Current Topics in Membranes and Transport, Volume 14

Current Topics in Membranes and Transport Volume 14 Carriers and Membrane Transport Proteins Advisory Board I . S . ...

Author: Felix Bronner | A. Kleinzeller

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Current Topics in Membranes and Transport Volume 14 Carriers and Membrane Transport Proteins

Advisory Board

I . S . Edelman Alvin Essig Franklin M . Harold James D. Jamieson Philip A. Knauf Anthony Martonosi Shmuel Razin Martin Rodbell Aser Rothstein Stanley G. Schultz

Contributors

J . P . Bennett L. I . Boguslavskp K . A . McGill S . H . P . Maddrell D . M . Matthews J . W . Papne Peter G. W . Plngeinann Adil E . Shainoo William F. Tivol G. B . Wurren W . F . Widdas Robert M. Wohlhueter

Current Topics in Membranes and Transport VOLUME 14

Carriers and Membrane Transport Proteins Edited b y Felix Bronner Department of Oral Biology University of Connecticut Health Center Fcirrnington, Connecticut und

Arnost Kleinzeller Depurtrnent of Physiology University of Pennsylvania School of Medicine Philadelphia, Pennsylvanio

1980 Academic Press A Subsidiary of Harcourt Brace Jovunovich, Publishers New York

London Toronto Sydney San Francisco

COPYRIGHT @ 1980, BY ACADEMIC PRESS,INC. ALL RIGHTS RESERVED. NO PART O F THIS PUBLICATION MAY BE REPRODUCED OR TRANSMITTED 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.

ACADEMIC PRESS, INC.

111 Fifth Avenue, New York, New York 10003

Utrited Kitrgdoiti Edifioir prrblislred by ACADEMIC PRESS, INC. ( L O N D O N ) LTD. 24/28 Oval Road, London N W 1 7DX

LIURAKY OF CONGRESS CATALOG CARD

N U M U E K :70-1 17091

ISBN 0-12-153314-X PRINTED IN THE UNITED STATES OF AMERICA 80 81 82 83

9 8 7 6 5 4 3 2 1

Contents List of Contributors, ix Preface, xi Yale Membrane Transport Volumes, xiii Erratum, xv Interface between Two Immiscible Liquic; as a Tool for Studying Membrane Enzyme Systems L. I. BOGUSLAVSKY

I. Introduction, 2 11. Potential Jumps at the Interface of Two Immiscible Liquids, 4 111. Experimental Equipment for Measuring the Volta Potential at the Oil/Water Interface, 1 1

IV. Compensation Potential in the Water/Oil Chain, 13 V. Some Approaches to the Study of Enzymatic Reactions Occurring at the Interface, 16 VI . Possible Mechanism of the Potential Generation at the Interface between Two Immiscible Liquids, 25 VII. Chlorophyll and Other Porphyrins at the Interface, 30 VIII. Study of Membrane Enzymatic Systems of the Respiratory Chain of Mitochondria, 42 IX. Rhodopsin and Bacteriorhodopsin at the Interface, 43 X . The Influence of the Dielectric Constant of the Oil Phase on the Efficiency of Charge Transfer through the Interface, 45 XI. Coupling of Membrane-Enzyme Systems, 46 XII. Conclusions, 48 Symbols and Abbreviations, 49 References. 51

Criteria for the Reconstitution of Ion Transport Systems ADIL E. S H A M 0 0 AND WILLIAM F. TIVOL

I. Introduction, 57 11. Reconstitution Experiments, 60 111. Conclusions and the Future of Reconstitution, 108 References, I I 1 V

vi

CONTENTS

The Role of Lipids in the Functioning of a Membrane Protein: The Sarcoplasmic Reticulum Calcium Pump J. P. BENNETT, K. A. McGILL, AND G. B. WARREN 1. 11. 111. IV. V. VI. VII. VIII. IX. X. XI. XII.

Introduction, 128 Sarcoplasmic Reticulum, 128 Purification of Ca-ATPase, 131 Equilibration of Lipid Pools, 133 Which Lipids Support ATPase Activity'?, 137 Reconstitution of Ca-ATPase into Sealed Vesicles, 144 Only 30 Lipid Molecules Modulate Ca-ATPase Function, 148 The Composition of the Lipid Annulus Is Not the Same as the Whole Bilayer, 150 Lipid Asymmetry, 154 Distribution of Lipids across the SR Membrane, 155 Transbilayer Disposition of the Phospholipid Annulus, 157 Concluding Remarks, 158 References. 159

The Asymmetry of the Hexose Transfer System in the Human Red Cell Membrane W. F. WIDDAS I. 11. 111. IV.

Kinetic Asymmetry, 166 Kinetics of Membrane Transfers with Asymmetric Affinities, 181 Morphological Asymmetry, 202 Implications of Asymmetry, 211 References, 215

Permeation of Nucleosides, Nucleic Acid Bases, and Nucleotides in Animal Cells PETER G. W. PLAGEMANN AND ROBERT M. WOHLHUETER I. Introduction and Technical Principles, 226 11. Carrier Model for Facilitated Diffusion and Tests for Its Applicability to

111. IV. V. VI. VII. VIII.

Nucleoside and Base Transport, 235 Uptake of Nucleosides and Purine Bases, 253 Properties of Nucleoside and Free Base Transport Systems, 271 Transport Inhibitors and Inactivation, 287 Regulation of Nucleoside and Free Base Transport and Uptake, 295 Permeation of Nucleotides, 303 Summary and Conclusions, 310 References. 313

Transmembrane Transport of Small Peptides D. M. MATTHEWS AND J. W. PAYNE I. General Introduction, 332 11. Peptide Transport in Animal Small Intestine, 333

CONTENTS

111. IV. V. VI.

Peptide Transport in Animal Tissues Other Than the Small Intestine, 365 Peptide Transport in Microorganisms, 367 Peptide Transport in Higher Plants, 397 Possible Physiological Advantages of Transmembrane Transport of Small Peptides, 403 VII. Concluding Remarks, 407 References, 408

Characteristics of Epithelial Transport in Insect Malpighian Tubules S. H . P. MADDRELL

I. The Route of Water Transport, 428 The Passive Epithelial Permeability of Malpighian Tubules, 438 Correlation of Structure with Function, 442 Regulatory Properties of Malpighian Tubules, 445 Malpighian Tubule Action in the Absorption of Water Vapor from the Air, 457 Summarizing Remarks, 459 References, 460

11. 111. IV. V. VI.

Subject Index, 465 Contents of Previous Volumes, 473

vii

This Page Intentionally Left Blank

List of Contributors Numbers in parentheses indicate the pages on which the authors’ contributions begin. J. P. Bennett, Department of Experimental Pathology, University College Hospital Medical School, London WClE 655, England (127)

L. 1. Boguslavsky, Institute of Electrochemistry, Academy of Sciences of the USSR, Leninsky Pr., 31, Moscow, V-71, USSR ( 1 ) K. A. McGill, Department of Biochemistry, University of Leeds, Leeds, England (127)

S. H. P. Maddrell, Agricultural Research Council Unit of Invertebrate Chemistry and Physiology, Department of Zoology, University of Cambridge, Cambridge CB2 3E5, England (427) D. M. Matthews, Department of Experimental Chemical Pathology, The Vincent Square

Laboratories of Westminster Hospital, London SWlV 2RH, England (331) J. W. Payne, Department of Botany, Science Laboratories, University of Durham, Durham DHI 3LE, England (331) Peter G. W. Plagemann, Department of Microbiology, University of Minnesota, Minneapolis, Minnesota 55455 (225) Adil E. Shamoo,* Department of Radiation Biology and Biophysics, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642 (57) William F. Tivol,i Department of Radiation Biology and Biophysics, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642 (57) G. B. Warren, European Molecular Biology Laboratory, Heidelberg, Federal Republic of Germany ( 127) W. F. Widdas, Department of Physiology, Bedford College (University of London), Regent’s Park, London NWI 4NS, England (165) Robert M. Wohlhueter, Department of Microbiology, University of Minnesota, Minneapolis, Minnesota 55455 (225)

* Present address: Department of Biological Chemistry, University of Maryland School of Medicine, Baltimore, Maryland 21201. t Present address: Department of Pharmacology and Toxicology, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642. ix


Preface Volume 14 continues the examination of problems relating to Carriers and Membrane Transport Proteins initiated in Volume 12. A major task in dissecting transport into its molecular components is to retain transport characteristics in the components. To achieve this, it is necessary to understand how transport proteins act at the interface between hydrophilic and hydrophobic domains. In the first chapter, L. I. Boguslavsky analyzes by electrochemical techniques the properties of enzymes and enzyme systems in interphases. Membrane vesicles have proved useful in elucidating some aspects of transport. A . E. Shamoo and W. F. Tivol, in the second chapter, have generalized from such studies in terms of ion transport, describing at the same time the various experimental approaches available to the investigator. Since the lipid environment of membranes profoundly affects membrane proteins, J. P. Bennett, K. A. McGill, and G. B. Warren have discussed in detail how membrane lipids influence the calcium pump in sarcoplasmic reticulum. Using hexose transport in erythrocytes as a model system, W. F. Widdas focuses attention on the evidence for an intrinsic asymmetry of the red blood cell membrane. The rapidly metabolizing cell takes up nucleic acids, nucleosides, and nucleotides; P. G. W. Plagemann and R. M. Wohlhueter have described the kinetic pitfalls one encounters in trying to characterize the entry step of cellular uptake of these important solutes. D. M. Matthews and J. W. Payne present an extensive treatment of how small peptides are transported by cells, both eukaryotic and prokaryotic. Finally, S. H. P. Maddrell analyzes the properties of electrolyte and water transport in the Malpighian tubules of the insect Rhodnius, demonstrating the problems and emphasizing the usefulness of the preparation for such studies.

xi


Yale Membrane Transport Processes Volumes These volumes originate from the Yale Department of Physiology under the editorial supervision of Joseph F. Hoffman and Gerhard Giebisch.

Joseph F. Hoffman (ed.). (1978). “Membrane Transport Processes,” Vol. I . Raven, New York. Daniel C. Tosteson, Yu. A. Ovchinnikov, and Ramon Latorre (eds.). (1978). “Membrane Transport Processes,” Vol. 2. Raven, New York. Charles F. Stevens and Richard W . Tsien (eds.). (1979). “Membrane Transport Processes,” Vol. 3: Ion Permeation through Membrane Channels. Raven, New York. Emile L. Boulpaep (ed.). (1980). “Cellular Mechanisms of Renal Tubular Ion Transport”: Volume 13 of Current Topics in Membranes and Transport (F. Bronner and A. Kleinzeller, eds.). Academic Press, New York.

xiii


ERRATUM Current Topics in Membranes and Transport, Volume 13 James B. Wade: Chapter 9, Hormonal Modulation of Epithelial Structure

Page 129 The seventh line in paragraph 3 should read: bladders do exist in cytoplasmic vacuoles of granular cells (13,28,

xv


CURRENT TOPICS I N M E M B R A N E S A N D TRANSPORT, V O L U M E

14

Interface between Two Immiscible Liquids as a Tool for Studying Membrane Enzyme Systems L . I . BOGUSLAVSK Y Institute of Electrochemistry Academy of Sciences of the USSR Moscow. USSR

I. Introduction . . . . . . . . . . . . . . . . . . . 11. Potential Jumps at the Interface of Two Immiscible Liquids . . . . . A. Distribution Potentials . . . . . . . . . . . . . . . B. Adsorption Potential . . . . . . . . . . . . . . . C. Dipole Component of Adsorption Potential . . . . . . . . . D. The Influence of Solution Composition on Adsorption Potential . . . E. Methods for Measuring the Potential at the Interface . . . . . . 111. Experimental Equipment for Measuring the Volta Potential at the Oil/Water Interface . . . . . . . . . . . . . . . . . I I IV. Compensation Potential in the WateriOil Chain . . . . . . . . . 13 V. Some Approaches to the Study of Enzymatic Reactions Occurring at the Interface . . . . . . . . . . . . . . . . . . 16 A. Adsorption of Enzymes at the Interface . . . . . . . . . . 16 9. Equipment for the Study of the Enzymatic Reaction Rate at the WateriAir Interface . . . . . . . . . . . . . . . . 19 C. Acetylcholinesterase at the Aidwater Interface . . . . . . . 20 D. Galactosyltransferase at the Aidwater Interface . . . . . . . 22 E. More Complex Systems Investigated at the Interface . . . . . . 24 VI. Possible Mechanism of the Potential Generation at the Interface between Two Immiscible Liquids . . . . . . . . . . . . . . . . 25 VII. Chlorophyll and Other Porphyrins at the Interface . . . . . . . . 30 A. Oxidation-Reduction Transformations of Chlorophyll and Porphyrins . 30 B. Electron Transfer by Chlorophyll across the Interface between Two Immiscible Liquids . . . . . . . . . . . . . . . . 33 C. Proton Phototransfer Chlorophyll . . . . . . . . . . . . 34 D. Photooxidation of Water in the Presence of Chlorophyll and Ferro Complex of Tetramethyl Ether of Coproporphyrin Adsorbed at the Octane/Water Interface . . . . . . . . . . . . . . . 35 E. Role of Water and DNP in Proton Transfer across the Interface . . . 40 Copyright 0 1980 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN @IZ-l533l4-X

1

2

L. I. BOGUSLAVSKY

VIII. Study of Membrane Enzymatic Systems of the Respiratory Chain of Mitochondria . . . . . . . . . . . . . . . . A. NADH Dehydrogenase . . . . . . . . . . . . . . . B. Succinate-Cytochrome c Reductase . . . . . . . . . . . C. Cytochrome Oxidase . . . . . . . . . . . . . . . IX. Rhodopsin and Bacteriorhodopsin at the Interface . . . . . . . . X. The Influence of the Dielectric Constant of the Oil Phase on the Efficiency of Charge Transfer through the Interface . . . . . . . . . . . . XI. Coupling of Membrane-Enzyme Systems . . . . . . . . . . . XII. Conclusions . . . . . . . . . . . . . . . . . . . Symbols and Abbreviations . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . .

1.

42 42 43 43 43 45 46 48 49 51

INTRODUCTION

One of the most important properties of biomembranes is their high catalytic activity which is determined by enzymes, some of which are inserted into membranes, others being adsorbed onto the membrane surface. The interface of phases with different dielectric constants facilitates a separation of reaction products provided the latter have different solubilities in contacting phases. The presence of a powerful electric field at the interface of the double layer and the specific orientation of molecules participating in the reaction endow an enzymatic reaction with a new property, which is described as a vectorial process. This allows a conversion of chemical or light energies into electrical energy. An interface between two immiscible liquids is the simplest model for studying the surface properties of biomembranes. Transport of ions through membranes has three stages. The first and third stages are the charge transfer through the membrane interface, whereas the second stage is diffusion through the membrane (Fig. 1). Electrochemical reactions accompanied by spatial separation of charges may occur at the interface. This can assist selective charge transfer through biomembranes. It is therefore necessary to study the ion and electron transport across the interface in order to clarify the selectivity mechanism of the membrane, the generation of transmembrane potential, and other processes which may be essential for understanding of membrane properties as a whole (Boguslavsky, 1971). The transport process through membranes as well as the interdependence of interfaces are supposed to be nonessential. Both interfaces are thought to be independent. This means that the results obtained for monolayers of a single interface are useful when the determining step of the process occurs at the interface of the membrane with an aqueous electrolyte solution. In this case one may

3

MEMBRANE ENZYME SYSTEMS

Three steps of charge transfer through a membrane

One step of charge transfer at the interphase

of two immiscible liquids

FIG. 1. Scheme of charge transfer through the membrane and across the interface of two immiscible liquids.

observe a similarity between some properties of molecules at the interface and their functions in the membrane. On the other hand, when the limiting stages of processes in the model and native systems do not coincide, the data disagree. This can be illustrated by the incompatibility of results obtained when measuring the rate of penetration of water molecules through the lipid monolayer, and the membrane of a squid giant axon (Shanes, 1963). Despite numerous studies of protein-lipid interaction in membranes, much effort is still required to achieve detailed and comprehensive knowledge of this interaction. A Coulomb interaction between charged groups on lipid and protein molecules has been studied in detail. This is much simpler than specific and hydrophobic interactions. Quite a number of investigations of enzymes at the water/oil interface deal with a special type of enzymatic system, i.e., phospholipase. These are water-soluble proteins whose substrates are lipids, i.e., the substances in a condensed state at the interface. This allows us to use the experimental procedure for monolayers to observe protein-lipid interaction and the products of this interaction (Colacicco, 1971). Henceforth, by interaction we mean not only the enzyme-substrate formation, but every chemical reaction occurring at the interface. In the region of a diffuse double layer at the lipid-water interface, when lipids and proteins are separated by more than 6 A, an electrostatic interaction occurs. The total charge of a protein globule is quite essential in this case. Such a conclusion was drawn, for example, when studying hemoglobin and albumin adsorption on a surface covered with lipid monolayers. Intermolecular interaction is essential at distances less than 6 A (Salem, 1962). The functioning of a phospholipase B was investigated by

4

L. I. BOGUSLAVSKY

measuring surface radioactivity of phospholipids containing 32P.The effective functioning of phospholipase was shown to proceed under specific conditions. It was particularly found that a surface-active substance with a negatively charged group should be present in the monolayer. The function of a special class of enzymes, i.e., the electrogenic enzymes at the interface, results in charge separation in the double electric layer. Together with the Laboratory of Bioorganic Chemistry of Moscow State University, we suggested a new approach to the problem of kinetics of enzymatic reactions accompanied by charge transfer across the interface between two immiscible liquids (Boguslavsky et al., 1974a, 197Sa; Kharkatz et NI., 1975). The change in the potential difference at the octane/water interface during enzymatic reactions accompanied by transfer of ions and electrons from one phase to another was measured by the vibrating electrode method. This approach has been used to study not only the enzymatic processes, but the ordinary catalytic reactions as well (Boguslavsky et ul., 1976~). Chlorophyll and other porphyrins were shown to be capable of transferring electrons between two redox systems in different phases (Boguslavsky et al., 1976~). Before discussing the results of the investigation of membrane enzymatic systems at the interface, we shall briefly consider some physicochemical properties of interfaces, which are necessary for the following discussion.

II. POTENTIAL JUMPS AT THE INTERFACE OF TWO IMMISCIBLE LIQUIDS

In the beginning of this century the problem of potential jumps arising at the interface of two immiscible liquids began to attract the attention of many investigators. The impetus to such investigations was given by the study of thick membranes represented by a layer of a polar organic liquid located between aqueous and nonaqueous phases. It was thought that a study of potentials in thick membranes would assist in understanding the function of more complex biological membranes. A thermodynamic theory for electrolyte solutions in two different solvents was developed by Nernst (1892), Luther (1896), Abel (1906), and van Laar (1907). By analyzing a chain of two different solvents the above authors came to a very important conclusion, namely, that the potential difference at the interface can be set up only if the coefficients of cation and anion distributions are unequal. Experimental measurements of po-

5


tential jumps at the interface of two immiscible liquids were performed by Haber (1908) and Beutner (1909). Beutner showed that the emf of those chains does not depend on the nature of the common ion. Frumkin (1919) demonstrated that the above results agree well with the theory of potential distribution. Beutner's ideas disagreed with those of Baur and Kornman (1916), who interpreted the origin of potentials in terms of the adsorption theory. The idea of the adsorption potential was introduced by Freundlich, who used it in order to treat electrocapillary phenomena. In Freundlich's opinion, the adsorption potential is superimposed on the potential, which is calculated from the Nernst formula. Frumkin compared experimental results obtained from investigations of solvent chains (I, 11) with data calculated on the basis of Nernst theory (Nernst, 1892). He showed that Baur's data are sufficiently well described by the Nernst theory. From this coincidence of theoretical and experimental results Frumkin concluded that a formal thermodynamic theory of ion distribution is quite sufficient for determining the potential difference between two immiscible liquids, and no additional assumptions about an adsorption potential jump are needed, as was proposed by Freundlich. A. Distribution Potentials For ideal solutions the distribution potential for a salt, M X , in a polar oil/water system depends on the coefficient of cation BM and anion B x distribution. If we express the coefficients of cation and anion distribution in terms of the difference of their standard chemical potentials in the aqueous and nonaqueous phases, then RTln B M=

-

Op."M

(1)

and RTln B x = " p i - ' p i

( 21

where "p0 and ' p 0 are the standard chemical potentials of ions in the aqueous and nonaqueous phases, respectively. The electrochemical potentials of individual ions can be expressed in terms of their standard chemical potentials. Under equilibrium conditions p M=

n-

OFM,

w px -

= "iix

(3)

When in each of the phases the electroneutrality is maintained, then "

c,, = c\,

oc2,

=

OC\

(4)

6

L. I. BOGUSLAVSKY

In order to determine the distribution potential, cpD, through the distribution coefficient for the salt, MX, S,,

OCMX

=-

CMX

it should be borne in mind that

S\n

=

(B\i.B\)''*

( 6)

For two systems containing the salts with a common anion, X

or

The process of ion distribution between phases is closely linked with resolvation, and is accompanied by interpenetration of the solvent molecules into both phases. The derived ratio shows the physical sense of the distribution coefficient as the power factor corresponding to the ion transfer from water into the saturated nonaqueous phase with a different polarity. Thus, cpD is the physical quantity resulting from differences of hydration and solvation energies of potential-determining ions. The concepts described here are particularly important for the interpretation of phenomena occurring at the octane/water interface in the case of membrane enzymatic systems. B. Adsorption Potential The contact of two immiscible liquids results in the interphase distribution of potential schematically shown in Fig. 2. It is assumed that the potential is measured near the aqueous phase, and the point of a test charge is separated from the interface by less than the Debye length. In this case not only the excess charges adsorbed at the interface, but the dipoles as well, will contribute to the measured value. At a n y point near the interface, where the electroneutrality conditions are not observed, the cation and anion concentration is determined by the Boltzmann equation:

(9)

7


'. I

/

x-m

t X

FIG.2. Potential distribution at the oil/water interface depending on the distance to the boundary.

Since the electroneutrality equation is not valid at the point near the interface inside the double electric layer, Eq. (7) can be expressed as

By substituting Eqs. (9) and (lo), instead of " C Mand "Cx in Eq. ( 1 I ) , at the condition that cplx+o ='Pads we have 'P =

'PO

-

('PO

-

'Pads)

= 'Pads

(12)

This means that at the interface of water with air or solvent of low polarity the Galvani potential is not determined by the coefficients of B , and B , ion distribution. The change of the observed potential can be attributed to the presence of particles adsorbed at the interface (Davies and Rideal, 1963). C. Dipole Component of Adsorption Potential

For a film composed of uncharged dipole molecules, the change of the potential jump at the interface can be assigned to a vector sum of indi-

8

L. I. BOGUSLAVSKY

vidual dipole moments (Davies and Rideal, 1963): Aqads = 4rrns1pd+ 4rrn,2pd

+ 4rrn,3pd

(13)

Here the effective dipole moment of the water molecule, ' p d , which is assumed to be specifically oriented at the water/air or water/hydrocarbon interface, is taken into account. Moreover, one should allow for the dipole moment of the bond 2 p d ,which connects the polar head with the hydrocarbon chain, or any other liposoluble part of the molecule. The bonds of H,C in the long-chain amines can serve as an example. Finally, in the case of a hydrocarbon chain, there is another component, 3pd, determined by the dipole component of the terminal bond C-H. In order to find the contribution of individual components to overall potential drop, Aqads, it is necessary to examine the adsorption potentials of molecules differing in any one functional group. By changing the terminal , hence Aqads. If the atom in the hydrocarbon chain, one can alter 3 p d and values of Ipd and 2pdare assumed to be constant when replacing the bond C-H by C-Br then the observed alteration of A q a d s may be attributed to the change of IAcPadsI

=

4.rrns*A3Pd

(14)

Gerovich and Frumkin (1936) were the first to show how considerable this alteration can be, taking as an example bromohexadecanoic acid. Insoluble monolayers of halogen-containing aliphatic compounds at the water/air interface are characterized by the negative jumps of the potential, which is explained by the orientation of the negative end of the dipole formed by the halogen atom toward air when an insoluble monolayer is formed by Br-(CH,),,-COOH molecules. A very high negative potential may be ascribed to C-Br bond orientation. The palmitic acid corresponding to this compound is characterized by a potential which is positively shifted by 0.39 V at the water/air interface. The replacement of the C-C bond by C-Br produces a general effect of 1.26 V . Since the dipole moment of C-Br bond p = 1.9 x (CGSE) with a 20 Az area per molecule, the effect would be -2.9 V , if the angle between C-Br dipoles and the water surface is taken into account. The difference of the observed potential jump from the calculated one can be due to the relative polarizability of the C-Br bond. When the surface pressure varies, i.e., the area per molecule changes, the vertical component 3pdalso undergoes certain alterations due to the change of the slope of the hydrophobic chains to the interface. Let a monolayer be adsorbed at the interface. If the polar heads of molecules adsorbed at the interface have an electric charge, then the monolayer is referred to as ionized.

9


D. The Influence of Solution Composition on Adsorption Potential

Ions whose charges are opposite to the charge of a monolayer are always active relative to those monolayers. The concepts of the effect of counterions on the properties of charged monolayers are based on the assumption of the existence of particle redistribution in the film-boundary layer due to their potential energy in this layer being different from that in the solution. (This assumption is valid both for charged and uncharged particles.) Therefore the concentration of uncharged particles at the surface may be expressed as

cs =

C.eUlkT

(15)

where U is the decrease of the potential energy of the system upon transition of the particles from the bulk to the surface layer. C', in accordance with the simplifying postulate of the theory, is the function of the surface excess, r:

cs = f(r)

(16)

If the ions on the surface have a charge n , then the potential energy should be altered by the value of the work of a single charge in the electric field:

Cs= Co.exp [ U

-

neAcpo]/kT

(17)

By assuming that a homogeneously charged surface is impenetrable to counterions, i.e., point charges, Gouy has found a solution for the Boltzmann equation of the type (17) for the potential distribution cpG at the interface :

I n principle, the absolute value A q G need not coincide with A v o . The concentration dependences of ApG and A p 0 are experimentally identical. The potential Acpo should not be identified with 30 mgiml) at a ratio of 0.5 mg cholate per mg SR protein, and the mixture is centrifuged (for 16 hours at 100,000 gav)through a 20-30% ( w h ) detergent-free sucrose gradient. As the Ca-ATPase-lipid-detergent micelles enter the sucrose gradient the detergent diffuses away and the micelles reassociate to become larger and sediment faster. The other proteins do not have this ability to form membrane-like oligomeric complexes and remain at the top of the gradient. The band containing purified Ca-ATPase together with its associated lipid is removed from the bottom of the gradient and dialyzed to remove any remaining cholate. IV.

EQUILIBRATION OF LIPID POOLS

When a membrane is disrupted by detergent the micelles formed are in dynamic equilibrium-in other words there is exchange of components

134

J. P.BENNETT ET AL.

between micelles. Metcalfe and co-workers showed that if exogenous lipid is added to solubilized SR and the Ca-ATPase is isolated by sucrose density gradient centrifugation then the lipid associated with the protein is found to have the same composition as the whole lipid pool (Warren et al., 1974a). In the experiment described in Table I, differing amounts of DOPC were added to Ca-ATPase in the presence of cholate. The proportion of DOPC in the isolated lipid-protein complexes was found to be the same as the fraction of lipids in the original incubation mixture that was DOPC. The lipids surrounding each protein in the membrane have become replaced in part by the exogenous lipids. This is a conservative process; at no time is the protein delipidated with a consequent danger of denaturation. The procedure whereby lipid-protein complexes were isolated from excess lipid and detergent in the incubation mixture by sucrose density gradient centrifugation (Warren et al., 1974a,b) was called "lipid substitution" and is shown schematically in Fig. 2. The lipid substitution technique provided a means for changing the lipid environment around the native Ca-ATPase for the lipids of choice. In one step over 90% of the lipid can be replaced by a synthetic lipid (Table I): if the process is repeated >98% of the lipid surrounding the

TABLE I THE EQUILIBRATION OF EXOGENOUS DoPC ENDOGENOUS LIPID POOL^

WITH THE

Percentage of total fatty acid content DOPC-substituted Ca-ATPase Fatty acid

Ca-ATPase

16:O

26 12 14 19

18:O 18: 1

18:2 20:4 22:5

Expected Observed

16

6

I:l

1:1

5:I

20: I

15 14 8 4 7 6 3 2 61 51 85 93 8 II 2 0.5 7 8 3 3 Percentage DOPC substitution SO 53

50 50

83 82

95 92

DOPC was added to Ca-ATPase in the presence of cholate at ratios to the endogenous lipid of 1: I (two experiments), 5: I , and 20: I . The complexes obtained by the lipid substitution technique were analyzed for fatty acid content by gas-liquid chromatography and the fraction of the lipid that was DOPC was calculated. From Warren e/ a / . (1974a).

135

SARCOPLASMIC RETICULUM CALCIUM PUMP

8

, I

EOUl L l BRAT I ON OF LIPID POOLS

ATPase Endogenous lipid Synthetic lipid Detergent

DETERGENT-FREE SUCROSE GRADlENl

FIG.2. A schematic illustration of the lipid substitution process. The lipids surrounding the membrane protein equilibrate with the exogenous synthetic lipid in the presence of detergent, and the protein-lipid complexes are then separated from excess lipid and detergent by sucrose density gradient centrifugation (see text).

Ca-ATPase will consist of the exogenous lipid. Using this technique Warren er al. (1974a) showed that a single synthetic lipid (DOPC) is sufficient to support the ATPase activity and calcium accumulating activity of the Ca-ATPase. For the purposes of examining the effect of different lipids on protein function, a much simpler method for removing the detergent from a lipidprotein-detergent complex can be used. If the incubation is diluted so that the total concentration falls below the critical micelle concentration, the detergent will largely dissociate from the complexes. This is the basis The experimenof the “lipid titration” procedure (Warren et al., 1974~). tal protocol is to incubate Ca-ATPase with a large excess of test lipid in the presence of cholate (at a concentration of 5-10 mg/ml). In these experiments protein function (Ca2+-dependent ATP hydrolysis) was measured by a coupled spectrophotometric assay. When an aliquot of the incubation mixture is diluted into the assay cuvette 200-400 times, most (> 96%) of the cholate diffuses away from the micelles. This has the effect of “freezing” the lipid composition of different micelles at the moment of dilution (for at least the duration of the assay). As a result a sequential series of samples removed from an incubation

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J. P. BENNETT ET AL.

mixture and assayed shows the time-course of equilibration of the lipid pools (Fig. 3). The rate of equilibration was found to depend upon the concentration of cholate used (Warren et al., 1974~).If the cholate concentration is 10 mg/ml or greater, equilibration takes place within a few seconds. If the cholate concentration is less than 5 mg/ml equilibration takes hours rather than minutes to occur. The interpretation that the activity changes observed in this sort of experiment are due to a change in the lipids interacting with the protein derives from a comparison of the properties of lipid titration complexes with those of Ca-ATPase associated with exogenous lipid isolated by the lipid substitution procedure (Warren ef al., 1974~). Figure 3 also shows that the cholate-mediated equilibration of lipid pools is a continuous process. After equilibration with the first lipid (DMPC) is complete and the enzyme activity has reached a stable level, the addition of a new exogenous lipid (DOPC) means further equilibration of pools and a resultant further change in enzyme activity. The fact that the enzyme activity can return to its original value makes it unlikely that the original decrease in activity was due to partial denaturation of the enzyme. One would anticipate that any detergent would allow equilibration of lipids between micelles, and could thus be used for lipid titrations. However, the ease of equilibration differs from detergent to detergent, depending upon the critical micelle concentration and the micellar structure. OMPC

I

"r

Incubation time (minutes)

FIG. 3. The time-course of lipid titration. Ca-ATPase was mixed with excess DMPC and cholate, and samples were assayed at various times for ATPase activity at 20°C. DMPC comprised -94% of the total lipid and the cholate concentration was 7.5 mg/ml. After 50 minutes an aliquot of this incubation was added to excess DOPC and cholate; in this second incubation DOPC comprised -90% of total lipid and the cholate concentration was 13.0 mg/ml (J. P. Bennett, unpublished results.)


137

Triton X- 100 is reported to facilitate exchange of the phospholipids associated with Ca-ATPase for lysolecithin (Peterson et al., 1978), although it is far less adept than cholate in the exchange of diacylphosphatidylcholines (G. B. Warren, unpublished results). Octyl glucoside, a nonionic detergent with a particularly high critical micelle concentration (Helenius et al., 1979), will readily exchange phospholipids (J. P. Bennett and G. B. Warren, unpublished results). A procedure that has been validated for one phospholipid cannot automatically be used for other lipids. The lipid titration method using cholate was shown to be valid for phosphatidylcholines by comparison with lipid substitution complexes (Warren et al., 1974~);for other phospholipids Bennett et al. (1978a) developed a protocol to validate the lipid titration results which did not require large quantities of lipid (see Section V). V. WHICH LIPIDS SUPPORT ATPase ACTIVITY?

In the case of the phosphatidylcholines it was demonstrable that cholate causes complete equilibration of lipid pools, by using the lipid substitution technique (Warren et al., 1974~).This means that we can begin to answer one of the questions originally asked: can lipids modulate the enzymatic activity of the proteins? One of the best known physical properties of pure phospholipids is the phase transition temperature. Above the transition temperature the fatty acid chains flex with respect to the lipid headgroup (Levine et al., 1972); below the transition their motional freedom is restricted. The temperature of this phase transition is principally dependent on the structure of the fatty acid chains, although it is also influenced by the phospholipid headgroup. Table I1 shows how the phase transition affects the enzyme activity of the Ca-ATPase. The lipid titration method was used to measure the ATPase activities (at 37°C) supported by a number of synthetic phosphatidylcholines. The activities fall into two groups. The lipids which contained unsaturated fatty acid chains, and which show a lipid phase transition well below 37"C, have a high activity, while the lipids with only saturated fatty acid chains and relatively high transition temperatures have lower activities. The distinction between the two classes is also clear when the way in which enzyme activity varies with assay temperature is examined (Fig. 4). The DOPC-ATPase lipid titration complex is most like the native CaATPase in its behavior. The unsaturated phospholipids, such as DMPC, have a much greater variation in activity with temperature, corresponding to a higher energy of activation for the ATPase reaction.

138

J.

P. BENNETT ET AL.

TABLE I1 ENZYMEACTIVITYOF Ca-ATPase I N LIPIDTITRATION COMPLEXES WITH DIFFERENT LCHOLINES” PHOSPHATIDY Transition temperature of pure lipid Lipid Dimyristoylphosphatidylcholine (DMPC) Dipalmitoylphosphatidylcholine (DPPC) Distearoylphosphatidylcholine (DSPC) Dioleoylphosphatidylcholine (DOPC) Dielaidoylphosphatidylcholine (DEPC) 2-Oleoyl-3-palmitoylphosphatidylcholine(OPPC)

Fatty acid chains

14:O

{

16:O 18:O 18: 1 cis 18: I trans 18. 6; I cis (posn 2)

(posn 3)

(“C)

ATPase activity at 37°C (pmolei minute/ mg)

23.7 41.7 58.2 -22

4.5 7.1 5.9 16. I

II

21.0

93% of the total lipid pool. ATPase activities were measured after the lipid pools had equilibrated (see Fig. 3). For comparison, the preparation of Ca-ATPase used in this experiment had an ATPase activity of 11.5 pmole/minute/mg at 37°C. (J. P. Bennett, unpublished results). (I

There is also evidence that the length of the fatty acid chains plays a role in the modulation of ATPase activity. Johannson e f al. (1980) have shown that lipids containing unsaturated 14-carbon chains do not support high activity despite a low phase transition temperature; however, the activity increases with the addition of agents which thicken the hydrocarbon region of the lipid bilayer. The low activity observed when Ca-ATPase is surrounded by DPPC below its transition temperature has been examined by Hidalgo et af. (1976) and by Nakamura et al. (1976). Both these studies concluded that the reduction in enzyme activity is due to a reduced rate of decomposition of the phosphorylated enzyme intermediate in the reaction mechanism (Makinose, 1973); the change of phospholipid affects to a lesser degree the rate of phosphorylation of the enzyme and the steady-state concentration of phospho-enzyme intermediate. Translocation of calcium ions from an outward-facing site on the Ca-ATPase (relative to the enzyme’s orientation in native SR) to an inward-facing site occurs subsequently to phospho-enzyme formation, and is necessary for the hydrolysis of phospho-enzyme (see, e.g., Ikemoto, 1976). This translocation of calcium is accompanied by a conformational change in the Ca-ATPase protein (Dupont and Leigh, 1978). It seems reasonable to suppose that the occurrence of this change of conformation will depend on the physical state of the

139


lipids with which the protein interacts, so that when the Ca-ATPase is in a “rigid” bilayer (below the transition temperature) calcium translocation is inhibited, with a consequent reduction in the rate of phospho-enzyme decomposition and hence ATPase activity. The distribution of Ca-ATPase molecules (visualized as intramembranous particles by freeze-fracture electron microscopy) is also dependent on the physical state of the phospholipid bilayer (Kleemann and McConnell, 1976). Ca-ATPase was incorporated into bilayers consisting almost entirely of DMPC and samples were prepared for freeze-fracture electron microscopy from temperatures either above or below the lipid phase transition of DMPC (Fig. 5). Above the phase transition temperature the protein molecules appear to be randomly distributed through the membrane. Below the transition temperature the protein is seen only in certain areas, leaving extensive regions of protein-free DMPC which show the banded appearance typical of a pure saturated lipid below its phase transition.

\ oYoMPC-ATPare \

l\

\

T [“C I

1 45 40 35 30 25 32

M 34

33 (OK-’.

l? IF\, 5

35 lo3)

36

FIG. 4. Arrhenius plots for lipid titration complexes. Ca-ATPase surrounded by its native SR lipids (SR-ATPase), or by DOPC or DMPC in a lipid titration complex (DOPCATPase and DMPC-ATPase), was assayed for calcium-dependent ATPase activity over the temperature range 4-48°C. The results are expressed as an Arrhenius plot so that the slope of the lines corresponds to the energy of activation for the ATPase reaction (J. P. Bennett, unpublished results.).

140

J. P. BENNETT ET AL

FIG.5 . Freeze-fracture of DMPC-ATPase vesicles. Vesicles comprising DMPC and CaATPase (at a ratio of 9:1 by weight) were quenched for freeze-fracture electron microscopy from (a) 30°C and (b) 13°C. The bar represents 0.1 pm. (From Kleeman and McConnell, 1976.)

One envisages that during the process of cooling the membrane through the phase transition (the change from fluid to rigid state takes place over a narrow temperature range for a single pure lipid) the rigid phase phospholipids form a two-dimensional "crystal" which excludes the protein: as the rigid phase increases in area the proteins are confined to a smaller area, and finally to tight clusters as seen in Fig. 5b. That this is likely to be the case is shown in experiments in which the membrane consists of a mixture of two saturated phosphatidylcholines (DMPC and DPPC) with different transition temperatures. For such a membrane the transition from all fluid to all rigid occurs over a wide temperature range, and at intermediate temperatures rigid and fluid phases coexist (Shimshick and McConnell, 1973). At such an intermediate temperature Kleemann and McConnell (1976) showed that Ca-ATPase is excluded from the rigid phase and randomly distributed in the fluid phase (Fig. 6). The clustering of Ca-ATPase molecules below the phase transition may itself affect the protein function, and it has been observed that the CaATPase in a DPPC membrane shows cooperative kinetics below the phase transition ( J . P. Bennett, unpublished results). This implies a specific interaction between the protein molecules when forced together in this way, which in turn raises the hope that conditions could be found in which the proteins would form a crystalline array in the membrane from which structural information about Ca-ATPase could be extracted by electron microscopy (Henderson and Unwin, 1975).


141

In order to investigate the role of different headgroups in supporting ATPase activity, Bennett et al. (1978a) devised a method for validating the lipid titration technique for lipids that were not available in sufficient quantity for isolation and analysis of lipid-protein complexes using the lipid substitution method. A lipid titration as shown in Fig. 3 involves starting with Ca-ATPase surrounded by a lipid which supports a high activity (DOPC or SR lipids)

FIG. 6 . Freeze-fracture (DMPC + DPPC)-ATPase vesicles. Vesicles comprising an equimolar ratio of DMPC and DPPC, and CA-ATPase (at a lipid-protein ratio of 3: I by weight), were quenched for freeze-fracture electron microscopy from (a) 37"C, (b) 32"C, (c) 20°C. The bar represents 0. I pm. (From Kleeman and McConnell, 1976.)

142


and following the decrease in activity on mixing with a test lipid in the presence of cholate; this type of experiment can be called a “forward titration.” However, one could also start with Ca-ATPase surrounded by a lipid which supported a very low activity and observe the increase in activity: this is a “back-titration.” Bennett et al. (1978a) argued that if a forward titration and a back-titration of Ca-ATPase with a given test lipid gave the same enzyme activity then cholate must be equilibrating the test lipid with the lipid surrounding the protein. As an illustration of this technique, the lipids surrounding the CaATPase were replaced with DOPC using the lipid substitution technique. When a 70-fold molar excess of cholesterol was added to the complex obtained (DOPC-ATPase) in the presence of a high concentration of cholate the enzyme activity fell to less than 2% of that of DOPC-ATPase within 1 minute of the addition. The ATPase activity could then be completely restored by adding excess DOPC in cholate (Fig. 7). The complete reversibility of the process indicates that no irreversible inactivation of the Ca-ATPase has occurred, and that cholate is acting to allow equilibration of lipid pools. If a test lipid is used to reactivate the cholesterol-ATPase complex and the ATPase activity rises to a value that is the same as that for a

10

ATPase activity (IU/mg at 37OC)

5

DOPC-ATPose

DOPC-ATPase Cholate Cholesterol

DOPC-ATPose Cholate Cholesterol DOPC

FIG. 7. The reversibility of the back-titration procedure. A DOPC-ATPase complex (prepared by the lipid substitution technique) was incubated with an excess of cholesterol in the presence of cholate, and then more DOPC in the presence of cholate was added, as described in the text. (From Bennett et a / . , 1978a.)

143


forward titration, then cholate facilitates exchange of that lipid just as it does for DOPC. If equilibration does not occur then the same lipid in cholate should not affect the ATPase activity-and this behavior was observed in the case of the cerebroside lipids ( G . B. Warren, unpublished results). By a similar argument, if cholate catalyzes only partial exchange of the test lipid then the forward and back titration procedures will not lead to the same ATPase activities. Figure 8 shows the results of forward and back titrations with seven classes of phospholipid, compared with DOPC. The lipids used all have fatty acid chains that will be fluid at 37°C and will not interfere with an analysis of headgroup specificity. All of the test lipids do lower the activity of DOPC-ATPase and raise the activity of cholesterol-ATPase to approximately the same level, so that lipid equilibration has occurred in all cases and the activities reflect the interaction of the protein with the test lipid. There is a clear correlation of enzyme activity with headgroup charge, as shown in Fig. 8. The zwitterionic dioleoylphospholipids support the highest ATPase activity while DOPA with two negative charges supports the lowest. Phospholipids with a single negative charge-DOPG and the

100

ToDOPC-ATPase activity

80

0

Forward litrotion Back titration

60 40

20 Test lipid headgroup charge

FIG.8. Phospholipid headgroup specificity for ATPase activity. Lipid titration complexes of Ca-ATPase with the indicated phospholipids were prepared by both the “forward titration” and “back titration” procedures (see text) and the ATPase activity was measured at 37°C. For lipid abbreviations, see Table 111. All lipids used had only oleoyl (18:l) fatty acid chains except cardiolipin; in this case gas-liquid chromatography analysis showed that 18:l and 18:2 fatty acid chains comprised 77% of the total so that the chains should be sufficiently fluid at 37°C not to interfere with analysis of headgroup specificity. The headgroup charge structures indicated assume that each ionizable group will be fully expressed: this will not be the case however for DOPS and DOPA under the assay conditions used. (From Bennett et al., 1978a.)

