Chloride in the human erythrocyte

Proo. Biophys. Molec. Biol., 1976, Vol. 3l, pp. 145-164. Pergamon Press. Printed in Great Britain.

CHLORIDE IN THE HUMAN ERYTHROCYTE DISTRIBUTION AND TRANSPORT BETWEEN CELLULAR AND EXTRACELLULAR FLUIDS AND STRUCTURAL FEATURES OF THE CELL MEMBRANE MADS DALMARK Department of Biophysics, duliane Mariesvej 28, II, DK-2100 Copenhacden O, Denmark

CONTENTS I. INTRODUCTION

DISTRmUTION III. CHLORIDETRANSPORT IV. STRUCTtmALFEATURESOFTim CELLMEMBRANEANDCHLORIDETRANSPORT II. CHLORIDE

V. CHLORIDE TRANSPORT MODELS

REi~ENCES

145 147 152 158 160 162

I. INTRODUCTION The cell and its surrounding fluid is a dynamic system. The structure of this system is schematized as two compartments (the cellular and extracellular fluids) separated by a third one (the cell membrane). The inorganic salt composition of the cellular and extracellular fluid is different, and the ability to regulate this difference by a transport of ions across the cell membrane is characteristic of the intact cell. The cellular salt concentration is a function of (1) the presence of cellular constituents other than inorganic salt, (2) the properties of the cell membrane and (3) the composition of the extracellular fluid. This paper reviews data on (1) chloride distribution between cellular and extracellular fluid, (2) chloride transport across the cell membrane, and (3) structural features of this membrane in order to evaluate to what extent these data contribute to our understanding of the mechanisms by which the cellular chloride concentration is regulated in the human erythrocyte. The human erythrocyte is the descendant of the haematopoietic stem cell in the red bone marrow (reviewed by Berlin and Berk, 1975). During differentiation and maturation the stem cell becomes smaller, the nucleus denser, and the cytoplasma fills with haemoglobin. Ultimately, the nucleus is extruded, and the cell enters the circulating blood. After 1 or 2 days in the circulation the cells have completely lost all remnants of the protein-synthesizing apparatus (reticulin) and the cell is a matured erythrocyte (red blood cell). The normal red cell life span in the circulation is of the order of 120 days. The senescent erythrocyte is removed from the circulation by the action of the reticuloendothelial cells in liver, bone marrow and spleen and sequestrated. Some metabolic products of the sequestrated cell, e.g. iron, are reused in the formation of new erythrocytes. Erythrocytes are convenient for studies of (1) distribution of material between cellular and extracellular fluids, (2) transport processes of material across the cell membrane, and (3) structural features of the cell membrane: a sample of red cells is easily obtained by puncture of an arm vein, a droplet of blood contains several millions of single cells with a rather uniform shape and size, chemical analyses of the cellular constituents are easily performed since the cells can be sampled almost without any extracellular fluid, and the membrane material can be analyzed after lysing the cells, e.g. by suspending the cells in hypotonic solutions. A disadvantage in such studies is the small size 145

146

MADSDALMARK

of the cell (8 × 2 #m in the rim) which impedes measurements of the electrical membrane potential difference. It is helpful in studies of chloride distribution and transport to be able to alter the cellular chloride concentration at a normal cell volume and in absence of other anions than chloride as this simplifies the experimental conditions. If this is to be done at a constant pH and temperature, then the cellular concentration of neutral salt like KC1 must be altered, so that electroneutrality is maintained. A change in KC1 concentration under these conditions is not readily performed, since the potassium permeability in human red cells is low (McConaghey and Maizels, 1962; Rinehart and Green, 1962; Garrahan and Rega, 1967). Only when the potassium permeability is increased by some technique, KC1 rapidly crosses the cell membrane and the ionic strength changes at a constant cell volume, pH and temperature without substituting other anions for chloride. Some lipophilic macromolecular compounds (ionophores) increase the cation permeability in red cells. One of these compounds is named nystatin. It is an antifungal agent with a molecular weight of approximately 900 produced by the bacteria of the genus Streptomyces. It is possible with nystatin to increase the potassium permeability for only a short period of time during the preparation of the cells, as nystatin can be removed from the cell membranes and a low potassium permeability is restored (Cass and Dalmark, 1973). The procedure for preparing human red cells with various KCI contents is described as follows (Cass and Dalmark, 1973): Nystatin (Mycostatin®--Squibb & Sons, London) is added as a methanolic solution (5 mg Mycostatin®/ml methanol) to 90 ml of a red cell suspension (hematocrit 0.03q3.05 v/v, pH 7.2) at 0°C to obtain a final nystatin concentration of 50 #M. At this concentration, pH and temperature the permeability coefficient of monovalent cations like potassium increases from 10 -11 cm/sec to 10-6 cm/sec. Under these conditions the cellular KC1 concentration is altered by dialysis of the cells against salt solutions of various KC1 concentrations. The volume of the cells is kept constant by addition of 27 mM of the impermeant sucrose to the medium, since the osmolality of the sucrose solution equals the osmolality of the impermeant cellular constituents. After equilibration of salt and water across the cell membrane, the nystatin is removed by washing the cells with nystatin-free salt solutions at room temperature, and a low permeability of the cell membrane towards cations is restored. Figure 1 demonstrates the cellular salt concentration of nystatin-treated cells as a func(a)

(b)

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FIG. l(a) and (b). Intracellular salt concentration as a function of extracellular salt concentration (0°C, pH 7.2) after reversible treatment of human red cells with nystatin: the sum of intracellular concentrations of K and Na against the sum of the (K + Na) concentrations in medium (Fig. la), and the intracellular chloride concentration plotted against the chloride concentration in medium (Fig. lb). The cells were modified by dialysis during a period of nystatin-induced increase of ion permeabilities at 0°C, and after establishment of ionic equilibrium nystatin was removed by washing cells with nystatin-free medium. Media contained 27 mM sucrose, 1 mM NaC1, and 0-500 mM KCI. (Cass and Dalmark, 1973.)

Chloride in the human erythrocyte

147

tion of extracellular salt concentration (0-500 rnM KC1) at constant cell volume, pH and temperature in absence of other anions than chloride. The preparation time is 40-60 min. Experiments by the present author with cells at ionic strengths different from 0.15 have been carried out with nystatin-treated red cells. It is not understood how nystatin exerts its action on the molecular level. Nystatin is a polyene compound which contains a polyhydroxylic lactone ring with a hydrophilic and a hydrophobic face. Experiments by Cass et al. (1970) and Finkelstein and Holz (1973) indicate that several nystatin molecules combine to form a pore which spans artificial lipid bilayers. They suggest that the hydroxylic groups line the pore, while the hydrophobic faces of the molecules are embedded in the lipids surrounding the pore. The radius of the pore allows passage of molecules with a Stokes-Einstein radius of less than 4 A. The lipid bilayers are anion selective when nystatin is added to the bulk phases on both sides of the membrane; but cation selective when nystatin is added to only one of the bulk phases (Marty and Finkelstein, 1975). A large increase of the cation permeability is demonstrated in red cells when nystatin is added to the suspension medium (Cass and Dalmark, 1973). The topics of the review will be presented in the following sections concerning (1) chloride distribution, (2) chloride transport, and (3) structural features of the cell membrane. In the last section, models of the chloride transport are discussed. II. CHLORIDE DISTRIBUTION This section deals with the steady-state distribution of chloride between cells and the surrounding medium. The distribution ratio (rcl) is defined as rc, = (Clc/l/V~)/(Clm/Wm),

