role in the structural latency of membrane-bound enzymes. For example, phospholipase C treatment of an arbovirus causes loss of viral stability and infectivity following loss of about 60-70% of lipid phosphorus (Friedman and Pastan, 1969). Inactivation of the virus seems to be an all-or-none reaction in the sense that all three viral structural proteins appear to dissociate from the virion upon its inactivation. Incubation with phospholipase C releases the lysosomal enzymes in mitochondria1 fractions of rat liver (Beaufay and De Duve, 1959), brain (Koening and Gray, 1964), and kidney (Koening and Gray, 1964). When FIG.4. Particle aggregation and removal during pronase digestion of red blood cells. (A) Control cell incubated in buffer only. (B-D) Digested cells incubated in pronase so m to remove (B) 30%, (C) 4574, or ( D ) 70% of the original membrane protein. (From Engstrom, 1970.) Figures obtained through the courtesy of Prof. D. Branton.

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purified renal lysosomes were incubated with phospholipase C, several acid hydrolases were liberated at a much more rapid rate than phospholipid phosphate (Koening, 1966). The bulk of the latent acid phosphatase and 0-glucuronidase (6040% of the total) was rendered active when only 7-15y0 of the lysosomal phospholipid phosphate was split. These enzymes were fully activated when 20-35% of the phospholipid was split. This treatment did not affect the fine structure of lysosomes as revealed by the electron microscope, but these particles did lose their ability to bind basic dyes (Koening and Gray, 1964; Koening, 1969). Disruption of lysosomal particles becomes noticeable only when approximately 50% of the lysosomal phospholipid is enzymically cleaved. Similar results have been obtained by phospholipase C treatment of the sarcoplasmic membranes of skeletal muscles (Fredman et al., 1969); AChase and AMP-deaminase activites are not affected even when 77% of the phospholipid is hydrolyzed; however, (Ca Mg)-ATPase is reduced by 25%. Cholinesterase activity of muscle is also liberated by incubation with bacteria of Cytophaga spp. (Lundin, 1967). Activation by phospholipase C of membrane-bound thymidine kinase in rat liver has been noted; phospholipase D is active to a lesser extent (Stirpe and La Placa, 1971). Solubilization of membrane-bound enzymes has also been observed following treatment with proteolytic enzymes. Release of active end plate AChase has been observed following treatment with trypsin or collagenase (Grafius and Millar, 1971; Hall and Kelly, 1971). The leucyl-0naphthylamidase activity of rat liver plasma membrane was found to be released in a fragment after papain treatment (Smith and Hill, 1960). Also, cytochrome b is released by nagarase (Ohnishi, 1966). I n fact, tryptic digestion appears to cleave cytochrome b6 into two roughly equal-sized moieties (It0 and Sato, 1968); the one carrying the bg activity is itself quite soluble, but the non-b6 portion confers hydrophobicity upon the entire molecule and presumably is responsible for its attachment to the membrane. Cell membranes from L. fermentii prepared by combined proteaselysozyme treatment release approximately 6&80% of the total ATPase activity in the soluble fractions. However, the specific activity of ATPase is much higher in the membranes than in the soluble cell fractions. When Mg was omitted from the medium employed during lysis of the cells, most of the ATPase was recovered in the soluble cell fractions (Neujahr, 1970). These observations are, however, a t variance with observations on the Micrococcus lysodeikticus membrane. In its membrane-associated state, the ATPase protein (10% of the total membrane protein) does not exhibit enzymic activity unless activated by trypsin (Munoz et al., 1969) or DNA (Ishikawa et al., 1965) in the presence of ATP. It appears that the dissocia-

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tion of ATPase from the membrane is dependent upon prior removal of poorly defined materials-cations, protein, lipid, and so on, with subsequent release upon exposure to a low ionic strength environment (cf. Munoz et al., 1968). In fact, proteolysis of myosin by trypsin has been found to be strongly dependent upon the ionic environment (Biro and Balint, 1966), and the fragments thus released contain almost complete ATPasc activity (Gergely, 1953; Go11 et al., 1971; Szent-Gyorgyi, 1953; Spudich and Watt, 1971; Mihalyi and Szent-Gyorgyi, 1953) without any modification of the catalytic or cation-binding site (Seidel, 1969). Similar attempts to libcrate Ca-ATPase from sarcoplasmic reticulum (SR) membranes by trypsin treatment have yielded particles containing only a fraction of the original activity (Inesi and Asai, 1968; Ikemoto et al., 1968; MacLennan et al., 1971). Activation of membrane-bound enzymes with or without solubilization has been noted following their treatment with phospholipases. Thus under appropriate conditions treatment of bovine liver microsomes with phospholipase A or C produces an activated form of glucose-6-phosphatase (G-6-Pase) (Zakim, 1970). These results are in discord with earlier observations of inactivation of G-6-Pase by phospholipases (see references in Zakim, 1970). The new data suggest that G-6-Pas, retains its activity after selective alteration of its phospholipid environment and that the normal phospholipid environment is not only nonessential for activity but acts to constrain the maximum potential activity of G-6-Pase. This hypothesis is further substantiated by the fact that a variety of treatments that exert their primary effects on the lipid portion of the microsomal membrane increase the activity of UDP-glucuronyl transferase with p-nitrophcnol as the glucuronide acceptor (Attwood et al., 1971; Vessey and Zakim, 1971). The enzyme phosphodiesterase loses activity during purification. Purified enzyme can, however, be activated by an exogenous activator or by incubating the purified enzyme with snake venoms or proteolytic enzymes. Stimulation by activator is not time-dependent and appears to be stoichiometric. In contrast, activation by proteolytic enzymes and snake venom is time-dependent and catalytic (Cheung, 1969). Thus a low conccntration of hydrolytic enzymes causes stimulation to the same extent as a high concentration, provided that the time of incubation is sufficient ; however, prolonged exposure of phosphodiesterase to these enzymes causes a diminished degree of stimulation or even complete inactivation. Both the activator and the hydrolytic enzymes induce identical end effects leading to an increase in K , of the enzyme. Many studies involving the use of phospholipases, detergents, solvent extraction, and reactivation by the addition of crude or purified lipids

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have furnished evidence that phospholipid is essential for maximum activity of various enzymes, Certain enzyme systems that require phospholipids for full activity exhibit specificities with respect to the nature of the hydrocarbon chains, as well as to the charged head groups of the lipid. Apart from the shape and size of the micelle, a given net charge or charge density of the surfaces may be involved, and this suggests the importance of evaluating the lipid requirement of these enzymes in physicochemical terms. The phenomenon of enzyme-induced release of membrane-bound proteins is best interpreted as a consequence of an enzymically or chemically induced disorganization of a lipoprotein matrix restricting the physical freedom of bound enzymes. In this model activation of microsomal enzymes by treatment with hydrolytic enzymes could be ascribed to changes in permeability of substrates so that the apparently inactive enzyme is “unmasked,” thus changing the accessibility of the enzyme for its substrate. However, this simplistic model can be rejected in quite a few cases. Organic solvents, detergents, or phospholipases which specifically perturb phospholipids can either alter the kinetic constants of certain enzymes (Tzagoloff and MacLennan, 1965; Zakim, 1970), or destroy their catalytic activity completely (Cater et al., 1970; Cerletti et al., 1969). This loss of activity can be restored by suitable lipid suspensions. Therefore these suspensions act either (1) by replacing essential membrane lipids, or (2) by removing bound detergents, or (3) by stabilizing one of the unstable apo forms of the enzyme. The lack of specificity by phospholipid to reverse the effects of phospholipases may indicate that the phospholipid is involved only in maintaining a generally hydrophobic environment. Most enzyme or enzyme complexes released by membrane disaggregation show differential tendencies toward inactivation by hydrolytic enzymes in both natural and delipidated states. Generally speaking, solubilized enzymes inactivate faster than membrane-bound enzymes (Ellingson et al., 1970; Lennarz, 1970; Zakim, 1970; Bresler et al., 1969). This may account for the observation that microsomal enzymes are inactivated in a reversible manner by removal of phospholipids. In some cases an enzyme may have an absolute requirement for lipid, and reactivation may not be possible. I n fact, not all lipids in biomembranes are bound with the same strength (Roelofsen, 1968; Roelofsen et al., 1966). Phospholipid present in both the loosely and strongly bound states may represent functionally different molecules. Thus removal of loosely bound lipid may not affect the activity of bound proteins, even though it may disaggregate the membrane lamella. Such studies emphasize the need for caution in interpreting data correlating the biochemical function of lipids in membrane structure and function. The role of reaction products that

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may also modify the structure of the enzymes remains largely undetermined in most cases.

IV. EFFECT OF ENZYMIC HYDROLYSIS ON TRANSPORT SYSTEMS

Modification of the transport characteristics of a membrane may involve either formation of leaks by modification of the diffusion barrier, or specific modifications of molecules involved in the appropriate transport system. It was noted in the preceding section that treatment of biomembranes with certain proteolytic and lipolytic enzymes may result in permeability changes which ultimately lead to lysis. Such nonspecific changes relate largely t o the system properties of the membrane and are of little interest with respect to transport mediated by specific membrane-bound molecules. Specific membrane transport systems as modified by hydrolytic enzymes are discussed below.

A. Excitable Membranes

Proteases and phospholipases modify the conductance, capacitance, resting potential, and excitability of axons (Condrea and Rosenberg, 1968; Narahashi and Tobias, 1964; Rosenberg, 1970; Rosenberg and Condrea, 1968; Tobias, 1955, 1958, 1960), heart fibers (De Mello, 1971), the node of Ranvier (Nelson, 1958), skeletal muscle (Albuquerque and Thesleff, 1967), sartorius muscle (Gainer, 1967), and the lateral geniculate body of the cat (Hafeman et al., 1970). Differences in the rate of action of hydrolytic enzymes have been noted in different tissues of the same animal. Thus, although skeletal muscle fibers of rat and chicken treated with phospholipases show a n increase in conductance and changes in associated phenomena, a difference has been noted in the kinetics of phospholipase action on different fibers of the same muscle. In rat the “fast” or “white” extensor digitorum longus muscle is the most sensitive, and the slow or “red” soleus is the least sensitive fiber to these enzymes (Albuquerque and Thesleff, 1967). Also, chronically denervated muscles are more resistant to the action of phospholipase D than are innervated fibers; but, even after denervation, the fast muscles remain more sensitive. Furthermore, phospholipase C digestion causes a gradual decrease in the threshold for excitation, and reduction in the rate of rise and amplitude of the action potential. The action

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potential-generating mechanism in both the fast and the slow muscles is completely and irreversibly blocked by 1.5 pg/ml of phospholipase C within 1 hour. The input resistance and the resting membrane potential are not reduced by this treatment. Similarly, the membrane conductance of the squid axon (Condrea and Rosenberg, 1968; Rosenberg and Condrea, 1968), lobster axon (Tobias, 1955), and node of Ranvier (Nelson, 1958) is altered to different degrees by phospholipase A and phospholipase C treatment. I n squid axon, for example, phospholipase C causes extensive phospholipid breakdown without any effect on conduction or on permeation of lipid-insoluble compounds. In contrast, treatment with phospholipase A blocks excitation and increases permeation (however, see Brzin et al., 1965). Electron micrographs of squid giant axons treated with snake venom revealed that the Schwann cells arc markedly affected; no structural change in the axon membrane was noticeable (Martin and Rosenberg, 1968). Proteolytic enzymes have selective effects on bioelectric properties of excitable membranes. External application of proteases does not alter membrane resistance significantly, and an effect on resting potential is noticeable only after extensive damage has occurred. Measurement of transport numbers of various ions across a crayfish axon treated with trypsin plus chymotrypsin or with papain alone show that there is a decrease in P c ~ / P ( P being permeability), which a t least initially accompanies a gradual increase in membrane resistance (Strickholm and Clark, 1971, and unpublished observations). The transport characteristics of red blood cells are also affected by trypsin (Jung, 1971) and pronase (Passow, 1971) treatment. Both proteases decrease permeability toward neutral molecules, and pronase decreases anion and increases cation fluxes. This observation and other data suggest that a protein amino group participates in the control of passive ion permeability across both erythrocyte and crayfish axon membranes. Intracellular microinjection of proteolytic enzymes such as trypsin blocks excitability, decrcases the resting potential and membrane resistance to hypcrpolarizing currents, and increases both Na and K efflux in squid axons (Rojas, 1965; Rojas and Armstrong, 1971; Rojas and Atwater, 1967; Rojas and Luxoro, 1963; Takenaka and Yamagishi, 1966; Tasalti et al., 1965, 1966). Before the action potential is abolished, the plateau phase is extended. These early effects have been further resolved by study of current-time curves under voltage-clamp conditions. The results suggest that the closing off (inactivation) of early current is selectively affected by internal perfusion with pronase. The peak inward current and steady-state current are also affected. However, the voltage a t which inward current is zero remains unchanged. These results suggest that the

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K-conducting mechanism is modified by proteolysis of the internal surface of axons. Hydrolytic enzymes have been shown to modify the bioelectric responses of various physiological and pharmacological receptors. 1. Bioelectric responses after fertilization of echinoderm eggs with spermatozoa or activation with pronase are identical (Steinhardt et al., 1971). This suggests involvement of a proteolytic step in the initial stages of fertilization. On the cap, or acrosome, of a spermatozoan, there is an enzyme called acrosin which chews through the protective layers around the ovum with an action rather like that of trypsin. Indeed, trypsin inhibitors have been found to inhibit fertilization (Stambaugh et al., 1969; Zaneveld et al., 1970). Changes in membrane conductivity have also been observed during the mitotic cycle (Malenkov et al., 1972, and references therein). 2. Treatment of isolated smooth muscles with neuraminidase and EDTA makes them insensitive to serotonin as measured by a lack of contraction (Woolley and Gommi, 1964). The treated tissue regains its sensitivity through the addition of gangliosides or sialic acid itself. Similarly, gangliosides can restore excitability to brain slices that have lost it in saline preparations (McIlwain, 1961). It has been suggested that the serotonin receptor may actually be a glycoprotein, and that the loss of sialic acid from this glycoprotein may result in conformational changes in the receptor area, which on the addition of free sialic acid or gangliosides is reversed (Carrol and Sereda, 1968; Offermeier, 1965; Sanford and Codington, 1971). Similarly, botulinum toxin is inactivated by gangliosides but not by other lipids. Neuraminidase partially restores the activity of the toxin inactivated by gangliosides. On the basis of such observations, it has been suggested gangliosides may be the receptors for botulinum toxin (Simpson and Rapport, 1971) and tetanus toxin (Van Heyningen and Mellanby, 1968). 3. Both phospholipases and proteases depolarize the smooth muscle cells of guinca pig Tueniu coli (Cuthbert, 1966) and frog sartorius muscle (Albuquerque et al., 1968). With a suitable degree of lypolysis, membrane potcntials remain normal, but the response to acetylcholine disappears (Cuthbert, 1966). The treated preparations respond to mechanical stretching with an action potential discharge and an increase in tension. In contrast, aftcr liniited proteolysis the preparations respond normally to acetylcholine by showing an electrical discharge, but fail to contract. Since the action of phospholipase C on ileal muscle is prevented by adding lithium to the bathing fluid, the effects of the enzyme are unlikely to be mediated by hydrolysis products. It is particularly interesting to note that

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AChase from a variety of sources is not inactivated by proteolytic or by lipolytic enzymes (Changeux et al., 1969; Eldefrawi et al., 1971; Dudai and Silman, 1971; Ruch and Green, 1971). Only proteolytic enzymes inactivate acetylcholine receptors in brain homogenates (Lu, 1952; Bosmann, 1972). Both pronase and phospholipase A reduce by more than 7501, the nicotine-binding activity of acetylcholine receptors from lobster axon (Denbury et al., 1972). Trypsin, chymotrypsin, and phospholipase C and D also reduce binding significantly. However, papain, collagenase, RNase, and DNase have no effect on binding. Similarly, treatment with pepsin or trypsin eliminates or drastically reduces the synaptic material in brain as observed in electron micrographs (Bloom and Aghajanian, 1968). Trypsin treated with the soybean inhibitor, hyaluronidase, or with neuraminidase did not affect synaptic material. Taken together, these observations suggest that the cholinergic receptor is a proteolipid and that the receptor action can be uncoupled from AChase activity. 4. When hyaluronidase is injected into the brain, it causes large impedance changes in the caudate nucleus, beginning about 1 hour after injection and lasting for several hours. If the hyaluronidase injections are accompanied by CaBrz, the impedance changes are brief and transient (Tarby and Adey, 1967; Tarby et al., 1968). The enzyme appears to interfere with sites involved in the mobility of charged elements. I n view of the effects of ions on impedance changes, and considering the fixed-charge profiles of the cell surface, it appears likely that the availability of ionized groups associated with macromolecules determines the patterns of conductivity in any specified region of the membrane. 5. Both phospholipase A and C inhibit histidine uptake in brain slices (Kirschmann et al., 1971). However, this inhibition may not be direct. In summary, all these effects can be rationalized in terms of what is known about the mechanism underlying bioelectric phenomena, but it is not possible to evaluate the molecular aspects and implications of these observations. B. Respiratory Chain

The action of snake venom (cf. Sarkar and Devi, 1968) or phospholipase A on mitochondria causes swelling (Condrea et al., 1965; Nils-Erik and Seppala, 1970; Earnshaw and Truelove, 1970; Earnshaw et al., 1970; Waite et al., 1969), inhibition of respiratory activity (Fleischer et al., 1962), uncoupling of phosphorylation (Arvindakshan and Braganca, 1959; Lenaz et al., 1968; Petrushka et al., 1957), separation of the inner membrane (Marks et al., 1970), inhibition of binding and translocation of

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Ca2+ (Carafoli et al., 1971), disruption of ATPase and the “F” particle (Racker, 1969), and relocation of enzymes from the inner to the outer membrane (Green et al., 1966; Parsons et al., 1966). The effect of phospholipase A is concentration-dependent. At low concentrations phospholipase A increases respiration in the absence of a phosphate acceptor; a t high concentrations it causes severe inhibition of electron transport; and a t intermediate concentrations it produces a stage of respiratory decline in which ADP acts as an inhibitor (Augustyn et al., 1970). Chronic ingestion of ethanol makes liver mitochondria fragile, presumably due to activation of endogenous phospholipase A (French and Morin, 1969). Most, but not all, of the effects of phospholipase A appear to be due to the uncoupling action of fatty acids produced in situ. Similar factors may be responsible for increased metabolism in polymorphonuclear leukocytes following phospholipolysis (Patriarca et al., 1971). Trypsin has been shown to disrupt the respiratory chain and to inactivate the rotenone-insensitive NADH-cytochrome c reductase system and the enzymes of the outer mitochondrial membrane (Juntti et al., 1970; Kuylenstierna et al., 1970). Nagarase inactivates palmitoyl-CoA synthetase on the outside of the outer membrane of mitochondria (De Jong, 1971). However, when mitochondria are oxidizing palmitate, they are protected from the inactivating action of nagarase. In fact, such protection, which is dependent upon the nature of the substrate and state of the mitochondria, has been observed in several other cases. Bacterial electron transport chains also respond to the action of trypsin (Eilermann et al., 1971), pronase, and phospholipase A (Boll, 1971). Racker and co-workers studied the effects of phospholipase A and C on Ca transport, oxidative phosphorylation, and the structure of bovine heart mitochondria (Burnstein et al., 1971a,b). Following treatment of intact mitochondria with phospholipase C or A (cf. Bachmann et al., 1967), the resulting structure appears to represent a n intermediate state between mitochondria and SMP; while they behave as mitochondria upon negative staining and in their sedimentation pattern, they respond like SMP in functional tests, including the sensitivity of phosphorylation to valinomycin, nigericin, or Ca. Serial sections reveal that these intermediate structures, indeed, consist of inner membrane S MP packaged within an outer mitochondrial membrane. By sequential exposure of the two sides of the mitochondrial membrane to phospholipase C, virtually all the P C and PE can be cleaved. The resulting particles, which have lost two-thirds of their phospholipid, catalyze oxidative phosphorylation with a n efficiency similar to untreated control particles, provided that coupling factors are added. I n these damaged particles the respiratory chain remains remarkably unimpaired. However, a t higher concentrations

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of hydrolytic enzymes, the site between cytochromcs c1 and aa3is affected, followed by blockage of thc site between cytochromes b and c, and that directly preceding coenzyme Q. In fact, a difference in the susceptibility of these various sites has been noted, depcnding upon whethcr or not the particles are incubated aerobically and whether or not the succinate oxidase system is connected with electron transfer (Luzikov et al., 1971) Trcatment with phospholipase C also decreases the inhibitory effect of 2-thenoyl-trifluoro-acetone on particulate preparations with succinate reductase activity (Cerletti et al., 1967). On exposurc of the outer face of the inner membrane to phospholipase C, the cristac break and form vesiclcs which have inner membrane spheres on the outside-similar to SMP. However, in contrast to rupture by sonic oscillation, phospholipase C does not rupture the mitochondrial membrane when it is intact (Bachmann et al., 1967). As much as 70% of thc total phospholipid may be cleaved by exposing both sides of the membrane to the enzyme, without damaging the potential capacity of the SMP to catalyze oxidativc phosphorylation (Racker et al. , 1970). However, digestion of the residual diglycerides with lipase results in a rapid loss of phospiiorylating activity. Some remarkable differences in susceptibility to phospholipase A digestion between mitochondria and SMP may be noted. Whereas respiration was found t o be relatively insensitive to this enzyme in mitochondria, it was highly sensitive in SMP, NADH oxidation being considerably more sensitive than succinate oxidation. On exposure of SMP to phospholipase C, NADH oxidase activity was lost, while neither succinoxidase nor NADH dehydrogenasc activity was markedly impaired (Burnstein et al., 1971b; Racker et al., 1970). This indicates a high sensitivity of the NADH-Qlo segment of the electron transport chain to lipolysis; this has also been observed with phospholipase A (Awasthi et al., 1970; Machinist and Singer, 1965). Quantitatively, phospholipase A seems to be more effective than phospholipase C in inhibiting various enzymes of the respiratory chain. This could be due to the fact that the products of phospholipase A action are more toxic t o mitochondrial processes (Dobiasov et al., 1971). Better preparations (ie., less leaky) of mitochondria are found to be rather refractory to lipolysis. Consistent with this is the observation that susceptibility of mitochondria to protcolysis (Weinbach and Garbus, 1968a) and lipolysis (Nils-Erik and Seppala, 1970; Weinbach and Garbus, 1968b) is decreased in the presencc of uncoupling agents. Significantly, 2,4,6-trinitropheno11 a reagent that interacts with mitochondria but does not uncouple phosphorylation, has no effect on the lysis or swelling of mitochondria by phospholipase A. Furthermore, various lipophilic,

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positively charged compounds (quoted in Nils-Erik and Seppala, 1970) and local anesthetics (Seppala et al., 1971) also inhibit phospholipase A-induced swelling of mitochondria. Thus it appears that it is lipid solubility that is responsible for the protective action of these various agents. However (see Nils-Erik and Seppala, 1970; Weinbach and Garbus, 1968b), the role of protein components cannot be altogether discarded as the locus of energy conservation that induces structural alterations which lead t o the loss of coupled phosphorylation. Trypsin (Selman and Bannister, 1971), phospholipase A (Gressel and Avron, 1965), and pancreatic lipase (Gressel and Avron, 1965; Okayama et al., 1971) have been shown to affect specific photosynthetic reactions in chloroplasts. It appears that all these enzymes inhibit electron transport in the vicinity of photosystem 11. Photosystem I is not affected significantly by any of these enzymes, except for some effect of trypsin in NADP+ reduction. These results suggest not only the importance of the lipoprotein matrix in maintaining electron transfer reactions in photosystem I1 but also indicate that the oxygen-producing mechanism of photosystem I1 is particularly susceptible to hydrolytic attack. Furthermore, phospholipase A treatment of chromatophores of Rhodospirillum rubrum inhibits ATPase and pyrophosphatase (PPase) activity which can be completely restored by soybean lecithin (Klemme et al., 1971). The partial digestion of chromatophores with phospholipase A also destroys their ability to catalyze the phosphorylation of ADP and the ADP-Pi exchange reaction; however, only 55% of the initial activity can be restored by soybean lecithin. The response to oligomycin and the uncoupler CCFP of the reactivated photophosphorylation system is essentially the same as that of native particles. The latter reactions are more sensitive to depletion of the membrane of phospholipids than is ATPase of PPase activity. The results suggest that an active ATPase is not an indicator of a coupled cyclic photophosphorylation system. The particle CFl is requircd for photophosphorylation and for the light-activated ATPases of chloroplasts. It appears that CF1 performs both these reactions by undergoing conformational changes. Treatments that irreversibly inactivate the ATPase of CF1, such as incubation with large amounts of trypsin (Bennum and Racker, 1969) or heat (Vambutas and Racker, 1965), result in complete loss of capability of CF1 to recombine with CF1-depleted chloroplast membranes. However, milder trypsin treatments of CF1 still allow partial enzyme inhibition by the membranes. The chloroplast membrane can be resolved and reconstituted for binding of CF1, so that inhibition of mild trypsin-activated ATPase b y the membrane seems to be a property of the complete membrane, rather than the result of a specific inhibitor in the membrane.

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C. Calcium Transport System

In addition to surface membranes, two distinct systems have been implicated in excitation-contraction coupling in skeletal muscle. The transverse system provides a channel for the inward conduction of the excitation wave which ultimately reaches the SR and acts as a regulatory signal for the operation of the Ca pump. The transverse tubules are represented as invaginations of the surface membrane of muscle cells, their lumen being continuous with and permeated by the external medium. Studies pertaining to the digestion of muscle fibers with hydrolytic enzymes have yielded insight into the mechanism of the operation of the Ca pump. Frog sartorius muscles incubated with phospholipase C lose their contractility to supramaximal tetanic stimuli within 10-30 minutes (Martonosi, 1968). The loss of contractile response is accompanied by a great increase in the rate of Kf released from phospholipase C-treated muscles. No detectable Ca release is observed. In contrast, the ATPase activity and Ca uptake of isolated SR fragments is highly sensitive to phospholipase C (Martonosi, 1964, 1968; Martonosi et al., 1968, 1971; Masoro and Yu, 1971), which may be due to differences in the side of the membrane exposed t o the action of this enzyme. (SR vesicles are formed from reticular membranes which seal with inside-out geometry.) Phospholipase A and D do not seem to have any effect on vesicles (Masoro and Yu, 1971) but only on membrane fragments (Martonosi et al., 1971). As observed in electron micrographs, tryptic digestion removes the surface particles from fragmented SR. However, both ATPase activity and Ca transport functions remain in the membrane proper (Ikemoto et al., 1971). In SR microsomes phospholipase C treatment causes nearly complete inhibition of Ca accumulation and ATPase activity, without comparable inhibition of formation of the phosphoprotein intermediate. The inhibited ATPase activity and Ca transport can be restored by readdition of lecithin or lysolecithin. Formation of phosphoprotein is irreversibly inhibited by extraction of lipids by organic solvents. In fact, the role of lipid in ATP-dependent Ca translocation has been demonstrated by a PMR study in which transition of a choline methyl group of lecithin from a rigid to a mobile state is correlated with hydrolysis of ATP (Davis and Inesi, 1971). In phospholipase C-treated preparations of SR microsomes (which are not optimally activated by Cazf), simultaneous measurement of the rate of ATP splitting and the rate of Ca uptake shows that the activity pattern changes in two steps (Diehl et al., 1965). The first observed change in membrane properties is a decline in the rate of Ca uptake. Nevertheless, the Cadependent ATPase is still fully active, that is, Ca uptake and Ca-ATPase

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are dissociated. Similar activation of ATPase can also be achieved by incubation a t alkaline pH, sonication, very mild treatment with phospholipase A, treatment with fluorodinitrobenzene, and acetone extraction of freeze-dried preparations. All these treatments lead to a n uncoupling of ATPase activity and Ca transport. Subsequent t o the cessation of Ca uptake, a continuous loss of activity of Ca-ATPase occurs. The time required for inactivation naturally depends on the activity of the enzyme. In the absence of the added Ca ions it takes 2-3 hours, while in their presence approximately 30-60 minutes are sufficient. Under the latter conditions it is difficult to demonstrate that Ca uptake ceases prior to inactivation of Ca-ATPase. The structural requirements for the activation of Ca transport and Ca-ATPase activity appear to be considerably different. While the ATPase activity of phospholipase C-treated microsomes is activated by both phospholipids and by a series of nonionic and anionic detergents, a rather specific requirement for lysolecithin, phosphatidic acid, or lecithin was noted with respect to the reactivation of Ca transport function. Similar results were obtained when the SR vesicles were treated with phospholipase D at pH 5.6 (W. Fiehn, quoted in Fiehn and Hasselbach, 1970). This is particularly interesting, since the products of phospholipid cleavage by phospholipase C are partially released from the membrane, whereas one of the products of cleavage by phospholipase D, phosphatidic acid, is retained in treated membranes. After treatment of SR microsomes with phospholipase A, the permeability of the membrane toward Ca increases, and ATP-driven Ca storage is abolished (Fiehn and Hasselbach, 1970; Hasselbach et al., 1970). Although ATPase activity is lost, the phosphoryl transfer reaction remains unaffected even after removal of 80-90% of the phospholipid (Meissner and Fleischer, 1972; however, see Hasselbach et al., 1970) Unsaturated fatty acids restore the phosphoryl transfer reaction and the Ca-dependent ATPase activity. Lysolecithin restores the ATPase activity to nearly the same extent, but the phosphoryl transfer reaction is not reactivated. This difference is also exhibited in tighter binding of the drug prenylamine by fatty acid-activated vesicles. The accumulation of Ca is not restored either by unsaturated fatty acids or by lysolecithin. Effects similar to those elicited by phospholipase A treatment are also obtained by treatment with low concentrations of ether or oleie acid. These results suggest either that an intact membrane is required to observe Ca transport activity, or that phospholipase-A treatment truly uncouples the transport function from the energy-supplying ATPase activity. It is difficult to distinguish between these alternatives unambiguously a t this time. It may, however, be noted that abolition of Ca

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uptake is complete when about 30% of the membrane phospholipid is hydrolyzed by phospholipase A. Similarly, decomposition of phosphoenzyme is inhibited when about 80% of the phospholipid is removed. Thus the rapid abolition of Ca accumulation by phospholipase is most reasonably explained by an increase in the Ca permeability of the membranes, which may wipe out any effect of the pump. As to the mechanism of leak formation by phospholipase A, it seems important that the products of phospholipid hydrolysis remain in the membrane. Their active role in leak formation is revealed by the observations that a t low levels of digestion their extraction by albumin can restore Ca storage, and that leaks are produced when both hydrolysis products are incorporated into intact membranes. For independent experimental evidence suggesting only a partial identity of Ca-activated ATPase with the Ca pump, see Martonosi and Feretos (1964). D. Sodium Plus Potassium Transport System

There is no clear-cut evidence defining the influence of various hydrolytic enzymes on the Na pump. However, treatment of red cells with phospholipase A impairs cation transport (Greig and Gibbons, 1956), and phospholipase C treatment impairs rubidium uptake in kidney cortex tubules (Malila et al., 1968). Similarly, short-circuit current (SCC) is inhibited in a reconstituted active transport system by trypsin (on both sides of the modified BLM) and phospholipase A (only on the side containing the enzyme and ATP) (Jain et al., 1972). Also, the increase in SCC and conductivity of the frog skin elicited by vasopressin is abolished by phospholipase C treatment, even though the resting SCC and conductivity are normal (Cuthbert, unpublished observations quoted in Cuthbert, 1967). The effects of vasoprcssin are not, however, abolished by chymotrypsin. Ever since (Na K Mg)-activated ATPase has been implicated as thc mediator for active transport of cations, a role for phospholipids in the functioning of transport ATPase has been suggested by the inactivating influence of phospholipase A (Schatzman, 1962; Tatibana, 1963; Ohnishi and Kawamura, 1964; Forte et al., 1966; Fischeretal., 1970; Hegyvary and Post, 1969; Taniguchi and Iida, 1971; Taniguchi and Tonomura, 1971), and phospholipase C (Schatzman, 1962; Emmelot and Bos, 1968; Taniguchi and Tonomura 1971; Sun et al., 1971; Fischer et al., 1970; Forte et al., 1966; Cavalotti, 1969; Kielley and Meyerhoff, 1950). Similarly, proteolytic enzymes (Cavalotti, 1969; Marchesi and Palade, 1967; Martin, 1970; Masiak and Green, 1967; Somogyi, 1968)

+ +

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and neuraminidase (Emmelot and Bos, 1965, 1968) have been shown to inactivate (Na K)-ATPase. It seems that there is little selectivity for the action of these various enzymes, and the differences observed by various investigators are most probably due to differences in the physical state of the preparations and to the nature of various ions and cofactors present in the medium (Jain and White, unpublished observations). Pronase treatment does not significantly alter ouabain-sensitive influx in intact red cells, although treatment of ghosts with pronase reduces the influx to a low value (Knauf et al., 1972). Incubation of (Na K)-ATPase with different proteases causes a progressive loss of the ATPase activity, which can be partially counteracted by Mg, Na, K , or Mg-ATP (Masiak and Green, 1967; Somogyi, 1968; Marchesi and Palade, 1967; Jain et al., unpublished observations). Furthermore, the presence of Mg-ATP lowers the protection by K , whereas the effect of Na is not influenced at all. These observations are consistent with the hypothesis that the conforrnation of ATPase is different in the presence of Mg-ATP plus Na than of Mg-ATP plus K. The loss of ATPase activity following incubation with phospholipase A seems to parallel the phospholipid content of the preparations (Hegyvary and Post, 1969), and inactivation is reversible if phospholipid is added prior to hydrolysis of more than 65% of the phospholipid. Ouabaininsensitive Mg-ATPase is not affected by treatment with phospholipase A (Ohnishi and Kawamura, 1964) ; however, phospholipase C inactivates both the ouabain-sensitive and ouabain-insensitive ATPase of erythrocyte ghosts (Schatzman, 1962). The initial rate of formation of the phosphorylated ATPase intermediate in the presence of Na is reduced to 40% of that of the control by treatment of the enzyme with snake venom (Taniguchi and Tonomura, 1971). The rate of decomposition of the intermediate in the presence of high concentrations of K ions is reduced to only 7% of that of the control by the same treatment. The rates of formation and decomposition of the phosphorylated intermediate of the venom-treated preparations were increased to 90 and 40% of the control levels, respectively, by adding PI PS. It may be noted that the steady-state concentration of the phosphorylated intermediate in the presence of Na alone is not affected by treatment with phospholipase A. The observations just described have implications in terms of the ATPase reaction cycle regulating the hydrolysis of ATP and, consequently, the transport of Na and K ions. The results suggest that the transport complex is a lipoprotein, and that the phospholipids participate in the formation of the phosphorylated intermediate and in its decomposition; phospholipids appear to be particularly important for the decomposition step. Further-

+

+

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more, the enzyme, when activated by various cofactors such as Na and K, assumes different configurations which have different susceptibilities to proteolysis. A greater decrease in (Na K)-ATPase activity is produced by phospholipase AZthan by phospholipase C treatment. This may reflect the fact that phospholipase Az cleaves all phospholipids but sphingomyelin, whereas phospholipase C appears to cleave all phospholipids but phosphatidylserine. Phospholipid-depleted ATPase is activated not only by PS PI (Taniguchi and Iida, 1971) but also by crude lecithin preparations (Emmelot and Bos, 1968; Hegyvary and Post, 1969; Hexum and Hokin, 1971) and alkyl phosphates (Tanaka et al., 1971, and references therein; Jain, unpublished observations). However, reactivation by the addition of phospholipids is obtained only with preparations that have been partially inactivated. Part of the activating effect of the phospholipids appears to be due to chelating action (Wheeler, 1971). The major role of phospholipids in the transport cycle is to provide negative charge and hydrophobicity in the vicinity of the active site, and thus maintain correct spatial orientation, rather than to act as a component of the active site itself. About 100 molecules of phospholipid are necessary for activation of one molecule of ATPase. In addition to the ATP-mediated transport systems for Ca and for Na and K just described, there are two other systems in which translocation of a substrate not only involves formation of a phosphorylated intermediate but also involves lipids as cofactors. A short discussion of these systems follows.

+

+

1. Glucose is known to be translocated across several bacterial membranes through a specific transport system which transfers a phosphate group from phosphoenolpyruvate (PEP) to the sugar. Phospholipase D specifically inhibits the vectorial phosphorylation of a-methylglucoside by isolated membrane preparations from E. coli, without increasing the efflux of intramembranal a-methylglucoside-phosphate (Milner and Kaback, 1970). This inhibition was abolished and activity restored following addition of phosphatidylglycerol ; other lipids had no effect. 2. The two proteins that constitute staphylococcal toxin, leucocidin, synergistically alter the permeability of the leukocyte to cations, which in the normal cell is regulated by a K-dependent ATPase. In the isolated membrane leucocidin specifically activates the K-stimulated p-nitrophenylphosphatase (PNPase). The action of leucocidin on leukocytes or isolated membranes is accompanied by conversion of leucocidin to an inactive form. However, if the isolated membrane is treated with phospholipase A, inactivation does not occur. Instead PNPase is stimulated and remains sensitive to K (Woodin and Wieneke, 1966). The PNPase activity

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in trypsinized membranes is stimulated by leucocidin, although the sensitivity to K is lost (Woodin and Wieneke, 1971). This suggests that the action of leucocidin is mediated through lipids. E. Hormone-Sensitive Transport Systems

Adipose tissues and isolated fat cells respond to hormones such as growth hormone, insulin, thyroid-stimulating hormone, luteinizing hormone, glucagon, ACTH, secretin, catecholamines, thyroid hormone, prostaglandins, and steroids. Of these glucagon, secretin, ACTH, LH, TSH, and catecholamines alter the metabolism of fat, glucagon, and protein by stimulating the production of cyclic AMP via a membrane-bound adenyl cyclase. Insulin affects cellular metabolism by some yet unknown process; however, the available evidence suggests that the primary site of action of insulin is a receptor on the plasma membrane, which presumably modulates glucose transport (Rieser, 1967; Illiano and Cuatrecasas, 1971a, b). The target system or the receptor for the action of insulin, glucagon, and other hormones seems to have two characteristic components : one component binds or reacts specifically with the hormone; a second component initiates changes in transport or in adenylcyclase activity. Studies pertaining to action of various hydrolytic enzymes on intact tissues, isolated cells, and isolated membrane vesicles have provided considerable information regarding the mode of binding and probably mode of action of these hormones. Some of the salient aspects are summarized in the following discussion. 1. GLUCOSE TRANSPORT SYSTEM

Altered binding of insulin-lZ6I, glucose transport and utilization, and antilipolytic activity in muscle and adipose cells have been noted following controlled treatment with phospholipase A (Blecher, 1967; Blecher and Carr, 1968; Cuatrecasas, 1971), phospholipase C* (Rodbell, 1966; Rodbell and Jones, 1966; Cuatrecasas, 1971b; Weiss and Narahara, 1969; Rosenthal and Fain, 1971), proteolytic enzymes (Kuo, 1968; Kuo et al., 1966, 1967; Kono, 1969, 1970; Weiss and Narahara, 1969; Cuatrecasas, 1971a; Kono and Barham, 1971), neuraminidase (Cuatrecasas and Illiano, 1971; however, see Rosenthal and Fain, 1971; Rodbell, 1966), and thiol compounds (Lavis and Williams, 1966). Sphingomyelinase (Blecher, 1967)

* I t has been suggested that the insulinlike action of phospholipase C and neuraminidase from Clostridium perfringens may be due to contaminants (Rosenthal and Fain, 1971).

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and phospholipase D (Cuatrecasas, 1971c) do not elicit comparable effects. Since the observed effects of insulin are dependent on complicated metabolic processes that may be distant consequences of the primary insulin-receptor interaction, it has been impossible to determine whether chemical or enzymic perturbations of the cell result in alteration of the initial recognition site or of the subsequent events. However, since glucose utilization is limited by transport in both muscle and adipose tissue, other processes may be considered secondary. The effect of hydrolytic enzymes on the insulin receptor system may be considered a t several levels. Thus, following treatment of isolated adipose cells with trypsin, the insulin-binding capacity (Cuatrecasas, 1971a) of these cells is destroyed. Howevcr, the membrane does not become leaky to glucose, and glucose uptake and utilization respond normally to changes in the external glucose concentration. The glucose metabolism, but not the insulin-binding capacity of the treated cells, is largely restored within 1 hour after inactivation with trypsin (Kono and Barham, 1971); restoration is, however, prevented by inhibitors of protein synthesis (Manchester, 1970). Thus protein synthesis is required for stimulation of glucose utilization by insulin because some specific protein has been lost and must be replaced before the hormone can act again. In fact, studies with isolated membranes have demonstrated that mild treatment of membranes with trypsin results in a profound decrease in the affinity of the membrane receptor for insulin (Cuatrecasas, 1971b). The effect of trypsin digestion is quite different at higher concentrations of this enzyme, which cause a marked stimulation of glucose transport (Kono, 1969) and metabolism (Kono and Barham, 1971). These effects of proteolytic enzymes may be more complex than they appear a t first sight (see Kuo, 1968). There may exist functionally distinct pathways for the basal and for the insulin- and protease-stimulated pathways for the transport of hexoses. Thus, for example, phlorizin inhibits all the pathways of glucose entry in the presence or absence of insulin and proteases but does not abolish the stimulatory effect of these agents. It appears that phlorizin and a-methylglucoside block only the process of sugar entry and do not affect the mechanisms whereby sugar entry is increased by insulin or proteases (Kuo, 1968; Kuo et al., 1967; Kono, 1970; Kono and Barham, 1971). Similarly, the antilipolytic action of proteases, similar to that of insulin, is independent of their ability to stimulate glucose uptake. Addition of protease to insulin-pretreated cells, or addition of insulin to protease-pretreated cells, does not result in a greater rate of utilization of hexoses than when insulin or protease alone is present in the incubation medium of adipose cells. However, the effect of trypsinization on the kinetic parameters of glucose uptake suggests a different mode of action

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of insulin and trypsin (Kuo, 1968; Kuo et al., 1966,1967).Trypsin enhances the initial rate of entry of sugar, but K , remains unchanged. Insulin exerts maximal stimulation on the utilization of sugars over a wide range of insulin concentrations. In contrast, maximal stimulation elicited by proteases is observed only within a very narrow range of the enzyme concentrations, and an excess of the enzyme causes a marked decrease in or complete abolition of the stimulation (Weiss and Narahara, 1969). Similar activation for accumulation of L-proline and sugars has been noted in proteolyzed rat diaphragm (Rieser and Rieser, 1964). The effects of other hydrolytic enzymes on fat cells are quite different from those already noted. Thus neuraminidase seems to interfere with insulin-stimulated glucose transport and antilipolysis, without interfering with the insulin-binding capacity of the cell or isolated membrane (Cuatrecasas and Illiano, 1971). Similarly, digestion of fat cells with phospholipase A or C abolishes the insulin-stimulated processes of glucose transport and antilipolysis. Furthermore, extensive digestion of fat cells appears to cause extensive cell damage and lysis ; however, their insulinbinding capacity is increased three- to six-fold (Cuatrecasas, 1971b, c). Similar increases can also be brought about by digitonin, vitamin Ks, and mellitin, which are thought to disrupt phospholipid structures in membranes. Thus phospholipase A and phospholipase C treatment seems t o unmask receptor structures which are normally inaccessible to insulin in the medium or to trypsin. There are striking similarities in the characteristics of insulin interaction with liver and fat cells (Cuatrecasas et al., 1971). These observations suggest that the insulin receptor systems of liver and adipose tissue may have similar or even identical structures. In summary, the insulinlike action of various hydrolytic enzymes, polyene antibiotics, detergents, and thiol agents appears to be nonspecific, and may be due to an increase in membrane permeability which may in turn affect the metabolic status of the cell (Rodbell et al., 1968). The possibility of selective action by trypsin, however, has a significant experimental basis. Recent experimental evidence suggests that TSH acts on membranes to control iodide transport, a t the same time influencing the transport of amino acids and sugars. Dog thyroid slices preincubated with TSH and then washed thoroughly show the same increase in the rate of oxidation of glucose to COz as found if TSH is present along with glucose (Pastan el al., 1966). This effect can be abolished, however, by exposing the tissue to either anti-TSH antibody or trypsin before incubating it with glucose. The action of TSH can also be prevented by adding phospholipase C (Maechia and Pastan, 1967) which also inhibits phospholipogenesis (Burke, 1969). These treated cells have normal basal glucose oxidation

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and show normal stimulation of glucose oxidation by acetylcholine. It has been shown that phospholipid synthesis in thyroid tissue is sensitive to TSH (Kogl and Van Deenen, 1961). Interestingly, thyroid phospholipids can bind iodide, and binding is lost after lipolysis with phospholipase A, C, and D (Schneider and Wolff, 1964). Synthetic lecithin, soybean lecithin, and calf brain lecithin are not capable of mimicking the effect of lecithinlike material from thyroid glands. Phospholipase C also decreases baseline iodide transport in thyroid slices (Larsen and Wolff, 1967; Macchia and Wolff, 1970), and so, presumably, it also modifies TSH stimulation of iodide transport. All these results emphasize the role of membrane lipids in the action of TSH, but give little information as to its mode of action (Macchia et al. 1970; Vilkki, 1962). CYCLASE MODULATED BY HORMONES 2. ADENYL A great number of hormones produce many of their effects by stimulating adenyl cyclase and thereby increasing the level of cyclic AMP within the cells of their target tissues (Robison et al., 1969, 1971). Adenyl cyclase catalyzes conversion of ATP to cyclic AMP in the presence of divalent cations and is stimulated by fluoride ions to the level where hormone stimulation can usually be seen. Hormone-sensitive adenyl cyclases are membrane-bound enzymes present mostly in the plasma membrane (Birnbaumer et al., 1970; Rodbell et al., 1971). Membrane localization, lability, and the complex molecular structure of hormone-sensitive adenyl cyclases have made their characterization very difficult. One of the striking features of adenyl cyclases is that they are sensitive to different hormones in different tissues. Many tissues are highly specific in that they contain adenyl cyclase that is stimulated by only one or a few hormones. For example, adenyl cyclase from fat cells can be activated by ACTH, epinephrine, secretin, and glucagon. However, it has been observed that pretreatment of adipose tissue with trypsin results in a total loss of the stimulatory effect of glucagon. The effects of ACTH and secretin are partially lost, and those of epinephrine and fluoride are not affected a t all. These results indicate that the different hormones do not act on a common binding or receptor site (Rodbell el al., 1970). Also, it appears that adenyl cyclase itself is not the initial site of action of hormones, but that there is a secondary site whose behavior reflects changes at the primary site. Trypsin, under the conditions described above, does not cause a loss of cellular integrity of fat cells (Kono, 1969; Rodbell et al., 1970); neither does it damage the metabolic response of fat cells to prostaglandins, oxytocin, epinephrine, theophylline, and growth hormones plus glucuronoids. However, the metabolic effects of insulin and glucagon are dimin-

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ished following trypsin treatment. Also, following incubation with trypsin insulin receptors can be regenerated, but the glucagon receptors cannot. Trypsin treatment of ghosts of fat cells inactivates both the hormone- and fluoride-stimulated adenyl oylase activities (Birnbaumer et al., 1969). Treatment with phospholipase A and C (Tomasi et al., 1970; Rethy et al., 1971) and solubilization in Lubrol-PX (Levey, 1971a, b) seem to have similar effects on adenyl cyclase activities. This provides suggestive evidence that the receptor sites for the peptide hormones are lipoprotein in character and localized on the external surface of the plasma membrane (Rodbell et al., 1970; Birnbaumer et al., 1970). Study of the effects of inorganic pyrophosphate, MnC12, certain drugs, digitonin, urea, and treatment by phospholipase A on the liver plasma membrane suggest that fluoride and glucagon modulate adenyl cyclase activity by different processes (Pohl et al., 1971). The action of phospholipase A inhibits binding of and activation by glucagon, but activation by fluoride is unaffected, thus suggesting that the hormone and fluoride ions activate the enzyme through different processes. Both the glueagon sensitivity of adenyl cyclase and binding of glucagon-126I can be partially restored by exposing treated membranes to aqueous suspensions of membrane lipids. Pure PS, PC, and PE are all effective; of these, PS is the most effective. Similarly, addition of PI totally restores the norepinephrine activation of solubilized adenyl cyclase (Levey, 1971a, b) ; the reconstituted preparation is, however, unresponsive to glucagon, histamine, and thyroxine, The responsiveness to glucagon and histamine can be restored by PS (Levey and Klein, quoted in Levey, 1971b). V. CATABOLISM OF MEMBRANE COMPONENTS BY ENDOGENOUS ENZYMES OR INTRACELLULAR CATABOLISM

Quite a few hydrolytic enzymes are important constituents of biomembranes or of the medium'surrounding them. These hydrolytic activities may be latent under normal conditions although, in a variety of cells and tissues, such enzymes have been shown to regulate important cellular functions in addition to the catabolism of membrane components. It is very difficult to establish the exact role in metabolism and its control played by a particular hydrolytic enzyme. However, there are several instances in which certain hydrolytic enzymes have been implicated rather strongly in physiological functions. Some of these examples are given below. 1. Tissue injuries caused by carbon tetrachloride intoxication and vitamin E deficiency lead to a 1.5 to 2-fold activation of lipoxygenase, neuraminidase, and phospholipase in cellular, mitochondrial, and lysosomal

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membranes. This change results in the formation of lipid peroxides, in the loss of membrane sialic acid, and in an increase in unesterified fatty acids, all of which are known to change membrane properties significantly (Linchevskaya et al., 1971). 2. It has been known for some time that biphasic inhibition of G-6-Pase occurs when rat liver microsomes are treated a t 5°C with phospholipase C (40-60% inhibition), and that the activity is completely lost when they are postincubated without the substrate in the presence of EGTA a t either 20” or 37°C. The loss of G-6-Pase activity under these conditions is completely restored by the addition of sonicated lecithin. The second phase of inhibition, which can also be brought about by addition of fatty acids, is not reversible by the addition of lecithin, although adding lecithin prior to heating a t 20” or 37°C protects completely against phase-I1 inhibition. It has been shown that phase-I1 inhibition is due to endogenous microsomal acyl hydrolase activity which produces fatty acids by the breakdown of diglycerides (Cater and Hallinan, 1971). 3. It has been shown that the exocrine parotid cell has two epinephrine receptors : &receptors control amylase secretion via adenyl cyclase, and or-receptor regulates K+ release via a different pathway (Batzri et al., 1971). Since secretion of amylase is associated with vacuole formation, the latter processes can be regulated by nervous activity. 4. In yeast (Saccharomyces cerevisiae), concomitant with the breakdown of the mitochondria1 membrane due t o glucose repression, there is an increase in the level of phospholipase D activity (Dharmalingam and Jayaraman, 1971). Such a mode of disintegration is particularly attractive since in the mitochondria lipid is involved in the barrier-matrix system and activation of enzymes of the respiratory chain, and the products of lipolysis cause lysis of membranes. 5. Brain, liver, and mammary glands of the rat contain two neuraminidases, one that occurs in soluble form in cytosol, and one firmly bound to the lysosomes. The activity of soluble brain neuraminidase increases during the second and third week of postnatal development, following a pattern similar to that for the accumulation of glycoproteins and gangliosides in brain (Carubelli and Tulsiani, 1971, and references therein). 6. It has been suggested that ATP activation of neutral proteolytic activity is a widespread phenomenon (Umana and Feldman, 1971, and references therein). ATP can release inhibition of some peptidases through its own products. Also, this activation could be linked to the presence of “amino acid-activating enzymes.” Under these conditions a close relationship between the rate of protein degradation and the rate of protein synthesis is expected; the net result is the phenomenon of regulated protein turnover.

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7. Protein degradation is also modulated by growth conditions (Goldberg, 1971) and the presence of certain drugs (Kuriyama et al., 1969). 8. Protein catabolism has been implicated in regulatory functions. Thus the hydrolysis of polynucleotide phosphorylase, leucyl-tRNA synthetase, and perhaps of methionyl-tRNA synthetase and DNA polymerase, by endogenous proteases of E. coli may be critical in determining the levels of these enzymes (Thang et al., 1971; also see Rechcigl, 1971). 9. Two of the endogenous proteases from human seminal fluid increase vascular permeability (Suominen and Niemi, 1971). This effect may be ascribed to liberated histamine and may be significant in human reproduction, since in addition to prostaglandins, histamines generated in situ can affect (uterine) smooth muscle. 10. I n sheep erythrocyte membranes significant complement-mediated proteolysis of membrane proteins has been observed (Knufermann et al., 1971). Similarly, a release of high proteolytic activity has been observed during protoplasmic incompatibility (Begueret, 1972). 11. Activation of endogenous phospholipase A has been observed after vagotomy in the rabbit (Imre, 1970) and treatment of E. coli with a variety of colicins (Cavard and Barbu, 1969). Endogenous phospholipase A has also been implicated in membrane fusion reactions (Poole et al., 1970; Lucy, 1970). 12. The effect of incorporation of ethanolamine on choline in the cell wall of Pneumococcus is such that cells with ethanolamine remain associated after division and can neither autolyze nor be lysed by deoxycholate treatment as readily as cells containing choline (Tomasz, 1968). In these cells ethanolamine or choline is incorporated into a macromolecular component, probably a polysaccharide, of the cell wall instead of being utilized for phospholipid biosynthesis. It has been suggested that, among other pleiotropic effects, the loss of autolysis and the lcss of deoxycholate solubility may all be consequences of a common primary effect ; for example, the ability of the autolytic system to digest ethanolamine-containing cell wall polymers. 13. Release of an aorta-contracting substance during inflammation may be associated with the activation of membrane phospholipase A (Vargaftig and Dao, 1971). 14. Several genetic disorders and inherited syndromes have been ascribed to in vivo degradation of sphingomyelin (Stoffel, 1971), glycoproteins (Ginshurg and Neufeld, 1969), mucopolysaccharides (Neufeld and Frantantoni, 1969), and lecithin (Bhagwanani et al., 1972). There appears to exist a causal relation between phospholipid and phospholipase concentration with age (Eisenberg et al., 1969). Similar factors may be

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responsible for the role of lysosomes in cellular processes (Weissmann, 1965). Also, Clostridium septicaemia produces a severe hemolytic anemia which occurs as a result of the lipolytic action of phospholipase C. This in turn may alter the structure of membrane proteins (Simpkins, 1971). 15. Penicillin inactivates both an endopeptidase and a glycosidase of E. coli (Hartmann et al., 1972). These two enzymes are essential for the normal growth of the sacculus, or fuzz, a netlike structure that encloses the membrane of the organism. The various observations presented in this section relate not only to aspects of metabolism of individual membrane components but also to the subtle dynamic and temporal aspects of their organization that are important in the functioning of an organism. These aspects relate to turnover, transport, and regulation of organization of various membrane components, as well of other cellular metabolites. An important aspect of developmental plasticity of membrane composition is environmentdependent (diet, drugs, abnormal growth) alteration of acyl chains or of polar residues. This brief discussion therefore illustrates that the intricacies of membrane behavior are modulated and regulated by in vivo hydrolytic enzymes. We are far from an understanding of these phenomena, less so for the mechanisms involved in these processes ;nonetheless, considerable insight is gained by the observations on the model systems and biosystems presented earlier in the article.

VI. CONCLUSIONS AND EPILOG

The results discussed in this article have been rather sketchy, but the breadth of coverage does emphasize various aspects, both topological and molecular, of membrane phenomena in their entirety. The various aspects of organization and topography, and the correlates of molecular processes, are probably best manifested in the rate and extent of hydrolysis of various membrane components by specific enzymes. The molecular aspects are best manifested while considering modification and modulation of various specific membrane functions following treatment of functional membrane with hydrolytic enzymes. Although not extensive, there is definite indication that the affinity and specificity of hydrolytic enzymes for membrane-bound substrates is significantly dependent upon the packing characteristics of the substrate in the membrane. Thus studies pertaining to the interaction of phospholipases with lamellar lipid layers bear not only on general aspects

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of lipid-protein interaction, but also on specific features of interfaces such as the electrical double layer and the orientation of polar head groups, including the glycerol backbone and the first few carbon atoms of the chains. These groups are rather rigidly oriented in a lipid bilayer and probably offer the most resistance to permeating solutes. The phenomenological correlates of the molecular process are not yet completely understood. However some caution must be exercised when interpreting differential effects of hydrolytic enzymes on intact cells as opposed to their effects on isolated membrane vesicles. These differences do not necessarily arise from the unmasking of buried groups or sites due to removal of some surface component but could also be due to altered conformation, mode of packing and organization, or mobility of various components in the membrane. This difference may be reflected both in the system properties and specific functional characteristics of a membrane. The approaches used by various investigators and those described herein constitute a potent conceptual and experimental armamentarium which may be used for isolation and characterization of specific membrane components. The dependence of the system properties and functional activity of biomembranes on the presence of certain proteins, lipids, and polysaccharides has been widely recognized. I n quite a few cases correlation of the nature of the functional molecule(s) has been established by modification with hydrolytic enzymes. These include : 1. Changes in the overall topography and viscoelastic properties of membrane-bound organelles 2. Changes in immunological and serological characteristics 3. Changes in surface-charge profile and consequently in mobility and motility 4. Asymmetry of distribution of various molecules a t the two interfaces, which gives rise to differential activity 5 . Changes in barrier characteristics 6. Changes in the specific catalytic behavior of membrane-bound proteins. Apparently, the bulk of membrane lipid can be modified without significantly affecting the bulk of membrane proteins-both structurally and functionally. However, there is definite evidence for the existence of specific lipid-protein interaction. Part of the evidence comes from the rate at which and the extent to which bulk lipids and protein-bound lipids undergo hydrolysis.

These apparently innocuous changes rather reflect on the whole range of membrane phenomena. Thus even qualitatively the results described in this article are in general consistent with a lipoprotein nature of bio-

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membranes and support the hypothesis that takes into consideration separation of phases, asymmetry of distribution of various surface components, and involvement of specific macromolecules in specialized functions along with other system properties expected from a lipoprotein matrix. The role of lipids in modulating the catalytic properties of aggregate systems, their spatial organization, and integration of their functions in membrane subunits, as well as in the membrane as a whole, is not yet clear but is hinted at by the studies described in Sections IV and V. The methodology of enzyme digestion of membrane components needs modification, and the use of pure enzyme preparations may alter some of the results quantitatively. Thus the catalytic activity of hydrolytic enzymes may be modified by extraneous contaminants, ions, solvents, detergents, or other hydrolytic enzymes of differing specificity or size which may expose buried groups. In our opinion one of the most important pieces of missing information is knowledge of the specificity pattern of various substrates of phospholipases, and the dependence of specificity on the state of the substrate and the presence of various activators and cofactors which may modulate and modify membrane structure and functions. From the standpoint of the studies relevant to biomembranes, a few other related immediate needs present themselves. The first is the availability of several pure isologous enzymes through which quantitative differences in the kinetics of hydrolysis can be meaningfully correlated to molecular and topological parameters. The second is the availability of suitable inhibitors of various hydrolytic enzymes, which may be used to regulate the extent of hydrolytic cleavage. Ultimately, it is hoped that our knowledge of the biochemistry of these various processes will be such as to allow us to modulate and regulate the extent of hydrolytic cleavage of mernbranc components, which may in turn be meaningfully correlated with molecular parameters. ACKNOWLEDGMENTS

I take this opportunity to express my sincere thanks to Professor Eugene Cordes for encouragement, comments on the manuscript, and financial support (grant no. AM08232-08). REFERENCES Adams, C. W. M., and Bayliss, 0. B. (1968). J . Histochem. Cytochem. 16, 119. Agostini, B.,and Hasselbach, W. (1971). Natumoissenschaflen 58, 148. Albuquerque, E. X., and Thesleff, S. (1967). J . Physiol. (London) 190, 123. Albuquerque, E. X.,Sokoll, M. D., Sonesson, B., and Thesleff, S. (1968). Eur. J . Pharmacol. 4 , 40. Alexander, A. E., and Rideal, E. K.(1937). Proc. Roy. SOC.,Ser. B 163, 70. Anonymous (1971).Nature (London)232,219.

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Vogt, W., Patzer, P., Lotte, L., Oldigs, H. D., and Wille, G. (1970). Naunyn-Schmiedebergs Arch. Pharmakol. Ezp. Pathol. 265, 442, Wahlstrom, A. (1971). Tozicon 9, 45. Waite, M., Van Deenen, L. L. M., Ruigrok, T. J. C., and Elbers, P. F. (1969). J . Lipid Res. 10, 599. Walborg, E. F., Jr., Lantz, R. S., and Wrey, V. P. (1969). Cancer Res. 29, 2034. Wallach, D. F. H. (1969). J. Gen. Physiol. 54, 3s. Wallach, D. F. H. (1972). Biochim. Biophys. Acta 265, 61. Warren, L. (1969). Cur. Top. Develop. Biol. 4, 197. Watkins, E., Jr., Ogata, Y., Anderson, L. L., Watkins, E., 111, and Waters, M. F. (1971). Nature (London),New Biol. 231, 83. Weed, R. I., LaCelle, P. L., and Merill, C. W. (1969). J . Clin. Invest. 48, 795. Weibull, C. (1958). Annu. Rev. Microbiol. 12, 1. Weidekamm, E., Wallach, D. F. H., and Fischer, H. (1971). Biochim. Biophys. Acta 241, 770. Weinbach, E. C., and Garbus, J. (1968a). Biochem. J. 106, 711. Weinbach, E. C., and Garbus, J. (1968b). Biochim. Biophys. Acta 162, 500. Weinbaum, G., Rich, R., and Fishman, D. A. (1967). J. Bacteriol. 93, 1693. Weinstein, D. B., Marsh, J. B., Glick, M. C., and Warren, L. (1970). J. Biol. Chem. 245, 3928. Weiss, L. (1961a). Ezp. Cell Res., Suppl. 8, 141. W e b , L. (1961b). Nature (London) 191, 1108. Weiss, L. (1963). Ezp. Cell Res. 30, 509. Weiss, L. (1965). J. Cell Biol. 26, 735. Weiss, L. (1966). J. Cell Biol. 30,39. Weiss, L. (1968). Ezp. Cell Res. 51, 609. Weiss, L. (1970a). In “Permeability and Function of Biological Membranes” (L. Bolis, ed.), p. 94. North-Holland Publ., Amsterdam. Weiss, L. (1970b). In “Adhesion in Biological Systems” (R. S. Manly, ed.),p. 1. Act+ demic Press, New York. Weiss, L., and Coombs, R. R. A. (1963). Ezp. Cell Res. 30, 331. Webs, L., and Mayhew, E. (1966). J . Cell. Physiol. 68,345. We&, L. S., and Narahara, H. T. (1969). J. Biol. Chem. 244,3084. Weissmann, G. (1965). New Engl. J. Med. 273, 1084, 1143. Wheeler, K. P. (1971). Biochem. J . 125, 71P. Wills, E. D. (1965). Advan. Lipid Res. 3, 197. Wintzer, G., and Uhlenbruck, G. (1967). 2. Immunitaetsjorsch. Allerg. Klin. Immunol. 133, 60. Winder, R. J. (1969). In “Red Cell Membrane” (G. A. Jamieson and T. J. Greenwalt, eds.), p. 157. Lippincott, Philadelphia, Pennsylvania. Winder, R. J. (1970). Int. Rev. Cytol. 29, 77. Woelk, H., and Debuch, H. (1971). Hoppe-Seyler’s Z. Physiol. Chem. 352, 1275. Wolff, K., and Schreiner, E. (1971). J. Ultrastruct. Res. 36, 437. Wolman, M., and Wiener, H. (1965). Biochim. Biophys. Acta 102, 269. Woodin, A. M., and Wieneke, A. A. (1966). Biochem. J. 99, 479. Woodin, A. M., and Wieneke, A. A. (1971). Biochim. Biophys. Acta 233, 702. Woodruff, J., and Geaner, B. M. (1967). J. Clin. Invest. 46, 1134. Woodruff, J., and Gesner, B. M. (1968). Science 161, 176. Woolley, D. W., and Gommi, B. W. (1964). Nature (London) 202, 1074. Woolley, D. W., and Gommi, B. W. (1965). Proc. Nat. Acad. Sci. U.S. 53,959.

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Regulation of Sugar Transport in Eukaryotic Cells* HOWARD E. MORGAN and CAROL F . WHITFIELD Department of Physiology, The Milton S. Hershey Medical Center, The Pennsylvania Stale University, Hershey, Pennsylvania

I. Kinetic Characterization of Passive Transport . . . . . . . . . 11. Nonhormonal Regulation of Sugar Transport . . . . . . . . . A. Effect of Anoxia on Sugar Transport . . . . . . . . . . B. Acceleration of Sugar Transport in Association with Muscular Contraction. . . . . . . . . . . . . . . . . . . C. Effects of Fatty Substrates on Sugar Transport . . . . . . . 111. Hormonal Control of Sugar Transport . . . . . . . . . . . A. Insulin . . . . . . . . . . . . . . . . . . . B. Epinephrine . . . . . . . . . . . . . . . . . C. Glucocorticoids . . . . . . . . . . . . . . . . D. Growth Hormone . . . . . . . . . . . . . . . . IV. Mechanisms of the Regulation of Transport . . . . . . . . . A. The Role of Ions in Transport Regulation. . . . . . . . . B. The Roles of Sulfhydryl Groups and “Energy-Charge’’ in Regulating Transport . . . . . . . . . . . . . . . . . . V. Summary . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . .

256 26 1 262 267 270 274 274 279 282 284 287 287 293 296 297

Regulation of sugar transport in eukaryotic cells provides an important mechanism for controlling the rate of glycolysis. A wide range of regulatory factors has been identified, including (1) hormones such as insulin, growth hormone, and others; (2) the degree of oxygenation of the cell; (3) the rate of energy utilization within the cell; and (4) the availability of alternative substrates for oxidation. In addition, these factors interact to provide a fine control of transport rate. In this article control of passive transport of sugar in erythrocytes, muscle, adipose tissue, and other tissues is described.

* Supported by grant HE-13029 from the National

Institutes of Health. 255

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HOWARD E. MORGAN AND CAROL F. WHITFIELD

Transport is passive in the sense that under ordinary conditions the sugar moves from a region of higher to one of lower concentration. Active transport of sugar has been the subject of several reviews (Kaback, 1970; Schultz and Curran, 1970).

1. KINETIC CHARACTERIZATION OF PASSIVE TRANSPORT

Entry of sugar into animal cells has been distinguished from simple diffusion by the following properties : saturation kinetics, stereospecificity, competitive inhibition, and countertransport. These properties suggested that penetration of sugar through a membrane involved binding of the sugar to a mobile membrane component, a carrier (Fig. 1). Kinetics of sugar transport have been studied in the greatest detail in human and rabbit erythrocytes. In constructing a model of transport, several simplifying assumptions were made (Widdas, 1952; Rosenberg and Wilbrandt, 1957). 1. Free carrier and sugar-carrier complex are confined to the membrane. The amount of carrier in an area of membrane is unchanged by synthesis or degradation of carrier during the period of the transport experiments.

MEMBRANE

FIQ. 1. The mobile carrier in the presence of either a nonmetabolied sugar or a nonmetabolized sugar plus glucose. S, Nonmetabolised sugar; G , glucose; C, free carrier; CS, sugar-carrier complex; CG, glucose-carrier complex; subscripts o and i, outside and inside of the cell, respectively.

SUGAR TRANSPORT IN EUKARYOTIC CELLS

257

2. The rates of diffusion of free carrier and sugar-carrier complex through the membrane are the same. 3. Formation and dissociation of sugar-carrier complex is rapid compared to the rate of transfer of carriers across the membrane. 4. The affinity of the carrier for a sugar is the same a t both surfaces of the membrane.

Subsequently, a kinetic treatment was developed by Regen and Morgan (1964) which avoided assumptions 2, 3, and 4. A test of the mobile carrier model of sugar transport in rabbit erythrocytes revealed that the simple carrier model adequately accounts for membrane penetration of glucose and 3-0-methylglucose (Regen and Morgan, 1964). I n addition, free carrier and sugar-carrier complex were found to move a t the same rate, and these movements were rate-limiting for transport. Movement of free carrier and sugar-carrier complex were compared in experiments in which outward movement of 3-0-methylglucose from preloaded cells was measured under conditions in which the sugar moved into buffer (1) that contained no extracellular sugar and (2) that contained the nonmetabolized glucose analog a t the same concentration present in the cells. In the first instance return of carrier to the inner surface of the membrane was as free carrier, while in the second case carrier returned to the inner surface as the sugar-carrier complex. Since the rate of outward movement of 3-0-methylglucose was unaffected by the presence of external sugar, free carrier and sugar-carrier complex appeared to move a t the same rate. These two forms of carrier also appeared to move a t the same rate in beef erythrocytes (Hoos et al., 1972). Countertransport provided evidence for involvement of a combining site not exposed to both surfaces of the membrane a t the same time and indicated that carrier movements were the rate-limiting events in the overall movement of sugar through the membrane (Regen and Morgan, 1964). Countertransport depended upon development of a gradient of free carrier across the membrane following addition of glucose to the extracellular phase of cell suspensions containing 3-0-methylglucose a t equal concentrations inside and outside the cells. Symmetry of transport in the rabbit red cells was established by showing that the Michaelis constant for exit of 3-0-methylglucose was equal to the Michaelis constant for entry of this sugar. These constants were assessed in experiments, which measured (1) uptake of 3-0-methylglucose into sugarfree cells, or (2) exit of 3-0-methylglucose from preloaded cells into sugarfree medium. Studies of sugar transport in rabbit erythrocytes indicated that the kinetics of transport can be described by the simple carrier model (1) in

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which free carrier and sugar-carrier complex moves a t the same rate, (2) in which carrier movements are the rate-limiting events for transport, and (3) in which transport is symmetrical. If sugar transport in human erythrocytes possesses these same propertics, the affinity constant and maximal rate of glucose transport would not vary whether the constants are measured in experiments in which glucose moves out of the cell into glucose-free medium or into medium containing glucose a t the same concentration as that found within the cells, or whether affinity is assessed by measuring the ability of glucose to compete with the transport of another sugar. As seen in Table I, however, the affinity constant and maximal rate of glucose transport varied markedly, depending upon the type of experiment in which the kinetic parameters were estimated. In the experiments of Miller (1971), however, the affinity constant for galactose and the maximal rate of galactose transport were the same whether egress of the sugar was into medium that was sugar-free or that contained galactose a t the same concentration present within the cells. The values for these parameters reported by Stein (1972) show a twofold difference in the maximal rate of transport in the two types of experiments. These results indicated that the simple carrier model not subject to regulation is not TABLE I KINETICPARAMETERS OF GLUCOSE AND GALACTOSE TRAXSPORT IN HUMAN ERYTHROCYTES

Type of experiment Efflux of glucose-l4C into varying extracellular concentrationsof glucose-'%! (Sen and Widdas, 1962) Glucose inhibition of sorbose transport Efflux of glucose-14C into the same concentration of extracellular glucose-1% Exit of glucose into sugar-free medium Efflux of galactose-14C into the same concentration of extracellular galactose-12C Exit of galactose into sugar-free medium

Data taken from Miller (196%). Data taken from Miller (1971). c Data taken from Stein (1972). b

Affinity constant (mM)

Maximal rate (mmoles/minute/cell unit)

1 . 8 f 0.3"

104 f 120

17 f 2 a 38 f 3. 7 . 4 f 1.4b 25 f 3" 73 f 13b 116c 58 f 16b 165O

260 f 300

112 f 8 b 139 f l l c 180 f 20b 46lC 140 f 34b 224O

SUGAR TRANSPORT I N EUKARYOTIC CELLS

259

adequate to account for sugar transport in human red cells. An explanation of the discrepancics in the data of Miller (1971) and Stein (1972) regarding kinetics of galactose transport is not apparent. Modifications of the carrier model and models that do not involve a mobile carricr have been Suggested to account for the kinetics of glucose transport in human red cells. Modifications of the carrier model that have been considered include: (1) different rates of diffusion of free carrier and sugar-carrier complex across the membrane (Lacko and Burger, 1963; Levine et al., 1965; Mawe and Hempling, 1965; Miller, 1968b); (2) restriction of diffusion of sugar to and from the carrier a t both membrane surfaces (Miller, 1968b); (3) a conformational change in the carrier molecule associated with movement of carrier from one surface of the membrane to the other (Miller, 1971); and (4) asymmetry of transport involving different rates of carrier movement and different affinity constants for entry and exit (Geck, 1971). A model involving different rates of diffusion of free carrier and sugar-carrier complex accounted for varying maximal rates of transport, but could not account for variations in the affinity constant (Miller, 1968b). A mechanism involving limitation in diffusion of sugar to the carrier did not yield values for affinity constants and maximal transport rates that gave simultaneous agreement for all experiments (Miller, 1968b). An asymmetrical model of transport (Geck, 1971) and a model involving a conformational change in the carrier (Miller, 1971) have been reported to account reasonably well for rates of sugar transport under all the experimental conditions listed in Table I. Support for a carrier model involving a substrate-induced conformational change has been provided by the demonstration that glucose and other readily transported sugars facilitate inactivation of the transport system by l-fluor-2,4-dinitrobenzene (FDNB) (Bowyer and Widdas, 1958; Krupka, 1971). However, maltose that was firmly bound t o the sugar site, but was not transported, protected the system from FDNB inhibition. These results suggested that binding of readily transported sugars exposed a chemical group which was attacked by FDNB by inducing a conformational change in the carrier. Noncarrier models of sugar transport have been proposed which fit the observed kinetics of sugar movement. A lattice membrane containing fixed binding sites for sugar, which are situated within water-filled channels spanning the membrane, readily accounted for saturation kinetics and stereospecificity (Naftalin, 1970). However, acceleration of glucose efflux by addition of extracellular sugar and countertransport could be accounted for only if an unstirred layer were present a t the membrane surface. Evidence for unstirred layers surrounding the cells was provided by demonstrating that glucose efflux was increased by agitation of the cell suspension and that acceleration of glucose efflux by addition of extracellular glucose was

260

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F.

