from the departamento de quimica biol6gica, facultad de

J. Physiol. (1972), 223, pp. 595-617 595With 7 text-figura9Printed in Great Britain

POTASSIUM ACTIVATEDPHOSPHATASE FROM HUMAN RED BLOOD CELLS. THE

EFFECTS OF p-NITROPHENYLPHOSPHATEON CATION FLUXES

By P. J. GARRAHAN AND A. F. REGA*From the Departamento de Quimica Biol6gica, Facultad deFarmacia y Bioquimica, Universidad de Buenos Aires,

Junin 956, Buenos Aires, Argentina

(Received 31 January 1972)

SUMFRY

1. When red cells are incubated in solutions containing p-nitrophenyl-phosphate (p-NPP), intracellular p-NPP quickly builds up reaching witha half-time of 3 min a concentration in cell water equal to one fourth theexternal concentration, which under the conditions used is the expectedvalue for a divalent anion in Gibbs-Donnan equilibrium. Hence p-NPPadded to the incubation media in red cells has quick access to the activecentre of the membrane phosphatase which is located at the inner surfaceof the cell membrane.

2. When p-NPP is added to the incubation media of ATP-free red cellsor reconstituted ghosts, no ouabain-sensitive cation movements aredetectable, suggesting that hydrolysis of p-NPP by the active transportsystem is unable to energize active ion translocation.

3. When p-NPP concentration in the incubation media of ATP-con-taining cells is progressively raised, both ouabain-sensitive Na loss andouabain-sensitive Rb uptake tend to zero along rectangular hyperbolae.For both movements inhibition is half-maximal at 77 mm external p-NPP(i.e. 19 mm internal p-NPP).

4. p-NPP inhibits with equal effectiveness the Na:K and the Na:Naexchanges catalysed by the Na pump.

5. The inhibitory effect ofp-NPP cannot be attributed to the productsof its hydrolysis, is inversely related to the intracellular ATP concen-tration and seems to be exerted at the inner surface of the cell membranewith an apparent affinity similar to that of the membrane phosphatase.

* The authors are established investigators of the Consejo Nacional de Investi-gaciones Cientificas y T6cnicas, Argentina.

22 PH Y 223

P. J. GARRAHAN AND A. F. REGA

These facts suggest that inhibition is mediated by the combination ofp-NPP with the active centre of the membrane phosphatase.

6. Apart from affecting the ouabain-sensitive cation movements,p-NPP increases the ouabain-resistant uptake and loss of both Na and Rb.This effect is about 4 times larger for Rb than for Na, and its kineticanalysis suggests that it is due to an increase in the passive permeabilityof the cell membrane.

7. The increase in passive cation permeability upon addition ofp-NPPcannot be attributed to the products of its hydrolysis. It seems to be dueto the combination of p-NPP with a site which, like the active centre ofthe ouabain-resistant membrane phosphatase, faces the inner surface ofthe cell membrane, is unaffected by ATP and is half saturated by about15 mM-p NPP.

INTRODUCTION

There is now little doubt that the K-activated phosphatase present incell membranes is a property of the Na pump (for references see Glynn,Hoffman & Lew, 1971). Current evidence suggests that hydrolysis ofphosphatase substrates by the Na pump requires, like hydrolysis of ATP,the formation of a phosphorylated intermediate, though there is disagree-ment concerning at which stage of the metabolic cycle of the Na pumpphosphorylation by phosphatase substrates takes place (Bond, Bader &Post, 1971; Dudding & Winter, 1971; Robinson, 1971).One of the possible approaches in understanding the interactions of

phosphatase substrates with the Na pump, is to look at their effects onthe ion movements mediated by the active transport system. This paperreports results of experiments on the effects of p-nitrophenylphosphate(p-NPP), one of the substrates of the membrane phosphatase, on activeNa and Rb movements in intact red cells. p-NPP was selected because,when added to the incubation media of intact cells, it has quick access tothe active centre of the membrane phosphatase which is located on theinner surface of the cell membrane (Garrahan, Pouchan & Rega, 1969).The experiments were designed to see: (i) if p-NPP is effective as anenergy source for the sodium pump in ATP-free cells and (ii) if p-NPP isable to modify active cation movements in ATP-containing cells.A preliminary account of some of the experiments reported here has

already been published (Garrahan & Rega, 1971).