144

J.

P.BENNETT ET AL.

unphysiological synthetic lipids OMeDORA and NAcDOPE-support an activity which is about 5-fold lower than DOPC, while DOPS which is zwitterionic with an additional negative charge supports an ATPase activity intermediate between that supported by zwitterionic phospholipids and those with a single negative charge. Cardiolipin has two negative charges, like DOPA, but supports an activity which is about the same as that supported by those phospholipids with a single negative charge. Cardiolipin is synthesized in vivo by condensing two molecules of phosphatidylglycerol (which has a single negative charge) so that the resulting molecule resembles two diacyl phospholipids joined by a glycerol bridge (Hirschberg and Kennedy, 1972). These results indicate that the bridge between the singly negatively charged headgroups on cardiolipin has very little effect on the activity supported by this lipid. The observation that cardiolipin, DOPG, OMeDOPA, and NAcDOPE which have structurally different headgroups all support a similar ATPase activity suggests that it is the charge structure of the headgroup that is the dominant factor in determining the phospholipid headgroup specificity of this membrane protein. Each of these lipids carries a single negative charge on each diacylglycerophosphate moiety. However, other factors must also be involved since DOPC and DOPE which are both zwitterionic support significantly different activities. VI.

RECONSTITUTION OF Ca-ATPase INTO SEALED VESICLES

The examination of the fatty acid chain and headgroup specificity of Ca-ATPase previously described considered only one functional assay: that of calcium-dependent ATPase activity. The requirements for optimum ATPase activity were that the protein should be surrounded by lipids with a zwitterionic headgroup and fatty acid chains that are in the fluid state. However, the functional requirements of SR in vivo are that the lipid environment should not only support ATPase activity but also allow accumulation of calcium. The “reconstitution” procedure first described by Racker allows one to incorporate Ca-ATPase into membrane vesicles comprising test lipid and measure the ability of these vesicles to accumulate calcium. Martonosi (1968) showed that after SR had been solubilized with deoxycholate, the detergent could be removed by simple dilution. The resulting material regained the appearance of consisting of membranous vesicles in the electron microscope and showed a partial restoration of the original calcium accumulating activity. Successful reconstitution of


145

SR which has been solubilized with detergent back into vesicles into which calcium uptake can be measured was later achieved by Meissner and Fleischer ( 1973). Racker ( 1972) described a simple and reproducible reconstitution procedure whereby SR (or Ca-ATPase), solubilized with cholate, is mixed with excess lipid in cholate solution. Slow removal of the detergent by dialysis allows the lipid to form sealed membrane vesicles incorporating Ca-ATPase into which uptake of calcium could be measured. The procedure readily allows investigation of the efficiency of different lipids in these reconstituted vesicles, and the conclusions of Racker and his colleagues (Racker, 1972; Racker et d., 1975; Knowles and Racker, 1975; Knowles et al., 1975, 1976) were that maximal calcium uptake activity was seen when using total purified phospholipids from soybean, or a mixture of purified phosphatidylcholine and phosphatidylethanolamine from natural sources. Phosphatidylethanolamine alone was found to support only a reduced rate of calcium uptake, while vesicles reconstituted with phosphatidylcholine did not support calcium uptake at all. Acetylated phosphatidylethanolamine, where the amine group has been blocked, did not support calcium uptake in reconstitution experiments unless alkylamines were added to the vesicles (Knowles et al., 1975). This led Racker and his colleagues to suggest that the presence of the free amine group in the membrane was an essential prerequisite for reconstitution of the Ca-ATPase. Warren el al. (1974a) demonstrated that purified Ca-ATPase and the synthetic phospholipid DOPC were sufficient to allow measurable calcium uptake in a reconstituted system. However it was clear that in this system the competence of the reconstituted vesicles in calcium uptake was greatly dependent upon the exact conditions used. In particular Warren et al. (1980a) showed that calcium uptake was sensitive to small changes in the ratio of lipid and detergent to protein (see Fig. 9). The use of SR instead of purified Ca-ATPase allows greater latitude of experimental conditions; since exogenous lipid added in the reconstitution procedure accounts for > 99% of the total lipid, SR was used as the starting material in studies of the lipid requirements of reconstituted vesicles (Bennett et al., 1978a). A series of synthetic phospholipids were tested in the reconstitution system (Table 111); all the lipids had fluid (oleoyl) fatty acid chains since the reconstitution methodology described seems to be inadequate for lipids with saturated fatty acid chains and high transition temperatures (J. P. Bennett and G. B. Warren, unpublished results). It is apparent from these results that several different phospholipid species can meet the requirements for calcium uptake. The complex lipid composition of

146


0 20 -

-

-

015 -

C a2'u pt ak e (tmoleslrninlrng) 010 005 -

-

--I

m g OOPC I m g ATPase m g tholate I m g OOPC

,

*O

*

5 0 loo 150

04

20

50 100 150

20

50 100 150

0.6

05

FIG.9. Conditions for reconstitution using DOPC-ATPase. Purified Ca-ATPase with lipids replaced by DOPC (by the lipid substitution technique) was reconstituted using DOPC and cholate in the ratios indicated, and the initial rates of calcium uptake at 25°C were determined. From Warren er ul (1980a).

TABLE I11 PROPERTIES OF MEMBRANES RECONSTITUTED FROM SARCOPLASMIC RETICULUM USING DIFFERENT LIPIDS"

Liuid Dioleoylphosphatid ylcholine (DOPC) Dioleoylphosphatidylet hanolamine (DOPE) Dioleoylphosphatid ylserine (DOPS) N-Acetyl-dioleoylphosphatidylethanolamine( N AcDOPE) Dioleoylphosphatidylgl ycerol (DOPG) 0-Methyl-dioleoylphosphatidic acid (OMeDOPA) Dioleoylphosphatidic acid (DOPA) DOPE + DOPC NAcDOPE + DOPC OMeDOPA + DOPC

Ca2+ accumulating ATPase activity activity 0.84 0 0.43 0 0. I7 0 0.24 0.68 0.05 1.20

I .55 2.03 0.37 0.32 0.32 0.38 0.15 0.06 0.45 1.01

a In each reconstitution experiment the total amount of lipid used was the same and added lipid comprised > 99% of the total; in the last three experiments equal amounts of the two lipids were used. The Ca2+accumulating and ATPase activities are in initial rates expressed in pnoleiminutelmg enzyme at 25°C. From Bennett et ul. (1978a).


147

native SR is not essential either for ATPase activity o r calcium accumulation. Since four of the lipids tested supported calcium uptake there can be no absolute specificity for a particular lipid species. The inability of certain lipids (which supported ATPase activity) to support calcium accumulation was a single consequence of their inability to form intact vesicles in these experiments. The morphology in negatively stained electron micrographs of vesicles reconstituted with DOPC and DOPE are shown in Fig. 10. The reconstituted vesicles containing DOPC showed high calcium uptake activity, and in the electron micrograph they appear to be smooth intact vesicles whose membranes bound an enclosed volume; some of these may be multishelled. In contrast the reconstituted vesicles containing DOPE, which are not capable of calcium uptake, appear to consist of irregularly stacked sheets of membrane. There is no evidence of a membrane-enclosed volume into which calcium could be pumped. The nature of the reconstitution technique means that it is difficult to interpret the inability of a particular lipid to form sealed vesicles. It may reflect a basic property of that lipid, or it may reflect a methodological failure of the reconstitution technique. For example, small changes in the proportions of components in the initial incubation can significantly alter the levels of calcium accumulation by the reconstituted vesicles (Warren et al., 1980). Although workers in Metcalfe’s laboratory have been unable

FIG.10. Electron micrographs of reconstituted membranes. Membranes reconstituted using SR were negatively stained with 2% phosphotungstate and examined at 40,OOOx magnification. The lipids used were (a) DOPC, (b) DOPE, and (c) an equal mixture of DOPC and DOPE. The bars represent 0.1 p m . (From Bennett et d., 1978a.)

148

J. P. B E N N E T ET AL.

to obtain successful reconstitutions with phosphatidylethanolamine from any source, Racker and co-workers have consistently had success with soybean phosphatidylethanolamine: this is presumably due to some small difference in methodology. The suggestion that a specific chemical group must be present in the phospholipids to allow the coupling of ATPase activity to calcium uptake in reconstituted membranes (Knowles et al., 1975) is clearly not borne out by the date in Table 111. Conversely the lipids which did not support calcium accumulation did not have any structural property that prevents coupling. Bennett et al. (1978a) carried out a reconstitution experiment using equal mixtures (by weight) of these lipids with DOPC: all these lipid mixtures can support measurable calcium accumulation in reconstitutions of lipid mixtures (Table 111). In the case of DOPE at least there is evidence (see Section VIII) that the DOPE molecules will interact with the Ca-ATPase protein, so that this direct interaction does not uncouple calcium uptake from ATP hydrolysis. Examination of these vesicles in the electron microscope (Fig. 10) shows that they are intact and apparently contain an enclosed volume. It seems to be this morphological requirement alone which allows the measurement of calcium uptake in reconstitution experiments. Racker and Eytan (1975) proposed that a proteolipid component of SR (MacLennan et al., 1972) acted as a “coupling factor” which might be the ionophoric component of the calcium pump (Racker, 1975): in its absence ATP hydrolysis could not be accompanied by calcium translocation. This model seems dubious because of the very small amount of proteolipid that is present in SR; Sigrist et al. (1977) found that it accounted for only 0.15% of the total protein. The molar ratio of proteo1ipid:Ca-ATPase is thus approximately I :50, which must preclude a direct stoichiometric interaction having an essential role in coupling. However, traces of proteolipid can always be detected in the preparations of purified Ca-ATPase [those of MacLennan (1970) and Warren et af. (1974a)l which have been used in reconstitution experiments so that it remains possible that the proteolipid has an indirect role in coupling-for example, by acting as the carrier for a counterion. VII.

ONLY 30 LIPID MOLECULES MODULATE Ca-ATPase FUNCTION

When Ca-ATPase is prepared using the sucrose density technique, the lipid-protein ratio in the isolated complex depends on the concentration of detergent used in the initial incubation. The higher the concentration

149


of the detergent above its critical micelle concentration the more detergent micelles there will be, and as a result lipid molecules will tend to partition out of the protein-lipid-detergent micelles to form more lipiddetergent micelles. At the same time there will be an irreversible loss of enzyme activity since lipid is required for the functioning of the CaATPase. When the ATPase activity is plotted as a function of the lipidprotein ratio it is apparent that there is a critical lipid-protein ratio that represents the minimum lipid content that will support maximal activity. Figure 1 1 shows the result of this experiment, using cholate as detergent (Warren et al., 1974~).The critical lipid content required to support ATPase activity is approximately 30 mole lipid per mole protein. The figure also shows data from a similar experiment (J. P. Bennett and G. B. Warren, unpublished results) in which a different detergent, octyl glucoside (see Helenius et al., 1979), was used: the behavior observed was the same. There is evidence that 30 lipids per protein are bound to the Ca-ATPase after solubilization with other nonionic detergents: Triton X-100, Tween 80, and C,,E, (LeMaire et af., 1976). These 30 molecules of lipid per molecule Ca-ATPase, which have been called the “lipid annulus” (Warren et af., 1975), are envisaged as the first bilayer shell of lipid molecules surrounding the Ca-ATPase protein. Experiments using spin-label lipid probes have identified the same number of lipid molecules as being immobilized relative to the rest of the bilayer,

% SR - ATPase

activity 0

Cholate

o Octyl - glucoside

0 J

0

20

40

60

80

100

moles lipid I mole ATPase

FIG. 11. The dependence of ATPase activity on 1ipid:protein ratio. Ca-ATPase was incubated with detergent (either potassium cholate or octyl glucoside) at various concentrations, and centrifuged through a sucrose gradient. The complexes obtained were analyzed for 1ipid:protein ratio and ATPase activity at 37°C. [Closed symbols, cholate from Warren el ul. ( 1 9 7 4 ~ )open ; symbols, octyl glucoside (J. P. Bennett and G . B. Warren, unpublished results).]

150


presumably because of direct interaction with the protein (Montecucco et al., 1977; see also Nakamura and Ohnishi, 1975). Even when the protein is embedded in a bilayer containing a great excess of lipids, it is the lipid annulus which determines the protein activity. Hesketh et al. (1976) showed this using Ca-ATPase with the lipids replaced by DPPC, in various lipid-protein ratios. The physical properties of the annulus lipids (examined using spin-label techniques) were different from the physical properties of the rest of the bilayer, and it was the physical properties of the lipid annulus that appeared to modulate enzyme activity. An attempt to make data from similar experiments fit quantitatively with a theoretical prediction of physical properties of lipids associated with Ca-ATPase (using a lipid annulus model) has been unsuccessful, however (Moore et al., 1978). Spectroscopic measurements can provide an estimate of the ability of annulus lipids to diffuse into the rest of the bilayer. Hesketh et al. (1976) point out that resolution of the annulus by electron spin resonance techniques provides an upper limit for the rate of exchange of annulus lipids into the free bilayer of about lo6 sec-I; Chapman ef al. (1979) report that deuterium nuclear magnetic resonance techniques fail to resolve the two lipid components and suggest that this implies a lower limit for the annulus-bilayer exchange rate of about l O5 sec-l. Thus annulus lipids can exchange rapidly into the bilayer and appear "immobilized" only by comparison with the extremely fast diffusion rates of lipids in the free bilayer.

VIII.

THE COMPOSITION OF THE LIPID ANNULUS IS NOT THE SAME AS THE WHOLE BILAYER

Cholesterol is a lipid which was found to support an extremely low ATPase activity. However, when cholesterol is added to Ca-ATPase in an equimolar mixture with a phospholipid (at higher ratios the lipid mixture does not form a bilayer) in a lipid titration experiment, it caused no loss in ATPase activity (Warren et al., 1975). This is interpreted as meaning that cholesterol is not entering the lipid annulus and interacting with the protein in these experiments. When phospholipid-cholesterol-protein complexes are isolated by the lipid substitution technique there is a reduced ATPase activity as long as there are fewer than 30 phospholipid molecules per Ca-ATPase (Fig. 12). If phospholipid was added back to these complexes so that the final stoichiometry exceeds 30 phospholipids per Ca-ATPase, then maximal ATPase activity is regained. These experiments suggest strongly that


151

Moles phospholipid I Mole ATPase FIG. 12. The ATPase activity of cholesterol- ATPase complexes. Ca-ATPase was incubated with various concentrations of cholesterol and potassium cholate, and centrifuged through a sucrose gradient. The complexes obtained were analyzed for phospholipid, cholesterol, and protein content and the ATPase activity was measured at 37°C. In every case the total lipid content (phospholipid + cholesterol) exceeded 30 mole per mole CaATPase. Open symbols are from a similar experiment on the absence of cholesterol, a s in Fig. 1 1 . (From Warren et a / . , 1975.)

cholesterol is rigorously excluded from the lipid annulus as long as there are sufficient phospholipids to complete the lipid annulus, and that cholesterol in the bilayer outside the annulus has little effect on enzyme activity. Cholesterol is not a major component of intracellular membranes such as SR in vivo, and it was a very different structure from the phospholipids. In order to find out whether there is a similar lateral segregation of different phospholipid types within the membrane Bennett et al. (1980) chose to use as a model system a binary mixture of DOPC and DOPA. These lipids differ only in their headgroups, and the differences are substantial. DOPC is zwitterionic while DOPA has two negative charges (although the second negative charge may not be fully expressed at physiological pH). They also support very different ATPase activities. The rationale was first to measure the way in which the observed ATPase activity of Ca-ATPase surrounded by just 30 phospholipid molecules depends on the proportion of DOPC and DOPA in the annulus. If all 30 annulus phospholipids have an equivalent role in the support of ATPase activity then the observed ATPase activity would be a linear function of annulus composition; however, this could not be assumed necessarily to be the case. Second, the dependence of ATPase activity on the lipid composition of a much larger pool surrounding the Ca-ATPase is measured: if the relationship between ATPase activity and lipid composition were the same as with a lipid pool consisting of only annulus lipids, then lateral segregation is not occurring. If, however, there is a

152


change with the pool size in the dependence of ATPase activity on phospholipid composition, this reflects lateral segregation occurring so that in the presence of excess lipids the composition of the annulus is not the same as that of the total lipid pool. In order to demonstrate the dependence of ATPase activity on the phospholipid composition in the annulus surrounding Ca-ATPase use was made of the fact that phospholipids will preferentially displace cholesterol from the lipid annulus (see previously). Ca-ATPase was prepared with its lipids replaced by cholesterol and phospholipid was added (in the presence of cholate) at a stoichiometry of 28 mole per mole Ca-ATPase. The enhancement of ATPase activity seen on addition of phospholipid (a 5-fold increase with DOPA and more than 30-fold with DOPC) is due to these phospholipids forming part of the phospholipid annulus. The ATPase activity was clearly found to be a linear function of the proportion of the two lipids in the annulus (Fig. 13). This implies that all the annulus lipids contribute equally in the modulation of enzyme activity. However, when the Ca-ATPase is embedded in a membrane containing an excess of phospholipid over and above that needed to form

o 28moles

lipid /mole ATPase

900 moles

0

20

100

80

40 60 %DOPA, 60 40

80

100 J

20

0

% DOPC

FIG. 13. ATPase activity supported by DOPC-DOPA mixtures. Phospholipid mixtures in the presence of cholate were incubated with Ca-ATPase at two different 1ipid:protein ratios and the ATPase activities were measured. Open symbols: phospholipid was added at a ratio of 28 mole/mole Ca ATPase to a cholesterol-ATPase complex prepared by the lipid substitution technique (which contained 27 mole cholesterol and 2 mole phospholipid per mole protein). Closed symbols: phospholipid was added at a ratio of 900 mole/mole CaATPase to DOPC-ATPase (which contained 38 mole phospholipid per mole protein). (From Bennett er d., 1980.)

153


the annulus, the response is nonlinear. In an experiment in which the total lipid pool was approximately 900 mole phospholipid per mole CaATPase (Fig. 13) it appeared that lateral segregation must be occurring such that when 80% of the total lipid pool consists of DOPA, the lipid annulus consists principally of DOPC. Similar experiments led us to speculate that lateral segregation occurs to a lesser degree to exclude a singly negatively charged phospholipid (DOPG) from the lipid annulus, but that the protein does not distinguish between DOPC and DOPE (Fig. 14). Lateral segregation occurs most strongly to exclude from the lipid annulus just those lipids which support the lowest ATPase activity (see Fig. 8). In other words, lateral segregation acts to optimize enzyme activity in a membrane comprising a mixture of phospholipid types. In these experiments, all the phospholipids had the same (oleoyl) fatty acid chains. Warren et al. (1980b) carried out a similar experiment using lipids with different fatty acid chains: DMPC and DOPA (Fig. 15). The result is striking. A mixture of these two phospholipids supported an ATPase activity which is much higher than that supported by either phospholipid alone; it is as if the protein is responding to the elements of DOPC. Similar synergism was observed with other mixtures of phospholipids differing in both chain and headgroup. The phenomenon may be accounted for by a combination of two effects. Lipid segregation as previously discussed will mean that DMPC interacts with the Ca-ATPase protein in preference to DOPA, but at the same time the presence of

a20 . I

b

0

20

100

80

40 60 96 DOPE 60 40 % DOPC

80

100

20

0

FIG. 14. ATPase activity supported by phospholipid mixtures. The dependence of ATPase activity on phospholipid composition for (a) DOPC-DOPG mixtures and (b) DOPC-DOPE mixtures. A DOPC-ATPase complex was incubated with excess phospholipids in cholate at a ratio of about 900 mole phospholipid per mole Ca-ATPase (J. P. Bennett, unpublished results).

154


I 0

20

40 /a '

60

80

100

20

0

DOPA

L

100

80

60

40

%DMPC

FIG. 15. ATPase activity supported by DMPC-DOPA mixtures. The dependence of ATPase activity on phospholipid composition for DMPC-DOPA mixtures, investigated as in Fig. 14. (From Warren et a / . , 1980b.)

DOPA in the membrane with oleoyl chains (18-carbon) will tend to overcome the inhibitory effect of the shorter chains (ICcarbon) of DMPC (Johannsson et af., 1980). IX.

LIPID ASYMMETRY

There is now a substantial body of evidence that there is an asymmetric distribution of phospholipids between the two halves of the bilayer for plasma membranes from various sources (see Rothman and Lenard, 1977), although the detailed distribution of the lipids is not the same for the different membranes examined. This asymmetry is maintained by the low rate of exchange of phospholipids between the two halves of the bilayer ("flip-flop"), and its origin may lie in the mode of membrane biosynthesis (Rothman and Kennedy, 1977a; Hirata er af., 1978). Despite the apparent universality of phospholipid asymmetry in plasma membranes no role for it in cell function has been identified, although one possible physiological outcome has been noted (Zwaal et af., 1977). The way in which phospholipids are distributed across the bilayer of intracellular membranes is rather less clear. Since in cells which secrete, at the moment of secretion the inner monolayer of an intracellular membrane becomes contiguous with the external monolayer of the plasma membrane while the cytoplasmic faces of each membrane join, one might


155

suppose that the lipid composition of the two cytoplasmic leaflets will resemble each other, as will those of the two luminal leaflets. If lipid asymmetry does in fact originate at biosynthesis then this relationship is necessary, since synthesis of phospholipids occurs primarily at an intracellular membrane, the endoplasmic reticulum (Jelsema and Morre, 1978), in eukaryotic cells and conservation of asymmetry during the membrane fusion event in secretion is the only way asymmetry arising from biosynthesis would be observed in the plasma membrane. However there is also the possibility that lipid asymmetry could be maintained by specific enzymes within the plasma membrane (Bretscher, 1973) in which case no such relationship of the transverse disposition of lipids across the bilayer in intracellular and plasma membranes is predicted. Published reports of measurements of the transverse disposition of phospholipids in intracellular membranes do not resolve the questions. For example, different workers using phospholipases as probes for lipid asymmetry in rat liver microsomes have variously proposed that phosphatidylethanolamine (PE) is found predominantly in the outer monolayer (Nilsson and Dallner, 1977), predominantly in the inner monolayer (Higgins and Dawson, 1977), or distributed equally between the two monolayers (Sundler et al., 1977). It is apparent that intracellular membranes are much less tractable than plasma membranes to analysis of lipid asymmetry. Rothman and Lenard (1977) have listed the experimental criteria necessary to establish lipid asymmetry. The membranes should be present as a pure preparation (no contaminating lipid from other membranes), and all the membranes should form closed vesicles with the same sidedness. It must be shown that the labeling reagent should under the conditions of the experiment neither penetrate the membrane nor lead to lysis. None of the above experiments on rat liver microsomes fulfill all these criteria. X.

DISTRIBUTION OF LIPIDS ACROSS THE SR MEMBRANE

Various chemical labeling reagents have been used to localize amino groups in SR. Hasselbach and Migala (1975) used fluorescamine, and came to the conclusion that the aminophospholipids are present mainly on the outer monolayer. However fluorescamine is by no means an ideal reagent since it decomposes rapidly, and being insoluble in water must be added to the sample as a solution in an organic solvent which may perturb the lipid bilayer. Hidalgo and Ikemoto (1977) used a stable, watersoluble, complex of fluorescamine with cycloheptaamylose which was

156


shown not to disrupt vesicles (as judged by the inaccessible [14C]inulin space). They also concluded that most of the PE (the predominant aminophospholipid) was situated in the exterior half of the lipid bilayer. Vale ( 1977) used 2,4,6-trinitrobenzenesulfonate(TNBS) as a label and again concluded that the majority of PE is external; electron microscopy was used to monitor vesicle integrity after labeling. Sarzala and Michalak (1978) used both TNBS and phospholipases and concluded again that most of the PE and PS was in the outer monolayer. In a different SR preparation, believed to consist of inside out vesicles, the majority of PE was, as predicted, on the inner monolayer. PC was approximately equally distributed between the two halves of the bilayer in both cases. In an attempt to fulfill the criteria listed by Rothman and Lenard (1977) for measurement of lipid asymmetry, McGill et al. (1980) made a preparation of sealed right-side out vesicles. The rationale was that only such vesicles will be able to accumulate calcium. SR was incubated with ATP and CaCI, in the presence of potassium oxalate so that within sealed vesicles calcium oxalate was precipitated. The dense calcium oxalatecontaining vesicles were separated from leaky vesicles and inside out vesicles by sucrose density gradient centrifugation. The precipitated calcium oxalate was finally removed by allowing the calcium pump to work in reverse in a solution containing ADP and EGTA. [For methodology, see Bennett et al. (1978b). A similar procedure has been used by Bonnet et al. (19781.1 These vesicles were found to be enriched in the Ca-ATPase protein as compared with the initial SR preparation, but to have a very similar phospholipid composition. TNBS labeling (using conditions described in Rothman and Kennedy, 1977b) indicated that only half (47 5 4%) of the PE in these membranes resides in the outer monolayer. PC is also distributed approximately equally in these vesicles (5 I k 4% external measured using a phospholipid exchange protein by a method similar to that of Bloj and Zilversmit, 1976). However the small amount of glycolipid in SR (Narasimhan et al., 1974) is present only on the inner surface where it is inaccessible to galactose oxidase (using the labeling technique of Gahmberg and Hakomori, 1973). It is not clear why the sealed right-side out preparation of SR should differ from unfractionated SR in its transbilayer distribution of phospholipids. The difference cannot be explained by the rate of flip-flop of lipids between leaflets of the bilayer. McGill et al. showed that there is neglible increase in the pool of PC accessible to a phospholipid exchange protein (method of Zilversmit and Hughes, 1977) over 4 days for sealed rightside out SR, so that the half-time for flip-flop is in excess of 10 days.


157

This is in contrast to rat liver microsomes where flip-flop is rapid with a half-time of 45 minutes (Zilversmit and Hughes, 1977); presumably this reflects the actively synthetic role of microsomes compared with the stability of SR as a specialized organelle.

XI.

TRANSBILAYER DISPOSITION OF THE PHOSPHOLIPID ANNULUS

The disposition of the annulus phospholipids between the two leaflets of the lipid bilayer has been investigated by Bennett et al. (1978b). It was observed that 30 lipids per Ca-ATPase protein were resistant to digestion by phospholipase D, regardless of the initial 1ipid:protein ratio. These lipids were believed to be the annulus lipids previously identified by other techniques (see Section VII). The annulus lipids will not be digested by phospholipase D because of their proximity to the Ca-ATPase protein; the reason they do not exchange into the rest of the bilayer and so become available for digestion is that phosphatidic acid (the product of phospholipase D lipolysis) will under the conditions of these experiments form a separate rigid phase in the lateral plane of the membrane (Galla and Sackmann, 1975). Phospholipase D in aqueous solution will digest the outer monolayer only of pure lipid vesicles, so that this method can be extended to determine how many of the annulus lipids reside in the outer half of the SR bilayer. A phospholipid exchange protein was used to insert into the outer monolayer only of sealed right-side out SR vesicles DOPC which was radiolabeled in the choline moiety. The proportion of these labeled lipids that are resistant to digestion by phospholipase D can be used to calculate the number of annulus lipids in the outer half of the bilayer. A representative experiment is shown in Fig. 16. When either the whole bilayer or only the outer monolayer is examined there is a pool of phospholipid which is not digested by phospholipase D. In the whole bilayer this corresponds to the 30 annulus phospholipids per Ca-ATPase protein. In the outer monolayer 15 mole lipid per mole Ca-ATPase remain undigested. This means that the annulus phospholipids are distributed approximately equally between the inner and outer leaflets of the membrane bilayer. Since the annulus phospholipids are all in contact with the penetrant hydrophobic surface of the protein, these experiments indicate that the part of the protein that penetrates the membrane must resemble a cylinder, and provide indirect evidence that the Ca-ATPase protein completely spans the bilayer.

158


W

0

a I-

U

--2 4o W

. n -

a

.-

\

Whole bilayer

‘.

30

’D

+

w (51 W .’D

3

-w 10 e

Outer monolayer

0)

I

FIG. 16. Phospholipase D digestion of the lipids surrounding Ca-ATPase. The time course of disappearance of radioactivity from the lipid fraction of Ca-ATPase associated with [3H-choline]-DOPC on treatment with phospholipase D in the presence of 40 mM CaC12. Closed symbols: unsealed fragments of DOPC-ATPase complex prepared used the lipid substitution technique. Open symbols: sealed right-side out SR labeled with [3H]DOPC in the outer monolayer only. (From Bennett ef u / . , 1978b.)

XII.

CONCLUDING REMARKS

Apart from a very few exceptional examples of membrane proteins susceptible to structural analysis by physical methods (such as bacteriorhodopsin; see Henderson and Unwin, 1975), information about the structural interactions involved in the functioning of a membrane protein must be inferred from less direct biochemical approaches. In the case of the sarcoplasmic reticulum Ca-ATPase these approaches have given a fairly detailed picture of the way the protein interacts with the lipid bilayer matrix in which it resides. The experiments reviewed here have depended largely on the use of synthetic (chemically homogeneous) lipids in conjunction with mild (nondenaturing) detergents. The techniques used have included sucrose density centrifugation, simple dilution and dialysis, phospholipid exchange proteins and phospholipases as well as physical methods such as electron spin resonance spectroscopy and electron microscopy. It turns out that Ca-ATPase is critically dependent upon its lipid environment for enzyme activity. The structure of the lipids which interact with the protein is important: they should be zwitterionic with “fluid”


159

fatty acid chains (which should be 16-20 carbons in length) for optimal protein function. Of the 90 or so lipids per Ca-ATPase molecule in native SR, only 30 lipid molecules-equally distributed between the halves of the bilayer-are required for maintaining and modulating the enzyme activity. The 30 “lipid annulus” phospholipids can be distinguished from the remainder of the bilayer both by physical probes and by biochemical criteria: most notably the protein can segregate into the annulus from the bilayer just those lipids which best support the protein’s function. While Ca-ATPase remains one of the best characterized membrane enzymes, a large body of work with many other examples shows that similar patterns of lipid-protein interactions occur generally (for access to the literature, see review by Sandermann, 1978). For example fluid fatty acid chains seem to be a general requirement and headgroup specificities, sometimes far more rigorous than for Ca-ATPase, are often observed. With other proteins amenable to suitable experiments a stoichiometry of lipid-protein interaction (a lipid annulus) has been identified. It is still true that our understanding of the behavior of membrane enzymes lags far behind what we know about soluble enzymes. However the efforts of the last decade, most profitably with “model” membrane systems such as sarcoplasmic reticulum, have given us sufficient factual knowledge for biochemists at least to dream of the nature of the molecular interactions within membranes that seem to be so crucial in cell function. REFERENCES Bennett, J. P., Smith, G. A , , Houslay, M. D., Hesketh, T. R . , Metcalfe, J. C., and Warren, G. B. (19784. The phospholipid headgroup specificity of an ATP-dependent calcium pump. Biochim. Biophys. Acra 513, 310-320. Bennett, J. P . , McGill, K. A., and Warren, G. B. (1978b). Transbilayer disposition of the phospholipid annulus surrounding a calcium transport protein. Nature (London) 274, 823-825. Bennett, J. P., Warren, G. B., Smith, G. A., Hesketh, T. R . , Houslay, M. D., and Metcalfe, J. C. (1980). Lateral segregation of binary phospholipid mixtures around a calcium transport protein. Submitted for publication. Bloj, B., and Zilversmit, D. B. (1976). Asymmetry and transposition rates of phosphatidylcholine in rat erythrocyte ghosts. Biochemistry 15, 1277- 1283. Bonnet, J. P . , Galante, M., Brethes, D., Dedien, J. C . , and Chevallier, J . (1978). Purification of sarcoplasmic reticulum vesicles through their loading with calcium phosphate. Arch. Biochem. Biophys. 191, 32-41. Bretscher, M. S. (1971). A major protein which spans the human erythrocyte membrane. J . Mol. B i d . 59, 351-357. Bretscher, M. S . (1973). Membrane structure: some general principles. Science 181, 622629.

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Louvard, D., Semeriva, M., and Maroux, S. (1976). The brush-border intestinal aminopeptidase, a transmembrane protein as probed by macromolecular photolabelling. J. Mol. B i d . 106, 1023- 1035. McFarland, B. H., and Inesi, G. (1970). Studies of solubilised sarcoplasmic reticulum. Biochem. Biophys. R e s . Commun. 41, 239-243. McGill, K . A., Smith, G . A,, Plumb, R. W., and Warren, G. B. (1980). Symmetry of phosphatidylcholine and phosphatidylethanolamine distribution in a population of sarcoplasmic reticulum vesicles sealed with their cytoplasmic side outwards. Submitted for publication. MacLennan, D. H. (1970). Purification and properties of an adenosine triphosphatase from sarcoplasic reticulum. J . B i d . C h e m . 245, 4508-4518. MacLennan, D. H., and Holland, P. C. (1975). Calcium transport in sarcoplasmic reticulum. Annu. R e v . Biophys. Bioeng. 4, 377-404. MacLennan, D. H., Yip, C. C., Iles, G. H., and Seeman, P. (1972). Isolation of sarcoplasmic reticulum proteins. Cold Spring Harbor Symp. Quani. Biol. 37, 469-477. Makinose, M. (1973). Possible functional states of the enzyme of the sarcoplasmic reticulum calcium pump. FEES L e u . 37, 140- 143. Martonosi, A. (1968). Sarcoplasmic reticulum. Solubilisation of microsomal adenosine triphosphatase. J . Biol. C h e m . 243, 71-81. Meissner, G., and Fleischer, S. (1971). Caracterisation of sarcoplasmic reticulum from skeletal muscle. Biochim. Biophys. Acta 241, 356-378. Meissner, G., and Fleischer, S. (1972). The role of phospholipid in Ca2+-stimulatedATPase activity of sarcoplasmic reticulum. Biochim. Biophys. Acia 255, 19-33. Meissner, G., and Fleischer, S. (1973). Ca2+uptake in reconstituted sarcoplasmic reticulum vesicles. Biochem. Biophys. R e s . Commun. 52, 913-920. Meissner, G., Connor, G. E., and Fleischer, S. (1973). Isolation of sarcoplasmic reticulum by zonal centrifugation and purification of Ca2+-pump and Caz+-binding proteins. Biochim. Biophys. Acra 298, 246-289. Montecucco, C., Smith, G . A., Warren, G. B . , and Metcalfe, J. C. (1977). In “Structure and Function of Energy Transducing Membranes” (K. van Damm and B . F. van Gelder, eds.), pp. 187- 192. Elsevier, Amsterdam. Moore, B. M., Lentz, B. R., and Meissner, G. (1978). Effects of sarcoplasmic reticulum CaZ+-ATPaseon phospholipid bilayer fluidity: boundary lipid. Biochemistry 17, 52485255. Nakamura, M., and Ohnishi, S. (1975). Organisation of lipids in sarcoplasmic reticulum membranes and Ca2+-dependentATPase activity. J. Biochem. (Tokyo) 78, 1039- 1045. Nakamura, H . , Jilka, R. L., Boland, R., and Martonosi, A. N. (1976). Mechanism of ATP hydrolysis by sarcoplasmic reticulum and the role of phospholipids. J . Biol. C h e m . 251, 5414-5423. Narasimhan, R., Murray, R. K., and MacLennan, D. H. (1974). Presence of glycosphingolipids in the sarcoplasmic reticulum fraction of rabbit skeletal muscle. FEES Leir. 43, 23-26. Nilsson, 0.S . , and Dallner, G. (1977). Transverse asymmetry of phospholipids in subcelM a r membranes of rat liver. Biochim. Biophys. Acia 464, 453-458. Packer, L., Mehard, C. W., Meissner, G., Zahler, W. L., and Fleischer, S . (1974). The structural role of lipids in mitochondria1 and sarcoplasmic reticulum membranes. Freeze-fracture electron microscopy studies. Biochim. Biophys. Acia 363, 159- 181. Peterson, S. W., Hanna, S . , and Deamer, D. W. (1978). Comparative studies of detergent effects on the calcium adenosine triphosphatase of sarcoplasmic reticulum: protein


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isolation, lipid exchange and enzyme transport function. Arch. Biochem. Bi o p h y~ .191, 224- 232. Racker, E. (1972). Reconstitution of a calcium pump with phospholipids and a purified Ca2+-adenosinetriphosphatase from sarcoplasmic reticulum. J . B i d . Chem. 247, 81988200. Racker, E . (1975). Reconstitution, mechanism of action and control of ion pumps. Biochem. Soc. Trcins. 3, 785-802. Racker, E., and Eytan, E. (1975). A coupling factor from sarcoplasmic reticulum required for the translocation of Ca2+ions in a reconstituted Ca2+-ATPasepump. J . B i d . Chem. 250, 7533-7534. Racker, E., Knowles, A . F., and Eytan, E. (1975). Resolution and reconstitution of iontransport systems. Ann. N.Y. Accid. Sci. 264, 17-31. Rothman, J. E., and Kennedy, E. P. (1977a). Rapid transbilayer movement of newly synthesised phospholipids during membrane assembly. Proc. N a t l . Accid. Sci. U . S . A . 74, 1821-1825. Rothman, J. E., and Kennedy, E. P. (1977b). Asymmetric distribution of phospholipids in the membrane of Beicillus meguterium. J . Mol. B i d . 110, 603-618. Rothman, J. E., and Lenard, J. (1977). Membrane asymmetry. Science 195, 743-753. Sandermann, H. (1978). Regulation of membrane enzymes by lipids. Biochim. Biophys. Acta 515, 209-237. Sarzala, M. G., and Michalak, M. (1978). Studies on the heterogeneity of sarcoplasmic reticulum vesicles. Biochim. Biophys. Acici. 513, 221-235. Shimshick, E. J., and McConnell, H. M. (1973). Lateral phase separation in phospholipid membranes. Biochemisiry 12, 2351-2360. Sigrist, H., Sigrist-Nelson, K . , and Gitler, C. (1977). Single phase butanol extraction: a new tool for proteolipid isolation. Biochem. Biophys. Res. Commun. 74, 178- 184. Sundler, R., Sarcione, S. L., Alberts, A . W., and Vagelos, P. R. (1977). Evidence against phospholipid asymmetry in intracellular membranes from liver. Proc. Ncitl. Accrd. Sci. U.S.A. 74, 3350-3354. Thorley-Lawson, D. A , , and Green, N. M. (1973). Studies on the location and orientation of proteins in the sarcoplasmic reticulum. Eur. J . Biochem. 40, 403-413. Vale, M . G . P. (1977). Localisation of the amino phospholipids in sarcoplasmic reticulum membranes revealed by trinitrobenzene-sulfonateand fluorodinitrobenzene. Biochim. Biophys. Acta 471, 39-48. Warren, G. B., Toon, P. A , , Birdsall, N. J. M., Lee, A. G., and Metcalfe, J. C. (1974a). Reconstitution of a calcium pump using defined membrane components. Proc. Nciil. Acud. Sci. U.S.A. 71, 622-626. Warren, G. B., Toon, P. A,, Birdsall, N. J. M., Lee, A . G., and Metcalfe, J. C. (1974b). Complete control of the lipid environment of membrane-bound proteins: application to a calcium transport system. FEBS Lett. 41, 122-124. Warren, G. B., Toon, P. A , , Birdsall, N. J. M., Lee, A. G., and Metcalfe, J . C. (1974~). Reversible lipid titrations of the activity of pure adenosine triphosphatase-lipid complexes. Biocherni,\try 13, 5501-5507. Warren, G. B., Houslay, M. D., Metcalfe, J. C., and Birdsall, N. J . M. (1975). Cholesterol is excluded from the phospholipid annulus surrounding an active calcium transport protein. Naiure (London) 255, 684-687. Warren, G. B., Hesketh, T. R., Smith, G. A , , Metcalfe, J. C., and Bennett, J . P. (1980a). Lipid requirements for the reconstitution of an active calcium pump. Submitted for publication.

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Warren, G. B., Bennett, J. P., Smith, G . A , , Hesketh, T. R., Houslay, M. D., and Metcalfe, J . C. (1980b). Optimal function of a reconstituted calcium pump. Submitted for publication. Zilversmit, D. B., and Hughes, M. E. (1977). Extensive exchange of rat liver microsomal phospholipids. Biochim. Biophys. Acttr 469, 99- 110. Zwaal, R. F. A . , Comfurius, P., and van Deenen, L. L. M. (1977). Membrane asymmetry and blood coagulation. NOIIII.E (London) 268, 358-360.

CURRENT TOPICS I N MEMBRANES AND TRANSPORT. VOLUME

14

The Asymmetry of the Hexose Transfer Svstem in the Human Red Cell Membrane W . F . WIDDAS Department of Physiology Bedford College (University of London) London. England

I . Kinetic Asymmetry . . . . . . . . . . . . . . . . . A . Historical . . . . . . . . . . . . . . . . . . B . Substrate Facilitated Transfers . . . . . . . . . . . . C . Affinity Constants for the Hexose Transfer System . . . . . . D . Heterologous Exchanges . . . . . . . . . . . . . . E . Initial Transfer Rates in Zero-trans Experiments . . . . . . . F . Flux Measurements under Different Conditions . . . . . . . G . Asymmetry of Affinities Derived from Inhibitor Studies . . . . . H . Asymmetric Affinity of Cytochalasin B . . . . . . . . . . I . Asymmetric Proteolytic Responses . . . . . . . . . . . I1 . Kinetics of Membrane Transfers with Asymmetric Affinities . . . . . A . The Basis of Kinetic Asymmetry . . . . . . . . . . . B . Asymmetric Affinities and the Two Site Model . . . . . . . C . Redistribution of Components in the Mobile Carrier Hypothesis . . D . The Regen and Tarpley Kinetics Applied to Nontransportable Inhibitors E . Applications of Asymmetric Carrier Kinetics . . . . . . . . F . The Effects of pH and Temperature . . . . . . . . . . . 111. Morphological Asymmetry . . . . . . . . . . . . . . . A . Asymmetry in the Erythrocyte Membrane . . . . . . . . . B . Asymmetry of the Membrane Environment . . . . . . . . C . Asymmetry of the Sugar Membrane Transfer System . . ? . . IV . Implications of Asymmetry . . . . . . . . . . . . . . . A . Consequences of Asymmetry . . . . . . . . . . . . . B . Physiological Implications . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . .

. 166 . 166 . 167

. . . . . . .

168 170 170 172 173 177 179 . 181 . 181 . 182 . 187 . 192 . 198 . 200 . 202 . 202 . 206 . 209 . 211 . 211 . 213 215

The present state of the Carrier Hypothesis was excellently reviewed in Volume 7 of this series by LeFevre (1975) and carrier-mediated glucose transport was critically discussed in the same year by Jung (1975). A I65 Copyright @ 1980 by Academic Press. Inc . All rights of reproduction in any form reserved ISBN C-12- 153314-X

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somewhat different approach to the same topic has since been given by Naftalin and Holman (1977) and readers have these and several earlier reviews to choose from to obtain an overall survey of the properties and complexities of the facilitated transfer of sugars across the human erythrocyte membrane and other sites. One of the complexities of the system in the human erythrocyte is that of asymmetry. This asymmetry of the hexose transfer system has presented itself in several ways. It is the intention in this article to review some of these and to trace their developments and to consider the implications to the concepts of facilitated transfers generally. 1.