WHITFIELD

reduced by vigorous stirring (Naftalin, 1971). However, Miller (1972) and Lieb and Stein (1972) have argued that diffusion in the external solution should not be limiting and that the effects of stirring (Naftalin, 1971) were due to an inefficient method of mixing in the control experiments. Since in the lattice model acceleration of glucose efflux by addition of extracellular sugar and countertransport depends upon unstirred layers surrounding the cells, both phenomena should be demonstrable if either is present. As noted earlier, countertransport was readily demonstrated in rabbit erythrocytes, while acceleration of glucose efflux was not found. These considerations suggest that the fixed site model does not account for sugar transport in rabbit erythrocytes. A model involving a tetrameric protein extending through the membrane has been suggested as an alternative to the mobile carrier (Lieb and Stein, 1970; Karlish et al., 1972; Eilam and Stein, 1972; Lieb and Stein, 1972; Stein, 1972). Each subunit of tetramer is assumed to bind sugar; two subunits have a high affinity for sugar, while two subunits have a low affinity. One high-affinity and one low-affinity subunit are present on each membrane surface. When extracellular sugar is bound to one of the subunits, a conformational change in the protein occurs which moves the sugar molecule into a pool within the membrane. Sugar within the membrane pool could bind to one of the pair of subunits on the inner membrane surface. Binding of sugar to one of these subunits again induces a conformational change and moves the sugar into the cell. This model can account for all the kinetic properties of transport that have been described. Regulation of affinity and movement of the carrier offers another possible solution to the kinetic anomalies observed in experiments on human red cells. I n beef erythrocytes glucose added along with 3-O-methylglucose competitively inhibited transport of the analog (Hoos et al., 1972). When glucose was added 1 hour before 3-O-methylglucose, a noncompetitive inhibition of transport was found. The possibility that varying rates of glycolysis in human red cells may affect transport kinetics is strengthened by the observation that exchange of mannose into glucose, or exchange of galactose into glucose, was slower than exchange of either mannose into mannose or galactose into galactose (Eilam and Stein, 1972). The major reservation in accepting more complicated models of transport in human red cells appears to be whether one requires that all variations be accounted for simultaneously or whether one is willing to accept that the properties of transport vary according to the metabolic state of the cell. If all the kinetic properties do not require simultaneous agreement, more complicated models of transport may be unnecessary. A det.ailed presentation of the kinetics of regulated transport has been published recently by Heinz et al. (1972). The kinetics of regulated transport in muscle, adipose tissue, nucleated

SUGAR TRANSPORT IN EUKARYOTIC CELLS

261

red cells, and others has not been studied in the same detail as transport in mammalian red cells. As a result, experimental findings inconsistent with the simple carrier model have not been reported. Detailed studies of kinetics of regulated transport have been hampered by a variety of factors: 1. Transport regulation occurrs most commonly in cells organized into tissues. Studies of transport in tissues require either that the tissue be perfused or be sufficiently thin to overcome restricted access of sugar to the cells in the preparation. Transport regulation has been observed, however, in isolated fat cells and avian erythrocytes. Fat cells have been difficult to study because of the small volume of intracellular water. 2. Since regulated transport is often a major restraint on glucose utilization in eukaryotic cells, intracellular glucose accumulates to only low levels in the absence of an acceleratory factor. Even in the presence of such factors, transport often remains slow relative to the rate of glucose phosphorylation, and only low levels of intracellular glucose are found. This situation prevents studies of the kinetics of glucose efflux. Efflux studies are among the most helpful in characterizing transport in human and rabbit erythrocytes. 3. An ideal nonmetabolized glucose analog has not been available. 3-0methylglucose, the most commonly employed analog, has a high affinity for the carrier but has the disadvantage that a significant fraction of its membrane penetration occurs by simple diffusion. In rabbit erythrocytes the diffusion constant for 3-0-methylglucose is eight times that for L-glucose. All other nonmetabolized glucose analogs have relatively low affinity for the carrier. As a result, sufficiently high concentrations often cannot be achieved to characterize the kinetic constants adequately. 2-Deoxyglucose, a glucose analog which is phosphorylated but not further metabolized, has the disadvantage that high concentrations of the phosphorylated product, 2-deoxyglucose-6-P, accumulates within the cell (Kipnis and Cori, 1959). The phosphorylated product affects the rate of enzymatic reactions and appears to affect sugar entry. As a result of these limitations, kinetics of regulated transport have been dealt with on the basis of the simple carrier model. A detailed kinetic study will be required to determine whether this model adequately accounts for sugar transport subject to regulation, and to characterize the effects of factors that act on various steps in the trandport process. II. NONHORMONAL REGULATION OF SUGAR TRANSPORT

Transport has been found to be affected (1) by interfering with energy production by induction of anoxia or addition of inhibitors of oxidative

262

HOWARD

E. MORGAN AND CAROL F. WHITFIELD

phosphorylation, (2) by acceleration of energy consumption as, for example, by contraction of muscles, by secretion in endocrine or exocrine glands, or by facilitation of other energy-requiring reactions, or (3) by provision of other readily oxidized substrates to the tissue. A. Effect of Anoxia on Sugar Transport

The importance of oxidative metabolism in the control of glucose utilization was first described by Pasteur, who observed that glycolysis in yeast was less rapid in the presence of oxygen than in its absence. Subsequently,'inhibition of glycolysis by respiration was found to be a property of most cells including yeast, muscle, and neural tissue. The wide range of cells involved in this type of regulation suggested that respiratory control of glucose utilization is the most primitive mechanism of glycolytic regulation and that hormonal regulation is superimposed on respiratory control (Randle, 1964). Respiratory control of glycolysis a t the membrane transport step was recognized by Randle and Smith (1958a, b) in diaphragm muscle, by Morgan et aZ. (1959a) in heart muscle, and by Kleinzeller and Kotyk (1965) in yeast. An example of the effects of anoxia on the transport of glucose in perfused rat heart appears in Table 11. As shown in the upper line of this table, entry of glucose into aerobic hearts was slow, resulting in low rates of glucose uptake and undetectable levels of intracellular glucose. Since under these conditions glucose phosphorylation was able to keep pace with entry of glucose into the cell, membrane transport was the major rate-limiting step for uptake. Anoxia increased glucose uptake approximately 10-fold ; this indicates that transport has been accelerated. The anaerobic stimulation of glucose transport was sufficient to lead to accumulation of intracellular glucose and to shift the major limitation on uptake to the phosphorylation step. Anoxia also accelerated glucose uptake in the heart treated with insulin, possibly because of more rapid glucose phosphorylation (Morgan et aZ., 1961a). Stimulation of transport in anaerobic muscle was substantiated by the finding of a more rapid entry of the nonmetabolized pentose, L-arabinose, into the anaerobic heart (Table 111). I n this case anoxia was found to enhance the stimulatory effect of a low concentration of insulin on the transport of L-arabinose. These studies indicated that sugar transport is a major rate-limiting step for glucose utilization in muscle tissue and that this step is markedly accelerated in anaerobic muscle. A similar conclusion was reached by Kleinzeller and Kotyk (1965) in yeast cells. The importance of transport acceleration in the anoxic effect is frequently overlooked. Attention has been given more often to factors controlling glycolytic

263

SUGAR TRANSPORT I N EUKARYOTIC CELLS

TABLE I1 EFFECTOF ANOXIAAND INSULIN ON THE ENTRY O F D-GLUCOSE INTO PERFUSED RAT HEARTO

Gas phase

oz/coz oz/co2 Nz/COz Nz/COz

Insulin (0.5 pg/ml) 0

+ 0 +

Glucose uptake (pmoles/gm/hour)b 60 373 638 814

f 26 f 17 f 39 f 52

Intracellular accumulation of glucose (% equi1ibrium)b N.D. 37 f 5 17&4 18 f 3

Hearts were removed from heparinized rats and perfused by a modified Langendorff technique with Krebs-Henseleit bicarbonate buffer gassed with either OZ/CO, (95:5%) or Nz/COZ (95:5%) (Morgan et al., 1961a). Hearts were perfused initially for 10 minutes under aerobic conditions with sugar-free buffer. Following this period glucose uptake was measured over a recirculation period of 30 minutes. The initial glucose concentration was 16 mM. Accumulation of intracellular glucose was calculated from measurements of glucose and sorbitol spaces (Morgan et al., 1961a). Accumulation of sugar was calculated from the following formula:

% Equilibrium

=

100 X Intracellular sugar concentration Extracellular sugar concentration

Values represent mean plus or minus standard errors. N.D., none detected.

enzymes including hexokinase, phosphofructokinase, and phosphorylase. In muscle and yeast, however, stimulation of glucose phosphorylation can result in large sustained changes in glucose utilization only after transport has been stimulated. The relationship between transport rate and energy levels of the cell has been studied in the greatest detail in avian erythrocytes. These cells provide a simpler experimental model in which transport is stimulated by a shift from aerobic to anaerobic conditions (Wood and Morgan, 1969). They offer the following advantages for studies of the mechanism of transport regulation: (1) large quantities of cells can readily be obtained; (2) barriers to extracellular diffusion of sugar are less likely to exist since the cells were free; (3) composition of the cells can be changed by the technique of reversiblc hemolysis (Hoffman, 1958; Whittam, 1962; Sen and Post, 1964; Vidaver, 1964) ; (4) transport is sufficiently slow to allow the rate to be evaluated by measurements of the flux of radioactive sugars; and

264

HOWARD E. MORGAN AND CAROL F. WHITFIELD

TABLE I11

EFFECT OF ANOXIAAND INSULINON THE ENTRY OF GARABINOBE INTO PERFUSED RAT HEART" ~

~

Gas phase 02/CO1

O2/COz

Nz/COz Nz/COz

Insulin (0.04 fig/ml)

0

+ 0 +

Intracellular accumulation of L-arabinose ( % equilibrium)b 4 f 4 29 f 4 28 f 3 46 f 3

a Hearts were perfused as described in Table I1 for 10 minutes with buffer containing 13 mM Larabinose. Accumulation of sugar was calculated aa described in Table 11. b Values represent mean plus or minus standard error.

( 5 ) intracellular water volume was large as compared to the isolated fat cell

(Crofford and Renold, 1965a). This allows for much easier estimation of intracellular sugar concentrations. Carrier-mediated transport of glucose in avian cells was stimulated approximately threefold by anoxia or by a variety of inhibitors of oxidative metabolism (Fig. 2). In both aerobic and cyanide-treated suspensions, glucose was distributed in a volume slightly less than that of sorbitol. This indicates that the sugar was restricted to the extracellular space. Since the intracellular concentration of glucose remained undetectable, glucose utilization was limited principally by entry of glucose into the cell. Under these c,onditionsthe kinetics of glucose utilization approximated the kinetics of glucose entry. In both aerobic and cyanide-treated cells, the entry of glucose followed MichaelisMenten kinetics (Fig. 3). Incubation with cyanide increased the VmaXthreefold and doubled the K,. An increased maximal transport rate could result from de now synthesis of carrier, activation of existing carrier, or accelerated diffusion of carrier and sugar-carrier complex through the membrane. De novo synthesis of carrier appeared unlikely, but the other possibilities were not easily resolved and were compatible with several mechanisms, as will be discussed presently. The conclusion that stimulation of glucose uptake involves an acceleration of sugar transport was confirmed by studying the entry and exit of nonmetabolized sugars. As shown in Fig. 4, entry of 3-O-methylglucose was accelerated by incubation of avian erythrocytes in a nitrogen atmosphere.

265

SUGAR TRANSPORT IN EUKARYOTIC CELLS

A lag of approximately 40 minutes preceded the onset of transport stimulation. This lag was due to washout of oxygen from the incubation bottle, as well as the time required to obtain the anaerobic stimulation. Entry of 3-0-methylglucose continued to accelerate as incubation of the anaerobic cells was prolonged. Depletion of ATP and accumulation of intracellular inorganic phosphate began in concert with the acceleration of transport rate and progressed as time went on. In other experiments transfer of 3-0-methylglucose across the membrane was found to involve both carrier-mediated transport and simple diffusion (Wood and Morgan, 1969). Evidence for stimulation of the carrier-mediated component of sugar transfer in anaerobic cells consisted of retention of transport specificity as 51

w h 41

CYANIDE -TRE ATE D

1

MEDIUM GLUCOSE, mM

I FREE INTRACELLULAR GLUCOSE PRESENT

I

0

I

1

I

5 10 15 MEDIUM GLUCOSE, m M

I

20

FIQ.2. Glucose uptake by goose erythrocytes. Suspensions (approximately 40%) of washed goose erythrocytes were incubated at 37°C in stoppered flasks flushed with either oxygen or nitrogen. NaCN (1 mM) was added to the suspensions exposed t o nitrogen. After a preliminary incubation of 15-20 minutes, aerobic suspensions were sampled a t 2-hour intervals and cyanide-treated suspensions at +hour intervals for estimation of glucose and sorbitol-SH disappearance and distribution. Results are expressed per gram of dry cell mass. Solid circles, values obtained with aerobic suspensions; open circles, data of cyanide-treated suspensions. Glucose uptake and space are plotted with the mean medium glucose concentration for the uptake period. (From Wood and Morgan, 1969.)

266

HOWARD E. MORGAN AND CAROL F. WHITFIELD

AEROBIC , , ,V = 1.04 pmoles / hourlgm Km=0.35 mM

l2

../

t

Vmox 3.36 pmoles /

8

4

0

8 12 16 MEDIUM GLUCOSE, mM

4

20

FIQ.3. Kinetic analysis of glucose uptake by goose erythrocytes. The data of Fig. 2 were plotted by the method of Lineweaver and Burk (1934). Regression lines were fitted by the method of least squares.

I 8C

E

2 a

m

i 3

60

6

s

s

E

a: > 40

a a 4

I I

a

W

-1

v)

3

0

-1 -1 W

V 3

5>

u

20

a 2 a

I

I-

t-

E

W

7 0 A

f

I

.-

I

F

r

0

I

0

0

160 0 TOTAL PERIOD OF I N C U B A T I O N , M I N U T E S

80

80

160

FIG.4. Effect of incubation under aerobic and anaerobic conditions on 3-0-methylglucose transport and ATP and Pi levels in goose erythrocytes. Cells were suspended in buffer a t 10% hematocrit and incubated at 37" in 2-liter roller bottles equipped with perfusion caps. Bottles were gassed continuously with either water-saturated oxygen or nitrogen. Twenty minutes before the end of incubation, sufficient 3-0-methylglucoseJ4C was added to give an extracellular concentration of 12.5 pM. Samples were used for estimation of the intracellular concentration of 3-O-methylglucose, ATP, and Pi.

SUGAR TRANSPORT IN EUKARYOTIC CELLS

267

evidenced by a very slow rate of L-glucose entry and continued ability to demonstrate competition and countertransport. The diffusion component of 3-0-methylglucose transfer did not appear to be affected by anoxia (Wood and Morgan, 1969). Anaerobic stimulation of transport is a major factor in- the accelerated glucose uptake and generation of ATP in muscle and avian erythrocytes. The avian erythrocyte appeared to be the best system currently available for investigating the mechanism of the anaerobic stimulation of transport. B. Acceleration of Sugar Transport in Association with Muscular Contraction

Acceleration of transport in working muscle has been observed in whole animals, skeletal muscle preparations, and perfused hearts. Ingle et al. (1950) found that stimulation of the leg muscles of diabetic or normal rats caused a rapid fall in blood glucose levels. Goldstein et al. (1953) implicated transport in this effect by showing that exercise lowered the concentration of nonmetabolized sugars, such as D-xylose and L-arabinose, in the blood of eviscerated animals. Helmreich and Cori (1957) found that accumulation of pentose within muscles of nephrectomized rats was increased by exercise. These results were confirmed by Sacks and Smith (1958) and Dulin and Clark (1961). Effects of contraction of skeletal muscle on sugar transport have been studied in the greatest detail in frog muscle by Holloszy and Narahara (1965, 1967a, b). In this tissue the rate of transport of 3-0-methylglucose reached a higher plateau as the frequency of stimulation was raised from 3 to 120 shocks per minute. The rate of rise of transport rate was faster a t the higher frequency of stimulation, Once the change in transport had occurred in response to contraction, it persisted for several hours. The kinetic effect of contraction was to increase the maximal rate of transport without modifying the apparent affinity of the carrier for 3-0-methylglucose. The effects of a rapid rate of contraction and of insulin on transport were not additive. This suggests that both factors affected the same transport system. In frog skeletal muscle the effect of a submaximal rate of contraction on transport was not enhanced by application of load to the muscle. This result suggested that frequency of contraction rather than rate of ATP-splitting is more directly related to transport acceleration. When contracture of frog skeletal muscle was induced by either caffeine or high extracellular levels of potassium, an accelerated rate of transport of 3-0-mcthylglucosc was found (Holloszy and Narahara, 1967a). The rate of sugar transport during potassium contracture depended upon the level of Ca2+in the buffer. Since caffeine did not depolarizc the membrane, these

268

HOWARD E. MORGAN AND CAROL F. WHITFIELD

experiments suggested that membrane depolarization was not required for acceleration of transport rate. Both the effects of caffeine and the dependence of transport rate on Ca2+levels during potassium contracture suggested that higher levels of cytoplasmic Ca2+might be related to the acceleration of transport. This suggestion was reinforced by experiments in which sodium nitrate was substituted for sodium chloride in the buffer used for incubation of frog muscle (Holloszy and Narahara, 1967b), Entry of 3-O-methylglucose was accelerated in the experiments in which nitrate was present. The ability of nitrate to induce more forceful contractions was thought to involve restraint of uptake of Ca2+by the sarcoplasmic reticulum (Sandow, 1965). In contrast to frog skeletal muscle, acceleration of glucose transport in perfused rat heart depended upon the rate of ventricular pressure development rather than the rate of contraction (Fig. 5). I n these experiments frequency of contraction was not significantly different a t any of the left atrial filling pressures (Neely et al., 196713). When left atrial pressure was increased from 0 to 20 mm Hg, peak systolic pressure rose from 61 to 106 mm Hg, and oxygen consumption was doubled (Neely et al., 1967a). In association with the higher level of pressure development, glucose uptake increased as perfusate glucose concentration was raised. Under all these conditions glucose space was not greater than sorbitol space. This indicates that the intracellular concentrations of glucose remained below detectable levels. When intracellular glucose concentrations were a t these low levels, glucose uptake was a good indicator of inward glucose transport. Kinetic analysis of these data indicated that the maximal rate of transport increased from 213 to 599 pmoleslgm per hour as atrial pressure was increased from 5 to 20 mm Hg. The apparent affinity constant ranged from 3.1 to 1.8 mM. The conclusion that sugar transport was accelerated in hearts developing increased levels of ventricular pressure was confirmed by measuring the accumulation of L-arabinose, a nonmetabolized analog of glucose (Fig. 6). In the absence of insulin, increased ventricular pressure development markedly accelerated the entry of L-arabinose (Neely et al., 1967b). As also shown by Fig. 6, transport was more sensitive to stimulation by insulin in hearts developing higher levels of ventricular pressure. In the working preparation 0.1 milliunit of insulin per milliliter significantly accelerated transport, whereas this level of hormone had no effect in the LangendorfT preparation. A comparable increase in insulin sensitivity was also found in the Langendorff preparation perfused under anaerobic conditions (Morgan et al., 1965). The increase in transport rate in hearts developing higher levels of ventricular pressure, and the greater sensitivity to insulin, led t o accelerated uptake of glucose by contracting muscles in contrast to welloxygenated and quiescent tissues.

SUGAR TRANSPORT IN EUKARYOTIC CELLS

1 O

t

Z k o\

+ 1.0

-

269

FREE I NTRA CELLU LAR GLUCOSE PRESENT

FIG. 5 . Effect of left atrial pressure on glucose uptake, glucose space, and sorbitol space of working rat hearts. Hearts were perfused in the working heart apparatus (Morgan et al., 1965; Neely et al., 1967a) a t various left atrial pressures as indicated on the figure. A 10-minute period of perfusion took place with buffer containing glucose a t the concentrations t o be used in the subsequent period of recirculation (60minutes). Sorbitol was used as a marker of extracellular space. Nine to twenty-six hearts were perfused in each group. If standard errors of the mean (S.E.M.) are not shown, the values did not extend beyond the data points. (Data from Neely et al., 196713).

These findings indicated that sugar transport in heart and skeletal muscle was accelerated in association with increased contractile activity. I n skeletal muscle the acceleration of transport was related to the frequency of contraction, not to the work that was performed. I n heart muscle the increase in ventricular pressure development was closely related to

270

HOWARD E. MORGAN AND CAROL F. WHITFIELD

INSULIN

A D D E D , milliunits/rnl

FIG.6. Effect of heart work on transport of L-arabinose and on sensitivity of transport t o stimulation by insulin. Following a 10-minute preliminary perfusion with plain buffer, hearts were perfused for an additional 10 minutes with buffer containing 13 m M barabinose and insulin. One group of hearts was perfused as Langendod preparations with a perfusion pressure of 60 mm Hg; a second group was perfused as working preparations with a left atrial pressure of 10 mm Hg. (From Neely et al., 1967b.)

transport acceleration. Possible mechanisms of these effects are discussed later in this article. C. Effects of Fatty Substrates on Sugar Transport

Fatty acids represent the major fuel of the body and are used in preference to glucose by several tissues. In the fasting human oxidation of fatty acids accounted for 80% of the total fuel consumed (Cahill, 1971). Brain and erythrocytes depend largely upon glucose as fuel, while muscle, adipose tissue, and liver oxidize a variety of substrates. During fasting sparing of glucose consumption is vital, since the supply of substrate for glucose production is limited. Under these circumstances the oxidative substrate of muscle is almost entirely fatty acids and ketone bodies. Since fatty acids spare glucose utilization, key reactions in glycolysis must have been inhibited. The first evidence that fatty substrates can inhibit glycolysis was provided by Drury and Wick (1953), who found that acetate and p-hydroxy-

SUGAR TRANSPORT I N EUKARYOTIC CELLS

27 1

butyrate reduced production of radioactive carbon dioxide from glucose in eviscerated nephrectomized rabbits treated with insulin. They concluded that these substrates inhibit oxidation of glucose, despite the presence of insulin, and that this represents preferential oxidation of fatty substrates by muscle. Similaily, the heart appeared to utilize primarily fatty acids or ketone bodies (Bing et al., 1954; Bing, 1965). Williamson and Krebs (1961) and Hall (1961) showed that ketone bodies inhibited glucose uptake by perfused rat heart, while other investigators found that long- and shortchain fatty acids had a similar effect (Shipp et al., 1961; Bowman, 1962; Newsholme et al., 1962; Randle el al., 1964). Phosphorylation of glucose rather than membrane transport appeared to be the major step affected by these substrates, since it was necessary to stimulate transport with insulin for the inhibition to be observed. When the hormone was absent and transport was the major restriction on glucose utilization, it was not clear whether fatty acids or ketone bodies had any significant inhibitory effect. The first evidence implicating transport as a site of inhibition by fatty acids was the observation that entry of L-arabinose into cardiac muscle cells was inhibited by fatty acids and ketone bodies (Randle et aE., 1964). Studies (Neely et al., 1969) have defined the role of fatty substrates as inhibitors of glucose transport in isolated rat heart. As shown in Table IV, palmitate did not significantly affect glucose uptake by hearts developing 64 mm Hg peak intraventricular pressure. When uptake was accelerated, however, by raising ventricular pressure development in either the Langendorff or working preparation, fatty acid was able to inhibit uptake markedly. Since intracellular free glucose did not accumulate in hearts perfused with buffer containing palmitate, entry of glucose was the major restraint on glucose uptake. Phosphorylation was restricted by the availability of intracellular glucose. These findings indicated that fatty acid was a potent inhibitor of the stimulation of transport that accompanied increased pressure development. Palmitate concentrations in the physiological range (0.37-0.65 mM) were effective. Studies of the accumulation of 3-O-methylglucose confirmed the conclusion that fatty acids are inhibitors of sugar transport (Table V). A range of fatty substrates, including acetate, 0-hydroxybutyrate, and palmitate inhibited the accelerated rate of transport that accompanies increased ventricular pressure development, Palmitate had a small inhibitory effect a t a low level of ventricular pressure development. Oxidation of the fatty acid appeared to be required for inhibition, since palmitate was ineffective in reducing the accelerated rate (of transport ,seen1 in (anaerobichearts. I n fact, fatty acid accelerated transport in the anoxic heart. In addition to blocking the effect of increased ventricular pressure development on transport, palmitate reduced the stimulatory effect of

272

HOWARD

E.

MORGAN AND CAROL F. WHITFIELD

TABLE IV

EFFECT OF PALMITATE ON GLUCOSE UPTAKEBY PERFUSED RAT HEART" Peak intraventricular pressure (mm Hg)

Average perfusate palmitate (mM)

Glucose uptake (pmoles/gm/hour)*

Intracellular glucose (mM)C

Langendorff preparation 64 64 106 106 106 106

f 14 f 10 f8

N.D. N.D. N.D.

f 14 f 15 f7

N.D. N.D.

0.09 1.25 0.04 0.37 0.65 1.19

82 58 232 124 66 35

0.1 1.4

351 f 30 113 f 12

-

Working preparation 110 110

Hearts were perfused for 75 minutes with buffer containing 13 mM glucose and 3% bovine serum albumin. Intracellular glucose was calculated from measurements of glucose and sorbitol space. Tissue weights are expressed per gram of dry tissue. (Data from Neely et al., 1969.) *Values represent the mean of 6 to 12 determinations plus or minus standard error. N.D., not detected.

insulin (Table V). Since a submaximal concentration of the hormone was employed, the fatty acid effect represented a reduction in insulin sensitivity (Randle et al., 1964). These studies of glucose and 3-O-methylglucose transport in isolated rat heart indicated that the stimulatory effects of insulin and ventricular pressure development on transport were inhibited by fatty substrates. The basal rate of transport was affected to a lesser degree. Oxidation of fatty acid was required for the effect. Although oxidation of fatty substrates by skeletal muscle has been well established (Fritz, 1961 ; Drummond, 1969; Ruderman et al., 1971), these substrates inhibit glucose transport either to a small extent or not a t all (Randle et al., 1964; Schonfeld and Kipnis, 1968). I n other studies glucose utilization by skeletal muscle was found to be unaffected by addition of either ketone bodies or fatty acids (Houghton and Ruderman, 1971; Beatty and Bocek, 1971; Jefferson et al., 1972). However, the following considerations suggest that additional studies are required before fatty acids are

273

SUGAR TRANSPORT IN EUKARYOTIC CELLS

eliminated as an important factor in controlling glucose transport in skeletal muscle.

1. In resting muscle glucose oxidation is minimal (Ruderman et al., 1971). Oxidation of ketone bodies accounts for 70-900/, of oxygen consumption. I n this case transport may have been inhibited markedly in the control muscles, so that addition of fatty acids could have only a small effect. 2. Fatty acid inhibition of transport in the perfused heart involves TABLE V

EFFECT O F FATTY SUBSTRATES ON TRANSPORT O F 3-O-METHYLGLUCOSE I N ISOLATED HEARTPERFUSED BY THE LANGENDORFF PREPARATION'

Additions to the perfusate

Perfusion pressure (mm Hg)

3-0-Methylglucose transport (% intracellular HzO equilibrated)b

60 60 100 100 100 100 60 60

17 f 2 11 f 2 64 f 3 21 f 2 16 f 3 11 f 2 76 f 1 53 f 3

60 60

59 f 4 82 f 2

Aerobic perfusions None Palmitate, 1.5 mM None Palmitate, 1.5 mM 6-Hydroxybutyrate, 5 mM Acetate, 5 m M Insulin, 0.3 mU/ml Insulin palmitate, 1.5 mM

+

Anaerobic perfusions None Palmitate, 1.5 mM

a After a preliminary perfusion of 10 minutes with plain buffer, recirculation of buffer gassed with either OZ/COZor Nz/COZ (95:5%) was begun. The perfusate contained 394 bovine serum albumin, 1 mM sorbitol- and the additions noted in the table. After 10 minutes of recirculation, a solution of 3-O-methylgluco~e-1~C was added to the perfusate to give a final concentration of 0.7 mM. After 7 minutes of perfusion with the sugar, hearts were analyzed for their content of intracellular 3-0-methylglucose. Hearts perfused by the Langendorff technique developed intraventricular pressures averaging about 5 mm Hg greater than the perfusion pressure (Neely et al., 1967a). (Data from Neely et al., 1969.) * Values represent the mean of 6 to 12 determinations plus or minus standard error.

274

HOWARD E. MORGAN AND CAROL F. WHITFIELD

restraint of the increase in transport resulting from increased mechanical activity or addition of insulin. Effects of fatty acids on sugar transport in skeletal muscle have not been investigated under these conditions. 3. Since many of the preparations that have been used were not perfused, the fatty acid-albumin complex may have been inaccessible to many of the cells in the preparation. As noted a t the beginning of this section, preferential utilization of fatty substrates by skeletal muscle is required for survival during fasting. The mechanism of this preference remains to be defined. 111. HORMONAL CONTROL

OF SUGAR TRANSPORT

Transport has been found to be regulated by several hormones including insulin, epinephrine, growth hormone, and corticosteroids. In the case of insulin, epinephrine, and growth hormone, increased activity of carrier already present within the membrane has been achieved by the hormone. However, effects of corticosteroids appear to involve induction of a n inhibitor of transport. A. Insulin

An effect of insulin on the passage of sugar through the cell membrane was first proposed by Lundsgaard (1939). This proposal was based on recognition of membrane penetration as a rate-limiting step for glucose uptake in perfused hindlimbs. The rate of transport appeared to restrict phosphorylation of glucose in heart and skeletal muscle (Lundsgaard, 1939; Park et al., 1955; Morgan et aZ., 1961a) and adipose tissue (Crofford and Renold, 1965a). An example of the effect of insulin on glucose uptake and intracellular glucose concentration in perfused rat heart is shown in Fig. 7. In control hearts intracellular glucose concentrations remained near zero over a large range of perfusate glucose concentrations. This indicates that transport restricted the rate of glucose uptake. Uptake increased as perfusate glucose concentration was raised but reached a plateau a t higher concentrations. Insulin increased glucose uptake (Bleehan and Fisher, 1954) and led t o accumulation of free intracellular glucosc. These findings indicated that insulin had stimulated the rate of sugar transport and had shifted the major restraint of glycolysis to glucose phosphorylation. The conclusion that insulin accelerates glucose transport was confirmed by demonstrating that the hormone hastened the entry of nonmetabolized sugars into the cell. Initially, Levine and co-workers (Levine et aZ., 1949, 1950; Goldstein et al., 1953) found that insulin increased the distribution of galactose in eviscerated nephrectomized dogs. Subsequently, the hormone

275

SUGAR TRANSPORT IN EUKARYOTIC CELLS ~

GLUCOSE UPTAKE

500

INTRACELLULAR

GLUCOSE

24 0

s

I

E 12 I

.,

9

I

0

m

I

-. Y l

-.,

12 PERFUSATE

.

CONTROL

I

24

36

GLUCOSE. mM

FIG.7. Effect of insulin on glucose uptake and intracellular glucose concentration in the perfused rat heart. Results are expressed per gram of dry heart. X, Intracellular glucose not detected. (From Morgan el al., 1961a.)

was found to accelerate entry of nonmetabolized sugar into heart (Tables I11 and V), skeletal muscle (Bleehan and Fisher, 1954; Park and Johnson, 1955; Fisher and Lindsay, 1956; Park et al., 1956; Helmreich and Cori, 1957; Kipnis and Cori, 1957; Randle and Smith, 1958b; Morgan et al., 1959b; Narahara and Ozand, 1963; Morgan et al., 1964; Bihler et al., 1965a, b; Fisher and Gilbert, 1970), and adipose tissue (Crofford and Renold, 1965b). In addition, the hormone was found to accelerate efflux of a nonmetabolized pentose from cardiac muscle (Fig. 8). Transport of pentose was inhibited in hearts of alloxan-diabetic animals that were insulindeficient, but a high level of insulin added to the perfusate induced the same rate of transport as in normal hearts.

276

HOWARD E. MORGAN AND CAROL F. WHITFIELD

NO I N S U L I N 0I N S U L I N I X 10'' U N I T S / M L .

INTRACELLULAR

L-

*---

ARAB1 NOSE

MG % FIQ.8. Effect of insulin and diabetes on the efflux of tarabinose from cardiac muscle. The hearts were first perfused with a high concentration of L-arabinose in order to accumulate a large amount within the cell. The perfusate was then changed to sugar-free buffer which passed through the heart a single time and was collected over successive intervals of 1 or 2 minutes. Perfusate appearing during the first 5 minutes was discarded, since this period was required to remove the extracellular sugar present at the beginning of the wash-through perfusion. (From Morgan et al., 1961b.)

Effects of insulin on kinetic parameters of transport have been investigated in several tissues. In perfused heart the hormone increased the maximal rate of transport and decreased the affinity somewhat (Post et al., 1961; Morgan et al., 1964; Bihler et al., 1965a; Fisher and Gilbert, 1970). An increase in the maximal rate was also observed in skeletal muscle and isolated fat cells (Narahara and Ozand, 1963; Letarte and Renold, 1969b). The biochemical mechanisms accounting for these changes are unknown, but an increase in the maximal rate of transport suggested that movement of carrier across the membrane was facilitated by insulin. Since the effect

SUGAR TRANSPORT IN EUKARYOTIC CELLS

277

occurred within 2 or 3 minutes (Morgan et al., 1961b), de novo synthesis of carrier appears unlikely. Insulin effects on the plasma membrane of adipocytes has been demonstrated in “ghosts” (Rodbell, 1967; Illiano and Cuatrecasas, 1971) and in microsomal particles (Carter and Martin, 1969; Martin and Carter, 1970). Entry and exit of glucose in the ghost preparation has been shown to be a carrier-mediated process by demonstration of saturation kinetics, stereospecificity, and counterflow (Illiano and Cuatrecasas, 1971). Insulin has been observed to increase the rate of transport in the ghost preparation but to have no effect on the microsomes. The rate of uptake by microsomes prepared from insulin-treated fat pads was more rapid than in microsomes from control tissue (Martin and Carter, 1970). These findings indicated that the insulin effect can persist in the membrane during the fractionation procedure. The findings of Cuatrecasas (1969) that insulin bound to agarose beads increased glucose utilization and inhibited lipolysis in fat cells also indicated that the hormone has only to interact with the membrane in order to affect transport. The agarose particle was larger than the fat cell and so restricted insulin to a surface action. Other workers have shown that insulin-agarose stimulates RNA synthesis (Turkington, 1970), accumulation of a-aminoisobutyric acid in isolated mammary cells (Oka and Topper, 1971), and that it activates glycogen synthetase in liver (Blatt, and Kim, 1971). The surface action of insulin suggested that the hormone binds to a “receptor” in the membrane. Cuatrecasas (1972) has suggested that the receptor has two functions. The first function is to recognize insulin, while the second is to convert this recognition to a more rapid rate of glucose transport. The recognition function has been intensively studied in membranes of fat and liver cells (Cuatrecasas, 1971a, b ; Freychet et al., 1971, 1972; Cuatrecasas et al., 1971). A reduced number of receptors has been found in liver membranes of obese-hyperglycemic mice (Kahn et al., 1972). This suggests that a deficiency of receptor sites may be a n important component in the etiology of some forms of diabetes. No alteration in the binding of insulin by isolated fat cells of rats treated with prednisone or streptozotocin, or of starved rats has been found (Bennett and Cuatrecasas, 1972). The suggestion has been made that binding of insulin to the membrane involved formation of disulfide bonds between the hormone and receptor. This suggestion arose from experiments in which sulfhydryl-blocking agents were found to inhibit the insulin effect (Cadenas et al., 1961; Fong et al., 1962; Edelman et al., 1963; Whitney et al., 1963). However, Wohltmann and Narahara (1968) were unable to relate the quantity of radioactive insulin that was covalently bound through disulfide bridges with the hormone

278

HOWARD E. MORGAN AND CAROL F. WHITFIELD

effect on transport. Recently, Cuatrecasas (1971b) found that insulinbinding by fat cell membranes was not impaired by addition of sulfhydrylblocking agents. Additional experiments are needed to identify the sulfhydryl-dependent steps involved in translating insulin-binding into accelerated sugar transport. The experiments of Wohltmann and Narahara (1968) and of Cuatrecasas (1971b) suggested that the binding step is not the step that is involved. Modification of the insulin effect has been achieved by digestion of muscle and adipose tissue with proteases and phospholipases. Treatment of adipose tissue or muscle either with low concentrations of trypsin, or with higher concentrations for short periods of time, resulted in an accelerated rate of glucose transport (Kuo, 1968; Kuo et al., 1966a, b, 1967; Weis and Narahara, 1969; Kono, 1969b; Kono and Barham, 1971a). The mechanism of this acceleration is not understood. Attempts to induce an accelerated transport rate with inactive derivatives of trypsin were not successful (Cuatrecasas, 1971~). Treatment of fat cells with trypsin as outlined above resulted in loss of insulin binding. Cuatrecasas (1971~)found that the affinity of the receptor for insulin was reduced, while Kono and Barham (1971b) reported a decreased number of binding sites. More extensive digestion of the membrane with trypsin resulted in a lower basal rate of glucose uptake and a loss of the insulin effect on transport (Fain and Hoken, 1969; Kono, 1969a; Kono and Barham, 1971a; Cuatrecasas, 1971~).Contrary to the initial report of Kono (1969b), the insulin “receptor” was not regenerated upon further incubation of cells in medium containing trypsin inhibitor (Kono and Barham, 1971b; Cuatrecasas, 1971~).Addition of insulin prior to trypsin treatment protected the receptor from proteolytic attack (Czech and Fain, 1970; Kono and Barham, 1971a). Since treatment of cells with trypsin released sialopeptides, effects of neuraminidase were also explored. Gentle digestion with this enzyme accelerated glucose transport in white and brown adipose tissue (Cuatrecasas and Illiano, 1971; Rosenthal and Fain, 1971). More intensive digestion reduced the basal rate of glucose uptake and abolished the insulin effect. In contrast to trypsin treatment, however, neuraminidase treatment left insulin binding unimpaired (Cuatrecasas and Illiano, 1971). Digestion of fat cells with phospholipases C or A accelerated glucose transport (Blecher, 1965, 1966, 1967, 1969; Rodbell, 1966; Rodbell and Jones, 1966; Rosenthal and Fain, 1971; Cuatrecasas, 1971d). The number of insulin binding sites was also increased by phospholipase treatment, but the affinity for the hormone was unchanged (Cuatrecasas, 1971d). However, digestion with phospholipase increased the sensitivity of the receptor to destruction by trypsin (Cuatrecasas, 1971~).