(1)

where Clc, Clm are the chloride content (mmol) and W~, Wm the water content (kg) in cells and medium. At constant chloride concentration in medium, the rci alters with a change in the cellular chloride content and/or in the cellular water content. As cellular chloride and water content depends on the presence of other cellular constituents, this section is divided into four paragraphs concerning (1) cellular constituents other than chloride and water, (2) cellular chloride content, (3) cellular water content, and finally (4) the chloride distribution ratio (to) and the electrical membrane potential difference as calculated from rm. The amount of cellular constituents is expressed per 3 x 1013 cells which contains 1 kg cell solids at normal ionic strength (Funder and Wieth, 1966a), since the dry weight of cells is easily determined and is linearly related to the number of cells. The dry weight of a given number of cells is determined as the residual weight of the cells after evaporation of the cell water (24 hr, 105°C). The dry weight of a given number of cells remains practically constant (less than + 1~) in contrast to the marked change in cell wet weight with pH and temperature. The cell solids constitute approximately 34~o of cell wet weight at normal ionic strength and neutral pH. The solids consist of haemoglobin, various enzymes, organic phosphates, inorganic ions and membrane material. (l) Haemoglobin constitutes 90~ (wt/wt) of the cell solids (cf. for example Funder and Wieth, 1967; Dalmark, 1975b). Haemoglobin is a protein with a molecular weight of 64450 (Braunitzer et al., 1961). The cellular concentration of haemoglobin is approximately 7 mM at normal cell water content. As haemoglobin contains several hydrogen ion titratable groups at neutral pH, the protein carries a net electrical charge--positive or negative--at various values of pH. This net electrical charge on the impermeant, macromolecular protein in the cells has important implications for the passive distribution of ions between cells and medium, since the driving forces tend to move ions towards their equilibrium, i.e. towards a distribution where the electrochemical potentials of ions are equal in cell water and medium. Only ions which permeate the cell membrane are able to redistribute themselves in accordance with the driving forces. Furthermore, the new equilibrium of permeable ions occurs after different time intervals in accordance

148

MADS DALMARK

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pH 9.4

7

pH 5.4

pH 7.2

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,

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I013 cells

FIG. 2. Influence of external pH on the cellular K + Na and C1 content of human red cells (0°C) from four donors (O, O, [~, ~) at constant composition of the medium (150mM KC1, 1 mM NaCI, 27 mM sucrose) (Dalmark, 1975b).

with their permeability coefficients after a perturbation of the cellular pH. The quantitatively most important inorganic ions in red cells are potassium, sodium, chloride and bicarbonate. The human red cell has a low cation permeability (cf. for example Dalmark and Wieth, 1970; Cass and Dalmark, 1973). Consistent with this is the observation that the cellular sodium and potassium content is unchanged after 8 hr at 0°C at various values of pH in the cell suspension medium (Fig. 2). The sum of the sodium and potassium content in a mean population of human red cells is fairly constant at 290 300 mmol/3 x 1013 cells or approximately 150 rnM at a normal cell water content. In contrast, the cellular chloride content changes within minutes with pH (Fig. 2) and temperature. (2) Cellular chloride content decreases with increasing pH at constant temperature (Warburg, 1922; Van Slyke et al., 1923). Cellular chloride content is determined by the total net charge on the cellular buffers when cells are titrated with base or acid in the absence of anions other than chloride. In nystatin-treated red cells the change of cellular chloride content with pH is estimated to be -134mmol/3 x 10 ~3 cells'pile (Dalmark, 1975b). This value corresponds to - 10 mmol/mmol haemoglobin" pHc assuming that haemoglobin is the only cellular buffer species. The estimated value is consistent with the buffer capacity of haemolyzed cells (Harris and Maizels, 1952; Siggard-Andersen and Sailing, 1971) and purified haemoglobin (Antonini et al., 1965; Tanford and Nozaki, 1966; Gary-Bobo and Solomon, 1968; Rollema et al., 1975). In the above calculation the cellular buffer capacity is expressed as the buffer capacity of haemoglobin. This represents, naturally, a simplification since the buffer capacity of other impermeants is ignored. However, these impermeants contribute but little to the cellular buffer capacity of freshly drawn cells (Harris and Maizels, 1952; Duhm, 1972), and during the preparation period the nystatin-treated cells may have lost some buffer species of low molecular weight (Deuticke et al., 1973). Thus, nystatin-treated cells may have a slightly lower cellular buffer capacity than freshly drawn cells. (3) The cellular water content depends on the osmolality in medium and the number of osmols in the cells. It is generally believed that water is at equilibrium between cells and medium, since (i) the water permeability of the cell membrane is high (approximately 10 -2 cm/sec, 25°C) (Sidel and Solomon, 1957; Paganelli and Solomon, 1957), (ii) the membrane is unable to resist a hydrostatic pressure difference of more than 2-3 mm water (Rand and Burton, 1964), and (iii) the water activity in cells and medium as defined by freezing point depression appears to be equal (Collins and Scott, 1932; Brodsky et al., 1956; Appelboom et al., 1958; Maffly and Leaf, 1958; Williams et al., 1959).

Chloride in the human erythrocyte

149

The cellular water content varies with pH and temperature in accordance with the change of chloride in the cells (Dalmark, 1975b). Van't Hoff's law predicts that the ratio (Dalmark, 1975b, appendix, eqn. 3) between the chloride (AClc) and the concomitant water shift (AW~) is proportional to the sum of the molalities of the dissolved molecules in the medium (nm/W.) ACIdAW~ =

ab(n,,/W,,),

(2)

where the proportionality factor, ab, is unity under the assumption that (i) the osmotic coefficients of chloride are equal in cell water and medium, (ii) all chloride is dissolved in the cell water, and (iii) all cell water acts as solvent water for chloride. Figure 3 demonstrates that the measured ratio is proportional to the sum of the molalities of the dissolved molecules in the medium at ionic strengths between 0.07-0.6. However, the proportionality factor, ab, is larger than unity. This means that (a) the cellular water shift following a given chloride shift decreases with increasing osmolality, but (b) the magnitude of the water shift is less than predicted. This observation is found both when the cells shrink or swell following titration of the cells with base or acid. In spite of considerable work the reason for the discrepancy between predicted and observed water distribution is still unknown (reviewed by Dalmark, 1975b). (4) The chloride distribution ratio at various values of pH and temperature is described by the equation ro = (CI~ + ACI~)/(W~+

AW~)/(CI./W.),

(3)

where the symbols are defined as in eqns. 1 and 2. The rci as a function of pH is depicted in Fig. 4 at various KC1 concentrations in cells and medium. The composition of the medium at each single KC1 concentration is kept constant at the various values of pH, so that only AC1c and AW~ vary with pH. At high osmolality in the medium

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FIG. 3. The chloride concentration (mmolal) of the transported fluid during changes of pH of a human red cell suspension (0°C) as a function of the sum of the mmolal concentrations of the dissolved molecules (K + Na + CI + sucrose) in medium at different KC1 concentrations in cells and medium. The cells--except at normal ionic strength--were prepared by the nystatin technique. The composition of the media (75--600 mM KCI, I 1 mM NaC1, 27 r t ~ sucrose) at each single KCI concentration was constant at the different values of pH. The following equation was obtained by regression analysis of the chloride concentration of the transported fluid (I0 vs the mmolal concentration of the dissolved molecules in the medium (X): Y = 1.37 X (S.D. 0.10) + 10(S.D. 78), r = 0.98. The dashed line has a slope of unity. (Dalmark, 1975b.)