KINETIC ASYMMETRY

By asymmetry we mean a lack of similarity of reactiveness of those parts of the hexose transfer system which may be approached from the outside or from the inside of the cell membrane. Since the transfer system can be approached effectively only by sugars which are substrates for transport or by sugar derivatives or other molecules acting as inhibitors of transfer, asymmetry shows itself as an anomaly either in the kinetics of sugar transfers or in the kinetics of inhibition of sugar transfer. A. Historical Wilbrandt (1954) first reported asymmetric behavior of the glucose transfer system in human erythrocytes. He reported that phloretin esters (particularly polyphloretin phosphate) showed a most definite preference for inhibiting glucose exit from cells as compared to the entrance. A 32Plabeled specimen could be shown not to penetrate the cells and in consequence the inhibitor could be presumed to be acting on the outside surface. On the hypothesis of enzymes being involved, one for inward transport and the other for outward transport, it was postulated that only the one involved in putward transport was inhibited. Wilbrandt pointed out however that the kinetics of inhibition could give rise to marked asymmetries even with identical enzymes cis and trans and varying with the type of inhibitor. This original observation of inhibitor asymmetry was readily confirmed by Bowyer and Widdas (1957, 1958), but these authors showed that if hexose-absorbing sites on both sides of the membrane were involved even when transfer was undirectional then competition could largely explain the differences observed. This is because in exits the outside

167

ASYMMETRY OF THE HEXOSE TRANSFER SYSTEM

sugar concentration is very small and cannot competitively displace the inhibitor as can occur during an entry experiment when the outside sugar concentration is large. However Bowyer and Widdas found that inhibition of exit developed more rapidly than inhibition of entry when cells were incubated with 1fluoro-2,4-dinitrobenzene(FDNB) as shown in Fig. 1. Since this inhibitor is noncompetitive and irreversible the asymmetric effect on exits and entrance called for an alternative explanation and although the possibility of FDNB causing “internal competition” was considered, subsequent work failed to find any supporting evidence (Sen and Widdas (1962b). The idea of internal competition was however revived by Regen and Tarpley ( 1974). Asymmetric inhibitory effects of irreversible and noncompetitive inhibitors have been further studied by Batt and Schachter (1973) and these will be discussed later. B. Substrate Facilitated Transfers

Sugars acting as substrates for the membrane transfer system appear to have a facilitatory effect on such transfer and one of the earliest kinetic anomalies to be observed was the more rapid rate of glucose-glucose

’41:1 0

1

1

1

1

1

1

100 200 300 Incubation time (min)

I

400

FIG. 1, Development of inhibition of glucose entry and exit on incubation with 1.4 m M FDNB at 20.5”C in the presence of 100 m M glucose. 0, Glucose exit; X , glucose entry. Exit and entry measured at 37°C. (From Bowyer and Widdas, 1958.)

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exchange, which could be measured with radioactively labeled sugar, relative to the maximal rate of net sugar transfer determined by osmotic swelling or shrinking methods. The first hint of this was given by Britton (1957) who observed a glucose exchange rate about 4-fold the maximum rate of net transfer found by Widdas (1954; see also Britton, 1964). This early observation was confirmed by LeFevre and McGinniss ( 1960) whose experiments also emphasized the different order of magnitude for glucose-glucose exchange relative to net accumulation. The effect was analyzed in more detail by Mawe and Hempling (1965) and by Levine et L I I . (1965) whose analyses concentrated on the accelerating effect of sugars on the trans side of the membrane in measurements of the flux of radioactive sugar. The hypothesis was that, in the mobile carrier model, there was a faster movement of carriers across the membrane if they carried a substrate sugar. Although acceleration of glucose efflux was seen with glucose (or galactose) in the outside medium sorbose and fructose appeared to have a retarding effect but there may have been osmotic complications at the high concentrations used with these sugars. From the low affinity of sorbose and fructose for the hexose system they would not be expected to show either acceleration or inhibition of glucose efflux.

C. Affinity Constants for the Hexose Transfer System

The earliest attempts at determining the half-saturation constant for glucose were based on competition experiments. Widdas (1953, 1954) based his estimates on the retardation of sorbose entry by glucose acting as a competitive inhibitor whereas LeFevre (1953, 1954) used the competition between glucose and phloretin. LeFevre estimated the half-saturation constant at between 7.5 and 10 mM while Widdas's results were between 7 and 17 mM. Thus both authors agreed that the magnitude of the half-saturation constant for glucose was about 10 mM at 37°C. Sen and Widdas (1960a,b, 1962a,b) introduced the method of following exits into low outside sugar concentrations to determine the half-saturation concentration (see Fig. 2a and b). They obtained a value of 4 mM at 37"C, i.e., less than half the best estimates based on sorbose and phloretin competition. The Sen- Widdas method involves following a saturated efflux into a series of outside solutions from which an influx is progressively built up as the concentration increases. The half-saturation determined may be


Q

I

$-

169

LL8.4mM

.

0 . 7 m ~ J2 . 7 m d 4.6mMJ 6.5mM

1 min

12.2mM

1.01

E 0.8

v

/

b

FIG. 2. (a) Tracings of a series of records from the photoelectric apparatus during "exit" experiments at 37°C and pH 7.4. Cells equilibrated in 76 m M glucose were losing glucose into media containing glucose at the concentrations shown. The linear part of each record has been produced to cut the baseline, and the time from injection of the cells to this intersection was measured for analysis of the results. (From Sen and Widdas, 1962a.) (b) "Exit" times obtained from records such as described in (a) plotted against the concentration of glucose in the outside media. The line (drawn by eye) gives two intercepts; that on the ordinate represents the time ( t o ) which would have been taken for exit into a glucose-free medium and that on the abscissa gives the concentration of glucose into which the exit time would be twice t o . (From Sen and Widdas, 1962a.)

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W.

F. WIDDAS

viewed as that outside concentration which engenders a half-maximal influx in opposition to the saturated efflux. Miller (1965a,b, 1968a,b) repeated estimations of the glucose affinity constants using the Sen and Widdas method and on the basis of sorbose inhibition but also showed how data from isotopic exchange experiments could be used to derive the affinity constant for exchange. Miller drew attention to the variability of the results and showed that this occurred even when the different procedures were used in the same laboratory and the divergent results were therefore ascribed to shortcomings of the kinetics of the simple carrier hypothesis. Thus at 20°C Miller found the affinity constant for glucose to be 1.8 mM by the Sen and Widdas method, 17-23 mM by the inhibition of sorbose transport, and 38 mM from a Lineweaver-Burk type plot of isotopic exchanges. D. Heterologous Exchanges

Allied to the acceleration of the sugar flux during the exchange situation (relative to the net transfers) is the observation by Miller (1968a) that the initial rate of radioactive exchange could be increased more by some sugars than others. Thus mannose and galactose in the outside medium accelerated the efflux of labeled glucose more than nonradioactive glucose itself. This result is also not to be expected from the kinetics of the simple carrier model. E. Initial Transfer Rates in Zero-trans Experiments

Zero-trans experiments involve measuring the rate of transfer of sugar from one side while there is no sugar at the other side. This is an ideal which in an entry experiment can be approached only in the initial stages but is more closely realized in exit experiments where the volume of medium can be greatly in excess of the volume of cells from which the sugar is egressing. Lacko et af. (1972b) measured the initial uptake from media of varying glucose concentrations by restricting their radioactive measurements to the first 4 seconds after adding the cells. They were able to estimate the half-saturation concentrations for this initial influx at 0 and 20°C. They compared these with the half-saturation concentrations for exchange and those determined by the Sen-Widdas exit method. In addition to the affinities they compared the maximal rates of influx with those for exit and for exchange.

171


They also measured exchange influx with an arbitrary inside concentration of 38 mM. Their results are summarized in Table I. The results confirmed the increased rate of exchange relative to net transfer but clearly showed that the maximal rate for initial entry was significantly less than that for exit or for entry under the arbitrary exchange condition used. Indeed the latter entry had parameters which approached those derived from the Sen and Widdas type of exit experiment which may be understood when it is appreciated that the Sen and Widdas procedure is directed to finding an influx which equals half the saturated efflux. The other finding was the confirmation of Miller's observation that the half-saturation for exchange is quite different from that for influx and remains remarkably constant over the 20°C temperature range studied. Whereas the higher rate of exchange could be explained on the basis of substrate facilitation of carrier transfers relative to transfers of unsaturated carriers the low rate of maximal initial entry relative to exit could not be so explained. This and the large value of the half-saturation for exchange required asymmetry of a more fundamental kind in the transfer process. Miller (1971) measured the parameters of glucose and galactose efflux into sugar-free solutions and compared these with the parameters from Sen and Widdas type experiments, from exchange experiments, and also from the inhibition of sorbose transfer. For the zero-trans efflux he estimated the half-saturation for glucose at 7.4 mM and for galactose at 58 mM, both substantially higher than the Sen and Widdas values but lower than for exchange. TABLE I PARAMETERS OF GLUCOSE TRANSFER UNDER DIFFERENT EXPERIMENTAL CONDITIONS DETERMINED B Y LACKO et a / . (1972b)" Concentrations Experiment type Equilibrium exchange

Temperature ("C)

Maximal flux (rnmole liter-' min-I)

Half-saturation concentration

(mM)

Out (mM)

5-37

5-37

0 20 0 20 0 20 20

22.5 264 0.18 36 12.6 159 I79

20 20 0.2 I .6 0.65 1.7 2.0

In

0

0.1-18

Exchange influx

38

0.1-37

Sen- Widdas exit

76

1-8

Initial entry

(mM)

' The experiments included equilibrium exchange, initial entry, exchange influx, and Sen-Widdas type exits.

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W. F. WIDDAS

Karlish et NI. (1972) also measured the parameters of glucose efflux under zero-trans conditions. By suspending preincubated cells in a large volume of medium they were able to follow the loss of radioactive sugar while the outside concentration was maintained very low. For cells preloaded with 80 mM glucose their outside medium was kept to 0.16 mM. Integrating the simplified kinetic equation and solving for K , and V,,, they obtained values of K , of 25.4 -+ 3.0 mM and V,,, of 139 2 I 1 mmole liter-' min-'. These authors showed a I0-fold difference between the K , for efflux and that obtained by the Sen and Widdas procedure at the same temperature. Since the Sen and Widdas procedure gives the halfsaturation concentration for influx and the zero-trans efflux experiments give the half-saturation concentration for efflux, the results indicated asymmetry of affinities of the transfer process. Miller (1969) deduced a similar asymmetry of affinities on the two sides of the membrane from the results of Levine et al. (1965) who had measured the egress of glucose from cells loaded in the range 0-100 mM at 25°C. Their results indicated that the internal half-saturation constant was about 20 mM and Miller contrasted this with the Sen-Widdas value of about 2 mM at the same temperature.

F. Flux Measurements under Different Conditions

The slow accumulation of kinetic anomalies and the tentative sugges. gathered momentum with the tion of asymmetry by Lacko et C J ~ (1972b) republication of asymmetric carrier kinetics by Geck ( 197 1). This together with earlier statements of kinetics to be expected with asymmetric affinities by, e.g., Regen and Morgan (1964) led investigators such as Batt and Schachter (1973) and Bloch (1974) to make direct comparisons of fluxes of sugars under a variety of experimental conditions including the initial entries and zero-trans effluxes referred to previously. These studies added to the evidence for an asymmetric transfer mechanism although the results were not wholly consistent. Thus while Bloch obtained results at 7°C which confirmed those of Lacko et al. in showing the correspondence of the half-saturation constant for initial influx (and influx with exchange) with those for Sen and Widdas exits, Batt and Schachter at 15°C found the half-saturation for influx to be higher than that for efflux. However, Batt and Schachter's influx measurements were based on data collected from 15-45 seconds as opposed to the 2-5 seconds by Bloch and 4 seconds by Lacko et al. and on theoretical grounds they are unlikely to be indicative of the initial entry rates. Both groups found high half-saturation constants for ex-


173

change and Bloch also found a high value for zero-trans efflux. Miller (1975) has drawn attention to the difficulties in following initial uptakes of glucose even in the short times used by Bloch. This casts doubt on the significance of low initial entry rates as evidence of asymmetry. Batt and Schachter (1973) also showed the asymmetric effect on fluxes of various noncompetitive inhibitors. Thus p-chloromercuribenzene sul(DCDS), and N fonate (PCMBS), 3,3’-di-2-chloroallyldiethylstilbestrol ethylmaleimide (NEM) inhibited efflux more than influx. None of these is a simple competitive inhibitor and thus these results extend the earlier observations of Bowyer and Widdas (1958) (Fig. 1) with FDNB and of Dawson and Widdas (1964) with NEM.

G. Asymmetry of Affinities Derived from Inhibitor Studies While it is suggestive that the half-saturation concentrations derived from entry and exit flux measurement denote different affinities of the hexose system on the outside and inside of the membrane, respectively, more direct evidence of this asymmetry has come from studies with nontransportable inhibitors. Baker and Widdas (1972, 1973a) showed that 4,6-O-ethylidene-a-~glucopyranose (ethylidene glucose) was a potent inhibitor of glucose exit ( K , ca. 5 mM at 37°C) although the compound was not transported on the hexose transfer system. Thus they found that the penetration of ethylidene glucose was much slower than would be expected of a sugar with a half-saturation close to that of glucose. The entry rate did not slow as the concentration was built up in steps and the curves fitted the integrated diffusion type equation. Neither glucose nor phloretin had an inhibitory effect on ethylidene glucose entry and incubation of cells with FDNB to inhibit glucose exit 95% was without effect on the rate of penetration of ethylidene glucose. On the other hand on incubation of cells with FDNB in the presence of ethylidene glucose the development of inhibition was retarded relative to a saline control. This was the opposite to the effects of transportable sugars (Krupka, 1971) but was similar to maltose which is a nontransportable compound. Baker and Widdas also showed that ethylidene glucose inside cells could not induce transient uphill influx as is readily seen with for instance 3-O-methyl glucose, a sugar with almost identical affinity as judged by the inhibition of glucose exits. Rosenberg et a / . (1956) had found that glucose benzoate penetrated cells rapidly and was less subject to inhibition by phlorizin. They found

174

W. F. WIDDAS

the compound penetrated bovine red cells which do not have a fast facilitated transfer system like human erythrocytes and penetration was presumably made possible by the increased lipid solubility conferred on the molecule by the benzoate grouping. In ethylidene glucose the dioxane ring serves the same purpose and the oil-water partition was found to be only a little less favorable than that of glycerol. Comparable rates of penetration of ethylidene glucose were found in adult guinea pig red cells to those in human red cells and penetration by diffusion through lipid parts of the cell membrane appeared to be the most probable explanation. That ethylidene glucose can penetrate the red cell membrane slowly but independently of the hexose system brings it into a special class of reagents which allows one to examine separately the reactions occurring inside and outside the cell. Thus it is possible to preload cells with ethylidene glucose together with a transportable sugar and subsequently to spin the cells down and resuspend them in a medium free of ethylidene glucose while the exchange flux of the transportable sugar is being measured. The egress of the preloaded ethylidene glucose is very slow and for the duration of the radioactive exchange of the transportable sugar the ethylidene glucose is exerting an inhibitory effect on the inside only. Similarly with ethylidene glucose in the outside medium the short time required to take samples for measuring the radioactive exchange of a transportable sugar does not allow appreciable entry of ethylidene glucose to the cell interior thus measurements with inhibitor only on the outside are also possible. It was found (Baker and Widdas, 1973b) that ethylidene glucose inside the cells was much less effective as an inhibitor of exchange than outside. Thus 200 mM ethylidene glucose inside was no more effective than 25 mM outside. Allowing for the lower affinity of the inside for glucose the asymmetry of affinity for ethylidene glucose was estimated at 40-fold whereas that for glucose at i6”C was 10-fold. Recent estimates with purified ethylidene glucose suggest the asymmetry of this compound may be as high as 60-fold (Baker er al., 1978). Figure 3 shows the asymmetric inhibition of glucose, galactose, and 3O-methyl glucose exchanges at 20 mM and 16°C by purified ethylidene glucose. In each case the asymmetry is similar in that 200 mM ethylidene glucose inside the cells has an inhibitory effect no larger than that exerted by 25 mM outside. The intersections of the lines occur where 1/J = 1/ V,,, and the value on the abscissa corresponds to the apparent halfsaturation constant for the inhibitor. This is an “apparent” and not a true half-saturation constant for reasons which are discussed later when the asymmetric kinetics are considered. A number of related compounds were also shown by Novak and

175


,120

--

Inside

,100

Glucose 160

1

160 5b 50 [Et hy I ide ne glucose]

100

150

200

(m M)

FIG.3. Asymmetric inhibition of galactose, 3-O-methyl glucose, and glucose exchange by purified ethylidene glucose. Lines and points represent the means of two similar results for each sugar for 20 m M exchange at 16°C. 0 , Glucose; 0, 3-0-methyl glucose; X , galactose. (From Baker et NI., 1978.)

LeFevre (1974) to be nontransportable inhibitors of the red cell hexose transfer system. 1,2-~-Isopropy~idene-~-g~ucofuranose (isopropylidene glucose) inhibits glucose exchange with a lesser degree of asymmetry from that of ethylidene glucose (Baker et nl., 1978). The asymmetry of methyl-2,3-di-O-methyl-c~-~-glucopyranoside (trimethyl glucoside) is less still. Barnett et al. (1973a,b) studied the inhibitory properties of a number of fluoride derivatives of glucose and other hexoses. Replacement of hydroxyl groups with hydrogen alters the affinity in a variety of ways: thus it increases the affinity at C-2 but decreases the affinity at C-3 of glucose. Bonding with the transfer site was presumed to involve the hydroxyls on C-I, C-3, and possibly C-4 with a complex effect at C-6 which may have an element of hydrophobic bonding. Essentially similar conclusions had been reached by Kahlenberg and Dolansky ( I 972) by studying the effect of glucose derivatives on the stereospecific uptake of D-glucose by isolated erythrocyte membranes. Barnett et al. (1975) found that 6-0-alkyl derivatives of galactose and glucose inhibited the glucose transport system when in the outside medium. The longer chain alkyl derivatives appeared to penetrate the red cells by a glucose independent pathway at rates proportional to their

176

W. F. WIDDAS

olive oil/water partition but when on the inside did not significantly inhibit sorbose entry or glucose exit. On the other hand propyl-P-D-glucopyranoside was an effective inhibitor when inside the cells. As a result of their studies Barnett et a l . (1975) have made the interesting suggestion that the reaction with the outside and inside sites for transfer through the cell membrane involves different ends of the glucose molecule. Thus they visualize a glucose molecule, approaching from the outside, entering a reactive cleft in the transfer protein with the C-1 end of the pyranose ring leading. The transfer protein is presumed to undergo a conformational change which opens up the protein around the C-1 end of the sugar while closing it behind the C-4-C-6 end (see Fig. 4). The cleft, initially open to the outside, is, by the conformational change, transformed to an inward facing mode. Figure 4 is a representation of the model for glucose transfer proposed by Barnett et al. (1975). In illustrating the hypothesis they point out that 6-O-propyl-~-glucose(R = C3H,; R' = H in Fig. 4) can bind to the transport system in conformation A but for steric reasons is not transported. Similarly propyl-P-D-glucopyranoside (R = H; R' = C3H,) can bind to conformation B without being transported. D-Glucose can of course bind to both conformations and the conformational change brings about the effective transfer of that sugar from one side to the other. The asymmetry of the two sides of the protein cleft was also seen as the explanation for the finding of Kahlenberg and Dolansky (1972) that methyl-a- and P-D-glucopyranosides were inhibitors of glucose binding to membrane fragments although showing no competitive inhibition for Conformation A membrane protein

Conformation B

FIG. 4. Possible model for sugar transport in the human erythrocyte. 6-O-Propyl-~glucose (R = C,H,; R' = H) can bind to the transport system in conformation A but cannot be transported for steric reasons. Similarly, propyl-P-D-ghcopyranoside (R = H; R' = C,H,) can bind to conformation B but cannot be transported. D-GhCoSe can bind to both conformations and is transported by the conformational change. (Modified from Barnett et (//., 1975.)


177

glucose transfer in intact cells. The inhibition in fragments was seen as due to these glycosides being able to approach from the inside and bind to the transfer system in conformation B. The suggestion that glucose molecules substituted in the C-1 position act as good competitive inhibitors on the cell interior but react poorly or not at all outside while molecules with substitution on C-6 react on the outside but, like ethylidene glucose, react poorly on the inside would support their hypothesis. However, the reactivity of several nontransportable inhibitors is not so clear cut as this. Thus Baker et al. (1974, 1978) find that isopropylidene glucose with its dioxane ring structure involving C-I and C-2 of the glucose molecule nevertheless has higher affinity for the outside than inside and this may also apply to methyl-2,3di-O-methyl-a-D-glucopyranoside if allowance is made for the fact that nontransportable inhibitors may have a higher true affinity for the outside than that indicated from the inhibition of exchange (Baker et al., 1978).

H. Asymmetric Affinity of Cytochalasin B The different reactivity of the internal and external sites of the hexose transfer system is shown to a most marked degree by the inhibitor cytochalasin B. This fungal metabolite was shown by Taverna and Langdon (1973a) and by Bloch (1973) to be a powerful inhibitor of glucose transfers in the human red cell. Studying its inhibition by the Sen and Widdas exit method they found that the sugar half-saturation constant was not increased by the inhibitor which they concluded was noncompetitive. However, Lin and Spudich ( I974a) found that the high-affinity binding of cytochalasin B was reduced by about 80% in the presence of a high concentration (ca. 500 mM) of D-glucose but not L-glucose. That there was competition for exchange in the range 10-100 m M glucose was shown by Jung and Rampal (1975, 1977), and Taylor and Gagneja (1975) reported competition for exits. Basketter and Widdas (1977, 1978) showed that 3-O-methyl glucose exchange was competitively inhibited (Fig. 5) but confirmed that there was no increase of the sugar half-saturation constant in exits. This competition for exchange but not for exits was seen as evidence for a reaction between cytochalasin B and the inside hexose transferring site only. The lack of reaction with the outside site would explain the unchanged sugar half-saturation constant in exit experiments whereas the reaction with the inside site would be sufficient to give the typical competitive results for equilibrium exchanges.

178

W. F. WIDDAS

300 500 1 D O - M e t h y l glucose]

100 0

100

b-’>

FIG.5. Lineweaver-Burk type plot of 3-O-methyl glucose exchange in the range 2-40 mM at 16°C in the absence (0)and in the presence of 0.25 p M cytochalasin B ( X ) . Points are means of two experiments with similar results. (From Basketter and Widdas, 1978.)

In conforming this interpretation Deves and Krupka (1978b) have advanced more direct experimental evidence of internal competition. They used zero-trans exit experiments in the absence and presence of cytochalasin B. From an analysis of the kinetics of inhibition (with nontransportable inhibitors) Deves and Krupka (1978a) have shown that, in a plot of the reciprocal of the transfer rate against the reciprocal of the inside sugar concentration, an inhibitory effect on the outside would cause a distinct shift of the intercept on the ordinate. This was not seen but the line had a steeper slope indicating competition on the inside (Fig. 6). Thus cytochalasin B appears to show marked asymmetry with a high affinity for the hexose sites inside and little or none for those outside. The asymmetry of binding of cytochalasin B offers a reinterpretation of the interesting observation of Masiak et al. (1977) that the inhibition of hexose transfers by dione compounds is protected by glucose (150 mM) but is not protected (and may be enhanced) by cytochalasin B. Rather than assume that cytochalasin B does not react with the hexose site per se it may be suggested that the dione compounds react with the outward facing components of the hexose transfer system where cytochalasin does not compete. Taylor and Gagneja (1975) have advanced stereochemical evidence suggesting that cytochalasin B probably reacts with the hexose site and the competitive experiments are in favor of there

179


being mutual interference between glucose and cytochalasin B either at the same site or at closely associated sites on the inside of the membrane (Basketter and Widdas, 1978). 1. Asymmetric Proteolytic Responses

Proteolytic enzyme digestion of ghosts at first tended to confirm that the hexose transfer system was an intrinsic component of the more hydrophobic parts of the red cell membrane. Thus ghosts, following Pronase treatment which caused the loss of over 50% of membrane proteins, retained the facilitated transfer system for sugars (Jung et a/., 1973). Following mild trypsin digestion (Avruch et a/., 1973; Carter et al., 1973) which resulted in selective hydrolysis of membrane proteins in Bands 3 and 4 the ghosts still retained many of the properties characteristic of glucose transport in intact cells.

I

1

2 loo [Glucose]

3

4

5

(rnM)

FIG.6. Initial rates of glucose exit in the presence or absence of 4.2 /.LM cytochalasin B determined from the appearance of uniformly labeled ['4C]glucose in the external medium (upper and lower lines, respectively). Rates are given as mmoles liter-' min-I. Exit medium, 0.9% NaCl + 5 m M sodium phosphate, pH 7.0, 25°C. Quenching medium, cold 2 m M HgCI, and 1.25 m M KI and 2% NaCl with centrifugation at 1°C. Cell concentration 0.76%. Radioactivity determined by scintillation counting in Aquasol. (From Deves and Krupka, 1978b.)

180

W. F. WIDDAS

However, Lin and Spudich (1974a) showed that the high-affinity binding of cytochalasin B was largely lost when trypsin or Pronase was incorporated into red cell ghosts but was unaffected by these enzymes when they were in the outside medium and the cells were intact. The high-affinity binding was not only competitively displaced by high concentrations of sugars such as D-ghCOSe, as referred to in the previous section, but with a series of eight different sugars the individual displacements corresponded to their relative affinity for the hexose transfer system. Treatment with p-chloromercuribenzoate, an inhibitor of the hexose transfer system, also prevented cytochalasin B binding. The authors therefore interpreted the loss of high-affinity binding as being due to a degradation or change in the protein involved in hexose transport. Using low and high ionic strength solutions they released most of the proteins found on electrophoresis in Bands 1 , 2, 5 (Fairbanks et nl., 1971) and in Band 6, respectively, but found that this had little effect on the high-affinity binding of cytochalasin B. Low and high salt treatments do not remove Bands 3, 4.1, 4.2, or the protein designated as PAS-1 which shows up with periodic acid and Schiff reagent. Masiak and LeFevre (1977) treated the outside surface of erythrocytes with the proteolytic enzymes trypsin and a-chymotrypsin but without observing any inhibition of the hexose transfer. The incorporation of either enzyme inside erythrocyte ghosts (which were then resealed) produced a progressive reduction of hexose transfer as the incubation time increased. These authors noted the gradual loss of the spectrin band with the proteolytic treatment used but degradation products of spectrin mask possible changes in later bands and the interpretation in terms of proteins involved in the hexose transfer system was difficult. These latter two sets of experiments with proteolytic enzymes are however complementary and taken with the evidence that cytochalasin B reacts only with the internal sites of the hexose transfer system assume a clearer significance. Thus it can be seen that the hexose transfer system in the membrane is susceptible to attack by proteolytic enzymes only from the inside of the cell and this indicates either a different chemical environment or a different chemical morphology in the transfer protein itself on the two sides of the membrane. The studies with cytochalasin B and proteolytic enzymes taken with those of the nontransportable inhibitors discussed earlier combine to provide strong indications of chemical as well as kinetic asymmetry in the hexose transfer system of the human red cell.


181

II. KINETICS OF MEMBRANE TRANSFERS WITH ASYMMETRIC AFFINITIES

A. The Basis of Kinetic Asymmetry Rosenberg and Wilbrandt (1955) reviewing the kinetics of membrane transports involving chemical reactions either of the simple carrier type or of a carrier in cooperation with an enzyme considered the possible bases of asymmetry. They pointed out that the system could be asymmetrical in two respects as regards either reaction velocities or the equilibria. They showed that velocities for entry or exit would not necessarily correspond if the inside and outside concentrations were reversed. This aspect was further developed by Schultz (1971) who made the point that either linear or nonlinear systems acting across a composite membrane may present asymmetric fluxes when the concentration gradient is reversed. However the asymmetry of affinities was not considered by Rosenberg and Wilbrandt except as a possible means of effecting uphill transfer in energy coupled systems. Regen and Morgan (1964) removed the restriction of symmetry from the kinetics of a simple carrier transfer and opened up the possibility of asymmetric affinities at the two sides of the membrane. They derived equations for the net transfer of sugars under these general conditions and since there should be no net transfer when the inside and outside concentrations were equal they could define the relationship which must hold between the various equilibrium constants. The rate constants governing individual steps in the process were grouped to give composite constants which could be determined and these could be used to test whether simplifying assumptions of the symmetrical carrier model were in fact justified. Regen and Morgan (1964) carried out experiments on rabbit red cells with the aid of this kinetic analysis but found no evidence of asymmetry in those cells. They did not use their method of analysis on human red cells. Similar kinetics were developed by Britton (1966) and these have been further extended to develop a series of tests and rejection criteria for the simple carrier (Lieb and Stein, 1971). The kinetics are currently formulated (Lieb and Stein, 1974) in relation to a number of resistance terms of a form extensively used by Geck (1971). An interesting conclusion from these kinetic studies is that a number of carrier models with either a single or several sugar complexing sites within the membrane will, from steady-state measurements, be indistinguishable and fit generally similar kinetic expressions. Wilbrandt (1972a) in an attempt to resolve kinetic discrepancies be-

182

W. F. WIDDAS

tween carrier affinities as determined from Lineweaver-Burk treatments of zero-trans exits and from exits using the Sen-Widdas (1962a) procedure considered that the reaction rates between sugar and carrier may be finite relative to the movements of the carrier in the membrane. He also considered the reaction rates could be different inside and outside though their ratio (from which the K , was derived) was assumed to be constant. With these assumptions Wilbrandt found that the resistance terms involving the reactions on the inside came out higher than resistance terms for the outside but that taking these differences into account a more consistent value for the affinity term ( K , 5 5.2 mM) was obtained by both the Lineweaver- Burk and Sen- Widdas approach. However using an indifferent tracer in the presence of equilibrated concentrations of glucose Wilbrandt observed different values of the K , for glucose. He used D-xylose and D-arabinose as tracer sugar. He therefore suggested there may also be diffusional resistances equivalent to unstirred layers contributing to the anomaly and concluded that the diffusional resistance inside the cell must be much higher than that outside. The complication of unstirred layers had been raised by Naftalin (1971) and commented on also by Miller (1972) and by Lieb and Stein (1972b). Wilbrandt (1977) in extending and reanalyzing this study found the results to be generally in favor of an explanation in terms of structural asymmetry such as might be explained by a transfer system deep in the membrane with diffusional resistances to be overcome in approaching the transfer sites from outside or from inside. Wilbrandt's approach however, assumed that the inside and outside K,s of the transfer system were symmetrical. Regen and Tarpley (1974) applied kinetics with asymmetric affinities to various data collected from the literature. They arrived at essentially the same conclusion as Wilbrandt, namely, that any diffusional resistances involved were much greater inside the cell than outside. Edwards (1974) also argued that a carrier model with a diffusion step inside the cell would explain many features of glucose transport kinetics. This aspect will be further considered in Section II1,B when discussing the asymmetric membrane environment.

B. Asymmetric Affinities and the Two Site Model

The underlying principles involved in the asymmetric affinity assumptions can be better appreciated from the simplest treatment and will first be exemplified with the use of kinetics described by Baker and Widdas ( 1973b).

183


In Fig. 7 is represented a two site transfer system with reactive sites in each interface which have different affinities for sugar in the associated medium. If the half-saturations for the sites are 4 and a4, respectively, a will be the asymmetry of affinities. The probabilities that at any time a site or component will be saturated with sugar is taken to be the same as the saturation fraction of all components in that interface as given by the Michaelis-Menten relationship. The probability of unsaturation is defined as the unsaturation fraction of all components in that interface. Transfer from side 1 to side 2 is presumed to require that side I is saturated with sugar and side 2 unsaturated. These probabilities are represented by C , / ( C , + $4 and a + / ( C 1 + a+), respectively, where C , and C 2 are the sugar concentrations at side 1 and 2 . The probability that transfer will occur is proportional to a rate constant multiplied by these two probabilities. Thus Transfer 1-2

=

Vl.--.

a4

Cl

c ,+ 4

c2

+ a+

FIG.7 . The schematic basis of the Two Site Model. Each site X and Y is presumed to be i n dynamic equilibrium with the sugar in the medium at sides 1 and 2, respectively, but with different affinities. The half-saturation constants are I$ and a $ , respectively, where a is the asymmetry factor. In (A) both sites are unsaturated. In (B) site X is saturated and the sugar either returns t o side I or vectorially transfers to site Y and may dissociate into side 2 effecting a net transfer from side I to side 2. In (C) site Y is saturated and as the reverse of ( B ) may effect a net transfer from side 2 to side 1. In (D) both sites X and Y are saturated but it is assumed an intramembranous molecular exchange may occur.

184

W. F. WIDDAS

where V , is the rate constant. Similarly the probability of transfer from side 2 -+ 1 is given by Transfer 241

=

V,'

c 2

-.

4

c, + a 4 c , + 4

where V , is the rate constant. However it is also proposed that if both sites are occupied an exchange can occur between the sugar molecules occupying the respective sites. The influx and efflux of this exchange reaction would be equal and cancel out when net transfer is being considered but is included for completeness. Thus the net transfer can be expressed as [Eq. ( I ) ] Net transfer 1-2

=

V I C I . a 4+ V l . \ C I C ,- V , C , + - V,.:\C,Cz (CI + 4)(C, + 04)

(1)

where VEXis the rate constant for the exchange reaction. One of the first principles of an asymmetric affinity scheme is that there should be no further accumulation of sugar once the inside concentration builds u p to be equal to the outside concentration. In this simple treatment the condition is met if V , = a V , , thus the rate constant for transfer from the low-affinity side must be higher than that from the highaffinity side and the ratio of this difference must equal the asymmetry factor. Why this relationship should hold is not clear but one could visualize that the glucose on the high-affinity side dissociates less readily than that from the low-affinity site and that transfer through the membrane involves a vectorially directed dissociation. In the presence of a competitive inhibitor the saturation by sugar is given by C c + 4(1 + Z/4J where I and 4, are the concentration and half-saturation constant of the inhibitor. Allowing for asymmetry of inhibitor affinities and making the assumption that the inhibitor cannot cross the membrane on the hexose system nor can it exchange across the membrane for a sugar molecule then the transfer in the presence of inhibitors can be represented by [Eq.

(211

where i is the asymmetry factor for the inhibitor's affinities.


185

These kinetics allow an interpretation of the effects of inhibitors in two practical types of experiments, namely, the Sen- Widdas exits (Sen and Widdas, 1962a,b) and equilibrium exchanges. 1 . SEN-WIDDASEXITS The principle of the Sen-Widdas experiments is to carry out exits from cells preloaded with a high concentration of sugar either into very low sugar concentrations at various inhibitor concentrations o r into various low sugar concentrations at constant or zero inhibitor concentrations. Thus Eq. (2) can be simplified by neglecting C , in the numerator and ignoring the exchange terms to give [Eq. ( 3 ) ]

In practice the exit time found by extrapolating the linear part of the exit record to the equilibrium level is used; this is proportional to the reciprocal of the exit rate so that

where S' is the content of sugar in the cells initially. This shows that the exit time is a linear function of the outside sugar concentration C , (if the inhibitor concentration is constant or zero) and also the exit time is a linear function of Z if C , is held constant. What is not so readily understood is that the apparent half-saturation constant (the value of C , which doubles the exit time relative t o that when C , = 0) is changed only with an outside competitive inhibitor and not with an inside competitive inhibitor. This can be seen however by a closer inspection of Eq. (4). Thus the uninhibited half-saturation constant would be a value of C , equal to the outside half-saturation constant, i.e.,

c , = +.

With an outside competitive inhibitor the value of C , would need to equal +( 1 + Z1/#q) to double the exit time. However with only an inside competitive inhibitor the exit time (when C = 0) would be doubled when

c , = 4.

Thus, on the basis of these simple asymmetric kinetics, the Sen- Widdas exit technique can be visualized as giving the half-saturation of the outside site and in the presence of inhibitors will show an increase in the half-saturation for the sugar only if the inhibitor is competing for the outside site.

186

W. F. WIDDAS

2. EQUILIBRIUM EXCHANGES

In carrying out equilibrium exchanges the sugar is at equal concentrations inside and outside the cells. The flux in one direction is measured by the use of radioactive tracer sugar molecules. To study the halfsaturation for exchange such flux measurements are carried out at a variety of increasing concentrations of sugar and the reciprocal of the flux plotted against the reciprocal of the sugar concentration in a typical Lineweaver- Burk type plot. The unidirectional flux is given by

J=

[ C + 441 +

V , a + C + VExC2 I1/h>l [ C + a+(l +

12/i+J1

(5)

The rate constant for exchange is larger than that for net transfer and as C increases the net transfer term may be neglected so that

L-['

VEX

1

+(:

1

+;)I

[ + %( 1

1

+$-)I

(6)

VEX

Equations (6) and (7) show that the Lineweaver-Burk plot of uninhibited flux measurements will be largely influenced by the large half1. For the same reason it will be more saturation value, i.e., a + if a influenced by inside inhibitor (unless i is correspondingly large). However if C is maintained large while the inhibitor concentration is varied the values of the inhibitor half-saturation constants should be determinable from the point on the line where 1/J = 1 / v E X . But as pointed out by Baker et a / . (1978)for the outside acting inhibitor the intercept occurs where

*

I

- -$1

-(1

+ a + a+/C) 1 + 10 + I 2 + a$/C 1+1

1

6

when dealing with an asymmetry of ca. 10 and with values of @ ca. 2 mM and C = 20 mM. Thus the intercept value of - I may be as large as 6-fold the half-saturation constant derived from Sen- Widdas exit measurements. From an inside acting inhibitor the corresponding intercept occurs

187


where

I -_ ic#lI

-(I

+ a + ac#l/C)

A

a+-

a4

1

+ 10 + 1 10+ I

+

1.1

C

and is a much closer approximation to the true inhibitor affinity on the inside. Thus on the basis of these kinetics the equilibrium exchange in the absence of any inhibitor can be interpreted as giving a half-saturation value approximating that of the higher valued site (that is the inside site for human red cells with common sugars). When inhibitors are present the discrepancy observed when using exchange experiments to estimate the outside affinity of competitive inhibitors compared with the SenWiddas method also receives an interpretation. Therefore Sen- Widdas experiments will given an indication of the affinity and reactiveness of the outside site and equilibrium exchanges will give an approximation to the affinity of the inside site for transported sugars. A measure of the asymmetry factor can thus be determined from a combination of these procedures (Widdas, 1974). The reactiveness of both sites with nontransportable inhibitors may be studied by equilibrium exchange experiments but the influence of asymmetric affinities is complex and caution is required in interpreting the quantitative results. C. Redistribution of Components in the Mobile Carrier Hypothesis

The simple kinetics described by Baker and Widdas (1973b) represent a special case where transfers and inhibitions are occurring without any significant redistribution of the components as between inward facing and outward facing conformations. With the original mobile carrier hypothesis different mobilities of free and complexed carriers through the membrane (different both for inward and outward transfers) were considered but it was proposed (Widdas, 1952) that provided there were no structural restrictions the components would rapidly come to a steady state in which equal numbers of carriers would leave each interface in unit time. Thus for every different experimental situation there would be an appropriate redistribution of carriers and the kinetics could be modified by this redistribution. With asymmetric affinities at the two sides uneven mobilities of the complexed and free membrane components are a necessary provisions to ensure that sugar is not accumulated beyond the equilibrium value (Regen and Morgan, 1964).

188

W. F. WIDDAS

A hypothetical example of redistribution was given by Baker et nl. (1978) and is reproduced as Tabel 11. An asymmetric affinity factor of 10 requires that the cycle of events involved in an efflux should occur 10 times more readily than the cycle of events for an influx. An efflux cycle can be visualized as an outward movement of the complexed carrier, followed by an inward return of the free carrier. The asymmetry factor can be divided up between these two events. Thus in

TABLE I1 STEADY STATEOF AN ASYMMETRIC TRANSFER SYSTEM W I T H REDISTRIBUTION OF COMPONENTS SHOWING THE CHANGE GIVING50% INHIBITIONDUE TO A N OUTSIDE NONTRANSPORTABLE INHIBITOR" Parameters

Outside

Half-saturation constant Allotted value Sugar concentration

Inside

4

04

2 mM 2 mM

20 m M 2 mM

4

2

2

7

Components unsaturated

u = asymmetry factor u = 10 Exchange condition

state of 30 components

Uninhibited-steady Components saturated

Comment

4

20 51

Rate constants ensure equal exchanges in unit time

7

I

Total

22

8 50% Inhibition-steady

Components saturated

2

I

state of 30 components I

L

Components unsaturated

2

zy

10

Exchanges are equal but half the previous value

51 7 L

1

Components inhibited

15

Total

Note half of all components inhibited

19

Saturation outside =

L

2

11 L

+ 2( I + I/bi) = 19

I Therefore - =7.5

41

" Fraction saturated = C/(C + 4,) where 4, is the appropriate half-saturation constant. Fraction unsaturated = 4J(C + 4,). In the presence of inhibitor the denominator becomes C + +,(I + I / + , ) . The ratio of y to 1 is irrelevant in exchange conditions but affects the kinetics in other situations. Modified from Baker et u l . (1978).