SUGAR TRANSPORT IN EUKARYOTIC CELLS

279

Studies with hydrolytic enzymes indicated that the insulin “receptor” was accessible from the outside of the mcrnbrane but that some insulin binding sites were masked. These sites were exposed by treatment with phospholipase. The effect of neuraminidase treatment indicated that insulin binding was not the only prerequisite for insulin stimulation of transport. Studies of the insulin stimulation of glucose uptake in adipose tissue, heart, and skeletal muscle have demonstrated that the hormone is a major factor affecting carrier-mediated transport of sugar through the membrane. The rate of carrier movement is increased, but not the affinity of carrier for sugar. Binding of hormone to the cell surface is necessary for the effect, but only a small percentage of the total available binding sites have to be occupied by the hormone before transport is accelerated.

B.

Epinephrine

Early work on the action of epinephrine indicated that the hormone increases glucose utilization by heart muscle. Patterson and Starling (1913) and Bogue and co-workers (1935) reported that epinephrine stimulated glucose uptake in dog hearts. Since the latter group also found that increased work of the heart stimulated glucose utilization, work load was maintained a t a constant level in experiments with epinephrine. Even so, the data could not be interpreted as a direct effect of epinephrine on glucose metabolism, since the heart rate was also increased. Williamson (1964) concluded that, in isolated perfused rat heart, increased oxidation of glucose is a direct result of a change in mechanical activity. As discussed earlier, Morgan et al. (1965) found that the rate of ventricular pressure development was an important factor regulating transport of glucose and 3-0-methylglucose. In a study of the effects of epinephrine in nonbeating hearts, Challoner (1970) inhibited contraction by addition of potassium and measured the entry of 3-0-methylglucose. Stimulation of transport by epinephrine was not found, but high potassium concentrations (16 and 31 mM) accelerated 3-0-methylglucose entry and may have obscured an effect of the hormone. In contrast to the stimulatory effects of epinephrine on glucose utilization in heart muscle, the hormone has been found either to stimulate or inhibit glucose uptake in skeletal muscle. Addition of epinephrine inhibited glucose uptake by the isolated diaphragm but stimulated uptake when injected in vivo (Walaas and Walaas, 1950). Epinephrine also prevented the stimulation of glucose uptake by insulin (Groen et al., 1958). Walaas and Walaas (1950) suggested that glucose 6-phosphate1 which increased as a result of the stimulation of glycogenolysis by epinephrine, is a n inhibitor of

280

HOWARD E. MORGAN AND CAROL F WHITFIELD

glucose uptake. This inhibition has been ascribed to restraint of the hexokinase reaction (Sols and Crane, 1954; Kipnis et al., 1959). Although epinephrine either stimulates or inhibits overall glucose utilization in skeletal muscle, the hormone consistently accelerates the rate of sugar transport. Newsholme and Randle (1961) found that epinephrine accelerated entry of 3-O-methylglucose into diaphragm muscle but had no effect on the transport of a nonmetabolized pentose, D-xylose. Involvement of the adrenergic receptor system in the effects of epinephrine on transport has been suggested by the observation that phenoxybenzamine, an a-adrenergic blocker, stimulated 3-O-methylglucose entry markedly in rat M ) was diaphragms (Ilse, 1971). In other experiments cyclic AMP found to stimulate glucose uptake, but to a lesser extent than insulin (Edelman and Schwartz, 1966). A stimulatory effect of epinephrine on sugar transport also has been obtained in frog sartorius muscle (Wohltmann et al., 1967; Saha et al., 1968). These workers found that entry of 3-O-methylglucose into resting muscle was increased by addition of epinephrine in concentrations of 10-7-10-6 M . After a lag period of 30 minutes, both influx and efflux were accelerated. When epinephrine was added after maximal stimulation of transport by insulin, sugar efflux decreased rather than increased. Inhibition of 3-0methylglucose efflux may have resulted from accumulation of free glucose that arose from acceleration of amylo-1 ,6-glucosidase following activation of phosphorylase by epinephrine. Therefore caution must be exercised in interpreting the stimulatory effects of epinephrine on 3-O-methylglucose entry, since an increase in intracellular free glucose might counterflow 3-O-methylglucose into the cells. The stimulatory effect of epinephrine on transport was completely inhibited by pronethalol, a 0-adrenergic blocking M cyclic AMP. Since agent, but in this case was not reproduced by both glucose-6-phosphate levels and sugar transport increased following addition of epinephrine to both diaphragm and sartorius muscle, this intermediate did not appear to be a likely inhibitor of glucose transport (Newsholme and Randle, 1961; Saha et al., 1968). Adipose tissue has been found to be very sensitive to epinephrine, but the large effect of the hormone on lipolysis has interfered with studies of its direct effects on transport. Cahill et al. (1960) found that glucose uptake was increased by epinephrine. Isoproterenol had the same effect (Love et al., 1963), and a blocking agent, 1-(2’-4’-dichlorophenyl)-2-t-butylaminoethanol, inhibited transport. Bray and Goodman (1968) attempted to dissociate the effects of epinephrine on glucose oxidation from effects on lipolysis and free fatty acid release. These workers found that propanolol, a 0-blocker, inhibited the effects of epinephrine on lipolysis and free fatty acid release but did not inhibit the hormone effect on glucose oxidation. An effect of

281

SUGAR TRANSPORT IN EUKARYOTIC CELLS

epinephrine on sugar transport was established by the finding that entry of L-arabinose, a nonmetabolized pentose, was accelerated. Dibutyryl cyclic AMP in low concentrations (0.3 mM) did not stimulate glucose uptake but did modify the fate of the glucose that was consumed. This result suggested to Bray and Goodman the possibility that the epinephrine effect on sugar transport is secondary to st,imulation of glycolysis by cyclic AMP. In other experiments, Clausen (1969) found a stimulatory effect of epinephrine on release of 3-O-methylglucose from epididymal adipose tissue. Epinephrine was also found to stimulate pinocytosis in adipose tissue (Cushman, 1970). Whether this effect occurs a t physiological concentrations of the hormone, or whether it is likely to be the basis for stimulation of carrier-mediated transport, has not been resolved. However, pinocytosis does not appear to provide the type of specificity demonstrated by sugar transport. Recently, epinephrine was found in our laboratory to accelerate the entry of 3-O-methylglucose into avian erythrocytes (Fig. 9). Since these cells do not contain a significant store of either glycogen or fat, they offer advantages to study the effects of epinephrine on transport. Pigeon red cells were reported to have an epinephrine-sensitive adenyl cyclase, and the hormone has been found to increase cyclic-AMP levels both in cells and incubation medium (Davoren and Sutherland, 1963; e)ye and Sutherland, 1966; Rosen et al., 1970~).The magnitude of the epinephrine effect on

-

'0

10 20 MINUTES

30

lap

I---

'0

0.4

0.0

EPINEPHRINE, m M

FIG.9. Effect of epinephrine on 3-O-methylglucose entry in goose erythrocytes. On the left, cells were preincubated for 60 minutes with 0.1 mM epinephrine. At 60 minutes 3-O-methylglucose was added, and entry measured over a period of 30 minutes. On the right, cells were preincubated for 60 minutes with various concentrations of epinephrine, and the entry of 3-O-methylglucose was measured after 30 minutes of incubation.

282

HOWARD E. MORGAN AND CAROL F. WHITFIELD

3-0-methylglucose transport in goose erythrocytes is both time- and dosedependent. When the cells were preincubated for 60 minutes with the hormone, the minimum effective concentration was 5 X 10-6M. This concentration elicited a maximal production of cyclic AMP in pigeon erythrocytes (Davoren and Sutherland, 1963). The stimulation of sugar entry is not blocked by propanolol and is not reproduced by addition of dibutyryl cyclic-AMP. Preliminary experiments have indicated that the stimulation of transport by epinephrine is accompanied by a small loss of ATP. The work that has been discussed indicates that epinephrine stimulates sugar transport in frog sartorius muscle and avian erythrocytes under conditions likely to result in some depletion of high-energy phosphates. In heart muscle much of the hormone effect may be secondary to changes in mechanical activity. In adipose tissue stimulation of sugar transport has been found following addition of epinephrine, but it has not been established whether or not this stimulation is accompanied by depletion of ATP, which could result from an increased rate of reesterification of fatty acids. C. Glucocorticoids

Glucocorticoids have inhibitory effects on glucose utilization in skin (Overell et al., 1960), adipose tissue (Munck, 1962; Yorke, 1967), tumor cells (Rosen et al., 1970a; Plagemann and Renner, 1972), diaphragm (Manchester et al., 1959), and thymocytes (Morita and Munck, 1964; Kattwinkel and Munck, 1966;Munck, 1968).Glucose utilization in skeletal muscle does not seem to be affected by cortisol (Munck and Koritz, 1962). The effects on glucose transport and metabolism in thymocytes have recently been reviewed (Munck, 1971; Munck et al., 1971). Glucose uptake in thymocytes was thought to be limited by transport through the membrane, since the rate of phosphorylation by hexokinase was greater than needed for the rates of glucose uptake normally seen in these cells. In addition, free glucose did not accumulate, even at high external sugar concentrations (Munck, 1971). In adipose tissue transport also appeared to be rate-limiting (Crofford and Renold, 1965a;Yorke, 1967). Cortisol and dexamethasone, whether administered in vivo or in vitro, decrease glucose uptake. In thymocytes the decreased uptake is accompanied by decreased levels of glucose 6-phosphate and decreased lactate output, without a change in levels of ATP (Munck, 1968). An inhibitory effect of glucocorticoids on sugar transport in adipose tissue is supported by the finding of lower levels of glycogen, glucose 6-phosphate, and glycerol 3-phosphate, along with a decreased output of lactate and

SUGAR TRANSPORT IN EUKARYOTIC CELLS

283

pyruvate. Levels of ATP, ADP, AMP, and citrate are unchanged and, in spite of some increase in free fatty acid release, there is no change in glycerol release (Yorke, 1967). The inhibition of uptake is linear when M (Kattwinkel and glucocorticoid concentrations ranged from lo-’ to Munck, 1966). The above findings suggested that decreased glucose uptake is a result of decreased transport (Munck, 1968). This conclusion was strengthened by the observation that cortisol also decreased 3-O-methylglucose or 2-deoxyglucose uptake (Rosen et al., 1970b; Makman et al., 1971; Munck et al., 1971). However, 3-O-methylglucose efflux from preloaded cells was not inhibited by cortisol. In adipose tissue dexamethasone decreased the VmaXof glucose uptake but did not change the K , (Yorke, 1967). Comparable results with a nonmetabolized sugar, xylose, could not be obtained because of the small amount of intracellular water available for equilibration with sugar. Although cortisol inhibited the basal rate of glucose uptake, the steroid did not interfere with the stimulation of uptake by anoxia in thymocytes or by insulin in adipose tissue. The lack of a steroid effect during anoxia was considered to be due to a lack of specific glucocorticoid binding which required ATP (Munck, 1968). To explain the lack of effect after addition of insulin, Yorke (1967) suggested that the glucocorticoid-sensitive mechanism responsible for basal glucose uptake is not the same as the transport mechanism stimulated by insulin. Munck (1971) concluded that the inhibition of glucose transport by cortisol (10-7-10-6 M ) was noncompetitive, since very high glucose concentrations did not prevent either the decrease in glucose uptake or the fall in glucose 6-phosphate levels. However, Plagemann and Renner (1972) reported kinetic studies on rat hepatoma cells which showed that prednisolone was a competitive inhibitor of glucose and 2-deoxy-~-glucose transport. However, these studies were made with prednisolone concentrations (0&5 mM) which were approximately equimolar with the sugar concentrations, and so the studies cannot be compared with the results from thymocytes. In addition, steroid effects on tumor cells were seen in 5 minutes, while a 20-minute lag was required for an effect on thymocytes. The inhibitory effect of cortisol on sugar transport may involve a n inducible protein with a short half-life. Makman et al. (1971) found t h a t the cortisol effect was inhibited if actinomycin D was added either before or with the hormone, but not if added afterward. Cycloheximide, however, inhibited the cortisol effect on transport when added either with the hormone or 60 minutes later, when the hormone effect was well developed. These data led to the suggestion that RNA synthesis was necessary for cortisol to induce a protein inhibitor of sugar transport. The ability of

2 84

HOWARD E. MORGAN AND CAROL F. WHITFIELD

cycloheximide to reverse the steroid effect was explained by suggesting that the inhibitor has a rapid turnover rate. Specific binding of cortisol to cytoplasmic and nuclear receptors was complete after 7 minutes. Since the first measurable effects on transport were seen after 15-20 minutes, there was sufficient time for protein synthesis to occur before transport was altered. Rosen et al. (1972) found a different result using tumor cells of thymus. They reported that cortisol-induced inhibition of 2-deoxyglucose uptake by lymphosarcoma cells preceded an effect on decreased incorporation of substrates into nucleic acid and protein. In addition, they found that cycloheximide and actinomycin D both depressed sugar uptake in the absence of cortisol. The above evidence indicates that glucose uptake is inhibited by the addition of cortisol. A major component of this effect appears to be a reduction in the rate of sugar transport. Restraint a t this step may involve induction of an inhibitory protein with a short half-life. Additional studies are required to identify this protein and to define the mechanism of its interaction with the transport system. D. Growth Hormone

Growth hormone has been shown to have a biphasic effect on membrane transport of glucose in muscle and adipose tissue (Krahl and Park, 1948; Park et al., 1952; Henderson et al., 1961; Goodman, 1967, 1968; Hjalmarson and Ahren, 1967a, b ; Hjalmarson, 1968a; Dawson and Beck, 1968). When growth hormone is injected into hypophysectomized animals, a hypoglycemic reaction results which is similar to the reaction seen following a small dose of insulin. Hypoglycemia is less marked when the hormone is injected into normal animals. After repeated injections of the hormone into either hypophysectomized or normal animals, hyperglycemia occurs which varies in severity depending upon the species (Cotes et al., 1949; Houssay and Anderson, 1951; Krahl, 1951). The latter effect was called the diabetogenic effect of growth hormone. This effect is potentiated by simultaneous injection of glucocorticoids and is characterized by impaired sensitivity of transport to stimulation by insulin and by a block in glucose phosphorylation (Morgan et al., 1959b, 1961c; Kipnis, 1959; Kipnis and Cori, 1960; Henderson et al., 1961). These effects have been explored by addition of growth hormone to in vitro preparations of muscle or adipose tissue, or by incubation of tissues from animals injected with the hormone. Effects of the hormone are variable, and depend upon the period of exposure of the tissue and the hormonal balance of the animal from which the tissue is obtained.

285

SUGAR TRANSPORT IN EUKARYOTIC CELLS

Since the diabetogenic effect of growth hormone appears to be of greater physiological importance than the hypoglycemic effect, initial studies with in vitro muscle preparations have attempted to reproduce the diabetogenic effect. However, addition of growth hormone, in vitro, to skeletal (Park et al., 1952; Manchester et al., 1959) or heart muscle (Henderson et al., 196l), or incubation of these tissues from animals that had recently been injected with growth hormone, resulted in acceleration of sugar transport. Representative data showing these effects in perfused heart are presented in Table VI. Injection of growth hormone 1 hour prior to death or addition to the perfusate accelerated entry of L-arabinose into hearts of hypophysectomized rats. Injection of the hormone accelerated transport in TABLE VI

EFFECT OF GROWTH HORMONE A N D CORTISONE ON TRANSPORT OF ~ARABINOSE AND O N THE SENSITIVITY OF TRANSPORT TO STIMULATION BY INSULIN IN THE ISOLATED HEARTOF HYPOPHYSECTOMIZED AND NORMAL RATS'

Heart donor rats Hypophysectomized Normal

Hypophysectomized

Normal

).

Treatment None GH, in vitro GH, in vivo, 1 hour None GH, in vitro GH, in vivo, 1 hour None None GH, in vivo, 4 days Cortisone, in vivo, 4 days GH plus cortisone, in vivo, 4 days None None

Intracellular LArabinose Larabinose Insulin space (% equilibrium (milliunits/ml) (ml/gm) concentration)

0 0 0

506 f 13 568 f 20b 580 f 20b 494 f 13 453 f 14 560 f 15b

45 86 67 41 29 61

0 0.1 0.1 0.1

509 713 659 664

i26 f 10 f lgb f 16*

45 103 88 89

0.1

603 f 12b

72

0 0.1

540 f 12 582 f 10

56 62

0 0

0

hearts from 18-hour fasted rats were perfused for 10 minutes at 37" with buffer containing 13 m M Larabinose. Intracellular Larabinose concentration, expressed as a percentage of the perfusate concentration, was calculated as described in Table 11. Growth hormone (GH) and cortisone were injected in a dosage of 0.1 mg per 100 gm and 2.5 mg per 100 gm of rat weight, respectively. Growth hormone was added to the perfusate in a concentration of 6 mg/ml. (From Henderson et al., 1961.) b Significantly different from control.

2 86

HOWARD E. MORGAN AND CAROL F. WHITFIELD

normal hearts, but addition to the perfusate was ineffective. The early stimulatory effect of growth hormone on glucose transport appeared to be due to a direct effect of the protein rather than to release of tissue-bound insulin (Ottaway, 1953a, b). This is so since the effect was observed in hypophysectomized diabetic rats (Henderson et al., 1961))depancreatized, hypophysectomized dogs (Kurtz el al., 1950), and hyFophysectomized, eviscerated rats (Park et al., 1952). Rapid inhibitory effects of growth hormone on sugar transport in heart muscle (Bronk and Fisher, 1957) and diaphragm (Ottaway and Bulbrook, 1955; Bolodia and Young, 1967) have been reported. However, these effects could not be reproduced by other investigators (Randle, 1954; Henderson et al., 1961; Ahren et al., 1970). As a result, the majority of the evidence indicates that the hormone does not have an immediate inhibitory effect on sugar transport. Inhibitory effects of growth hormone on sugar transport were obtained by prolonging the interval between injection of the hormone into hypophysectomized rats and removal of the tissue for in vitro perfusion, or by prolonging the period of exposure in vitro. As seen in Table VI, injection of growth hormone and particularly a combination of growth hormone and cortisone into hypophysectomized rats returned the increased insulin sensitivity of transport toward normal. This level of insulin had no effect on arabinose transport in hearts of normal rats. These observations fit well with a decrease in insulin sensitivity in the whole animal following growth hormone injection (deBodo et al., 1950). More recently, Hjalmarson (1968b) found that the increased insulin sensitivity of muscle from hypophysectomized rats was reduced in vitro by pretreatment of diaphragm muscle with a combination of growth hormone and dexamethazone. Pretreatment with these hormones inhibits both the basal and insulin-stimulated rates of xylose transport. Inclusion of inhibitors of protein synthesis in the incubation medium blocks the effect of pretreatment with growth hormone and cortisone. This indicates that restoration of normal insulin sensitivity involves synthesis of new protein. The action of growth hormone on transport may involve binding of the hormone to the cell membrane. Sonenberg (1969, 1971) found that human growth hormone induced a change in the ellipticity of human erythrocyte membranes and that the membranes altered the near-ultraviolet spectrum of human growth hormone. In addition, the hormone modified the intrinsic fluorescence of erythrocyte membranes. Oski et al. (1967) found that human growth hormone inhibited glucose uptake of human red cells by blocking a step in glycolysis at or beyond the phosphofructokinase reaction. This effect was characterized by accumulation of glucose 6-phosphate and fructose 6-phosphate (Ludescher and Hohenwallner, 1969; Hohenwallner and Ludescher, 1970). No evidence has been presented to indicate that

SUGAR TRANSPORT IN EUKARYOTIC CELLS

287

growth hormone interferes with glucose transport in human erythrocytes. Bornstein et al. (1969) reported fractionation of acid-treated growth hormone into two components; one fraction inhibited glucose uptake in diaphragm and soleus muscle, while the other reversed this effect. Inhibition of glyceraldehyde-3-phosphate dehydrogenase was suggested as the major factor accounting for the reduction in glucose uptake. Additional studies are needed to determine whether binding of growth hormone to membranes precedes the effects on transport and insulin sensitivity in muscle, and to describe the action of growth hormone moieties on these effects. IV. MECHANISMS OF THE REGULATION OF TRANSPORT A. The Role of Ions in Transport Regulation

Discovery of the cotransport of sodium and glucose in the intestine has led to a search for similar ion dependencies in tissues possessing passive carrier-mediated transport. Experiments have utilized isolated diaphragm or heart muscle, isolated adipose tissue, and avian erythrocytes as test systems. The ability of muscle and adipose tissue to take up glucose or rionmetabolizable glucose analogs has been reported to be altered in the absence of sodium or potassium, or in the absence of a functioning sodium pump. However, evidence directly linking the effects of ions to a change in carrier-mediated transport is slim. Relationships between availability of ions and sugar entry into cells have been evaluated in the basal state and also when sugar entry was increased, such as during anoxia or in the presence of insulin. A general conclusion that can be reached from earlier work, and from recent experiments using avian red cells, is that the presence of sodium ions in the surrounding fluid is not essential for the entry of glucose into muscle, adipose tissue, or avian red cells. Accelerated uptake of glucose with variations in ionic composition has been reported in several tissues. In rat hearts perfused with ouabain a t concentrations below 10-6 M , glucose uptake increased approximately 30% (Kreisberg and Williamson, 1964; Hoeschen, 1971). Higher ouabain concentrations led to decreased glucose uptake. Crone (1966) reported an increased glucose uptake in perfused cat hindlimb when the potassium concentration was increased. Oxygen consumption was increased, however, suggesting that ATP splitting had been accelerated. These changes were similar to those in the perfused rat heart that developed increased levels of ventricular pressure. Gould and Chaudry (1970) found that glucose uptake was stimulated in soleus muscle when sodium but not potassium was added to an ion-free medium. Addition of potassium alone led to a rate that was

288

HOWARD E. MORGAN AND CAROL F. WHITFIELD

one-half the value found in muscle in ion-free media. However, the rate of glucose uptake in ion-free media was not different from the rate found in the presence of sodium (110 rnM) plus potassium (6 mM). Increased uptake of glucose in diaphragm has been found in the presence of (1) a hyperosmolar medium (Clausen, 1968a, 1970), (2) lithium, in place of sodium (Clausen, 1968a, b, 1970), (3) ouabain M ) , for 1hour (Kypson et al., 1968), and (4) increased sodium concentration in the medium (Clausen, 1965). I n each of the above cases, metabolism of glucose by diaphragm was altered in the direction of decreased catabolism to lactate and carbon dioxide, and toward increased deposition of glycogen. In another series of articles, glucose uptake was unchanged by variations in ionic composition. Clausen (1965, 1966) reported that glucose uptake by diaphragm was unaffected by ouabain (10-3-10-7 M ) or by low potassium concentrations. In these experiments decreased production of carbon dioxide and increased glycogen deposition were noted. Randle and Smith (1958a) tried to relate the rate of potassium release from diaphragm to glucose uptake and found no correlation. In the studies discussed thus far, alterations of ion pumping or extracellular ion concentrations lead to a marked change in glucose metabolism, perhaps due to changes in the use of energy for ion pumping, but may or may not actually change glucose transport. Further attempts to resolve this problem were made by studying the transport of nonmetabolizable sugars. Parrish and Kipnis (1964) found no decrease in uptake of 2-deoxyglucose or galactose by diaphragm when sodium was absent from the incubation medium. Bihler (1968) found that 3-0-methylglucose entry into diaphragm was increased by ouabain when the tissue was preincubated with the drug and glucose and then incubated with ouabain and 3-0-methylglucose. He also reported that there was increased entry of sugar in potassium-free media, which was additive with the incr,ease produced by ouabain. Phlorizin and N-ethylmaleimide were found to block the increased 3-0-methylglucose entry. Bihler and Sawh (1971a, b, c) have reported that high extracellular potassium (16 mM) inhibited 3-0-methylglucose entry, and that diphenylhydantoin (DPH) had the opposite effect. This compound acted to stimulate the sodium pump and to increase intracellular potassium. Ouabain was found to inhibit the effect of DPH. Efflux of 3-0-methylglucose from diaphragm was found to be stimulated under the same conditions as influx, i.e., in potassium-free media and with ouabain. Bihler and Sawh (1971~)concluded that stimulation of sugar transport in diaphragm is correlated with intracellular sodium and potassium concentrations but not with the activity of the sodium pump per se or with the extracellular ion concentration. This is in agreement with the previous conclusion that ions are not cotransported with sugar.

SUGAR TRANSPORT IN EUKARYOTIC CELLS

289

Variations in water content of the muscle cells may account for some of the effects of ions on transport. Although substitution of potassium for sodium did not lead to a change in efflux of 3-O-methylglucose, high potassium levels prevented stimulation of 3-0-methylglucose efflux by hyperosmolarity, 2,4-dinitrophenol (DNP), and muscle work. Isosmolar solutions containing high amounts of potassium were found by Kohn and Clausen (1972) to cause cell swelling, as were hypoosmolar solutions. In solutions containing high potassium, but which were also hyperosmolar, cell volume and transport rate did not change. These investigators concluded that the inhibition of the stimulation of sugar transport by potassium seen by them, and by Bihler and Sawh (1971a), may have been due to changes in cell volume, and not to a direct potassium effect on the regulatory process. Bihler and Sawh (1971~)suggested that intracellular potassium concentrations are inversely related to transport rate. However, Randle and Smith (1958a) were not able to correlate potassium loss from diaphragm with glucose uptake. Kohn and Clausen (1971) showed that a fall in potassium content of rat soleus muscle following treatment with DNP was associated with an increase in 3-0-methylglucose efflux. However, ouabain (10-3 M ) caused loss of potassium and did not increase sugar efflux until after 90 minutes of incubation, when a small stimulation of efflux was seen. This effect may have been due to contraction of the muscle after this period of incubation. Kohn and Clausen (1971) have suggested that the effect of ouabain may be related to increased cell calcium (also Holloszy and Narahara, 1967a), and that the difference between their findings and those of Bihler (1968) may be related to the longer period of time the muscles were incubated with ouabain. A report by Ilse and Ong (1970) indicated that diaphragm incubated in a medium in which 65 mM sodium was replaced with isosmolar mannitol showed an increased uptake of both 3-0-methylglucose and calcium. The increase was related t o a higher V,,,, with no change seen in the K , for the sugar. In these studies complete replacement of sodium with mannitol did not lead to a change in transport rate, This again demonstrates that sodium ions are not essential for basal sugar uptake in muscle. The ionic dependence of the stimulatory effect of insulin in muscle has also been investigated. Bhattacharya (1961) found that insulin did not stimulate glucose uptake in diaphragm when the incubation medium was an isosmotic sucrose solution. Addition of Na+, K+, Li+, Rb+, or Csf led to an effect of insulin. Magnesium was found to increase the insulin effect and the basal uptake rate as well. In soleus muscle the insulin effect was also found to depend upon magnesium, but not on calcium (Gould and Chaudry, 1970). In this tissue insulin was effective in the

290

HOWARD

E. MORGAN AND CAROL F. WHITFIELD

absence of sodium and potassium. Insulin stimulation of transport in muscle has been found by other investigators to be independent of sodium ions (Parrish and Kipnis, 1964; Clausen, 1970; Clausen and Kohn, 1970; Kohn and Clausen, 1972). In these studies the effect of insulin was found to be suppressed by high concentrations of potassium, but Kohn and Clausen (1972) attributed this to swelling of the cells. As noted above, cell volume increased in cells exposed to high concentrations of potassium, and in other experiments the insulin effect was abolished in cells exposed to hypoosmolar solutions. Bihler (1968) found that ouabain produced a greater stimulation of 3-O-methylglucose penetration in diaphragm when submaximal doses of insulin were present. However, Kohn and Clausen (1971) reported that ouabain did not increase the insulin-stimulated efflux of 3-O-methylglucose from soleus muscle. As mentioned above, these differences may be related to the shorter time of incubation in the former experiments. From the above experiments i t seems that insulin stimulation of sugar transport in muscle does not require the presence of sodium or potassium, but that magnesium may be necessary. The situation in adipose tissue with regard to the effects of ions on sugar transport is similarly complicated, since glucose metabolism and lipolysis were also markedly changed. Glucose conversion to carbon dioxide and lipids in isolated fat cells has been reported to be stimulated by (1) lack of potassium (Ho et al., 1966; Letarte et al., 1969; Clausen, 1969; Touabi and Jeanrenaud, 1970; Clausen et al., 1969), (2) decreased sodium (Letarte and Renold, 1967, 1969a, b ; Clausen et al., 1969), (3) ouabain (Ho et al., 1966, 1967; Ho and Jeanrenaud, 1967; Letarte and Renold, 1969b; Clausen, 1969; Clausen et al., 1969), (4) and hyperosmolarity (Clausen et al., 1970). Glucose uptake has becn reported to increase after incubation with ouabain (Ho et al., 1966; Clausen et al., 1969), in potassium-free media (Ho et al., 1966; Rodbell, 1967), or in a low sodium-high potassium medium (Letarte and Renold, 1969a). Other reports indicated that glucose uptake did not change in isolated fat cells incubated in potassium-free media (Rodbell, 1965) or with ouabain (Ho and Jeanrenaud, 1967). Kujalova and Mosinger (1966) also found no change in basal glucose uptake in intact fat pads incubated with ouabain. Ho and Jeanrenaud (1967) did not find consistent effects of ouabain in isolated cells but did find a stimulation of glucose uptake in intact fat tissue. This brings up the possibility that some of the apparently conflicting findings in regard to effect on glucose uptake may be due to differences in the preparation of isolated fat cells. Two recent reports on the transport of 3-O-methylglucose in intact fa t pads indicate that efflux of the sugar from preloaded tissue was increased by ouabain or incubation in a potassium-frce medium (Clausen, 1969, 1970). The effect was seen only after 50-60 minutes, and the ouabain effect was

SUGAR TRANSPORT IN EUKARYOTIC CELLS

291

observed only when potassium was also present. Bihler and Jeanrenaud (1970) found that both ouabain and lack of potassium partially prevented loss of ATP in isolated fat cells. The sparing of ATP under these conditions was not expected to result in increased conversion of glucose to carbon dioxide and an accelerated rate of sugar transport. At present, it is difficult to show a direct effect of any ion on carrier-mediated transport in fat cells and, as concluded by Clausen (1970), it is most likely that changes in transport are secondary to changes in the route of glucose metabolism in the cell. In adipose tissue insulin stimulated sugar entry, but it also inhibited lipolysis. A change in lipolysis may have affected sugar transport and intracellular ion concentrations. Rodbell (1965, 1967) first reported that potassium was required for insulin stimulation of glucose uptake in isolated ghosts, even though the basal rate of glucose uptake was higher in the absence of potassium. Letarte and co-workers (Letarte and Renold, 1969a; Letarte et al., 1969), however, found no consistent effect of potassium lack on the response of isolated cells to insulin. In contrast to muscle, adipose tissue did not appear to require magnesium for insulin to act. Several investigators have reported that the insulin effect on glucose uptake and glucose metabolism to carbon dioxide and lipids was inhibited in a medium low in sodium (Hagen et al., 1959; Letarte and Renold, 1967, 1969b; Clausen et al., 1969; Clausen, 1970). The conclusion reached by Clausen (1970) suggests that sodium is not essential for insulin-stimulated glucose transport, but may be required for the increase in glucose conversion to carbon dioxide and triglycerides. Letarte and Renold (196913) concluded that insulin-stimulated glucose uptake is sodium-dependent. In their study, however, lack of sodium depressed but did not completely abolish the effect. This suggests that some component of the effect was sodium-independent. Thus there is no conclusive evidence that ions are a n absolute requirement for the insulin stimulation of sugar transport in adipose tissue. Since the avian erythrocyte is not complicated by actin-myosin interaction, lipolysis, or variability due to the preparation of isolated cells from intact tissue, i t seems to be a better cell in which to study the effects of ions on transport ratc. Transport in this cell is not sensitive to insulin but is accelerated by anoxia. When goose erythrocytes were incubated under anaerobic conditions, in sodium or potassium-free buffer, no significant change in 3-O-methylglucose entry was found (Table VII). Changes in ion composition of the buffer did not modify the water content. These experiments demonstrate that the presence of sodium is not required for the stimulated rate of 3-O-methylglucose entry in anoxic cells. Although ouabain slowed the rate of 3-O-methylglucose entry in anoxic cells, the rate was still above the aerobic value. Addition of ouabain also reduced the rate of ATP

292

HOWARD E. MORGAN AND CAROL F. WHITFIELD

TABLE VII

EFFECT O F IONS ON 3-0-METHYLGLUCOSE ENTRY I N AVIAN ERYTHROCYTES" Equilibration of cell water ( % of anoxic control)

ATP (%of anoxic control)

Aerobic cells Control

43.9 f 1.0

(4)

424 i 3 4

(4)

100i 10 99.0 f 8 . 0 87.4 f 7 . 2 76.3 f 6.0

(11) (7) (9) (7)b

100f 10 170 f 8 164 f 2 6 220 f 54

(11) (7)b (9)b (7)b

Anoxic cells Control Sodium-free Potassium-free Ouabain, 5 X 10-6 M

a Cells were washed three to five times in the buffer and incubated under oxygen or nitrogen. Entry of 3-O-methylglucose was calculated as the percent to which intracellular water had equilibrated with extracellular sugar. Values are those reached 30 minutes after addition of sugar to cells preincubated 45 minutes with the respective buffers. All ion substitutions were made isosmotically, with choline, potassium or lithium in place of sodium, and sodium in place of potassium. Values are given as the percent of the value for anoxic cells incubated in control buffer. * Significantly different from control.

utilization. These results suggest that the effect of ouabain on sugar transport is to decrease the rate a t which the cell becomes energy-poor and thus indirectly to delay the anoxic stimulation of transport. I n the studies of 3-O-methylglucose transport in muscle reviewed above, little attempt was made to assess the metabolic state of the cells after incubation in the ion-substituted buffers. However, this is necessary in view of the known effect of cellular energy levels on sugar transport rate. Other evidence suggests that ions may control glycolysis and, perhaps through this mechanism, change transport rates. Whittam and Ager (1965) found that intracellular sodium and extracellular potassium stimulated glycolysis in erythrocytes, and that ouabain inhibited it. Similar findings were reported by Parker and Hoffman (1967). The latter investigators suggested that ouabain inhibits glycolysis directly, a t the phosphoglycerate kinase step. Others have suggested that ouabain inhibits phosphofructokinase (Minakami et al., 1964). I n Ehrlich ascites tumor cells, addition of potassium to potassium-depleted cells immediately stimulated glycolysis (Gordon and DeHartog, 1968). The ADP and Pi produced from active ion pumping may be the agents that stimulate glycolysis.

SUGAR TRANSPORT IN EUKARYOTIC CELLS

293

Whether slowing down or inhibiting the ion pump will affect sugar uptake or metabolism may depend on what proportion of the synthesized ATP is used to support the pump. With an agent such as ouabain, ATP consumption may be decreased and so may ATP synthesis by inhibition of phosphofructokinase or phosphoglycerate kinase. When sodium or potassium is omitted, ATP utilization is decreased. Higher levels of ATP, together with lower levels of ADP and Pi, inhibit glycolysis. The evidence presently available does not indicate a direct effect of ions on sugar transport in muscle, adipose tissue, or nucleated erythrocytes. All the effects that have been described may be explained by variations in ATP consumption or production which lead to changes in energy levels and associated variations in transport rate. B. The Roles of Sulfhydryl Groups and "Energy-Charge" in Regulating Transport

Involvement of sulfhydryl groups in transport and its control was indicated by (1) inhibition of glucose transport in human red cells by mercuric chloride and other sulfhydryl-blocking agents (LeFevre, 1947), and (2) by interference with the insulin effect by pretreatment with a sulfhydryl-blocking agent (Cadenas et al., 1961). Subsequently, sugar transport in rabbit cells was found to be unaffected by sulfhydryl blockers (Park el al., 1968). In avian erythrocytes (Wood and Morgan, 1969) and isolated fat cells (Minemura and Crofford, 1969), these blocking agents accelerated transport and mimicked other effects of insulin. Surprisingly, thiol compounds such as 2,3-dimercaptopropanol, 1 ,.l-dithiothreitol, and reduced glutathione also accelerated transport in fat cells (Minemura and Crofford, 1969; Lavis and Williams, 1970). An example of the effect of sulfhydryl-blocking agents on sugar transport in avian erythrocytes is shown in Table VIII. Aerobic restraint of carrier activity was removed by addition of Hgz+, N-ethylmaleimide, or other sulfhydryl-blocking agents (Wood and Morgan, 1969). Addition of these compounds resulted in a threefold acceleration of transport rate which was competitively inhibited by addition of glucose The stimulation of transport resulting from addition of Hg2+ was abolished rapidly by addition of dithiothreitol and was partially reversed by other thiol compounds. I n other experiments addition of 1 ,4-dithiothreitol partially inhibited the accelerated rate of 3-O-methylglucose transport found in anaerobic cells. The signal for a change in transport during the onset of the anaerobic effect must arise either from variations in levels of metabolites within the cells or through entry of some material from the extracellular phase. As

294

HOWARD E. MORGAN AND CAROL F. WHITFIELD

TABLE VIII

EFFECTS OF SULFHYDRYL-BLOCKING AGENTS,GLUCOSE, AND CYANIDE ON THE ENTRY O F 3-O-METHYLGLUCOSE INTO GOOSEERYTHROCYTES"

Additions to incubation medium None HgCl2 (1 pmole/ml cells) HgC12 glucose (20 mM) HgCl2 dithiothreitol ( 5 mM) N-Ethylmaleimide (10 pmoles/ml cells) NaCN (1 mM)

+ +

3-O-Methylglucose entry ( % equilibrium) 14 40 6 22 28 29

A 10% suspension of goose red cells was incubated a t 37°C with the additions listed. After 15 minutes 10 pl of a solution of 3-O-methylglucose-14C were added to each flask. Dithiothreitol was added 5 minutes after the addition of the methylglucose. The suspensions were sampled for determination of degree of equilibration of intracellular and extracellular sugar concentrations after 20 minutes of incubation with the methylglucose. Similar results were obtained in a t least two other experiments. (From Wood and Morgan, 1969.)

noted earlier, the accelerated rate of transport during anoxia in avian erythrocytes did not appear to be dependent upon the ionic composition of the incubation medium. Similarly, addition of metabolites that were potential regulators of transport to the extracellular phase had little or no effect on transport rate. These additions included Pi (5-25 mM), citratc (4 mM) and cyclic AMP (0.1 mM). Additional support for involvement of a signal for the anaerobic effect that originated from within the cell was provided by reversible hemolysis experiments (Wood and Morgan, 1969). I n these studies cells were suspended in solutions of low osmolarity, followed by restoration of membrane integrity in solutions containing 300 mosmoles/liter. Restoration of integrity was indicated by demonstrations of saturation of 3-O-methylglucose efflux and by lack of sorbitol permeability. Proteins and metabolites were lost from cells in which the rate of transport increased. These results suggested that some inhibitor of transport present in aerobic cells had been lost by the hemolysis procedure. Several mechanisms of regulation of glucose transport are consistent with the data presently available from studies with avian erythrocytes.