150

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FIG. 4. Comparison between calculated (--) and measured (e) chloride distribution ratios after titration of human red cell suspensions with base or acid at 4 different KCI concentrations in cells and medium (0°C). The cells--except at normal ionic strength--were prepared by the nystatin technique. The composition of the media (75~500mM KCI, 1 mM NaCI, 27 mM sucrose) at each single KC1 concentration were constant at the different values of pH. The calculated chloride distribution ratios were determined from the measured ra at pH 7.2 (0'~C), a buffer capacity of the cell constituents of 134 mmol/3 × 1013 cells pHc~ (~ 10 mmol/mmol haemoglobin pH,.,,), a hyperosmotic fluid transport (cf. Fig. 3) during titration of the cells, and thc assumption that the hydroxyl-ion distribution ratio between cell water and medium was of the size of the chloride distribution ratio. (Dalmark, 1975b.) (300-600 mM KCI) the cellular water shift is small (Fig. 3). In this case almost linear rct curves are observed, since the chloride shift is determined by a pH-independent cellular buffer capacity in this p H range. At low KCI concentrations (75-150mM) the rcl is strongly pH-dependent with S-shaped curves, since (a) the magnitude of the buffer capacity is large relative to the total cellular chloride content, (b) the cellular water shift is large at low osmolality, and (c) the difference between cellular and medium p H increases as ro differs from unity (Funder and Wieth, 1966b). The r a curves shift towards lower values of p H with increasing temperature (Dalmark, 1975b), since the cellular chloride content decreases with temperature and the buffer capacity of the cellular buffers is temperature-independent between 0-38°C (Antonini et al., 1965). The isoelectric p H of the cellular constituents can be estimated from the rc] graphs. The is oelectric p H of a protein solution is defined as the p H at which'the net electrical charge on the protein is zero (Sorensen et al., 1928). The isoelectric pH of the cellular constituents is estimated as the external p H at which rcl equals unity, when (1) cellular chloride content (concentration) equals the potassium and sodium content (concentration), (2) cellular constituents other than NaCI and KC1 are balanced osmotically by a non-electrolyte in medium, and (3) chloride is the only anion in medium. It is generally believed that chloride is adsorbed only to a minor extent to cellular constituents, i.e. cell water volume is the major cellular chloride c o m p a r t m e n t (Warburg, 1922; Van Slyke et al., 1923; Adair and Adair, 1934; Harris and Maizels, 1952; Funder and Wieth, 1966b; Tandford and Nozaki, 1966; Chiancone et al., 1972; Antonini et al., 1972; Rollema et al., 1975). Anions other than chloride are adsorbed to the cellular constituents, since r a is a function of the concomitant anions in medium and cells (Dalmark and Wieth, 1972). The isoelectric p H in these cases decreases as a function of inorganic anions adsorbed. The isoelectric p H of the cellular constituents in 150mM KC1 has been estimated to be 7.2 (0°C) by Cass and D a l m a r k (1973). This value is close to the isoelectric p H

Chloride in the human erythrocyte

151

of oxyhaemoglobin (7.15) at the same ionic strength at 4°C (Winterhalter and Colosimo, 1971). The isoelectric pH decreases with temperature to 6.6 at 38°C (Dalmark, 1975b). The isoelectric pH of oxyhaemoglobin has been estimated to 6.6 (38°C) by Van Slyke et al. (1923). Change of rct with pH in whole blood has been determined several times at body temperature (reviewed by Funder and Wieth, 1966b). These experiments are carried out under conditions where the electrolyte concentration and osmolality of the medium varied with pH. Consequently, these results are not strictly comparable with those of Dalmark (1975b). However, the isoelectric pH of the cellular constituents in whole blood experiments is the cellular pH where rc~ equals approximately 0.9, since the concentrations of inorganic salts (NaC1, KCI, NaHCO3) in medium have to exceed the cellular concentrations of inorganic salts (KC1, NaC1, KHCO3) with approximately 15 mM in order to balance the osmotic pressure of cellular constituents other than inorganic salts. The cellular impermeants in the presence of nystatin are osmotically balanced by a solution of 27 mM sucrose, which has an osmolality equal to that of a solution containing approximately 15 mra NaC1. Using this calculation procedure the average isoelectric pH is estimated to 6.6 in the four whole blood experiments reviewed by Funder and Wieth (1966b). This value agrees with those given by Van Slyke et al. (1923) and Dalmark (1975b). The ionization enthalpy of the groups which determine the pH dependence of rcl can be estimated from the temperature shift of the pH where rc~ has a given value. The ionization enthalpy is estimated to 6 kcal/mol (~25 kJ/mol.) between 0-38°C at neutral pH (Dalmark 1975b). The ionization enthalpy of the hydrogen ion titratable groups of purified haemoglobin in the same pH range has been estimated to 6.0-6.5 kcal/ mol (Rossi et al., 1963; Antonini et al., 1965). This indicates that the change of cellular chloride content with pH and temperature is dominated by the buffer characteristics of haemoglobin which contains imidazole groups (Tanford and Nozaki, 1966). The steady-state distribution of chloride across the cell membrane is generally believed to express a thermodynamic equilibrium of chloride, i.e. the electrochemical potentials of chloride in cell water and medium are equal. In this case the electrical membrane potential difference equals the chloride equilibrium potential as calculated from the Nernst equation: V,. = (RT/zF)ln[(CI)m/(C1)~],

(4)

where (C1)m, (C1)c are the activities of chloride in medium and cell water, R the universal gas constant, T the absolute temperature, z the valency of chloride, and F the Faraday constant. Measurement of membrane potential difference and knowledge of chloride activities in cells and medium are required in order to test the validity of this statement. The single ion activity coefficients are not measurable, but the variation of rn and rc~ with pH indicates that the activity coefficient of chloride in cell water is close to the coefficient in medium at normal ionic strength (Funder and Wieth, 1966b). Direct measurement of the membrane potential difference in mammalian red cells is technically difficult because impalement of the tiny cells with electrodes may lead to relatively severe ionic leaks (Lassen, 1972). In order to minimize the problem of a rapid discharge of the membrane potential difference an electrophysiological study of the large red cell (50 x 80/zm) from the giant salamander, Amphiuma, was performed by Hoffman and Lassen (1971). In these cells the measured potential differences at various values of pH can be calculated from the Nernst equation and the chloride distribution ratio under the assumption that the activity coefficients of chloride in cell water and medium are g.qual (Lassen, 1972). In spite of the technical difficulties measurements of the membrane potential difference across the human red cell membrane have been performed. The membrane potential difference agrees apparently with the predicted value from the Nernst equation and