189

the example of Table I1 the efflux of a complexed carrier has a rate constant twice that of influx whereas for the free carrier the rate constant for influx is 5-fold that for efflux. Examples can be prepared in which the ratios are 10 and 1 , 3 and 3.3, o r any other arbitrary pairs of values chosen so that their product is 10, i.e., equal to the asymmetry of affinities. The way in which redistribution affects the kinetic properties is shown by the hypothetical case of an inhibitor fixing 50% of all the components (drawn from both interfaces) on the outside. This reduces the exchange flux to half of the uninhibited value but in doing so has brought about a major redistribution of the components between inward facing and outward facing modes. In consequence of this redistribution the inhibitor is actually complexing 79% of the outside components (considered separately) and there is no simple relation between the percentage of outside components complexed and the percentage of inhibition of exchange. In this example 50% inhibition of exchange would actually require an inhibitor concentration of Z = 7.54,.This illustrates how an asymmetric transfer involving redistribution of components would, for 50% inhibition of exchange, require an outside inhibitor concentration several fold larger than that determined for 50% inhibition of exit in a Sen-Widdas experiment. Table I11 shows the distribution of components between outside and inside at different exchange concentrations and illustrates the case of an outside inhibitor which complexes only 50% of the outside components (considered separately). The asymmetry factor and ratios of the mobilities are arbitrarily taken to be the same as for Table 11. Figure 8 shows these distributions graphically and emphasizes how at low exchange concentrations the actual inhibition falls short of the 50% which might be expected from the inhibition of outside components. Taking a point close t o zero sugar concentration as the extreme example the components outside ( 16.7% in the uninhibited state) increase t o 28.6% when 50% inhibited. Of these 28.6% half are complexed with inhibitor but that still leaves 14.3% for sugar exchange relative to 16.7% in the uninhibited state. This represents a reduction of only 14.3% and corresponds to the actual inhibition of exchange. It should be noted that it is also the percentage of all the components complexed by the inhibitor. The inside components initially 83.3% are reduced to 71.4%; this is also a reduction of 14.3%. As more of the components become distributed to the outside then 50% inhibition of the outside components has a correspondingly larger effect in spite of the further redistribution of components which this brings about. This is because the proportion of components left facing

190

W. F. WIDDAS

TABLE I11 CALCULATED DISTRIBUTION OF COMPONENTS BETWEEN INSIDE A N D

EXCHANGE CONCENTRATIONS AND THE EXCHANGE WHEN 50% OF THE OUTSIDE COMPONENTS ARECOMBINED WITH ~NHIBITORa

OUTSIDE AT DIFFERENT INHIBITION OF

Exchange sugar conc ( 0 in and out

0- 0 2-2 10-10

20-20 30-30

Uninhibited distribution of components

Inhibited distribution of

Inhibition of exchange which would be observed (%)

In (9%)

out (7%)

components In Out (9%) (%I

83.3 73.3 55.6 47.6 43.9

16.7 26.7 44.4 52.4 56.1

71.4 58 48.5 41.2 28.1

28.6 42 61.5 68.8 71.9

14.3 21 31 34.4 36

a The redistribution is calculated o n the basis of 10-fold asymmetry and rate constants a s illustrated in Table 11. The half-saturation for the sugar is set at 6 = 2 m M a s is the half-saturation for the inhibitor 4, = 2 m M . Of the outside components 50% are considered t o be combined with a nontransportable inhibitor of concentration I such that for the outside components I / C + b(l + I / I $ ~ )= 1/2 or I = C + 2. Note that the percentage inhibition is equal to the percentage of all components complexed by the inhibitor, i.e., half those distributed outside in the inhibited state.

inside are reduced and the redistribution (which occurs on the development of outside inhibition) brings about a larger percentage fall on the inside. It follows that the actual inhibition is also larger. The same sort of redistribution could be brought about by increasing the inside concentration of transportable sugar while keeping the outside sugar at a low level. The effect is illustrated in Fig. 9 in which the redistribution is calculated on the basis used by Baker et nl. (1978). This illustration also assumes a 10-fold difference in the basic rate constant for saturated components relative to unsaturated components. Figure 9 shows that 50% inhibition of outside components (which would be obtained at a constant outside inhibitor concentration) would have an increasingly potent inhibitory effect on efflux as the inside concentration of transportable sugar was increased. The same pattern would apply to other levels of outside inhibition. The greater inhibitory effect of ethylidene glucose in competition with 2 mM 3-O-methyl glucose when the inside concentration was raised from 2 to 30 mM was demonstrated by Baker et al. (1978). Batt and Schachter (1973) observed differences in the kinetics of initial

191


influx and efflux which were most marked when the inside concentration was changed and these results also indicated effects of redistribution as well as asymmetry. They discussed an interpretation based on two carrier states the conversion from one to the other depending on intracellular glucose or other sugar. With an asymmetric system the redistribution effects complicate the interpretation of inhibitory affinities as illustrated in Tables I1 and 111 and lead to the interesting conclusion that an inhibitor acting at one side only brings about a percentage inhibition equal to the percentage of all the components which it complexes at that side and is not simply related to the percentage of components complexed in the particular interface. Redistribution of components between an inward facing and outward facing mode is inherent in the kinetic treatments of Regen and Morgan (1964). Britton (1966), Geck (1971), Lieb and Stein (1974), and Regen and Tarpley (1974) all based on the mobile carrier model. However actual translation of the components between the interfaces of the membrane is not necessarily involved. A conformational change which might be as

8oi

180

I

I

I

k 60 -+%

1Inhibiition 140

___

/*-

__ - - - - *--- - - - -- - -4 Exchange

1

;20 I

I

Inside conc (rnM)

r I I

01

0

10

20

to

30

FIG.8. Redistribution of membrane components a s a function of sugar concentration in equilibrium exchanges. Continuous lines (and left-hand scale) represent the components with an outward facing mode in the uninhibited state and when inhibited to the extent that one-half of the outward facing components are complexed by a nontransportable inhibitor. Interrupted line (and right-hand scale) represents the inhibition of exchange which would actually be observed. Note that inhibition of exchange is lowest at low sugar concentrations and is the same a s the overall percentage of components combined with inhibitor. Calculations based on the parameters used for Tables I1 and 111.

192

W.

80 -

F. WlDDAS

80 a I

L I

I

60

I % Inhibition

I - - _ - -_ _~

Efflux reduction

41

I

:!+ 140

------a-.,

,

L

L

Inside c o n c (mM)

I

FIG.9. The effect of changing the inside sugar concentration on the redistribution of membrane components. Continuous line (and left-hand scale) represent the components with an outward facing mode in the uninhibited state and when inhibited to the extent that one-half of the outward facing components are complexed by a nontransportable inhibitor. The sugar concentration on the outside is assumed to be 2 m M throughout while that to which the cells have been equilibrated varies from 2 up to 80 mM. Interrupted line (and right-hand scale) represents the inhibition of eMux actually observed. Calculations based on the parameters of Table I1 but with y = 10 x 1.

minimal as a swing of one or two hydrogen bonds (Vidaver, 1966) may be all that is required to change the sugar-reacting sites from an inward facing to an outward facing mode. The asymmetric kinetics published by the authors quoted above are described in a wealth of mathematical terms which tend to obscure the underlying mechanisms to all but the expert kineticist and the principles of redistribution of components and their effects are particularly difficult to appreciate without working through examples as illustrated here. Nevertheless some of the parameters, expressed as groups of rate constants, can be interpreted with fair precision and compared with the parameters based on the simpler kinetic treatments. In this article only the wider interpretations will be discussed and for kinetic details the reader must refer to the original articles. D. The Regen and Tarpley Kinetics Applied to Nontransportable Inhibitors

Lieb and Stein (1974) reviewed the kinetics of asymmetric carriers and showed that there was an overall consistency in the kinetics, whether


193

these were based on a single complex in the membrane-that is, one formed from reaction of sugar and empty carrier at either side without providing for translocation of the complex between the interfaces-or whether the kinetics were based on the more conventional two complex model in which both empty carriers and sugar-carrier complexes have kinetic terms for mobility across the membrane. Although they point out that steady-state kinetics do not enable one to distinguish between the various formulations the single complex model presents difficulties in interpretation of the reactions with nontransportable inhibitors which can react from either interface. The complexes so formed cannot be kinetically identical and one would need to postulate three complexes-one at either side when nontransportable inhibitors were involved and an internal one which was formed by transportable sugars. The two complex model on the other hand provides for mobilities in the membrane of the sugar-carrier complexes which depend upon the particular sugar involved and for nontransportable inhibitors it is plausible and convenient to set the rate constant for transfer across the membrane at zero. Thus the kinetics published earlier in the same year by Regen and Tarpley (1974) which were based on the two complex model and which provided for the case of sugars in competition are suitable for illustrating the more complex asymmetric kinetics. The kinetics provide for redistribution between the interfaces as well as asymmetry of the internal and external affinities. In the following discussion detailed reference to the individual rate constants will be omitted. In the presence of a competing sugar of concentration So and Si outside and inside the cell, respectively, the transfer of glucose (Ug) is given by

where Go G,

concentration G at outside surface = G , - U , / D , concentration in the external medium and D o is a diffusion constant for movement between the interface and bulk volume G i = concentration G at inside surface = G , + U , / D i G , = concentration in the cell and D i is the internal diffusion constant S o and S i are similarly defined for sugar S F , = activity constant for G transport = V / K , for G transport K , , = Michaelis constant for G entry K g i = Michaelis constant for G exit R , = flux ratio constant for G R , = flux ratio constant for S Bg = half-maximal constant for G exchange =

=

194

W. F. WIDDAS

(9) B s = half-maximal constant for S exchange K , , = Michaelis constant for S entry K S i = Michaelis constant for S exit

Thus the entry of glucose involves four independent parameters F,, K g o , R , , and B , [the fifth parameter K g i is related to the others through Eq. (9)]. But if unstirred layers are involved there are two further parameters D, and Di making six in all. The same four independent parameters would apply to sugar S , i.e., F,, K,,, R , , and B s so that in competitive situations the position would be more complex. Where the competitor is nontransportable R s = m. This arises since the rate constant for transfer through the membrane (and which is set at zero) is one of the terms in the denominator of R , . It follows that 1 - 1 Ngs

KsiRg

and 1 - 1 Ns, KsoR, We can therefore rewrite Eq. (8) as

ug=

Fg[Go(1 + G i / R g ) - Gi(1 + Go/Rg)I 1 + G , / K , , + G i / K g i+ G o G i / R , B g + [So/Kso(I + Gi/Rg)I + [Si/Ksi(1 + Go/Rg)I

(13)

The terms involving the nontransportable inhibitor occur only in the denominator and are enclosed in square brackets for convenience since one or the other or both terms may be zero in the various experimental conditions. For a Sen- Widdas type experiment the next exit, for the case where there is a nontransportable inhibitor only on the outside, may be written


195

as follows:

Fg(Go - Gi)

u, =

[2

1 + Gi/Kgi + -( 1 + Gi/Rg)

1

(14)

+ Go(l/Kgo + Gi/RgBg)

Considering G, small relative to G i the value of G,, necessary to reduce the maximal exit rate (when G, = 0) to half that value will be approximately determined by the value of G,, which doubles the value of the denominator. Thus in the absence of inhibitor the Sen-Widdas constant will be obtained when GII/K,(,--c I and G,,/R,B, fi 1/Kgi.Indeed since G i is large this last relationship, i.e., G, = R,B,/Kgi will determine the Sen- Widdas constant (Regen and Tarpley, 1974). In the presence of an outside inhibitor the value of G, necessary to double the value of the denominator must be larger so that the inhibitor terms are also compensated for. With a nontransportable inhibitor only on the inside Eq. (13) can be rewritten as Fg(G(J- Gi)

u, =

1

+ Gi/K,i +

[&] +

G, {l/Kgo + Gi/RgBg + [ ~ i / ~ g ~ s i ~ )

(15) In this case G o multiplies one of the inhibitor terms and indeed if Go/ R g fi 1 when G,, = R,B,/Kgi the denominator would be doubled without any increase in G, relative to the uninhibited value (where S i = 0). Thus an inside competitive inhibitor need not affect the Sen- Widdas constant (Basketter and Widdas, 1978). These considerations particularly apply where K,, fi R, and therefore B , fi Kgi. This type of asymmetry was deduced for the human red cell sugar transferring system by Regen and Tarpley (1974) and while applicable it follows that the Sen- Widdas constant is related to the outside K,(K,,) and the half-saturation for exchange is related to the inside K,,,(Kgi). The effect of nontransportable inhibitors on equilibrium exchange can be seen by making G, = G i = G in Eq. (13). The positive terms on the right-hand side represent influx and the negative terms efflux. Since from Eq. (9) 1 - = - +1 1 1 -+ K,,, K,i B, R, Eq. (13) simplifies to give the exchange flux JEx

196

W. F. WIDDAS

The reciprocal can be written a s 1 and where an outside inhibitor o r an inside inhibitor is present on its own the change in the slope and the intercept where l/JEx= 0 can be used to derive K,,, and KSi. However the inhibitor will appear t o have a greater affinity in the inhibition of exit compared with the inhibition of exchange and the halfsaturation concentration of the inhibitor derived from the inhibition of exit into a sugar-free medium will be smaller than that derived from inhibition of exchange. This is because the exit will appear to be half inhibited when Sv/KsV(l+ Gi/Rg) = 1 + Gi/Kgi that is the condition which will bring about a doubling of the denominator in Eq. (14) when G,, = 0. Since G i is large this will occur when So -

Rg Kgi + Gi __.

Ksv

Kgi R g + Gi

Rg Kgi

2-

thus the concentration which half inhibits exit will be smaller than K , , in proportion a s R g is smaller than Kgi. On the basis of the simpler kinetics the concentration which half inhibits exit into a sugar-free medium is a measure of the affinity for the outside component whereas on the Regen and Tarpley kinetics the socalled Michaelis constant is that derived from inhibition of exchange and the higher apparent affinity from exit measurements follows from the kinetics. This difference in interpretation affects the magnitude of the asymmetry factor to be allocated to the affinities of nontransportable inhibitors (Table IV) but does not affect the relative differences in asymmetry found for different compounds. Thus on either interpretation there is a 20-fold range in asymmetries among the three nontransportable inhibitors studied by Baker et al. (1978). Deves and Krupka (1978a) have reviewed the kinetics of inhibition in the conventional two complex model and have drawn attention t o the extra information which can be obtained from zero-trans entry and zerotrans exit experiments. The points they make can also be illustrated from the Regen and Tarpley kinetics. Thus for a nontransportable inhibitor the zero-trans entry of test sugar can be written as Fg.G v ug= (18) 1 + Gv/Kgv + [Sv/KsvI + [Si/Ksi(1 + Gv/Rg)I

197


TABLE IV ASYMMETRY OF PARAMETERS OF NONTRANSPORTABLE INHIBITORS~

Compound Ethylidene glucose Isopropylidene glucose Trimethyl glucoside

Apparent K ,

Asymmetry ratio

Inhibitor Inhibitor inside outside (mM (mM)

Regen and Tarpley Baker and Widdas kinetics kinetics ( K 5 >K: J (41,:4J

110 76

I35

II 59 290

10: 1 1.3: 1 0.47: 1

60:I 7.7: I 2.8: I

a The apparent K , s are obtained where the lines in plots such as Fig. 3 intercept IIJ = 1/Vmah. The intercepts correspond to K,, and K,,, in the Regen and Tarpley kinetics but the apparent K , obtained from exchange experiments with inhibitor on the outside is ca. 6-fold larger than 6 of the Baker and Widdas kinetics. The half-saturation concentration for an inhibitor in a Sen-Widdas exit experiment would be less than the Regen and Tarpley Michaelis constant K,,, by a similar factor a s explained in the text. Data from Baker et a / . (1978).

and the reciprocal flux can be written as 1 This would give a linear plot of 1/J versus l / G o in which the ordinate intercept would be increased with an inside acting inhibitor (Siterms) but not with an outside acting inhibitor. The slope would however be affected by both types of inhibitor. In a comparable approach the zero-trans exit or efflux can be shown to yield

(20) which would also be a linear plot of 1/J vs l / G i but one in which the ordinate intercept would be increased with an inhibitor acting on the outside. Deves and Krupka (1978b) point out that in showing up an inhibitor which acts only on one side of the membrane the zero-trans entry and exit approaches are the most unambiguous and they used the absence of a shift in intercept in zero-trans exit experiments with cytochalasin B to prove that that inhibitor is competitive for the internal sites in the membrane and does not react with the external sites (see Fig. 6 ) . This confirmation of the hypothesis proposed by Basketter and Widdas (1977, 1978) also marks an interesting development in the application of asymmetric carrier kinetics to problems of inhibition of hexose transfers.

198

W. F. WIDDAS

Deves and Krupka (1978a) summarizes the main results of their kinetic survey as showing that:

I . Competitive inhibitors acting at only one side of the membrane may give results (in some experiments) which are interpretable as being due to noncompetitive inhibitors but the reverse does not hold. Noncompetitive inhibitors cannot mimic competitive ones. 2. To test whether an inhibitor is competitive or not the only unambiguous single experiment is that of inhibition of exchange. 3. Zero-trans exit and entry experiments have advantages in showing up an inhibitor which competes only at one side of the membrane. 4. A combination of experiments enables one to determine whether the inhibitor is competitive and also whether it is transportable on the hexose system. E. Applications of Asymmetric Carrier Kinetics The application of the asymmetric kinetics to human red cells presents problems by virtue of the speed of the transfer process. Regen and Tarpley (1974) illustrated their original approach with data from the literature notably the anomalous findings of Miller (l968a) and the galactose data of Levine and Stein (1966). Kinetic studies designed to exploit the asymmetric kinetic approach were made on rat thymocytes by Whitesell et al. (1977a) and by Whitesell et al. (1977b). The kinetics were also used to analyze the properties of the sugar transfer process in avian erythrocytes in the same laboratory by Cheung et al. (1977). Readers should refer to the original articles for full details but it is interesting to note that emphasis was placed on the use of influx measurements into empty cells-the zero-trans influx-and also influx measurements into cells preloaded with a fixed internal concentration of sugar. These types of measurements can be more informative than the equilibrium exchange experiments alone. Thus writing Eq. (8) in the simplified form where there is no competing sugar or inhibitor gives

For the influx of sugar into empty cells G i = 0 and the equation can be put in the form of a Hanes plot (Hanes, 1932) as

199


the intercepts are I/F, and - K , , and the reciprocal of the slope is F,K,, = V for entry. The conditions for exchange are G , = G i = G and the equation can be written as

in the Hanes plot form. IIF, and - B , can be determined as the respective intercepts. In the influx measurements at fixed internal concentrations Whitesell e f al. (1977b) point out that the equation takes the form

with intercepts of 1/P and - H where

P

=

F g [( 1

+

?)/( + -5)] 1

and

H=

( $: / ($ &) I+-

+

Having derived F , and K , , from Eq. (22) and B , from Eq. (23), Eq. (24) allows one to evaluate the remaining parameters by making use of the relationship in Eq. (9). Thus

and

Techniques such as these are particularly powerful in analyzing the asymmetric carrier parameters in cells in which the transfers of sugar are relatively slow as in rat thymocytes and avian erythrocytes. Indeed the work with avian erythrocytes has some similarities to the original application of asymmetric kinetics to the case of rabbit erythrocytes (Regen and Morgan, 1964) in that asymmetry is not marked. In avian erythrocytes however there is a difference between aerobic and anaerobic conditions. Sugar transport is stimulated by anoxia (Wood and Morgan, 1969; Whirfield and Morgan, 1973; Cheungef al., 1977). The latter authors ascribe the effect to increased efficiency of preexisting carriers which in the aerobic state may be bound by some immobilizer or have depressed

200

W. F. WIDDAS

mobility. There may also be effects on carrier affinity which shows clearer evidence of asymmetry in the aerobic state. Ginsburg (1978) has applied asymmetric carrier kinetics to the problem of galactose transfers in human erythrocytes. He collected data for zerotrans entry and exit and for equilibrium exchange. His analysis followed the lines recommended by Lieb and Stein (1974) and by Eilam and Stein (1974), but some of the data were analyzed on the basis of a pair of antiparallel carriers (Ginsburg and Stein, 1975; Eilam, 1975). Thus zerotrans entry was best resolved on the basis of an asymmetric carrier with high affinity outside and low affinity inside which accounted for 85% of the maximal transfer together with an asymmetric carrier with high affinity inside and low affinity outside which accounted for 15% of the maximal transfer. Exit and exchange could be analyzed on the basis of a single asymmetric carrier since it was shown that these measurements would not be affected by the 15% of components presumed to be arranged in an antiparallel fashion.

F. The Effects of pH and Temperature The use of influx measurements into preloaded cells has been extensively used by Lacko et al. (1972a, 1973, 1974, 1975, 1977a,b) for a different reason. By preloading cells with glucose the initial uptake from experimental solutions of low glucose concentration has the nature of an exchange uptake but under standardized conditions. The K , and the maximal uptake rate can be determined and the changes analyzed in terms of the experimental variables being studied. This approach is particularly well illustrated in their experiments on the effects of pH which they studied over the range from pH 2 to pH 11. An important consideration was that the cells were in the abnormal pH solutions only for the 5-second period of the initial uptake after which the solution was diluted by the large volume of stopping solution. Thus these experiments may be taken to refer to the effects of pH mainly on the outside components of the red cell transfer process rather than those parts more deeply situated. The results showed that there were two regions in which the transfer rate was decreased corresponding to pK, = 5.2 and pK, = 9.5, but the K , value was constant throughout. The interpretation given was that the glucose carrier if protonated or deprotonated became immobile but that the protons were not lost or gained from the glucose binding site itself. There is little effect of pH in the range 6-8 which was the region studied by exits by Sen and Widdas (1962a).


201

Lacko et al. (1973) used the initial uptake into preloaded cells to investigate the effect of temperature on the parameters of the initial uptake. The K , showed a marked dependence on temperature and their results confirmed the observations of Sen and Widdas (1962a) but their analysis of the change in V,,, with temperature went further. Sen and Widdas (1962a) found that V,,, had a steeper temperature coefficient at lower temperatures than at higher temperatures. The Arrhenius activation energy was of the order 20 kcal/mole at 20°C and 7- 10 kcal/mole at 37°C. Dawson and Widdas (1964) found that in fetal guinea pig red cells the K , from exits did not progressively decrease at lower temperatures. Since the Sen-Widdas exit K , could be shown to contain both a rate term for transit through the membrane as well as the association and dissociation rate terms of a true Michaelis constant Dawson and Widdas examined the possibility that a change in the rate-determining step may occur as the relative magnitudes of the three rate terms changed with temperature. Although this line of analysis went someway to explain the curvature of the Arrhenius plot of V,,, for both human and fetal guinea pig cells the results on human cells particularly the linear relation between log, K , and l/Tabs could not be explained. This point was emphasized by Bolis et 01. (1970) who confirmed the curvature of the Arrhenius plot of V,,, but interpreted the change as being due to the interplay of different heats of activation of loaded and unloaded carriers in the symmetrical carrier model with substrate facilitated mobilities of the complexed form. Lacko et al. (1973) however make the hypothesis of a definite phase transition effect round about 20°C with a sharp change in the heat of activation from 9.3 to 21.5 kcal/mole at pH 7.5. Their Arrhenius activation energies were 9.9 and 22.1 kcalhole and are comparable to the results from exits reported by Sen and Widdas (1962a). The similarity in the K , results is not surprising when it is realized that the Sen- Widdas exit technique is essentially based on finding a rate of entry which retards the exit to half its maximal value. Thus the parameter concerned is closely related to that of an entry into preloaded cells. The technique used by Lacko et al. is to look at the initial part of this entry process. The V,,, term derived by Sen-Widdas exits is however different from the V,,, derived by Lacko et al. In the latter's experiments V,,, is more akin to the V,,, for exchange apart from the fact that the glucose concentrations are very dissimilar across the membrane and thus will affect the redistribution of components between the two sides. The variation in the redistribution of components may be partly re-

202

W. F. WIDDAS

sponsible for differences between these results and the values for exchange obtained by Hankin and Stein (1972). The latter authors found no curvature in the Arrhenius plot of V,,, derived either from the exchange or from zero-trans effluxes. Their activation energies were 18 kcalhole for zero-trans effluxes and 16 kcalhole for exchange over the temperature range 5-45°C. Ginsburg and Yeroushalmy (1978) have extended the use of asymmetric carrier kinetics to study the individual parameters for galactose transfer over a range of temperatures. Their temperature range 0-25°C was somewhat limited by the techniques involved but over that range they found relatively little change in the affinity parameters which are equivalent but not identical to K,,, Kgi,and B , in the Regen and Tarpley kinetics. Thus the asymmetric affinities reported earlier (Ginsburg, 1978) are maintained over this temperature range. There was a marked temperature dependence of the transfer rate constants and the pattern was similar to that reported by Lacko et al. (1973) but suggested a further break in continuity between 5 and 15°C with much higher activation energies being required at the lower temperatures. Since the asymmetric affinities were maintained over the whole temperature range studied (0-25°C) they may be seen as a feature of the human red cell which is not peculiar to one temperature. They may therefore be assumed to apply also in vivo at 37°C.

III. MORPHOLOGICAL ASYMMETRY A. Asymmetry in the Erythrocyte Membrane

Asymmetry is recognized to be a structural feature of erythrocyte membranes affecting both lipids and proteins. Articles by Bretscher (1973), Zwaal et al. (1973), and Bretscher and Raff (1975) review the main points which for the present purposes may be summarized as follows: 1. Phospholipid molecules appear to be unevenly distributed in the red cell membrane. Thus while the lipid double layer proposed by Gorter and Grendel (1925) and Danielli and Davson (1935) is supported by X-ray evidence, enzymatic and other studies suggest that the lipids in the outermost layer tend to be phosphatidylcholine and sphingomyelin (the choline lipids) with some cholesterol whereas the amino phospholipids, phosphatidylethanolamine and phosphatidylserine, are almost wholly


203

situated in the inner layer. Glycolipids appear to be in the outer layer (Steck and Dawson, 1974). For a recent review see Bergelson and Barsukov (1977). 2 . Most of the lipids have two paraffin chains of variable unsaturation and a polar head group which in the water interface may have ionized groups which are charged. The majority are zwitterions but phosphatidylserine has a net negative charge, thus the asymmetric distribution of the phospholipids conveys an electrical asymmetry to the lipid bilayer. 3 . The paraffin chains of the lipids, especially where unsaturation is present, are in a liquid state with greater mobility toward the center of the bilayer. The lipid molecules also have considerable translational mobility within their particular half of the bilayer but transfer across the membrane from layer to layer-a process termed “flip-flop” by Kornberg and McConnell (1971)-is very slow. Thus whereas lipids may change position with their neighbors in less than a microsecond the flipflop motion may require hours or longer. 4. Membrane proteins may be embedded in the lipid bilayer as proposed by Singer and Nicholson (1972) so as either to traverse the bilayer completely and expose polypeptide chains at both interfaces or be partially embedded in the inner lipid membrane with free polypeptide chains approachable from the inside of the cell but not from the outside. Asymmetry of such proteins is emphasized in the review by Rothman and Lenard ( 1977). 5. Of the proteins which penetrate through the membrane one (referred to as component a by Bretscher) is a large protein with molecular weight about 100,000 and fairly globular in shape. This protein is the same as that of “Band 3” in the classification of Fairbanks et a / . (1971) and its isolation has been achieved by Furthmayr et al. (1976). It is thought that the (80 A) particles seen on freeze-fracture electron micrographs may represent molecules of this protein which among other functions may be responsible for the chloride-bicarbonate exchange function of the erythrocyte membrane (Cabantchik and Rothstein, 1974). Rothstein et a / . (1978) have drawn attention to the asymmetry of functional sites approachable from outside and inside the cell in the case of the anion transport protein. The other major protein which traverses the whole membrane is a lower molecular weight (30,000) glycoprotein which has its sugar residues exposed in the external interface. These residues include the sialic acid which gives the external surface of the erythrocyte its negative charge. The intramembranous segment of glycophorin A has been isolated by Furthmayr et a / . (1978). Both these proteins are estimated to be present in amounts sufficient

204

W. F. WIDDAS

to provide about half a million molecules per red cell and the techniques so far used would not separate minor components present in smaller quantities such as the Na-K-ATPase. 6. The proteins which partially embed in the membrane from the inner side are thought to do so by hydrophobic interaction with the amino phospholipids. Numerically the amino phospholipids are deficient relative to the choline lipids in the outer layer. Either some choline lipids must be on the inside or these partially embedded proteins fill in for the deficiency in the inner half bilayer. It may be that both factors apply. Recent x-ray studies (Pape et al., 1977) confirm the asymmetric arrangement of protein and lipid constituents and correlate with electron micrograph interpretations (Tilney and Detmers, 1975; McMillan and Luftig, 1975). 7. There has been an accumulation of experimental evidence that the Band 3 group of proteins may also include the protein responsible for glucose transport. Lin and Spudich (l974b) studied the cytochalasin B binding by ghosts from which various fractions of the membrane proteins had been released by treatment with solutions of high and low ionic strength. These experiments pointed to the involvement of Band 3, 4.1, 4.2, or the periodic acid-Schiff (PAS) sensitive material. Kasahara and Hinkle ( 1976) incorporated proteins solubilized with Triton X-100 into liposomes made from soya bean lipids and were able to show that they took up D-glucose with some of the characteristics of the hexose transfer system in intact erythrocytes. The most effective material was Band 3 protein with some material from Zones 4.1 and 4.2. I n a red cell membrane study Kahlenberg and Walker (1975) found that by crosslinking the sulfhydryl groups of Band 3 proteins there was an inhibition of glucose transfer into the membrane vesicles. This inhibition could be reversed by 2-mercaptoethanol. In a further study Zala and Kahlenberg ( 1976) used 2,3-dimethyl maleic anhydride (DMMA) to dissolve “extrinsic” proteins, i.e., those in Bands I , 2 , 2.1, 2.2, 4.1, 4.2, 5 , and most of 6. This material and the pellet left behind were tested in liposomes prepared from sonicated erythrocyte lipids and glucose uptake was promoted only by the intrinsic proteins left in the pellet. These constituted Bands 3,4.5,7, and some of 6 together with the PAS-sensitive material. Following DMMA with Triton X-100 it was possible to solubilize and remove Bands 5 , 6, and the PAS-sensitive material. The pellet left behind with Bands 3, 4.5, and 7 had enhanced sugar-transferring properties when tested in the liposomes. Band 7 is a protein of the cytoplasmic surface and for this and other reasons is thought unlikely to


205

be involved in the sugar transfer system (Lin and Spudich, 1974b; Kahlenberg and Walker, 1976) leaving Band 3 and Zone 4.5. The lower molecular weight proteins of Zone 4.5 were shown to induce glucose transport in further experiments by Kasahara and Hinkle (1977) and by Kahlenberg and Zala (1977). More recently Jones and Nickson (1978) have been able to incorporate red cell protein extracts into thin lipid bilayers which then showed an increase in conductance and of Dglucose permeability. The major components of their most effective extract were again Bands 3 and 4.2. That the transport protein may originally be in Band 3 has been suggested by Phutrakul and Jones (1979). What was of interest in the context of asymmetry was the finding that stable bilayers were formed only if the protein extract was added on one or the other sides but not if present on both sides. This is consistent with the view that the transmembrane proteins are themselves asymmetrical and so arrange themselves in the lipid membrane in a definite orientation. Symmetrical presentation in the lipid may lead to association with consequent instability. Van Steveninck et al. (1965) from their studies with sulfhydryl reagents concluded that there was a large asymmetry in the distribution of such groups in the membrane. Sulfhydryl groups on the outside reacted with p-chloromercuribenzene sulfonate (PCMBS) and there was some correlation with the inhibition of glucose entry. However thiol groups deeper in the membrane were also implicated in this and the work of Smith and Ellman (1973) using maleimide derivatives. The latter authors found that the reagents with highest lipid solubility inhibited glucose transfer at lowest concentrations. They considered that this supported other evidence that blockage of thiols in different membrane locations was involved in the inhibition of glucose transfer. Batt et a / . (1976) used impermeant maleimides as developed by Abbott and Schachter (1976) to label the exofacial surface of erythrocyte proteins and found labeling in four bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The major band corresponded with proteins in the 40,000 to 70,000 molecular weight range. Since the impermeant maleimides also inhibited glucose transfer there was the possibility of further identification of the hexose transfer system. In an earlier study of the same kind Taverna and Langdon ( 1973d) used D-glucosyl isothiocyanate as a covalent probe, which is also an irreversible inhibitor of glucose transport, to try and label the active site on the glucose-transferring protein. This probe was found predominantly in proteins of 70,000 and 100,000 daltons, respectively. The asymmetric orientation of Band 3 (or other protein concerned with

206

W. F. WIDDAS

glucose transfer, e.g., Zone 4.5) within the erythrocyte membrane and the hydrophobic interactions with the membrane would preclude any rotation in the plane of the membrane (Kahlenberg and Walker, 1976) and this led Kahlenberg to suggest that the transport of glucose occurred through water-filled channels formed by specific subunit aggregates of the transport protein. By progressively removing cholesterol from the lipid membrane of human erythrocytes Masiak and LeFevre (1974) found no change in the K , for glucose transport. The rate of transport was at first increased but as more cholesterol was removed the rate became inhibited. Read and McElhaney (1976) confirmed these findings and since the removal of cholesterol increased the fluidity of the lipid bilayer they argued that the absence of an increase in the rate of glucose transport was against a model for transport which involved movement of the transport protein in the lipid environment. What is clear from this brief summary is that there is an asymmetric polarization of the red cell lipid bilayer and also of the proteins traversing it. The evidence is strongly against a flip-flop mechanism involving either lipids or proteins and a mobile carrier for glucose is unlikely to be of the nature of a membrane ferryboat (Ussing, 1952). An allosteric model for membrane transport was suggested by Jardetzky (1966) and similar models have been reviewed by Singer (1974). In its simplest form such a model involves an aggregate of integral proteins with a slit or cavity large enough to admit a small molecule. The membrane molecule is presumed to be capable of adopting two different conformations with the cavity opening to one or other side of the membrane, respectively, and to contain a binding site for the transported species within the cavity. The general problem of the way in which integral proteins are incorporated into the lipid environment of the membrane and take part in transport has been further reviewed by Singer (1977). It has already been pointed out that a minimal conformational change within a transporting protein may provide the “mobile” element which is a kinetic requirement of the facilitated transfers of sugars (Vidaver, 1966).

B. Asymmetry of the Membrane Environment Besides structural asymmetry the intact red cell is peculiar in having an asymmetric environment in the majority of laboratory experiments. This is due to the high concentration of protein (hemoglobin) inside the


207

cell and the fact that most experiments are carried out in media free of protein. Taverna and Langdon (1973b) developed a new technique for following glucose transfer which involved incorporating glucose oxidase into red cell ghosts which were then resealed. On removal of glucose oxidase from the outside medium the consumption of oxygen measured with an oxygen electrode was shown to be dependent on the rate of penetration of glucose to the inside of the resealed ghosts. They showed (Taverna and Langdon, 1973~)that the maximum rates of entry into resealed pink ghosts was similar to that into white ghosts and also into inside-out vesicles and thus they deduced that the transport across the ghost membrane was symmetrical. However the K , for glucose was ca. 10 mM for both ghosts and vesicles which at 15.5"C corresponds more closely to the half-saturation of the inside sites of intact erythrocytes (Baker and Widdas. 1973b) and there may have been some reorganization of membrane components in preparing the ghosts. Naftalin et NI. (1974) have shown that sugars which are rapidly transported across the red cell membrane also protect red cells against osmotic hemolysis and from sugar-dependent increases in the viscosity of hemoglobin solution they deduce that glucose and other sugars can induce hemoglobin molecules to form a gel in which glucose itself is nonspecifically bound. Naftalin and Holman (1977) estimate that in the range 10100 mM glucose as much as 80% of intracellular glucose may be bound loosely to hemoglobin. The same authors have advanced a hypothesis for glucose transport incorporating the slow sorption and desorption of sugars to and from hemoglobin into the kinetics of an otherwise symmetrical membrane transport based on a gated pore model first proposed by Adair (1956) and expanded upon by Jung (1975). In this model there are recognition sites at each end of the pore which on combination with sugar induce the conformational changes on which transfer through the membrane largely depends. As in the tetramer model of Lieb and Stein (1972a) the rate constant for transfer is presumed dependent on the saturation by sugar and the rate is multiplied by the sum of fractional saturations at the two sides to give the higher rate for exchange when both sides are saturated. They advance the interesting suggestion that in this state, i.e., when the gates at both ends of the protein pore are open exchange may actually occur without a conformational change accompanying each molecule of glucose transferred. This would explain the lower activation energy for the exchange process as opposed to net transfers.

208

W. F. WIDDAS

A gated pore for glucose through which smaller molecules such as polyols (e.g., erythritol) may penetrate in a noninhibitable manner as the pore gates open and close was also proposed by Bowman and Levitt (1977). The asymmetry in the Naftalin and Holman model arises from the fact that 85% of the cell water is presumed to be bound to hemoglobin and takes up glucose only slowly. The 15% of free water therefore fills up rapidly during an entry experiment and creates a back flux which slows the entry rate relative to what might be expected if all the cell water was instantaneously available to sugar. Readers should refer to the text for a full explanation of the kinetics which involve the numerical solution of nonlinear differential equations and are not therefore immediately applicable to problems other than those illustrated by the authors. They are able to show that computer solutions predict operational parameters (K,s and Vmaxs)of the right order for the different experimental procedures for exits and entries and for exchange. One interesting example is their explanation of the asymmetry observed by Bowyer and Widdas with FDNB and illustrated in Fig. 1 . In the Naftalin and Holman model the exit of glucose is rate limited by the membrane transport system and is thus inhibited progressively as the membrane components are taken out of action by reaction with FDNB. The rate-limiting step for entry is the complexing of glucose by hemoglobin and consequently the rate of entry of glucose will not be lowered until the rate of transfer across the membrane is reduced below that for complexing by the hemoglobin. This explanation also fits in with the observations of Sen and Widdas (1962b) who showed that entry of glucose in the range 38-76 mM was more inhibited by incubation with FDNB than the initial entry from 0.7 to 38 mM. Naftalin and Holman point out that at the higher concentrations the rate of entry is again limited by transport across the membrane and consequently the inhibition is dependent on the inactivation of membrane components and is more comparable with that for inhibition of glucose exit. The sorption and desorption of sugar to the hemoglobin-bound water may well be the explanation for the apparent diffusional resistances ascribed to the inside of the cell by Wilbrandt (1972a) and by Regen and Tarpley (1974). Wilbrandt estimated that not more than 10% of the allowance he had to make for diffusional resistances could be outside the cell but that the estimated "unstirred layer" within the cell would have to be an impossible value of 0.034 cm. Regen and Tarpley (1974) also estimated the diffusional resistance inside the cell to be very much larger than that


209

outside and their value of l/Di = 0.062 minute cell-' ml-' was approximately half the minimal resistance for overall glucose transport. There is thus a very significant environmental factor operating inside the red cell contributing to the anomalies on which kinetic asymmetry is based.

C. Asymmetry of the Sugar Membrane Transfer System

In view of the structural asymmetry of the membrane and the asymmetry of the environment is there any need to look for asymmetry in the sugar transfer process itself? A11 the authors referred to in the previous section were drawn toward the possibility that the sugar transfer process in the membrane may in fact be symmetrical. Naftalin and Holman (1977) assumed the membrane transfer was essentially symmetrical with equal half-saturation concentrations ( K , ca. 2 mM) at the two sides. In their model the different operational K,s arise from the interplay of the rate-limiting processes for membrane transfer with those for sorption into and desorption from the hemoglobin. Wilbrandt ( 1972a) in his analysis allowing for diffusional resistances arrived at a K , of 1.95 mM at 20°C again for a symmetrical membrane transfer. Regen and Tarpley acknowledging that the kinetic analysis involved asymmetric affinities considered that these may arise due to the presence of a nontransportable inhibitor on the inside of the membrane which by competing for the carrier raises the apparent half-saturation constant for the sugar on the inside. On this basis the inherently symmetrical transfer process would have a K , determined by the outside site which would approximate the Sen- Widdas value at 20°C (1.86 mM) and be comparable with the values used by Naftalin and Holman and by Wilbrandt. Experiments which tended to show the presence of high-affinity sites on the inside of the human red cell membrane were reported by Hankin et al. (1972). Their results, which were based on the analysis of the slowing of net entry of glucose, were criticized by Foster and Jacquez (1976) but have been defended by Lieb and Stein (1977). Baker and Naftalin (1977, 1979) have also reported an experiment in which glucose exits into a constant high concentration of galactose indicated a higher internal affinity (lower K , ) than that obtained from equilibrium exchange. A symmetrical carrier in which interactions of the internal environment or redistribution of components can, under some experimental conditions, mimic the presence of low-affinity sites, might go some way to fit these results in with those that point to there being low-affinity sites on

21 0

W. F. WIDDAS

the inside. On the other hand the same factors might cause a system with low-affinity sites inside to mimic one with high affinity. However the idea of inherent symmetry whereby the hexose transfer system in the human red cell has identical sites with similar affinities facing outward and inward has the attraction that it would tend to conform with the “principle of the uniformity of nature” in postulating a similar transfer system in red cells of a variety of species. Thus asymmetry was looked for and not found in rabbit red cells (Regen and Morgan, 1964) nor is it a feature of avian erythrocytes (Cheung ef al., 1977). In avian erythrocytes there is the suggestion of preexisting “carriers” being immobilized under ordinary conditions but made available by anoxia or intracellular Ca2+ions (Carruthers and Simons, 1978) and this lends support to the possibility of there being an internal inhibitor or some other mechanism operating in human red cells which could leave the transfer process inherently symmetrical. However, the “principle of uniformity of nature” has led biologists astray in the field of muscle physiology (Huxley, 1977) and is beset with difficulties in this present context. Thus there are stereospecific differences between the sugar transfer system in rabbit and human red cells (Regen and Morgan, 1964) in respect to the handling of ketose sugars and in the response to inhibitors and such differences must be in the recognition sites or neighboring groups on the transfer protein. In beef erythrocytes although a saturable facilitated transfer system is involved in the transfer of sugars it is one with an unusually high affinity for glucose and 3-0-methyl glucose (Hoos et al., 1972). Fetal blood from a number of farm and laboratory animals have a rapid facilitated transfer of sugars similar to human red cells (Widdas, 1955). This rapid transfer is also present in blood of the newborn rabbit (Augustin et af., 1967), pig (Zeidler et nf., 1976) and dog (Lee et nf., 1976) but rapidly declines after birth. It has recently been observed that in fetal shows a similar guinea pig red cells, 4,6- 0-ethylidene-a-D-glucopyranose asymmetric inhibition of 3- O-methyl glucose exchange to that of human cells though the estimated concentrations which half inhibit the exchange are not identical (Aubby and Widdas, 1979). The small differences in inhibitor affinity could be due to the different environment of carriers in human and fetal guinea pig cells but taken with the different temperature dependence of the Sen- Widdas constant for glucose (Dawson and Widdas, 1964) it is more likely that there are small molecular differences in the transfer protein. There is no a priori reason to expect the hexose transfer system to be molecularly identical in different species. Although kinetic anomalies and kinetic asymmetries are not unambiguously in favor of a basic asymmetry in the hexose transfer mechanism


21 1

itself the evidence from nontransportable inhibitors is stronger and is reinforced by the demonstration that cytochalasin B reacts only with the internally situated sites of the hexose transfer system (Basketter and Widdas, 1977, 1978: Deves and Krupka. 1978a,b) and by the different susceptibility of the inside and outside sites to proteolytic enzymes (Lin and Spudich, 1974a; Masiak and LeFevre, 1977) discussed previously. Proteins which traverse the cell membrane are presumably stabilized in it by having an intramembranous structure which is in some way complementary to the asymmetrically arranged lipid environment and capable of hydrophobic interactions with it (Wickner, 1977). Looked at in the light of the overall asymmetry of the membrane involving the bulk of the lipids and proteins it would be remarkable if the components responsible for hexose transfer were not themselves asymmetrical. However, the final answer to this question of asymmetry of the membrane transfer system for sugars and some of the kinetic problems associated with it may have to wait until the molecular structure can be determined and the overall mechanism of transfer clearly described.

IV.

IMPLICATIONS OF ASYMMETRY

A. Consequences of Asymmetry

Assuming a chemical and dynamic asymmetry as previously discussed one can consider some general implications. The first consequence of asymmetry of affinities will be that the parameters of transfer, particularly the apparent K , or half-saturation constant, will vary with the type of experiment. The variation in K , will also be influenced by factors such as the sorption of sugar into the hemoglobin-bound water which may be kinetically equivalent to the diffusional resistances of unstirred layers. for entry and exit taken at their initial stages when they The V,, approach the zero-trans condition will be different principally because of the redistribution of membrane components between outward and inward facing modes coupled with the different translation "velocities" of components and sugar complexes. Indeed redistribution will largely be due to the asymmetric rate constants for the transfer of sugar complexes (and for the rearrangement of the uncomplexed components within the membrane) which are an essential corollary to the concept of asymmetric affinities if the overall system is to be nonaccumulating and not to contravene the second Law of Thermodynamics. for exchange may be interpreted as arising from the The higher V,,

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greater mobilities of sugar complexes in the membrane but there are other possibilities along the lines of gated pores which may need to be examined. A pore in which the gates remain open however would not meet the requirements for uphill transfer by counterflow (Park et a/., 1956, 1968; Rosenberg and Wilbrandt, 1957; Wilbrandt, 1972b) unless the sites within the pore are given the kinetic properties of mobile carriers. Asymmetric affinities also introduce complications to the interpretation of inhibitor studies particularly in respect of the K , s obtained from inhibition of equilibrium exchange. The analysis of Deves and Krupka (1978a, suggests some new approaches to the inhibitor problems which will help in resolving the properties of inhibitors which act only at one side of the membrane. In general asymmetry of affinities brings with it a large increase in complexity of the overall framework for the facilitated transfer of sugars and its kinetic analysis. Simple treatments are still useful in describing the results of experiments in which the conditions are clearly defined and are not radically changed as between experiments. The exchange transport into red blood cells used by Lacko (Lackoer al., 1972a,b, 1973, 1974, 1975, 1977a,b, 1978a,b) may be quoted as an example. This technique has been used to study the effects of pH, temperature, alcohols, various drugs, and local anesthetics on the transport system and in general the procedure will respond to factors affecting the outward facing sites and the influx process. It may be assumed that, over the very short times involved in making the influx measurements, complications, due, for example, to the sorption of sugars into hemoglobin-bound water, are either absent or are so similar in the various experiments as to play no rate-determining part relative to the factors under investigation. The Sen and Widdas (1962a,b) procedure, like the exchange transport used by Lacko, remains useful for investigating factors affecting the outside sites of the hexose transfer system. The inhibition of the overall system can also be studied by the Sen-Widdas procedures but caution is required in interpreting inhibition in terms of its being competitive or noncompetitive. The fuller analysis of Sen-Widdas exits shows that the competitive characteristic of being able to demonstrate a n increase in the apparent half-saturation constant of the transported sugar may be given only by inhibitors which are in competition for the outside sites. Competition only for the inside sites of the hexose transfer system may give all the appearances of a noncompetitive inhibitor (Basketter and Widdas, 1978; Deves and Krupka 1978a). This surprising result is not due to asymmetry per se but considerations of asymmetry have brought it to light and of course without asymmetry of affinities it would be technically difficult to observe since the inhibitor is usually present in the outside


213

medium and in a symmetrical transfer system would compete for external sites as well as for internal sites. The simple asymmetric kinetics used by Baker and Widdas (1973b) are sufficient for the interpretation of many experiments, e.g., on the inhibition of equilibrium exchanges where the concentrations are not widely varied. But if the concentrations are changed over a range where substantial redistribution of components is brought about or if a variety of entry, exit, and exchange experiments are to be analyzed then it is essential to use more sophisticated kinetic treatments which may need t o allow for diffusional resistances. Although an outline of such treatments has been given in this article reference to the original articles is recommended. B. Physiological Implications Apart from the increase in complexity of the kinetic treatments which asymmetry of affinities creates it would be interesting to consider if there were any physiological implications involved. Krupka and Deves (1979) have discussed the valve-like properties of asymmetric transfer systems. These could arise from an inherently asymmetric facilitated transfer, from a symmetrical transfer system which is asymmetrically inhibited, o r from an obligatory exchange system also inhibited asymmetrically. In all three cases the maximal rates of influx and efflux are different and it can be shown that the net transfer rate for an inwardly directed concentration gradient would be different from the transfer rate for the same gradient outwardly directed. An inhibitor acting asymmetrically outside the cell is inwardly directing in the sense that exit from the cell is inhibited more than entry and in a fluctuating medium with the sugar concentration rising and falling the level of sugar inside the cell would reach a steady state which would be higher than the case for a symmetrical and uninhibited transfer system. If the asymmetrical inhibition is exerted on the inside of the cell then the reverse situation would hold, that is, the sugar would be rapidly lost from the cell when the concentration in the medium was low but would be less rapidly accumulated when the concentration in the medium was high. Thus with a fluctuating medium concentration the steady-state level in the cell would be lower than for a cell with a symmetrical transfer system which is not inhibited. This valve-like property makes it possible to use asymmetric inhibition as a means of control of the intracellular substrate concentration where such a carrier system exists and Krupka and Deves (1979) draw attention t o the possible physiological significance of this type of mechanism.

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On the other hand if one considers a membrane transfer system delivering sugar to a cell interior under conditions of low sugar concentration one can see that utilizing enzymes inside the cell, with their high affinity for sugars, could retain a high saturation at the expense of a low saturation of the inward facing sites of the transfer protein. Indeed Coleman (1973) has argued that membrane enzymes impose a geography on the functions of the cell (see also Schrier, 1977). If the glucose concentration in the cell was close to zero and the utilizing enzymes were arranged adjacent to the transfer proteins one could visualize a vectorial movement from one to the other. However, the human red cell membrane transfers sugar so rapidly that the sugar effectively equilibrates across the cell membrane and consequently the utilizing enzymes would be in an environment of plasma-determined concentration irrespective of the affinities of the transfer protein. Teleologically one might have expected asymmetry of affinities across the rabbit red cell and not the human red cell whereas the reverse is the case. The rapid transfer of sugars across red cells is a feature not only of primate red cells but of the fetal and neonatal red cells of a considerable number of nonprimate mammals. The rapid transfer in fetal red cells may be seen as offering a distinct biological advantage in the carriage of glucose from the placenta to fetal tissues. Goodwin (1954, 1956) pointed out how the blood glucose may be approximately the same in adult and fetal animals in species like the rabbit and guinea pig but because the sugar is practically all in the plasma in the adult, whereas it is equilibrated between cells and plasma in the fetus, there is nevertheless an appreciable plasma-to-plasma gradient across the placenta. The red cells by permitting a rapid charging and discharging of sugar across their membranes during the short time (of a second or so) that they take to traverse the capillaries of the placenta and tissues will have a small buffering-type action preserving the gradient both in the placenta and between the plasma and the tissue cells. If the rapid transfer property is an adaptation primarily for fetal existence then the persistence in the adult red cells in humans and other primates may be a reflection of their evolution (Goodwin, 1954), that is, it may be an example of “biochemical” pedomorphosis. Pedomorphosis-the persistence of morphological characteristics which were embryonic or juvenile features of ancestral types-is a recognized phenomenon in primate evolution (Le Gros Clark 1959). Whether the persistence in the adult primate serves any physiological purpose such as by buffering the gradient between the blood and tissue cells has not so far been the subject of investigation. Even if it were so there seems n o advantage to be fulfilled by the asymmetry of affinities though the greater rate of maximal exit, one of the consequences of

ASYMMETRY

OF THE HEXOSE TRANSFER SYSTEM

21 5

asymmetry, could be advantageous if blood was circulating through a capillary bed which behaved like a glucose "sink." The buffering-type action would help to maintain the gradient across the primate placenta from both the maternal and fetal sides and one naturally thinks of the primate brain, which is largely dependent on glucose metabolism, as another possible site where such a factor might apply but in the absence of experimental evidence further speculation along these lines is unwarranted. What is remarkable is that in the three or four decades over which the glucose transfer of human red cells has been extensively studied there have been no discoveries of gross abnormalities in the process or of its anomalous absence in contrast to abnormalities of several membranebound enzymes (Schrier, 1977) and proteins (Anselstetter, 1978). Bang and Orskov (1937) reported a reduced sugar permeability in the red blood cells from cases of pernicious anemia and this has been confirmed (Widdas, unpublished observations), but it was found that if allowance was made for the large volume of the cells from the pernicious anemia patients the membrane transfer was within normal limits. It would appear that the hexose transfer system in red cells is either genetically very stable or else abnormalities, if they occur, are lethal to the organism at an early stage of development. Thus in the human red cell the arrangement of components, necessary for the facilitated transfer of sugars with asymmetric affinities, serves only an obscure physiological function to the body as a whole. At present its study is therefore an example of pure research but such study opens up the possibility of getting a fuller insight into the concepts and properties of facilitated membrane transfers which may apply to other sites and to other substrates. REFERENCES Abbott, R. E., and Schachter, D. (1976). Imperrneant maleimides. Oriented probes of erythrocyte membrane proteins. J . Biol. Chem. 251, 7176-7183. Adair, G. S. (1956). A general discussion on membrane phenomena. Discuss. Furridoy Soc. 21, 285-286. Anselstetter, V. (1978). Gel electrophoresis of the human erythrocyte membrane proteins: Aberrant patterns in haematological and nonhaernatological diseases. Blut 36, 135- 144. Aubby, D. S . , and Widdas, W. F. (1979). Asymmetry in the hexose transfer system of erythrocytes from new-born guinea-pigs. J. Physiol. 293, 73P. Augustin, H . W., Rohden, L. V . , and Hacker, M. R. (1967). Uber einige eigenschaften des mono saccharid transport systems in erythrozyten neuge borenen und envachsener kaninchen. A c / o B i d . Med. Ger. 19, 723-735. Avruch, J., Price, H. D., Martin, D. B . , and Carter, J. R. (1973). Effect of low levels of trypsin on erythrocyte membranes. Biochim. Biophys. Actri 291, 494-505.

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Bowman, R. J . , and Levitt, D. G . (1977). Polyol permeability of the human red cell. Interpretation of glucose transport in terms of a pore. Biochim. Biophys. Aciu 466, 6883. Bowyer, F., and Widdas, W. F. (1957). The importance of the reaction at both interfaces in facilitated transfer. J . Physiol. (London) 136, 13P- 14P. Bowyer, F . , and Widdas, W. F. (1958). The action of inhibitors on the facilitated hexose transfer system in erythrocytes. J . Physiol. (London) 141, 219-232. Bretscher, M. S. (1973). Membrane structure: Some general principles. Science 181, 622629. Bretscher, M. S.,and Raff, M. C. (1975). Mammalian plasma membranes. Nuiure (London) 258, 43-49. Britton, H. G. (1957). The permeability of the human red cell to labelled glucose,/. Physiol. (London) 135, 61P-62P. Britton, H. G. (1964). Permeability of the human red cell to labelled glucose. J . Physiol. (London) 170, 1-20. Britton, H . G. (1966). Fluxes in passive monovalent and polyvalent carrier systems. J . Theor. B i d . 10, 28-52. Cabantchik, Z. I . , and Rothstein, A. (1974). Membrane proteins related to anion permeability of human red blood cells. I. Localization of disulfonic stilbene binding sites in proteins involved in permeation. J . Memhr. Biol. 15, 207-226. Carruthers, A., and Simons, T. J. B. (1978). Calcium and ionophore A23187 stimulate sugar transport in pigeon red cells. J . Physiol. (London) 284, 49P. Carter, J . R., Avruch, J . , Jr., and Martin, D. B. (1973). Glucose transport by trypsintreated red blood cell ghosts. Biochim. Biophys. Aciu 291, 506-518. Cheung, 3. Y., Regen, D. M., Schworer, M. E., Whitfield, C. F . , and Morgan, H. E. (1977). Anaerobic stimulation of sugar transport in avian erythrocytes. Biochim. Biophys. Aciu 470, 212-229. Coleman, R. (1973). Membrane-bound enzymes and membrane ultrastructure. Biochim. Biophys. Acici 300, 1-30. Danielli, J. F., and Davson, H. (1935). A contribution to the theory of permeability of thin films. J . Cell. C o m p . Physiol. 5, 495-508. Dawson, A. C., and Widdas, W. F . (1964). Variations with temperature and pH of the parameters of glucose transfer across the erythrocyte membrane in the foetal guinea pig. J . Physiol. (London) 172, 107- 122. Deves, R., and Krupka, R. M. (1978a). A new approach in the kinetics of biological transport. The potential of reversible inhibition studies. Biochim. Biophys. Acta 510, 186-200. Deves, R., and Krupka, R. M. (1978b). Cytochalasin B and the kinetics of inhibition of biological transport. A case of asymmetric binding to the glucose carrier. Biochim. Biophys. Actu 510, 339-348. Deves, R., and Krupka, R. M. (1978~).Testing transport models with substrates and reversible inhibitors. Biochim. Biophys. Acta 513, 156- 172. Edwards, P. A. W. (1974). A test for non-specific diffusion steps in transport across cell membranes and its application t o red cell glucose transport. Biochim. Biophys. Aciu 345, 373-386. Eilam, Y. (1975). Two-carrier models for mediated transport. I. Theoretical analysis of several two-carrier models. Biochim. Biophys. Acia 401, 349-363. Eilam, Y . , and Stein, W. D. (1974). Kinetic studies of transport across red blood cell membranes. In “Methods in Membrane Biology” (E. D. Korn, ed.), Vol. 2, pp. 283354. Plenum, New York.

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Fairbanks, G., Steck, T. L., and Wallach, D. F. H. (1971). Electrophoretic analysis of the major polypeptides of the human erythrocyte membrane. Biochemistry 10, 2606-2617. Foster, D. M., and Jacquez, J. A. (1976). An analysis of the adequacy of the asymmetric carrier model for sugar transport. Biochim. Biophys. Acta 436, 210-221. Furthmayr, H., Kahane, I., and Marchesi, V. T. (1976). Isolation of the major intrinsic transmembrane protein of the human erythrocyte membrane. J. Membr. B i d . 26, 173187. Furthmayr, H., Galardy, R. E., Tomita, M., and Marchesi, V. T. (1978). The intramembranous segment of human erythrocyte Glycophorin A. Arch. Biochem. Biophys. 185, 21-29. Geck, P. (1971). Eigenschaften eines asymmetrischen carrier-modells fur den zuckertransport am menschlichen erythrozyten. Biochim. Biophys. Acta 241, 462-472. Ginsburg, H. (1978). Galactose transport in human erythrocytes. The transport mechanism is resolved into two simple asymmetric antiparallel carriers. Biochim. Biophys. Acta 506, 119- 135. Ginsburg, H., and Stein, W. D. (1975). Zero-trans and infinite-cis uptake of galactose in human erythrocytes. Biochim. Biophys. Actu 382, 353-368. Ginsburg, H., and Yeroushalmy, S. (1978). Effects of temperature on the transport of galactose in human erythrocytes. J. Physiol. (London) 282, 399-417. Goodwin, R. F. W. (1954). Blood-sugar in foetal and neonatal mammals. Nuture (London) 173, 777-778. Goodwin, R. F. W. (1956). The distribution of sugar between red cells and plasma: Variations associated with age and species. J. Physiol. (London) 134, 88- 101. Gorter, E., and Grendel, F. (1925). On bimolecular layers of lipoids on the chromocytes of the blood. J. Exp. Med. 41, 439-443. Hanes, C. S. (1932). Studies on plant amylases. I. The effect of starch concentration upon the velocity of hydrolysis by the amylase of germinated barley. Biochem. J. 26, 14061421. Hankin, B. L., and Stein, W. D. (1972). On the temperature dependence of initial velocities of glucose transport in the human red blood cell. Biochim. Biophys. Acra 288, 127136. Hankin, B. L., Lieb, W. R., and Stein, W. D. (1972). Rejection criteria for the asymmetric carrier and their application to glucose transport in the human red blood cell. Biochim. Biophys. Acta 288, 114- 126. Hoos, R. T., Tarpley, H. L., and Regen, D. M. (1972). Sugar transport in beef erythrocytes. Biochim. Biophys. Actu 266, 174- 181. Huxley, A. F. (1977). Evidence, clues and motive in science. Presidential Address to the British Association for the Advancement of Science. Times Higher Educ. Suppl. Sept. 23, pp. 61-66. Jardetzky, 0. (1966). Simple allosteric model for membrane pumps. Nature (London) 211, 969- 970. Jones, M. N . , and Nickson, J. K. (1978). Electrical properties and glucose permeability of bilayer lipid membranes on incorporation of erythrocyte membrane extracts. Biochim. Biophys. Acta 509, 260-271. Jung, C. Y. (1975). Carrier-mediated glucose transport across human red cell membranes. In “The Red Blood Cell” (D. M. Surgenor, ed.), 2nd ed., pp. 705-749. Academic Press, New York. Jung, C. Y., and Rampal, A. L. (1975). Effects of Cytochalasin B on equilibrium exchange flux of D-glucose across human erythrocyte membranes. Fed. P r o c . , Fed. A m . Soc. Exp. Biol. 34, 238.

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OF THE HEXOSE TRANSFER SYSTEM

21 9

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C U R R E N T TOPICS I N M E M B R A N E S A N D TRANSPORT. VOLUME

14

Permeation of Nucleosides. Nucleic Acid Bases. and Nucleotides in Animal Cells PETER G . W . PLAGEMANN A N D ROBERT M . WOHLHUETER Department of Microbiology University of Minnesota Minneapolis. Minnesota

I . Introduction and Technical Principles . . . . . . . . . . . I1 . Carrier Model for Facilitated Diffusion and Tests for Its Applicability to Nucleoside and Base Transport . . . . . . . . . . . . . A . Zero-trans Influx and Efflux . . . . . . . . . . . . B . Equilibrium Exchange Inward and Outward . . . . . . . . C . Infinite-trans Procedure . . . . . . . . . . . . . . D . Infinite-cis Procedure . . . . . . . . . . . . . . E . Interpretation of Experimental Data . . . . . . . . . . 111 . Uptake of Nucleosides and Purine Bases . . . . . . . . . . A . General Considerations . . . . . . . . . . . . . . B . Relationship between Transport and Metabolism Operating in Tandem C . Estimation of Zero-trans Transport Kinetic Parameters from Substrate Uptake Curves . . . . . . . . . . . . . D . Uptake into Vesicles of Mammalian Cells . . . . . . . . E . Contributions of Transport and Nonmediated Permeation to Overall Uptake . . . . . . . . . . . . . . . . IV . Properties of Nucleoside and Free Base Transport Systems . . . . A . Specificity for Natural Substrates . . . . . . . . . . . B . Transport of Substrate Analogs . . . . . . . . . . . C . Effect of Temperature . . . . . . . . . . . . . . D . Effect of pH . . . . . . . . . . . . . . . . . E . Presumptive Cell Clones Defective in Transport . . . . . . F . Comparison to Transport in Other Types of Organisms . . . . V . Transport Inhibitors and Inactivation . . . . . . . . . . . A . Effects of Sulfhydryl Reagents . . . . . . . . . . . . B . Effect of Other Nonspecific Inhibitors . . . . . . . . . C . Inhibition of Nucleoside Transport by p-Nitrobenzylthiopurine Nucleosides . . . . . . . . . . D . Heat Shock . . . . . . . . . . . . . . . . . E . Effect of Hydrolytic Enzymes . . . . . . . . . . . .

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VI. Regulation of Nucleoside and Free Base Transport and Uptake . . . . 295 VII. Permeation of Nucleotides . . . . . . . . . . . . . . . 303 VIII. Summary and Conclusions . . . . . . . . . . . . . . . 310 References . . . . . . . . . . . . . . . . . . . . 3 13

1.

INTRODUCTION AND TECHNICAL PRINCIPLES

Greater knowledge of the mechanism of permeation of nucleosides, nucleobases, and nucleotides through the cell membranes of eukaryotes is important for several reasons. First, cells of certain tissues in animals and man are deficient in the pathway for de novo synthesis of purines and thus need to take up from the circulation purines that have been synthesized and released by other body cells or derived from food (Murray, 1971). Evidence accumulated during the last 10 years has indicated that the uptake of nucleosides and nucleobases involves their transport through the plasma membrane by specific carriers and their subsequent intracellular phosphorylation (the salvage pathways). Second, many anticancer and immunosuppressive agents presently in use or under development are nucleoside, nucleotide, or nucleobase analogs and a clear understanding of their mode of entry into cells and metabolism is important in the assessment of their mode of action, efficacy, and optimal administration, and of the development of drug-resistant mutants (Sirotnak ef al., 1979). Third, radioactively labeled nucleosides and nucleic acid bases are widely used as precursors to label specifically the nucleic acids of various types of organisms or of the viruses or plasmids replicating therein as well as to assess the rates of nucleic acid synthesis. An interpretation of the rates of nucleoside and base incorporation into nucleic acids, be it R N A or DNA, depends on a clear understanding of the extent to which these rates may reflect the rates of the conversion of the extracellular substrate to intracellular nucleotides which are the direct precursors in nucleic acid synthesis. In cultured animal cells the incorporation of nucleosides and bases into the nucleotide pool seems to be the main ratedetermining step in their incorporation into nucleic acids. Alterations in either their transport or phosphorylation have been found to cause proportional changes in the rates of incorporation into nucleic acid and can occur independently of changes in the rate of nucleic acid synthesis per se. Thus great care is required in equating rates of nucleoside or base incorporation into acid-insoluble cell material with rates of nucleic acid synthesis.

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The relationship between nucleoside and base transport, phosphorylation, and other metabolism by animal cells has only been recently evaluated and only in a few cell systems (see Section I I I ) , but is important for an understanding of the uptake process as well as for measuring nucleoside and base transport rates in metabolizing cells. The question has acquired additional significance because of the finding that the incorporation rates for nucleosides, bases, and other substrates by animal cells vary as the function of the growth stage and that tumor cells often exhibit higher incorporation rates than untransformed cells (see Section VI). In fact, it has been proposed that an enhanced nutrient transport capacity may be an essential aspect of the altered growth potential of tumor cells (Holley, 1972; Pardee, 1971). Numerous studies have assessed the kinetics of uptake of radioactively labeled nucleosides and bases by cultured animal cells (see Section 111). “Uptake” here denotes the accumulation of radioactivity derived from exogenous, labeled substrate within the cell, regardless of metabolic conversion, in the same sense as used by Berlin and Oliver (1975). It is a composite phenomenon, which, in the case of nucleosides and nucleic acid bases, results from the tandem operation of a nonconcentrative transport system and of various cytoplasmic enzymes, including kinases, hydrolases, phosphoribosyltransferases, and nucleic acid polymerases. “Transport,” in contrast, here denotes only the transfer of a substance (or the translocation of a chemical substituent) across the plasma membrane in either direction, as mediated by a saturable, selective carrier. “Incorporation” denotes the appearance of radioactivity derived from substrate in a specified cellular compartment, chemical compound, or class of compounds. Uptake of nucleosides and bases by cultured cells has generally been found to be approximately linear with time for between 1 and 10 minutes and initial uptake velocities have been assumed to reflect those of transport of the substrates into the cell. Some indirect lines of evidence reviewed previously (Plagemann and Richey, 1974) supported this assumption. The prime evidence was ( I ) that uptake obeyed simple Michaelian kinetics suggestive of a single, saturable, rate-determining step in the overall uptake process: (2) that the kinetic constants for uptake by whole cells were much lower than those for the phosphorylation of the substrates in cell lysates: (3) that the intracellular steady-state concentration of free substrate appeared to be far below that in the extracellular fluid: and (4) that uptake was inhibited in an apparent competitive manner by various substances which were presumed to act on the transport step since they did not affect the intracellular metabolism of the substrate. Recent evidence, however, has indicated that, although these observa-

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tions are correct, their interpretation is more complicated and that the uptake rates estimated from time intervals of minutes (here referred to as long-term rate) reflect the intracellular accumulation of phosphorylated intermediates. Transport, on the other hand, has been found to be so rapid that intracellular steady-state concentrations of free substrate are attained within seconds, at least at concentrations below the MichaelisMenten constant of transport. Catabolic reactions which occur in most types of cells also complicate studies of transport of several nucleosides. Adenosine, deoxyadenosine, cytidine, and deoxycytidine are deaminated, and guanosine, deoxyguanosine, inosine, deoxyinosine, uridine, deoxyuridine, and thymidine are subject to phosphorolysis, the products of which may be further catabolized. Hypoxanthine and guanine, on the other hand, are converted to nucleosides by purine nucleoside phosphorylase. In animal cells these reactions occur intracellularly and the products, if not phosphorylated, are subject to exit transport. Nucleotides, in contrast, are largely retained by the cells, since the plasma membrane is relatively impermeable to most phosphorylated compounds (see Section VII). The distinction between long-term rates of nucleoside uptake and rates of transport is illustrated most strikingly by data on the uptake and metabolism of [3H]adenosine in P388 mouse leukemia cells. At an extracellular adenosine concentration of 100 p M , long-term uptake was approximately linear for at least 30 minutes (Fig. 1A). Though the uptake curve appears to extrapolate near the origin, closer inspection shows that, at the first time point (30 seconds), the intracellular concentration of radioactivity already exceeded that in the medium. Furthermore, analysis of the culture fluid (Fig. 1B) showed that adenosine disappeared from the medium 70 times more rapidly than radioactivity accumulated within the cells. By 30 minutes most of the radioactivity accumulated in the medium was in the form of inosine and hypoxanthine, a consequence of the deamination of adenosine and the phosphorolysis of the product inosine. Since all evidence indicates that deamination is solely an intracellular process (Lum et a / . , 1979), it is obvious that the rate of uptake of radioactivity by the cells from exogenous labeled adenosine represents at best 1.5% of the transport rate. In fact, it was shown that upon exposure of P388 cells to adenosine at concentrations of 100 p M and above, the intracellular rate of deamination approaches that of the transport rate, since deaminase and transport have similar Michaelis-Menten constants with respect to adenosine, while the maximum velocity of deamination (as measured in cell lysates and expressed per cell) exceeds that of transport 2- to 3-fold (Lum et al., 1979; see also Table IV). The need to sample at short intervals if one is to estimate initial


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r-B

CULTURE FLUID

TOTAL

v,, =63pmole/pl CELL H 2 0 . sec

0 60

ADENOSINE

W

z 0

10

20

3(

TIME I MIN)

FIG. I . Adenosine uptake and metabolism by P388 mouse leukemia cells. (A) A suspension of I .5 X 10' cellsiml of serum-free basal medium was supplemented (0 time) with 100 p M [3H]adenosine (3.4 cpm/pmole) and incubated at 37°C. At various times, duplicate 0.5-ml samples of suspension were centrifuged through an oil layer (see text; Section I) and the cell pellets were analyzed for radioactivity. All values are averages of the duplicate samples corrected for nonspecific substrate trapping as estimated with [14C]inulin. (B) Adenosine, inosine, and hypoxanthine in the cell-free culture fluid were separated chromatographically a s described by Lum et a / . (1979). The velocities of uptake into cell material (A) and of the disappearance of adenosine from the medium (B) were estimated graphically from the linear portions of the curves and are based on an intracellular water volume of 1.3 pl/106 cells as estimated with 3H20 (Wohlhueter et a/., 1978a). The broken line in (A) indicates the intracellular concentration of radioactivity equivalent to that in the medium. Data are similar to those reported by Lum et ( I / . (1979).

transport velocities in metabolizing cells has been emphasized previously by Berlin and Oliver (1975). The validity of this admonition, and a quantitative appreciation of "short ," is becoming increasingly clear. The data in Fig. 1, for example, reveal that a 30-second sample would grossly underestimate the adenosine transport rate. In fact, at 24°C samples at 3-5 seconds were the longest that yielded reasonably accurate estimates of initial velocities of adenosine transport in P388 cells (Lum et a / . , 1979). Even so, identification of the main rate-determining step in long-term uptake may be contingent on the concentration of exogenous substrate. For example, at extracellular nucleoside concentrations far below the Michaelis constant for transport, the rates of intracellular phosphorylation of some nucleosides and of hypoxanthine approach those of trans-

230


port, whereas at concentrations comparable to or exceeding the Michaelis constant for transport, the transport rate far exceeds the phosphorylation rate (for detailed discussion see Section 111). Elimination of the ambiguities inherent in metabolizing cells is of clear advantage to transport studies. Nucleoside and purine transpmt has been studied successfully in the absence of intracellular metabolism by the use of erythrocytes or of mutant clones of cultured animal cells which are deficient in specific metabolic enzymes and by the use of cellisubstrate systems in which substrate metabolism is blocked in some other manner. For example, uridine and thymidine transport has been studied in human erythrocytes which lack enzymes for the phosphorylation and phosphorolysis of pyrimidine nucleosides (Oliver and Paterson, 1971; Lieu et a / . , 1971). The use of enzyme-deficient mutants of cultured cells for transport studies was introduced by Kessel and Shurin (1968), who studied the uptake of deoxycytidine and cytosine arabinoside in a deoxycytidine kinase-deficient line of L 12 10 mouse leukemia cells. Subsequently, mutant cell lines deficient in thymidine kinase, uridine kinase, hypoxanthinei guanine phosphoribosyltransferase and adenine phosphoribosyltransferase have been employed to study the transport of thymidine, uridine, hypoxanthineiguanine, and adenine, respectively (Schuster and Hare, 1971: Cunningham and Remo, 1973; Zylka and Plagemann, 1975; Plagemann et a / . , 1976; Wohlhueter et a / . , 1976; Alford and Barnes, 1976; Plagemann et a / . , 1978b: Witney and Taylor, 1978: Murphy et u / . , 1977). Another approach to the prevention of substrate phosphorylation has been to deplete cells of ATP by preincubation in a glucose-free medium containing KCN and iodoacetate. This approach has the advantage of general applicability, since it prevents all phosphorylation reactions (Plagemann and Erbe, 1973; Plagemann et a / . , 1976; Wohlhueter et n / . , 1978a). Such treatment renders cells somewhat more fragile osmotically (Plagemann el "/., 1976), perhaps because ATP depletion causes the aggregation of integral membrane proteins (Gazitt et a / . , 1976), and, in some cell lines, has been observed to cause a slight decrease in transport capacity (Plagemann et a / . , 1978b). Overall, however, the kinetics of transport of nucleosides and hypoxanthine in enzyme-deficient and ATPdepleted cells are comparable (Plagemann et u / . , 1978b; Wohlhueter et a / . , 1978a, 1979a; Marz et a / . , 1979). Substrate metabolism other than phosphorylation can sometimes be inhibited by treatment of cells with appropriate inhibitors. For example, adenosine deamination has been blocked by preincubation of the cells with deoxycoformycin thus permitting an accurate assessment of adenosine transport (Lum et [ I / . , 1979). A third general approach for measuring substrate transport in the ab-


231

sence of metabolism is the use of natural nucleosides or nucleobases or analogs thereof that are not subject to enzymatic modification, but still possess substrate activity for the respective transport systems. This approach has been successfully employed in the case of hexose transport by the use of 3-O-methyl-~-glucose, but has been applied to the study of nucleoside transport only recently. Kessel(l978) has shown that 5’-deoxyadenosine is transported by the nucleoside transport system in L 12 10 mouse leukemia cells, but is neither phosphorylated nor deaminated by the cells. Yoshida sarcoma cells (Mulder and Harrap, 1975),golden hamster fibroblasts (Heichal et u / . , 1978, 1979), and rat uterus (Oliver, 1971) do not phosphorylate cytosine arabinoside or do so only slowly, and thus lend themselves to a study of transport of this nucleoside. Thymine likewise is metabolically inert, and uracil is converted to nucleosides and nucleotides only very slowly in many animal cells and both have been used in transport studies (Zylka and Plagemann, 1975; Plagemann et ( I / . , 1978b). Thymidine transport has been examined in primary cultures of rat hepatoma cells which phosphorylate this nucleoside to only a limited extent (Ungemach and Hegner, 1978). Studies with cells in which the substrate was not modified intracellularly clearly indicated that nucleoside, purine, and uracil transport in most animal cells is energy independent and nonconcentrative, i.e., the intracellular concentration at equilibrium equals that in the extracellular fluid. This type of transport is generally referred to as facilitated diffusion or facilitated transport. The availability of cells incapable of substrate metabolism by itself, however, does not assure success in transport measurements. Initial studies with enzyme-deficient or ATP-depleted cultured animal cells clearly indicated the great rapidity of the nucleoside and purine transport systems, but it was just this rapidity of transport which presented technical difficulties in the estimation of accurate transport velocities from substrate accumulation curves (see Section 11,E). These difficulties have been successfully solved only recently by the development of various technical improvements in measuring substrate accumulation by both cells in suspension and attached to supporting media. These technical improvements can be divided into three categories: ( 1 ) the rapid separation of cells and extracellular fluid; ( 2 ) the development of rapid mixing/ sampling procedures; and (3) the estimation of transport velocities and assessment of transport models from the entire time course of attainment of substrate transmembrane equilibrium. For studies of nucleoside and base influx, cells in suspension have been rapidly separated from the extracellular medium containing labeled

232

PETER G. W. PLAGEMANN AND ROBERT M . WOHLHUETER

substrate by centrifugation through or into an inert oil layer which has a density higher than that of the aqueous suspension medium, but lower than that of the cells themselves. Such a procedure has long been applied to study the distribution of substrate across the membrane of mitochondria (Werkheiser and Bartley, 1957).To study uridine influx human erythrocytes have been separated from the medium by centrifugation into dibutylphthalate and the transport rate was estimated from the rate of disappearance of radioactivity from the medium (Oliver and Paterson, 1971). To study nucleoside and base influx in suspensions of cultured animal cells (Wohlhueter et ( I / . , 1976, 1978a: Ungemach and Hegner, 1978), mouse lymphocytes (Strauss et [ I / . , 1976), and mouse lung macrophages (Pofit and Strauss, 1977) cells have been centrifuged into silicone-oil mixtures and influx measured by the appearance of radioactivity in the pelleted cells. If high-speed centrifugation is employed cells can be separated from the medium in less than 2 seconds (Wohlhueter et i l l . , 1978a). The time resolution of this approach has been further improved by use of a rapid mixing technique similar to those of the stop-flow kineticist, with which the intracellular accumulation (and metabolism) of substrate can be followed in intervals as short as 1 second (Wohlhueter et i l l . , 1976, 1978a). Fixed aliquots of a suspension of cells are rapidly mixed with a solution of radioactively labeled substrate at short time intervals by means of a hand-operated dual-syringe apparatus. Cell substrate mixtures emerging from the mixing chamber are dispensed into 12 tubes which contain an oil mixture and are mounted in a microcentrifuge. After the last sample is mixed the centrifuge is started and within 2 seconds the cells have entered the oil phase, thus terminating transport. Thus it is possible to obtain 12 time points of substrate accumulation within a time period as short as 15 seconds (see Section 111). The apparatus and methodology have been described in detail by Wohlhueter et id. ( 1978a). In substrate efflux measurements from erythrocytes preloaded with radioactive substrate a rapid separation of the medium from the cells has been achieved by filtration through Millipore filters, followed by analysis of the filtrate for residual substrate concentration (Mawe and Hempling, 1965; Lassen, 1967; Cabantchik and Ginsburg, 1977). I t has been estimated that the sampling time can be reduced with practice to 1 second (Cabantchik and Ginsburg, 1977). Cells attached to solid substrate lend themselves to another approach to the rapid removal of extracellular medium in substrate influx studies. For example, cells attached to glass coverslips have been immersed in substrate solution and then rapidly rinsed by repeated immersion in cold


233

buffer rinse solutions (Hawkins and Berlin, 1969; Foster and Pardee, 1969: Sander and Pardee, 1972). In other studies monolayer cultures of animal cells in Petri plates have been used and the substrate solution and rinse fluids have been removed by rapid aspiration (Rozengurt et a / . , 1977b; Plagemann et al., 1978b). In this procedure cell monolayers can be washed free of extracellular substrate within about 6 seconds (Plagemann et u / . , 1978b). Each of these experimental approaches has certain advantages and disadvantages. Separation of cells from the substrate solution by centrifugation into an inert oil layer necessitates corrections for trapping of extracellular substrate in an aqueous layer surrounding the cells which is not removed by centrifugation through oil and which represents between 10 and 20% of the intracellular aqueous space in various types of cultured cells (Wohlhueter et a / . , 1976, 1978a). The trapped extracellular water space relative to the intracellular water space increases progressively with a decrease in cell size. Such substrate trapping has been assessed by the use of substances to which cells are impermeable or only slowly permeable such as inulin or L-glucose (see Wohlhueter et a / . , 1978a). Such corrections are not required when removing extracellular substrate by rinsing cell layers attached to dishes or coverslips with aqueous buffers, but in this procedure the possible loss of intracellular substrate during the rinsing period needs to be considered. Low-temperature rinses minimize, but do not abolish, such losses. For example, it has been calculated that Novikoff cells would be expected to lose half their intracellular thymidine pool, if it is less than 100 p M , during a 43second rinse with buffer at 4°C (Wohlhueter et a / . , 1979a). However, such losses can be completely prevented in the case of nucleosides and purines by inclusion of high concentrations of a transport inhibitor, such as dipyridamole (Persantin) (Plagemann et a / ., 1978b) or 6-([4-nitroben(nitrobenzylthioinosine) (Rozengurt zyl] thio)-9-p-~-ribofuranosylpurine et u / . , 1978) in the rinse fluid. The use of “stopper solutions” to quench transmembrane fluxes rapidly has earlier been applied to measure nucleoside transport in cells in suspension. Cass and Paterson (1972) introduced the use of 2-hydroxylnitrobenzylthioguanosine in this capacity followed by the centrifugal separation of the cells from the medium to demonstrate nucleoside accelerated exchange diffusion in human erythrocytes. Cabantchik and Ginsburg (1977) used the same inhibitor, as well as nitrobenzylthioinosine, followed by centrifugal separation of the cells from the medium and removal of residual extracellular substrate by washing in aqueous solutions containing the inhibitor to study uridine influx in human erythrocytes. A similar approach using dipyridamole has been

234


employed successfully to measure uridine accumulation in uridine kinasedeficient cultured Novikoff rat hepatoma cells (Plagemann et a / . , 1978b) and of 5’-deoxyadenosine in L1210 cells (Kessel, 1978). In the latter study a solution of mercuric chloride plus sodium iodide was also used as a stopper solution. The filtration method, on the other hand, appears useful only where the experimental protocol permits measurement of radioactivity in cell-free filtrate, as in efflux studies (Cabantchik and Ginsburg, 1977; Wohlhueter et ul., 1978a). A disadvantage encountered in transport studies with cells attached to culture dishes or coverglasses is the small intracellular volume available relative to the total medium volume required to cover the cell layer. At best, the available intracellular volume represents 0.5% of the extracellular volume. In nonphosphorylating cells, therefore, the amount of substrate present intracellularly at equilibrium is severely limited and incomplete removal of extracellular substrate introduces large errors in uptake values. Because of this relatively small intracellular volume available in culture dishes some investigators failed to detect nucleoside and purine transport in cells lacking the enzymes responsible for their conversion to nucleotides (see Section 11, E). Furthermore, it is difficult to estimate accurately the intracellular volume of attached cells, knowledge of which is needed to assess the absolute intracellular concentration of substrate. These volume limitations do not apply to suspensions of cells which can be prepared at any desired density. Erythrocyte suspensions with hematocrits of up to 40% (v/v) have been used (Oliver and Paterson, 1971), in which case one can estimate substrate uptake by measuring its disappearance from the medium. Much lower cell densities (1.5-7%, v/v) have been used with suspensions of cultured cells (Wohlhueter et d., 1978a) which, though precluding measurement of substrate disappearance from medium, assures accurate direct measurement of accumulation within cells. Intracellular volumes of suspended cells can be readily estimated by exposing cells to 3 H 2 0 and then separating the cells from the extracellular fluid by centrifugation through an oil layer (Wohlhueter et a / . , 1978a). Regardless of the experimental approach employed, transport velocities have generally been estimated graphically from the initial linear phases of time courses of intracellular accumulation of substrate (influx), of appearance of substrate in extracellular fluid from preloaded cells (efflux), or of the movement across the membrane of isotopically labeled substrate at chemical equilibrium (equilibrium exchange). In general, initial entry or exit rates have been based on only few early time points. Often, because of the rapidity of nucleoside and base transport, the


235

initial, linear phase is impractically short, especially at low extracellular substrate concentrations. These facts, together with theoretical considerations discussed in the next section, limit the accuracy of graphical estimation of initial velocities. More accurate estimates of transport velocities are obtainable by an analysis of the entire time course of attainment of transmembrane equilibrium in terms of integrated rate equations describing various transport models (Section 11). In addition, such an approach yields considerable information on the mechanism of transport per se. Though we have stressed here transport measurements in the absence of metabolism, we consider in Section I11 the relationship of transport to phosphorylation, and their operation in tandem. This topic is developed more fully by Wohlhueter and Plagemann (1980). Other reviews pertinent to nucleoside and/or purine uptake in eukaryotic cells are those by Plagemann and Richey (1974), Berlin and Oliver (1973, Hochstadt (1974), and Perdue (1978). Studies on nucleoside and base uptake by prokaryotic cells have been reviewed by Hochstadt (1974). II. CARRIER MODEL FOR FACILITATED DIFFUSION AND TESTS FOR ITS APPLICABILITY TO NUCLEOSIDE AND BASE TRANSPORT

Recent studies in which the transport of nucleoside and purine base transport by animal cells has been kinetically separated from subsequent metabolic steps are clearly compatible with the view that the movement of uridine across the human erythrocyte membrane (Cabantchik and Ginsburg, 1977) and the movement of various nucleosides and purines across the membrane of animal cells in culture (Wohlhueter et al., 1979a; Marz ei al., 1979; Lum ei al., 1979) proceeds via the simple carrier mechanism formulated mathematically by Lieb and Stein ( 1974) and Edam and Stein (1974). The model is summarized in Fig. 2 along with the various experimental protocols which have been used to test its applicability to the transport of nucleosides and purines in human erythrocytes and cultured animal cells. Edam and Stein (1974) developed unidirectional flux, initial velocity, and integrated rate equations corresponding to these experimental protocols. It should be noted that these experimental designs are strictly applicable only to cells which fail to metabolize the substrate or in which metabolism is blocked in some manner. In general, we follow the terminology of Eilam and Stein (1974).

236

PETER G . W. PLAGEMANN AND ROBERT M. WOHLHUETER

EXPERIMENTAL DESIGN =

0, s2 = 0

S2 V A R I E D (CELLS PRELOADED) s2 VARIED AS s1 IS VARIED (CELLS PRELOADED)

s2’

VARIED (CELLS PRELOADED)

S2 ’’ K

(CELLS PRELOADED)

S2 V A R I E D 0 * S1 (CELLS PRELOADED)

S2

>>

K (CELLS PRELOADEU)

S2 V A R I E D 0 *

I MEMBRANE I

S1

(CELLS

PRELOADED)

FIG.2 . The simple carrier model in various experimental configurations. Nomenclature is that of Eilam and Stein (1974) and pertains to the conditions set at the beginning of the experiment: S, = extracellular substrate concentration; S, = intracellular substrate concentration; S* = isotopically labeled substrate; K = a fundamental constant reflecting substrate-carrier affinity: C = carrier; zt = zero-trans: ee = equilibrium exchange: it = infinite-trans; ic = infinite-cis. The external face of the plasma membrane is designated face 1 , and the internal face as face 2: k , , k , , and g , and g , are the rate constants for the “movement” of the unloaded and loaded carrier, respectively.

A. Zero-trans Influx and Efflux

In the zero-trans (zt) procedure one measures the transport of a substrate from one side of the membrane (the cis side), where its concentration is varied, to the other side (the trans side) where its concentration is initially zero (Fig. 2 ) . The intracellular concentrations of nucleosides and purines in animal cells are generally very low ( < 1 pM) and in influx studies (transport from face I to face 2 ) , therefore, the initial intracellular substrate concentration is considered to be zero. Initial rates of uridine entry (v;:) into human erythrocytes and exit ( ~ 5 : )from these cells have been estimated by graphical or linear regression analysis of initial time courses of change in intracellular ( S , ) or extracellular ( S uridine concentration (Oliver and Paterson, 1971 : Cabantchik and Ginsburg, 1977). Michaelis constants for uridine influx ( K ; ; ) and efflux ( K ; : ) and the corresponding maximum velocities @and Vzi) were computed from plots of the estimated slopes as a

237


function of S on the cis side. The estimated kinetic constants are summarized in Table I and are further discussed in Section II,E. Because of the rapidity of nucleoside and purine transport in most animal cells, however, it has often proved technically infeasible to obtain accurate, direct estimates of initial velocities, thus necessitating the use of integrated rate equations. The application of nonlinear equations not only permits estimation of true initial velocities when the linear portion of the curve is impractically short, but also can, theoretically at least, yield valuable information about the molecular properties of the transport system (Eilam and Stein, 1974; Wohlhueter et a / . , 1978a, I979a). Edam and Stein ( 1974) have developed integrated rate equations to describe the time course of transport beginning at zero-trans and proceeding to equilibrium. They detail a graphical method for estimating the various rate and association constants. The graphical method relies on a logarithmic transformation in which the relative errors become large as the trans substrate concentration approaches the cis concentration and statistical weighting becomes problematic. The approach has been rarely used. Wohlhueter et a / . (1978a, 1979a) and Heichal et al. (1979) have arrived at somewhat more simplified, integrated rate equations (for zero-trans in the I to 2 direction) by assuming that the exogenous substrate concentration ( S , ) is constant. This assumption holds for zero-trans influx studies if the intracellular H,O volume represents not more than about 5% of the total volume of suspension, a condition generally met in studies with suspensions of cultured animal cells. Heichal et a/. (1979) have applied the equation in a linearized form to evaluate the transport kinetics of cytosine arabinoside. Wohlhueter et a / . (1978a, 1979a) have employed the equation in exponential form to fit transport data by nonlinear regression. They write the equation in a form analogous to the equation for first-order approach to equilibrium, although implicit with respect to intracellular concentration (S'J:

f

1

(S1)

(1)

where S,J = concentration of S , at time t (Sz,o= 0); and f l and f 2 are functions of various kinetic parameters as defined by Eilam and Stein ( 1974):

f,(S)= KRoo + R12S1 + R21S1 + ST ReeIK f z ( S 1 , Sz,t) = (Rz1 + ReeSlIK)

Sz,t

(2)

TABLE I KINETICPARAMETERS FOR ZERO-TRANS, EQUILIBRIUM EXCHANGE, A N D INFINITE-CIS INFLUX ERYTHROCYTES~ Michaelis-Menten constant

(WW

Protocol zt zt ee ee ic ic

(12) (21) (12) (21) (12) (21)

400 2 122 73 2 63 1310 2 92 1280 2 142 252 ? 90b 937 2 226b

Maximum velocity (pmoleipl H,O.second)

33 8.9 130 120 14' 11.5'

?

2 k 2

5.2 0.6 20 6.1

Michaelis- Menten constant (P1Z.T)

710d N De 530V ND ND 7000

AND

EFFLUXOF U R I D I N IEN HUMAN

Maximum velocity (pmole/pl packed cells.second)

8.4d 3Ih 100 156h ND 1700

Temperature ("C) 15

25 25 25 37

Data are from Cabantchik and Ginsburg (1977), and were collected at 25°C. Sources of other data are indicated in individual footnotes, and were collected at the temperature given in the final column. * K Z and KFl values calculated from the experimentally determined ee and zt maximum velocities according to K V e e = K i12c V"' or = KF1V;:, were 231 and 1080, respectively. Equals V ; : or V ; : , respectively; calculated from the Y-intercepts of Figs. 7 and 9 of Cabantchik and Ginsburg (1977) [see Eq. @)I. From Oliver and Paterson (1971). N D = not determined. From Pickard and Paterson (1972). From Lieu et ( I / . (1971). Estimated from data i n Fig. 6 . a

'


239

K is a measure of the affinity of the carrier for its substrate, and is related directly to the Michaelis-Menten constants apparent in various experimental protocols (see Table I); with zero-trans influx, for example, K?: = K ( R c J R l z ) .The R-terms are resistance factors, proportional to the time of round-trip for the carrier in one of four modes; ( I ) loaded on the inbound trip and empty on the outbound trip (Rlz);( 2 ) empty on the inbound and loaded on the outbound trip (Rzl); (3) empty in both directions (Roo);and (4) loaded in both directions (Ree). Necessarily, Roo + Re, = R l 2 + RZl. Functional symmetry of the carrier is manifest as equivalence of the various R-terms. If carrier movement is indifferent with respect to direction (idout symmetry) R,, = Rzl. If loaded carrier moves as rapidly as the unloaded carrier Re, = Roo,and for a cmpletely symmetrical carrier all R-terms are equal (and represented by R ) , and the Michaelis- Menten constants apparent in various experimental protocols are all equal to K . The R constants are the reciprocals of the corresponding maximum velocities, for example, R 12 = I/VZ,:. As t approaches 0, fz approaches 0, and as t approaches CQ, fz becomes a constant 6 t . If one assumes f z to be negligible at all t , as Wohlhueter er al. did in earlier studies (Wohlhueter et al., 1976, 1978a) for the sake of mathematical simplicity, Eq. (1) is reduced to the integrated equation for a first-order reaction: Sz,t = S , [ l - exp(-k’t)]

(4)

where k’ = pseudo-first-order rate constant = l / f l ( S l ) .In these studies initial zero-trans velocities were calculated from computed k’ values according to the first derivative of Eq. (4) at t = 0: vZ,\= k ’ S 1 . When S l 4 K?:, f z is indeed insignificant, so that Eq. (4) describes the influx of substrate under these conditions; this of course is the condition in which influx is first order with respect to substrate concentration. Subsequently, computational procedures have been developed (Wohlhueter er al., 1979a; Marz er al., 1979) to permit exact solutions of Eq. ( I ) , thus allowing least-squares fits of Eq. (1) to time courses of the approach to transmembrane equilibrium, whereby t and S , are treated as independent variables and Sz,t as dependent variable (based on the experimentally determined substrate radioactivity and intracellular HzO space).’ This non-linear, multivariable regression procedure determines the values of K , the substrate: carrier affinity constant, and of the various

’

All computations were carried out on a Hewlett-Packard (Loveland, CO) 9825A desktop computer equipped with a 9871A printer. Curve fitting programs were developed on the algorithm of Dietrich and Rothmann (1975); the authors will honor requests for program listings.

240


R-constants (and associated standard errors of estimate) best fitting a set of data comprising complete time courses at 6 or more substrate concentrations. Application of this procedure to uridine transport in uridine kinasedeficient Novikoff rat hepatoma cells is illustrated in Fig. 3 . Kinetic parameters for the transport of thymidine, uridine, adenosine, cytidine, deoxycytidine, adenine, and hypoxanthine by this and other cell lines are summarized in Table I1 and discussed further in Section II,E. The data in Fig. 3 also emphasize the technical difficulties encountered in estimating accurate uf: values by graphical or linear regression methods [see Eq. (5) in legend to Fig. 31. The curves described by the integrated zero-trans equation, Eq. ( l ) , deviated from linearity within the first few seconds of incubation at 24°C. The hazards involved in regarding these early time points as representing initial, linear substrate entry are indicated by linear regression analysis of the data (not shown). The linear regression lines fit reasonably well (correlation coefficient ru,B = 0.920.97), but do not pass through the origin-a frequent observation made by other investigators using linear regression-and the slopes of the lines underestimate the true u:; by 65-70%. The velocities estimated from such slopes yield reasonable Michaelis-Menten hyperbolae, but the estimated maximum velocities grossly underestimate V B and, consequently, Kq;.

B. Equilibrium Exchange Inward and Outward In the equilibrium exchange procedure, the substrate concentration at the two faces of the membrane is held equal and the movement of radioactively labeled substrate from one face to the other ( 1 to 2 or vice versa) is followed as a function of time and substrate concentration (Fig. 2). For the equilibrium exchange procedure, too, an integrated rate equation has been solved by Eilam and Stein (1974) for the simple carrier model. For isotope initially on side 1 of the membrane it is:

where N z , t = intracellular concentration of radioactivity at time t , which is proportional to the specific radioactivity of intracellular substrate; N z , , = intracellular concentration of radioactivity at f = co which is equal to N the concentration of radioactivity per equivalent volume of medium; S = concentration of substrate and K e e and V"" are the apparent Michaelis-Menten constants for equilibrium exchange. Equation (6) as-

241

PERMEATION IN ANIMAL CELLS 8otC

1

SI=80pM

2 10

20 TIME

20

20

40

I SEC)

FIG.3. Kinetics of zero-trans uridine transport i n uridine kinase-deficient Novikoff cells at 24°C. Samples of 448 pI of cell suspension ( I .6 x lo7 cells) were mixed in rapid succession with 61 pI of solutions of [S3H]uridine, the mixtures were centrifuged through oil layers, and the cell pellets were analyzed for radioactivity (for detailed description of methodology, see Wohlhueter et a / . , 1978a, 1979a). The final uridine concentrations were 20, 40, 80, 160, 320, 640, and 1280 p M (240 cpm/pl, irrespective of concentration) and the ambient temperature was 24°C. All values were corrected for trapping of substrate in extracellular H 2 0 space. The intracellular and extracellular water spaces in cell pellets were 13 and 1.3 pl/107 cells, respectively, i.e., 20.5 and 2.0 p1/509-pl sample, respectively. Data are from Table I by Plagemann et a / . (1978b), but have been reanalyzed by fitting Eq. (1) to the pooled data with all R parameters held equal; time ( t ) and S , were treated a s independent variables and S2,tas independent variable. The best fitting parameters were K = 261 f 12 p M and V = 25.8 f 0.5 pmole/pl cell H,O.second (= UR). The correlation coefficient ( r , 3 was 0.9918. The theoretical curves for S , = 20, 40, 80, 160, 320, and 640 pLM are illustrated in (A-F), respectively. Initial zt velocities (of:) in pmole/pl cell H20.second were calculated from the computed kinetic parameters according to the zero-trans rate equation of Eilam and Stein (1974) at S, = 0:

sumes that the volume available to substrate within the cell is negligible relative to the total volume of cell suspension, a condition that generally applies to such transport studies with animal cells (as discussed already). A similar equation, with subscripts identifying membrane face reversal, holds for equilibrium exchange in the 2 to 1 direction. Equation (6) reduces to a first-order equation by substituting V e e / ( K e " + S) = k'. Cabantchik and Ginsburg (1977) used this formulation to measure the equilibrium exchange of uridine by human erythrocytes in

TABLE I1 KINETICPARAMETERS FOR ZERO-TRANS, EQUILIBRIUM EXCHANGE, A N D INFINITE-TRANS INFLUXOF VARIW- NUCLEOSIDES A N D PURINES IN CULTURED MAMMALIAN CELLSAT 24"Ca,b Michaelis-Menten constant (zt,12) Cell line Novikoff

P388

CHO

Substrate Thymidine Uridine Deoxycytidine Cytosine arabinoside Cytidine Inosine Adenosine Hypoxanthine Adenine Uracil Thymidine Uridine Adenosine Hypoxanthine Thymidine Uridine Hypoxanthine Adenine

(ee,12)

(it,12)

(it,12)

cell H,O.second)

(PM

228 t 14(9) 250 ? 13(12) 626 t 52(4) 762 2 57(1) 2,425 f 497(4) 1 5 0 5 9(1) 103 2 8(1) 349 & 17(4) 3,300 2 524(1) 14,200 2 950(1) 125 f l O ( 1 ) 230 t 17(1) 123 ? 9(1) 445 t 31(1) 103 t S(5) 1 6 9 ? 12(1) 1,463 t 69(1) 2,109 ? SOO(1)

273 2 42(4) 241 2 22(1)

246 2 44( 1) 254 2 55(2)

7-

1 I-5

2.5

r_'

1

46 2 9 .'5 2 0.8

71 2 4 26 2 2

Ji + i; 59 2. 2

J8 t

136 ? 27(1) 551

?

85(1)

:-/

13 f E: ? 17 2 O . i 53 2 10

!! t 1.0 68 t 5

1 1 .z 1 1 164 i 5

31

--: c1:7

19 z

138 ? 14(1)

0.3

295 07 18 ?: 1.3 6.8 f 0.8

18 t 0.6

- .

0.1 1.5 t 0.3 39 f 5 3 . b i

' Data are from Plagemann et al. (1978a,b); Wohlhueter et al. (1979a,b); Marz et al. (1979): Luni P I ul. (1979) o: pr-eviousiy unpublished in the case of cytidine, inosine, and adenosine transport in Novikoff cells. Those for uridine and deoxpcytidine (PI:.iscmann ei u!., 1978a,b) were recalculated by fitting Eq. (1) to the data originally reported (see Fig. 3). Values for uridine equilibrium exchar?ge transpwi are from Fig. 4. Values for uridine infinite-trans transport are averages of data from Fig. 5 and from Fig. 8 in Plagclmann r i (11. i i978bi. * Michaelis-Menten constants apparent in zero-trans, equilibrium exchange and infinite-trans protoculs ( K l ; , ,K"'. and K\\) and the corresponding maximum velocities ( V f : , Veeand V',$). To evaluate zero-trans data, Eq. (1) was fit to combined data with Seven or eight substrate concentrations with all R-parameters held equal (see Fig. 3), so that K f : = K and U V f ; = R. Equilibrium exchangc and infinite-trans data were evaluated as described in Sections II,B and C. Values are either means of the numbcr of experiments indiraied in parentheses ? standard error (of the mean), or from single experiments where the kinetic parameters are stated ? standard error of thc csriinate (as defined in Wohlhueter et al., 1979a).


243

both directions. k' was estimated by linear regression of plots of experimental data where k' = u " / S at t = 0 and ue" expresses the unidirectional flux of isotope. For equilibrium exchange experiments in the 1 to 2 direction uee was also estimated graphically from the initial rate of entry of radioactivity into the cells; both methods gave similar results. K"" and V e ewere computed by conventional plots of wee versus S, as in the zero-trans procedure. Computed values are summarized in Table I and are discussed further in Section I1,E. Wohlhueter et d.(1978a, 1979a) rearranged Eq. (6) in exponential form:

in which form V"' and K e e can be estimated directly by nonlinear regression to pooled time courses of attainment of radioactivity equilibrium at several substrate concentrations, whereby t and S are treated as independent variables and N2,t as dependent variable ( N 2 , tis based on the experimentally determined 3 H 2 0 space of the cells or on the apparent intracellular radioactivity space as t+m, N2,m;Wohlhueter, Erbe and Plagemann, previously unpublished procedure). This approach is exemplified in Fig. 4A-D for uridine equilibrium exchange in the I to 2 direction in uridine kinase-deficient Novikoff cells. The kinetic parameters are summarized in Table I 1 and discussed further in Section II,E. I n a previous study on thymidine equilibrium exchange in thymidine kinase-deficient Novikoff cells (Wohlhueter et al., 1979a) V"" and K'" (see Table 11) were estimated in a different, though statistically less defensible, manner: k' = V e e / ( K e e+ S ) was substituted in Eq. (7) and k' was computed by nonlinear regression procedures for the individual time courses of attainment of radioactivity equilibrium at each substrate concentration. V'" and Kee were evaluated by replots of k' versus S or of the product k ' S = uee versus S (Fig. 4E and F). These alternative methods of fitting data do not necessarily give identical results for K"' and V"', for in essence each weights the data differently. C. Infinite-trans Procedure

In the infinite-trans (it) procedure, one measures the movement of radiolabeled substrate at a given concentration from one face of the membrane (the cis side) to the other face (the trans side) where unlabeled substrate is present at a concentration + K of the transport system (i'e., S2+m) (Fig. 2).

244


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

-

w

r 0 05

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

O

.... 1

................... 2

3

{"

loo0

5oo

..,

"

0

S = 80 pM

$lZft:pM

,

ry,; = 0 9 9 3 6

S =1280vM OO TIME (SEC)

100

200 URlDlNE ( p M 1

FIG.4. Kinetics of uridine equilibrium exchange in uridine kinase-deficient Novikoff rat hepatoma cells. Samples of a suspension of about 3.6 x 10' cells/ml of basal medium were preincubated with 20, 40, 80, 160, 320, 640, 1280, or 2560 F M nonradioactive uridine at 37°C for 30 minutes and then brought to 24°C. Samples of 448 pI of each suspension were mixed in rapid succession with 61 ~1 of a solution of an equivalent concentration of [SH]uridine, the mixtures were centrifuged through an oil layer, and the pellets were analyzed for radioactivity. The radioactivity concentration of the final mixtures was 380 c p d p l , irrespective of concentration. All values were corrected for uridine trapped in extracellular space of cell pellets (3.4 pl). The intracellular water space was 22.5 pCLYcell pellet. Equation (7) was fitted by nonlinear regression to the pooled data; time ( t ) and S were treated as independent variables and N2,f as dependent variable. The best fitting parameters were K" = 24 I + 22 p M and V" = 34.5 ? 0.8 pmoleipl cell H,O-second. The correlation coefficient (ry.r) was 0.9936. The theoretical curves for S = 40, 80, 640, and 1280 pM are illustrated in (A-D), respectively. In addition, a single-variable version of Eq. (7), N 2 , *= N 2 . - [I-exp(-k't)] (Wohlhueter et al., 1979a) was fitted to each individual time course of attainment of radioactivity equilibrium by nonlinear regression procedures. V"' and Kee were then computed by replots of the computed k' versus S ( E ) or of the product k'S = uee versus S (Michaelis-Menten plot, F). The best fitting kinetic parameters are listed for each plot. Vee is in pmole/pI cell H,O.second. (Previously unpublished data of Plagemann and Wohlhueter.)

The rapid kinetic technique developed by Wohlhueter et ul. (1976, 1978a) is not suited to an infinite-trans protocol, nor is rapid sampling as essential as with other experimental protocols. In the infinite-trans procedure radioactivity accumulates on the trans side against a concentration gradient (generally referred to as countertransport), because the transferred radiolabeled substrate becomes extensively diluted by the high concentration of unlabeled substrate on the trans face. Due to the reduced specific radioactivity of the labeled substrate on the trans side backflow of radioactivity is initially minimal and the linearity of radioactivity move-

245


ment from the cis to the trans face is prolonged. Indeed, this fact is the unique feature of the infinite-trans procedure and countertransport is generally considered one of the unequivocal criteria for establishing carrier-mediated transport of a specific substrate. Figure 5 illustrates these principles. Infinite-trans accumulation of uridine in uridine kinase-deficient Novikoff hepatoma cells was estimated by conventional sampling methods at 15-second intervals, and is compared to zero-trans influx measured with rapid sampling techniques. It should be emphasized in connection with the data in Fig. 5 that an underestimation of v;: which is inherent in linear graphical methods for estimating v;: from substrate accumulation curves would make 0; appear to be significantly lower than vl;t,, when, in fact, they are the same. Such underestimation of vf; could lead to the conclusion of carrier asymmetry. Computed V & and K & values for nucleoside transport in cultured animal cells are summarized in Table I1 and are discussed further in Section II,E. D. Infinite-cis Procedure

Two different infinite-cis (ic) protocols have been used to study nucleoside transport in animal cells. One protocol was originally developed for measuring hexose transport by Sen and Widdas (1962). It measures the net movement of substrate from one face of the membrane, where it is present at a concentration % K of the transport system, to the trans face, where the substrate concentration is varied (Fig. 2). Sen and Widdas (1962) extrapolate the initial slope of the efflux curve to its intersection with the asymptote corresponding to equilibrium, thereby defining a time (re), which relates linearly to S, and the kinetic constants apparent in infinite-cis experiments. This procedure was followed by Lieu er al. (1971) to measure the efflux of various nucleosides from human erythrocytes. Their results are summarized in Table I and discussed further in Section II,E. Cabantchik and Ginsburg (1977) applied the infinite-cis protocol to the measurement of net uridine transport in human erythrocytes in both the 1 to 2 and 2 to 1 directions. uie was estimated by measuring the initial rate of transfer of radioactivity from one side of the membrane (cis face), where radioactively labeled substrate was present at a very high concentration, to the trans side, where the concentration of labeled substrate of the same specific radioactivity was varied (Fig. 2). These investigators converted the infinite-cis net flux equation of Eilam and Stein (1974) to a linear form: SZ -- 1 - - 1 vk(net) V:; KiC 12 VZt 12

+-

246


TIME I S E C l

FIG. 5 . Infinite-trans analysis of uridine transport i n uridine kinase-deficient Novikoff rat hepatoma cells. Samples of a suspension of 2 x lo' cellsiml of basal medium were incubated with and without 10 mM unlabeled uridine at 37°C for 20 minutes and then thermally equilibrated at 25°C for at least 5 minutes. Cells from 5-ml samples of uridinepreloaded suspensions were collected by centrifugation and washed rapidly once in 10 ml cold (0°C) basal medium. The cell pellets were rapidly warmed to 25°C and then suspended in 5 ml of basal medium (at 25°C) and containing 20, 40, 80, 160, 240, 320, 480, 640, 1280, or 2560 p M [3H]uridine (430 cpm/pl, irrespective of concentration). At 15, 30, 45, and 60 seconds of incubation at 25°C duplicate 509-pI samples of each suspension were centrifuged through oil and the pellets were analyzed for radioactivity. All values are averages of duplicate samples (infinite-trans procedure). Cells from the control suspension (not preloaded with uridine) were washed and suspended to the same density in basal medium and then analyzed for uridine transport by the rapid kinetic technique as described in the legend to Fig. 3, but with the substrate concentrations indicated above (zero-trans procedure). The intracellular H,O space was 22.5 pV509-pI sample. All values were corrected for substrate trapping in extracellular space of the cell pellet (3.6 p1/509-p1 sample). The time courses of zero-trans and infinite-trans accumulation of radioactivity at S , = 20 p M are compared in (A). Equation ( I ) was fitted to the pooled zero-trans data and uf: was calculated from the computed parameters as in Fig. 3. The best fitting parameters of transport for all seven concentrations were K = 290 9 p M and V = 26 ? 3 pmole/pI cell H,O.second. Initial infinite-trans velocities ( u & ) were estimated from the initial linear portion of the uptake curves (B) and were subjected to Michaelis-Menten analysis. The best fitting parameters were K\\ = 332 87 p M and V\: = 24 ? 3 pmole/pl cell H,O.second. The broken lines indicate the intracellular radioactivity concentration equivalent to the extracellular concentration.

*

*

247


In a plot of l/uf; versus S, the X-intercept yields directly - K $ and the Y-intercept is l/VSk. Corresponding relationships with 1 and 2 interchanged apply to infinite-cis in the 2 to 1 direction. Kic Kic V B , and Vgl values for uridine transport in human erythro12, cytes, computed by linear regression analysis from such plots, are summarized in Table I and are discussed further in Section II,E. In the second protocol, also referred to as "accelerated exchange diffusion" (Cass and Paterson, 1972, 1973), the unidirectional flux of substrate is measured. One measures the movement of radioactivity from the cis side of the membrane, where labeled substrate is present at a concentration * K of the transport system, to the trans side, where the concentration of unlabeled substrate is varied. For unidirectional infinitecis transport in the 1 to 2 direction the unidirectional flux equation of Eilam and Stein (1974) reduces to (Wohlhueter et al., 1978a): vlc

-

K + S, ( K / V ? ' , )+ S , / P )

(9)

The subscripts are altered accordingly for the opposite direction. These equations are not Michaelian in form, so that KY2 and K',", are not defined, and moreover, as Vq', and VZ,:approach V e e(that is, as for a carrier symmetrical with respect to the mobility of loaded and unloaded carrier) the equations degenerate into identities. Only if the movement of loaded and unloaded carrier differs, i.e., V e e # Vzt, does the presence of unlabeled substrate at the trans face cause an accelerated transfer of radioactivity from the cis face. In fact, one of the useful features of the infinitecis procedure is that it allows the unequivocal demonstration of differences between VZtand V"". For example, such differences were observed by Cass and Paterson (1972) from infinite-cis transport experiments in the 2 to 1 direction for the transport of uridine and thymidine in human erythrocytes. uic was estimated from initial rates (20 to 30 seconds) of release of radioactivity from cells preloaded with "infinitely" high concentrations of labeled substrate into the medium containing varying concentrations of the same (or an alternate) substrate in unlabeled form. These investigators observed a linear relationship between I / v & and I! S1, and calculated apparent K , and V,,, from these plots. But, as previously mentioned, these parameters probably have little meaning with respect to the simple carrier model (Fig. 2). However, if Vz' and V" are sufficiently different Eq. (9) takes on some useful attributes. A plot of u';i versus S , (or v!$ versus S l ) , for example, is hyperbolic with a horizontal asymptote Vee, with Y-intercept = V;; and X-intercept = - K . Thus, theoretically, the infinite-cis protocol can directly yield values for

248


K as well as for V;", V;:, and V e e .In practice, however, this approach may not be satisfactory (Wohlhueter et al., 1978a). First, because the concentration of labeled substrate on the cis side is infinitely high, the relative rates of transfer of radioactivity are slow, and the radioactivity on the cis face is high in comparison to that on the trans face. The unfavorable "signal-to-noise ratio" results in imprecise estimations of vie and, consequently, in unsatisfactory fits of Eq. (9). Second, the hyperbolic extrapolation to the X-axis, and hence the value of K , is quite imprecise. Furthermore, this problem of extrapolation is accentuated with decrease in difference between V z tand V". For example, whatever difference there may be between V z t and Veefor nucleoside transport in cultured animal cells seems to be too small (see Section II,E) to allow this kind of analysis (Wohlhueter rt af., 1978a). Our attempts to fit Eq. (9) to some data of Cass and Paterson (1972) for the accelerated exchange diffusion of uridine by cytidine in the 2 to 1 direction in human erythrocytes did not converge satisfactorily, even though in this instance VZ:, and V" clearly differed (Fig. 6). Only when V;: was fixed at a graphically estimated value ( 3 1 pmole/pI cell H,O*second) was a successful fit of Eq. (9) to the experimental data obtained, with K = 336 & 58 p M and V e e = 156 k 12 pmole/pl cell H,O.second.

I.

l

I

I

ry,g = O . S E J ~ I 1 I

1

0

20

40

60

S, I CYTIDINE, m M )

FIG.6. Infinite-cis transport (accelerated exchange diffusion) of uridine in human erythrocytes. Data have been calculated from Fig. 6 of Cass and Paterson (1973). Initial rates of appearance of radioactivity in the medium were determined at 25°C when cells preloaded with 6 m M [2-I4C]uridine were incubated in media containing different concentrations of nonradioactive cytidine. Attempted regression of Eq. (9) for the 2 to 1 direction whereby S , was treated as independent variable and IJ& as dependent variable did not satisfactorily converge. Only when Vg: was fixed at a value estimated graphically (31 pmoleipl cell H,O.second) was a successful fit of the equation obtained with K = 326 ? 58 p M and V e e = 156 % 12 pmole/pI cell H,O.second.


249

E. Interpretation of Experimental Data The kinetic parameters reported by Cabantchik and Ginsburg (1977) (Table I) are consistent with the operation of a simple carrier mechanism in the transport of uridine in human erythrocytes. First, as required by the simple carrier model (Fig. 2), the ratios of apparent maximum velocities/Michaelis-Menten constants for the various experimental protocols were all equal (to about 0.1) within the experimental error. Second, the KiCvalues calculated from the fundamental constant K and maximum velocities of equilibrium exchange and zero-trans influx and efflux agreed well with Kic values determined experimentally (Table I, footnote b). On the basis of these kinetic parameters, Cabantchik and Ginsburg (1977) suggested that the uridine carrier of human red blood cells exhibits two types of asymmetry. First, in either direction the loaded carrier moves more rapidly than the unloaded carrier, implying that the movement of unloaded carrier is the rate-limiting step in uridine transport. Second, on the basis of the zero-trans data, they suggested that the unloaded carrier moves about four times more rapidly in the 1 to 2 direction than in the 2 to 1 direction, whereas the loaded carrier moves equally rapidly in either direction. Thus, influx would be faster than efflux. Not all experimental data seem to support the conclusion that the unloaded carrier moves more rapidly in the I to 2 direction than in the 2 to 1 direction. For example, the VZtvalues calculated from the infinitecis data of Cabantchik and Ginsburg (1977) according to Eq. (8) (see Table I, footnote c ) , are not in agreement with this conclusion. Furthermore, the V;: and VZt, for uridine transport in human erythrocytes estimated from the zero-trans and infinite-cis experiments of Paterson and co-workers differ in the opposite direction (Table I). However, zero-trans velocities estimated directly in the zero-trans procedure may be more accurate than those calculated from infinite-cis kinetic parameters. Furthermore, because of differences in experimental conditions and methodology for estimating initial velocities a comparison of data from different laboratories may not be entirely appropriate. Such differences in methodology and conditions might account for the discrepancies between the kinetic parameters (Table I) reported by Oliver and Paterson (1971), Pickard and Paterson (1972), Lieu et al. (1971), and Cabantchik and Ginsburg (1977). Nevertheless, most values are consistent with the conclusion that the loaded carrier moves faster than the unloaded carrier, as was first indicated by the data of Oliver and Paterson (1971) and Pickard and Paterson (1972). Overall, the suggested asymmetry of the nucleoside

250


carrier of human erythrocytes is similar to that for the hexose carrier of these cells (Miller, 1971; Geck, 1971; Bloch, 1974; LeFevre, 1976). The nucleoside carrier of cultured mammalian cells, on the other hand, exhibits little, if any, asymmetry. The kinetic parameters for zero-trans, equilibrium exchange and infinite-trans transport of thymidine, uridine, and adenosine in Novikoff hepatoma and P388 leukemia cells are the same within the experimental errors (Table 11). Furthermore, the fully symmetrical version of the integrated rate equation (see Eqs. 1-3) well describes the transport of thymidine and uridine, as is evident in Wohlhueter et al. (1979a) and in Fig. 3 , respectively. Similarly, Heichal et al. ( 1 979) calculated from zero-trans data of cytosine arabinoside transport in transformed hamster cells (Nil 8 HSV) that the carrier half saturates at similar substrate concentrations at the outer (250 pA4) and inner (150 pM) faces of the membrane. The kinetic parameters for the transport of various nucleosides-with the exception of cytidine-are similar for the three different cell lines represented in Table 11. The exceptional status of cytidine has not been explored further. This generalization holds also for the transport of nucleic acid bases, but with more qualifications. For example, hypoxanthine transport in CHO cells exhibited a much higher K than those observed with other cell lines (Table 11; see also Marz et al., 1979). The basis for this difference is unknown. Furthermore, except in CHO cells, the K values for adenine and uracil transport are much higher than that for hypoxanthine (or those for nucleosides). The results for adenine tabulated here do not agree with those reported by Witney and Taylor (1978), who observed two Kqe values (6- 10 p M and 80 pA4) for the transport of adenine in an adenine phosphoribosyltransferase-deficient line of CHO cells. Marz et al. (1979), on the other hand, detected only a low-affinity adenine transport system in these same cells ( K = 2-3 mM, similar to that of wild type CHO cells), as well as in an adenine phosphoribosyltransferasedeficient mouse L cell line ( K = 3058 -+ 569 pM; V = 53 4 6 pmole/pl cell H,O-second; P. Plagemann and R. Wohlheuter, unpublished data). No evidence for additional systems with higher affinity was found. The reason for this discrepancy is not known, but it may be related to the fact that in the study of Witney and Taylor (1978) transport velocities were estimated from single 15-second time points at 30°C, which may have underestimated true, initial transport velocities. Other studies pioneering the use of enzyme-deficient mutant cell lines for nucleoside and purine zero-trans transport studies are subject to the same uncertainty. Kessel and Shurin (1968) found the transport of deoxycytidine and cytosine arabinoside in deoxycytidine kinase-deficient


251

L1210 murine leukemia cells to be saturable and, based on I-minute uptake time points, obtained a K ; ; = 7.5 mMat 0°C for both nucleosides. At 37"C, however, transport seemed to become saturable only above 20 mM. Nevertheless, these studies showed that deoxycytidine transport was nonconcentrative, was inhibited by various other nucleosides and by dipyridamole, but not by free bases or sugars, and that transport was not affected by inhibitors of energy production. Saturable, temperature-dependent efflux of deoxycytidine and its inhibition by dipyridamole was also demonstrated (Kessel and Hall, 1970). Similar results have recently been reported for the transport of the nonmetabolizable nucleoside, 5'deoxyadenosine in L1210 cells (Kessel, 1978). A K ; ; = 115 p M was estimated on the basis of single 18-second time points at 20"C, which were corrected for a nonsaturable uptake component. Plagemann and Erbe (1973) and Zylka and Plagemann (1975) studied the transport of uridine in ATP-depleted Novikoff cells, of hypoxanthine and guanine in hypoxanthine/guanine phosphoribosyltransferase-deficient Novikoff cells and of uracil in the same cell line, but the estimated Kqi (40, 9, and 9 F M , respectively) and V ; ; values for transport, based on 0.5- or 1-minute uptake values, were greatly underestimated due to the lack of methodology for measuring accurate initial transport velocities. Schuster and Hare (1971) examined thymidine transport in thymidine kinase-deficient BHK2, cells. Five-minute uptake values were directly proportional to the extracellular thymidine concentration, but these values probably represented equilibrium levels, and the conclusion that thymidine entry into these cells was solely by nonmediated permeation is, therefore, unwarranted. The 10-minute uptake values for thymidine at 37°C in thymidine kinase-deficient 3T3 cells measured in a study by Cunningham and Remo (1973) must have also represented equilibrium levels. Ungemach and Hegner (1978) observed two saturable processes for the accumulation of thymidine at 37°C in cultured rat hepatocytes, in which little thymidine phosphorylation occurred. Based on 30-second accumulations, values of two K ; ; of 5.3 and 480 p M were estimated. In several recent studies the transport of cytosine arabinoside has been investigated in cell lines that either fail to phosphorylate this nucleoside or do so only slowly. For example, Mulder and Harrap (1975) observed two saturable components for cytosine arabinoside and deoxycytidine accumulation in Yoshida sarcoma cells with K ; ; of 400-600 p M and 2 mM, as well as a passive diffusion component. Transport rates were estimated from uptake slopes between 15 and 120 seconds of incubation at 19°C with labeled substrate. Uptake of these nucleosides was inhibited by each other and other nucleosides in an apparent simple competitive manner. Heichal ef a / . (1978) measured cytosine arabinoside influx and

252


outward equilibrium exchange at 20°C in chemically transformed golden hamster fibroblasts (MTC) which phosphorylate the nucleoside only very slowly as compared to its influx. Influx was measured graphically from apparently linear initial 10-20 second uptake curves and yielded Ktk = 350 pM and V ; ; = 13 pmole/pl cell H,O.second. Equilibrium exchange efflux, on the other hand, involved a saturable component ( K e e = 503 p M ; V" = 21 pmole/pl cell H,O.second) and an unsaturable component ( k = O.OOS/second).Cytosine arabinoside influx was inhibited by various other nucleosides and itself inhibited the uptake of other nucleosides. Using similar methodology, Koren et a / . (1979) analyzed the kinetic parameters for cytosine arabinoside influx in B77 rat cells (Kik = 500 pM, V;; = 3.9 pmole/pI cell H,O.second). Fitting the linear form of the integrated zero-trans rate equation [Eq. ( I ) ] to data on cytosine arabinoside influx in murine sarcoma virus-transformed golden hamster cells (Nil 8 HSV) yielded KTk = 250 p M and V;; = 3 . 5 pmole/pI cell H,O.second (Heichal ez a / . , 1979). These kinetic parameters are similar to those determined for cytosine arabinoside transport in Novikoff rat hepatoma cells (see Table 11), which are based on nonlinear regression analysis of uptake curves to substrate equilibrium, although the estimated V t ; values are somewhat lower. Cytosine arabinoside is also not metabolized in rat uterus. Cytosine arabinoside uptake by this tissue was saturable, nonconcentrative and competitively inhibited by other nucleosides, and countertransport by uridine was demonstrated (Oliver, 197I). Only sheep erythrocytes homozygous for a specific gene locus ( N u ' ) possess a functional nucleoside carrier (Jarvis and Young, 1978a; see Section IV,E), but even these cells transport various nucleosides at only about 0.3% the rate observed with human erythrocytes (Young, 1978). The Michaelis constants for zero-trans influx of inosine, adenosine, and uridine ( K ? ; = 200, 130, and 470 p M , respectively; Young, 1978), on the other hand, are similar to those reported for human erythrocytes (Table I) and cultured animal cells (Table 11). Benke et a / . (1973) failed to detect significant transport of hypoxanthine in cultures of hypoxanthine/guanine phosphoribosyltransferase-deficient human fibroblasts from Lesch-Nyhan patients, but the facilitated transport of hypoxanthine has been unequivocally demonstrated in transferase-deficient Novikoff hepatoma cells, Chinese hamster lung cells, and human Lesch-Nyhan fibroblasts by Zylka and Plagemann (1975), Alford and Barnes (1976), and Murphy ez a / . (1977), respectively, by observing the countertransport of hypoxanthine in these cells. Based on 15-second uptake time points, Alford and Barnes (1976) observed two saturable transport components with K;: = 17-26 and 530-710 p M and found that transport was inhibited by p-hydroxymercuribenzoate. Epstein and Lit-


253

tlefield ( 1977) reported that hypoxanthine accumulation by hypoxanthine/ guanine phosphoribosyltransferase-deficient diploid human lymphoblast lines was nonsaturable at 20 or 30°C. However, initial transport velocities were estimated from 1-minute uptake time points and the highest hypoxanthine concentration tested was 560 p M . Thus, the conclusion of these investigators that the transferase is involved in the saturable transport of hypoxanthine is not warranted. It is only the accumulation of the phosphorylated products that is transferase dependent and responsible for long-term uptake of hypoxanthine (see Section 111,A). Many of the above studies with enzyme-deficient cells clearly indicated the existence of facilitated diffusion systems for nucleosides and free bases in animal cells other than erythrocytes, but the reported kinetic parameters, with the exception of those for cytosine arabinoside transport, varied greatly and are inconsistent with values that have been obtained more recently by more rigorous analyses (Table 11). The low K:: systems detected in some of the above studies may reflect either residual phosphorylating activity of the cells or, more likely, an inadequacy in estimating true initial zero-trans transport velocities. The observation of unusually high or nonsaturable permeation components might also result from inaccurate initial velocity measurements, but more often seems to be due to the analysis of relatively long uptake time points, which measured merely the intracellular equilibrium concentrations of substrate. Necessarily for facilitated diffusion systems such equilibrium concentrations are a direct function of the extracellular concentration (see Section 111,E).

111.

UPTAKE OF NUCLEOSIDES AND PURINE BASES

A. General Considerations

The kinetic parameters for nucleoside and purine uptake (as defined earlier) in cultured Novikoff cells are much lower than those for the corresponding transport systems (compare Tables I1 and 111) (uptake data are for 37°C transport data for 24-25°C). In all reported uptake studies (Table III), uptake velocities were estimated from 0.5- to 10-minute substrate uptake time points and, with a few exceptions, similar substrate K , values were obtained for different types of cells in numerous laboratories. The exceptions were K , values for thymidine uptake of 40-50 p M for rabbit polymorphonuclear leukocytes (Taube and Berlin, 1972) and for CHO cells (Sander and Pardee, 1972). In both studies cells were propagated on glass coverslips, but uptake velocities estimated from 45-second

254


TABLE 111 KINETICPARAMETERS FOR THE UPTAKEOF NUCLEOSIDES A N D FREEBASESBY NOVIKOFF RAT HEPATOMA CELLSA N D OTHERTYPESOF ANIMAL CELLS Other types of cells

Novikoff cells"

-

Vmax

Substrate Thymidine

K, (wM) 0.4-0.5

Deoxycytidine 0.8- I .6 Deoxyadenosine 40-80 Uridine 12-16

Cytidine Adenosine

Guanosine Hypoxanthine Adenine

(pmolelpl cell H,O.second) 0.15-0.3

0.04-0.08 2-4 1-2.6

K m

(WW

Reference"

0.2- I .5

3,8,9,10,20,22,25,27, 30.31-36.47 43,56 17 2,10,30,45

4@50 400 0.7-8 4-30

1-3 2-4

8- 9 1-20

8-15 4-8

2-4 1.5-2.2

30-50

3-4.5

40 102 I- 10 3-36 400 0.2- 100

15-25 6-10

3,4,13,14,18-21.24, 26,27,29,3 1-34,37, 39,42-45,50- 5 4 3 7 44 7,16,22,32-34,38,44, 45,46,49,54,56,58 40 41 33,34,44 1,5, I I , 1 2 , 6 2 2 3 9 28 6,1 1,22,23,48,55,59

~~

a From Plagemann and Roth (1969), Plagemann (1970, 1971a,b), Plagemann and Erbe (1972, 1974a), Plagemann and Richey (1974), and Zylka and Plagemann (1975). In all instances uptake rates were estimated from 1-5 minute time points of uptake of various concentrations of radioactively labeled substrate at 37°C by cell suspensions of 1-3 x lo6 cellslml of a serum-free medium. Previously reported V,,, values expressed on a lo6 cell basis were converted for comparative purpose to pmolelpl cell H,O.second on the basis of an average cell H 2 0 space of 1.3 pVI06 Novikoff cells (Wohlhueter et al., 1978a). References: 1. Alford and Barnes (1976); 2. Barlow (1976); 3. Barlow and Ord (1975); 4. Benedetto and Casson (1974); 5. Benke et a / . (1973); 6. Berlin (1970); 7. Berlin (1973); 8. Bittlingmaier et al. (1977a); 9. Cass and Paterson (1977); 10. Cunningham and Remo (1973); I I . Dybing (1974a); 12. Dybing (1974b); 13. Eilam and Bibi (1977); 14. Eilam and Cabantchik (1977); 15. Epstein and Littlefield (1977); 16. Fleit et al. (1975); 17. Freed and Mezger-Freed (1973); 18. Goldenberg and Stein (1978); 19. Hakala et al. (1975); 20. Hand (1976); 21. Hare (1972a); 22. Harris and Whitmore (1974); 23. Hawkins and Berlin (1969); 24. Heichal et al. (1979); 25. Hopwood et a / . (1975); 26. Jiminez de Asua et a/. (1974); 27. Kunimoto et al. (1974); 28. Lassen (1967); 29. Lemkin and Hare (1973); 30. Leung and Visser (1976); 31. Loike and Horwitz (1976); 32. Lynch et al. (1977); 33. Lynch et al. (1978); 34. Mizel and Wilson (1972); 35. Myers and Feinendegen (1975); 36. Paterson et a/. (1975); 37. Paterson et a / . (1977a); 38. Paterson et al. (l977b); 39. Peters and Hausen (1971); 40. Pofit and Strauss (1977); 41. Roos and Plfeger (1972); 42. Rozengurt and Stein (1977); 43. Sander and Pardee (1972); 44. Scholtissek (1968); 45. Scholtissek (1972); 46. Schrader et al. (1972); 47. Schuster and Hare (1971); 48. Sixma et al. (1973); 49. Sixmaet al. (1976); 50. Skehel et a / . (1967); 51. Stambrook et al. (1973); 52. Steck et a / . (1969); 53. Stein and Rozengurt (1975); 54. Strauss et al. (1976); 55. Suresch et al. (1979); 56. Taube and Berlin (1972); 57. Turnheim et a / . (1978); 58. Weber and Rubin (1971); 59. Yang and Visser (1977).


255

and 15-minute uptake time points, respectively. A K , of 400 p M has been reported for thymidine uptake by haploid frog cells (Freed and Mezger-Freed, 1973)and thymidine uptake by mouse spleen lymphocytes seemed to be nonsaturable (Strauss et al., 1976). Other extraordinarily high K,s are those for adenosine uptake of 40 and 120 p M in mouse macrophages (Pofit and Strauss, 1977) and guinea pig erythrocytes (Roos and Pfleger, 1972), respectively, and of 400 p M for hypoxanthine uptake by human erythrocytes (Lassen, 1967). Reported K , values for adenine uptake are extremely variable ranging from 0.2 p M for human blood platelets (Sixma et al., 1973) to 100 p M for CHO cells (Harris and Whitmore, 1974). The maximum uptake velocities reported in various studies are also highly variable, but are difficult to compare, since different units were used to express the uptake capacity of the cells. The meaning of these kinetic parameters for uptake has recently come into question, since studies on the uptake of thymidine (Wohlhueter et al., 1976, 1979a; Marz e f al., 1977a), uridine (Plagemann et al., 1978b), adenosine (Lum et al., 1979), and hypoxanthine (Marz et al., 1979) by cultured cells have clearly shown that such long-term uptake velocities pertain to the period of steady-state intracellular substrate concentrations and measure the intracellular formation of nucleotides derived from exogenous substrate. A similar conclusion is indicated by the studies on uridine uptake in various other cultured cell lines (Rozengurt et al., 1977b, 1978; Koren et al., 1978; Heichal et al., 1979). Nucleoside or nucleobase transport systems with low K,s similar to those reported for the uptake of these substrates by phosphorylating cells have not been detected in Novikoff or P388 cells. The initial velocities of transport (us:) of adenosine (Lum et al., 1979), hypoxanthine, and adenine (Marz et al., 1979), and thymidine and uridine (R. Wohlheuter and P. Plagemann, unpublished data) by ATP-depleted cells or cells deficient in the appropriate phosphorylating enzymes increase in direct proportion to intracellular substrate concentration between 0.1 and 10 PM.

