Garrahan, 1975; Hexum et al. 1970; Robinson et al. 1978). As pointed out by Garay

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1 J. Physiol. (1982), 326, pp With 3 text-figures Printed in Great Britain INHIBITION OF THE SODIUM PUMP BY INORGANIC PHOSPHATE IN RESEALED RED CELL GHOSTS BY D. A. EISNER* AND D. E. RICHARDS From the Physiological Laboratory, Downing Street, Cambridge CB2 3EG (Received 11 June 1981) SUMMARY 1. Ouabain-sensitive Rb influx was measured into K-free resealed red cell ghosts. The effects of inorganic phosphate (Pi) were examined. 2. Phosphate decreased the magnitude of the influx. Increasing Pi lowered the apparent affinity for both ATP and external rubidium ions. The effects of Pi on the affinity for external Rb were greatest at low ATP concentrations. 3. In the nominal absence of phosphate, increasing ATP from 1 to 1 gsm had little effect on the Rb influx from solutions of low (1 AM) rubidium concentrations. In the presence of Pi, ATP increased Rb uptake markedly, even from solutions of low Rb concentration. 4. The above interactions between Pi, ATP and external Rb are consistent with a consecutive scheme for the Na pump in which phosphate is released after potassium binds at the external surface and before ATP binds to release potassium ions to the internal solution. 5. Previous failures to find an effect of phosphate on either the affinity for ATP or that for external potassium (rubidium) ions are shown to be equally consistent with the model. The lack of change of apparent affinity is shown to result from the 'restricted range of concentrations used in these previous experiments. INTRODUCTION The products of an enzyme reaction can inhibit that reaction by driving it backwards. Kinetic analysis of such product inhibition has provided much evidence about reaction mechanisms (Cleland, 197). Inorganic phosphate (Pi) is released in the normal working of the Na pump and therefore the effects of Pi as a product inhibitor of the Na pump have been investigated. Pi inhibits the Na pump in red cells (Garay & Garrahan, 1975) and also in rat brain Na-K-ATPase (Hexum, Samson & Himes, 197; Robinson, Flashner & Marin, 1978). Pi has been reported to have no effect on the apparent affinity for K ions (Garay & Garrhan, 1975; Robinson et al. 1978) and similarly to have little or no effect on the affinity for ATP (Garay & Garrahan, 1975; Hexum et al. 197; Robinson et al. 1978). As pointed out by Garay & Garrhan (1975) these results are inconsistent with a consecutive model for the Na * Present address: Department of Physiology, University College London, Gower Street, London WC1E 6BT. PrY 326

2 2 D. A. EISNER AND D. E. RICHARDS pump in which potassium ions bind before phosphate is released. On such a scheme Pi should lower the apparent affinity for external K. The observed independence of the K affinity is therefore worth investigating. It has been suggested that K ions remain occluded within the enzyme following dephosphorylation (Post, Hegyvary & Kume, 1972; Karlish, Yates & Glynn, 1978). Such occlusion (following dephosphorylation) has recently been demonstrated (Glynn & Richards, 1981). ATP is required at a low affinity, non-phosphorylating site to accelerate the release of occluded K ions to the inside surface (Beauge & Glynn, 1979). If this ATP dependent reaction follows dephosphorylation, the affinity for ATP at this site should be reduced by Pi. The observation that Pi has no effect on the ATP affinity is therefore inconsistent with this scheme. The results of this paper show, in contrast to previous work, that phosphate lowers the affinity for both ATP and potassium ions. Reasons for the previous failure to see this effect are discussed. In a previous paper (Eisner & Richards, 1981) we showed that ATP lowered the affinity for external potassium ions. We suggested that the failure of other studies to show this effect could be attributed to the presence of inorganic phosphate. This prediction is confirmed in the present paper. METHODS The methods used to prepare ghosts have been described previously (Eisner & Richards, 1981). The cells were depleted of ATP by incubation in a substrate-free medium followed by exposure to iodoacetamide and inosine. The K content of the cells was reduced to less then 1 EM by nystatin treatment in a K-free solution. Resealed ghosts, containing an ATP regenerating system, were prepared from these cells. Preparing phosphate-containing ghosts produced two additional problems. Firstly, early experiments showed that if the ghosts were prepared by lysing in solutions containing different phosphate concentrations, variable numbers of ghosts were obtained. This was overcome by lysing in a phosphate-free solution and then adding the requisite amount of phosphate on restoring tonicity. Secondly, the ATP concentration in the ghosts was held constant with a system in which creatine kinase is used to regenerate ATP from ADP and creatine phosphate (Glynn & Karlish, 1976). In previous work we have used 5 units of creatine kinase per millilitre which is sufficient to ensure that the creatine kinase reaction is at equilibrium. However, phosphate ions inhibit creatine kinase (Noda, Nihei & Morales, 196) and preliminary experiments showed that the ATP concentration could be reduced by 1 mm-phosphate by up to 4 %. This problem was overcome by using a creatine kinase concentration of 25 u./ml. Under these conditions Pi had no measureable effect on the ATP concentration. The flux measurements were made in the following solution: 155 mm-choline Cl; 5 mm-(tris) HEPES (ph 7*5, 2 C); 5 mm-nacl; 1 mm-mgcl2; 1 mm-(tris) EGTA. The incubation solution also contained the same phosphate concentration as the ghosts. Extra Mg was added to all solutions to make the free Mg concentration 1 mm after allowing for the chelating properties of ATP, Pi and creatine phosphate (Dawson, Elliott, Elliott & Jones, 1969). Rubidium influxes were measured as described by Eisner & Richards (1981). All measurements were made in triplicate. Ouabain insensitive fluxes were measured in the presence of 1-3 M-ouabain. The ATP concentration in the ghosts was checked with a firefly method (cf. Eisner & Richards, 1981). The values for ATP concentration given in the Figures and Tables are those in the lysing solution. The measured values always agreed with these expected values to within 1%. The phosphate concentrations are those in the lysing (and incubation) solutions. Nominally phosphatefree ghosts were prepared by including inosine in the lysing and incubation media (Glynn, Lew & Liithi, 197). The flux measurements have all been expressed with respect to the volume of ghosts resealed to Na ions. This was calculated by measuring the concentrations of Na in a suspension of ghosts

3 Pi INHIBITION OF Na PUMP in a Na-free solution. Knowing the Na concentration of the lysing solution allows one to calculate the resealed volume. When the resealed ghost volume is calculated in this manner the ouabaininsensitive Rb uptake is found to be unaffected by ATP or Pi (e.g. Fig. 1). The fact that the ouabain-insensitive fluxes are independent of ATP and Pi suggests that the effects of ATP and Pi on the oubain-sensitive fluxes are real and are not seriously affected by differences in the number of resealed ghosts. Source of materials. Blood was obtained from the Regional Transfusion Centre, Cambridge and was a few days old. 86Rb was from the Radiochemical Centre, Amersham. Creatine kinase, creatine phosphate, Tris, ouabain, nystatin, choline chloride and inosine were from Sigma. NaCl was of Speepure grade (Johnson Matthey). Choline chloride was recrystallized from hot absolute ethanol (Garrahan & Glynn, 1967). 3 RESULTS Effect of Pi on the affinity for ATP The experiment of Fig. 1 was designed to investigate the effects of inorganic phosphate on the Na pump. Ghosts were prepared to contain various concentrations of ATP and either or 1 mm-phosphate. The nominally zero phosphate ghosts had inosine (5 mm) present to reduce further any phosphate contamination (Glynn et al. 197). It is clear that Pi inhibits the Rb influx at all concentrations ofatp examined. It is also obvious that the inhibition by Pi is greatest at the lower range of ATP concentrations. The inset shows the two curves normalized to the same value in 3 mm-atp. The apparent affinity for ATP is lower in the presence of phosphate than in the control oase. This contrasts with previous work on the red cell where Pi was reported to have no effect on the ATP affinity (Garay & Garrahan, 1974). Effects of Pi on the affinity for external K Fig. 2 shows Rb influx as a function of the external Rb concentration at two levels of phosphate: and 5 mm. 5 mm-pi inhibits the Rb influx at all Rb concentrations examined. The magnitude of the inhibition decreases as [Rb]o is increased. This is emphasized in the inset which shows the two curves normalized to the same value in 32 /SM-[Rb]. It is obvious that the affinity for external Rb is decreased in the phosphate-containing ghosts. This effect of phosphate on the Rb affinity is at odds with the results of Garay & Garrahan (1975) and Robinson et al. (1978). Both these groups reported no effect of Pi on the affinity for K (Rb). While there are many differences between the conditions of Fig. 2 and those used by previous workers, an obvious difference concerns the ATP concentration used. The level of 1 jum is much less than that used in previous work. It therefore seemed worthwhile to see if changing ATP had any effect on the inhibition by phosphate. In particular it is important to know if changing ATP has any action on the changes in the affinity for external K induced by Pi. Interactions between ATP and Pi on the external K affinity. The experiment of Fig. 3 shows Lineweaver-Burk plots of the effects of 5 mm-phosphate at two different ATP levels (1 and 1 SM) on the activation of Rb influx by external Rb. The ordinate plots v1 (see Eisner & Richards, 1981) to allow for the fact that two rubidium ions are transported per cycle. Pi lowers the maximum velocity (Vmax) and increases the apparent Km (Kapp) at both ATP concentrations. The effect on Kapp is, however, more obvious at the lower ATP concentration. This point is emphasized in Table 1 which 1-2

4 4 D. A. EISNER AND D. E. RICHARDS S o Pi -5 B Yo1 mm-pi -@ E, OPiI ATP (#M) I C. / /Y ~~~~~~~1 MaM-Pi ATP (OM) Fig. 1. The effects of phosphate on the ouabain-sensitive Rb influx into ghosts containing various ATP concentrations. The main figure shows Rb uptake from a solution containing 5 /SM-RbCI into ghosts prepared to contain either () or 1 mm (@) Pi. The inset shows the two curves normalized to the same value at 3 mm-atp. Error bars show +s.e.m. (n = 3). For comparison the ouabain insensitive Rb influx was m-mole/i. hr averaged over the Pi tubes and m-mole/l. hr averaged over the 1 mm-pi tubes. In neither case did the ouabain insensitive influx vary with ATP concentration. Measurements of ATP with the firefly technique showed that the ATP concentration was unaffected by adding either 5 mm-inosine or 1 mm-pi. Detailed procedure. After depleting the cells of ATP and nystatin treatment to remove K, the cells were lysed in a solution containing: 1 mm-creatine phosphate (di Na salt) to give a final [Na] of 2 mm; 25 u./ml. creatine kinase; 1 mm-mgcl2; 5 mm-(tris) HEPES (ph 7 5); -2 mm-(tris) EGTA plus the desired concentration of ATP. Tonicity was restored by adding a small volume of solution containing 2-5 M choline chloride plus the desired concentration of phosphate and enough MgCl2 to give a final free Mg of 1 mm. After washing the ghosts were resuspended with and without ouabain (1-3 M) in the incubating solution described in the Methods plus 5,M-86RbCl at a final haematocrit of -3 %: Further details are given by Eisner & Richards (1981). gives the results of a least squares analysis of Fig. 3. Values for both the calculated Kapp and also the slope of the regression lines (Kapp/V/ Vmax) are given. Increasing Pi has a greater effect on Kapp at 1 /M than at 1 /sm-atp. Table 1 also demonstrates that increasing ATP in the nominal absence of Pi has no significant effect on the slope of the regression line. However, in the presence of 5 mm-pi, increasing ATP over this range halves the value of the slope. This conversion of a parallel pair of lines to divergence by the addition of a product (Pi) is diagnostic of a consecutive or 'ping-pong' mechanism (Cleland, 197). This analysis requires fitting the data to Michaelis kinetics. Since two potassium ions are transported the values of Kapp obtained should be regarded as descriptive and of limited physical significance (Eisner & Richards, 1981). We pointed out that it is better to measure the influx at low

5 Pi INHIBITION OF Na PUMP 5 8 O Pi x 1- -C._ E x. i -5 E Z - 16 [Rb] (pm) [ R b] (MM) Fig. 2. The effects of phosphate on the activation of ouabain-sensitive Rb influx by external Rb. The main figure shows Rb uptake as a function of external Rb concentration into ghosts prepared to contain either () or 5 mm (@) Pi. The inset shows the two curves nomalized to their values at 32,uM-[Rb]. ATP was 1 #SM. Detailed procedure. Ghosts were prepared in a similar manner to the experiment of Fig. 1 except that only two lots of ghosts ( and 5 mm-pi) were required. After washing they were incubated in the solution described in the Methods containing the appropriate amount of 86RbCl pm-atp + Pi L- -l, E 1 E /[Rblo (mm)-' Fig. 3. Interactions between Pi and ATP on the affinity for external Rb. The graph shows: ordinate, (ouabain-sensitive Rb influx)i; abscissa, 1/[Rb]o. Ghosts were prepared to contain: 1,LM-ATP, Pi (); 1 /SM-ATP, 5 mm-pi (); 11uM-ATP, Pi (V); 1,uM-ATP, 5 mm-pi (V). As in the other Figures the data are given as mean + S.E.M. The regression lines are, however, calculated from the original data points (see legend to Table 1). The experimental methods are identical to those described for Figs. 1 and 2.

6 6 D. A. EISNER AND D. E. RICHARDS external rubidium rather than the slope of the Lineweaver-Burk plot. A consecutive mechanism should be characterized by the influx at very low external Rb being independent of the ATP concentration. This is equivalent to Lineweaver-Burk plots at different ATP concentrations being parallel (Eisner & Richards, 1981). Table 2 shows the Rb influx from a solution containing 1 /LM-Rb into ghosts prepared to contain 1 or 1 /sm-atp and or 5 mm-pi. In the nominal absence of phosphate, TABLE 1. Effect of ATP and Pi on the apparent affinity for external Rb and the slope of the Lineweaver-Burk plot ATP Pi Kapp Kapp/A/ Vmac (#am) (mm) (,mm) (mm/(m-mole/l. hr)1) ± ± P9± The values of Kapp and Kapp/V Vmax are obtained by fitting linear regression lines to the original data of Fig. 3. K8pp/V- Vm.. is the gradient of the Lineweaver-Burk plot. The regression lines were fitted with an unweighted least squares method. A Lineweaver-Burk plot was used in preference to other transforms of the Michaelis-Menten equation. The usual objection to this double reciprocal plot is that it emphasizes the points at lower substrate concentrations which are generally less accurate. In the present experiments both the absolute and fractional error increase with increasing [Rb]o (Fig. 2) and the double reciprocal plot is appropriate. TABLE 2. The effects of ATP and Pi on ouabain-sensitive Rb influx at 1 M-external Rb ATP Pi Influx ('M) (mm) (#-mole./l. hr) Ghosts were prepared as described in the legend to Fig. 1 and ouabain sensitive rubidium uptake measured from a solution containing 1 /tm-8rbcl. increasing ATP over this range increases the influx by only about 5 %. Under these conditions the influx at high external Rb was increased by about twenty-fold on increasing ATP (not shown). In the presence of Pi the same increase of ATP increases the influx at 1 /mm-[rb]o by at least 3 %. Thus, in the presence of phosphate, increasing ATP has a large effect on the Na pump rate even at low external Rb. This supports the analysis of the slopes of the double reciprocal plot (Table 1). DISCUSSION Model The model used to analyse these results is essentially the same as that used previously (Eisner & Richards, 1981). Only three forms of the enzyme are considered: E, EP and E2K. The total enzyme concentration is ET. Transitions between these forms are governed by the following rate constants: a, that of phosphorylation, a

7 Pi INHIBITION OF Na PUMP function of ATP and internal Na; b, that of dephosphorylation, a function of external K; c, that of the conformational change which releases K ions to the internal surface and depends on ATP at the low affinity, non-phosphorylating site; and e, the rate constant for reversal of dephosphorylation which is a function of the inorganic phosphate concentration. We assume that, in the range of ATP concentrations examined, changes of a can be ignored (Eisner & Richards, 1981). b E. EP a E2 (K) 7 A steady-state analysis gives: C K influx = ce2(k) = ab+ac+ea+bcet Effects of Pi on the apparent ATP affinity We must first consider a trivial explanation for the decrease of ATP affinity produced by Pi: that Pi competes with ATP at the low affinity site and thereby removes enzyme from the reaction sequence. Although there is evidence that Pi does not compete at the phosphorylation site (Hegyvary & Post, 1971; Post, Toda & Rogers, 1975) no such evidence is available for the low affinity site. We can, however, exclude the possibility that all the inhibitory effects of Pi are produced by competition with ATP to remove enzyme from the reaction sequence. If Pi inhibits by competing with ATP for a site on E2(K) the inhibitory effects of Pi should be greater the higher is E2(K). Therefore increasing external K (Rb) should increase the inhibition. In other words phosphate would increase the apparent affinity for external K (Rb). The fact that the inhibitory effects of Pi are least evident at high (K) (Rb) and that Pi lowers the apparent affinity for external K argues against a mechanism in which Pi merely acts as a competitive inhibitor. If the rate of the conformational reaction c can be described as a simple Michaelis function: c = Cmax x/(x+,f) where x is the ATP concentration, fi the real Km and cmax the maximum velocity, the apparent affinity is given by: (b+e) Kapp = (b (e) Cmax(l +b/a)+b+e (2) Similarly m Vmax mx mx1bax+be)et. (3) Cma~x(l + b/a) + b + e) It is obvious from eqns. (2) and (3) that (i) increasing e (Pi) increases Kapp and decreases Vmax and (ii) these effects are reduced by increasing b (external Rb). In the limit when dephosphorylation is infinitely fast e would have no effect on either Kapp or Vmax. The point of immediate interest is the relative effect on Kapp and Vmax. Equation (3) shows that to halve Vmax requires a concentration of Pi such that e = b +cmax (1 + b/a). Substituting in eqn. (2) gives the ratio of Kapp with this concentration of phosphate to that without phosphate as 1 + cmax(a + b)/2ab. The

8 8 D. A. EISNER AND D. E. RICHARDS relevance of this to the present experiments is seen by considering two limiting cases. (i) When external Rb is very high and therefore b is large. In this case the ratio of the Kapp becomes 1 + cmax/2a. Depending on the relative values of cmax and a this value can vary between unity (no effect of phosphate on ATP affinity) and much higher values. Taking the values used by Karlish & Yates (1978) of45 and 324 minrespectively for a and cmax gives a value of 1-36 for the ratio of Kapp. Therefore, since previous work has used saturating K concentrations (Garay & Garrahan, 1975; Hexum et al. 197; Robinson et al. 1978) it is not surprising that either no effect or very small effects were seen on the ATP affinity. The above result shows that under these conditions, a concentration of phosphate sufficient to change Vmax by a factor of 2 should have less than a 4% effect on Kapp. (ii) In the present study (Fig. 1), a low concentration of Rb was used and b was therefore small. Under these conditions the above result predicts the large observed change in affinity. It is, however, not worth quantifying this result further since the assumption that c depends in a hyperbolic manner on ATP concentration remains to be validated and is certainly not true at very low ATP concentrations where an ATP independent component persists (Karlish & Yates, 1978). Effect of Pi on the K affinity Another difference between this and previous studies is the finding that phosphate lowers the affinity for external rubidium ions. A similar argument to that used above predicts that, for a given reduction of Vmax, Kapp for external Rb will be increased more at low ATP than at high ATP. As shown by Eisner & Richards (1981) the fact that two K (Rb) ions are transported further reduces the size of the expected change of Kapp, In previous studies ATP has always been in the millimolar range. It is therefore reasonable that no effect of phosphate on K affinity was found before. Fig. 3 and Tables 1 and 2 show that Pi complicates the effects of ATP on the external Rb affinity. In the presence of Pi, ATP increases the influx even from very low external Rb concentrations. At these low levels of external Rb eqn. (1) gives the influx as ETbc/(c+e). Therefore an increase of ATP (c) is expected to increase the influx even from a vanishingly low external Rb (K) concentration. This can be intuitively seen as follows. In the absence of phosphate at very low external Rb, the rate limiting step is the dephosphorylation and the overall reaction rate should be independent of ATP. In the presence of phosphate, however, following dephosphorylation the occluded form can either (i) release Rb (K) to the inside or (ii) be driven back to EP by phosphate. The higher is ATP the smaller the probability that the occluded form will be phosphorylated by Pi and therefore the forward rate increases with ATP. Unfortunately the influx at 1 gm-external Rb does depend slightly on ATP even in the nominal absence of phosphate. It is not clear whether this reflects some Pi contamination in the nominally phosphate-free ghosts or some other cause. The fact that, in the presence of phosphate, increasing ATP (i) decreases the slope ofthe Lineweaver-Burk plot and (ii) increases the Rb influx, even at low [Rb], means that ATP changes in the presence of phosphate have more effect on V/ Vmax than on Kapp. This result lends support to the suggestion made in a previous paper that the failure of previous studies to find effects of ATP on the external K (Rb) affinity was partly due to the presence of intracellular Pi (Eisner & Richards, 1981).

9 P. INHIBITION OF Na PUMP 9 Conclusion The results of this paper show that the product inhibition patterns produced by phosphate are consistent with the scheme of Post et al. (1972) and Karlish et al. (1978). Garay & Garrahan (1975) concluded from the apparent lack of effect of Pi on K and ATP affinities that some sort of simultaneous model should be used to describe the sodium pump. This conclusion is not warranted since, as shown above, under the restricted range of conditions previously used, even a consecutive scheme would produce no effect on these affinities. It is interesting to note that other pieces of evidence in favour of a simultaneous model such as the effects of the transported cation on one side of the membrane on the affinity for the other cation on the other side of the membrane (Chipperfield & Whittam, 1974; Sachs, 1977) can no longer be regarded as unequivocal proof for a simultaneous model (Sachs, 198). Similarly, recent results showing that the apparent affinity for external K depends on the ATP concentration (Beauge & Di Polo, 1979; Eisner & Richards, 1981) suggest that a consecutive or ping-pong model is a more suitable description of the sodium pump. This work was supported by grants from the M.R.C. to Professor I. M. Glynn. We thank Professor Glynn for his help and advice. We are grateful to Dr. J. C. Ellory for drawing our attention to the effects of phosphate on creatine kinase. We thank Dr V. L. Lew for useful comments. REFERENCES BEAUG9, L. A. & Di POLO, R. (1979). Sidedness of the ATP-Na+-K+ interactions with the Na+ pump in squid axons. Biochim. biophys. Acta 55, BEAUGi, L. A. & GLYNN, I. M. (1979). Occlusion of K ions in the unphosphorylated sodium pump. Nature, Lond. 28, 51G-512. CHIPPERFIELD, A. R. & WHITTAM, R. (1974). Evidence that ATP is hydrolysed in a one-step reaction of the sodium pump. Proc. R. Soc. B 187, CLELAND, W. W. (197). Steady state kinetics. In The Enzynes, vol. II, 3rd edn., ed. BOYER, P. D. New York: Academic Press. DAWSON, R. M. C., ELLIOTT, D. C., ELLIOTT, W. H. & JONES, K. M. (1969). Data for Biochemical Research. Oxford: Clarendon. EISNER, D. A. & RICHARDS, D. E. (1981). The interaction of potassium ions and ATP on the sodium pump of resealed red cell ghosts. J. Physiol. 319, GARAY, R. P. & GARRAHAN, P. J. (1975). The interaction of adenosinetriphosphate and inorganic phosphate with the sodium pump in red cells. J. Physiol. 249, GARRAHAN, P. J. & GLYNN, I. M. (1967). The behaviour of the sodium pump in red cells in the absence of external potassium. J. Physiol. 192, GLYNN, I. M. & KARLISH, S. J. D. (1976). ATP hydrolysis associated with an uncoupled sodium flux through the sodium pump: evidence for allosteric effects of intracellular ATP and extracellular sodium. J. Physiol. 256, GLYNN, I. M., LEW, V. L. & LUTHI, U. (197). Reversal of the potassium entry mechanism in red cells, with and without reversal of the entire pump cycle. J. Physiol. 27, GLYNN, I. M. & RICHARDS, D. E. (1981). Two routes to the occluded K+ form of the sodium pump. J. Physiol. 313, 31P. HEXUM, T., SAMSON, F. E. & HIMES, R. H. (197). Kinetic studies of membrane (Na+-K+- Mg2+)-ATPase. Biochim. biophys. Acta 212, HEGYVARY, C. & POST, R. L. (1971). Binding of adenosine triphosphate to sodium and potassium ion-stimulated adenosine triphosphatase. J. biol. Chem. 246, KARLISH, S. J. D. & YATES, D. W. (1978). Tryptophan fluorescence of (Na+ + K+)-ATPase as a tool for study of the enzyme mechanism. Biochim. biophys. Acta 527,

10 1 D. A. EISNER AND D. E. RICHARDS KARLISH, S. J. D., YATES, D. W. & GLYNN, I. M. (1978). Conformational transitions between Na+-bound and K+-bound forms of (Na++K+)-ATPase, studied with formycin nucleotides. Biochim. biophy8. Acta 525, NODA, L., NIHni, T. & MORALES, M. F. (196). The enzymatic activity and inhibition of adenosine 5'-triphosphate-creatine transphosphorylase. J. biol. Chem. 235, POST, R. L., HEGYVARY, C. & KUME, S. (1972). Activation by adenosine triphosphate in the phosphorylation kinetics of sodium and potassium ion transport adenosine triphosphatase. J. biol. Chem. 247, POST, R. L., TODA, G. & ROGERS, F. N. (1975). Phosphorylation by inorganic phosphate of sodium plus potassium ion transport adenosine triphosphatase. J. biol. Chem. 25, ROBINSON, J. D., FLASHNER, M. S. & MARIN, G. K. (1978). Inhibition of the (Na+ +K+)-dependent ATPase by inorganic phosphate. Biochim. biophys. Acda 59, SACHS, J. R. (1977). Kinetic evaluation of the Na-K pump reaction mechanism. J. Physiol. 273, SACHS, J. R. (198). The order of release of sodium and addition of potassium in the sodiumpotassium pump reaction mechanism. J. Physiol. 32,