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The observation that addition of Hg2+ and N-ethylmaleimide stimulated 5-O-methylglucose transport can be interpreted to mean that the aerobic restraint of transport involves interaction of the carrier with an intracellular regulatory protein sensitive to sulfhydryl-blocking agents (Fig. 10). If a second protein component were not involved, sulfhydrylblocking agents would be required to react directly with the carrier to accelerate transport. Direct reaction of sulfhydryl-blocking agents with the carrier appears to be more liltcly to inhibit carrier activity than to stimulate it. As noted above, sulfhydryl-blocking agents inhibited transport in human erythrocytes but had no inhibitory effect on rabbit erythrocytes. One could suppose that the avian cell resembles the rabbit cells in that the carrier is insensitive to addition of sulfhydryl-blocking agents and that an additional intracellular protein is responsible for regulation. In this model the regulatory protein would be bound to the carrier in aerobic cells and would inhibit carrier movement. The substance signaling the onset of anoxia would interact with the regulatory protein and would displace it from the carrier. Several intraccllular substances could be suggested to serve as the signal to displace the regulatory protein. A metabolite of glucose may act as a feedback inhibitor of transport in yeast (Kleinzeller and Kotyk, 1965). More specifically, glucose 6-phosphate has been suggested as the signal that mediates regulation of transport in Baker’s yeast (Azam and Kotyk, 1969). Substances such as ADP, AMP and Pi involved in the ‘Lenergy-charge”(Atkinson, 1965, 1968; Shen et al., 1968; Klungsoyr el al., 1968) of the cell could also serve to displace the regulatory protein. Extension of this model to control of sugar transport in muscle suggests that a factor in working muscle displaces the regulatory protein, while the utilization of fatty acid by cardiac muscle results in association of the regulatory protein with the carrier. This model also is consistent with the observation that reversibly liemolyzed cells have a fast rate of transport, since in these cells the regulatory protein would have been lost along with the other soluble proteins of the cell. Another modrl (Fig. 10) involving phosphorylation of the carrier was suggested by Randle and Smith (1958a) in their original description of the anaerobic effect on sugar transport in muscle. In this model regulation involves a soluble protein kinase that phosphorylates the carrier using a high-energy compound as a phosphate donor. Phosphorylation of the carricr would immobilize it and would be responsible for the aerobic restraint of transport. A protein phosphatase would account for carrier activation. Activity of protein kinase would be inhibited by sulfhydrylblocking agents and would be lost from the cell by reversible hemolysis. Activity of protein kinase would be low in anaerobic cells because of low levels of ATP or because of allosteric regulation by a compound related to

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REGULATORY -PROTEIN MODEL OUT

IN

OUT

IN

PROTEIN PHOSPHORYLATION MODEL OUT

IN

OUT

MOBILE CARRIER

IN

IMMOBILE CARRIER

FIG.10. Models accounting for the control of sugar transport. The models are described in the text.

“energy-charge.” Phosphorylation of membrane proteins by a cyclic-AMPdependent protein kinase has been reported (Schlats and Marinetti, 1971), but the effect of phosphorylation on permeability properties of the membrane has not been investigated.

V. SUMMARY

Transport is a major rate-limiting step for glucose uptake in a large variety of eukaryotic cells. Existing kinetic data are compatible with a simple carrier model for passage of the sugar through the membrane. Nonhormonal control of transport is the most basic type of regulation, since it is present in both unicellular organisms and in cells of mammalian tissues. The conditions involved in this type of regulation are decreased

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energy production, increased energy expenditure, or utilization of other substrates. These conditions are brought about by anoxia and inhibitors of oxidative phosphorylation, by increased ATP consumption from muscle contraction and greater rates of ion pumping, and by providing fatty acids as substrates. The anoxic effect on transport is related to ATP depletion, but the cffcct cannot be considered to be due to ATP levels alone. Increased transport rates due to muscle contraction are not related to increased rate of ventricular pressure development in heart, but to increased frequency of contraction in skeletal muscle. Fatty substrates have a greater inhibitory effect after transport is stimulated by increased mechanical activity. The inhibition is clearly seen in heart but is not demonstrable in vitro in skeletal muscle preparations. Insulin is the major hormone that accelerates transport in muscle and adipose tissue. Growth hormone modifies insulin sensitivity, as does cortisol. The inhibitory effect of cortisol in adipose tissue, diaphragm, and thymocytes may involve synthesis of an inhibitory protein. Epinephrine increases sugar transport in heart, adipose tissue, skeletal muscle, and avian red blood cclls. In each case there is difficulty in distinguishing a direct effect of transport from ATP depletion due to the inotropic and lipolytic effects of the hormone, and to production of free intracellular glucose from glycogenolysis. Avian red cells offer the best opportunity to demonstrate a direct effect on transport. Control of sugar transport in avian red cells and probably in muscle can be accounted for by models that include (1) a regulatory protein in soluble form within thc cell, or (2) phosphorylation of the carrier by a protein kinase. At prescnt, there is no way to distinguish between these models. Avian red cells offer a system in which these models can be studied, since transport is regulated by hormonal and nonhormonal factors, and since the cells are unattached and available in large amounts. REFERENCES Ahren, K., Hjalmarson, A., and Isaksson, 0. (1970). Actu Physiol. Scand. 78, 574. Atkinson, D. E. (1965). Science 150, 851. Atkinson, D. E. (1968). Biochemistry 7, 4030. Azam, F., and Kotyk, A. (1969). FEBS (Fed. Eur. Biochem. Sac.), Lett. 2, 333. Beatty, C. H., and Bocek, R. M. (1971). Amer. J . Physiol. 220, 1928. Bennett, G. V., and Cuatrecasas, P. (1972). Science 176, 805. Bhattzcharya, G. (1961). Biochem. J . 79, 369. Bihler, I. (1968). Biochim. Biophys. Acta 163, 401. Bihler, I., and Jeanrenaud, B. (1970). Biochim. Biophys. Actu 202, 496. Bihler, I., and Sawh, P. C. (1971a). Biochim. Biophys. Actu 241, 302. Bihler, I., and Sawh, P. C. (1971b). Biochim. Biophys. Actu 249, 240. Bihler, I., and Sawh, P. C. (1971~).Biochim. Biophys. Actu 225, 56. Bihler, I., Cavert, H, M., and Fisher, R. B. (1965a). J . Physiol. (London) 180, 157.

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Shipp, J. C., Opie, L. H., and Challoner, D. (1961).Nature (London) 189, 1018. Schlatz, L.,and Marinetti, G. V. (1971).Biochem. Biophys. Res. Commun. 45, 51. Sols, A., and Crane, R. K. (1954).J . Biol. Chem. 210, 581. Sonenberg, M.(1969). Bioehem. Biophys. Res. Commun. 36, 450. Sonenberg, M.(1971).Proc. Nut. Acad. Sci. U.X. 68, 1051. Stein, W. D.(1972).Ann. N . Y . Acad. Sci. 195, 412. Touabi, M.,and Jeanrenaud, B. (1970).Biochim. Biophys. Acta 202, 486. Turkington, R. W. (1970).Biochem. Biophys. Res. Commun. 41, 1362. Vidaver, G.A. (1964).Biochemistry 3, 795. Wallaas, O.,and Walaas, E. (1950).J . Biol. Chem. 187, 769. Weis, L.S.,and Narahara, H. T. (1969).J. Biol. Chem. 244, 3084. Whitney, J. E.,Cutlar, 0. E., and Wright, F. E. (1963).Metub., Clin. Ezp. 12, 352. Whittam, R.(1962).Biochem. J.84, 110. Whittam, R.,and Ager, M. E.(1965).Biochem. J . 97, 214. Widdas, W. F. (1952).J. Physiol. (London) 118, 23. Williamson, J. R.(1964).J . Biol. Chem. 239, 2721. Williamson, J. R.,and Krebs, H. A. (1961).Biochem. J. 80, 540. Wohltmann, H.J., and Narahara, H. T. (1968).Biochim. Biophys. Acta 150, 550. Wohltmann, H.J., Narahara, H. T., and Wesley, M. E. (1967).Diabetes 16, 26. Wood, R.E.,and Morgan, H. E. (1969).J . Biol. Chem. 244, 1451. Yorke, R.E.(1967).J.Endocrinol. 39, 329.

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Secretory Events in Gastric Mucosa* RICHARD P. DURBIN Cardiovascular Research Institute and Department of Physiology, University of California, S a n Francisco, California

I. Introduction . . . . . . . 11. Comparative Aspects . . . . . 111. Structural Aspects . . . . . . A. Morphology . . . . . . B. Gastric Barrier. . . . . . C. Water Flow . . . . . . IV. Coupling of Secretion to Metabolism A. ATPase. . . . . . . . B. Cytochromes . . . . . . C. High-Energy Intermediates . . V. Conclusion . . . . . . . . References . . . . . . . .

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305 305 307 307 309 312 313 313 314 317 319 319

I. INTRODUCTION

The field of gastric secretion has been extensively reviewed in recent years (Forte, 1971; Makhlouf and Rehm, 1973; Rehm, 1972). The proceedings of a recent symposium on H ion secretion have appeared, edited by Sachs et al. (1972a). Accordingly, this article is not intended as a comprehensive review. Instead, discussion and speculation are focused on some topics of particular interest to this writer. II. COMPARATIVE ASPECTS

The study of gastric secretion encompasses the movement of a group of ions, including a t least H, C1, HC03, and OH ions. It is comforting to the

* Preparation of this report was supported by the National Institutes of Health grant HL-06285. 305

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RICHARD P. DURBIN

small band of workers in this field that many others now share their interest. As in the case of gastric mucosa, identification of the presence of carbonic anhydrase, the enzyme catalyzing the reaction of COz with water, may precede further elucidation of the transport processes, e.g., in the shell-boring mechanism of gastropods (Ch6tail and FourniB, 1969) or in the unicellular algae (Graham and Reed, 1971). Several recent reviews have amassed evidence for active transport of H, C1, and related ions in a variety of plant and animal cells (MacRobbie, 1970; Keynes, 1969; Motais and Garcia-Romeu, 1972). Note that the presence of one kind of transport does not exclude another. Two epithelia, reputed for active transport of Na ion, have been shown to secrete H ion as well. These are frog skin (Emilio et al., 1970) and urinary bladder of the toad (Fanestil and Ludens, 1971; Frazier and Vanatta, 1971). It is of special interest to gastric physiologists that electron transfer can give rise to a n H ion gradient across chloroplast (Neumann and Jagendorf, 1964), mitochondria1 (Mitchell and Moyle, 1969a), and bacterial membranes (Scholes and Mitchell, 1970). It is still not clear to what extent these observations support the chemiosmotic hypothesis of Mitchell (1961). However, the prediction of Mitchell (1961) that agents that uncouple electron transfer from phosphorylation (i.e., uncouplers) do so by increasing the permeability of an associated membrane to H ions, appears to be correct. Uncouplers increase H ion permeability in artificial lipid membranes (Bielawski et al., 1966; Liberman et al., 1969). Uncoupling by various phenols correlated well with an increase in proton conduction but not with stimulation of mitochondrial ATPase (Stockdale and Selwyn, 1971). An important advance in the understanding of ion transport has been the discovery of ionophores (Moore and Pressman, 1964). Of these, a group represented by valinomycin is composed of relatively specific mediators of K ion transport, while the class represented by nigericin promotes exchange of K ion for H ion (Pressman et al., 1967). These effects can be shown in artificial as well as biological membranes. There is now evidence for another category of agents that promote the exchange of C1 ion for OH ion (Watling and Selwyn, 1970; Selwyn et al., 1970). These include trialkyltin compounds [e.g., trimethyltin or (CH1)aSnCl] and phenylmercuric acetate. Thus closed lipid vesicles, suspended in a KC1 medium containing phenylmercuric acetate, exhibited a limited swelling until both valinomycin and an uncoupler (carbonylcyanide p-trifluoromethoxyphenylhydrazone or FCCP) were added, a t which point the vesicles swelled rapidly (Watling and Selwyn, 1970). These results showed that a net uptake of KC1 and water had occurred, with valinomycin and FCCP allowing the exchange of external K for internal H ion, and phenylmercuric acetate external C1 for internal OH ion.

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A hypothesis invoking exchange of C1 for HCOI ion a t the secretory surface of the gastric mucosa antedated the above results (Hogben, 1952). Coupled exchange in this manner would lead to formation and secretion of HCl, while partial exchange with either HC03 or C1 absent would yield electrogenic C1 or H ion transport, respectively (Durbin' and Kasbekar, 1965). At present there is little direct evidence in support of such an anion exchange mechanism at the secretory surface in stomach. Recent experiments of Solberg and Forte (1971) may bear upon the hypothesis, however. They instilled the sulfhydryl reagent, p-chloromercuribenaene sulfonate (PCMBS), on the secretory surface of isolated frog gastric mucosa and observed a decrease in secretory rate which closely paralleled a stoichiometrically equivalent increase in electrogenic C1 ion transport. The correspondence was more apparent during the first 20 minutes after addition of PCMBS. Since this agent probably does not penetrate the oxyntic cells to an appreciable extent (Solberg and Forte, 1971), i t is possible that conversion of secretory to electrical activity was due to displacement of HC03 from an exchange mechanism a t the secretory surface. Rehm and Sanders (1972) presented convincing evidence that a nonelectrogenic exchange of anions occurs a t the serosa-facing surface of the oxyntic cell. I n this process base formed as a result of H ion secretion exits to the blood or serosal solution in exchange for entering C1 ion; the latter (in a steady state) moves across the mucosal surface in the secreted acid. This discovery was prompted by experiments in which barium ion was added to the serosal solution of isolated gastric mucosa (Schwartx et al., 1968). This agent greatly increased mucosal resistance, with little change in acid secretion. Later work showed that barium decreased the K ion conductance of the serosal membrane; this effect could be negated by increasing serosal K+ from 4 to 79 mM (Pacific0 et al., 1969). The rapidity of its action in these experiments showed that barium primarily affected the serosal membrane. The proton (or base) conductance a t this surface is negligible (Sanders et al., 1972). The conductance remaining in the presence of barium is probably associated with electrogenic movement of C1 ion. 111. STRUCTURAL ASPECTS A, Morphology

Forte and Forte (1971) used the freexe-etch technique with glutaraldehyde-fixed gastric mucosa to secure a more three-dimensional view of the

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FIG.1. Apical surface membrane of an oxyntic cell showing possible interconnections with tubules. Apical plasma membrane (B) adjacent to the gland lumen (L). A few tubular or vesicular structures are seen (arrows) in a conformation suggesting continuity with the surface. More densely packed tubular elements are found beneath the granular cortical cytoplasm in the lower right quadrant. X46,OOO. (Forte and Forte, 1971.)

oxyntic cell. An extensive, tightly packed array of tubular elements was seen to occupy virtually the entire apical region of the cell. These tubules often terminated in dilated, bulbous structures, and the investigators conjectured that sections of these structures might have appeared as vesicles in prior studies with conventional electron microscopy. Figure 1, taken from their work, indicates that the smooth inner surface of the cytoplasmic tubule (bordering its lumen) appears to be continuous with the smooth surface of the apical plasma membrane. Profound changes in the secretory surface of the oxyntic cell occur with the onset of secretion (cf. Forte, 1971). Helander et al. (1972) applied stereological techniques to quantitate these changes. Biopsies were taken from the stomachs of conscious dogs, in the fasting, nonsecreting state, and an hour later while acid secretion was stimulated with intravenous histamine. Measurements were made on 20 parietal cells, 5 from each of four animals, as summarized in Table I. The results indicate that stimulation did not change the total area of the basal and lateral surfaces of the parietal cell. The plasma membrane lining the secretory surface of the parietal cell increased in area by more than a factor of 10, largely at the expense of the membrane lining vacuoles and tubules within the cytoplasm. During secretion a massive proliferation of canaliculi, studded with microvilli, is largely responsible for the increase in secretory area. Upon withdrawing

309

SECRETORY EVENTS IN GASTRIC MUCOSA

histamine acid secretion ceased, and the morphological changes were reversed (Helander and Hirschowitz, 1972). In what may be another approach to the study of these events, we measured p-nitrophenyl phosphatase activity of intact gastric mucosa (Durbin and Kircher, 1973). This enzyme was previously demonstrated to occur in the microsomal fraction obtained from homogenates of gastric mucosa (Forte et al., 1967a). We placed p-nitrophenyl phosphate (PNPP) in the solution bathing the secretory surface and measured the products of hydrolysis appearing in the external solutions. Stimulation of resting mucosae (Kasbekar, 1967) greatly increased acid secretion and approximately doubled the rate of PNPP breakdown. In other experiments histaminestimulated secretion was inhibited by anoxia, and the rate of PNPP hydrolysis dropped by about half. We interpret these results to indicate that less secretory membrane, and consequently less membrane-bound enzyme, was exposed in the resting state or during anoxia than in the aerobic, secreting state. B. Gastric Barrier

In recent years attention has been directed toward the role of the gastric mucosa as a barrier to substances ingested or instilled in the lumen of the stomach. The subject, which is of interest to physiologists as well as TABLE I

MEMBRANE SURFACES PER UNITMACROSCOPIC SURFACE IN PARIETAL CELLSOF DOGGASTRIC MUCOSA~~~

Cytoplasmic (vacuoles tubules) Secretory canalicular) (apical Nutrient (lateral basal)

+

+

Sum

+

Fasted

Histaminestimulated

1513 f 118

595 f 138

53 f 33

568 f 188

228 f 18

240 f 40

1691 f 128

1400 f 157

From Helander el al. (1972). Mean value plus or minus standard deviation. Vestopalembedded tissue. Values corrected for estimated membrane lost due to oblique sectioning. a

b

310

RICHARD

P. DURBIN

clinicians, may be said to have begun with the pioneering work of Teorell (1939) and Grant (1945). Despite the large area of the gastric tubules and the abundant secretory surface of the oxyntic cells, the principal route for passive diffusion is by way of the surface epithelium, bypassing thc cells lining the tubules (the gastric tubules referred to here, as viewed in the light microscope, should not be confused with the cytoplasmic tubules of the oxyntic cell discussed in the preceding section). I n the studies of Davenport et a2. (1967), the rate of absorption of several substances (e.g., ethanol) was observed to be the same in the presence or absence of vigorous secretion of gastric juice. Their calculations showed that the volume flow of secretion should have effectively swept the test substance out of the tubules. Since this factor did not affect absorption, these workers were forced to conclude that the gastric glands were not an important site for absorption. In addition, their results appear to indicate that the tubules are closed in the resting state. The gastric mucosa (hence the surface epithelium) is normally an adequate barrier to diffusion of H ions from the lumen, or Na ions to the lumen (cf. Ivey, 1971; Davenport, 1967a, 1972). A variety of agents can damage the barrier. Prominent among these are weak acids, including acetic and acetylsalicylic acids, but these are effective only when present in acid solution in the lumen (Davenport, 1964). Acetic acid has a p K of 4.8, and acetylsalicylic acid 3.5. At the pH of gastric acid, these substances are primarily in nonionixed, lipid-soluble form and readily permeate the mucosal barrier (Teorell, 1939 ; Davenport, 1965). Martin (1963) suggested that the damage caused by weak acids reflects dissociation and consequent trapping inside the mucosal cell at the ambient intracellular pH. Accumulation of such products may lead to disturbance of intracellular pH or metabolism, or to osmotic swelling. Evidence for the penetration of weak acids was advanced by Flemstrom (1971). He found that acetate, propionate, and L-lactate (but not D-lactate), when instilled in a solution of pH 4 on the mucosal surface, led to the potentiation of acid secretion in the presence of secretagogue. There is also direct morphological evidence that weak acids affect the surface epithelium. Hingson and Ito (1971) placed 20 mM acetylsalicylic acid in HCl in the lumen of mouse stomach in viva, and observed marked swelling and damagc to the surface epithelial cells as early as 1-2 minutes after instillation. At these times the gastric glands appeared normal. Frenning and obrink (1971) used the scanning electron microscope for a direct view of the surface, and Figs. 2 and 3 are taken from their work. Exposure of the luminal surface of cat stomach to 6 ml of 170 mM acetic acid for 30 minutes produced cells that were swollen (Fig. 3) compared to normal (Fig. 2). Use of a larger volume of acetic acid gave surfaces that

SECRETORY EVENTS IN GASTRIC M U C O S A

31 1

FIG.2. Normal surface epithelial cells of cat stomach. X3000. (Frenning and Obrink, 1971.)

FIG.3. Surface epithelial cells after instillation of G ml of 170 mM acetic acid in cat stomach. The cells are swollen, and the intercellular connections appear partly disrupted. X3000. (Frenning and Obrink, 1971.)

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RICHARD P. DURBIN

were hardly recognizable. Instillation of acetylsalicylic acid (17 mM) produced rounded, swollen surface epithelial cells which showed indications of strained or broken intercellular bridges. Hyperosmotic solutions in the lumen of stomach also break the mucosal barrier. Davenport (1968) found that 1-4 M urea increased Na ion entry into, and H ion efflux from Heidenhain pouches of dog. I n experiments with a mounted segment of canine mucosa, Altamirano (1969) observed that 1.2-2 M instillates containing urea, glucose, or sucrose increased the output of Na and HCO, ions and protein t o the lumen. Electrolytes are also effective in disrupting the surface epithelium in hypertonic concentrations (Forte et al., 1972). These effects may be compared to the increase in permeability produced by hyperosmotic solutions on the outside of frog skin (Ussing, 1965). In the latter case tight junctions between cells appear to be opened (Erlij and Martinez-Palomo, 1972). Many agents that disrupt the mucosal barrier clearly do so by chemical, rather than csmotic action, e.g., digitonin (Davenport, 1970). The action of ethanol is interesting and less obvious. It must be used a t a concentration of a t least 14% w/v (3 M ) for consistent effects on H and Na ion movements (Davenport, 1967b)* Ethanol had similar effects in neutral and acid instillates (Davenport, 196713). We found that ethanol in the secretory solution led to swelling of isolated frog gastric mucosa; ethanol added to the nutrient solution had the opposite effect of shrinking the tissue (Durbin et al., 1973). In addition to these osmotic effects, ethanol appears to inhibit active C1 ion transport (Shanbour, 1972). The surface epithelial cells may also be damaged by stress (Kim et al., 1967) and eating (Grant, 1944; Willems et al., 1971). The cause in the latter instance may be the hyperosmotic nature of food or drink (Altamirano, 1969). C. Water Flow

The considerations discussed in the preceding section are unfavorable for the experimenter seeking to demonstrate osmotic flow across the gastric mucosa. Hyperosmotic solutions placed in the lumen may not reach the tubules in full strength, and they are likely to cause damage and other undesirable effects. For whatever reason, osmotic flow induced by exogenous solute is small, lending little support to the commonly accepted view that the water component of gastric juice moves in response to a n osmotic gradient due to secreted acid. One approach to the problem has been to use hydrostatic rather than osmotic pressure (Moody and Durbin, 1969; Moody, 1971, 1972). If the

SECRETORY EVENTS IN GASTRIC MUCOSA

313

serosa is supported mechanically, a pressure of 50 cm HzO can be applied to the luminal surface of dog gastric mucosa without inhibiting H ion output or gastric blood flow (Moody, 1972). The hydrostatic conductivity (net volume flow per unit pressure) is about two orders of magnitude greater than the corresponding osmotic parameter. It is not known, however, whether the volume filtered is water or isosmotic instillate, or some intermediate concentration. Experiments in which gastric arterial or venous pressures were controlled yielded less equivocal results (Altamirano and Durbin, 1972). Increasing gastric blood pressure by either means led to net movement toward the lumen of a solution which resembled an ultrafiltrate of plasma. Effects due to moderate changes in pressure were fully reversible. Presumably, the volume filtered in these experiments passed between mucosal cells (cf. Fromter and Diamond, 1972). The change in blood pressure of the gastric capillary bed due to changes in arterial or venous pressure is not precisely known. However, a reasonable estimate shows that the hydrostatic conductivity from changes in blood pressure is of the same order of magnitude as that obtained when the hydrostatic pressure on the luminal surface is changed. I n turn this suggests that an appreciable flow between cclls must have been recorded by Moody and Durbin (1969). An experimental technique for separating this flow from flow across mueosal cell membranes would be of great value. Presumably, physiological flow (during secretion) corresponds to the latter, since the reflection coefficient (Kedem and Katchalsky, 1958) for HC1 in the region of the tight junctions must be quite small, reducing the effective osmotic gradient there.

IV. COUPLING OF SECRETION TO METABOLISM A. ATPase

Secretory and electrical activities of gastric mucosa are well known to depend on aerobic metabolism for their support. The nature of this linkage has been the subject of many investigations, but the problem has not yet been solved. In the case of tissues that primarily transport Na and K ions, the essential link is ATP supplied by mitochondria to a membrane-bound Na K-activated ATPase (Skou, 1957; Baker, 1965 ; Bonting, 1970). A membrane-bound ATPase was also isolated from gastric mucosa (Kasbekar and Durbin, 1965; Sachs et al., 1965, 1972b). This Mg2+-de-

+

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RICHARD P. DURBIN

pendent enzyme is inhibited by SCN and stimulated by HCOI ions, properties that suggest it may play an essential role in acid secretion. This hypothesis is by no means universally accepted (cf. Rehm, 1972). In the following sections some important findings are discussed, which must be fitted into any satisfactory theory of acid secretion. B. Cytochromer

Hersey (1971) has emphasized the value of nondestructive techniques which enable the experimenter to observe a functioning system. Among the most valuable are the spectrophotometric methods of B. Chance and his collaborators, first applied to gastric mucosa by Kidder et al. (1966). Their use of the split-beam apparatus to measure the reduced versus the oxidized difference spectrum revealed all the usual members of the respiratory chain. From their results, Kidder et al. (1966) estimated that the molar ratio of cytochrome (cyt)b to cyt c(+cl) was about 0.29. Since this was less than values previously quoted for skeletal muscle and liver mitochondria, Kidder et al. concluded that an appreciable quantity of cyt c(+cl) was extramitochondrial and might be involved in ion transport. The question was reexamined by Hersey et al. (1972), who also used the split-beam technique. These investigators found the ratio of cyt b to cyt c(+cl) to be 0.70 for intact frog gastric mucosa, and 0.68 for mitochondria obtained from gastric mucosa. Such close agreement in a direct comparison for the same tissue argues against significant amounts of extramitochondrial cyt c(+cl), unless it was accompanied by cyt b. Kidder et al. (1966) directed their attention primarily toward the behavior of cyt c, observing that this component became reduced upon resumption of acid secretion after a period of anoxia, and oxidized with inhibition of acid secretion by SCN. Their experiments were performed prior to the introduction of the resting mucosal preparation by Kasbekar (1967). Hersey and Jobsis (1969), using the latter technique, found that the entire respiratory chain from NAD to cyt a became reduced upon stimulation of acid secretion. Subsequent addition of SCN led t o oxidation of the whole respiratory chain. These results are consistent with (and extend) those of Kidder el al. (1966). Hersey and Jobsis (1969) pointed out that the foregoing results are unexpected if ATP is the immediate energy donor for secretion, Utilization of ATP should elevate intracellular ADP, in turn stimulating phosphorylation. I n the terminology of Chance and Williams (1955), initiation of secretion should correspond to a transition from state 4 (substrate and 02 available, respiration limited by ADP) to state 3 (ADP, 02, and substrate all available).

SECRETORY EVENTS IN GASTRIC MUCOSA

315

Upon increasing ADP, components toward substrate from particular points in the respiratory chain become oxidized, and components toward 0 2 more reduced. ADP thus acts as if it removes a barrier to the passage of electrons a t such a point, called a crossover point by Chance and Williams (1955). The location of the crossover point depends on several factors, but for rat liver mitochondria utilizing succinate or 0-hydroxybutyrate, i t fell between cyt c and cyt a (Chance and Williams, 1955). That is, addition of ADP led to oxidation of the entire respiratory chain, except for cyt a(+ar). In contrast, it was noted above that the respiratory chain of gastric mucosa became reduced with the onset of secretion. The contradiction may mean that direct comparison of isolated mitochondria and intact gastric mucosa is unwarranted, however. Substrate may be rate-limiting in the latter, since @-hydroxybutyrateand other fatty acids increased secretion in unstimulated mucosae (Alonso et al., 1967). I n addition, secretagogues may be expected to affect substrate utilization (Alonso et al., 1968). The role of substrate in isolated mitochondria may be emphasized by various manipulations. Increasing the concentration of succinate with liver mitochondria in state 3 led to increasing reduction of NAD, cyt b, and cyt c (Muraoka and Slater, 1969a). The crossover point disappeared with 1-10 mM succinate; that is, the whole respiratory chain became oxidized in the transition from state 4 to state 3 (Muraoka and Slater, 196910). The crossover point can be moved in the other direction with azide which inhibits cytochrome oxidase. Mitochondria oxidizing P-hydroxybutyrate in the presence of 0.5 mdd azide showed a crossover point between NAD and flavoprotein, so that the respiratory chain from flavoprotein to cyt a( +aa) became reduced in the transition from state 4 to state 3 (Muraoka and Slater, 196913). Except for NAD the latter behavior is similar to that of gastric mucosa upon stimulation. Aeide is not present, of course, but High and Hersey (1972) have speculated that a factor limiting the reaction of cytochrome oxidase with 0 2 in state 3 may appear in gastric mucosa under normal circumstances. Initiation of active transport leads to increased reduction of NAD in other tissues in addition to gastric mucosa. Direct observation of NADH fluorescence showed reduction following electrical stimulation in the eel electric organ (Aubert et al., 1964; Williamson et al., 1967b), in frog dorsal root ganglion (Rodriguez-Estrada, 1967), and in rabbit vagus nerve held near its physiological temperature (Landowne and Ritchie, 1971b). The increase in NADH in the electric organ was shown to be due to activation of glycolysis a t the steps involving phosphorylase and phosphofructokinase (Williamson et al., 1967a). Prior to reduction of NAD, a transient oxidation occurred with stimula-

316

RICHARD P. DURBIN

tion in the above experiments. This was abolished in rabbit vagus nerve by inhibitors of mitochondria1 oxidative phosphorylation (Landowne and Ritchie, 1971a). The oxidation could be explained by assuming that stimulation led to ATP hydrolysis, releasing ADP which removed a block to electron flow between NADH and 0 2 (crossover point between NADH and 0 2 ) . Subsequent mobilization of substrate then presumably reversed the initial oxidation of NADH to a more reduced state. This sequence of oxidation and then reduction may be a clue to the changes in cyt c reported by Kidder et al. (1966) in gastric mucosa. Upon reoxygenation from the anaerobic state, they observed an overshoot in the oxidation level of cyt c, followed by a reduction. The overshoot did not appear to be due to the shift from an anaerobic to an aerobic state, since it was abolished by SCN, NOz, OCN, and NH, ions (Kidder, 1970), which inhibit gastric acid secretion (LeFevre et al., 1964). This class of inhibitors is characterized by the presence of a nitrogen with an unshared pair of electrons. To account for the inhibition of the overshoot in oxidation of cyt c, we might postulate that the SCN class of ions blocks the gastric ATPase in viva The subsequent diminished levels of ADP and inorganic phosphate would inhibit electron flow (respiration) associated with phosphorylation, reverse or block substrate mobilization, and thereby yield an oxidized respiratory chain, as observed by Hersey and Jobsis (1969). It appears that this explanation is overly simplified. Sachs et al. (1969) have pointed out that it does not account for the modest effects of SCN on respiration. Moody (1968) found little or no inhibition by SCN of 0 2 uptake in dog stomach a t concentrations that practically abolished acid secretion. In sacs of spontaneously secreting frog mucosa, 6 or 12 mM SCN reduced acid secretion by 78%, but OZ uptake by only 23% (Bannister, 1964). It should be kept in mind that not all mucosal respiration is associated with secretion (Davenport, 1952; Bannister, 1966). Nevertheless, it appears that SCN to some extent uncouples respiration and acid secretion in the intact mucosa. In this connection it is perhaps relevant that large concentrations of classic uncoupling agents (e.g., dicumarol) cause progressively greater oxidation of the whole respiratory chain in isolated mitochondria (Chance et al., 1963; Van Dam, 1967). Experiments with isolated mitochondria do not support the thesis that SCN is a conventional uncoupler, however. SCK, in concentrations of the order of 40 mM, appreciably inhibited respiration in gastric mitochondria (Forte et al., 196713) and liver mitochondria (Kidder, 1968). Neither group observed the decrease in the ratio of phosphorylation to 0 2 uptake (P/O ratio) that would have been expected for an uncoupling agent. A somewhat different picture emerges from the work of Sachs et al. (1970) with rat liver

SECRETORY EVENTS IN GASTRIC MUCOSA

317

mitochondria. Two effects were found; 10 mM SCN inhibited state-3 respiration with P-hydroxybutyrate as substrate by 25%, and i t reduced the P/O ratio by 20-25oJ,. Increasing the ADP/SCN ratio restored the oxidation rate. This suggested that SCN was behaving like atractylate, inhibiting the binding of ATP and ADP to the mitochondria under study, and Sachs et al. (1970) confirmed the latter by direct measurement of nucleotide binding. Atractylate is known to inhibit oxidative phosphorylation by competitive inhibition of nuclrotide binding and translocation (Bruni et al., 1964; Klingenberg, 1970). It also inhibits respiration and secretion in gastric mucosa (Sachs et al., 1968). The partial uncoupling noted by Sachs et al. (1970) requires further explanation. A finding that may be relevant is the observation by Mitchell and Moyle (196913) that SCN is a rapidly permeating ion in rat liver mitochondria. This property was used by Papa et al. (1970), who showed in “inside-out” submitochondrial particles that respiration-driven proton uptake was accelerated by SCN (1 mM) and other permeant anions. At the same time SCN decreased the velocity constant describing H ion efflux. Papa et al. suggested that the permeant anions had reduced the transmembrane electrical potential (by making the inside less positive), facilitating proton uptake and hindering efflux. Mitchell and Moyle (1969a) have presented evidence indicating that the inside of an intact mitochondrion is electrically negative with respect to the outside, opposite the polarity of the submitochondrial particle mentioned above. In their interpretation, valinomycin-induced K uptake is passive, leading to equilibration of K ion across the mitochondria1 membrane. The interesting observation of Sachs et al. (1970), shown in Fig. 4, may be explicable in this context. Partial collapse of the electrical gradient by SCN should rcduce the electrogenic valinomycin-induced K uptake, and this was observed. The nonelectrogenic exchange of K for H ion was unaffected by SCN (Fig. 4). Partial uncoupling of mitochondria by SCN might therefore arise from the increased expenditure of energy to maintain pH or electrical gradients, or both. C. High-Energy Intermediates

In another approach to the problem of energy coupling, we measured various intermediates in isolated frog gastric mucosae which had been rapidly frozen under different conditions (Durbin and Michelangeli, 1972; A. Nickel, F. Michelangeli, and R. P. Durbin, unpublished observations, 1973). A ratio of tissue levels, e.g., ATP/ADP, is particularly reliable in such studies, since errors due to adherent fluid or mucus cancel out.

318

RICHARD P. DURBIN

Wilhoul SCN-

--e -250

r n I-I rn1n-l

-245

-.255

050pglrnl

3

-0 -

Nigericin

I

..

T 1

0

-250

0

n

25mMK'

. IMilochadrio 45 mg prolem

+l rnm -I -245

FIG.4. Effect of SCN ion on K ion uptake and efflux in rat liver mitochondria induced by valinomycin and nigericin. Upward deflection] K uptake; downward deflection, K efflux. (Sachs et al., 1970.)

Maximal stimulation of resting mucosae significantly depressed the ATP/ADP ratio in these experiments. The preponderance of mitochondria in oxyntic cells supports the assumption that the observed change took place primarily in that cell type. Depression of ATP/ADP by stimulation of secretion then indicates (1) phosphorylation is intimately involved with acid secretion; (2) the primary effect of secretagogues is to increase ATP utilization. Substrate mobilization follows, not precedes, the onset of secretory activity. Treatment with oubain (Davenport, 1962) or removal of K ion from the bathing solutions (Davis et al., 1965) procedures that inhibit acid secretion, reversed the decrease in ATP/ADP ratio due to histamine (A. Nickel, F. Michelangeli, and R. P. Durbin, unpublished observations, 1973). Both modes of inhibition reduce intracellular K levels, leading to a quasiresting mucosa. SCN produced no significant change in ATP/ADP ratio in our experiments, in agreement with the prior findings of Forte el al. (1965). These results exclude the possibility that SCN acts only as an uncoupler a t the mitochondria1 level in stomach.

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319

V. CONCLUSION

The diverse aspects of gastric physiology and pathology touched upon here find unification in considering the role of the cell membranes. They act as an osmotic barrier and osmotic link, accomplishing the translocation of solutes or chemical groups between the phases on either side (Mitchell, 1970). Most of the problems mentioned have not been solved; yet progress has been made. We may anticipate that in future years the field of gastric secretion will be successfully integrated with studies of proton translocation throughout nature. REFERENCES Alonso, D., Nigon, K., Dorr, I., and Harris, J. B. (1967). Amer. J . Physiol. 212, 992. Alonso, D., Park, 0. H., and Harris, J. B. (1968). Amer. J . Physiol. 215, 1305. Altamirano, M. (1969). Amer. J . Physiol. 216, 33. Altamirano, M.,and Durbin, R. P. (1972). Gastroenterology 62, 716. Aubert, X.,Charice, B., and Keynes, R. D. (1964). Proc. Roy. Soc., Ser. B 160, 211. Baker, P. F. (1965). J . Physiol. (London) 180, 383. Bannister, W. H. (1964). Nature (London) 203, 978. Bannister, W.H. (1966). J . Physiol. (London) 186, 89. Bielawski, J., Thompson, T. E., and Lehninger, A. L. (1966). Biochem. Biophys. Res. Commun. 24, 948. Bonting, S. L. (1970). I n “Membranes and Ion Transport ”(E. E. Bittar, ed.), Vol. 1, pp. 257-363. Wiley, New York. Bruni, A., Luciani, S., and Contessa, A. R. (1964). Nature (London) 201, 1219. Chance, B., and Williams, G. R. (1955). J. Biol. Chem. 217, 409. Chance, B., Williams, G. R., and Hollunger, G. (1963). J . Biol. Chem. 278, 439. ChBtail, M., and FourniB, J. (1969). Amer. 2001.9, 983. Davenport, H.W. (1952). Fed. PTOC.,Fed. Amer. SOC.Exp. Biol. 11, 715. Davenport, H.W. (1962). PTOC. SOC.Exp. Biol. Med. 110, 613. Davenport, H. W. (1964). Gastroenterology 46, 245. Davenport, H.W. (1965). Gastroenterology 49, 189. Davenport, H. W. (1967a). New Engl. J . Med. 276, 1307. Davenport, H.W. (1967b). PTOC. Soc. Exp. Biol. Med. 126, 657. Davenport, H.W. (1968). Gastroenterology 54, 175. Davenport, H. W. (1970). Gastroenterology 59, 505. Davenport, H. W. (1972). Sci. Amer. 226, 86. Davenport, H. W., Rehm, W. S., and Overholt, B. F. (1967). PTOC. SOC.Exp. Biol. Med. 126, 841. Davis, T. L., Rutledge, J. R., Keesee, D. C., Bajandas, F. J., and Rehm, W. s. (1965). Amer. J . Physiol. 209, 146. Durbin, R. P., and Kasbekar, D. K. (1965). Fed. PTOC.,Fed. Amer. SOC.Exp. Biol. 24, 1377. Durbin, R. P., and Kircher, A. B. (1973). Biochim. Biophys. Acta (in press). Durbin, R. P., and Michelangeli, F. (1972). In “Gastric Secretion” (G. Sachs, E. Heinz, and K. J. Ullrich, eds.), pp. 307-318. Academic Press, New York. Durbin, R. P., Carson, L., and Nickel, A. (1973). GaStTOenterOlOgy 64, 721.

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Martin, B. K. (1963). Nature (London) 198, 896. Mitchell, P. (1961). Nature (London) 191, 144. Mitchell, P. (1970). I n “Organization and Control in Prokaryotic and Eukaryotic Cells” (H. P. Charles and B. C. J. G. Knight, eds.), pp. 121-166. Cambridge Univ. Press, London and New York. Mitchell, P., and Moyle, J. (1969a). EUT.J . Biochem. 7, 471. Mitchell, P., and Moyle, J. (1969b). EUT.J . Biochem. 9, 149. Moody, F. G. (1968). Amer. J . Physiol. 215, 127. Moody, F. G. (1971). Amer. J. Physiol. 220, 467. Moody, F. G. (1972). I n “Gastric Secretion” (G. Sachs, E. Heinz, and K. J. Ullrich, eds.), pp. 432-452. Academic Press, New York. Moody, F. G., and Durbin, R. P. (1969). Amer. J. Physiol. 217, 255 Moore, C., and Pressman, B. C. (1964). Biochem. Biophys. Res. Commun. 15, 562. Motais, R., and Garcia-Romeu, F. (1972). Annu. Reu. Physiol. 34, 141. Muraoka, S., and Slater, E. C. (1969a). Biochim. Biophys. Acta 180, 221. Muraoka, S., and Slater, E. C. (1969b). Biochim. Biophys. Acta 180, 227. Neumann, J., and Jagendorf, A. T. (1964). Arch. Biochem. Biophys. 107, 109. Pacifico, A. D., Schwartz, M., MacKrell, T. N., Spangler, S. G., Sanders, S. S., and Rehm, W. S. (1969). Amer. J. Physiol. 216, 536. Papa, S., Guerrieri, E., Lorusso, M., and Quagliariello, E. (1970). FEBS (Fed. Eur. Biochem. SOC.),Lett. 10, 295. Pressman, B. C., Harris, E. J., Jagger, W. S., and Johnson, J. H. (1967). Proc. Nat. Acad. Sci. U.S. 58, 1949. Rehm, W. S. (1972). In “Metabolic Pathways” (L. E. Hokin, ed.), third ed., Vol. VI, pp. 187-241. Academic Press, New York. Rehm, W. S., and Sanders, S. S. (1972). I n “Gastric Secretion” (G. Sachs, E. Heinz, and K. J. Ullrich, eds.), pp. 91-110. Academic Press, New York. Rodriguez-Estrada, C. (1967). Brain Res. 6, 217. Sachs, G., Mitch, W. E., and Hirschowite, B. I. (1965). Proc. SOC.Exp. Biol. Med. 119, 1023. Sachs, G., Collier, R. H., Shoemaker, R. L., and Hirschowitz, B. I. (1968). Biochim. Biophys. Acta 162, 210. Sachs, G., Collier, R. H., Pacifico, A., Shoemaker, R. L., Zweig, R. A., and Hirschowitz, B. I. (1969). Biochim. Biophys. Acta 173, 509. Sachs, G., Collier, R. H., and Hirschowitz, B. I. (1970). PTOC.SOC.Exp. Biol. Med. 133, 456. Sachs, G., Heinz, E., and Ullrich, K. J., eds. (1972a). “Gastric Secretion.” Academic Press, New York. Sachs, G., Shah, G., Strych, A., Cline, G., and Hirschowite, B. I. (1972b). Biochim. Biophys. Acta 266, 625. Sanders, S. S., O’Callaghan, J., Butler, C. F., and Rehm, W. S. (1972). Amer. J. Physiol. 222, 1348. Scholes, P., and Mitchell, P. (1970). J . Bioenerg. 1, 309. Schwartz, M., Pacifico, A. D., MacKrell, T. N., Jacobson, A., and Rehm, W. S. (1968). Proc. SOC.Exp. Biol. Med. 127, 223. Selwyn, M. J., Dawson, A. P., Stockdale, M., and Gains, N. (1970). Eur. J . Biochem. 14, 120. Shanbour, L. L. (1972). Gastroenterology 62, 809. Skou, J. C. (1957). Biochim. Biophys. Acta 23, 394. Solberg, L. A., Jr., and Forte, J. G. (1971). Amer. J. Physiol. 220, 1404.

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AUTHOR INDEX Numbers in italics refer to the page on which the complete references are listed.

A Abrams, A., 94, 166 Abrams, A., 94, 95, 108, 109, 140, 166, 161, 168, 170 Adams, C. W. M., 208, 838 Adams, P. H., 318, 380 Adelberg, E. A., 15, 128, 168 Adey, H. R., 220, 268 Adler, J., 56, 57, 162 Adye, J. C., 79, 166 Aganon, M. A., 76, 170 Ager, M. E., 292, SO3 Aghajanian, G. K., 220, 240 Agostini, B., 204, 838 Ahlgren, 5. K., 111, 140, 166, 166 Ahmed, K. A., 127, 166 Ahren, K., 284, 286, 297, 899 Alaupovic, P., 180, 248 Albrecht, I., 42, 43, 45, 46, 168, 169 Albuquerque, E. X., 217, 219, 838 Alexander, A. E., 185, 838 Allmann, D. W., 213, 221, 239, 844 Alonso, D., 315, 319 Aloof, S., 179, 205, 846 Alpen, E. L., 43, 169 Alpers, D. H., 3, 171 Altamirano, M., 312, 313, 319 Altwerger, L., 35, 169 Ambrose, E. J., 195, 848 Ames, B, N., 19, 92, 93, 130, 131, 166, 168, 170 Ames, G. F., 18, 19, 21, 130, 135, 136, 137, 139, 140, 147, 151, 152, 166, 170 Amos, D. B., 200, 944 Amsden, T., 103, 169 Ancowits, A. A,, 286, 899, 300

Anderson, B., 64, 69, 166, 171 Anderson, E., 284, SO0 Anderson, L. L., 200, 863 Anderson, R. L., 70, 71, 161, 169 Anderson, R. S., 103, 166, 165 Ando, T., 47, 51, 167, 171 Andreeva, I. V.,63, 67, 167 Andrews, E. P., 192, 847 Andrews, H., 199, 844 Anraku, Y., 14, 56, 166 Ansell, G. B., 178, 839 Antener, I., 47, 129, 163 Appleman, M. M., 180, 939 Appleton, J., 113, 160 Arakawa, T., 47, 61, 167 Araki, S., 214, 846 Arceneaux, J. L., 97, 166 Archibald, A. R., 191, 844 Arima, K., 109, 131, 166 Arky, I., 206, 861 Armstrong, C., 218, 860 Armstrong, J. McD., 287, 898 Arnaud, C., 113, 168 Arrow, V. K., 43, 166 Arst, H. N., Jr., 74, 75, 111, 131, 166, 169 Arstib, A. U., 202, 868 Arvindakshan, I., 220, 839 Asai, H., 215, 846 Asatoor, A. M., 42, 42, 166, 166 Asensio, C., 63, 166 Ashmore, J., 280, 301 Atkinson, D. E., 697, 300, 308 Attwood, D., 189, 215, 139 Atwater, I., 218, 860 Aubert, X., 315, 319 Augenstein, L. G., 189, 846 Augustyn, J. M., 221, 839 Avi-Dor, Y., 206, 220, 839, ,241 323

324

AUTHOR INDEX

Avigad, G., 63, 156 Avioli, L. V., 113, 156 Avron, M., 223, 244 Awasthi, Y. C., 213, 222, 239 Ayad, S., 182, 251 Azam, F., 297 Azzi, A., 204, 239

B Baadenhuysen, H., 216, 249 Baarda, J. R., 94, 95, 108, 109, 161 Babcock, D., 110, 112, 113, 157 Bachmann, B. J., 193, 239 Bachmann, E., 213, 221, 222, 239, 244 Baddiley, J., 191, 244 Baerlocher, K. E., 48, 156 Bagshawe, K. D., 201, I39 Bajandas, F. J., 318, 319 Bajwa, G. S., 201, 243 Baker, P. F., 313, 319 Balantine, R., 205, 206, 239 Baldini, I., 208, 248 Balint, M., 215, 2.40 Ball, E. G., 291, 299 Baloeet, L., 206, 207, 239 Bangham, A. D., 178, 180, 181, 185, 186, 188, 198, 239 Bannister, T. T., 223, 250 Bannister, W. H., 316, 319 Barash, H., 6, 166 Barbanti-Brodano, G., 199, 239 Barber, A. J., 196, 199, 239, 245 Barbu, E., 235, 241 Bard, M., 127, 156 Barenholz, Y., 180, 243 Barham, F. W., 229, 230, 246, 278, 300 Barman, T. E., 178, ,939 Barnes, E. M., Jr., 121, 123, 153, 156, 160, 163 Baron, C., 94, 95, 140, 165, 156, 161, 162 Baron, D. N., 45, 156 Barratt, R. W., 28, 156 Barzilay, M., 205, Z 4 f Bassel, J., 75, 156 Batterham, T. J., 173 Battersby, E. J., 268, 269, 273, 301 Batzri, S., 234, 239 Bauerle, R. H., 30, 138, 156 Baxter, A. W., 25, 156

Bayliss, 0. B., 208, 238 Beare, J. L., 180, 245 Beatty, C. H., 272, 297 Beaufay, H., 213, 239 Bechet, J., 38, 39, 128, 130, 135, 147, 151, 156, 161 Beck, J. C., 284, 299 Becker, A., 117, 169 Becker, F. F., 150, 156 Beckwith, J. R., 107, 170 Beebe, J. L., 123, 156 Begueret, J., 235, 239 Bellman, C., 220, 847 Ben-Bassat, H., 196, $39, 245 Bender, W. W., 205, 239 Benedetti, E. I,., 203, 208, 209, 210, 211, 239, 243 Bennett, G. V., 277, 297 Bennett, R. L., 104, 105, 131, 134, 156 Bennum, A., 223, 239 Benson, C. E., 81, 157 Benzonana, G., 178, 180, 239 Beppu, M., 109, 131, 156 Berg, C. M., 13, 130, 158 Berg, H. C., 205, 239 Berger, D., 205, 245 Berger, E. A., 16, 17, 157 Berkowitz, D., 69, 71, f 5 7 Berlin, I., 194, 239 Berman, M., 65, 157 (see Berman-Kurtz,

M.) Berman-Kurtz, M., 61, 65, 157 (see Berman, M.) Berndt, W. O., 193, 241 Bernhard, W., 192, 248 Bernstein, A., 117, 157, 167, 169 Bernstein, H., 149, 157 Berry, D. H., 239 Berwick, L., 195, 239 Betz, W., 192, 239 Beug, H., 194, 239 Beverin, S., 53, 117, 157 Bhagwanani, S. G., 235, 239 Bhattacharya, G., 289, 297 Bhattacharya, P., 88, 91, 167 Bielawski, J., 306, 319 Bigalli, G., 183, 247 Bihler, I., 275, 276, 288, 289, 290, 291, 297,298 Bils, R. F., 226, 243

AUTHOR INDEX

Binder, H. J., 112, 170 I k g , R. J., 271, 298 Bird, P. R., 179, 2.40 Birge, C. H., 121, 157, 158 Birnbaunier, L., 231, 232, 233, 240, 249 Biro, N. A,, 215, 240 Blair, A., 2, 42, 43, 1 Y 1 Blatt, L. M., 277, 298 I3laustein, M., 99, 172 Blecher, M., 229, 240, 278, 298 IHeehan, N. M., 274, 275, 298 Bloom, B. A,, 125, 168 Bloom, F. E., 220, 240 Blostein, R., 99, 101, 103, 157, 1Y2 Blumberg, J. W., 202, 251 Blumenthal, R., X X I Bocek, R. M., 272, 297 Bodlund, K., 101, 158 Boezi, J. A., 10, 1,5Y Bogue, J. Y., 270, 298 Bohmann, V. R., 99, 164 Bohonos, N., 278, 300 Bolinger, R. E., 279, 299 Boll, M., 221, 240 Bolling, H., 127, ZYO Bolodia, G., 286, 298 Boman, H. G., 115, 16Y Bonner, I).M., 193, 239 Bonsen, P. P. M., 178, 251 Bonting, S. L., 313, 319 Boorman, K. E., 197, 240 Boos, W., 56, 57, 59, 132, 139, 157, 165, 1 YO, 1 Y S Bordes, A. M., 41, 130, 1Y1 Borland, J. L., 2, 42, 43, 166 Bornstein, J., 274, 287, 298, 302 Bos, C. J., 210, 226, 227, 228, 242, 245 Bosmann, H. B., 220, 240 Boucrot, P., 180, 240 Bourd, G. I., 63, 67, 15Y, 161 Bowman, M. H., 202, 206, 240, 248 Bowman, R. H., 271, 272, 272, 273, 298, 301

Bowyer, F., 259, 298 Boyd, N. D., 186, 246 Boyer, P., 178, 240 Braasch, D., 206, 240 Brackett, B. G., 219, 251 Bradbeer, C., 84, 8.5, 159 Bradfield, G., 110, 112, 113, 157

325 Bradley, D., 110, 112, 113, 15Y Bradlow, B., 205, 240 Braganca, B. M., 220, 239 Bramley, T. A., 203, 243 Branton, D., 211, 240 Braun, V., 192, 240 Bray, G. A., 280, 298 Breiman, A , , 211, 251 Brenner, R. R., 193, 252 Brenner, S., 107, 1YO Bresler, S. E., 216, 240 Breslow, R. E., 85, 132, 157 Bretscher, M. S., 199, 208, 211, 240 Brierley, G., 220, 243 Briggs, D. R., 213, 240 Brink, N. G., 27, 29, 157 Britten, A. G., 220, 242 Brockerhoff, H., 179, 180, 240 Brockman, R. W., 81, 157 Brodehl, J., 45, 157 Brody, 0. V., 197, 198, 199, 243 Bronk, M. S., 286, 298 Brot, N., 92, 157 Brown, C. E., 50, 51, 52, 53, 74, 157, 169 Brown, D. H., 284, 285, 286, 302 Brown, K. D., 10, 11, 130, 157 Brown, K. K., 72, 172 Bruce, A. L., 211, 244 Bruni, A., 317, 319 Brutlag, U., 211, 251 Bryce, G. F., 92, 157 Bryne, W. L., 202, 252 Brzin, M., 218, 240 Budreau, A., 13, 17, 129, 132, 165 Bulhrook, R. D., 286, 302 Burhach-Westerhuis, G. J., 178, 251 Burger, M., 259, 300 Burger, M. M., 196, 197, 840 Burk, I)., 266, 301 Burke, G., 231, 240 Burmeister, M., 89, 167 Burnett, C. H., 113, 147, 157, 173 Burnstein, C . , 208, 221, 222, 240, 249 Burrous, S. E., 10, 157 Burt, D. H., 205, 240 Burton, E. G., 110, 111, 140, 150, 157 Busse, D., 76, 79, 157, 160 Bussey, H., 39, 41, 157, 158 Butcher, R. W., 232, 249 Butler, C. F., 307, 321

326

AUTHOR INDEX

Butler, R. D., 220, 242 Butler, W. L., 223, 248 Butlin, J. B., 114, 168 Buttin, G., 2, 52, 54, 55, 56, 57, 168, 168 Byers, B. R., 97, 131, 169 Byrne, W. L., 211, 243

C Cadenas, E., 276, 277, 293, 298, 301 Cahill, G. F., Jr., 270, 280, 298 Caiafa, P., 213, 216, 241 Caldwell, E. J., 201, 240 Calvet, J., 125, 161 Calvo, J. M., 144, 168 Camejo, G., 189, 240 Campbell, A., 84, 85, 168 Campbell, J. N., 194, 246 Canessa-Fischer, M., 226, 243 Carafoli, E., 221, 240 Carr, J., 229, 240 Cam, L., 280, 301 Carraway, K. L., 204, 840 Carroll, P. M., 219, 2.41 Carsenti, H., 197, 841 Carson, L., 312, 319 Carter, J. R., 52, 53, 54, 55, 139, 168, 160, 277,298,301 Carton, D., 47, 163 Carubelli, R., 234, 241 Case, M. E., 138, 168 Caetorph, H., 127, 170 Casu, A., 202, 205, 208, 213, 241, 243, 248 Cater, B. R., 216, 234, 241 Cavalotti, C., 226, ,841 Cavanaugh, D. J., 193, 241 Cavard, D., 235, 241 Cavert, H. M., 275, 276, 297, 298 Celada, F., 101, 168, 169 Cellino, M., 226, 243 Cerletti, P., 213, 216, 222, 2.41 Chaiet, L., 61, 131, 182 Challoner, D. R., 271, 279, 298, 303 Chan, S. I., 189, 203, 204, g43, 261 Chance, B., 221, 248, 314, 315, 316, 319 Chang, R., 84, 85, 168 Changeux, J-P., 220, $41 Chapman, D., 204, 246 Charamella, L. J., 13, 130, 168

Chariot, J., 241 Chaudhuri, S., 197, 241 Chaudry, H., 287, 289, 299 Cherest, H., 41, 130, 168, 169 Chernick, S. S., 178, 260 Chernyak, E. A., 193, 241 Chesney, R. E., 76, 168 Chetail, M., 319 Cheung, W. Y., 215, 241, 315, 322 Chiappe de Cingolani, G. E., 231, 249 Chinard, F. P., 76, 170 Chojnacki, T., 75, 160 Choke, H. C., 31, 33, 168 Christensen, M. S., 74, 75, 166 Christensen, R. O., 208, 222, 249 Chung, Y. S., 63, 168 Cirillo, V. P., 74, 75, 168, 166 Clark, D., 90, 91, 170 Clark, J. J., 267, 299 Clausen, T., 281, 288, 289, 290, 291, 298, 300 Clement, J. R., 180, 240 Clewe, T. H., 201, 261 Cline, G., 313, 321 Cocking, E. C., 193, 841 Codington, J. F., 193, 202, 219, 241, 260 Cohen, B. L., 99, 147,160 Cohen, G. N., 2, 52, 168, 164, 168 Cohn, M., 62, 178 Colacicco, G., 185, 186, 187, 189, 190, 240, 241 Coleman, R., 202, 203, 209, 2-41, 243 Coles, H. S., 315, 322 Colley, C. M., 202, 241 Collier, R. H., 316, 317, 318, 321 Coman, 11. R., 195, 239 Comfurius, P., 181, 202, 203, 205, 660,264 Condie, F., 75, 169 Condon, 8. E., 282, 308 Condrea, E., 179, 180, 188, 190, 205, 206, 217, 218, 220, 239, 2.41, 246, 260 Contessa, A. R., 317, 319 Cook, G. M. W., 197, 198, 199, 241 Cook, P., 99, 101, 166 Coombs, R. R. A., 663 Cooper, D. A., 248 Cooper, O., 291, 299 Cooper, R. A., 103, 163 Cordaro, J. S., 69, 168 Cordes, E. H., 180, 189, 226, 246

327

AUTHOR INDEX

Cori, C. F., 261, 267, 275, 280, 284, 299, 300

Cornblath, M., 284, 285, 286, 302 Corwin, L. M., 11,23, 130, 172 Cosloy, S. D., 13, 130, 158 Costin, A., 217, 220, 244, 252 Cotes, P. M., 284, 298 Cottier, P., 129, 163 COX, G. B., 92,93, ii4,168,16r, 173 Cozzarelli, N. R., 61, 63, 158, 164 Crabeel, M., 38, 39, 41, 130, 135, 151, 158, 161 Cramer, R., 221, 248 Crandall, M., 114, 115, 168 Crane, F. L., 213, 222, 239 Crane, R. K., 76, 158, 166, 280, 303 Crapo, L., 55, 131, 167 Crawford, M. A., 45, 166 Crawhall, J. C., 43, 168 Crocken, B., 72, 168 Crofford, 0. R., 264, 274, 275, 282, 293, 298, 301, 302 Cronan, J. E., Jr., 121, 138, 168 Crone, C., 287, 298 Csuzi, S., 184, 241 Cuatrecasas, P., 178, 229, 230, 231, 241, 245, 277, 278, 297, 298, 300 Curran, P. F.,xxi, 256, 302, 314, 316, 320 Currie, G. A., 201, 239 Curtis, A. S. G., 193, 194, 195, 241 Curtiss, R., 111, 13, 130, 168 Cushman, S. W., 281, 298 Cuthbert, A. W., 219, 226, 241, 2.62 Cutlar, 0 . E., 277, 303 Czech, M. P., 278, 299

D Dahl, R., 65, 168 Dahlqvist, A,, 77, 166 Ilamadian, R., 89, 158 Danon, I)., 198, 206, 207, 248, 243, 24Y Dao, N., 235, 252 Darlington, A. J., 81, 131, 158 Darrow, 1). C., 113, 158 Ilaughaday, W. H., 284, 285, 286, 302 Ilavenport, H. W., 310, 312,316, 318, 319 David, J. R., 200, 242, 249 navies, M., 89, 160

navies, R. E., 318, 320 Davis, B. D., 13, 17, 129, 130, 168, 169, 166

Davis, D. G., 224, 848 Davis, R. H., 35, 129, 159 Davis, T. L., 318, 319 Davis, W. B., 97, 131, 169 Davoren, P. R., 281, 282, 299 Dawson, A. P., 306, 321 Dawson, E. C., 74, 172 Dawson, K. G., 284, 299 Dawson, R. M. C., 179, 180, 181, 185, 186, 187, 188, 189, 239, 2.42, 249 Day, P. R., 149, 160 Deak, G., 208, 250 Debavadi, C. S., 81, 167 deBodo, R. C., 286, 299, 300 DeBons, A. F., 277, 299 Debuch, H., 179, 253 DeBusk, A. G., 26, 27, 29, 30, 31, 37, 130, 159, 168, 171, 172, 1Y3 DeBusk, B. G., 26, 169 DeCicco, B. T., 95, 171 decrombrugghe, B., 65, 159 DeDuve, C., 213, 239 DeHartog, M., 292, 299 De Hass, G. H., 178, 179, 181, 184, 188, 189, 239, 840, 246, 261, 262 DeJong, J. W., 221, 2.42 Ilelbauffe, D., 211, 239 del Campillo-Campbell, A , , 84, 85, 168 DeLuca, H. F., 113, 166, 159 DeMartinis, F. D., 226, 247' 1)e Mello, W. C., 217, 242 Demerec, M., 107, 109, 159, 166 DeMoss R. D., 10, 157 Denbury, J. L., 220, 84% Ilenduchis, B., 194, 246 Denhardt, G. H., 149, 167 Dennert, G., 121, 162 Dent, C. E., 2, 42, 43, 45, 113, 127, 166, 157,169 Deppe, G., 121,162 de Robichon-Szulmajster, H., 41, 74, 75, 130, 158, 1 6 9 , ' m Desbuquois, B., 231, ,241, 277, 298 Desnuelle, P., 178, 180, 185, 239, 243 Dettbarn, W. D., 218, 240 Deuel, T. F., 17, 163 Devi, A., 205, 220, 260

328

AUTHOR INDEX

De Vries, A,, 179, 180, 188, 190, 205, 206, 207, 220, 239, 841, 243, 246 de Zeeuw, J. R., 132, 173 Dharmalingam, K., 234, 24%' Diamond, J., 313, 320 Diana, A. L., 203, 262 Diehl, F., 224, 242 Dietz, G. W., 58, 61, 169 Di Girolamo, P. M., 84, 85, 159 Dill, I. K., 229, 230, 231, 246, 278, 300 D'Monte, B., 220, 247 Dobiasov, M., 222, 242 Dodd, B. E., 197, 240 Doggenweiler, C. F., 208, 242 Dondon, L., 235, 952 Donley, J., 224, 247 Doolan, P. D., 43, 159 Dorn, F., 91, 161 Dorn, G. L., 111, 169 Dorr, I., 315, 319 Douglas, H. C., 75, 159 Downing, S., 43, 147, 150, 169 Doy, C. H., 138, 144, 163 Iloyle, M. E., 53, 159 Drahota, Z., 222, 242 Iheyfuss, J., 106, 107, 131, 134, 154, 159, 168

Drillien, R., 86, 87, 169 Drummond, G., 272, 299 Drummond, K. N., 47, 169 Drury, D. R., 270, 299 Dudai, Y., 220, 2.42 Dulin, W. E., 267, 299 Dunham, P. B., 99, 101, 159, 165 Dunnick, J., 233, 252 Durant, J. L., 42, 43, 47, 49, 51, 147, 152, 169

Durbin, R. P., 307, 309, 312, 313, 317, 319, 3.20, 321

Duttera, S. M., 202, 252 Dvorkin, B., 283, 301 Dwyer, J. M., 200, 2.48 Dyadyusha, G. P., 214, 243 Dzhanbaeva, N. R., 249

E Eagleton, G. E., 99, 159 Eaglstein, W. H., 206, 242 Earnshaw, M. J., 220, 242

Easty, D. M., 195, 242 Easty, G. C., 194, 195, 242 Ebashi, S., 242 Ebert, W., 200, 242 Eddy, A. A., 193, 242 Edelman, P. M., 277, 280, 299 Edgar, R. S., 149, 167 Edidin, M., 195, 248 Edwards, K. D. G., 42, 43, 166 Efron, M. L., 46, 170 Egan, J. B., 71, 159, 162, 167 Egelrud, T., 180, $42 Eggermont, E., 77, 169 Eichholz, A., 210, 242 Eilam, Y., 260, 299 Eilermann, L. J. M., 221, 242 Eisenberg, S., 235, 242 Eklund, J., 113, 168 Elbers, P. F., 220, 263 Eldefrawi, A. T., 220, 242 Eldefrawi, M. E., 220, 242 Elliker, P. R., 70, 71, 166 Ellingson, J. S., 216, 242 Elliot, W. B., 221, 239 Ellis, E. H., 211, 243 Ellory, J. C., 99, 101, 169, 172 Elorza, M. V., 74, 75, 131, 169 Elsas, L. J., 47, 49, 51, 76, 77, 79, 152, 157, 160, 169

Emerson, M. R., 125, 160 Emerson, S., 125, 160 Emilio, M. G., 306, 320 Emmelot, P., 203, 208, 209, 210, 226, 227, 228, 239, 242, 243 Endo, H., 119, 173 Englesberg, E., 50, 51, 52, 53, 56, 66, 129, 140, 160, 162, 163, 167, 170, 171 Engstrom, L. H., 213, 243 Ennis, H. L., 91, 97, 119. 160, 166, 173 Epel, B. L., 223, 248 Epshtein, Y. A., 234, 247 Epstein, W., 63, 65, 67, 88, 89, 91, 104, 121, 134, 135, 139, 147, 167, 160, 170, 172

Erlichman, J., 281, 302 Erlij, D., 312, 320 Emster, L., 221, 246, 246 Ershoff, B. H., 201, 243 Esaki, K., 84, 86, 163, 164 Esfahani, M., 121, 123, 160

329

AUTHOR INDEX

Esposito, S., 185, 243 Essex, H. E., 207, 243 Evans, J. V., 99, 147, 160 Evanson, J. M., 113, 160 Even-Shoshan, A., 4, 6, 7, 161 Eylar, E. H., 197, 198, 199, 841, 243

F Fabian, I., 180, 188, 190, 241 Fahey, K. R., 113, 160 Fahmy, I)., 235, 239 Fain, J. N., 229, 250,278, 299, 302 Fairbanks, G., 207, 261 Falcoz-Kelly, F., 58, 63, 160 Fall, L., 300, 302 Fanestil, D. D., 306, 320 Farquhar, M. G., 208, 243 Feenstra, M., 221, 242 Feldman, G., 234, 252 Fellows, R. E., 43, 160 Ferenci, T., 58, 61, 67, 160 Feretos, R., 226, 247 Ferro, M., 208, 248 Fiehn, W., 225, 243, 244 Fina, J. J., 282, 302 Fincham, J. R. S., 149, 160 Finean, J. B., 202, 203, 209, 241,243 Finer, E. G., 189, 243 Finerman, G. A. M., 46, 160 Fink, G. R., 144, 158 Fischer, H., 204, 235, 246, 253 Fischer, S., 226, 243 Fisher, R. B., 274, 275, 276, 286, 297, 298, 299 Fishman, D. A , , 207, 263 Fleischer, B., 208, 209, 213, 2.43 Fleischer, S., 208, 209, 213, 220, 225, 243, 247 Flemstrom, G., 310, 320 Flinn, R. B., 280, 298 Flook, A. G., 189, 243 Flye, M. W., 200, 244 Fogel, S., 127, 162 Foltz, E. L., 61, 131, 162 Fong, C. T. O., 277, 299 Forte, G. M., 226, 243, 309, 316, 320 Forte, J. G., 226, 243, 305, 307, 308, 309, 312, 316, 318, 320, 321 Forte, T., 307, 308, 3.20

Fournib, J., 319 Fox, C. F., 2, 52, 53, 54, 55, 63, 65, 67, 121, 123, 137, 139, 148, 153, 158,160, 163,i r y FOX, M., 2, 42, 43, 160, i r i Fraenkel, D. G., 58,.63, 65, 160, 171 Frank, L.,7, 160 Fratantoni, J. C., 235, 248 Frasier, L. W., 306, 320 Fredman, D. L., 214, 243 Fredrickson, D. S., 3, 171 Freedberg, W. B., 61, 63, 158 Freer, J. H., 203, 251 French, S. W., 221, 243 Frenk, S., 208, 242 Frenning, B., 310, 311, 320 Freychet, P., 277, 279, 300 Friede, J. D., 24, 25, 130, 160 Friedman, R. M., 199, 213, 243 Frimpter, G. W., 43, 160 Fritz, I. B., 272, 299 Fromter, E., 313, 320 Frumkin, S., 58, 168 Furlong, C. E., 6, 7, 9, 12, 14, 160, 1'72 Furth, E., 43, 160

0 Gaham, A. G., 215, 239 Gainer, H., 217, 243 Gains, N., 306, 321 Gajewski, W., 75, 160 Gallai-Hachard, J., 202, 206, 243 Galloway, W. H., 89, 133, 166 Galsworthy, P. R., 107, 109, 131, 154, 167 Gamble, J. L., 113, 160 Gammack, D. B., 189, 239 Ganesan, A. K., 56, 57, 58, 59, 160, 169 Garan, H., 205, $39 Garbus, J., 206, 222, 223, 263 Garcia-Romeu, F., 306, 321 Gardos, G., 206, 251,262 Garen, A., 149, 161 Garen, S., 149, 161 Garland, P. B., 271, 272, 302 Garner, H. R., 30, 166 Gatt, S., 180, 181, 183, 243, 246 Gaudy, E. T., 72, 172 Gauffin, M. L., 206, 223, 250 Gazsotti, P., 221, 240

3 30 Geck, P., 259, 260, 299 Gee, R., 316, 320 Geld, H., 279, 299 Geller, A. M., 211, 243 Gellissen, K., 45, 167 Gergely, J., 215, 224, 243, 246 Gerisch, G., 194, $39 Gershanovitch, V. N., 63, 67, 167, 161 Gesner, B. M., 201, 243, 263 Ghosh, S,, 64, 166 Ghuysen, J. M., 192, 194, 243, 261 Gibbons, A. J., 226, 244 Gibbons, I. R., 194, 261 Gibson, F., 92, 93, 114, 140, 168, 166, 167, 173, 174 Gilbert, J. C., 275, 276, 299 Gilbert, M., 271, 298 Gilbert, W., 55, 131, 147, 149, 161, 167 Gilboe, I). P., 24, 25, 130, 160 Giles, N. H., 138, 144, 168, 163 Gillespie, D. H., 107, 109, 169, 166 Gilvarg, C., 16, 17, 131, 168, 171 Gimmel, N. G., 214, 243 Ginsburg, V., 197, 201, 235, 243 Ginzburg, B. Z., xxi Giordano, M. G., 213, 216, 222, 2.41 Giovenco, M. A , , 213, 216, 222, 841 Giovenco, S., 222, $41 Girtio, C. B., 45, 166 Gits, J. J., 38, 39, 130, 135, 147, 151, 161 Gitter, S., 206, 207, 242, 243 Glaeser, R. M., 198, 202, 243 Glaser, M., 203, 204, 243, 261 Glassey, M., 125, 168 Glauert, A. M., 210, 243 Glazunov, E. A., 216,240 Gleason, M. K., 111, 166 Glick, J. L., 201, 243 Glick, M. C., 193, 197, 199, 263 Gliemann, J., 290, 298 Glorieux, F., 112, 113, 166, 161 Glover, S. W., 15, 128, 181 Glynn, I. M., 98, 161 Godefroy-Colburn, T., 235, 262 Godfrei, D. G., 262 Godson, G. N., 121, 168, 161 Gohmann, E. J., Jr., 316, 320 Goldberg, A. L., 201, 235, 243 Goldfine, H., 162 Goldsby, R. A,, 85, 132, 167

AUTHOR INDEX

Goldstein, M. S., 267, 274, 299, 301 Goll, D. E., 215, 244 Gommi, B. W., 219, 263 Good, R. A., 47, 169 Goodman, H. M., 280,284, 298,299 Goodman, S. I., 47, 51, 147, 161 Goodwin, B. L., 79, 173 Goodwin, J., 92, 167 Gordin, R., 89, 133, 161 Gordon, A. S., 16, 56, 167, 161, 204, 244 Gordon, E. E., 292, 299 Gordon, P., 300 Gots, J. S., 79, 81, 131, 166, 167, 163 Gottfried, E. L., 187, 244 Gottschalk, A., 183, 199, 244 Gouin, B., 197, 241 Gould, M. K., 287, 289, 298, 299 Govons, S., 152,166 Griisbeck, R., 89, 133, 161 Graf, E., 244 Grafius, M. A,, 214, 244 Graham, D., 306,320 Graham, J. B., 113, 147, 161, 173 Grande, E. F., 279, 298 Grant, M. E., 182, 261 Grant, R., 310, 312, 320 Gray, G. M., 202, 206,243 Gray, J. L., 248 Gray, R., 213, 214, 246 Green, D. E., 213, 220,221,222,239, 244, 246

Green, H., 150 166 Green, J. W., 205, 220, 226, 227, 240, 247, 260 Greenberg, J., 63, 168 Greene, M. L., 49, 161 Gregolin, C., 213, 244 Greig, M. E., 226, 244 Grenson, M., 37, 38, 39, 41, 80, 81, 83, 128, 130, 131, 135, 147, 148, 151, 152, 166, 168, 161, 162, 163

Gressel, J., 223, 244 Groen, J., 279, 299 Gronlund, A. F., 22, 23, 25, 130, 164 Gross, J. D., 13, 17, 129, 132, 166 Gross, S. R.,132, 166 Grossman, I. W., 202, 244 Groth, U., 151, 161 Grothaus, E. A., 200, 244 Guardiola, J., 12, 13, 15, 128, 161

33 1

AUTHOR INDEX

Giinther, T., 91, 161 Guerrieri, E., 317, 321 Guest, J. R., 5, 162 Guzman, R., 56, 58, 169 Gwatkin, R. B. L., 193, 244

H Habermann, E., 205, 206, 244 Haddock, B. A., 114, 170 Hafeman, D. R., 217, 244 Hageman, J. T., 800 Hagen, J. M., 291, 299 Haggerty, D., 193, 262 Hagihara, B., 66, 161, 244 Hagihira, H., 46, 166 Hagstrom, B., 207, 244 Hagstrom, B., 207, 244 Hakamori, S., 196, 199, 244, 246 Haley, A. B., 127, 164 Hall, J. G., 99, 169, 168 Hall, L. M., 271, 299 Hall, R. E., 56, 167 Hall, 2. W., 214, 244 Hallinan, T., 216, 234, 2.41 Halpern, Y. S., 4, 5, 6, 7, 140, 166, 161, 166 Halpin, R. A., 224, 247 Hamilton, J. G., 125, 161 Han, S. C . H., 180, 260 Hanel, K. H., 103, 162 Hanna, M. L., 84, 171 Hanson, T. E., 70, 71, 161, 169 Harcourt, J. A., 287, 298 Harold, F. M., 94, 95, 108, 109, 134, 153, 161, 162, 168 Harold, R. L., 95, 108, 109, 161 Harper, C., 113, 167 Harper, H. A., 43, I69 Harris, E. D., 193, 244 Harris, E. J., 306, 321 Harris, H., 43, 45, 99, 127, 147, 166, 169, 160, 169 Harris, J. B., 315, 319 Harris, P. E., 13, 130, 168 Hart, B. A., 138, 144, 163 Hart, E. W., 45, 166 Hartman, P. E., 69, 1'71 Hartmann, R., 236, 244 Harvald, B., 103, 162

Hasselbach,

W.,204, 224, 225,

238, 242,

243,944

Hasunuma, K., 111,168,163 Hatch, F. T., 198, 244 Hatfield, D., 63, 162, 163 Hauser, H., 180, 181, 185, 189, 242, 243 Haverland, L. H., 99, 164 Hawthorne, D. C., 75, 169, 167 Hawthorne, J. N., 178, 239 Hayashi, S., 60, 61, 168 Haydon, D. A., 197,204, 244 Hays, J. B., 72, 162, 167 Hazelbauer, G. L., 56, 57, 162 Heard, D. H., 197, 198, 199, 242, 244 Heath, E. C., 191, 192, 194, 244 Hechemy, K., 16.9 Hechtman, P., 3, 24, 25, 45, 162, 170 Heegen, H., 197, 262 Heemskerk, C. H. T., 179, 205, 240, 244, 260 Hegdekar, B. M., 213, 246 Hegyvary, C., 226, 227, 228, 244 Heidrich, H. G., 208, 244 Heinz, E., 260, 299, 305, 321 Heistoe, H., 244 Heitkamp, D. H., 202, 244 Helander, H. F., 308, 309, 320 Helling, R. B., 53, 169 Helmreich, E., 260, 280, 299, 300 Hemington, N., 180, 187, 188, 242 Hempling, H. G., 259, 301 Hems, R., 272, 273, 302 Henderson, L. M., 24, 25, 130, 160 Henderson, M. J., 262, 263, 274, 275, 284, 285, 286, 299, 301 Hendlin, D., 61, 131, 162 Hendrickson, H. S., 188, 244 Hengstenberg, W., 70, 71, 72, 162, 167, 170 Hennaut, C., 41, 130, 148, 152, 161, 162 Henning, U., 121, 162 Henrikso, K. P., 188, 244 Henrikso, R. C., 188, 244 Henry, S. A., 125, 127, 162 Heppel, L. A., 3, 6, 7, 9, 16, 17, 50, 58, 61, 130, 167, 169, 168, 167, 172 Heptinstall, S., 191, 244 Herbert, A. A., 5, 162 Herczeg, B. E., 315, 322 Hermann, K. O., 59, 166

332

AUTHOR INDEX

Hernandez, S., 61, 131, 162 Hersey, S. J., 314, 315, 316, 320 Here, F., 204, 244 Herzl, A., 180, 243 Hexum, T., 228, 244 Higashi, T., 117, 167 High, W. L., 314, 315, 820 Hilger, F., 148, 162 Hill, C., 117, 162 Hill, E. E., 216, 242 Hill, K. L., 70, 71, 72, 162, 167 Hill, R. L., 214, 251 Hillman, R. E., 46, 76, 77, 160, 162 Hingson, D. J., 310, 320 Hirota, Y., 119, 121, 162, 168 Hirsch, C. A., 61, 164 Hirschhorn, R., 203, 251 Hirschowite, B. I., 308, 309, 313, 316, 317, 318, 320, 821 Hjalmarson, A., 284, 286, 297, 299 Hlmeida, J. D., 199, 245 €10,M. W., 180, 244 Ho, R. J., 290, 299 Hochberg, A., 43, 130, 162 Hochstadt-Oeer, J., 78, 83, 137, 162 Hochstein, P., 239 Hoeschen, R. J., 287, 299 Hoffee, P. A., 66, 71, 160, 162 Hoffman, J. F., 99, 101, 159, 172, 227, 246, 263, 292, 299, 302 Hoffman, P. G., 99, 101, 162, 166 Hofnung, M., 63, 162, 163, 172 Hogben, C. A. M., 307, 320 Hogg, R. W., 50, 51, 52, 53, 74, 129, 132, 167,169,163

Hohenwallner, W., 286, 299, 301 Hoken, S. C., 278, 299 Hokin, L. F., 228, 244 Holby, M., 110, 112, 113, 167 Holden, J. T., 8, 24, 25, 132, 168, 172, 173 Holland, I. B., 117, 162 Holland, J. M., 42, 43, 169 Hollosey, J. O., 267, 268, 289, 299 Hollunger, G., 316, 319 Holmlund, C. E., 229, 230, 231, 246, 278, so0

Holtje, J.-V., 236, 244 Honig, G. R., 103, 163 Hooft, C., 47, 163 Hoos, R. T., 257, 260,299

Hopkins, I., 7, 160 Horecker, B. L., 56, 58, 63, 156, 160, 163, 167

Horne, R. W., 188, 289 Horwith, M., 43, 160 HOLI,C., 38, 39, 41, 130, 135, 151, 161 Houghton, C. R. S., 272, 273, 300, 302 Houssay, B. A., 284, 300 Houston, R. B., 211, 246 Hovmoller, S., 221, 246 Howard, R. L., 213, 246 Howell, J. I., 202, 235, 249 Hoyle, L., 199, 245 Hsia, J. C., 204, 246 Hsia, S. L., 206, 242 Hsu, C. C., 121, 148, 163 Hsu, F. Y., 279, 298 Hubbell, W. L., 204, 245 Huddlestun, B., 267, 274, 299, 301 Huestis, R. R., 103, 156. 163 Hughes, A., 185, 187, 246 Huh, H., 72, 164 Humphreys, T., 195, 248 Hunkeler, F. L., 211, 246 Hunter, J. E., 99, 164 Hurwitz, J., 121, 162 Hussey, C., 111, 134, 163 (see Hussey, E. C.) Hussey, E. C., 111, 171 (see Hussey, C.) Hutchin, M. E., 43, 169 Hutchison, D. J., 81, 157

I Iaccarino, M., 12, 13, 15, 128, 161 Ibrahini, S. A., 179, 180, 190, 245 Iida, S., 117, 170, 226, 228, 252 Ikemoto, N., 215, 224, 245 Iles, G. H., 215, 247 Illiano, G., 178, 229, 231, 241, 245, 277, 278, 298, 300 Ilse, D., 280, 289, 300 Imae, Y., 117, 163 Imerslund, O., 89, 133, 163 Irnre, S., 235, 246 Inbar, M., 196, 239, 246 Inesi, G., 215, 224, 242, 246 Ingle, 11. J., 267, SO0 Ioneda, T., 123, 160 Irr, J., 53, 140, 160

333

AUTHOR INDEX

Isaacson, D., 53, 129, 163 Isaksson, O., 286, 297 Ishikawa, S., 214, 245 Ishikawa, T., 111, 162, 163, lY2 Ishisawa, M., 119, 173 Ito, A., 211, 214, 245 Ito, J., 138, 1 Y3 Ito, S., 193, 245, 310, 320 Ivey, K. J., 310, 520 Iwashima, A., 86, 163

Joiris, C. R., 39, 163 Jolly, W., 220, 246 Jones, A. B., 229, 231, 249, 278, 302 Jones, M. D., 287, 298 Jones, T. H. D., 54, 163 Jones-Mortimer, M. C., 107, 163 Jund, R., 81, 83, 131, 163 Jung, C. Y., 218, 845 Juntti, K., 221, 245 Jurovitzkaya, N. V., 67, 161

J

K

Jackman, 1,. M., 173 Jackson, D. S., 182, 251 Jackson, L. J., 199, 245, 250 Jackson, M., 61, 131, 162 Jacob, F., 55, 119, 121, 140, 141, 162, 163, 168 Jacob, H. S., 103, 163 Jacobson, A,, 307, 321 Jacobson, E. S., 35, 130, 163 Jacobson, J. W., 138, 144, 163 Jagendorf, A. T., 306, 321 Jagger, W. S., 306, 321 Jain, M. K., 176, 178, 180, 189, 206, 226, 245 James, C. G., 198,24Y James, L. K., 189, 245 Jamieson, G. A , , 196, 199, 239, 248 Jandl, J. H., 103, 163 Jardillier, J. C., 197, 241 Jasaitis, A. A., 306, 320 Jayaraman, J., 234, 242 Jeanlos, R. W., 193, 199, $41, 246 Jeanrenaud, B., 290, 291, 297, 299, 300, 303 Jefferson, L. S., 272, 300 Jensen, L., 24.4 Jepson, J. B., 45, 156, 163 Jervis, H. H., 26, 1Y3 Jewett, S., 63, 65, 67, 139, 160 Jiji, R. M., 89, 133, 171 Jobsis, F. F., 314, 316, 320 Johnseine, P., 91, 170 Johnson, C. A,, 193, 844 Johnson, F. B., 94, 155 Johnson, J. H., 306,321 Johnson, K. G., 194,245 Johnson, L. H., 275, 302

Kabnck, H. R., 3, 16, 17, 137, 138, 153, 166, 161, 163, 228, 248, 256, 300 Kadner, R. J., 85, 159 Kaser, H., 129, 163 Kahan, F. M., 61, 131, 162 Kahn, C. R., 277, 299, SO0 Kaji, H., 277, 293, 298 Kakinuma, K., 292, 301 Kalckar, H. M., 56, 57, 163, 173 Kalle, G. P., 79, 131, 165, 171 Kalnian, C . F., 275, 302 Kamat, V. B., 204, 245 Kandrach, A,, 221, 222, 240 Kanegasaki, S., 192, 264 Kantero, I., 89, 133, 161 Kaplan, A., 180, 245 Kaplan, E., 204, 244 Kappy, M. S., 35,163 Kariya, B., 27, 29, 33, 167, l Y l Kariya, M., 180, 245 Karlish, S. J. D., 260, SO0 Kasarov, L. B., 205, 245 Kasbekar, D. K., 307, 309, 313, 314, 319, 320 Kashket, E. R., 55, 131, 132, 153, 154, lY3 Katchalsky, A., xxi, xxii, 198, 24Y, 313, 320 Kntes, M., 180, 181, 188, 245 Kattwinkel, J., 282, 283, 300 Kats, W., 205, 245 Katsen, R., 181, 248 Kaudewits, F., 131, 164 Kawamata, J., 117, 16Y Kawamura, H., 226,227, 248 Kawasaki, T., 84, 85, 86, 163, 164, 166 *-' Kay, W. W., 4, 5,22,23, 25, 130, 136 164

334 Kearney, E. B., 213, 240 Kedem, O., x x i , 313, S20 Keesee, D. C., 318, S19 Kefalides, N. A., 194, 246 Keith, A. D., 125, 127, 162, 164, 173 Kekornaki, M., 45, 164 Kelly, K. A., 26, 168 Kelly, R. B., 214, 244 Kemp, R. B., 197, 246 Kennedy, E. P., 2, 52, 53, 54, 55, 70, 90, 91, 123, 131, 139, 148, 153, 168, 160, 16S, 164, 166, 167, 169 Kepes, A., 52, 54, 164 Keret, R., 190, 241 Kerets, S., 233, 262 Kerr, R., 312, 520 Kessel, D. H., 13, 16, 17, 91, 129, 130, 132, 134, 164, 166 Kettman, J., Jr., 213, 246 Keynes, R. D., 306, 315, Sl9, S20 Kezdp, F. J., 185, 186, 246 Khaiat, A., 187, 245 Kiang, S. P., 286, 299, 300 Kidder, G. W., 111, 314, 316, 320 Kidwell, J. F., 99, 164 Kielley, W. W., 226, 246 Kim, B. S., 88, 91, 134, 135, 147,160 Kim, K. H., 277, 298 Kim, Y. S., 312,320 King, J. W. B., 99, 147, 160 King, T. E., 213, 246 Kinsey, J. A., 35, 130, 164 Kinter, W. B., 77, 170 Kipnis, D. M., 261, 272, 275, 280, 284, 288, 290, 2300, SO2 Kircher, A. B., 309, 319 Kirschmann, C., 179, 205, 220, 246 Kiser, W., 42, 43, 160 Kistler, W. S., 61, 164 Kito, M., 121, 164 Klein, S. P., 274, SO1 Kleinseller, A., 262, 300 Klemme, B., 223, 246 Klemme, J.-H., 223, 246 Klibansky, C., 190, 241 Kline, B. C . , 109, 164 Klingenberg, M., 317, S20 Klingmuller, W., 72, 131, 164 Klopotowski, T., 21, 135, 164 Klouwen, H., 220, 24s

AUTHOR INDEX

Klungsoyr, L., SO0 Klyutchova, V. V., 67, 161 Knauert, F. K., 37, 172 Knauf, P. A., 227, 246 Knuds, F., 244 Knusel, F., 97, 131, 164, 174 Knufermann, H., 211,235,246, 251 Knutton, S., 202, 241 Koch, A. L., 114, 115, 168 Koch, J. P., 60, 61, 162 Kocholaty, W. F., 248 Kochwa, S., 207, 24s Koehler, J. O., 272, SO0 Koening, H., 213, 214, 246 Kogl, F., 232, 246 Kohn, A., 198, 242 Kohn, P. G . , 289, 290, 298, SO0 Kojo, N., 113, 168 K O ~A. , H. J., 221,242 Kondo, T., 206, 262 Kono, T., 211, 229, 230, 232, 246, 278, 293, SOO, 302 Koritz, S. B., 282, SO1 Kornberg, A., 211, 261 Kornberg, H. L., 4, 5, 58, 59, 61, 67, 129, 136, 160, 164 Koscielak, J. 199, 246 Kostellow, A. B., 163 Kotyk, A., 262, 297, SO0 Kowalewski, S., 45, 167 Kosyreff, V., 233, 249 Krahl, M. E., 284, SO0 Krajewska-Grynkiewicz, K., 21, 135, 164 Krans, H. M. J., 232, 233, 240 Krebs, E. G., 211, 246 Krebs, H. A , 271, 30s Kreisberg, R. A., 287, 300 Krixon, K., 223, 248 Krug, F., 231, 241, 277, 298 Krupa, R. M., 259, SO0 Krusche, B., 205, 244 Kuboyama, M., 213, 246 Kuhn, L., 127, 170 Kuhlbiick, B., 89, 133, 161 Kuhn, J., 135, 164 Kujalova, V., 290, SO0 Kundig, F. D., 69, 171 Kundig, W., 64, 68, 69, 138, 166, 164, 166, 171

335

AUTHOR INDEX

Kuo, J. F., 229, 230, 231, 246, 278, 300 KUO,S.-C., 74, 75, 166 Kuriyama, Y., 235, 246 Kurtz, M., 286, 299, SO0 Kusch, M., 55, 131, 132, 153, 154, 173 Kushner, D. J., 192, 846 Kuwana, H., 125,166 Kuylenstierna, B., 221, 246 Kypson, J., 288, 300

1 LaCelle, P. L., 206, 263 Lacey, B. W., 42, 43, 166 Lacko, L., 259, 300 Lacroute, F., 81, 83, 86, 87, 131, 169, 163, 166

Lacson, P. S., 103, 163 Lagocki, J. W., 185, 186, 246 Lajtha, A,, 220, 247 Lamy, F., 66, 162 LaNauze, J. M., 108, 109, 131, 134, 147, 169

Landowne, D., 315, 316, 320 Lands, W. E., 216, 242 Langman, L., 93, 140, 174 Langridge, R., 106, 166 Lanier, W., 87, 166 Lankford, C. E., 97, 166 Lankisch, P. G., 205, 246 Lantr, R. S., 193, 263 La Placa, M., 199, 214, 239, 261 Larsen. P. R., 232, 246 Laster, Y., 205, 246 Laue, P., 70, 166 Lauf, P. K., 99, 101, 103, 167, 166, 200, 246

Lavis, V. R., 229, 246, 293, 500 Law, J. H., 123, 160, 185, 186, 246 Lawrence, H. S., 200, 842 Leaf, A., 126, 127, 166 Leavitt, R. I., 13, 128, 166 LeBlond, C. P., 191, 208, 249 Leboeuf, B., 280, 298 Lecocq, J. P., 211, 246 Leder, I. G., 54, 166 Lederberg, E. M., 63, 166 Lederberg, J., 129, 166 Lee, H. S., 183, 184, 248

Lee, N., 53, 140, 160 Lee, P., 99, 166 LeFevre, M. E., 316, 320 LeFevre, P. G., 293, 300 Lehman, J. F., 111, 166 Lehninger, A. L., 306, 319 Leibecq, C., 268, 269, 279, 301 Leibovitch, S. J., 182, 261 Leibovite, Z., 183, 246 Lein, J., 125, 166 Lein, P. S., 125, 166 Leive, L., 17, 166 Lenaz, G., 220, 221, 222, 239, 246 Lengeler, J., 59, 166 Lennarz, W. J., 178, 216, 246' Lennox, E. S., 107,173 Leonard, J., 202, 204, 246 Lesseps, R. J., 208, 846 Lesslauer, W., 188, 246 Lester, G., 26, 27, 130, 166 Lester, H. E., 132, 166 Letarte, J., 276, 290, 291, 298, 300 Lever, J . E., 18, 19, 21, 135, 139, 140, 151, 152, 166, 166 Levey, G. S., 233, 246, 847 Levine, E . M., 166 Levine, M., 259, 301 Levine, R., 267, 274, 299, 301 Levinthal, M . , 69, 166, 171 Liberman, E. A., 306, 380 Liberman, I., 197, $41 Lieb, W. R., 260, 300, 301 Liebermeister, H., 268, 269, 270, 273, 279, so1

Lietman, P. S., 49, 161 Lilien, J. E., 195, 24'7 Limbrick, A. R., 202, 241 Lin, E. C . C., 3, 46, 55, 60, 61, 63, 65, 66, 67, 69, 71, 132, 167, 168, 161, 162, 164, 166, 169, 171, 174

Linchevskaya, A. A., 234, 247 Lindsay, D. B., 275, 299 Lineweaver, H., 266, 301 Lipkin, M., 312, 320 Lipovac, V., 183, 247 Litwinska, J., 75, 160 Livingston, L. R., 125, 145, 166 Lo, T. C. Y., 4, 5, 166 Lobeck, C. C., 113, 169 Loeb, H., 77, 169

336

AUTHOR INDEX

Lombardi, F. J., 16, 161 London, D. R., 42, 43, 156 Lopez-Mondragon, R., 280, SO2 Lorusso, M., 317, 321 Lotte, L., 205, 2*53 Loughridge, L. W., 42, 43, 45, 166 Lovatt, C., 279, 298 Love, W. C., 280, SO1 Lowe, C. U., 127, 166 Lowendorf, H. S., 110, 165 Loyter, 205, 246 Loyter, A., 208, 221, 222, 240, 246, 249 Loaier, R., 223, 248 Lu, F. C., 220, 247 Lubin, M., 13, 16, 17, 91, 97, 121, 129, 130, 132, 134, 164, 165 Lubochinsky, B., 91, 166 Luciani, S., 317, 319 Luckey, M., 92, 93, 131, 166 LUCY,F. C., 220, 247 LUCY, J. A., 202, 210, 235, 243, 249 Ludens, J. H., 306, 320 Luder, J., 129, 166 Ludescher, E., 286, 299, 301 Luke, R. K. J., 92, 93, 140, 158, 166, 174 Lund, J., 113, 156, 159 Lundin, L., 219, 251 Lundin, S. J., 214, 247 Lundsgaard, E., 274, 301 Lupo, M., 5, 7, 161 Luria, S. E., 117, 167 Lusk, J. E., 90, 123, 166 Luxoro, M., 218, 250 Luzikov, V. N., 222, 2467 Lynch, H. J., 2, 42, 43, 166 Lynen, F., 127, 166 Lyon, I., 76, 166

M Maas, W. K., 9, 13, 66, 67, 130, 159, 166, 170 McCarthy, C. F., 2, 42, 43, 166 McCauley, M. J., 97, 159 Macchia, V., 181, 231, 232, 247, 248 McClatchy, J. K., 166 McConnell, H. J., 204, 245 MacDonald, R. E., 70, 165 McFall, E., 13, 130, 168 McFalls, V. W., 113, 147, 161, 173

Machado, M. M., 306, 320 Machinist, J. M., 222, 247 McIlwain, I). L., 181, 202, 203, 247 McIlwain, H., 219, 847 McIntyre, C. A., Jr., 47, 51, 147, 161 McKay, E., 89, 133, 166 Mackay, I. R., 200, 2.42 McKay, L., 70, 71, 166 McKillen, M. N., 87, 166 MacKrell, T. N., 307, 321 MacLachlan, E. A., 113, 127, 160, 165 McLaren, L. C., 198, 247 McLellan, W. L., Jr., 56, 167 MacLennan, D. H., 204, 215, 216, 24,5, 247, 252

McNamara, P., 94, 155 MacRobbie, E. A. C., 306, 320 Madeley, J. R., 180, 245 Madoff, M. A., 197, 198, 199, 243 Magasanik, B., 79, 174 Magee, W. L., 179, 180, 190, 205, 252 Mager, J., 190, 205, 220, 241 Magill, C. W., 26, 29, 31, 135, 166 Maity, B. R., 58, 168 Makhlouf, G. M., 305, 320 Makinose, M., 224, 225, 242, 244 Makman, M. H., 283, 301 Malamy, M. H., 104, 105, 131, 134, 166 Malenkov, A. G., 219, 247 Malila, A,, 226, 247 Maling, B. D., 35, 169 Manchester, K. L., 230, 247, 271, 282, 285, 301, 302 Manus, D., 226, 243 Marchesi, V. T., 192, 204, 226, 227, 247 Marcus, A. J., 205, 240 Marcus, M., 4, 5, 7, 140, 166 Marcus, P. I., 199, 247 Margolin, P., 138, 156 Marikovsky, Y., 198, Z4Z,247 Marinari, U. M., 208, 248 Marinetti, G. V., 180, 190, 233, 247, 252, 296, SO3 Mark, C. G., 74, 75, 166 Marko, 0. W., 183, 184, 248 Markowita, J., 207, 243 Marks, N., 220, 247 Marsh, J. B., 193, 197, 199, 253 Martin, B. K., 310, 321 Martin, 1).B., 277, 298, 301

337

AUTHOR INDEX

Martin, H. H., 205, 245 Martin, K., 226, 247 Martin, R., 218, 247 Martinez, L., 28, 31, 169 Martfnez-Palomo, A., 191, 247, 312, 320 Martonosi, A., 202, 224, 226, 243, 247 Marussi, M., 221, 248 Marzluf, G. A , , 108, 109, 110, 111, 131, 140, 1.50, 166, 168 Masiak, S., 226, 227, 247 Masoro, E. J., 224, 226, 247 Mastroianni, L., 219, 251 Mata, J. M., 61, 131, 162 Matchett, W. H., 26, 173 Matsson, E., 115, 167 Matsuhashi, M., 117, 171 Matsuura, A , 86, 163 Mattman, J. R., 194, 247 Maurer, H. S., 103, 163 Mavrides, C. A., 180, 250 Mawe, R. C., 259, 301 Mayhew, E., 198, 247, 253 Mays, L. L., 125, 145, 166 Mazia, I)., 103, 163, 219, 251 Medveczky, N., 104, 105, 108, 166, 169 Meeuaisse, G. W., 77, 166 Meissner, G., 225, 247 Mel, H. C., 198, 202, 243 Melchers, F., 166 Mellanby, J., 219, 252 Melloni, E., 211, 249 Menano, 11. P., 306, 320 Mengel, C. E., 202, 206, 240 Menon, I. A,, 211, 247 Merill, C. W., 206, 253 Meronk, F., Jr., 53, 140, 160 Messer, W., 166 Mete, J., 200, 242 Metzenherg, R. L., 35, 110, 111, 125, 130, 140, 150, 157, 163, 165, 166, 172 Meunier, J. C., 220, 241 Meury, J., 91, 166 Meyerhoff, O., 226, 245 Meyn, T., 110, 112, 113, 157 Michael, A. F., 47, 159 Michalec, C., 181, 251 Michelangeli, F., 317, 319 Michiel, H., 232, 233, 240 Mihalyi, E., 215, 248 Milholland, R. J., 282, 283, 284, 302

Millar, Miller, Miller, Miller, Miller,

D. B., 214, 244 A., 111, 70, 71, 166 A. K., 61, 131, 162 D. M., 258, 259, 260, 301 I. R., 185, 186, 187, 189, 190, 245,

248

Miller, T. W., 61, 131, 162 Milne, M. D., 42, 43, 45, 133, 166, 166 Milner, L. S., 138, 163, 228, 248 Minagawa, A., 47, 171 Minakami, S., 213, 248, 249, 292, 301 Mindich, L., 123, 125, 148, 166, 173 Minemura, T., 293, 301 Mitch, W. E., 313, 321 Mitchell, P., 306, 317, 319, 321 Mittwoch, U., 43, 162 Miurt, R., 45, 167 Miyata, I., 84, 85, 164, 166 Mizohuchi, K., 107, 109, 159, 166 Mizutani, H., 199, 248 Mizutani, H., 199, 248 Mochales, S., 61, 131, 162 Mohamed, S. D., 89, 133, 166 Mohyuddin, F., 48, 156 Molzahn, S. W., 127, 167 Moncalvo, S., 221, 248 Monneron, A., 192, 248 Monod, J., 2, 52, 55, 56, 140, 141, 163, 168 Montagnier, L., 197, 248 Monty, K. J., 107, 134, 159 Moody, F. G., 312, 313, 316, 321 Moor, N., 179, 205, 246 Moore, C., 306, 321 Moore, G. L., 248 Mora, F., 26, 167 Mora, J., 28, 31, 169 Morgan, H. E., 257, 262, 263, 265, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 279, 284, 285, 286, 293, 299, 300, 301, 302, 303

Morikawa, T., 47, 51, 267, 171 Morin, R. J., 221, 2-48 Morita, Y., 282, 301 Morowitz, H. J., 211, 248 Morrison, M., 208, 249 Morse, H. G., 63, 65, 67, 167, 172 Morse, M. L., 63, 65, 67, 70, 71, 72, 158, 169, 162, 167, 172 Mortimer, R. K., 75, 127, 156, 167, 168 Moscona, A., 193, 194, 195, 248

338

AUTHOR INDEX

Mosher, K. M., 282, 283, 301 Mosinger, B., 290, 300 Motais, R., 306, 321 Motulsky, A. G., 103, 165, 163 Moulton, R. H., 99, 172 Mousset, M., 38, 39, 130, 147, 151, 161 Moyle, J., 306, 317, 321 Mueller, P. S., 127, 169 Mtiller-Hill, B. S., 55, 131, 147, 149, 161, 167 Muhlethaler, K., 211, 848 Mulleck, V., 267, 274, 299 Muller, E., 194, 239 Muller, L., 180, 248 Mullis, K. B., 96, 167 Munck, A,, 282, 283, 300, 301 Munoz, E., 214, 215, 248 Murakami, W. T., 196, 244 Muraoka, S., 315, 321 Murphy, J. T., 86, 87, 147, 167 Mutolo, V., 194, 195, 242

N

Neufeld, E. F., 197, 235, 243, 248 Neuhaus, F. C., 3, 12, 13, 26, 27, 130, 168, 172 Neujahr, H. Y., 194,214,239, 248 Neumann, E., xxif Neumann, J., 306, 821 Neutra, M., 191, 249 Neville, I). M., 277, 299, 300 Neville, M. M., 72, 167 Newsholme, E. A., 271, 272, 280, 802 Newton, A., 119, 172 Newton, N. A., 92, 93, 158 Nezamis, E., 267, 300 Ng, M. H., 214, 248 Nichoalds, G. E., 14, 167, 168 Nicholls, D. G., 221, 246 Nickel, A., 312, 319 Nickel, K. S., 213, 246 Niemi, M., 235, 261 Nigon, K., 315, 319 Nils-Erik, L. S., 206, 220, 222, 223, 848 Nomura, M., 117, 167 Normark, S., 115, 16Y Norrell, S. A., 84, 171 Northrop, J. H., 207, 248 Nose, Y., 84, 85, 86, 163, 164, 166 Novotny, C. P., 53, 140, 167

Nachbar, M. S., 215, 248 Nachmansohn, D., 218, 240 Naftalin, R. J., 259, 260, 301 Nagaeva, L. I., 193, 241 Nakel, M. T., 226, 243 0 Nagel de Zwaig, R., 117, 167 Nahaa, G. G., 288,300 O’Brien, D., 47, 51, 147, 161 Nakamura, A., 215, 246 O’Brien, I. G., 92, 93, 168, 167 Nakamura, H., 115, 167, 213, 240 O’Brien, R. D., 220, 242 Nakane, P. K., 14, 167 O’Callaghan, J., 307, 321 Nakazawa, T., 72, 167 Oda, T., 210, 248 Nanni, G., 202, 205, 208, ,841, 248 Obrink, K. J., 310, 311, 320 Nanninga, N., 202, 248 pye, I., 281, 302 Narahara, H. T., 229, 231, 263, 268, 275, Offermeier, J., 219, 248 276, 277, 278, 280, 289, 299, 301, 308, Ogata, Y., 200, 253 303 Ohnishi, K., 214, 248 Narahashi, T., 217, 248 Ohnishi, T., 226, 227, 248 Neale, S., 16, 17, 131, 172 Ohta, N., 107, 109, 131, 154,167 Neely, J. R., 268, 269, 270, 271, 272, 273, Oka, T., 277, 302 279,301 Okayama, S., 223, 248 Neilands, J. B., 92, 93, 96, 131, lse, 167, Okunuki, K., 250 168 Oldigs, H. D., 205, 263 Nelson, D. L., 90, 91, 131, 167 Olivecrona, T., 180, 242 Nelson, P. G., 248 Omura, T., 235, 246 Ness, H., 183, 184, 248 Oncley, J. L., 197, 198, 199, 843 Neu, H. C., 50, 167 Ong, S., 289, 300

339

AUTHOR INDEX

Onishi, H., 192, 246 Ono, Y., 181, 248 Onodera, K., 117, 167, 167, 169 Op Den Kamp, J. A. F., 202,248 Opie, L. H., 271, 302 Oppenheimer, S. B., 195, 248 Orme-Johnson, N., 221, 222, 239 Ornston, L. N., 87, 167 Ornston, M. K., 87, 167 Orr, C. W., 195, 248 Orsi, B. A., 111, 134, 163, 171 Oshorn, M. J., 56, 167 Oski, F. A., 286, 302 Oster, G., xxi, xxii Otsuji, N., 117, 167 Ottaway, J. H., 286, 302 Ottolenghi, A. C., 202, 206, 240, 248 Ovchinnikov, Y. A., 219, 247 Overath, P., 121, 123, 137, 167, 170 Overell, G. B., 282, 302 Overholt, B. F., 310, 319 Overland, E. S., 103, 163 Owen, E. E., 2, 42, 43, 166 Oxender, D. L., 3, 11, 12, 13, 14, 15, 119, 130, 135, 167, 168,259, 301 Oyaert, W., 47, 163 Oyanagi, K., 45, 167 Ozand, P., 275, 276, 301

P Pacifico, A. D., 307, 316, 321 Pala, V., 202, 205, 241 Palade, G. E., 204, 208, 226, 227, 235, 243, 246,247 Pall, M. L., 26, 27, 28, 29, 30, 31, 32, 135, 150, 151, 167, 168 Pandit-Hovenkamp, H. G., 221, 242 Panko, E., 202,203, 204,205,261 Papa, S., 317, 321 Papineau, D., 94, 95, 134, 161 Pardee, A. B., 52, 54, 57, 87, 106, 107, 109, 123, 131, 154, 169, 166, 166, 167, 168, 173,201,243 Pardoe, G . I., 197, 862 Park, C. R., 262, 263, 268, 269, 274, 275, 276, 277, 279, 284, 285, 286, 293, 298, 299, 300, 301, 302 Park, 0. H., 315, 319 Park, S. S., 53, 117, 167

Parker, J. C., 292, 302 Parks, L. W., 41, 127, 171, 172 Parmelee, M. L., 99, 101,166,200,246 Parpart, A. K., 205, 206, 239 Parrish, J. E., 288, 290, 309 Parsa, B., 221, 239 Parson, J. W., 110, 166 Parsons, D. F., 221, 248 Passow, H., 218, 248 Pastan, I., 65, 169,168, 181, 199, 213, 231, 232, 243, 247, 248 Paszewski, A., 75, 160 Patriarca, P., 221, 248 Patterson, J. H., 76, 77, 160 Patterson, N. K., 208, 248 Patterson, S. W., 279, 302 Patzer, P., 205, 263 Pavlasova, E., 94, 95, 153, 182, 168 Payne, J. W., 16, 17, 131, 168 Paysant, M., 262 Pease, D. C., 191, 249 Penberthy, W. K., 65,70,71,72, 162,167 Pendyala, L., 31, 172 Penrose, W. R., 14, 168 Perdue, J. .R., 221, 222, 239, 244 Perelson, A., xxi Perheentupa, J., 45, 113, 164, 168 Perkins, D. D., 28, 125, 168, 168 Perlman, R. L., 65, 169, 168 Perry, J. W., 54, 166 Pethica, B. A., 198, 289 Petrow, V., 282, SO2 Petrushka, E., 220, 249 Phelps, C. E., 79, 173 Phillips, D. R., 208, 249 Pickering, W. R., 82, 83, 131, 168 Pilwat, G., 91, 168, 174 Pimstein, R., 43, 130, 162 Piperno, J. R., 12, 14, 168 Pittard, J., 87, 168 Pizer, L. I., 121, 164 Plagemann, P. G. W., 282, 283, 302 Podelski, T. R., 220,241 Pogell, B. M., 58, 168, 211, 262 Pohl, S. L., 232, 233, 240, 249 Pokerny, V., 222, 242 Pollack, J. R., 92, 93, 96, 131, 166, 167, 168 Polonovski, J., 262 Pontrernoli, S., 211, 249

340

AUTHOR INDEX

Ponz de Posadas, G., 193, 2566 Poole, A. R., 202, 235, 249 Popenoe, E. A,, 277, 299 POPOV, A. G., 216,240 Posch, E., 211, 250 Possati, L., 199, 239 Possehel, E. A., 213, 245 Post, R. L., 226, 227, 228, 244, 263, 274, 275, 276, 302 Poste, G., 193, 195, 249 Postema, N. M., 188, 246 Posternak, T., 290, 299 Poulter, J., 216, 241 Poulter, J. L., 312, 520 Pousada, M., 181, 250 Power, J., 53, 140, 160 Pressman, B. C., 306, 321 Prestidge, L. S., 54, 57, 106, 107, 131, 154, 168 Price, H. D., 56, 15Y Proverbio, F., 227, 246 Pucell, A. G., 224, 247 Purkiss, P., 43, 168

0 Quagliariello, E., 317, 321 Quarles, R. H., 180, 186, 249 Quastel, J. H., 220, 249 Quinn, P. J., 189, 246

R Raanan-Ashkenazi, O., 63, 169 Rachmeler, M., 35, 169 Racker, E., 208, 221, 222, 223, 239, 240, 249,252 Radford, A., 145, 168 Radojkovic, J., 56, 169 Rahat, M., 43, 130, 162 Rahmanian, M., 15, 130, 168 Rajagopalan, G. T., 211, 24s Rakhimov, M. M., 249 Ram, D., 260, SO0 Ramakrishnan, T., 15, 128, 168 Rambourg, A., 191, 192, 208, 249 Randle, P. J., 262, 271, 272, 275, 280, 282, 285, 286, 288, 289, 301, 302 Rapport, M. M., 181, 185, 186, 187, 189, 202, 203, 219, 240, 241, 244,647, 251 Rasmusen, B. A., 99, 101, 166,168

Rasmussen, H., 277, 293, 298 Ray, P. K., 193, 200, 249, 251 Ray, T . K., 121, 168, 233, 262, 312, 320 Rayman, M. K., 4, 5, 165 Read, C. P., 29, 31, 136, 1Y3 Reasor, H. S., 206, 642 Rechcigl, M., Jr., 183, 235, 249 Reed, M. L., 306, 320 Reeves, R. E., 67, 164 Regen, 13. M., 257, 260, 262, 263, 274, 275, 276, 277, 284,299, 301, SO2 Rehm, W. S., 305, 307,308, 309, 310, 314, 316, 318, 319, 320, 321 Rehn, K., 121, 162, 192, 240 Reid, E., 284, 298 Reid, K. G., 24, 25, 132, 168, 172 Reitz, R. H., 3, 26, 27, 30, 168 Remold, H. G., 249 Rendina, G., 213, 249 Renner, E. D., 282, 283, 302 Renold, A. E., 264, 274,275,276, 282, 290, 291, 298, 299, 300 Resnick, M. A., 127, 168 Resnick, M. R., 127, 164, 1YS Rethy, A., 213, 249 Ricard, M., 119, 168 Rice, K. L., 267, SO0 Rich, R., 207, 263 Richey, 1).P., 61, 65, 157 Rickenberg, H. V., 2, 52, 168, 168 Rideal, E. K., 185, 258 Rieser, C. H., 231, 249 Rieser, P., 229, 231, 249 Rinaldini, L. M. J., 191, 249 Ringler, R. L., 213, 248, 249 Rios, A., 200, 261 Ritchie, J. M., 315, 316, 520 Roberts, K. R., 108, 109, 150, 168 Robertson, R. T., 219, 264 Robinson, S. L., 248 Robison, G. A., 232, 249 Robson, E. B., 43, 162, 168 Robson, R. M., 215, 244 Rocca, E., 213, 240 Rodbell, M., 229, 231, 232, 233, 240, 249, 277, 278, 290, 291, 298, 302 Rodriguez-Estrada, C., 315, 321 Roelcke, D,, 200, 242 Roelofsen, B., 181, 202, 203, 205, 216, ,241, 249, 250, 254

341

AUTHOR INDEX

Rorsch, A., 115, 172 Roess, W. B., 30, 31, 130, 168 Rogers, D., 66, 168 Rojas, E., 218, 260 Rolfe, B., 117, 167, 167, 169 Romano, A. H., 74,75,167,166 Romashina, L. V., 222, 247 Romhanyi, G., 208, 260 Ronen, A., 63, 169 Root, A., 286, 302 Rose, G. A., 2, 42, 43, 169, 168 Rose, S. P., 121, 123, 137, 173 Roseman, S., 3, 63, 64, 65, 68, 69, 70, 72, 138, 166, 168, 164, 166, 167, 169, 171, 181, 195, 196, 248,260 Rosen, B. P., 8, 9, 130, 169 Rosen, F., 282, 283, 284, 302 Rosen, J. M., 281, 282, 283, 284, 302 Rosen, M., 206, 242 Rosenberg, A., 183, 247 Rosenberg, H. 92, 93, 104, 105, 108, 109, 131, 134, 147, 168, 166, 169 Rosenberg, L. E., 2, 3, 42, 43, 45, 46, 47, 49, 51, 76, 77, 79, 112, 113, 126, 127, 133, 137, 147, 149, 150, 151, 152, 167, 160, 161, 162, 169, 170, 171 Rosenberg, P., 217,218,240, 84l,247,260 Rosenberg, T., 256, 302 Rosenbloom, A., 113, 169 Rosenblum, E. D., 166 Rosenthal, A. F., 180, 181, 260 Rosenthal, J. W., 229, 260, 278, 302 Rosenthal, S. L., 277, 299 Rossi, C. S., 221, 240 Rossi, F., 221, 248 Roth, I., 231, 248 Roth, J., 277, 299, 300 Roth, J. R., 18, 19, 130, 147, 166 Roth, S.,196, 206, 260 Rothstein, A., 74, 172 Rotman, B., 56, 57, 58, 59, 101, 160, 169 Rowley, P. T., 127, 169 Roy, A. C., 206, 260 Rubenchik, A. L., 193, 2.41 Rubin, H., 196, 198, 260 Ruby, A., 103, 163 Ruch, F., 121, 170 Ruch, G. A., 220, 260 Rudenburg, F. H., 198, 260 Ruderman, N. B., 272, 273, 300, 302

Ruesink, A. W., 193, 260 Ruigrok, T. J. C., 220, 263 Rumley, M. K., 153, 169 Russell, R. R. B., 13, 169 Russell, W. S., 99, 169 Rutledge, J. R., 318, 319 Ruysschaert, J. M., 185, 186, 248 Ruzicka, F. J., 213, 222, 239 Ryan, M. T., 180, 260 Ryter, A., 121, 162

S Sabban, E., 205, 246 Saburova, A. M., 234,247 Sachs, G., 305, 313, 316, 317, 318, 321 Sachs, J. R., 99, 101, 169 Sachs, L., 196, 239, 246 Sacks, J., 267, 302 Sacks, M; S., 89, 133, 171 Sackton, B., 202, 2.44 Saha, J., 280, 302 Saier, M. H., Jr., 63, 65, 69, 169 St. Amand, G. S., 193, 260 St. Lawrence, P., 35, 169 Sakamoto, T., 228, 262 Sakamoto, Y., 228, 262 Sakmann, B., 192, 239 Salach, J. I., 179, 260 Saltman, P., 309, 316, 320 Salton, M. R. J., 191, 214, 215, 248, 260 Samorajski, T., 226, 261 Sanchez, S., 28, 31, 169 Sanders, H., 179, 180, 190, 246 Sanders, S. S., 307, 308, 309, 320, 321 Sanderson, K. E., 71, 107, 109, 141, 169 Sandine, W. E., 70, 71, 166 Sandow, A,, 268,302 Sanford, B. H., 193, 202, 219, 241, 260 Sanno, Y., 60,169 SanPietro, A., 223, 246 Sanwal, B. D., 4, 5, 166 Sapico, V., 71, 169 Saris, N.-E. L., 206, 223, 260 Sarkar, N. K., 205, 220, 260 Sarvas, M. O., 57, 132, 167 Sato, R., 211, 214, 246 Sato, T., 117, 171 Saunders, L., 188, 189, 239,260 Sawh, P. C . , 288, 289, 297

342

AUTHOR INDEX

Sawyer, T. K., 284, 301 S a x h , L., 45, 164 Scaletti, J. V., 198, 247 Scarborough, G. A., 70, 72, 153, 164, 169 Scarpa, A., 208, 260 Scattergood, E. M., 188, 244 Schaedel, P., 56, 167 Schaefler, S., 66, 67, 170 Schafer, I. A., 46, 170 Schairer, H. U., 114, 121, 123, 137, 167, 170 Schatzman, H. I., 226, 227, 260 Schazzochio, C., 81, 131, 168 Scherphof, G. L., 208, 260 Schiess, B., 97, 131, 164 Schlatz, L., 296, 303 Schleif, R., 50, 51, 52, 53, 170 Schmitt, R., 54, 55, 170 Schnaitman, C. A., 114, 170 Schnebli, H. P., 94, 170 Schneider, A. J., 77, 170 Schneider, P., 232, 260 Schneider, R. P., 72, 74, 170 Schoenhard, D. E., 109, 164 Scholefield, P. G., 220, 849 Scholes, P., 306, 321 Schonfeld, G., 272, 302 Schor, M. T., 214, 215,248 Schramm, M., 234, 239 Schrecker, O., 72, 170 Schreiner, E., 192, 263 Schulman, J. H., 180, 185, 186, 187, 261 Schultz, S. G., 88, 89, 94, 104, 135, 170. 172, 174, 256, SO2 Schwartz, F. E., 277, 299 Schwartz, I. L., 280, 299 Schwartz, J. H., 9, 130, 170 Schwarts, M., 63, 162, 163, 170, 307, 321 Schwartz, V. G., 199, 247 Schwars, U., 236, 244 Schweizer, E., 127, 170 Scott, J. M., 111, 134, 163, 171 Scott, M . T., 201, 260 Scow, R. O., 178, 260 Scriver, C. R., 3, 24, 26,43, 46, 46, 47, 48, 49,51,52,112,113,133,166,161,162, 169,170, 173 Seaman, G. V. F., 197, 198, 199, 241, 244, 246,260 Sears, B., 189, 260

iw,

Seeds, N. W., 193, 195, 260 Seegmiller, J. E., 49, 161 Seeman, P., 206, 215, 247, 260 Sefton, B. M., 198, 260 Segal, S., 2, 42, 43, 45, 147, 160, 169, 17'1 Segel, I . H., 110, 112, 113, 167, 172 Seidel, J. C., 215, 260 Seki, S., 210, 248 Sekiguchi, M., 117, 170 Sekuzu, L., 260 Seldin, D. W., 105, 170 Selinger, Z., 234, 239 Selman, B. R., 223, 260 Selmeci, L., 211, 260 Selwyn, M. J., 306, 321, 322 Semeriva, M . , 185, 243 Sen, A. K., 258, 263,302 Seng, R., 179, 860 Senior, 13.) 43, 169 Seppala, A. J., 206, 220, 222, 223, 248, 250 Sereda, D. D., 219, 241 Sessa, G., 203, 261 Setlow, P., 211, 261 Sevilla, C. L., 180, 239 Shabolenko, V. P., 63, 67, 167, 161 Shadur, C . A , , 12, 13, 130, 172 Shah, D. O., 180, 185, 186, 187, 261 Shah, G., 313, 321 Shallenberger, M. K., 114, 171 Shanbour, L. L., 312,321 Shapiro, B., 121, 162 Shapiro, S., 58, 168 Shapiro, S. K., 87, 171 Shaw, K. N . F., 45, 170 Sheard, B., 189, 204, 261 Sheetz, M. P., 189, 203, 204, 243, 261 Sheldon, W., 129, 166 Shen, L. C., 302 Sheppard, D.E., 53,117,140,167,160,170 Shifrin, S., 19, 130, 170 Shimazono, N., 214, 246 Shinagawa, H., 106, 166 Shipp, J. C., 271, SO3 Shoemaker, R. L., 316, 317, 321 Short, E. M., 112, 170 Shumas, S. R., 81, 167 Siegel, A., 271, 298 Sieglin, U., 192, 840 Siekevitz, P., 235, 246 Signer, E. R., 107, 170

343

AUTHOR INDEX

Silbert, D. F., 121, 123, 168, 170 Silhavy, T . J., 59, 139, 170 Silman, I., 220, 242 Silver, L., 277, 299 Silver, S., 88, 90, 91, 167, lY0 Silverman, M . , 76, 1YO Simmonds, S., 165 Simmons, R. L., 193, 200, 249, 261 Simon, E . J., 9, 130, 170 Simoni, R. D., 3, 63, 64, 65, 69, 70, 72, 114,166,162, 166,167,169, i r i Simpkins, H., 202, 203, 204, 205, 206, 236, 243, 261 Simpson, L. L., 219, 261 Singer, I., 218, 262 Singer, J., 56, 171 Singer, S. J., 202, 203, 204, 243, 246, 261 Singer, T . P., 179, 213, 222, 240, 244, 247, 248, 249, 260 Sinha, U., 43, 130, 171 Sire, J., 41, 130, 171 Skavronskaya, A. G., 67, 161 Skou, J. C., 98, 171, 313, 321 Skulachev, V. P., 306, 320 Slade, H. D., 3, 26, 27, 130, 168 Slater, E. C., 315, 321 Slautterback, D. B., 220, 243 Slaynian, C. L., 96, 125, 145, 1'71 Slayman, C. W., 96, 97, 110, 125, 132, 134, 145, 146, 147,166, i r i Slotboom, A. J., 178, 188, 246, 261 sly, w., 41, 130, i r i Sniets, P., 312, 322 Smith, A. D., 261 Smith, A. J., 47, l Y l Smith, C. W., 202, 261 Smith, D. A,, 22, 23, 131, 171 Smith, E. L., 214, 261 Smith, G. H., 262,275,288,289, 302 Smith, J., 58, 59, 61, 67, 129, 160, 164 Smith, J . B., 94, 166 Smith, J. F., 267, 302 Smith, S. I., 37, 17.8 Smith, T. E., 193, 241 Smorodinski, I., 220, 246 Snell, E. E., 26, 167 Snoeck, J., 47, 163 Snyder, J. J., 101, 166,200, 246 Sokoll, M. D., 219, 238 Solberg, L. A., Jr., 307, 321

Solomon, A. K., 88, 135, 270 Solomon, E., 67, 69, 171 Sols, A., 280, SO3 Somerville, R. L., 135, 164 Sommerfield, P., 110, 112, 113, 167 Somogyi, J., 226, 227, 261 Sonenberg, M., 286, SO3 Sonesson, B., 219, 238 Sorsoli, W. A., 41, 171 Soucek, A., 181, 261 Souckova, A., 181, 261 Soupart, P., 201, 261 Spangler, R., x x i Spangler, S. G., 307, 321 Spence, K. D., 41, 86, 87, 147, 167, 171 Spencer, B., 111, 134, 163, 171 Speth, V., 211,261 Spiro, R. G . , 194, 261 Spitnik-Elson, P., 211, 261 Spudich, J. A., 215, 261 Spurling, C. L., 89, 133, 171 Squires, C., 53, 140, 160 Squires, R., 103, 16% Srb, A. M., 132, 173 Stadler, D. R., 26, 27,29, 33, 35, 130, 147, 167, 164, 171 Stadtman, E. R., 17, 78, 83, 162, 163 Stahl, W . L., 181, 202, 261 Stambaugh, R., 219, 261 Stanbury, J. B., 3, 171 Stanbury, S. W., 113, 160 Stapley, E. O., 61, 131, 16% Starling, E. H., 279, SO2 Starr, P. R., 127, 172 Steck, T. L., 207, 261 Stein, O., 235, 242 Stein, W. D., 258, 259, 260, 299, 300, 301, so3

Stein, Y., 235, 242, 244 Steinhardt, R. A., 219, 261 Steven, F. S., 182, 261 Stevenson, J. H., 204, 244 Stewart, J. A., 183, 184, 248 Stirling, C. E., 77, 1'70 Stirpe, F., 214, 261 Stockdale, M., 306, 321, 322 Stoeckenius, W., 208, 209, 843 Stoffel, W., 123,167, 197,235,261 Stolkowski, J., 91, 166 Strang, L. B., 47, 171

344

AUTHOR INDEX

Straughn, W. R., 25, 156 Straus, J. H., 204, 244 Strenkoski, L. F., 95, 171 Streter, F. A., 215, 224, 245 Strickholm, A., 218, 226, 245, 251 Strom, R., 101, 158, 222, 241 Stromer, M. H., 215, 244 Strunk, S. W., 202, 251 Strych, A., 313, 321 Stuart, W. D., 26, 27, 171 Stutts, P., 81, 157 Suganuma, A , , 115,167 Sugino, Y., 117, 171 Sukhudolova, A. T., 216, 240 Summers, K. E., 194, 251 Sun, A. Y., 226, 251 Sun, G. Y., 226, 251 Sun, R., 183, 184, 248 Suominen, J. J. O., 235, 251 Surdin, Y., 41, 130, 171 Suskind, S. R., 72, 167 Sussman, A. J., 16, 171 Sutherland, E. W., 232, 249, 281, 282, 299,502

Svennerholm, L., 183, 251 Sweeney, H., 26, 29, 31, 135, 166 Szasz, I., 206, 251, 252 Szent-Gyorgyi, A. G., 215, 248, 252

T Tacconi de Alaniz, M. J., 193, 252 Tada, K., 47, 51, 167, 171 Takenaka, T., 218, 252 Taketa, K., 211, 252 Tamaki, S., 117, 171 Tamburrini, O., 232, 247 Tanaka, R., 228, 252 Taniguchi, K., 226, 227, 228, 252 Tarby, T. J., 217, 220, 244, 252 Tarpley, H. L., 257, 260, 299 Tasaki, I., 218, 252 Tatibana, M., 226, 252 Tatum, E. L., 72, 97, 132, 134, 146, 147, 158,171 Tay, S., 202, 203, 204, 251 Taylor, A. L., 9, 79, 107, 115, 119, 131, 139, 143, 144,171 Taylor, J., 204, 244 Taylor, R. T., 84, 171

Teather, C., 199, 244 Teilor, A. E. R., 252 Teitel, P., 252 Temin, H. M., 196, 252 Temple, J., 215, 244 Ten-Ami, I., 220, 246 Tenforde, T., 197, 252 Teorell, T., 310, 322 Terrey, M., 127, 165 Terry, T. M., 211, 248 Thakar, J. H., 79, 131, 171 Thang, M. N., 235, 252 Thesleff, S., 217, 219, 238 Thier, S. O . , 2, 3, 42, 43, 160, 171 Thimann, K. V., 193, 250 Thirion, J. P., 63, 172 Thomas, J., 56, 163 Thomas, L., 200, 242 Thompson, R. H. S., 179, 180, 190, 245 Thompson, D. D., 43, 160 Thompson, E. D., 127, 172 Thompson, G. A., Jr., 178, 181, 852 Thompson, T. E., 306, 319 Thomson, J. L., 193, 244 Thorne, G. M., 11, 23, 130, 172 Thwaites, W. M., 31, 37, 129, 172 Tien, H. T., 203, 252 Tijssen, F. C., 202, 248 Till, J. E., 167 Timmermans, J., 47, 163 Tipton, S. R., 193, 250 Tisdale, H., 179, 250 Tisdale, J. H., 37, 172 Tizzo, R., 221, 240 Tobias, J. M., 198, 217, 218, 248, 250, 252 Toh-e, A., 111, 163, 172 Tomasi, V., 233, 249, 152 Tomasz, A., 191, 235, 252 Tomizawa, M., 206, 252 Tonomura, Y., 226, 227, 252 rropaiy, v. P., 306,szo Topper, Y. J., 277, 302 Torndal, U.-B., 221, 245 Torquebiau-Collard, O., 252 Tosteson, D. C., 99, 101, 103, 157, 162, 165, 172, 200, 246 Touabi, M., 290, 303 Touster, O., 208, 248 Traniello, S., 211, 849 Trap-Jensen, T., 103, 162

345

AUTHOR INDEX

Trevisani, A,, 233, 249 Trevithick, J. R., 125, 172 Triebwasser, K. C., 24, 25, 130, 160 Triner, L., 288, 300 Triplett, R. B., 204, 240 Tristram, H., 16, 17, 131, 172 Truelove, B., 220, 242 Trump, B. F., 202, 252 Tsay, S.-S., 72, 172 Tsofina, L. M., 306, 320 Tsukagoshi, N., 123, 160 Tucker, E. M., 99, 101, 169,172 Tulsiani, D. R. P., 234, 841 Turkington, R. W., 277, 303 Turnbull, A. C., 235, 239 Tweedie, J. W., 110, 112, 113, 172 Tyor, M. P., 2, 42, 43, 166 Tzagoloff, A., 216, 252

U Uhlenbruck, G., 197, 208, 260, 262, 253 Ullrich, K . J., 305, 521 Ulstrom, R. A., 47, 159 IJmana, C. R., 234, 262 Umbarger, H. E., 3, 5, 7, 13, 39, 41, 128, 157, 158, 161, 166, 172 Ungar, I., 271, 298 Uno, I., 111, 163 Unsold, H. M . , 59, 165 Urban, C. L., 196, 245 Ussirig, H. H . , 312, 322 Utech, N. M., 24, 25, 132, 168, 172 Uthe, J . F., 179, 180, 190, 205,252

v Vagelos, P. R., 121, 138, 157, 158, 170 Vainio, H . , 204, 239 Vambutas, V. K., 223, 252 Vanatta, J . C., 306, 320 Van Dam, K., 316, 322 Van Deenen, L. L. M., 178, 179, 181, 184, 188, 189, 202, 203, 205, 216, 220, 232, 239, 240, 244, 246, 249, 250, 261, 252, 253,254 van den Hende, C., 47, 163 Van de Putte, P., 115, 172 Van der Meer-Van Buren, M., 221, 242 Van Dillewijn, J., 115, 172

Van Heyningen, W. E., 219, 252 Vansteenkiste, Y., 312, 322 Van Steveninck, J., 74, 172 Vargaftig, B. B., 235, 262 Varmus, H. E., 65, 169 Vatter, A. E., 94, 170, 193, 195, 260 Vessey, D. A., 215, 252 Vidaver, G. A., 263, 303 Vilkki, P., 232, 262 Vinten, J., 290, 298 Visakorpi, J. K., 45, 164, 168 Visser, A., 210, 243 Vocikov, V. L., 219, 247 Vogt, W., 205, 246, 253 von Meyenburg, K., 114, 115, 172 Voznaya, N. M., 222,247

W Wada, C., 115, 143, I74 Wade, M. A., 99, 164 Wahlstrom, A., 205, 253 Waite, M., 220, 263 Wakil, S. J., 121, 123, 160, 162 Walaas, E., 279, 303 Walborg, E. F., Jr., 193, 253 Walczak, W., 21, 135, 164 Wald, R., 262 Wallaas, O., 279, 303 Wallace, B. J., 87, 168 Wallach, D. F. H., 204,207,208,211,235, 244, 246, 251, 253 Wallenfels, K., 56, 57, 167 Wallick, H., 61, 131, 162 Walshe, J. M., 43, 159 Walter, R. W., 71, 169 Walton, G. M., 302 Wang, C. C., 119, 172 Wang, R. J., 63, 65, 67, 158, 167, 172 Wargel, R. J., 12, 13, 130, 172 Warland, B. J., 113, 157 Warren, F. L., 43, 99, 147, 160, 162 Warren, L., 192, 193, 197, 199, 253 Watanabe, A , , 215, 262 Watanabe, K., 106, 154, 168 Waters, M. F., 200, 253 Watkin, D. M., 127, 169 Watkins, E., Jr., 200, 253 Watkins, E., 111, 200, 253 Watling, A. S., 306, 322

346

AUTHOR INDEX

Watson, J. A., 66, 160 Watt, S., 215, 261 Watts, R. W. E., 43, 168 Wayne, R., 92, 93, 131, 166 Webb, M., 92, 93, 131, 172 Webbs, S. J., 194, 247 Weed, R. I., 206, 263 Weibull, C., 192, 263 Weicker, H., 200, 242 Weidekamm, E., 204,211, 261,263 Weiden, P. L., 89, 104, 172 Weinbach, E. C., 206, 222, 223, 263 Weinbaum, G., 207, 263 Weiner, J. H., 6, 7, 9, 12, 14, 16, 17, 130, 167, 160, 172 Weinstein, D. B., 193, 197, 199, 263 Weis, L. S., 278, 303 Weiss, J. B., 182, 262 Weiss, L., 191, 195, 197, 198, 247, 263 Weiss, L. S., 229, 231, 263 Weissmann, G., 203, 236,261, 263 Wesley, M. E., 280, 303 Westall, R. G., 43, 166 Westling, B., 115, 167 Wheeler, K. P., 228, 263 Whelan, D. T., 45, 172 Whipple, M. B., 106, 107, 131, 154, 168 White, A., 283, 301 White, D., 196, 260 White, D. C., 181, 248 White, F. P., 226, 246 White, J., 103, 163 Whitney, E. N., 91, 117, 270, 1'72 Whitney, J. E., 277, 303 Whittam, R., 263, 292, 303 Whittington, E. S., 99, 101, 103, 16'7, 172 Wiame, J. M., 38, 39, 128, 130, 135, 147, 151, 166, 161 Wick, A. N., 270, 299 Widdas, W. F., 256, 258, 250, 298, 302, 303

Wieneke, A. A., 228, 229,263 Wiener, H., 191, 263 Wiesmeyer, H., 62, 272 Wijsman, H. J. W., 117, 172 Wilbrandt, W., 256, 260, 299, 302 Wiley, W. R., 26, 27, 72, 74, 170, 173 Wille, G., 205, 263 Willebrands, A. F., 279, 299 Willecke, K., 86,87, 123, 148,166,173

Willems, G., 312, 322 Williams, E., 226, 246 Williams, G. R., 221, 248, 314, 315, 316, 319 Williams, R. H., 229, 246, 293, 300 Williams, R. J. P., 123, 168 Williams, T. F., 113, 147, 166, 1'73 Williams, W. L., 219, 664 Williamson, D. H., 193, 242 Williamson, J. R., 271, 279, 287, 300, 303, 315,322 Willis, D. R., 97, 1'73 Wills, E. D., 178, 263 Wilson, G., 63, 65, 67, 121, 123, 137, 148, 160,173 Wilson, J. D., 105, 1'70 Wilson, 0. H., 8, 46, 170,173 Wilson, T. H., 46, 52, 54, 55, 60, 66, 131, 132, 153, 154, 161, 166, 169, 173 Winegard, A. I., 286, 302 Winkler, H., 261 Winkler, H. H., 52, 54, 55, 58, 66, 153, 173 Winters, R. W., 113, 147, 161, 173 Wintzer, G., 208, 263 Winder, R. J., 191, 211, 263 Wira, C. R., 282, 283, 301 Wisnieski, B. J., 127, 173 Witten, C., 117, 167 Woelk, H., 179, 263 Wohltmann, H. J., 277, 278, 280, 303 Wolf, F. J., 61, 131, 162 Wolfinbarger, L. Jr., 26, 29, 30, 31, 130, 173 Wolff, H., 192, 240 Wolff, J., 232, 246, 247, 260 Wolff, K., 192, 263 Wolman, M., 191, 263 Wong, P. T. S., 55, 131, 153, 154,173,204, 246 Woo, A,, 99, 166 Wood, G. C., 215,239 Wood, R. E., 263, 265, 267, 268, 269, 279, 293, 301, 303 Woodin, A. M., 228, 229, 263 Woodruff, H. B., 61, 131, 162 Woodruff, J., 201, 263 Woods, R. A., 82, 83, 127, 131, 166, 167, 168, 173 Woodward, C. B., 181, 205, 260

347

AUTHOR INDEX

Woodward, C. K., 29, 31, 135, lY3 Woodward, V. W., 26, 29, 31, 132, 135, 166, 1Y3 Woolf, L. I., 79, lY3 Woolley, D. W., 219, 263 Wrey, V. P., 193, 263 Wright, A., 192, 264 Wright, F. E., 277, 303 Wright, J . H., Jr., 275, 302 Wu, H. C. P., 56, 57, 173 Wulff, D. L., 121, 168 Wyngaarden, J. B., 3, 1 Y l

Young, E. P., 43, 168 Young, F. G., 282, 284,285, 286,298, 301 Young, I. G., 92, 93, 140, lY3, 174 Young, W. S., 111, 69, 169 Yourno, J., 211, 246 Yu, B. P., 224, 247 Yu, S.-H., 66, 168 Yuldashev, P. K., 249 Yunis, E., 200, 244 Yura, T., 115, 143, 174

Y

Zakim, D., 215, 216, 262,264 Zambrano, F., 226, 243 Zampighi, G., 226, 243 Zand, R., 14, 168 Zaneveld, L. J. D., 219, 264 Zarlengo, M. H., 94, 174 Zimmerman, E. F., 79, lY4 Zimmerman, J. D., 35, 169 Zimmerman, W., 97, 131, 164, lY4 Zimmermann, U., 91, 168, 174 Zinder, N., 129, 166 Zwaal, R. F. A., 181, 202, 203, 205, 241, 260,264 Zwaig, N., 55, 65, 132, 167, 17.4 Zweig, R. A., 316, 321

Yamada, K., 86, 164 Yamagishi, S., 218, 262 Yamamoto, M., 119, 173 Yamanouchi, T., 45, 16Y Yamashita, S., 214, 246 Yanofsky, C., 107, 138, 173 Yip, C. C., 215, 247 Yokoyama, Y., 47, 51, 16Y Yon, J., 183, 264 Yorke, R. E., 282, 283, 303 Yoshida, T., 47, 51, 16Y, 171 Yoshikawa, H., 292, 301 Young, D. A., 282, 283, 301

Z

SUBJECT INDEX A Acetate, membrane transport of mutations affecting, 86-87 Acidosis, renal tubular type, genetic aspects of, 102-105 Adenine, membrane transport of, mutations affecting, 80-83 S-Adenosylmethionine, membrane transport of, mutations affecting, 8&87 Adenyl cyclase, hydrolytic enzyme effects on, 232-233 Aerobacter aerogenes, mutations of, affecting membrane transport, 70-71,92-93 Alanine, membrane transport of, mutations affecting, 12-13, 24-25, 26-27 Amino acids, membrane transport of, mutations affecting, 4-51 Amino aciduria, genetic defect in, membrane transport effects, 44-45 a-Aminoadipate, membrane transport of, mutations affecting, 38-39 Ammonium ion, membrane transport of, mutations affecting, 94-95 L-Arabinose, membrane transport of, mutations affecting, 50-53 Arginine, membrane transport of, mutations affecting, 8-11, 24-25, 36-39 Arsenate, membrane transport of, mutations affecting, 108-109 Aspartate, membrane transport of, mutations affecting, 4, 24-25, 32-37, 38-39 Aspergillus nidulans, mutations of, affecting membrane transport, 40-43, 7475, 80-81, 86-87, 110-113 ATPase-deficient hemolytic anemia, hereditary, genetic aspects of, 102-103

B Bacillus cereus, mutations of, affecting membrane transport, 108-109 348

Bacillus megalerium, mutations of, affecting membrane transport, 96-97 Bacillus subtilis, mutations of, affecting membrane transport, 86-87, 9 6 9 7 , 122-123 Biomembranes asymmetry across, 207-208 hydrolytic enzymes in, 175-254 hyperstructure of, 209-210 protein release from, 210-217 Biotin, membrane transport of, mutations affecting, 84-85 Busby syndrome, genetic aspects of, 126127

C Calcium transport system, enzyme hydrolysis effects on, 224-226 Carbohydrates, membrane transport of, mutations affecting, 50-79 Cell wall, hydrolytic enzyme effects on, 202-207 Chloride, membrane transport of, mutations affecting, 112-114 Citrate, membrane transport of, mutations affecting, 86-87 Cystine, membrane transport of, mutations affecting, 16-17 Cystinuria, genetic defect in, membrane transport effects, 42-45

D Deoxyribose, membrane trmsport of, mutations affecting, 70-71 Diaminopimelic acid, membrane transport of, mutations affecting, 16-17 Dipeptides, membrane transport of, mutations affecting, 16-17

349

SUBJECT INDEX

E Enzymes, hydrolytic, in biomembranes, 175-254 Epinephrine, effects on sugar transport, 279-282 Escherichia coli, mutations of, affecting membrane transport, 4-17,50-69,8493, 104-107, 114-123, 141-144 Excitable membranes, enzyme hydrolysis effects on, 217-220

F Fanconi syndrome, genetic aspects of, 126-127 Fructose, membrane transport of, mutations affecting, 66-67, 70-71

G Galactose, membrane transport of, mutations affecting, 58-59, 74-75 Genetics, of membrane transport, 1-174 Glucoaminoaciduria, genetic aspects of, 128-129 Glucocorticoids, effects on sugar transport, 282-284 Glucoglycinuria, genetic aspects of, 128129 Glucose, membrane transport of, mutations affecting, 66-67, 72-75 Glucose-galactose malabsorption, genetic aspects of, 76-77 Glucose transport system, enzyme hydrolysis effects on, 229-232 @-Glucosides, membrane transport of, mutations affecting, 66-67 Glutamate, membrane transport of, mutations affecting, 6-9,24-25,32-37 Glutamine, membrane transport of, mutations affecting, 6-8 Glycerol, membrane transport of, mutations affecting, 60-63, 72-73 L-a-Glycerophosphate, membrane transport of, mutations affecting, 60-61 Glycine, membrane transport of, mutations affecting, 12-13, 26-27 Glycolate, membrane transport of, mutations affecting, 86-87

Glycolytic enzymes, in biomembranes, 181-183 Glycosuria, genetic aspects of, 76-79 Growth hormone, effects on sugar transport, 284-287 Guanine, membrane transport of, mutations affecting, 78-81

H Hartnup disease, genetic defect in, membrane transport effects, 44-47 Hexose phosphates, membrane transport of, mutations affecting, 58-61 Hexoses, membrane transport of, mutations affecting, 74-75 Histidine, membrane transport of, mutations affecting, 12-13, 18-21, 38-39 Hormones, effect on sugar transport, 274287 Hydrogen ion, membrane transport of, mutations affecting, 94-97 Hydrogenmas eulropha, mutations of, affecting membrane transport, 94-95 Hydrolytic enzymes, in biomembranes, 175-254 action of, on model systems, 184-191 types of, 178-184 effects of catabolism of membrane components, 233-236 on cell wall, 191-202 membrane transport, 217-233 protein release, 210-217 Hypoxanthine, membrane transport of, mutations affecting, 78-83

I Iminoglycinuria, genetic aspects of, 46-51 Insulin, effects on sugar transport, 274-279 Iron, membrane transport of, mutations affecting, 92-93, 96-99 Isoleucine, membrane transport of, mutations affecting, 12-15, 24-25

K Katzir-Katchalsky, Aharon, 1913-1972 bibliography of publications of, xxi-xldi memorial for, xv-xx

350

SUBJECT INDEX

Lactose, membrane transport of, mutations affecting, 52-55, 72-75 Leucine, membrane transport of, mutations affecting, 12-15, 22-23 Lipolytic enzymes in biomembranes, 178-181 action of, 184-189 Lowe’s syndrome, genetic aspects of, 126127 Lysine, membrane transport of, mutations affecting, 8-11, 24-25, 36-39

M Magnesium, membrane transport of, mutations affecting, 90-91 Maltose, membrane transport of, mutations affecting, 62-63 Mannitol, membrane transport of, mutations affecting, 66-71 Melibiose, membrane transport of, mutations affecting, 54-57 Membrane phenomena, Katzir-Katchalsky publications on, xxi, xxii Membrane transport enzyme hydrolysis effects on, 217-233 genetic control of, 1-174 dominance and recessiveness in, 146149 gene identification, 136-140 isolation of mutants, 3-135 linkage relationships, 140-146 mutant use in study of, 151-155 regulation of, 150-151 Methionine malabsorption syndrome of, genetic aspects, 46-47 membrane transport of, mutations affecting, 22-23, 38-39 p-Methylgalactoside, membrane transport of, mutations affecting, 56-59

Ochromonas danica, mutations of, affecting membrane transport, 42-43 Oligopeptides, membrane transport of, mutations affecting, 16-17 Ornithine, membrane transport of, mutations affecting, 8-11

P Peptides, membrane transport of, mufations affecting, 4-51 Phenylalanine, membrane transport of, mutations affecting, 10-13, 18-21, 2225 Phosphate, membrane transport of, mutations affecting, 104-113 Phosphotransferase system, mutations affecting, 62-69,70-71 Potassium, membrane transport of, mutations affecting, 88-91, 94-103 Proline, membrane transport of, mutations affecting, 16-17, 22-23, 24-25 Proteolytic enzymes in biomembranes, 183-184 action of, 189-191 Pseudomonas aeruginosa, mutations of, affecting membrane transport, 22-25, 72-73 Pseudomonas fluorescens, mutations of, affecting membrane transport, 24-25 Pseudomonas pseudomallei, mutations of, affecting membrane transport, 108109 Purines and pyrimidines, membrane transport of, mutations affecting, 78-85

R Respiratory chain, enzyme hydrolysis effects on, 220-223

S N Neurospora crassa, mutations of, affecting membrane transport, 26-37, 72-75, 96-99, 108-111, 124-125, 144-146

Saccharomyces cerevisiae, mutations of, affecting membrane transport, 36-41, 74-75, 80-87, 126-127 Salmonella pullorum, mutations of, affecting membrane transport, 108-109

35 1

SUBJECT INDEX

Salmonella typhimurium, mutations of, affecting membrane transport, 18-23, 68-71, 78-81, 92-93, 106-109, 122123, 141-144 Serine, membrane transport of, mutations affecting, 12-13 Shikimic acid, membrane transport of, mutations affecting, 86-87 Sodium, membrane transport of, mutations affecting, 94-95, 102-105 Sodium-potassium transport system, enzyme hydrolysis effects on, 226-229 Spherocytosis, hereditary, genetic aspects of, 102-103 Staphylococcus aureus, mutations of, affecting membrane transport, 70-73, 96-97, 124-125 Streptococcus faecalis, mutations of, affecting membrane transport, 24-27, 80-81, 94-97, 108-109 Streptococcus lactis, mutations of, affecting membrane transport, 70-71 Streptococcus strain Challis, mutations of, affecting membrane transport, 26-27 Sugar transport anoxia effects on, 262-267 epinephrine effects on, 279-282 fatty substrate effects on, 270-274 glucowrticoid effects on, 282-284 growth hormone effects on, 284-287 hormonal control of, 274-287 insulin effects on, 274-279 muscular contraction effects on, 267-270 nonhormonal regulation of, 261-274 pmsive, kinetics of, 256-261 regulation of, in eukaryotic cells, 255322 mechanisms of, 287-296

Sulfate, membrane transport of, mutations affecting, 106-109 Sulfhydryl groups, in sugar transport regulation, 293-296

T Thiamine, membrane transport of, mutations affecting, 84-87 Thymidine, membrane transport of, mutations affecting, 84-85 Tryptophan malabsorption syndrome of, genetic aspects, 46-47 membrane transport of, mutations affecting, 10-13, 18-23 Tyrosine, membrane transport of, mutations affecting, 10-13, 18-21, 22-25

U Uracil, membrane transport of, mutations affecting, 78-79, 80-83 Ureidosuccinic acid, membrane transport of, mutations affecting, 86-87 Uric acid, membrane transport of, mutations affecting, 80-81 Urindine, membrane transport of, mutations affecting, 82-83

V Valine, membrane transport of, mutations affecting, 12-15 Vitamin BI*, membrane transport of, mutations affecting, 84-85,88-89

X Xanthine, membrane transport of, mutations affecting, 78-81

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