6. Relationship between Transport and Metabolism Operating in Tandem

Typical results for the uptake of hypoxanthine by wild-type Novikoff rat hepatoma cells at 25°C are illustrated in Fig. 7. Free hypoxanthine accumulates intracellularly to steady-state levels within seconds; within 2.5 seconds at an extracellular concentration of 0.5 p M , and within 30 seconds with 160 p M exogenous hypoxanthine. Thereafter, the uptake of radioactivity into total cell material reflects the accumulation of nucleo-

256


SEC

SEC

MIN

MIN

TIME

FIG.7. Time courses of intracellular accumulation of labeled free hypoxanthine, inosine, and nucleotides in untreated wild-type Novikoff cells incubated at 24°C with 0.5 p M (A) or 160 p M (B) [3H]hypoxanthine. Short-term time courses of [3H]hypoxanthine uptake were measured by the rapid kinetic procedure as described in the legend to Fig. 3. For long-term uptake measurements, cell suspension and substrate solution were mixed in the same proportion as i n the rapid kinetic method, incubated at 24°C and 509-pI samples were centrifuged through oil at the indicated times. The final concentrations of [3H]hypoxanthine were 0.5 p M or 160 p M (1400 cpmipl, irrespective of concentration). The cell HZO and extracellular H,O spaces were 22.8 and 2.9 plkell pellet, respectively. Initial velocities of uptake ( v , , ) at 0.5 p M hypoxanthine ( A ) were estimated graphically from the linear portions of the short-term and long-term uptake curves. In parallel experiments duplicate samples of cells were collected by centrifugation through oil directly into an acid layer, the acid extracts were further processed and chromatographed as described by Marz et a / . (1979). Time courses of intracellular accumulation of labeled hypoxanthine ( 0 - O ) , inosine (AA), and nucleotides (A-A) were constructed on the basis of these chromatographic analyses and time courses of uptake i n t o total cell material (0-0). All values were corrected for trapping of extracellular [3H]hypoxanthine in cell pellets and acid extracts. The broken lines indicate equality between extracellular and intracellular radioactivity concentrations. (Data are from Marz et a / . , 1979.)

tides. Since these nucleotides consist of over 90% of triphosphates, phosphoribosylation of hypoxanthine seems to be the rate-determining step in the long-term formation of nucleotides from exogenous hypoxanthine. Furthermore, computer simulations have shown that. the intracellular steady-state levels of free hypoxanthine and the time courses of intra-

257


cellular formation of nucleotides at both 0.5 and 160 p M exogenous hypoxanthine are consistent with the kinetic parameters for the hypoxanthine transport system and the hypoxanthine/guanine phosphoribosyltransferase operating in tandem [Fig. 8; see legend for Eqs. (10) and ( I I ) ] . Differences in uptake at low and high exogenous hypoxanthine concentrations are inherent in the fact that the Michaelis constant and maximum velocity for the transport system are at least 50 times higher than the corresponding values for the transferase, whereas the ratio V / K , for transport is somewhat lower than that for phosphorylation (Marz et al., 1979; see also Table IV). At extracellular concentrations of hy-

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FIG. 8. Simulated time courses of hypoxanthine transport and phosphoribosylation. The intracellular accumulation of hypoxanthine (S,, solid line) and phosphorylated products (P,dotted line) at a given exogenous concentration of hypoxanthine (S,, broken line) were computed by numerical integration of the following rate equations: dS,ldt

=

vI2 - vZl- dP/dt

dPldt = V'"" S,/(Ke,"Z + S,)

(10)

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where v I 2is calculated as in Eq. (9,and u2, by an analogous efflux equation. In panel A, S , = 0.5 p M at time zero; in panel B, S , = 160 p M at time zero. For both cases the kinetic parameters for transport and phosphoribosyltransferase with respect to hypoxanthine used are those determined experimentally for Novikoff cells (see Marz et al., 1979; see also Table 11): K E Z = 2 pM, VenZ= 1 pmole/second.pI cell H,O, and for transport K ( K ? = 350 p M and V (V? = 50 pmole/second.pl cell H,O. Cellular space was taken to comprise 1% of total water space.

258


TABLE IV KINETICPARAMETERS FOR TRANSPORT A N D METABOLIC REACTIONS I N ADENOSINE UPTAKEI N P388 CELLS INVOLVED Process Adenosine (inosine) transport Adenosine kinase Adenosine deaminase Nucleoside phosphorylase HGPRT Hypoxanthine transport

Km (pM)

Vmax" (pmole/pl cell H,O.second)

130' 0.4- 6 .0' 3 5- 400'

70-100 0.5- 1 160-220 40-60 1-3 80-150

13-30d

2- 6" 45oe

All V,,, values are for P388 cells at 37°C and from Lum et a / . (1979), except those for hypoxanthineiguanine phosphoribosyltransferase (HGPRT) and hypoxanthine transport which are from Marz et a / . (1979). * From Lum et (11. (1979). Range of values observed in many different mammalian cell systems and summarized by Fox and Kelley (1978). Range of values for different mammalian species; from Agarwal et a / . (1975) and Milman et a / . (1976). From Marz et a / . (1979).

poxanthine much higher than the K , of the transferase for hypoxanthine, the rate of transport of hypoxanthine greatly exceeds the maximum velocity of intracellular phosphoribosylation, so that the concentration of free hypoxanthine in the cell approaches that in the extracellular space. At extracellular concentrations of hypoxanthine within the first-order range for both transport and phosphoribosylation ( 1 p M and below), transport is slower than phosphoribosylation. Thus at such low extracellular concentrations the steady-state intracellular concentration of free hypoxanthine attains not more than a few percent of that in the extracellular fluid. It is clear that in this concentration range, the overall uptake rate for hypoxanthine is largely determined by its rate of transport into the cell; for example, increases and decreases in transport rate alone would cause similar, although not strictly proportional, changes in the overall uptake rate, whereas an increase in the phosphoribosylation capacity of the cells would have little effect on overall uptake rate. In contrast, at hypoxanthine concentrations in excess of the K , of the transferase for hypoxanthine, the overall long-term uptake rate is strictly a function of the phosphoribosylation capacity of the cells. Similar relationships between transport and phosphorylation pertain to the uptake of various nucleosides in a number of cultured cell systems. All seem to have in common that the transport carrier: substrate affinity constants and the maximum velocities for the transport systems greatly exceed the


259

corresponding values for the in s i f u phosphorylation reactions (Wohlhueter et al., 1976; Marz et al., 1977a; Plagemann et al., 1978b; Lum e f al., 1979; Koren et al., 1978; Heichal et al., 1979). Thus whether transport or phorphorylation capacity is the main determinant of overall substrate uptake may often be a function of the extracellular substrate concentration. These results support the view that the kinetic parameters for the longterm uptake of nucleosides and purines (Table 111) reflect those of the in situ phosphorylation of the substrate. It is not surprising, therefore, that the substrate specificities of the uptake processes have generally been found to be the same as those of the corresponding phosphorylation reactions (see Section IV,A). In the case of hypoxanthine, a good agreement between the kinetic parameters of hypoxanthine uptake by whole cells and those of the hypoxanthine/guanine phosphoribosyltransferase as measured in cell-free preparations has been observed (Epstein and Littlefield, 1977; Marz e f al., 1979). N o such agreement has generally been observed with all nucleosides. For uridine and thymidine, for example, the K , values for uptake by whole cells (Table 111) are about one to two orders of magnitude lower than those of the corresponding kinases for the nucleoside substrate as measured in cell-free preparations (Cleaver, 1967; &hak and Rada, 1976; Wohlhueter and Plagemann, 1980). This observation originally contributed to the erroneous conclusion that nucleoside uptake rates reflected those of the transport system (Plagemann and Richey, 1974). One possibility that might account for this discrepancy is that the in vitro analyses might not measure the kinetic properties of the kinase operative in situ (Plagemann et al., 1978b). Support for this conclusion comes from the work of Goldenberg and Stein (1978) and others to be discussed in Section VI. The interrelationship between transport and metabolism operating in tandem is even more complex for substrates that are catabolized in addition to being phosphorylated. This, for example, is the case with adenosine, which is rapidly deaminated to inosine by many types of cells (see Fig. I). Inosine, in turn, is phosphorolyzed to hypoxanthine, which is then converted to IMP. Recent studies (Lum et al., 1979) have shown that the metabolism of adenosine by P388 leukemia cells as a function of extracellular adenosine concentration is entirely consistent with the kinetic properties of the nucleoside transport system and those of the intracellular enzymes involved in adenosine metabolism (Table IV). In the case of adenosine, intracellular steady-state levels of free substrate remain very low regardless of the extracellular concentration of adenosine. This is a consequence of the facts that adenosine deaminase and the nucleoside carrier exhibit similar affinities for adenosine, and that the

260


maximum velocity for adenosine deaminatibn as measured in cell lysates is several times higher than that for adenosine transport (Table IV). In contrast to transport and deamination the maximum velocities of adenosine kinase and hypoxanthine/guanine phosphoribosyltransferase are much lower, but their affinities (for adenosine and hypoxanthine, respectively) are higher. Thus when adenosine enters the cells at a relatively low rate, either because of a low ( 1 p M or below) extracellular adenosine concentration or because transport is inhibited in some manner, most of the entering adenosine becomes directly phosphorylated. With an increase in the rate of adenosine entry, consequent to an increase in extracellular concentration, the intracellular steady-state levels of adenosine are sufficient to saturate adenosine kinase, and adenosine deamination becomes the predominant reaction. However, nucleoside phosphorylase and hypoxanthine/guanine phosphoribosyltransferase also become saturated as inosine and hypoxanthine pools swell, with the result that these products exit the cell and accumulate in the medium (see Fig. I ) . It should be emphasized that the comparable rates of nucleoside and hypoxanthine transport and phosphorylation observed at low substrate concentration (below 1-10 p1M) pertain to growing cultured cells. Thus salvage activities seem to be operating maximally in these growing cells at concentrations at which certain nucleosides and purines are normally found in blood and other body fluids (Murray, 1971; Hughes et a/., 1973; Schaer et a / . , 1978; Nattebrock and Then, 1977; Kuttesch et a!., 1978; Schrader et a/., 1978). Salvage activity, however, seems to be much lower in quiescent than growing cells, not because of lowered transport capacity, but because of lowered phosphorylation capacity. This situation is further discussed in Section VI on regulation of transport, but in the context of the present discussion it should be pointed out that, under such conditions, an increase in phosphorylating capacity alone would result in an increase in nucleoside and purine uptake, even at low substrate concentrations. Little information is available on nucleoside and purine uptake by tissue cells in the body. Cornford and Oldendorf (1975) injected various concentrations of 14C-labeledpurines and pyrimidines and their nucleosides into the common carotid artery of rats, decapitated the animals 15 seconds later, and determined the amounts of 14C in the brain. Two saturable uptake processes were defined; one for adenine ( K , = 27 p.M), subject to inhibition by hypoxanthine and one for adenosine ( K , = 18 p M ) , guanosine, inosine, and uridine, subject to mutual inhibition by these nucleosides. Uptake into the brain was equated with carrier mediated transport through the blood-brain barrier. The entry of cytosine,


261

uracil, thymine, thymidine, and cytidine was considered not to be significant.

C. Estimation of Zero-trans Transport Kinetic Parameters from Substrate Uptake Curves

Comparative studies with kinase-deficient or ATP-depleted Novikoff and P388 cells, in which, when necessary, substrate metabolism other than phosphorylation was also inhibited by appropriate inhibitors, have shown that the kinetics of transport of nucleosides and purines can be approximated in some instances from uptake studies with cells in which the substrate is metabolized provided that very early initial uptake time courses are analyzed (Plagemann et al., 1978b; Marz et al., 1979; Lum et al., 1979). For example, Fig. 9 shows that Eq. (1) fits quite well to early time courses of uridine and hypoxanthine uptake by wild type Novikoff rat hepatoma cells in which both substrates are rapidly converted to phosphorylated intermediates. Fitting Eq. (1) to pooled data with seven substrate concentrations (20- 1280 pM) yielded kinetic paramters similar to those estimated for uridine and hypoxanthine transport in enzyme-deficient or ATP-depleted cells (see Table 11). Equation (I), of course, applies strictly only to substrate influx and must, therefore, be confined to early time points at which the intracellular concentration of radioactivity is still well below equilibrium levels and at which phosphorylated products do not make a large contribution. It is obvious from Fig. 9 that, within these constraints, fitting Eq. (1) to uptake time courses yields more accurate estimates of initial uptake velocities-which reflect substrate influx-than a linear graphical estimation. This is true because, as with accumulation of these substrates in nonphosphorylating cells, significant backflux and a consequent downward deviation from linearity of the uptake curve occurs within the first few seconds of incubation. The greater the phosphorylating capacity of the cells, however, the sooner does the accumulation of phosphorylated products cause the uptake curves to deviate upward from the curve described by Eq. (I), particularly at the lower substrate concentrations (see Fig. 9A and E). Thus, although possible in some situations, the estimation of initial permeation rates of substrates that are rapidly phosphorylated intracellularly by graphical methods or curve-fitting of transport rate equations is fraught with problems and must be approached, if at all, with extreme caution. A possible remedy to these problems is to develop integrated rate equations describing flux in a two-step pathway, comprising carrier-mediated

262

PETER G. W. PLAGEMANN AND ROBERT M. WOHLHUETER UR I D l N E

O IN

HY POXANTHINE

1

10

$;r.;

.:

..*

_,....'

1280 IrM

,,.*'

.*"

0.

00

.

..'

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TIME I S E C )

FIG.9. Kinetic analyses of uridine (A-D) and hypoxanthine (E-H) transport as estimated from early uptake time courses in untreated, wild-type Novikoff rat hepatoma cells. The experiments were conducted as described in the legend to Fig. 3, except that untreated cells were used in which both substrates are converted to phosphorylated products. Final substrate concentrations were 20, 40, 80, 160, 320, 640, and 1280 p M (280 cpmipl for [3H]uridine and 470 cpmipl for [3H]hypoxanthine, irrespective of concentration). The intracellular H 2 0 spaces and trapped HzO spaces were in (A-D) 21.9 and 2.7 plicell pellet, respectively, and in (E-H) 22 and 6.5 plicell pellet, respectively. Data are from Plagemann et a / . (1978b. Table I) and Marz er a / . (1979, Fig. 9), but reanalyzed as follows. Equation ( I ) was fitted to the pooled uptake data in each experiment with all R parameters held equal. Only four of the theoretical curves are shown for each substrate. The best fitting kinetic parameters were for uridine: K = 230 ? 8 p M and V = 15 ? 0.2 pmole/pl cell H,O.second, and for hypoxanthine: K = 474 2 30 p M and V = 77 ? 2 pmoleipl cell H,O.second. The correlation coefficients (r,,g) were 0.9889 and 0.9868, respectively.

permeation and Michaelian phosphorylation. We discuss this approach in detail elsewhere (Wohlhueter and Plagemann, 1980). So far, such flux equations have been successfully fitted to uptake time courses only where some of the kinetic parameters describing the pathway are supplied known values-too severe a limitation for our present purposes. Koren et ul. (1978) and Heichal et al. (1979) have recently attempted to estimate the kinetics of uridine transport in monolayer cultures of untransformed and transformed mouse 3T3 and Nil 8 hamster cells, respectively, by estimating initial uptake rates by linear regression analysis of four to five time points taken during the first 20-30 seconds of incubation at 20°C. The estimated kinetic parameters were similar for quiescent and serum-stimulated untransformed and Simian virus 40-trans-


263

formed 3T3 cells ( K , = 220-340 p M , V,,, = 27-46 pmole/106 cells*second) and for untransformed and murine sarcoma virus transformed Nil 8 hamster cells ( K , = 400-530 p M , V,,, = 25-55 pmole/106 cells second) and much higher than those estimated from long-term uptake time courses (see Table 111). It was concluded, therefore, that these kinetic parameters are reasonable estimates of Kqg and V?; for uridine transport in these cells. Since uridine influx seems to be at least as fast in these cells as in suspensions of animal cells (Fig. 9) it seems likely, however, that 20-30 second slopes underestimated true initial transport velocities. In early uptake studies it was shown that the incorporation of extracellular nucleosides and purines into cellular nucleic acids follows simple Michaelis-Menten kinetics and that the apparent K , values were similar to those estimated for the uptake of the respective substrates into total cellular material (Plagemann and Richey, 1974). Since the uptake rates were considered to reflect those of transport, the data were interpreted to indicate substrate transport to be the rate-limiting step in the incorporation into nucleic acids. These observations are still important in relation to the use of these precursors to assess rates of nucleic acid synthesis, but the interpretation with respect to rate-limiting steps needs to be modified in light of the newer information discussed already. Although, at very low extracellular substrate concentration, influx may contribute in a major way to determining the rate of its incorporation in growing cells in culture, with increase in extracellular concentration it is certainly the conversion of these substrates to nucleotides that becomes rate-determining, rather than transport. This conclusion also pertains to the uptake and incorporation into nucleic acids of various nucleoside and purine analogs that are toxic to cells and might be employed in cancer chemotherapy (Plagemann et al., 1978a). Other aspects of the relationship between transport of nutrients and their metabolism have been reviewed and discussed by Wohlhueter and Plagemann (1980).

D. Uptake into Vesicles of Mammalian Cells

Hochstadt and her collaborators (Quinlan and Hochstadt, 1974, 1976; Li and Hochstadt, 1976a,b; Hochstadt and Quinlan, 1976; Dowd at al., 1977; Hochstadt, 1974) introduced the use of membrane vesicles from cultured animal cells for the study of nucleoside and purine permeation through membranes. Although the study of substrate permeation into membrane vesicles could be potentially very informative with respect to the mechanism of transport, the particular preparations employed by

264


these investigators are compromised by the fact that they contained, although at a reduced level, most of the enzymes present in the cytoplasm of mammalian cells, including adenosine deaminase, nucleoside phosphorylase, adenosine kinase, uridine kinase, and hypoxanthine/guanine phosphoribosyltransferase, and are also capable of some conversion of extravesicular nucleosides to intravesicular phosphorylated intermediates. Because of differences in recovery of various enzymes in vesicular preparations it was reasoned that small proportions of the total cellular content of these enzymes were associated with the plasma membrane. But, whatever the subcellular location of the enzymes, in their metabolic activities these vesicles resemble, in many respects, metabolizing whole cells and thus cannot be handled as if they were metabolically inert entities. Accumulation of radioactivity from [U-14C]uridine, -adenosine, -inosine, and -guanosine by the vesicles from a number of different cell lines (LgZ9,3T3, and BHK,,) has been found to be linear for at least several minutes. Radioactivity accumulated intravesicularly in 10-20 minutes to concentrations 5- to 20-fold higher than those in the medium. Accumulation of radioactivity bore a simple, Michaelis-Menten relation to exogenous substrate concentration, and the apparent Michaelis-Menten constants (7-12 p M for uridine, 7-19 p M for adenosine, and 35-55 p M for inosine) resemble those estimated for the uptake of nucleosides by metabolizing cells (Table 111), rather than those for nucleoside transport (Table 11). However, in contrast to intact cells which accumulate mainly nucleoside triphosphates, the intravesicular content consisted mainly of labeled ribose-I-phosphate and small amounts of labeled nucleoside monophosphates. The concentrations of free nucleosides were found regularly to be very low, well below those in the extravesicular fluid. Free hypoxanthine was not detected inside vesicles of L,,, cells after incubation with labeled inosine, whereas free hypoxanthine accumulated extravesicularly in amounts similar to the amounts of ribose-lphosphate inside (Li and Hochstadt, 1976a,b). Some free hypoxanthine, on the other hand, accumulated in vesicles of 3T3 (Quinlan and Hochstadt, 1976) and BHKzl cells (Dowd et al., 1977). On the basis of these results and the finding that a small proportion of the cell’s purine nucleoside phosphorylase activity was recovered in purified membrane preparations these investigators proposed that the main route of entry of radioactivity from uniformly labeled inosine is a kind of group translocation, catalyzed by membrane-bound purine nucleoside phosphorylase, whereby ribose- I-phosphate is transferred to the intravesicular space, while hypoxanthine is released to the outside. The finding of free hypoxanthine in vesicles of some of the cell lines was


265

interpreted to indicate the additional operation in these cell lines of direct permeation of inosine with subsequent phosphorolysis. The rapidity of the nucleoside and purine transport systems of mammalian cells suggests a more plausible alternate explanation for these results, namely, that inosine entered the vesicles via the nucleoside transport system and was phosphorolyzed inside the vesicles, but that most of the intravesicular hypoxanthine was transported out of the vesicles or lost from the vesicles during their extensive washing before radioactivity analysis, whereas the labeled ribose-I-phosphate was retained because the membrane is largely impermeable to phosphorylated intermediates (see Section VII). That nucleoside metabolism rather than transport has been measured in these vesicles is best illustrated by the following data. Uridine uptake over a 20-minute period was found to be about six times higher in vesicles from growing than quiescent 3T3 cells (Quinlan and Hochstadt, 1974) just as is observed in whole cells (see Section VI). However, in studies with whole cells it has been demonstrated that this difference in uptake rates is due to differences in rates of uridine phosphorylation rather than of transport (Rozengurt et al., 1977b). An involvement of purine nucleoside phosphorylase in inosine permeation has also been postulated by Cohen and Martin (1977) on the grounds that purine nucleoside phosphorylase-deficient human fibroblasts took up only 3 1% as much [8-14C]inosineat an exogenous concentration of 100 p M as normal fibroblasts. However, this conclusion was based on a single 30-minute time point, and no difference in uptake of either [8-14C]- or [U-14C]inosine between the two types of cells was detected when the exogenous inosine concentration was 10 p M . Furthermore, cells of a purine nucleoside phosphorylase-deficient subline (NSU-1) of the mouse T cell lymphoma line S49 seem to take up inosine and guanosine unabated (Ullman et al., 1979), whereas inosine uptake by a nucleoside transport mutant (AE,) of the same cell line (see Section IV,E) is reduced at least 98% when compared to wild type S49 cells, even though the cells possess normal purine nucleoside phosphorylase levels (Cohen et al., 1979). The correlation between adenosine deaminase activity of vesicles and their capacity to accumulate radioactivity from uniformly labeled adenosine, which led Li and Hochstadt (1976b) to postulate a function of adenosine deaminase in adenosine transport, has also a more plausible, alternative explanation along the lines discussed already. Since the direct phosphorylation of adenosine is minimal in these vesicles, transported intravesicular adenosine is rapidly lost during washing of the vesicles. The major form of radioactivity deriving from it, and remaining in the cell, is ribose- 1-phosphate, the formation of which, via inosine, requires

266


the presence of intracellular adenosine deaminase. An association of adenosine deaminase with the nucleoside carrier in mammalian cell membranes has also been suggested by Agarwal and Parks (1975), but detailed studies with P388 mouse leukemia cells have shown that the deamination of adenosine by these cells can be accounted for totally by the tandem operation of nucleoside influx, intracellular deamination, and subsequent efflux of inosine (Lum et af., 1979). The suggestion that adenosine kinase may be involved in group translocation of adenosine in human blood platelets (Sixma et af., 1976) is also not supported by studies with other types of cells. Hochstadt and Quinlan (1976) have suggested that hypoxanthine enters animal cells, as bacteria, by group translocation catalyzed by membraneassociated hypoxanthine/guanine phosphoribosyltransferase. Membrane vesicles from both wild type and transferase-deficient (thioguanine-resistant) 3T3 cells accumulated little labeled hypoxanthine, but accumulation of radioactivity by vesicles from wild type cells was stimulated about 10-fold by addition of phosphoribosylpyrophosphate (PRPP). Whether phosphoribosylation of hypoxanthine was intra- or extravesicular, or occurred during passage through the membrane, however, cannot be distinguished from these data. That transferase-deficient cells have normal transport capacity has clearly been shown in studies with whole cells (see Section 11,E).

E. Contributions of Transport and Nonmediated Permeation to Overall Uptake

In the context of uptake studies the entry of nucleosides and nucleic acid bases by nonmediated permeation also needs to be considered. As we have seen, the transport of nucleosides and bases can be adequately described by rate equations for carrier-mediated permeation, without invoking nonmediated components. Yet, studies of the kinetics of longterm uptake of these substrates by metabolizing cells have invariably suggested a nonsaturable uptake component (see Fig. 10B) or at least a component with a K , two to three orders of magnitude higher than that of the high-affinity uptake system (Jacquez, 1962; Lassen, 1967; Hawkins and Berlin, 1969; Hare, 1970; Schuster and Hare, 1971; Plagemann, 1971a,b,c; Roos and Pfleger, 1972; Schrader et af., 1972; Plagemann and Erbe, 1972, 1975; Stein and Rozengurt, 1975; Zylka and Plagemann, 1975; Sixma et al., 1976; Cass and Paterson, 1977; Yang and Visser, 1977; Epstein and Littlefield, 1977; Paterson et al., 1977a,b; Heichal et af., 1978; Turnheim et af., 1978). For example, in the case of hypoxanthine

267


uptake, “velocities” estimated from 2-minute uptake time points (Fig. 10A) yielded apparent biphasic uptake kinetics. There was clearly a saturable uptake component with a K , of about 4 p M (Fig. IOB, upper frame), but in the range of 20 to 1250 p M the apparent rate of uptake increased in direct proportion to the extracellular concentration of hypoxanthine (Fig. IOB, lower frame). Such relationships have generally been interpreted as reflecting substrate entry by nonmediated permeation (Roos and F’fleger, 1972; Schrader et a / . , 1972; Lassen, 1967; Plagemann, 1971a,b; Plagemann and Erbe, 1972, 1975; Paterson et a / . , 1977a,b; Schuster and Hare, 1971; Stein and Rozengurt, 1975). In some studies

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- 0 0 HYPOXANTHINE ( U M I

TIME

FIG. 10. Uptake of hypoxanthine by untreated wild-type Novikoff rat hepatoma cells as function of substrate concentration at 24°C. Short-term hypoxanthine uptake (A) was measured by the rapid kinetic technique as described in the legend to Fig. 3, except that untreated wild-type cells were used and hypoxanthine was the substrate. The [3H]hypoxanthine concentrations were 1, 2, 4, 8, 20, 40, 80, 160, 320, 640, and 1280 p M (390 cpm/pl, irrespective of Concentration). For long-term uptake measurements cell suspension and substrate solution were mixed in the same proportion as in the rapid kinetic technique ( 0 time) and after the indicated time periods equivalent samples of mixture were centrifuged through oil and the cell pellets were analyzed for radioactivity. All values were corrected for substrate trapped in extracellular space in cell pellets (4.0 11.1).The intracellular H,O space was 21.5 pl. The broken lines in (A) indicate the intracellular concentration of radioactivity equal to that in the medium at 0 time. Uptake “velocities” ( u J were estimated from 2-minute time points i n (A; long-term) and plotted as a function of hypoxanthine concentration (20-1280 p M ) in (B; lower frame) or subjected (1-20 p M ) to MichaelisMenten analysis in (B; upper frame). V,,, is expressed in pmoleipl cell H,O.second. (Previously unpublished data of Plagemann and Wohlhueter.)

268


total uptake rates were corrected for this nonsaturable component in order to estimate the contribution of transport to the overall uptake rate. However, recent studies of the uptake of cytosine (Graff et al., 1977) and of the transport of other substrates in the absence of metabolism indicate that these interpretations may have been incorrect. Cytosine is not metabolized by cultured cells, its uptake is not saturable up to concentrations of 10 mM (Zylka and Plagemann, 1979, and its influx is about two orders of magnitude slower than that of transported nucleosides and purine bases at equivalent concentrations in the first-order range for the transport system (see Table V). The first-order rate constant for cytosine influx is lower than the apparent first-order rate constant of nonsaturable permeation components for transportable and metabolizable substrates estimated from curves similar to that in Fig. 10B (lower frame). For example, at a concentration of 10 p M , cytosine enters Novikoff cells at 24°C at about 0.017 pmole/pl cell H,O.second (Zylka and Plagemann, 1975; Graff er al., 1977), whereas the rate of uptake of uridine (Plagemann, I97 la,b), adenosine (Plagemann, 197 1b), thymidine (Plagemann and Erbe, 1972), hypoxanthine, and guanine (Zylka and Plagemann, 1975; See Fig. IOB) attributed to nonsaturable uptake fell in the range of 0.04 to 0.18 pmolelpl cell H,O-second. Furthermore, the apparently nonsaturable uptake components were found to be independent of temperature between 6 and 27°C (Jacquez, 1962; Plagemann and Richey, 1974), whereas the nonsaturable permeation of cytosine, like nonmediated permeation of substances through membranes in general (Lieb and Stein, 1971), is highly temperature dependent (Graff et al., 1977). According to the formulation of Lieb and Stein (1971), the permeation coefficient (0 of a substance through a lipid bilayer membrane is a function of the solubility of the substance in membrane lipids relative to that in an aqueous medium ( Z ) , z its diffusion constant describing its movement within the membrane (Dmem)and the reciprocal of the thickness of the membrane (1): Z X Dmem

P=

1

where D,,, is a complex function of the molecular properties of the substance such as molecular weight, shape, and charge. Thus for uncharged substances with similar molecular dimensions, the lipid solubility of a substance is the main determinant of its rate of permeation. Indeed, it has been found that the rates of nonsaturable uptake of L-glucose,

* Partition coefficients have generally been designated K (Lieb and Stein, 1971). For clarity, we have used Z , since K has been reserved to designate the substrate:camer affinity constant.

269


TABLE V

PARTITION COEFFICIENTS AND MOLECULAR WEIGHTSFOR L-GLUCOSE, NUCLEOSIDES, AND NUCLEIC ACIDBASESAND VELOCITIES OF THEIR ZERO-TRANS TRANSPORT AND NONMEDIATED PERMEATION AT AN EXOGENOUS CONCENTRATION OF 10 p M AND 24°C Transportb Substrate L-Glucose Cytosine 8-Azaguanine DMO Thymidine Uracil Adenosine H ypoxanthine Uridine

Molecular weight I80 Ill I50 129 242 112 267 I36 244

Z“ 0.00404 f 0.0004 0.0352 f 0.00067 0.173 2 0.0064 0.983 ? 0.078 0.0730 f 0.003 0.778 f 0.0004 0.105

0.1 15 0.185

f 0.015 f 0.009 f 0.0004

Nonsaturable permeationc

9: 0 9; (pmole/pl cell H&l.second) 0.00017 0.017 0.035 0.27

u:;/Z

0.24 0.21 0.49 0.28

1.13 0.115

2.2 I .48 0.96

” Z = partition coefficient: substrate concentration in octanoYaqueous buffer solution as determined by Graff et a / . (1977).Data are from Graff et a / . (1977)and unpublished (P. Plagemann and R. Wohlheuter). Z for 8-azaguanine and 5’,5’-dimethyl-2,4-oxazolidinedione (DMO) is at pH 6.0,all others are independent of pH between 6 and 8. S, = 10 p M (24°C).Calculated from average K and V (see Table 11). ‘ S, = 10 p M (24°C).uf: were computed by first fitting Eq. (4)to time courses of substrate accumulation to equilibrium and then calculating u;: = S , k . The uptake of 8azaguanine and DMO were determined at pH 5.8-6.0(unpublished data). Other data are from Graff ef a / . (1977),but have been corrected for temperature differences (24 versus 37°C).

cytosine, 8-azaguanine, and 5 ’ ,5’-dimethyl-2,4-oxazolidinedione are similarly dependent on their lipid solubility (see vT:/Z ratios: Table V). This finding, and other evidence discussed by Graff et al. (1977), supports the view that the main mode of entry of these substances is nonmediated permeation. Because various other nucleosides and nucleic acid bases have molecular weights and lipid solubilities similar to cytosine and 8azaguanine (Table V), one would expect all these substances to enter by nonmediated permeation at about the same rate. A resolution of these discrepancies is as follows. The apparently nonsaturable uptake component becomes significant in Michaelis-Menten plots in a range of extracellular substrate concentrations that suffice to saturate the nucleoside kinases or purine phosphoribosyltransferases (see Fig. 1OB). At these substrate concentrations the formation of nucleotides is slow relative to the transport rate and free substrate rapidly accumulates intracellularly to equilibrium levels (see, e.g., Figs. 7B and 10A).

270


Thus, measurements at 2- 10 minutes encompass these equilibrium levels, which, of course, increase in direct proportion to extracellular concentration (Fig. 10A; the total concentration of radioactivity was the same for all hypoxanthine concentrations and only its specific radioactivity was decreased by addition of unlabeled h ypoxanthine). If such steadystate accumulations, divided by time, are erroneously construed as "velocities," this "velocity" component would be directly proportional to exogenous substrate concentration and would be independent of temperature. The data in Table V indicate that, in the first-order range of substrate for transport (at 10 p M , for example), less than 2% of the total rate of thymidine, adenosine, hypoxanthine, and uridine entry can be attributed to nonmediated permeation. This proportion, of course, increases as the transport carrier becomes saturated, but, in view of the relatively high substrate : carrier affinity constants of nucleoside and base transport, it would make a large (and therefore measurable) contribution, only at very high, and certainly unphysiological, concentrations. No method is presently available to estimate accurately this entry component in cultured cells, except empirically from rates of entry of substances, such as cytosine, that enter solely by a nonsaturable process. Sheep erythrocytes, however, provide confirmation of these views. Nucleosides enter sheep erythrocytes that lack a functional nucleoside transport carrier only very slowly (Young, 1978; see Section IV,E). For example, at an extracellular concentration of 5 mM, the rate of inosine uptake by these cells was estimated to be about 0.025 pmole/pl cell H,O*second at 37"C, which is appreciably slower than the rate of cytosine permeation into cultured animal cells under equivalent conditions. That nonmediated permeation plays only a minor role in nucleoside and purine uptake is also indicated by the finding that the nucleoside transport inhibitors, nitrobenzylthiopurine riboside and nitrobenzylthioguanosine (see Section V,C), protect L5 178Y leukemia cells against the toxic effects of various nucleoside analogs that seem to enter the cells via the nucleoside carrier (Warnick et al., 1972). Transport inhibitors have been employed in attempts to dissect Michaelis-Menten curves for uptake of various nucleosides and purines into transport and nonsaturable components (Plagemann, 197la; Plagemann and Erbe, 1972; Plagemann and Richey, 1974; Cass and Paterson, 1977; Paterson et al., 1977a; Young, 1978). The residual rate of uptake in the presence of excess of inhibitor was considered to represent the nonsaturable component. Such an approach might be valid with inhibitors that specifically interact with the carrier, such as the nitrobenzylthiopurine nucleosides (Cass and Paterson, 1977), but interpretation of data with nonspecific inhibitors, such as dipyridamole and cytochalasin B, is com-


271

plicated by the finding that the nonsaturable uptake of cytosine and glucose is also inhibited by these inhibitors (Graff et af., 1977). IV.

L-

PROPERTIES OF NUCLEOSIDE AND FREE BASE TRANSPORT SYSTEMS

A. Specificity for Natural Substrates

T h e zero-trans influx of uridine by human erythrocytes is inhibited in ,Ipparent competitive manner by forrnqcin €3 (Oliver and Paterson, 197 1 !, and many natural nucleosides and nucleoside analogs cause ?he acielerated exchange diffusion of uridine and thymidine (Oliver and Pa!erson, 1971; Cass and Paterson, 1972, 1973). These findings suggest that human erythrocytes possess only a single nucleoside transport system with broad substrate specificity. As pointed out already, only substrates that are transported by the same carrier are expected to cause accelerated exchange diffusion, but this test is limited to asymmetric transport systems with significant difference in the rate of movement of loaded and unloaded carrier. Although quantitative values are not available from the work of Paterson and co-workers, the data suggest that many nucleosides are transported with similar efficiency. However, purine nucleosides accelerate the efflux of uridine slightly less than do pyrimidine nucleosides (Cass and Paterson, 1973). Ribose and nucleic acid bases are not substrates for the carrier. Initial studies of the long-term uptake of nucleosides by cultured cells, on the other hand, resulted in confusion and misinterpretations. Some nucleosides were found to inhibit the uptake of a given substrate-the inhibition was mutual and apparently competitive, but the K , values as inhibitor differed significantly from the K , values as substrate. Other nucleosides showed little or no inhibition with a given substrate (Plagemann, 1971a,c; Plagemann and Erbe, 1974a; Steck et al., 1969; Hare, 1970; Cass and Paterson, 1977; Paterson et af., 1977b). These findings supported the view of the existence of several nucleoside transport systems in cultured cells; one for uridine and cytidine, one for adenosine, one for thymidine, one for inosine and guanosine, and possibly others for other deoxyribonucleosides (Plagemann and Richey, 1974). Additional support for this view were the findings that certain experimental conditions resulted in changes in uridine, but not thymidine, uptake and vice versa (see Section VI), and that certain mutant cell clones exhibited defects in uptake affecting all nucleosides in a presumptive substrate class (see Section IV,E). However, it is clear now that the uptake rates measured in these studies largely reflected the rates of intracellular accumulation of nucleo-

-

‘i

272


tides derived from extracellular substrate (see Section 111), and consequently, that the observed substrate specificities reflected those of the phosphorylating enzymes involved: uridine, thymidine and adenosine kinase, and hypoxanthine/guanine phosphoribosyltransferase, respecti~ely.~ A dissenting view on the existence of several specific or overlapping nucleoside transport systems in cells other than human erythrocytes was that of Taube and Berlin (1972) based on results with monolayer cultures of nonproliferating rabbit polymorphonuclear leukocytes. These investigators reported that uridine, adenosine, and thymidine inhibited the uptake of each other in an apparently competitive manner with apparent Kis similar to the K,s of their uptake (10-40 pIM). They concluded that all purine and pyrimidine nucleosides are transported by a single system. They also explored rather thoroughly the structural requisites for inhibition of adenosine uptake in their leukocyte system. They found that, although 2’-deoxyadenosine strongly inhibited adenosine uptake, most other structural alterations in the sugar moiety greatly reduced the inhibition. Adenine itself had no significant effect. Differences in the base moiety, on the other hand, were less effective in reducing the inhibitory potency of ribonucleosides, but were more difficult to categorize on a rational basis. From spatial considerations of the nucleosides, it was concluded that in order to accommodate the structurally distinct molecules, substrate-induced conformational changes of the carrier must occur (see also review in Berlin and Oliver, 1975). However, a quantitative comparison of inhibition of adenosine uptake-on which this conclusion was based-involved some uncertainties: (1) the reported K i values were calculated from data obtained with single concentrations of adenosine (7 pkf) and of inhibitors on the assumption of competitive inhibition: ( 2 ) relatively long uptake time points (45 seconds) were used to estimate initial uptake rates in cells in which the substrates were rapidly converted to nucleotides; and (3) adenosine was rapidly deaminated in the cells. Such uncertainties may account for the fact that the K , for adenosine uptake in uninhibited controls, as well as the K,s for uptake of other substrates tested, were much lower than the nucleoside :carrier affinity constants estimated with cultured cell lines lacking phosphorylating activity or in human red blood cells (Tables I and 11). In fact, values of uptake K , for polymorphonuclear leukocytes, except for thymidine, are similar to those for the uptake of nucleosides There is no evidence that a direct phosphorylation of inosine occurs in P388 cells or most mammalian cells (Friedmann ct ( I / . , 1969: Burke et ( I / . , 1977: Fox and Kelley, 1978) as has been reported for some types of cells (Pierre and LaPage, 1968: Schaffer er ( I / . , 1973). Recently it has been demonstrated that deoxyguanosine is phosphorylated in S49 human T lymphoma cells by deoxycytidine kinase (Gudas ef ( I / . , 1978).


273

by other phosphorylating, cultured cells (Table III), which are now known to reflect the saturation of the phosphorylating systems (see Section 111). The same correlation holds for the K , for adenine transport estimated in these polymorphonuclear leukocytes (7 p M ; Hawkins and Berlin, 1969; Berlin, 1970). In contrast, the K , for the uptake of xanthine which is not phosphorylated by the cells was found to be 2.3 mM, thus similar to K for adenine transport in ATP-depleted or adenine phosphoribosyltransferase-deficient cultured animal cells (Table 11). More direct evidence for a single nucleoside carrier with broad substrate specificity in cultured animal cells comes from recent studies in which transport per se was measured without complications of metabolism. Two lines of evidence support this conclusion. First, various riboand deoxyribonucleosides countertransport with uridine (Fig. 1 l), deoxycytidine, and thymidine (Plagemann er a / . , 1976) in ATP-depleted and/or kinase-deficient Novikoff cells. Second, deoxycytidine transport in a deoxycytidine kinase-deficient line of L12 10 leukemia is effectively inhibited by other purine and pyrimidine nucleosides (Kessel and Shurin, 1968),and cytosine arabinoside transport is inhibited by other nucleosides in a variety of cell lines (Mulder and Harrap, 1975; Heichal et a/., 1978). Similarly, various nucleosides inhibit the transport of each other in either ATP-depleted Novikoff cells or in cells deficient in the kinase specific for the substrate whose transport is measured (Plagemann et al., 1978a,b; Wohlhueter et al., 1979a). Though such data strongly suggest a common carrier, a molecular interpretation of these inhibitions is difficult at present, since the inhibitions have been found to be of a mixed type, involving both increases in carrier-substrate affinity constant and decreases in maximum velocity (Wohlhueter er al., 1979a; Marz et al., 1979). Detailed analyses of the inhibition of thymidine transport in thymidine kinase-deficient Novikoff = 192, 395, cells by inosine, uridine, and deoxycytidine yielded Ki,intercept and 1620, and Ki,sloPe= 64, 156, and 232 p M , respectively (Wohlhueter er al., 1979a). Mixed-type inhibition might be considered unexpected for alternative substrates from analogy to enzymatic reactions, but needs to be accounted for in a n y molecular and mathematical concepts of the mechanism of facilitated transport. Similar mixed-type inhibitions have been observed for the transport of hypoxanthine by apparently alternate substrates in cultured cells (Marz et al., 1979). Nevertheless, the degree of inhibition of the transport of nucleosides by each other seems to be inversely related to their carrier-substrate affinity constants. For example, K seems to be lowest for purine nucleosides and these seem to inhibit the most, whereas the opposite seems true for deoxycytidine and cytidine (Table 11; Wohlhueter et a/.,

274


v)

j:

0-

W V

PRELOADED ( I O m M ) NONE

o- URlDlNE &

DEOXYURIDINE

& CYTlDlNE P DEOXYCYTIDINE

F GUANINE D

URACIL

60

~

OO

I0

20

30

40

50

TIME ( M I N I

FIG. I I . Countertransport of [3H]uridine in ATP-depleted Novikoff cells. Samples of a suspension of 1 x lo' ATP-depleted cells/ml of glucose-free basal medium containing 5 mM KCN and 5 mM iodoacetate were supplemented where indicated with 10 mM uridine, deoxyuridine, cytidine, deoxycytidine, guanine, or uracil and incubated at 37°C for 20 minutes. The cells were collected by centrifugation and suspended to the same density in glucose-free basal medium with KCN and iodoacetate and containing 5 p M [3H]uridine (57 cpmipmole) at 18°C. The suspensions were incubated at 18°C and duplicate I-ml samples were analyzed for radioactivity in total cell material. All points are averages of the duplicate samples. (From Plagemann et a / . , 1978b.)

1979a; Marz et al., 1979; Wohlhueter and Plagemann, unpublished data). These findings suggest that some nucleosides are transported with different efficiencies. This conclusion needs to be considered in relation to the apparent symmetry of the nucleoside transport carrier. If, indeed, loaded and empty carrier move equally rapidly, the carrier movement should be indifferent toward the particular substrate with which it is loaded. Thus, although the substrate affinity of the carrier might differ for different substrates, the maximum substrate transport velocity should be the same for all substrates carried by the carrier of a specific cell line. This requirement seems to be met by the data for the limited number of substrates examined and within the experimental errors (Table 11), but more extensive data, such as kinetic analysis of the transport of different nucleosides with a single population of cells, are required to allow un-

275


equivocal conclusions on this point. It is generally observed that the maximum velocities for a single substrate may vary up to 80% among cell populations of a cell line analyzed at various times (Plagemann et al., 1978b; Wohlhueter et a l . , 1979a; Marz et al., 1979). The reason for these variations is not clear, since the transport capacity of the cells varies little with the growth stage of the cells (see Section VI), but it makes quantitative comparisons of transport rates between different batches of cells difficult. The number of transport systems involved in nucleic acid base transport is less certain. Hypoxanthine and guanine seem to be transported by the same system in Novikoff hepatoma cells, since they inhibit the transport of each other about equally (Marz et a/., 1979). The kinetic parameters for their transport in these and P388 cells are similar to those of nucleoside transport (Marz et ul., 1979; see Table 11). Hypoxanthine transport is strongly inhibited by nucleosides (Marz et al., 1979) and nucleoside transport is similarly inhibited by hypoxanthine (Fig. 12A; Plagemann et al., 1978b; Wohlhueter et a / . , 1979a). These inhibitions are

1

0.021

/K i , slope =

420 pM

-500 TIME ISEC)

0

500

I000

HYPOXANTHINE ( p M 1

FIG.12. Kinetics of inhibition of uridine transport in uridine kinase-deficient Novikoff cells by hypoxanthine. The experiment was conducted as described in the legend to Fig. 3, except that where indicated the [3H]uridine solutions were supplemented with hypoxanthine to yield final concentrations of 365 and 920 pM. The final concentrations of [3H]uridine were 20, 40, 80, 160, 320, 640, and 1280 pM (350 cpm/pI, irrespective of concentration) and the intracellular and extracellular trapped water spaces were 14.6 and I .8 plisample pellet, respectively. Equation ( 1 ) was fitted to pooled corrected uptake data for each hypoxanthine concentration with all R parameters held equal. The theoretical curves for S , = 320 p M uridine plus 0, 365, and 930 p M hypoxanthine are shown in ( A ) . The best fitting parameters for 0, 365, and 930 p M hypoxanthine were K = 284 ? 13,400 ? 16, and 649 2 47, respectively; V = 30.7 ? 0.5, 25.7 ? 1 . 1 , and 24.3 0.9, respectively. The correlation coefficients (ru,G)were 0.9873, 0.9895, and 0.9722, respectively. The kinetic parameters were replotted in (B) as described by Segel (1975). (Previously unpublished data of Plagemann and Wohlhueter.)

+

276


also of mixed type (Fig. 12B). Nucleosides also countertransport with hypoxanthine in both hypoxanthine/guanine phosphoribosyltransferasedeficient and ATP(PRPP)-depleted wild type Novikoff cells (Zylka and Plagemann, 1975). These results indicate some overlap between nucleoside and hypoxanthine transport. Nucleoside and hypoxanthine transport are also similarly inhibited by p-hydroxymercuribenzoate and dipyridamole (see Sections V , A and B). Clear differences between nucleoside and hypoxanthine transport, however, exist in their sensitivity to inhibition by nitrobenzylthionucleosides. These compounds strongly inhibit the transport of nucleosides in many animal cells ( K i about 0.1-1 nM), whereas hypoxanthine transport is not affected by concentrations much higher than those affecting nucleoside transport (see Section V,C). That nucleosides and hypoxanthine/guanine are transported by different carriers is most strongly indicated by the recent isolation of single-step mutants from S49 lymphoma cells with defects in nucleoside transport, but not purine or pyrimidine uptake (Cohen et al., 1979). These studies are discussed further in Section IV,E. Adenine, on the other hand, seems to be transported in cultured animal cells by a system different from that transporting hypoxanthine, but here, too, there seems to be some overlap with the nucleoside transport system. These conclusions were anticipated on the basis of studies on adenine uptake by various types of cells. Some studies showed that adenine uptake is little affected by relatively high concentrations of hypoxanthine, guanine, cytosine, and uracil in hepatoma cells, and that similarly, adenine had little effect on the uptake of hypoxanthine and nucleosides (Plagemann, 1971a; Zylka and Plagemann, 1975; Dybing, 1974a). Adenine uptake in Novikoff hepatoma cells was also far less inhibited by p hydroxymercuribenzoate or dipyridamole than hypoxanthine or nucleoside uptake and not significantly affected by dipyridamole in various other types of cells (see Sections V,A and B). In other studies, however, inhibition of adenine uptake by guanine and hypoxanthine was observed, as in rabbit polymorphonuclear leukocytes (Hawkins and Berlin, 1969) and human blood platelets (Sixma et al., 1973), and adenine inhibited adenosine uptake in polymorphonuclear leukocytes (Strauss et al., 1976). Nucleosides, on the other hand, strongly inhibited adenine uptake (Hawkins and Berlin, 1969; Sixma et al., 1973; Zylka and Plagemann, 1975). Recently it has been shown (Suresch et al., 1979) that folate competitively inhibits ( K i = 450 pM) the uptake of adenine by L1210 mouse leukemia cells ( K , = 21 pM), while adenine competitively inhibits ( K i = 17 pM) the uptake of folate ( K , = 100-00 pM). Because of the similarity of the apparent K , of uptake and K i of inhibition the authors suggested that folate enters the cells via the same transport system as adenine. Studies in which adenine transport per se was measured in ATP-de-


277

pleted or adenine phosphoribosyltransferase-deficient cells have confirmed the lack of effect of other nucleic acid bases on adenine transport in Novikoff hepatoma cells, but have failed to detect any effect of thymidine or inosine on adenine transport (Marz et af., 1979). Adenine, on the other hand, inhibited the transport of uridine in ATP-depleted cells (Plagemann et af., 1978b). This effect could not have been due to the formation of adenosine from adenine, since animal cells are devoid of adenosine phosphorylase. The specificity of the carriers transporting uracil and/or thymine is equally uncertain. Uracil causes a slight countertransport of uridine (Fig. 11) and hypoxanthine (Zylka and Plagemann, 1975) and weakly inhibits uridine transport (Plagemann et al., 1978b), hypoxanthine transport (Marz et af., 1979), and thymidine transport (Wohlhueter et af., 1979a), but it is not certain that these effects are caused by uracil itself and not by some uridine formed from uracil by uridine phosphorylase. Thymine, which is not converted to thymidine in these cells, however, also slightly inhibits thymidine transport (Wohlhueter et al., 1979a) and uridine transport (Plagemann et al., 1978b). Furthermore, the uracil: carrier affinity constant is very high (Table 11), approaching the solubility of uracil in aqueous solutions. Pyrimidine transport is also distinct from nucleoside transport in that thymine transport, like hypoxanthine transport, is not inhibited by nitrobenzylthioinosine (Wohlhueter et af., 1978b). The apparent competitive nature of the inhibition of the uptake of various nucleosides and purines by each other (Plagemann and Richey , 1974) warrants special consideration, particularly in view of the fact that inhibition of transport per se by alternate substrates is of a mixed type. The apparent competitive inhibition of uptake seems to result from the fact that the maximum velocities of substrate uptake reflect those of substrate phosphorylation rather than of transport. Thus, alternate transport substrates that are not substrates for the phosphorylation reaction would be expected to inhibit substrate uptake at low substrate concentrations, due to effects on transport, while the maximum velocity of uptake should not be affected-a situation tantamount to competitive inhibition (see later Fig. 15). B. Transport of Substrate Analogs

Most nucleoside and nucleic acid base analogs tested seem to be transported by the same system(s) as their natural counterparts. This conclusion was first indicated by the finding that many nucleoside analogs when present extracellularly accelerate the efflux of uridine from human erythrocytes (Oliver and Paterson, 1971; Cass and Paterson, 1972, 1973), and

278


are presumed, therefore, to permeate the membrane via the same carrier as uridine. Among the several structurally diverse analogs of uridine tested, 2‘deoxyuridine and 5-bromouridine accelerated uridine efflux to about the same extent as uridine, whereas dihydrouridine and pseudouridine were less effective. Since the C-glycosides, pseudouridine and formycin B accelerated uridine efflux, it appears that the N-glycosidic linkage is not crucial for interaction of permeant with the carrier. On the other hand, 6-azauridine and orotidine were found not to accelerate uridine efflux, indicating that the presence of charged groups on the base is not accommodated by the carrier. Cytosine arabinoside and 2’-deoxynucleosides seem to be effective substrates, but other sugar substitutions, including substitutions at either the 2‘ or 3’-hydroxyl groups of ribose, were found to reduce greatly the ability of uridine or cytidine to accelerate uridine efflux. The authors concluded that the sugar moiety is more important than the base in recognition of substrate by the carrier. Paul et al. (1975) further studied the specificity of the nucleoside carrier of human erythrocytes by determining the effect on nucleoside transport of 70 different 9-/3-~-ribofuranosylpurine derivatives containing S, 0, or N atoms at the purine C-6 position and bearing various arylalkyl substitutions. The derivatives were all strongly inhibitory and the degree of inhibition increased with increase in hydrophobicity of the molecules. Many analogs have also been found to inhibit the uptake of nucleosides and purines or of each other by cultured animal cells (Taube and Berlin, 1972; Berlin, 1970, 1973; Strauss, 1974; Zylka, 1976; Plagemann and Erbe, 1974a; Plagemann, 1976; Yang and Visser, 1977; Hakala et al., 1975; Hare and Hacker, 1972; Turnheim et al., 1978; Grunicke et al., 1975). The list of effective substances is too long to be stated here, but some of the more common inhibitory analogs are as follows: cordycepin (3’-deoxyadenosine), 6-mercaptopurine riboside, 6-methyladenosine, 6chloropurine riboside, 6-dimethyladenosine, purine riboside, puromycin, showdomycin, tubercidin, 6-thioguanosine, 6-thioinosine, 5-bromodeoxyuridine, 5-bromouridine, cytosine arabinoside, 5-fluorouridine, 5fluorodeoxyuridine, tricyclic 7-deazanucleoside, formycin B, isoguanine, thioguanine, 2-amino-6-mercaptopurine, 6-mercaptopurine. For additional compounds the reader is referred to Berlin (1970) and Berlin and Oliver (1975). Where investigated, the inhibitions of uptake were of the simple competitive type, as also observed with natural alternate substrates, but such kinetic analyses are difficult to interpret in quantitative terms because of lack of information on the rate-determining step in uptake measurements (see Section 111). Direct information on the transport of these analogs in cultured animal


279

cells is rather limited. Cytosine arabinoside seems to be transported with about the same efficiency as deoxycytidine (Table 11; Mulder and Harrap, 1975; Plagemann et al., 1978a). 5’-Bromodeoxyuridine, purine riboside, 6-mercaptopurine riboside, 6-methylmercaptopurine riboside, and 2’,3’,5’-trideoxyadenosineinhibit thymidine transport in thymidine kinase-deficient Novikoff cells about as effectively as deoxyuridine or deoxyinosine (Wohlhueter ot al., 1979a). The transport of 5’-deoxyadenosine, which is neither deaminated nor phosphorylated, has been directly demonstrated in L1210 mouse leukemia cells (Kqk = 115 F M ; Kessel, 1978). These studies point to the suitability of 5’-deoxyribosides as a class of chemically inert nucleosides for transport studies. 6-([4-Nitrobenzyl]thio)9-P-~-ribofuranosylpurine(nitrobenzylthioinosine)and similar nitrobenzylthionucleosides are potent and specific inhibitors of nucleoside transport; their action will be discussed in detail in Section V,C. 2’-Deoxycoformycin seems to be transported by the nucleoside carriers of mammalian cells, since extracellular uridine and nitrobenzylthioguanine prevent the inhibition of adenosine deaminase activity in whole human erythrocytes or mouse sarcoma 180 cells by 2-deoxycoformycin (RoglerBrown et id., 1978). The drug, however, seems to have relatively low affinity for the nucleoside carrier, since it does not affect the transport of adenosine in ATP-depleted P388 cells, even at a concentration of I mM (Lum et ( I / . , 1979). Slow entry into cells probably is responsible for the relatively slow effect of 2-deoxycoformycin on adenosine deaminase activity in whole cells (Rogler-Brown et a / . , 1978; Lum et a / . , 1979). Similarly, 8-azapurine analogs, such as 8-azaguanine, 8-azaadenine, etc., do not inhibit the uptake of either adenine or hypoxanthine in Novikoff cells (Zylka, 1976). Moreover, the influx of 8-azaguanine into hypoxanthine/guanine phosphoribosyltransferase-deficient cells shows no indication of saturation up to extracellular concentrations of 5 mM (Plagemann and Zylka, unpublished data), suggesting that entry might be solely by nonmediated permeation (see Section 111,E). These results indicate that the C-8 is essential for transport activity of purines. Comparison of the effect of various analogs on the uptake of adenine by rabbit polymorphonuclear leukocytes and its phosphoribosylation in cell lysates indicate that C-2 is equally important, although C-2 substituent groups can be accommodated. Furthermore, the electronic configuration about C-9 was found to be critical for uptake by whole cells, whereas wbonding or interaction with positions 7 and 8 seem more critical to the phosphoribosyltransferase (Berlin, 1970; Berlin and Oliver, 1975). C. Effect of Temperature Arrhenius plots of the uptake of adenosine by rabbit alveolar macrophages show a break at about 25°C with a change in activation energy

280


from 27 kcal/mole below 25°C to 15.7 kcal/mole above 25°C (Berlin, 1973). Similar breaks in Arrhenius plots between 15 and 23°C have been observed for the uptake of uridine and thymidine (Plagemann and Erbe, 1975), and of hypoxanthine and guanine (Zylka and Plagemann, 1975) by Novikoff rat hepatoma cells with corresponding shifts in activation energy from 15-26 to 4-7 kcalhole. Arrhenius plots of uridine uptake by quiescent and serum-stimulated 3T3 cells exhibited a similar type of curvature (Stein and Rozengurt, 1975). These findings suggested the possibility that membrane lipid transitions might affect the movement of transport carriers: greater membrane fluidity facilitating the movement of the carrier. However, no temperature transition was observed for adenosine uptake by polymorphonuclear leukocytes, and partial inhibition of adenosine uptake by macrophages by treatment with colchicine eliminated the break in the Arrhenius curve (Berlin, 1973). More informative, however, is the failure to detect any temperature discontinuities when thymidine transport per se was measured in thymidine kinasedeficient Novikoff cells (Fig. 13). The reason for the discrepancy between transport and uptake determinations has not been elucidated, but is probably related to the fact that uptake rates can be a function of both the rate of transport or phosphorylation (see Section 111). For example, the temperature discontinuity in uptake could reflect a transition from permeation-limited uptake at low temperatures to phosphorylation-limited uptake at higher temperatures. It should be noted that the activation energy estimated for thymidine transport per se (18.3 kcalhole) is similar to those observed for substrate uptake below the transition temperature. Furthermore, relatively low substrate concentrations were employed for uptake measurements (50 p M and below), but it has become apparent that the thymidine:carrier affinity constant as measured by both the zero-trans and equilibrium exchange protocols decreases markedly with decrease in temperature (see Fig. 13; Wohlhueter et a / . , 1979a). Assessment of the temperature response of transport, therefore, requires substrate concentrations well above K . Velocities of thymidine transport measured at a low substrate concentration ( 5 pkf) also yielded a curved Arrhenius plot in conformity with a dissimilar temperature dependence of Vqi and K (Wohlhueter et a / . , 1979a). In addition, linearity of Van’t Hoff plots for the thymidine :carrier affinity constant indicated an apparent enthalpy of 9.3 kcalhole and a lack of thermal discontinuities over the temperature range 4 to 37°C. That the transition temperatures for nucleoside and purine uptake are unrelated to membrane lipid changes is also suggested by the finding that no breaks are detectable in Arrhenius plots for the nonsaturable influx of prednisolone, cytosine, and L-glucose in Novikoff cells (Graff et ul., 1977; activation energies = 20-24 kcalhole).

281

PERMEATION IN ANIMAL CELLS T E M P E R A T U R E I'C)

40

35

30

20

25

10

I5

5

A 32

I

I

33

34 lo3/ T

I

35

36

IOK-')

FIG. 13. Temperature dependence of thymidine influx and exchange. Suspensions of thymidine kinase-deficient Novikoff cells (at about 3 x lo' cells/ml basal medium) were assayed for zero-trans influx of [3H]thymidine at 800 p M ( 0 )and for isotopic exchange with cells preincubated at 1330 p M (A)as described by Wohlhueter et a / . (1979a) (see Figs. 3 and 4). Cell suspensions, substrate solutions, and apparatus were thermally equilibrated before assay at the temperature indicated on the upper abscissa. The time course of appearance of radioisotope in the cell pellets was followed with 12 samplings encompassing 39 to 234 seconds, depending on temperature. Initial velocities were computed by fitting Eq. (7) to the equilibrium exchange data or Eq. ( 1 ) to the zero-trans influx data. For details, see Wohlhueter et al. (1979a). The computed initial velocities are plotted against inverse absolute temperature. The curve is the regression line on pooled influx and exchange data and corresponds to an Arrhenius activation energy of 18.3 kcaVmole (from Wohlhueter et al., 1979a). The listed K ; ; and K'" values as a function of temperature are also from Wohlhueter et al. (1979a).

D. Effect of pH The effect of extracellular pH on the uptake or transport of nucleosides and nucleic acid bases has not been investigated extensively. One recent study (Wohlhueter et al., 1979a)has shown that the velocity of thymidine transport, when expressed on the basis of intracellular HzO space in thymidine kinase-deficient Novikoff cells, increased about 90% with increase in pH between from 6.2 to 7.5. However, the HzO space of the cells was also found to increase somewhat with pH so that the increase in thymidine transport velocity when expressed on a number of cell basis increased only 40% with increase in pH. These data not only raise the question of which dimensions are most appropriate for expressing cellular transport data, but also suggest that observed differences in velocity may perhaps reflect changes in cell volume, rather than actual pH dependence of the thymidine transporter. Conversion to absolute dimensions of per-

282


meation (pmole/second-cm2of cell surface) necessitates relating cell volume to surface area. Assuming a spherical relationship yielded values of 2.18 (at pH 6.2) and 2.25 (at pH 7.5) pmole/second*cm2for thymidine permeation (Wohlhueter et al., 1979a). It should be noted that none of the natural nucleosides have pKs within the range of pH studied. E. Presumptive Cell Clones Defective in Transport

A number of drug-resistant cell lines have been isolated whose resistance has been considered to stem from a defect in nucleoside or purine transport. Breslow and Goldsby (1969) isolated a line of Chinese hamster cells partially resistant to 5-bromodeoxyuridine and unable to take up thymidine, though it possessed as much as 40% of the thymidine kinase activity of wild-type cells. Similarly, Lynch et al. (1977) isolated a 5bromodeoxyuridine/5-fluorodeoxyuridine-resistantline of HeLa cells with normal thymidine kinase activity, but with markedly reduced capacity to take up thymidine and 5-bromodeoxyuridine. In both instances it was concluded that the drug resistance was due to a defect in thymidine/ 5-bromodeoxyuridine transport. Furthermore, the finding that these lines took up other nucleosides, such as uridine, at an unabated rate supported the view that uridine and thymidine are transported by different carriers. Freed and Mezger-Freed (1973) isolated a similar, partially 5-bromodeoxyuridine-resistant, subline of haploid frog cells with apparently normal thymidine kinase activity, but reduced ability to take up thymidine. Furthermore, the cells failed to grow in HAT medium (containing hypoxanthine, aminopterin, and thymidine), but took up uridine, cytidine, and adenine normally. This finding again suggested that thymidine is normally transported by a specific carrier which was defective in the mutant subline, so that the line was referred to as thymidine transport negative (TT-). These investigators also found that it was possible to isolate, in a single step, thymidine kinase-deficient (TK-) clones from the TT- cells, but not from wild-type cells, by exposure to an even higher concentration of 5-bromodeoxyuridine, and suggested that the TT- genotype might be an obligatory intermediate in the isolation of the TKgenotype. In mammalian cells too, it has been found impossible to obtain, in a single step, thymidine kinase-deficient mutant clones by exposure to high concentrations of 5-bromodeoxyuridine (Thompson and Baker, 1973). In a subsequent study, however, Freed and Hames (1976) observed that the TT- frog cells had lost a heat-labile thymidine kinase, which made up about 30% of the total activity of wild-type cells; they suggested that this thymidine kinase participates in transport and is responsible for the reduced uptake of thymidine and the resistance to low concentrations of 5-bromodeoxyuridine. Since it is now clear that the long-term uptake

PERMEATION IN ANIMAL

CELLS

283

rates determined by all these investigators measure the accumulation of phosphorylated intermediates rather than transport, and that all nucleosides are transported by a single carrier in all cultured lines of animal cells investigated, the validity of the interpretation that these three similar 5-bromodeoxyuridine-resistant cell lines represent thymidine transport mutants seems doubtful. It seems more likely that the partial resistance to 5-bromodeoxyuridine and the reduced uptake of thymidine is related to some alteration in thymidine kinase activity of the cells. For example, one could envision that the heat-sensitive thymidine kinase absent from the resistant haploid frog cells is the cytoplasmic enzyme, whereas the remaining activity might represent mitochondrial thymidine kinase, not readily available for the phosphorylation of thymidine entering the cells from extracellular fluid via the plasma membrane-associated nucleoside transport system. Mammalian and chicken cells possess at least one cytoplasmic and one mitochondrial thymidine kinase which differ in kinetic, electrophoretic, and other properties (Littlefield, 1979; W. C. Leung et al., 1975). Similar uncertainties pertain to the interpretation that a line of Chinese hamster ovary cells highly resistant to 8-azaguanine possesses a defective hypoxanthine transport system (Harris and Whitmore, 1974). This line is 300 times more resistant to 8-azaguanine than wild-type cells, but only five to six times more resistant to 6-thioguanine, and exhibits unaltered sensitivity to 6-mercaptopurine and azaadenine. The kinetic properties of the hypoxanthine/guanine phosphoribosyltransferase of the cells ( K , = 1.5 pcLM) and the V,,, for hypoxanthine uptake by whole cells were reported to be about the same as in wild-type cells. Only the K , for hypoxanthine uptake by whole cells differed: 18 p M compared to 7 p M for wild-type cells. These results are difficult to interpret, but are not consistent with the existence of a hypoxanthine transport defect in the mutant cells. First, as pointed out already (Section II1,E) 8-azaguanine is, at best, a very poor substrate for the hypoxanthine transport system, whereas 6-mercaptopurine and 6-thioguanine strongly inhibit hypoxanthine uptake (Zylka and Plagemann, 1975; Zylka, 1976) and transport (Plagemann, unpublished data) and thus seem to be transported by the same carrier as hypoxanthine. Thus, hypoxanthine transport mutants ought to exhibit resistance to 6-mercaptopurine and 6-thioguanine rather than to 8-azaguanine. Second, the reported uptake K,s probably reflect the saturation of the transferase reaction and not the hypoxanthine : carrier affinity constant, which was found to be greater than 1 mM for Chinese hamster ovary cells (see Table 11). Third, a high resistance of cells to a substrate analog is difficult to explain by a slight change in uptake K , without change in V,,,, particularly if the resistance level exceeds several fold the estimated uptake K , of the line. Thus, further

284


work is required to elucidate the mechanism of resistance of this line to 8-azaguanine. Convincing evidence for a nucleoside carrier defect has only been recently reported for a single-step mutant subline (AE,) of the S49 mouse T cell lymphoma line which was isolated on the basis of its resistance to adenosine in t h e presence of an adenosine deaminase inhibitor (Cohen et al., 1979). These mutant cells take up all ribo- and deoxyribonucleosides examined at less than 2% of the rate obtained with wild-type cells and exhibit cross-resistance to various toxic nucleosides, but not to toxic purines or pyrimidines. Hybrids between the mutant and wild-type S49 cells exhibit normal uptake of and sensitivity to nucleosides. It has been reported that some 5-fluorouridine-resistant clones of S49 exhibit a similar nucleoside transport defect (Ullman et a l . , 1979). The properties of these mutant cell lines are strong evidence for the existence of only a single nucleoside carrier in these cells, as well as for the existence of separate purine and pyrimidine carriers. A single gene locus also seems to control expression of a functional nucleoside carrier in sheep erythrocytes (Jarvis and Young, 1978a). Erythrocytes from most, but not all, sheep lack nucleoside transport activity, but surprisingly cells from heterozygotes also fail to take up nucleosides. Thus, the lack of nucleoside carrier activity seems to be a dominant trait and the authors suggest that the gene (Nu') may code for an inhibitor of nucleoside transport rather than for the nucleoside carrier itself. The failure of [35S]nitrobenzylthioinosineto bind to sheep erythrocytes lacking nucleoside transport activity indicates the absence of functional nucleoside binding sites in membranes of these cells (Jarvis and Young, 1978b). F. Comparison to Transport in Other Types of Organisms

Results comparable to those obtained with cultured animal cells or erythrocytes in which nucleoside and purine transport was measured uncomplicated by intracellular metabolism are not available for any other organisms. In most studies, long-term uptake of substrates into cells was measured and little distinction between transport and intracellular metabolism was possible. From analogy to the results obtained with animal cells it seems, therefore, likely that in most studies substrate uptake rates reflected the accumulation of nucleotides rather than of transport. Several studies to which this analogy probably pertains are summarized in the following paragraphs. Based on a 2-minute uptake time-points, uridine uptake in Tetrahynzena


285

is saturable with a K , = 2.3 p M , and competitively inhibited by other ribo- and deoxyribonucleosides, nucleoside analogs, as well as by dipyridamole, but not by uracil or ribose (Freeman and Moner, 1976; Wolfe, 1975). Most intracellular radioactivity was found to be associated with nucleotides. The uptake of thymidine and deoxyadenosine increased during germination of Neurospora crassa conidia (Schiltz and Terry, 1970). Based on 6-minute uptake time points, the K , for thymidine and deoxyadenosine uptake were 30 and 65 p M , respectively. Since thymidine inhibited competitively the uptake of deoxyadenosine, whereas deoxyadenosine had little effect on thymidine uptake, it was concluded that two different uptake systems are involved. Magill and Magill (1973) assessed the effects of various nucleosides on the growth of an adenosine auxotroph of N . crassa. They found that growth of this mutant on adenosine was inhibited by all other nucleosides tested, but not by adenine. They concluded that the results are contrary to the existence of separate purine and pyrimidine nucleoside uptake systems. This conclusion was supported by the finding that many purine and pyrimidine nucleosides competitively inhibited the uptake of labeled adenosine by this auxotroph with Kis between 7 and 28 p M (Magill et a / . , 1974). Based on 5-minute uptake values, the K,s for adenosine and uridine uptake were estimated as 6.2 and 16 p M , respectively. Foury and Goffeau (1975) reported that treatment with cyclic AMP caused an increase in the V,,, for uridine uptake by the yeast Schizosaccharomyces pombe without affecting the uptake K , of 14 p M . These values were based on 5-minute uptake time points. Since the inhibition of energy production by the cells caused a decrease in uridine uptake it was concluded that uridine enters by active transport, but this conclusion is unwarranted, since the estimated uptake rate probably reflected the accumulation of nucleotides rather than of transport. K,s for adenosine and guanosine uptake were found to be much higher (2 and >5 mM, respectively) than that for uridine uptake. Housset and Nagy (1977) reported that the K,s for guanine phosphoribosyltransferase and adenine phosphoribosyltransferase of Schizosaccharomyces pombe (28 and 69 p M , respectively) were much higher than those for the uptake of guanine and adenine by whole cells (0.66 and 0.25 p M , respectively) and that the pH optima for the enzymes and uptake also differed greatly. They concluded these data to rule out the involvement of group translocation in purine transport. They also found that the uptake K,s for adenine and guanine were higher and the V,,, values were lower in adenine phosphoribosyltransferase-deficient and guanine phosphoribosyltransferasedeficient mutants, respectively, than in wild-type cells. These results

286


again suggest that the uptake velocities measured by these investigators represented rates of accumulation of phosphorylated products rather than transport rates. Polak and Grenson (1973) studied purine and pyrimidine uptake in purine and pyrimidine-requiring mutants of Saccharomyces cerevisiae , respectively. Uptake of cytosine was competitively inhibited by hypoxanthine and adenine, and cytosine inhibited purine uptake. The Kis of inhibition were similar to the K,s of their uptake (5-15 pn/J). Uridine and uracil, on the other hand, were without effect. It was concluded that hypoxanthine, adenine, and cytosine are transported by a single carrier, whereas uracil is transported by a different carrier. The literature on nucleoside transport in bacteria, mainly Escherichia coli, is voluminous, but definitive information on the nature of the transport systems is still limited. Transport studies with E . coli and other gram-negative bacteria are complicated not only by the phosphorylation of nucleosides in the cells, but also by the rapid catabolism (deamination and phosphorolysis) of nucleosides in the periplasmic space (Beck et al., 1972; Hochstadt, 1974; Munch-Petersen and Mygind, 1976). Detailed discussion of this work is beyond the scope of the present article and the reader is referred to the review by Hochstadt (1974) and recent publications by the main groups of investigators working in the field (K. K. Leung et al., 1975; Roy-Burman and Visser, 1975; von Dippe et al., 1975; Leung and Visser, 1977; Munch-Petersen and Mygind, 1976; Mygind and Munch-Petersen, 1975; Komatsu and Tanaka, 1973; Doskotil, 1976; McKeown et al., 1976; Munch-Petersen et al., 1979). Because E . coli concentrates radioactivity from labeled nucleosides several hundred fold, nucleoside transport in bacteria has generally been considered an active process, and the operation of at least two nucleoside transport systems (nupC and nupG) has been indicated on the basis of substrate specificity and antibiotic resistance (Munch-Petersen et al., 1979). On the other hand, Rader and Hochstadt (1976) postulate that the ribose moiety of uridine and adenosine is transferred through the membrane as ribose- 1-phosphate by group translocation catalyzed by the appropriate nucleoside phosphorylases. It is thought that the nucleoside phosphorylases have a transmembrane orientation with the base release site on the external face and the ribose-I-phosphate release site on the internal face. The uptake of purines is similarly believed to occur by group translocation involving purine phosphoribosyltransferases located in the periplasmic space and PRPP as cofactor (Hochstadt-Ozer and Stadtman, 1971; Hochstadt, 1974). The uptake of pyrimidines has been postulated to proceed via a similar mechanism involving uracil phosphoribosyltransferase (Hochstadt, 1974). However, here, too, a tandem


287

operation of periplasmic phosphorylases, nonconcentrative transport, and intracellular phosphoribosylation may account for the experimental data, and confirmation of the operation of the postulated group translocations is still needed. One exception to the generalization that nucleoside uptake in all eukaryotes investigated resembles that in cultured animal cells might be studies with the tapeworm Hynienolepidid cestodes. This parasite lacks a mouth and alimentary tract and nutrients enter through the outer surface. Entry of uridine has been reported to occur via an active transport system, since uptake was inhibited by iodoacetate, even though little phosphorylation occurred during 2 minutes of incubation with substrate (Page and MacInnis, 1975). Based on 2-minute uptake values, MichaelisMenten constants between 100 and 200 p M were estimated for the uptake of uridine, thymidine, adenosine, deoxyadenosine, and guanosine. These nucleosides inhibited the uptake of each other in a similar manner, whereas thymine and uracil stimulated the uptake of uridine and thymidine, but not of the purine nucleosides. It was concluded that the purine and pyrimidine nucleosides are transported by different, but overlapping, transport system.

V.

TRANSPORT INHIBITORS AND NACTIVATION

A. Effects of Sulfhydryl Reagents

The preincubation of various types of cultu ed cells with sulfhydryl reagents, such as p-hydroxymercuribenzoate and p-hydroxymercuribenzenesulfonate, causes a marked inhibition of the uptake of various nucleosides and purines (Schuster and Hare, 1970, 1971; Hare, 1975; Plagemann and Richey, 1974; Plagemann and Erbe, 1972; Zylka and Plagemann, 1975; Alford and Barnes, 1976; Barlow and Ord, 1975; Tsan and Berlin, 1971). Attainment of inhibition is concentration, time, and temperature dependent. For example, a maximum inhibition of 85-90% of uridine, thymidine, and hypoxanthine uptake was caused by 70- 100 p M p-hydroxymercuribenzoate, but only after an incubation period of about 5 minutes (Plagemann and Richey, 1974). Adenine uptake was much more resistant to inactivation by p-hydroxymercuribenzoate than nucleoside or hypoxanthine uptake (Zylka and Plagemann, 1975). Since in many of the studies it was demonstrated that the treatment had no effect on the phosphorylating activity of the cells as measured in lysates of treated cells, it was concluded that the observed inhibitions of uptake were due to an effect on substrate transport (Plagemann and Richey,

288


1974). This conclusion seems correct, since p-hydroxymercuribenzoate causes a similar inhibition when transport of uridine was measured in uridine kinase-deficient Novikoff cells (Plagemann et a / ., 1978b). Although the mechanism of inhibition has not been elucidated in detail, the inhibitions are probably a consequence of an interaction of these reagents with sulfhydryl groups of membrane proteins, presumably the carrier. The effect of p-hydroxymercuribenzoate treatment on thymidine uptake is almost completely reversed after 1 hour of incubation of the cells in fresh medium containing 1 mM dithiothreitol (Plagemann and Erbe, 1972). It has been reported that the efficacy of various sulfhydryl reagents in inhibiting nucleoside uptake by cultured hamster and mouse cells is related to their hydrophobicity (Schuster and Hare, 1971; Hare, 1975). This may indicate that the affected sulfhydryl groups are deeply embedded in the membrane, but it is also possible that, in part at least, the inhibition of uptake reflects an inactivation of the respective phosphorylating enzymes. The function of sulfhydryl groups in transport is not understood, but all mechanisms considered for the inactivation of enzymes by sulfhydryl reagents (Boyer, 1959; Webb, 1966) may also apply to effects on transport. I n contrast to the results previously discussed, Eilam and Cabantchik (1976, 1977), Heichal et ul. (1978), and Bibi et al. (1978) reported that treatment of golden hamster MCT cells with p-hydroxymercuribenzene sulfonate (20 p M ) stimulated the uptake of uridine and cytosine arabinoside. On the other hand, N-ethylmaleimide was inhibitory, and p hydroxymercuribenzene sulfonate and N-ethylmaleimide caused a synergistic inhibition of cytosi,ie arabinoside uptake. On the basis of these and other similar results the authors proposed the operation of a complex carrier with four different organomercurial binding sites, one of which was postulated to represent the substrate binding site and two to be SHcontaining modifier sites. This model cannot be generalized at present and seems premature, since contrary results have been reported by other investigators with other cell lines (see above).

B. Effect of Other Nonspecific Inhibitors The uptake of nucleosides and purines by various types of animal cells is directly inhibited by numerous substances which structurally have nothing in common with each other or with the substrate whose uptake they inhibit. By direct inhibition we mean that the inhibition is very rapid (within 1 minute or less) and readily reversed by removal of the inhibitors. Dipyridamole is the classical inhibitor of this type, and its effect on


289

nucleoside and hypoxanthine uptake has been demonstrated in many studies (Scholtissek, 1968: Plagemann and Roth, 1969; Plagemann, 1971a; Plagemann and Erbe, 1972, 1974a; Zylka and Plagemann, 1975; Peters and Hausen, 1971; Schrader et a / . , 1972; Koos and Pfleger, 1972; Rau and Schlotissek, 1970; Crifo et al., 1973; Kessel and Hall, 1970; Kessel and Dodd, 1972; Turnheim et a / . , 1978). Its effect on nucleoside transport was first deduced from its effect on adenosine deamination in dog erythrocytes (Kiibler and Bretschneider, 1963, 1964). The effect of dipyridamole on adenine uptake observed in various animal cells has been more variable. Adenine uptake was found to be inhibited (although to a lesser extent than nucleoside and hypoxanthine transport) in human platelets (Sixma et a / . , 1973) and in Novikoff rat hepatoma cells (Zylka and Plagemann, 1975), but little or no significant effect was detected in chicken embryo cell cultures (Schlotissek, 1968), human platelets (Rozenberg et d . , 1971), perfused guinea pig and rat heart (Kolassa et d., 1970), L1210 mouse leukemia cells (Kessel and Dodd, 1972), and various transplantable mouse ascites tumors in in vitro suspension (Henderson and Zomber, 1977). Other general inhibitors of nucleoside and purine uptake are cytochalasin B (Plagemann and Estensen, 1972; Takana et al., 1975; Plagemann et a / . , 1975b, 1978c), papaverine (Plagemann and Sheppard, 1974; Sixma et a / . , 1973, 1976; Woo et a/., 1974), theophylline (Plagemann and Sheppard, 1974; Rozengurt and Jiminez de Asua, 1973; Benedetto and Casson, 1974: Woo et al., 1974), prostaglandins (Plagemann and Sheppard, 1974; Rozengurt and Jiminez de Asua, 1973), colchicine (Mizel and Wilson, 1972; Berlin, 1973; Plagemann and Erbe, 1974b; Zylka and Plagemann, 1975), 2-mercapto- 1-/3-4-pyridethylbenzirnidazole(Skehel et al., 1967; Nakata and Bader, 1969), streptovaracin (Tan and McAuslan, 1971), phloretin and phloridzin (Lemkin and Hare, 1973), aflatoxins and sterigmatocystin (Kunimoto et al., 1974), podophyllotoxin (Loike and Horwitz, 1976), and acronycin (Dunn et al., 1973). The inhibitions of substrate uptake by cultured cells were considered to be caused by an inhibition of the transport step since, where investigated in detail, the inhibitors had little if any effect on the phosphorylation of the substrates, either in whole cells or in cell lysates (for additional details, see Plagemann and Richey, 1974). This conclusion has been confirmed by direct transport measurements for some of these inhibitors. It has been shown that dipyridamole inhibits the transport of deoxycytidine in deoxycytidine kinase-deficient L 12 10 leukemia cells (Kessel and Hall, 1970), that dipyridamole and papaverine inhibit the transport of hypoxanthine in hypoxanthine-guanine phosphoribosyltransferase-deficient Novikoff cells (Zylka and Plagemann, 1975), and that dipyridamole,

290


papaverine, and cytochalasin B inhibit the transport of uridine and thymidine in kinase-deficient or ATP-depleted Novikoff cells (Plagemann et al., 1976, 1978b,c). Recent studies have shown that dipyridamole causes a mixed type of inhibition of uridine transport with a Ki,int= 12 p M and Ki,slope= 3 p M (Plagemann et al., 1978b; see Fig. 14). This finding is in contrast to the simple competitive inhibition reported earlier for the inhibitions of nucleoside and purine uptake by various of the above substances (Plagemann and Richey, 1974). This competitive inhibition can now be explained on the basis of the finding that substrate uptake rates reflect rates of phosphorylation rather than transport rates (see Section 111). These inhibitors render transport the rate-determining step in uptake, but with increase in substrate concentration the effect of inhibitor is overcome, phosphorylation again becomes rate-determining, and the same apparent

T I M E ISECI

DIPYRIDAMOLE IVM I

FIG. 14. Kinetics of inhibition of uridine transport by dipyridamole in uridine kinasedeficient Novikoff rat hepatoma cells. The experiment was conducted as described in the legend to Fig. 3, except that, where indicated, the [3H]uridine solutions were supplemented with dipyridamole to yield final concentrations of 5 and 15 p M . The final concentrations of [3H]uridine were 20, 40, 80, 160, 320, 640, and 1280 p M (400 cpm/pI, irrespective of concentration) and the intracellular and extracellular trapped water spaces were 28 and 3.7 $sample pellet, respectively. Data are from Plagemann et (11. (1978b), but have been reanalyzed by fitting Eq. ( I ) to the pooled data for each dipyridamole concentration with all R parameters held equal. The theoretical curves for S, = 80 p M uridine plus 0, 5 , and 15 pM dipyridamole are illustrated in (A). The best fitting parameters for 0, 5 , and 15 pM dipyridamole were K = 202 2 15, 598 2 24, and 648 2 101 pM,respectively; V = I I .8 2 0.3, 9.3 2 0.2, and 5.3 2 0.5 pmoleipl cell H,O.second, respectively. The correlation coefficients (r,,o) were 0.9422, 0.9893, and 0.8632, respectively. The kinetic parameters were replotted in (B) as described by Segel (1975).


291

maximum uptake velocity is attained as in the absence of inhibitor. A computer simulation of the effect of a theoretical transport inhibitor on thymidine transport and phosphorylation operating in tandem is illustrated in Fig. 15 [see legend for Eqs. ( l 3 ) - ( 17)]. This subject is discussed in more detail by Wohlhueter and Plagemann (1980). The molecular basis of the inhibition of transport by these substances is not known. The finding that dipyridamole causes a decrease in maximum velocity of nucleoside transport (Fig. 14) suggests that it affects the movement of the carrier, but even this conclusion is not unequivocal, since alternate substrates have also been observed to give a mixed type of inhibition (see Section IV,A). One possibility suggested by the lipid solubility and broad specificity of the inhibitors-many inhibit hexose and phosphate uptake as well as nucleoside and base transport (Plagemann and Richey, 1974)-is that they either affect the structure of the membrane or directly interact rather nonspecifically with integral membrane proteins including transport carriers (Plagemann et al., 1977). This view is supported by the finding that dipyridamole and cytochalasin B also inhibit the nonsaturable permeation of cytosine and L-glucose (Graff et a / . , 1977). Any relationship between lipid solubility of these substances and their efficacy as transport inhibitors, however, has not been ascertained as yet. Many of the substances in question have additional toxic affects on cells, but none of these effects seem to result from or be related to an inhibition of nutrient transport by these inhibitors (Plagemann and Richey, 1974). Because of the wide range of different substances already found to inhibit the transport of various substrates it is predictable that many other substances will be found to have such effect. Thus, great caution is required in studies involving the use of radioactively labeled nucleosides and purines and other precursors to assess the effect of inhibitors on metabolism and macromolecular synthesis. Another group of substances that has been found to inhibit the uptake of nucleosides, purines, and other substrates by animal cells are organic solvents such as ethanol (Scholtissek, 1974; Plagemann and Erbe, 1974b), phenethyl alcohol (Plagemann and Roth, 1969; Plagemann, 1970; Crifo et al., 1973), and dimethyl sulfoxide (Scholtissek, 1974; Collins and Roberts, 1971). Here again the finding that the phosphorylating activity of treated cells was unaltered has led to the view that the effect on uptake is mediated at the transport step, but direct evidence is still lacking. These substances have in common the ability to inhibit substrate uptake only at concentrations approaching those that cause outright lysis of the cell. Thus, it seems likely that a transport inhibition is caused by a perturbation in membrane structure, probably due to an effect of these compounds on

292


3

2

0

5

llS,

FIG. 15. Computer simulation of transport and phosphorylation operating in tandem. As a simple model we take: t

s , e s,-

P

s-P,

(13)

in which reaction "t" is a symmetrical, facilitated transport system, operating bidirectionally ( K , ,

Current Topics in Membranes and Transport, Volume 14

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