152

MADS DALMARK

the measured chloride distribution ratio at pH 7.4 (37°C) (Lassen and Sten-Knudsen, 1968; Jay and Burton, 1969). In conclusion, (1) chloride is apparently at thermodynamic equilibrium across the red cell membrane, and (2) in bicarbonate-free, nystatin-treated human red cells the change of the cellular chloride content with pH and temperature appears to be dominated by the acid-base properties of haemoglobin. III. CHLORIDE TRANSPORT Considerations of electroneutrality dictate that transport of chloride across the cell membrane is followed by a transport of anions in the opposite direction or a transport of cations in the same direction. In the former case chloride might exchange for another chloride (chloride self-exchange) or for another anion (chloride net transport by exchange). In the latter case chloride is transported together with counterions (chloride net transport with counterions). The human red cell exchanges anions more readily than cations. This is demonstrated by Dalmark and Wieth (1970) with red cells suspended in a plasma-like medium (0°C). The half-time of Z2Na uptake is in the order of days, but the half-time of 36C1 efflux is in the order of seconds. The chloride permeability is 104 times the sodium permeability at 0°C (pH 7.4). The selectivity of the cell membrane increases with temperature being 105 at 10°C (Dalmark and Wieth, 1970) and about 107 between room and body temperature (Tosteson, 1959; Wieth, 1970; Brahm, 1975). The permeability coefficients for sodium and potassium (cf. for example, Cass and Dalmark, 1973) are close to values found in artificial lipid bilayers prepared from red cell membrane lipids (Andreoli et al., 1967). The low permeability coefficients for cations means that the salt diffusion of KC1 and NaC1 is 104 times smaller than the chloride transport by self-exchange. The data indicate that the human red cell membrane has a structural feature which facilitates chloride exchange relative to cation exchange. This facilitated chloride exchange has the following characteristics: (1) a marked temperature dependence (Dalmark and Wieth, 1972), (2) saturation kinetics (Gunn et al., 1973; Cass and Dalmark, 1973), (3) inhibition by other halides which indicates substrate specificity (Dalmark, 1976), (4) an unexpected pH dependence (Gunn et al., 1973; Dalmark, 1975a; Brahm, 1975), (5) a distinct discrepancy between the chloride permeability coefficients calculated from (a) chloride exchange studies with radioactive chloride at anion equilibrium and (b) salt diffusion studies in the presence of a concentration gradient of salt with modified red cells having a high cation permeability (Harris and Pressman, 1967; Hunter, 1971), consistent with (6) an electrical resistance of the cell membrane which is several orders of magnitude higher than predicted from chloride exchange studies (Hoffman and Lassen, 1971; Lassen, 1972; Lassen et al., 1974). The chloride transport has mainly been studied by measuring the specific activity in the medium as cellular 36C1 exchanges for nonradioactive chloride in medium under steady-state distribution of chloride between cells and medium. The temperature dependence of chloride exchange is such that below 10°C the half-time of isotope movement is greater than 2 sec. It is therefore possible to obtain samples of cell-free medium by the manual filtration technique of Mawe and Hempling (1965), modified to reduce hemolysis (Dalmark and Wieth, 1972). At higher temperatures this method gives poor resolution and a filter-flow apparatus must be used (Tosteson, 1959; Brahm, 1975). The progress of such a manual filtration experiment is described as follows (Dalmark and Wieth, 1972): Freshly drawn human red cells are washed and incubated with 36C1 in a given salt solution at a given pH and temperature. After equilibration of 36C1 across the cell membrane, the cells are packed by centrifugation. The efflux experiments are carried out with these labelled cells. The set-up of the efflux experiments is shown in Fig. 5. The efflux medium has a composition, a pH, and a temperature identical with the labelling medium. At time zero approximately 200 mg of labelled, packed cells are injected into 40 ml of vigorously

Chloride in the human erythrocyte

153

Scherno'l'ic diogrorn of the fUtro'fion ossernbly

3

[2J FIG. 5. Schematic diagram of the filtration assembly. The inset (A) shows the sampling of filtrate (0.5-1.0ml) from the dilute cell suspension through the Luer-Lock needle (1), and the filter holder (3) and (4), into a 10 ml syringe (9). (1) Blunt Luer-Lock needle (ga. 13), (2) ring of ice in the external trough of filter holder, (3) lower half of filter holder (Swinnex-25, Millipore), (4) upper half of filter holder, (5) silicone rubber gasket for filters, (6) micro-fibre glass disc prefilter (Millipore AP 2502500), (7) cellulose ester filter with a mean pore size of 1.2/~m (Millipore RAWP 02500), (8) porous filter support, (9) 10 ml syringe employed for filtration, (10) plunger of syringe. The gasket (5) and the filters (6) and (7) are tightly fitted to the filter support (8) when assembled. (Dalmark and Wieth, 1972.)

stirred efflux medium, and the 36C1 in the cells begins to exchange with nonradioactive chloride in the medium. At intervals, cell-free etttux medium is sampled by rapid filtration of the cell suspension: the cell suspension is sucked through the cannula by pulling the plunger of the syringe, the cells are kept back in the Millipore ® filter, and cell-free efflux medium is sampled in the syringe. During one experiment about 8 samples of cell-free efflux medium are collected at appropriate time intervals. The radioactivity in the samples is measured and the results expressed as shown in Fig. 6. This figure shows the rate of 36C1 efflux towards isotopic equilibrium. The ordinate indicates on a log scale that fraction of the initial radioactivity which is retained in the cells at the time of sampling. The abscissa indicates time after injection of labelled cells into the efflux medium. The linearity of the curves demonstrates that the system is well described by a dosed, two-compartment model with constant volumes. The halftime of chloride efflux is determined from the curves as the interval between injection of the cells and the point at which radioactivity in the cells reaches half of its initial value. The numerical value of the slope, b, of a curve is equal to the sum of the rate coefficients for isotope efflux (k°) and influx (ki). The slope b approaches k °, when the haematocrit is low. The rate coefficient k ° (time-1) indicates that fraction of the cellular 36C1 content which leaves the cells per unit time. The chloride equilibrium flux (mmol C1/3 x 1013 cells min) is defined as the product of the rate coefficient of 36C1 etttux (min-1) and the chloride content in the ceils (mmol C1/3 x 1013 cells). Figure 6 demonstrates the marked temperature dependence of chloride exchange. The half-time of 36C1 efflux is 18 sec at 0°C and decreases to approximately 2 sec at 10°C (Qlo ~ 8) (Dalmark and Wieth, 1970). The half-time of 36C1 efflux at room temperature has been estimated to 0.2 sec by Tosteson (1959) and at 38°C to 0.05 sec by Brahm (1975) with a rapid flow technique. Thus, the marked temperature dependence is also

MADS DALMARK

154 IC

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8 D I

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,

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FIG. 6. The rates of 36C1 efflux from human red cells towards isotopic equilibrium with steadystate distribution of chemically measurable chloride between cells and medium at 0 and 10°C (pH 7.4). The medium had the following composition (mM): 142 Na, 3.7 K, 1.5 Ca, 1 Mg, 126.5 CI, 22 HCO3, 1.1 phosphate, and 5 glucose. The cells were loaded with 36C1 during incubation in the medium at pH 7.4 and the appropriate temperature. The appearance of 36C1 in the extracellular phase was determined after injecting approx 200 mg packed, labelled cells into 40 ml of vigorously stirred medium (haematocrit less than 0.01), kept at constant temperature and pH. The 36C1 efl]ux from cells to medium was followed by serially isolating cell-free medium by rapid filtration of the cell suspension. The rate coefficient of 36C1 efftux (time -1) was determined from the slope of ( l ~ / a ~ ) vs time, a being the specific activity in the effiux medium at the time of sampling, and a~ the specific activity in the medium at isotopic equilibrium. (Dalmark and Wieth, 1970.)

present at higher temperatures. This is demonstrated with the manual filtration technique, since addition of inhibitory anions increases the half-time of 36C1 et~ux to such an extent that the half-time can be measured in the temperature range from 0 to 38°C (Dalmark and Wieth, 1972). The Arrhenius activation energy in absence or presence of other anions is 30-40 kcal/mol (--~130-170kJ/mol). This temperature dependence has to be compared with the low temperature dependence of KC1 diffusion in water (4 kcal/mol, Qlo ~ 1.3). Thus, chloride transport across the human red cell membrane has a marked temperature dependence in contrast to chloride diffusion in water. The chloride transport shows saturation kinetics (Gunn et al., 1973; Cass and Dalmark, 1973). Figure 7 demonstrates the concentration dependence of chloride equilibrium flux in the absence of other anions than chloride (Dalmark, 1975a). The cells were prepared by the nystatin technique, and the KC1 concentration (ionic strength)

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Chloride in the human erythrocyte

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F[o. 8. The dependence of chloride equilibrium flux on chloride concentration in the cell water (O) and in the medium (t) at different values of pH (0°C). The cells were modified with the nystatin technique (cf. Fig. 1), and the KCI concentration (ionic strength) in cells and medium varied. The media had the following compositions (mr,t): 5-150 KC1, 1 NaCI, and 27 sucrose. (Dalmark, 1975a.)

in cells and medium varied. The chloride transport increases more and more slowly with concentration to reach a maximum after which the transport decreases: chloride transport shows saturation kinetics with self-inhibition in the pH range between 6.2-9.2 (0°C). The saturation curve (pH 7.2) is described by a maximum chloride transport (740 mmol C1/3 x 1013 cells min) at an optimum chloride concentration (150 mM) and a half-saturation constant (18 mM). The saturation kinetics are apparently not a simple ionic strength effect since the concentration dependence is unchanged in the presence of 150 mM sodium acetate under conditions where the chloride concentration is varied by addition of ammonium chloride (Wieth et al., 1973). The distribution of chloride between cells and medium varies with pH (Dalmark, 1975b). This means that the concentration dependence of chloride transport is different when the flux is plotted as a function of the concentration in cell water or medium at chloride distribution ratios different from unity. Figure 8 shows that the dependence of transport on concentration in cell water and medium is almost uniform between pH 7-8, but outside this pH range the dependence is different: the half-saturation constant increases with pH when the transport is regarded as a function of the concentration in medium, and decreases with pH when the transport is regarded as a function of the concentration in cell water. Thus, chloride transport is not a simple Michaelis-Menten function, since the transport shows (1) self-inhibition, and (2) a concentration dependence with S-shaped or steeper graphs than predicted from simple Michaelis-Menten kinetics at various values of pH. The concentration dependence is apparently a function of a saturable component of the transport apparatus and the asymmetrical distribution of chloride at various values of pH (Dalmark, 1975a). Chloride transport is inhibited by other anions. The inhibitory action depends on properties other than the valency: acetate mainly decreases the maximum chloride transport (Gunn et al., 1973), bicarbonate mainly increases the half-saturation constant (Gunn

156

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FIG. 9(a) and (b). The dependence of chloride equilibrium flux (0°C, pH 7.2) on chloride concentration in the presence of fluoride (V), bromide (O), and iodide (@) at concentrations of 40 mM (Fig. 9a) and 160 mM (Fig. 9b). The cells were modified with the nystatin technique (cf. Fig. 1), and the salt concentration (ionic strength) in cells and medium varied. The curves were drawn by eye. (Dalmark, 1976.)

et al., 1973; Dalmark, 1976), and halides like F, Br, and I affect both the maximum

transport and the half-saturation constant (Dalmark, 1976). This demonstrates that some steps in the transport process are substrate-specific with different affinities for various anions. The inhibitory action is not a simple effect of the ionic strength in the bulk phases, since the observed chloride transport is markedly different when this parameter is kept constant (Fig. 9). The inhibition kinetics can be interpreted as competition between anions for two membrane binding sites: a transport site (competitive inhibition) and a second site which modifies maximum chloride transport (non-competitive inhibition). The chloride selfinhibition is explained as a non-competitive effect of chloride on its own transport in analogy with the non-competitive effects of other halides on chloride transport. Furthermore, the results indicate that chloride self-inhibition also takes place at low chloride concentration. It is possible from such a model to account for the chloride saturation graph in absence of other anions than chloride (Dalmark, 1976). The inhibitory action of halides on chloride transport increases through the sequence F, CI, Br, and I (Dalmark, 1976). A similar sequence has been demonstrated for halide binding to intracellular constituents (Tosteson, 1959; Dalmark and Wieth, 1972). This sequence is identical with Eisenman's sequence I (Eisenman, 1965). Thermodynamic considerations of the interaction between the four halides and positively charged sites predict seven selectivity sequences out of twenty-four possible permutations. Sequence I indicates interaction of halides with weak binding sites by coulombic forces. In this case the attraction between anions and binding sites is weaker than the hydration energies of the various halides. The meaning of this sequence for the transport rates is not clear, since the transport rates for the various halide ions themselves decreased through CI, Br, and I (Tosteson, 1959; Dalmark and Wieth, 1972). The slower transport of larger halides is not explained simply on the basis that they are more tightly bound. Quantitative considerations predict that iodide should be transported at half the rate of chloride under the assumptions that (1) all halides share the same carrier-mediated transport system, and (2) the rate of movement of the loaded carrier through the membrane is independent of the species of halide whiCh is bound to it. In fact, iodide is transported at one hundredth the rate predicted by these considerations. This indicates that the rate-limiting step of the halide transport process is dominated by other factors than those which dominate the inhibition of the halide transport (Dalmark, 1976).

Chloride in the human erythrocyte

157

Chloride transport has a bell-shaped pH dependence at normal ionic strength (Gunn et al., 1973; Dalmark, 1975a; Brahm, 1975) (Fig. 10). The interpretation of the graph is difficult since chloride transport depends on the chloride concentration, and the cellular concentration varies with pH (Fig. 4). It is predictable that the transport of a nontitratable molecule across a membrane with pH-independent properties is independent of pH under conditions where the concentrations of the molecule are constant on both sides of the membrane. The distribution of chloride between cells and medium is almost pH-independent in (1) haemoglobin-containing red cells (Fig. 4) at high KC1 concentrations (ionic strengths) and (2) haemoglobin-depleted cells (ghosts) at 150mM KC1 (normal ionic strength) (Funder and Wieth, 1975). It is observed with both types of cell preparations that chloride transport increases with pH from 5.8 to 7.5 (0°C); but at higher pH the transport is constant. This demonstrates that protons have a direct effect on the membrane below pH 7.5. A close relationship between the asymmetrical distribution of chloride across the cell membrane and the appearance and position of the alkaline branch of the pH graphs is observed when the KC1 (150-5 mM) in medium and cells is varied by the nystatin technique (Dalmark, 1975a) (Figs. 4 and 10). In these cases the pH-dependent changes of the cellular chloride concentration and the calculated membrane potential difference are more pronounced at lower than at higher KC1 concentrations (ionic strengths). This means that the bell-shaped pH graph observed in intact cells at normal ionic strength is the result of two different mechanisms: (1) an effect of protons on the membrane properties below pH 7.5, and (2) the asymmetrical distribution of chloride across the membrane. Chloride transport in red cells is limited to exchange with other anions, since the cation permeability is low. It is not possible from such studies in normal cells to decide whether chloride efflux and influx is linked together in a 1:1 relationship by interaction between chloride and groups attached to the membrane, or whether chloride is transported by independent migration under conditions of electroneutrality. The properties of the cell membrane are altered by ionophores. Addition of ionophores like gramicidin or valinomycin increases the cation permeability in human red cells. Chloride transport across these modified cell membranes is not limited to exchange reactions: net movements of chloride may occur in conjunction with counterions like potassium. At high concentrations of ionophores the net transport of KC1 along a

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158

MADS DALMARK

concentration gradient is supposed to be rate-limited by the independent migration of chloride ions through the membrane (electrodiffusion). The chloride permeability under these conditions is 10-4-10 -5 the chloride permeability in unmodified cell membranes calculated from chloride exchange studies with radioactive chloride at anion equilibrium (Harris and Pressman, 1967; Scarpa et al., 1968; Hunter, 1971; Tosteson et al., 1973). Unpublished results by Dr. Albert Cass and myself indicate a chloride permeability coefficient in the presence of a KC1 concentration gradient of 10-s cm/sec (25°C) in gramicidin-modified human red cells (normal ionic strength, pH 6.8). The chloride permeability coefficient determined from chloride tracer studies is more than 10 -4 cm/sec (25°C)in the presence of gramicidin, since the chloride tracer is at equilibrium within less than 1 sec. These experiments with modified membranes indicate that chloride transport in unmodified cells is a facilitated and tightly coupled exchange transport process. The coupling apparently involves interactions between chloride and specific groups in the membrane and is not a result of the electrical driving forces. This acceleration of chloride transport by exchange relative to independent migration of single ions has also been demonstrated in electrophysiological studies of unmodified cell membranes. The conductance of the cell membrane is much less than calculated from tracer experiments, since a large fraction of the chloride transport is via "electrically silent", tightly coupled exchange. The specific resistance of the red cell membrane from Amphiuma (Hoffman and Lassen, 1971; Lassen et al., 1974; Vestergaard-Bogind and Lassen, 1974) is estimated to more than 2000 ohm cm 2. This value has to be compared with a membrane resistance of 1 ohm c m 2 calculated from chloride tracer experiments. These data demonstrate that chloride transport determined from tracer studies is at least more than 10 3 times the chloride transport by independent migration of single ions which is able to carry current through the membrane. This factor may represent an underestimate, since a specific resistance as high as 107 ohm cm 2 has been reported for the Amphiuma membrane (Lassen et al., 1974). In that case, chloride transport in red cells is one of the most tightly coupled exchange processes so far described in nature. Such a transport process is an example of "exchange diffusion" as defined and discussed by Ussing at the Cold Spring Harbour meeting in 1948. In conclusion, transport studies and electrophysiological data indicate that (1) chloride transport in human red cells is a tightly coupled exchange transport process, (2) chloride transport via this exchange mechanism is accelerated relative to chloride transport by independent migration of single ions by a factor of 104-107. Thus~ chloride transport depends on the chloride concentration on both sides of the membrane. Cellular chloride concentration is determined by the concentration of non-titratable cations like K, the osmotic properties of the cells, and the ionization of cellular buffers. In this sense. chloride transport depends on (1) the properties of the cell membrane and (2) the properties of the cellular constituents. This anion exchange mechanism is well qualified to facilitate the transport of carbon dioxide from tissue to blood and from blood to alveolar air, since it allows a rapid exchange of chloride for bicarbonate across the red cell membrane (Dalmark, 1972). The carbon dioxide is produced in the tissue and flows along a tension gradient into the red blood cells, where it is rapidly hydrated in the presence of carbonic anhydrase. This hydration reaction would go to equilibrium if it wasn't for the anion exchange mechanism, which keeps the intracellular bicarbonate concentration low by the chloridebicarbonate shift (Nasse, 1878; Hamburger, 1892; Roughton, 1935). Bicarbonate is allowed to distribute itself in the much larger extracellular volume by this anion exchange process. Thus, plasma acts as a sink for the hydration product of the carbon dioxide generated in the tissue, as red blood cells contain carbonic anhydrase and a rapid anion exchange transport system. IV. STRUCTURAL FEATURES OF THE CELL MEMBRANE AND CHLORIDE TRANSPORT Chemical studies of the red cell membrane show that both lipids and proteins are major components. The membrane consists of 40% lipids, 50% proteins and 10~,,, carbo-

Chloride in the human erythrocyte

159

hydrates (wt/wt). The gross structure of the membrane is a lipid bilayer leaflet in which particulate proteins are embedded. Several excellent papers of the structural features have been published in recent years (Winzler, 1969; Bretscher, 1973; Singer, 1974; Van Deenen and de Gier, 1974) and only a number of selected topics are dealt with here, The lipids consist of 60% phospholipids, 30% neutral lipids mainly cholesterol, and 10% glycolipids (wt/wt) (Van Deenen and de Gier, 1974). The membrane lipids constitute more than 70% of the cell membrane surface area (Finean et al., 1971). The phospholipids appear to be asymmetrically distributed in the cell membrane, since phosphatidylcholine and sphingomyelin are preferentially located in the outer half and amino phospholipids like phosphatidylethanolamine and phosphatidylserine are preferentially located in the inner half of the cell membrane (Van Deenen et al., 1974). The mobility of phospholipids in the membrane indicates that probably only translational and in-plane rotational mobility takes place, and that "flip-flop" of phospholipids from one side of the membrane to the other is an extremely slow process with a half-time of 6 hr at 30°C (Kornberg and McConnell, 1971). The membrane proteins can be solubilized with sodium dodecyl sulphate and separated on polyacrylamide gels according to their molecular weight (mol. wt) into three groups: 33% with a mol. wt in excess of 200,000, 25% with a tool. wt of 100,000, and the rest with a lower mol. wt (Weber and Osborn, 1969). However, only one major band is observed when the proteins are stained for carbohydrates instead of polypeptides. This band consists of two glycoproteins: component a with a mol. wt of 100,000 and a low carbohydrate content (8%), and component b (glycophorin) with a tool. wt of 30,000 and a high carbohydrate content (66%) (Bretscher, 1971). The number of copies of component a and b per single cell is of the same order of magnitude (105 per single cell) (Bretscher, 1971). Component b accounts for 80% of the carbohydrate and 90% of the sialic acid on the cell surface. The large negative electrical charge of the cell membrane at neutral pH, and a variety of cell antigens like MN antigens, are attributable to this molecule (Winzler, 1969). It appears that all carbohydrates of the glycoproteins are located on the outer surface of the membrane, and in this sense the proteins show polarity in their orientation in the membrane (Winzler, 1969; Nicolson and Singer, 1974). The remainder of the glycoprotein molecules span the membrane from the outer to inner surface (Bretscher, 1971). The orientation of these proteins has been demonstrated by comparing the patterns of labelling or digesting intact red cells and permeable ghosts using chemical or enzymatic methods. The interaction between glycoproteins and the lipid bilayer is unknown. It has been suggested that the proteins interact with the membrane lipids through hydrophobic associations between lipophilic segments of the proteins and the lipid bilayer, i.e. the proteins consist of lipophilic, nonpolar segments which span the membrane, and two ends with hydrophilic, charged residues which stick out of the membrane bilayer on both surfaces (Winzler, 1969). These residues prevent rotational movements of the proteins, since a large amount of energy is required to carry charged groups through the low dielectric bilayer (Marchesi, 1974). The transmembranous glycoproteins may form the major components of the particles observed by freeze-fracture microscopy. By this technique the fracture plane passes through the middle of the lipid bilayer leaflet and each half of the membrane can be studied separately. The fracture faces are covered with small (80 A) particles, rather more of which are attached to the inner half of the bilayer (da Silva and Branton, 1970). The particles are randomly distributed at neutral pH with 3 x 10a particles per #2 on the inner half of the bilayer (fracture face A) or approximately 105 per single cell (Bretscher, 1971; da Silva et al., 1971; Tillack et al., 1972; Kirk and Tosteson, 1973). The distribution of particles appears to be a function of pH (da Silva, 1972; Elgsaeter and Branton, 1974; Rothstein and Cabantchik, 1974). The particles cluster around pH J.P.B. 31/2--~

160

MADS DALMARK

5.5. It appears that the particles are limited in their translational mobility by the molecular meshwork of spectrin on the inside of the cell membrane (Elgsaeter and Branton, 1974). Spectrin is a rod-like molecule (tool. wt 220,000) 2000A long which apparently anchors the particles in their random distribution since removal of spectrin increases the particle aggregation observed by lowering the pH to 5.5. Thus, some membrane proteins form a structure which spans the hydrophobic core of the lipid bilayer leaflet. This structure of the membrane may have important implications for the permeability characteristics. In recent years several attempts have been made to correlate the transmembranous proteins to anion transport after Passow (1969) demonstrated the inhibitory action of amino reagents on anion transport. The inhibitory action of various derivatives of stilbenedisulfonic acid, e.g. SITS and DIDS, are of special interest, since complexes of these reagents covalently bounded to membrane proteins can be extracted from the cells. Binding of these reagents apparently involves interaction with positively charged groups on the outer surface of the membrane located close to some hydrophobic regions (Cabantchik and Rothstein, 1972). The chloride transport is inhibited in a 1:1 relationship with 3H-DIDS binding to membrane proteins, predominantly component a. In contrast, the DIDS has no effect on cation transport (Rothstein and Cabantchik, 1974). The binding of DIDS to component a is mainly on the outside of the membrane, since only a few additional sites on component a are labelled from the inside in ghost experiments. The asymmetrical labelling of component a by DIDS may be correlated with the asymmetrical effect of phlorizin, one of the classic sugar transport inhibitors, on chloride transport (Schnell et al., 1973). Phlorizin inhibits chloride transport when added to a suspension of intact cells. No inhibition of chloride flux is observed when phlorizin is incorporated in resealed ghosts, although sugar transport is strongly inhibited with phlorizin inside. Thus, apparently proteins mediating chloride transport have an asymmetrical structure. Recent data suggest that component a forms tetramers with a mol. wt of 450,000, and that this tetramer constitutes a proteinous part of the chloride transport apparatus (Wang and Richards, 1974; Rothstein and Cabantchik, 1974). v. CHLORIDE TRANSPORT MODELS The experimental data restrict the number of models which applies to a given transport system. Until now the red cell membrane has been regarded as a "black box", and the purpose of transport studies has been to give phenomenological description of the anion transport as a function of the properties of the bulk phases on both sides of the cell membrane, i.e. concentration of anions and inhibitors, pH and temperature. The composition of the bulk phases affects the properties of the cell membrane, which is the rate limiting structure of anion transport between cellular and extracellular fluids. Topics of investigations in the future are to describe the relationships between the properties of the bulk phases and the cell membrane in order to answer the following questions: (1) what do the concentration profiles in the membrane and at its surfaces look like under various conditions? (2) what is the magnitude of the rate coefficients of anion transport within the cell membrane and of anion transport at the surfaces of the cell membrane? When these questions are answered further restrictions are placed on anion transport models. At this point of anion transport research all models are tentative descriptions based on some structural properties of the "black box" which are not experimentally proven. The models mentioned below are valuable as working hypotheses which all account for important observations on anion transport in red cells. It has been suggested by Passow (1969) that the red cell membrane is an anion selective membrane with fixed, positively charged groups in front of a rate limiting barrier through which anions permeate by free diffusion. The simple fixed charge theory apparently does not apply to chloride transport as this transport is a fast, tightly coupled exchange process across a membrane with a low electrical conductance. It appears that

Chloride in the human erythrocyte

161

interactions between anions and membrane groups with different affinities for various anions take place in anion exchange, since some steps in the transport process show substrate specificity. These groups could be placed in front of the rate limiting barrier on both surfaces of the cell membrane as suggested by Passow (1969). If so, the tightly coupled chloride exchange is described by the law of joint probabilities, i.e. chloride exchange is proportional to the probability of finding chloride attached to groups on both sides of the membrane simultaneously M ° = A x Pc x Pm = A x {1/[1 + Kc/(C1)c]} x {1/11 + K,J(C1),,,]},

(5)

where A is the chloride exchange transport capacity of the red cells (mmol C1/3 x 10 la cells min), Pc, Pm are the probabilities of finding chloride attached to groups on the inside and the outside of the cell membrane in such positions that chloride exchange takes place, Ko Km are the dissociation constants of complexes between chloride and chloride binding groups on the inside and the outside of the cell membrane, and (C1)c, (C1),, are the chloride concentrations in cell water and medium. A similar equation has been given by Baker and Widdas (1973) to account for glucose transport in red cells. Equation 5 describes that chloride exchange shows saturation kinetics at low values of K/(C1), but a squared concentration dependence at high values of K/(C1). Equation 5 accounts for the pH dependence of chloride exchange: chloride exchange depends on the asymmetrical distribution and saturation of membrane groups at constant value of the factor A as observed in the alkaline pH range at normal ionic strength, but at pH below 7.5 the factor A decreases with decreasing pH. The decreasing value of A may be related to an altered structure of the cell membrane which is observed at low values of pH (da Silva, 1972). Equation 5 describes a tightly coupled exchange transport process, but the equation offers no explanation of the coupling mechanism. Several models on molecular basis have been suggested in order to account for the coupling mechanism (Wieth, 1971, 1972; Gunn, 1972; Passow and Wood, 1974). Equation 5 accounts for a rotational carrier model as well as for a non-rotational, gate model (Patlak, 1956; Vidaver, 1966). Both models can be envisioned as having a rod-like, transmembranous molecule with a chloride binding group at both ends projecting into the water phases at the two surfaces of the membrane. The very slow rate of rotation of membrane components like phospholipids (Kornberg and McConnell, 1971; Toyoshima and Thompson, 1975) and proteins (Bretscher, 1971; Marchesi, 1974) speaks against a rotating carrier. The transport process of a gate model can be envisioned as follows: the ends of the transmembranous molecule move a few AngstrSms translationally when chloride is attached to both ends simultaneously, and channels are opened which allow two chloride to exchange for each other through the rate limiting barrier of the cell membrane. The model predicts that the sequence of the various halides for attachment to the anion binding groups on the transmembranous molecule and for the rate of transport through the cell membrane could be different. This would account for the discrepancy between inhibitory actions and transport rates of various halides. There are phenomenological similarities between the transport properties of the red cell membrane and artificial liquid anion exchange membranes (Wieth, 1971, 1972). The coupling mechanism of anion exchange in such systems is created by the difference in solubility of charged and uncharged molecules in low dielectric media, i.e. only electroneutral (lipophilic) complexes of anions with positively charged membrane components are rapidly transported across the membrane. Shean and Sollner (1966) demonstrated that membranes of organic solvents turned into highly anion selective membranes after addition of secondary amines to the organic solvents. Such membranes show fast anion exchange reactions with saturation kinetics and competition between anions. The secondary amines carry positive charges which are able to combine with anions to

162

MADS DALMARK

form electroneutral, lipid-soluble complexes. The a m i n e s act as cycling carriers, since they transfer a n i o n s from one a q u e o u s phase to the other. The electrical c o n d u c t a n c e of these m e m b r a n e s is low since electrical driving forces do n o t affect the t r a n s p o r t of electroneutral complexes. Such a m o d e l a c c o u n t s for the observed chloride t r a n s p o r t data in red cells ( D a l m a r k , 1975a, 1976). Passow a n d W o o d (1974) suggest that the c o u p l e d exchange reactions take place at the two surfaces of the m e m b r a n e , a n d that these surface reactions, rather t h a n the diffusion process across the m e m b r a n e , are rate-limiting for a n i o n transport. This suggestion is b a s e d o n the o b s e r v a t i o n that fluoride a n d chloride are able to increase the rate c o n s t a n t of iodide t r a n s p o r t in ghosts experiments. A l t h o u g h the c o u p l e d exchange reactions m a y take place at the surfaces of the cell m e m b r a n e , these surface reactions are a p p a r e n t l y n o t rate-limiting for chloride exchange transport, as no acceleration of chloride t r a n s p o r t is observed in fluoride m e d i a ( D a l m a r k , 1976). REFERENCES ADAIR,G. S. and ADMP,,M. E. (1934) The determination of the isoelectric and isoionic points of haemoglobin from measurements of membrane potentials. Biochem. J. 28, 1230-1258. ANDP,EOLI,TH. E., TIEFFENBERG,M. and TOSTESON,D. C. (1967) The effect of valinomycin on the ionic permeability of thin lipid membranes. J. gen. Physiol. 50, 2527 2545. ANTONINI,E., AMICONI,G. and BP,UNOP,I,M. (1972) The effect of anions and cations on the oxygen equilibrium of human hemoglobin. In Oxygen Affinity of Hcmoylohin and Red Cell Acid Base Status (eds. M. RtJRTH and P. ASTRUP,A. Benzon Symp. IV, pp. 121-129. Munksgaard, Copenhagen. ANTONINI,E., WYMAN,J., BP,UNOP,I,M., FROMTICELL1,C., Bucc1, E. and ROSSI-FANELLI,A. (1965) The influence of temperature on the Bohr effect in human and in horse hemoglobin. J. biol. Chem. 240, 1096-1103. APPELBOOM, J. W., BP,ODSKY,W. A., TUTTLE,W. S. and DIAMOND, I. (1958) The freezing point depression of mammalian tissue after sudden beating in boiling distilled water. J. gen. Physiol. 41, 1153-1169. BAKER, G. F. and WIDDAS,W. F. (1973) The asymmetry of the facilitated transfer system for hexoses in human red cells and the simple kinetics of a two-component model. J. Physiol. Lond. 231, 143-165. B~l~LIN, N. I. and BERK, P. D. (1975) The biological life of the red cell. In The Red Blood Cell (ed. D. MACN. SURGENOR),Chapt. 24, pp. 957-1019. Academic Press, New York. BRAHM, J. (1975) Chloride permeability in human red cells at 0-38°C. Abstract, P-319, Vth Int. Biophys. Congress, Copenhagen. BRAUNITZER,G., GEHRING-Mi]LLER,R., HILSCHMANN,N., HILSE,K., HOBOM,G., RUDLOFF,V. and WITTMANNLIEBOLD,B. (1961) Die Konstitution des normalen adulten humanhiimoglobins. Hoppe-Seyler's Z. Physiol. Chem. 325, 283-286. BRETSCHER, M. S. (1971) Major human erythrocyte glycoprotein spans the cell membrane. Nature New Biol. 231, 229-232. BP,ETSCHEP,,M. S. (1973) Membrane structure: some general principles. Science N.Y. 181, 622-629. BP,ODSKY, W. A., APPELBOOM, J. W., DENNIS, W. H., REHM, W. S., MILEY, J. F. and DIAMOND, I. (1956) The freezing point depression of mammalian tissue in relation to the question of osmotic activity of cell fluid. J. gen. Physiol. 40, 183-199. CABANTCHIK,Z. I. and ROTHS~IN,A. (1972) The nature of the membrane sites controlling anion permeability of human red blood cells as determined by studies with disulfonic stilbene derivatives. J. membr. Biol. 10, 311-330. CASS, A. and DALMAP,R,M. (1973) Equilibrium dialysis of ions in nystatin-treated red cells. Nature New Biol. 244, 47~49. CASS, A., FINKELSTErN,A. and KRESPI,V. (1970) The ion permeability induced in thin lipid membranes by the polyene antibiotics nystatin and Amphotericin B. J. gen. Physiol. 56, 100-124. CH1ANCONE,E., NOP,NE, J. E., FOP-SEN,S., ANTONINI,E. and WYMAN, J. (1972) Nuclear magnetic resonance quadrupole relaxation studies of chloride binding to human oxy- and deoxyhaemoglobin. J. molec. Biol. 70, 675-688. COLLINS,D. A. and SCOTT, F. H. (1932) The freezing-points of serum and corpuscles. Am. d. Physiol. 101, 21-22. DALMARK,M. (1972) The effect of temperature, bicarbonate-carbon dioxide, and pH on the chloride transport across the human red cell membrane. In Oxygen Affinity of Hemoglobin and Red Cell Acid Base Status. pp. 320-332 (eds. M. ROP,THand P. ASTRUP),A. Benzon Symp. IV, Munksgaard, Copenhagen. DALMARK,M. (1975a) Chloride transport in human red cells. J. Physiol. Lond. 250. 39-64. DALMARK, M. (1975b) Chloride and water distribution in human red cells. J. Physiol. Lond. 250, 65-84. DALMARK, M. (1976) Effects of halides and bicarbonate on chloride transport in human red cells. J. gen. Physiol. 67, 223-234. DALMARK, M. and WIETH, J. O. (1970) Chloride and sodium permeabilities of human red cells. Biochim. biophys. Acta 219, 525-527. DALMARK, M. and WIETH, J. O. (1972) Temperature dependence of chloride, bromide, iodide, thiocyanate and salicylate transport in human red cells. J. Physiol. Lond. 224, 583-610. DA SILVA,P. P. (1972) Translational mobility of the membrane intercalated particles of human erythrocyte ghosts. J. cell. Biol. 53, 777-787. DA SILVA, P. P. and BRANTON, D. (1970) Membrane splitting in freeze-etching: covalently bound ferritin as a membrane marker. J. cell. Biol. 45, 598-605.

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