METHODS

Freshly drawn human blood from haematologically normal adults was used.Coagulation was prevented with either heparin or acid-citrate-dextrose solution.The blood was centrifuged at 1750 g for 10 min and the plasma and buffy coat were

596

p-NITROPHENYLPHOSPHATE AND CATION FLUXES 597

removed by aspiration. The remaining cells were washed 3 times with about 10volumes of a 1% (w/v) NaCl solution. After the last wash the cells were spun downfor 15 min at 10,000 g.

Preincubation of cells. A known volume of washed and packed cells was suspendedin an adequate volume of the pre-incubation medium to give an haematocrit of10-30 %. Unless otherwise stated in Results, the composition of the pre-incubationmedium was (mM): NaCl, 40; KCl, 100; MgCl2, 1; orthophosphoric acid titrated withTris base to pH 7-4, 2-5; Tris-HCl (pH 7-4 at 370 C), 45. When pre-incubation wasprolonged for 24 hr, the pre-incubation medium was supplemented with: bovineserum albumin, 5 g/100 ml.; Na-penicillin G, 100,000 u/100 ml.; L-streptomycinsulphate, 0.1 g/100 ml. Pre-incubations were performed at 370 C with intermittentshaking. The low Na and high K content of the pre-incubation medium insured thatno large changes in intracellular cation concentration would take place due to theabsence of substrates for glycolysis. When Na loss was to be measured, 10 uc/ml. of[22Na]Cl were present. At the end of the pre-incubation period the cells were spundown and washed 3 times with at least 10 volumes of iced-cold 160 mm cholinechloride solution. If necessary, a sample of the washed cells was haemolysed in dis-tilled water for measurement of intracellular Na and haemoglobin. After this step thewashed cells were suspended in the final incubation solution ready for use.

Measurement of cation movements. Na loss, Na uptake and Rb uptake were measuredby estimating the radioactivity lost or taken up by the cells during a 20 min incuba-tion at 370 C. Estimations were performed at least in triplicate, following a pro-cedure essentially similar to that described by Garrahan & Glynn (1967 a, b).

Unless otherwise stated in Results, the incubation medium for measuring Na losscontained (mM): KCl, 20; NaCl, 10; MgCl2, 1; choline chloride, 90; orthophosphoricacid titrated with Tris base to pH 7-8, 2-5; Tris-HCl, 70 (pH 7-8 at 370 C). When Nauptake was estimated [22Na]Cl was omitted from the pre-incubation medium andadded to the above-mentioned medium. Rb uptake experiments were performed inmedia similar to those for Na uptake except that KCl was replaced by RbCl labelledwith 86Rb. When present p-NPP (Tris salt, pH 7-8 at 370 C) replaced an equimolaramount of Tris-HCl. Ouabain-sensitive cation movements were calculated as thedifference between the movements in the above mentioned media and in media con-taining 10-3 M ouabain. Preliminary experiments demonstrated that Rb was aseffective as K in activating membrane phosphatase activity. From data in recon-stituted ghosts (Rega, Garrahan & Pouchan, 1970) it can be calculated that 10 mMexternal Na in the presence of 20 mm external K only gives a 5% inhibition of K-dependent phosphatase activity.Though the conditions under which the cation movements were estimated insured

that unidirectional movements were measured, the fact that in the course of theexperiment the intracellular concentration of p-NPP is increasing (see Results)implies that the measured movements in the presence of p-NPP are not steady-statefluxes, but rather represent an average loss or uptake during the incubation period.This experimental approach was adopted since to wait before performing the fluxmeasurements for p-NPP to reach a steady intracellular level would have resultedin a large accumulation of the products of its hydrolysis.

Estimation of intracellular p-NPP, nitrophenol and inorganic phosphate. The redcell suspension was centrifuged for 15 min at 18,000 g and at 20 C. The supernatantwas carefully removed and a measured volume of the pellet was haemolysed in dis-tilled water. Ice-cold trichloroacetic acid (TCA) was added to give a final concen-tration of 5 g/100 ml., and the precipitated material was removed by centrifugation.The TCA-soluble fraction was separated into three portions. One of them was usedto measure inorganic phosphate (Pj) by the method of Fiske & Subbarow (1925), a

22-2

P. J. GARRAHAN AND A. F. REGAsecond was treated with enough NaOH to raise the pH above 9 and its nitrophenol(p-NP) concentration estimated by its absorbance at 410 nm, and the last portionreceived enough concentrated HCO to give a final concentration of 1 N and was thenheated for 2 hr at 1000 C. This sample was then brought to pH 9 with NaOH and itsabsorbance was read at 410 nm to estimate its p-NP concentration. The intracellularp-NPP was calculated as the difference between the content ofp-NP before and afteracid hydrolysis. Preliminary experiments demonstrated that the procedure ofhydrolysis employed was sufficient to hydrolyse the p-NPP completely. The con-centration of P,, p-NP and p-NPP in cell water was calculated assuming a watercontent of red cells of 70% (v/v).Measurement of radioactivity. 22Na and 86Rb were estimated by liquid scintillation

counting using Bray (1960) solution. Before counting, all samples were depro-teinized with TCA (final concentration 5 g/100 ml.).

Sources of materials. [22Na]Cl and [86Rb]Cl were obtained as sterile isotonic solutionsfrom the Comision Nacional de Energia Atomica, Argentina.The Tris salt of p-NPP was prepared from Sigma 104 Phosphatase Substrate

(Sigma Chemical Co. U.S.A.) by the procedure previously described (Garrahan et al.1969).p-NP was crystalline p-nitrophenol 'spectrophotometer grade' (Sigma Chemical

Co. U.S.A.).Ouabain (Strophanthin-G octahydrate, Sigma Chemical Co. U.S.A.), was dissolved

on the day of use in the appropriate salt solution.Iodoacetamide and inosine were obtained from Sigma Chemical Co. U.S.A.Bovine serum albumin was Albumin Bovine (96-99% albumin) (Sigma Chemical

Co. U.S.A.).Antibiotics were obtained from E. R. Squibb & Sons, Argentina.All other salts and reagents were A.R. grade. The solutions were prepared in

doubly glass-distilled water.Calculations of the concentrations offree My and of ATP-Mg. The concentration of

free Mg and of Mg-ATP within the cells was calculated taking into account all pos-sible equilibria between Mg2+, ATP, Pi, organic phosphate esters and p-NPP. Thefollowing apparent formation constants were used: ATP-Mg, 2 x 10 M-1, P,-Mg,102 M-1; organic phosphate esters-Mg, 5 x 102 M-1; and p-NPP-Mg, 102 M-1.

RESULTS

Penetration ofp-NPP into intact red cellsSince the active centre of the membrane phosphatase is accessible only

from the intracellular medium (Garrahan et al. 1969), before undertakingthe flux experiments it was necessary to analyse in detail the uptake ofp-NPP by red cells and its distribution between the cells and the sus-pending medium.

Fig. 1 shows the results of an experiment in which intracellular p-NPPwas measured in intact red cells after incubation for 5 and 20 min at 370 Cin media containing from 17 to 70 mM-p-NPP. It is clear that, regardlessof the concentration in the incubation medium, p-NPP reaches with a half-time of 3 min a concentration in cell water equal to one fourth the con-centration in the external medium. At the pH in which the experiment was

598

p-NITROPHENYLPHOSPHATE AND CATION FLUXES 599performed the ratio of intra- to extracellular chloride is 0-5 (Funder &Wieth, 1966) and almost all the p-NPP is in its divalent form. Hence, thedistribution of p-NPP reached after 20 min incubation is in accord withthe Gibbs-Donnan relationship.The results of the experiment in Fig. 1 were used during the flux

measurements to adjust the extracellular concentration ofp-NPP as to getan intracellular concentration close to the Km of the membrane phos-phatase which, according to the experimental conditions, may vary from5 to 25 mm (Garrahan et al. 1969; Garrahan, