reflected by the low dissociation constant of 0-28 x 108 M. erythrocyte. Thus one receptor site corresponds to less than 1,um2 of the

Transcription

1 J. Physiol. (1975), 251, pp With 6 text-figure8 Printed in Great Britain QUANTITATIVE ASPECTS OF OUABAIN BINDING TO HUMAN ERYTHROCYTE AND CARDIAC MEMBRANES BY ERLAND ERDMANN AND WERNER HASSE From the Medizinische Klinik I, Klinikum Grof3hadern, Univer8itdt Munchen, D-8 Minchen, Germany (Received 13 December 1974) SUMMARY 1. [3H]ouabain binding to human erythrocyte membranes is a time- and temperature-dependent process. The association of ouabain to the membrane-bound receptor follows second-order kinetics, while the dissociation is a monomolecular reaction. An association rate constant of 4-6 x 104 M-1 sec-1 and a dissociation rate constant of 1-4 x 10-4 sec-1 were measured at 370 C. The dissociation constant calculated from these data agrees with that determined from equilibrium binding experiments. There is only one type of ouabain binding sites with high affinity for the drug as reflected by the low dissociation constant of 0-28 x 108 M. 2. The dissociation constants of the ouabain-receptor complexes from human erythrocyte and cardiac membranes are identical. 3. The maximal number of membrane-bound ouabain binding sites was measured from equilibrium binding experiments as per single erythrocyte. Thus one receptor site corresponds to less than 1,um2 of the membrane, provided the receptors are diffusely distributed on the surface of the membrane. 4. Neither the maximal number of ouabain receptors nor the affinity for the drug changes with the age or sex of the blood donor. 5. A maximal transport capacity for sodium of 5-6 m-equiv/hr.1. is calculated from the number of receptor sites per erythrocyte and from the turn-over number of the (Na+ + K+)-ATPase. INTRODUCTION Cardiac glycosides in binding to the outer surface of the erythrocyte membrane (Hoffman, 1966) inhibit specifically the (Na+ + K+)-activated ATPase system (Dunham & Hoffman, 1970) and the active transport of sodium and potassium across the cell membrane (Schatzmann, 1953). Recently it could clearly be established that ouabain binding correlates directly with inhibition of erythrocyte potassium influx (Gardner & Kiino,

2 67262ERLAND ERDMANN AND WERNER HASSE 1973). Furthermore it was demonstrated that the specific binding of cardiac glycosides to cell membranes involves a glycoside binding site or 'receptor' and a cation site, both of which are closely attached to or are parts of the (Na+ +K+)-ATPase system (Gardner & Frantz, 1974; Kyte, 1972; Erdmann & Schoner, 1973b). Recent experiments performed by Ruoho & Kyte (1974) indicate that a purified and solubilized (Na+ + K+)-ATPase preparation from canine renal medulla is composed of two polypeptide chains. The glycoside binding site, which is accessible from the outside surface of the plasma membrane and the enzymically active site, which is accessible from the inside surface (Jorgensen, Hansen, Glynn & Cavieres, 1973), are both on one, the larger polypeptide subunit of the (Na+ + K+)-ATPase. This means that this polypeptide chain is exposed to both sides of the membrane and probably spans the membrane (Ruoho & Kyte, 1974). The apparent intimate relationship between the ouabain binding site and (Na+ + K+)-ATPase activity is further emphasized by the direct correlation of the amount of cardiac glycoside bound to human cardiac cell membranes and the extent of the inhibition of enzymic activity if both are measured at equilibrium (Erdmann, Patzelt & Schoner, 1974). In the light of these findings it is of great interest to know the exact number of receptor sites for glycosides (being equivalent to the number of sodium-pump sites) on such a welldefined surface as is the erythrocyte membrane. Data reported for human erythrocytes range from 200 (Dunham & Hoffman, 1970), 650 (Baker & Willis, 1972) to 1200 ouabain binding sites per cell (Gardner & Frantz, 1974), or per#,m2 of the erythrocyte membrane surface (Solomon, 1958). Earlier equilibrium binding experiments (Erdmann & Schoner, 1973 a) revealed that the glycoside receptors from different tissues but the same species apparently have the same affinity for ouabain, although there are considerable differences among different species. In this paper we report experimental evidence that the affinity for ouabain of this receptor from human erythrocyte and cardiac membranes is identical in different agegroups and that the number of receptor sites of human erythrocytes does not change with the person's age or sex either. METHODS Material8 [3H]ouabain with a specific activity of 13 c/rn-mole was obtained from New England Nuclear, Dreieichenhain, Germany. The liquid scintillation fluid used was Insta-Gel (Packard Instruments GmbH, Frankfurt, Germany). All other chemicals were of analytical grade and obtained through E. Merck A.G., Darmstadt or Boehringer Mannheim, Germany.

3 NUMBER OF OUABAIN BINDING SITES 673 Methods 1. Erythrocyte counting. Erythrocyes were counted routinely in a Coulter Counter Modell S, Coulter Electronics, Dunsta ble; Beds., U.S.A. 2. Erythrocyte membrane preparation. Usually 20 ml. freshly drawn human venous blood from haematologically normal adults were used. Coagulation was prevented by addition of 0-1 ml. of a solution of heparin (5000 U.S.P.-units heparin/ml, Liquemin, Hoffman-La Roche A.G., Grenzach, Germany). The blood was centrifuged at 2000 g for 10 min and the plasma and buffy coat were delicately removed by aspiration. The remaining cells were lysed in 150 ml. distilled water ph 6-5 (ph was adjusted with imidazole/hcl, 1 mm). After 15 min the membranes were spun down at 35,000 g for 30 min. The pellet was homogenized thoroughly in a Teflon/glaspotter. The homogenate was made up to 150 ml. with distilled water, ph 6-5, and again centrifuged at 35,000 g for 30 mm. The resulting sediment was homogenized in ml M imidazole/hcl, ph 6-5. All procedures were performed at 0-4 C. Precaution was taken to save all membrane particles. c.p.m _ Time (min) Fig. 1. Binding of [3H]ouabain to human erythrocyte membranes. The incubation medium contained 50 mm imidazole/hcl, ph 7-25, 3 mm-mgcl2, 3 mm imidazole-po4, 8-5 x mole [3H]ouabain and 1 ml. erythrocyte membrane suspension originating from 1 ml. venous blood; 370 C, total volume 2 ml. The amount of [3H]ouabain, which could not be displaced by 10-3 M unlabelled ouabain (unspecific binding) was subtracted. It amounted to 18%. 3. A8say of ouabain binding. Unless otherwise indicated ml. of the membrane suspension usually representing the erythrocyte membranes of ml. venous blood were incubated in a medium containing 50 mm imidazole/hcl buffer ph 7-25, 3 mm-mgcl2, 3 mm imidazole-phosphate, 8-5 x mole [2H]ouabain and various amounts of unlabelled ouabain at 370 C until equilibrium was reached (Fig. 1). The incubation was carried out in polypropylene tubes suited for the rotor 50 Ti of a Beckman ultracentrifuge L2 65 B. The incubation was started by adding the membranes and it was stopped by rapid cooling of the tubes in liquid air. After thawing in ice, the membranes were sedimented at 80,000 g for 30 min at 00 C. The

4 67464ER".7LAND ERDMANN AND WERNER HASSE supernatant was decanted and the centrifugation tubes were kim-wiped thoroughly; 0-2 ml. 1 m-naoh was added to the pellet and it was heated in a water-bath at 500 C until the precipitate was dissolved. Then two drops of concentrated HCI were added and 10 ml. scintillation fluid (Insta-Gel). The radioactivity was counted in a Packard Tri-Carb Specific [3H]ouabain binding is obtained by subtracting from the total radioactive uptake the amount that is not displaced by high concentrations (10-3 M) of unlabelled ouabain. 4. Release of [3H]ouabain from the [3H]ouabain-receptor complex. To measure the dissociation rate constant (k-,) the amount of erythrocyte membrane suspension needed was allowed to bind to [3H]ouabain in 50 mm imidazole/hc1 ph 7-25, 3 mmv- MgCl2, 3 mm~imidazole-phosphate until equilibrium was attained. The membranes were spun down at 80,000 g for 30 min. The pellet was resuspended in 0-01 m imidazole/hci, ph 6-5. One ml. of this [3H]ouabain-receptor complex was incubated for the indicated time and temperature in 50 mm~imidazole/hci ph 7-25, 3 mm- MgCl2, 3 mm~imidazole-phosphate and 10-3 M unlabelled ouabain in a total volume of 2 ml. The reaction was stopped by rapid cooling in liquid air and immediate centrifugation. 5. Preparation and quantitation of (Na+ +K+)-activated ATPase from human ventricular myocardium. Cardiac (Na+ +K+)-ATPase preparations from human myocardium. were isolated according to Matsui & Schwartz (1968). (Na+ +K+)- ATPase activity was measured with the coupled optical assay (Schoner, von Jlberg, Kramer & Seubert, 1967). One enzyme unit is defined as the amount of enzyme activity hydrolysing 1 gtmole ATP/min at 370 C. Protein was quantitated by the procedure of Lowry, Rosebrough, Farr & Randall (1951). RESULTS Guabain binding to its specific, membrane-bound receptor is a temperature- and time-dependent process (Fig. 2). The kinetics of these drugreceptor-interactions have been described in great detail in a previous paper for heart, kidney and brain-cell membranes (Erdmann & Schoner, 1973a). The binding of ouabain (0) to the glycoside receptor (R) from erythrocyte membranes follows the mass law equation (Hansen, 1971): k+1 O+R _-_ k-i OR, where OR stands for the ouabain-receptor complex. According to this equation the binding process follows second-order kinetics while the dissociation is a monomolecular reaction. The association rate constant (k±1) can be calculated, if the initial receptor concentration (b) and the initial ouabain concentration (a) are known according to the second-order equation: k lgb(a -x) +1 (a-b) t a(io-x)' where x is the amount of ouabain bound to the receptor per time (t). The temperature-dependent association rate constants (k±1,) calculated from Fig. 2B are given in Table 1.

5 NUMBER OF OUABAIN BINDING SITES 675 S U 1000 / i,1-0, 2 0 LLX4--.,-4--2roc l-x -. C4 co ~~~~~~220 C 00 C-, Time (min) Time (min) Fig. 2. Time- and temperature-dependent binding of [3H]ouabain to human erythrocyte membranes. Membrane suspension, 0-5 ml. (initial receptor concentration = b = 1-02 x mole), were incubated in 50 mm imidazole/hcl, ph 7-25, 3 mm-mgcl2, 3 mm imidazole-po4 and 9 x mole [3H]ouabain (initial ouabain concentration = a) at the indicated temperature and time. Total volume 2 ml. Each point was done in duplicate. B, the experimental points along the curves in A when substituted into the secondorder equation: b(a -x) = (a-b)t a(b-x) C 0.0 C.0 0 oi L:i: C0 300 Time (min) Fig. 3. Rates of dissociation of [3H]ouabain-receptor complex of human erythrocyte membranes. One ml. [3H]ouabain-receptor complex (preparation see Methods) was incubated for the indicated time and temperatures in 50 mm imidazole/hcl, ph 7-25, 3 mm-mgcl2, 3 mm imidazole-po4 and 10-3 M unlabelled ouabain, total volume 2 ml.

6 676 ERLAND ERDMANN AND WERNER HASSE TABLE 1. Kinetic constants for specific ouabain binding to human erythrocyte membranes 370 C 220 C 00 C Association rate constant k+1 (M-1 sec-1) 4-6 x x x 104 Dissociation rate constant k-l (sec-1) 1-4 x x x 10-4 Dissociation constant KD (M) From k-1/k+, 0 3 x x x 108 From Scatchard plots 0-28 x 10-8 The dissociation of the ouabain-receptor complex ofhuman erythrocytes follows first-order kinetics (Fig. 3). The temperature-dependent dissociation rate constants calculated from Fig. 3 are given in Table 1. At 00 C the [3H]ouabain-receptor complex is very stable. Less than 5 % of the radioactivity was lost after four times washing at 80,000 g. This indicates that neither the number of binding sites of the erythrocyte membranes changes nor the radioactivity bound ([3H]ouabain) dissociates during the washing procedure. The dissociation constant (KD) may be calculated from the ratio of the dissociation rate constant (k-,) and the association rate constant (k+1): K The dissociation constant (KD) obtained by this method is 0 3 x 10-8 M at 370 C (Table 1). A second way to measure the dissociation constant is to perform an equilibrium binding experiment (Cuatrecasas, 1971; Erdmann & Schoner, 1973a). Ouabain binding at equilibrium is shown in Fig. 4. The concentration-dependent binding of ouabain to its receptor proceeds until all receptors are saturated. The maximal binding sites of the erythrocyte membranes incubated may be approximately obtained in this way. If these data (Fig. 4A) are plotted according to Scatchard (1969) (Fig. 4B), the intercept with the ordinate gives the maximal number of receptor sites. The dissociation constant (KD) can be calculated from the slope of the plot. It is 0-27 x 10- M. This value agrees within experimental error with that calculated from the ratio of the rate constants (Table 1). The straight line of the plot indicates that there is only one type of ouabain receptors in human erythrocyte membranes with high affinity for the drug. The total number of ouabain binding sites per erythrocyte calculated from the respective Scatchard plots at 370 C was determined for nineteen male and twelve female people between 18 and 88 years of age (Fig. 5). There are ouabain-receptor sites per erythrocyte irrespective of the persons age or sex. The dissociation constants were x k1c

7 NUMBER OF OUABAIN BINDING SITES E 0 E V 2._.0 C (8 M Free ouabain (10-9M) Ouabain bound Ouabain free Fig. 4. Binding of [3H]ouabain to human erythrocyte membranes at equilibrium. One ml. membrane suspension (representing 7 9 x 109 erythrocytes) were incubated for 180 min in 50 mm imidazole/hcl, ph 7-25, 3 mm-mgci2. 3 mm imidazole-po4 and various concentrations of [3H]ouabain at 37' C. Total volume 2 ml. The experiments were performed in duplicate. B, the experimental data from A are plotted according to Scatchard (1969). The maximal binding sites calculated from the intercept on the ordinate are 242 per single erythrocyte. The dissociation constant calculated from the slope of the plot is 0-27 x 10-8 M. I I I I I I I I I VD GD U U E- GD z I1 I I I I I I I I Years Fig. 5. The maximal numbers of [3H]ouabain receptor sites per single erythrocyte were calculated from equilibrium binding experiments (Fig. 4) of thirty-one blood donors between 18 and 88 years of age.

8 678 ERLAND ERDMANN AND WERNER HASSE 10-8 M (n = 31). There was no age- or sex-dependent alteration of the KD either. The affinity of ouabain for the membrane-bound glycoside receptor from human ventricular myocardium was determined from similar binding experiments (Fig. 6). There was only one type of receptor sites in the cardiac membrane preparation, too. The dissociation constants of the ouabain-receptor complexes were 0*21 x 10-8 M and 0-25 x 10-8 M (Fig. 6) in cardiac cell membranes obtained from a 34- and a 67-year-old (males). The ouabain binding capacity of these myocardial cell membranes were 129 x moles of ouabain per (Na+ +K+)-ATPase unit (i.e. 1 unit = 1 molee ATP hydrolysed/min) of the cell membranes. This latter value agrees well with the ouabain binding capacity of cardiac membranes from beef and dog (Erdmann & Schoner, 1973a). I I I I _ D20 KD=0-25 x 10- M C C E\.0~~~~~ _ ~~~~~~~~ Fig Ouabain bound/free Binding of [3H]ouabain to human cardiac membranes. Membrane protein, 2-3 mg ((Na+ + K+)-ATPase activity = #rmole ADP/mg protein = 10.5 x mole receptor sites), were incubated in 50 mm imidazole/hcl, ph 7-25, 3 mm-mgcl2, 3 mm imidazole-po4 and various [3H]ouabain concentrations for 120 min at 370 C. Total volume 2 ml. The dissociation constant is calculated from the slope of the plot. It is 0-25 x 10-8 M. DISCUSSION The ability of cardiac glycosides to inhibit membrane-bound (Na+ + K+)- activated ATPase and the transmembrane active transport of sodium and potassium suggests that the locus of ouabain action is the cell membrane.

9 NUMBER OF OUABAIN BINDING SITES 679 This, however, does not exclude the presence of additional binding structures for ouabain inside the cell. The object of our studies was to quantitate and to analyse the membrane-bound binding sites for ouabain in human erythrocytes and to correlate some of their properties with those of human myocardium. Experiments performed by other investigators (Gardner & Kiino, 1973; Dunham & Hoffman, 1970; Gardner & Frantz, 1974; Chipperfield & Whittam, 1973) indicate that there is a finite number of glycoside binding sites in erythrocyte membranes, whose affinity for cardiac glycosides is depending on the cation composition of the incubation medium. The number of binding sites or 'receptors' calculated varies considerably, between 200 per erythrocyte (Dunham & Hoffman, 1970) and 1200 (Gardner & Frantz, 1974). Our experiments performed at equilibrium and at optimal binding conditions disclose but one type of ouabain binding sites in human erythrocyte and cardiac membranes. This agrees with previous data (Gardner & Conlon, 1972; Gardner & Kiino, 1973; Erdmann & Schoner, 1973a). Using intact human erythrocytes Baker & Willis (1972) measured two components of binding, one component that saturated at low glycoside concentrations and a second component that increased up to the highest ouabain concentration used. An unsaturable component of ouabain binding could not be detected in our erythrocyte or cardiac membrane preparations. Unspecific binding of ouabain (i.e. [3H]ouabain not being displaced by high concentrations of unlabelled ouabain) amounted to % in erythrocyte membranes and 1-3 % in cardiac membranes. Optimal ouabain binding to human erythrocyte or cardiac membranes occurs in the presence of either 3 mm-mgcl2 plus 3 Mm-ATP plus 150 mm- NaCl or 3 mm-mgcl2 plus 3 mm-po4. At both conditions identical results are obtained in respect to the maximal number of binding sites as well as to the affinity of the receptor for the drug. The remarkably high affinity for ouabain is reflected in the low dissociation constant (KD) of the ouabain-receptor-complex (0-28 x 10- M). These dissociation constants did not change with the age or sex of the blood donors. These values obtained at equilibrium and from the ratio of the rate constants are somewhat lower but in the same range as those reported by other groups and obtained by different methods (0.625 x 10- M (Gardner & Conlon, 1972) and 0-78 x 10-8 M (Gardner, Kiino, Swartz & Butler, 1973)). There were membrane bound ouabain binding sites per erythrocyte as calculated from equilibrium binding experiments (Scatchard plots) at multiple ouabain concentrations (Fig. 6). This rather low number of receptors with high affinity for the drug was determined by Dunham & Hoffman (1970), too. Due to our experimental set up of using erythrocyte fragments and not intact cells (Baker & Willis, 1972; Gardner & Kiino,

10 68080ERLAND ERDMANN AND WERNER HASSE 1973; Gardner & Frantz, 1974) it is safe to assume that there was little or no entrapment of [3H]ouabain in our binding experiments. This latter phenomenon might be responsible for the higher number of receptor sites found by other investigators. The possible entrapment of ouabain within the cell also explains the unsaturable component of [3H]ouabain 'binding' found by Baker & Willis (1972). These experimental results lead to some interesting reflexions. The surface of a single erythrocyte amounts to about 250 /tm2. The 228 ouabain receptors, which are supposed to be located on the same polypeptide chain as the (Na+ + K+)-ATPase (Ruoho & Kyte, 1974) and as such being a measure of the sodium-pump sites (Sachs, Ellory, Kropp, Dunham & Hoffmann, 1974) might be diffusely distributed on the cell surface. Then one receptor site - equal to one sodium pumping site - corresponds to about 1 gim2 of the membrane. According to Cuatrecasas (1973) a single normal fat cell of a rat contains about 10 insulin receptors per JiM2 and 105 concanavalin A binding sites per /im2. There are cytochalasin B high affinity binding sites per human red cell (Lin, Santi & Spudich, 1974). The quantitation and determination of different hormone or drug binding sites on the surface of the cell (erythrocyte) membrane and the subsequent analysis of the binding structures might lead to a 'mapping' of the membrane. (Na+ + K+)-ATPase activity is directly related to the quantity of nucleotide and ouabain binding sites per mg membrane protein (Kaniike, Erdmann & Schoner, 1974; Erdmann & Schoner, 1973c). There are 129 x 10-12mole ouabain binding sites per (Na+ + K+)-ATPase unit in human cardiac membranes. The ouabain binding capacity of erythrocyte membranes being of the same order ( x mole per enzyme unit) indicates a turn-over number of about 8000 molecules ATP hydrolysed/min and per enzymic site. This value agrees well with previous data obtained for different tissues and species (Erdmann & Schoner, 1973a). If the sodium pump of the erythrocyte membrane moves sodium and potassium ions with a stoicheiometry of 3Na+: 2K+ per ATP hydrolysed (Sen & Post, 1964; see Schoner, 1971 and Skou, 1973) then 24,000 molecules sodium/gm2 membrane or 5-5 x 106 molecules sodium/erythrocyte could maximally be pumped out actively per minute or about 5-6 m-equiv/l. hr. According to Solomon (1952), human erythrocytes have to pump out 3-1 m-equiv/l.hr to maintain the measured intra-extracellular cation difference. Another reflexion involves the identical dissociation constant of the ouabain-receptor complex in human myocardium and in human erythrocyte membranes. The same result was obtained for other species (Erdmann & Schoner, 1973a). The membrane-bound receptor having the same affinity for the drug in both tissues might be composed of the same

11 NUMBER OF OUABAIN BINDING SITES 681 protein structures in one species. This is underlined by the fact that there seems to be only one single class of binding sites according to our data and others (Gardner & Kiino, 1973). The nature of this 'receptor' remains highly uncertain, although ouabain binding to this site is very specific as expressed by the low dissociation constant of 0-28 x 1o-8 M. The question remains to be answered - which hormone or substance naturally binds to this receptor and by this possibly influences or regulates the active transport of sodium and potassium. This work was supported by Deutsche Forschungsgemeinschaft (SFB 89 Kardiologie, Gdttingen). REFERENCES BAKER, P. F. & WRUs, J. S. (1972). Binding of the cardiac glycoside ouabain to intact cells. J. Physiol. 224, CHIPPERFIELD, A. R. & WHITTAM, R. (1973). Reconstitution of the sodium pump from protein and phosphatidylserine: features of ouabain binding. J. Physiol. 230, CUATRECASAS, P. (1971). Insulin-receptor interactions in adipose tissue cells: direct measurements and properties. Proc. natn. Acad. Sci. U.S.A. 68, CUATRECASAS, P. (1973). Insulin receptor of liver and fat cell membranes. Fedn Proc. 32, DUNHAM, P. B. & HOFFMAN, J. F. (1970). Partial purification of the ouabain-binding component and of Na, K-ATPase from human red cell membranes. Proc. natn. Acad. Sci. U.S.A. 66, ERDMANN, E. & SCHONER, W. (1973 a). Ouabain-receptor interactions in (Na+ + K+)- ATPase preparations from different tissues and species. I. Determinations of kinetic constants and dissociation constants. Biochim. biophy8. Acta 307, ERDMANN, E. & SCHONER, W. ( 1973 b). Ouabain-receptor interactions in (Na+ + K+)- ATPase preparations. II. Effect of cations and nucleotides on rate constants and dissociation constants. Biochim. biophys. Acta 330, ERDMANN, E. & SCHONER, W. (1973 c). Ouabain -receptor interactions in (Na+ + K+)- ATPase preparations. III. On the stability of the ouabain receptor against physical treatment, hydrolases, and SH-reagents. Biochim. biophy8. Acta 330, ERDMANN, E., PATZELT, R. & SCHONER, W. (1974). The cardiac glycoside receptor: its properties and its correlation to nucleotide binding sites, phosphointermediate and (Na+ + K+)-ATPase activity. In Recent Advances in Myocardiology. Baltimore, U.S.A.: University Park Press. (In the Press.) GARDNER, J. D. & CONLON, T. P. (1972). The effects of sodium and potassium on ouabain binding by human erythrocytes. J. yen. Physiol. 60, GARDNER, J. D. & FRANTZ, C. (1974). Effects of cations on ouabain binding by intact human erythrocytes. J. membrane Biol. 16, GARDNER, J. D. & KIINO, D. R. (1973). Ouabain binding and cation transport in human erythrocytes. J. clin. Invest. 52, GARDNER, J. D., KIINO, D. R., SWARTZ, T. J. & BUTLER, V. P. (1973). Effects of digoxin-specific antibodies on accumulation and binding of digoxin by human erythrocytes. J. clin. Invest. 52,

12 682 ERLAND ERDMANN AND WERNER HASSE HANSEN, 0. (1971). The relationship between g-strophanthin binding capacity and ATPase activity in plasma membrane fragments from ox brain. Biochim. biophy8. Acta 233, HOFFMAN, J. F. (1966). The red cell membrane and the transport of sodium and potassium. Am. J. Med. 41, JORGENSEN, P. L., HANSEN, O., GLYNN, I. M. & CAVIERES, J. D. (1973). Antibodies to pig kidney (Na++K+)-ATPase inhibit the Na+ pump in human red cells provided they have access to the inner surface of the red cell membrane. Biochim. biophy8. Acta 291, KANI=E, K., ERDMANN, E. & SCHONER, W. (1974). Study on the differential modifications of (Nat + K+)-ATPase and its partial reactions by dimethylsulfoxide. Biochim. biophys. Acta 352, KYTE, J. (1972). Properties of the two polypeptides of sodium- and potassiumdependent adenosine triphosphatase. J. biol. Chem. 247, LIN, S., SANTI, D. V. & SPUDICE, J. A. (1974). Biochemical studies on the mode of action of cytochalasin B. J. biol. Chem. 249, LowRY, 0. H., ROSEBROUGH, N. J., FARR, A. L. & RANDALL, R. J. (1951). Protein measurements with the folin phenol reagent. J. biol. Chem. 193, MATsuI, H. & SCHWARTZ, A. (1968). Mechanism of cardiac glycoside inhibition of the (Na++K+)-dependent ATPase from cardiac tissue. Biochim. biophy8. Acta 151, RUOHO, A. & KYTE, J. (1974). Photoaffinity labeling of the ouabain binding site on (Na+ + K+)-adenosine-triphosphatase. Proc. natn. Acad. Sci. U.S.A. 71, SACHS, J. R., ELLORY, J. C., KROPP, D. L., DUNHAM, P. B. & HOFFMAN, J. F. (1974). Antibody-induced alterations in the kinetic characteristics of the Na:K pump in goat red blood cells. J. gen. Physiol. 63, SCATCHARD, G. (1969). The attractions of proteins for small molecules and ions. Ann. N.Y. Acad. Sci. 51, SCHATZMANN, H. J. (1953). Herzglycoside als Hemmstoffe fur den aktiven Kaliumund Natriumtransport durch die Erythrocytenmembran. Helv. physiol. pharmac. Acta 11, SCHONER, W., VON ILBERG, C., KRAMER, R. & SEUBERT, W. (1967). On the mechanism of Na+- and K+-stimulated hydrolysis of adenosinetriphosphate. 1. Purifications and properties of a Na+- and K+-activated ATPase from ox brain. Eur. Jnl. Biochem. 1, SCHONER, W. (1974 1). Zum aktiven Na+, K+-Transport durch die Membran tierischer Zellen. Angew. Chem. 83, SEN, A. K. & POST, R. L. (1964). Stoichiometry and localization of adenosine triphosphate-dependent sodium and potassium transport in the erythrocyte. J. biol. Chem. 239, SKOU, J. C. (1973). The relationship of the (Na++K+)-activated enzyme system to transport of sodium and potassium across the cell membrane. Bioenergetic8 4, SOLOMON, A. K. (1952). The permeability of the human erythrocyte to sodium and potassium. J. gen. Physiol. 36, 57-i10. SOLOMON, A. K. (1958). The permeability of red cells to water and ions. Ann. N.Y. Acad. Sci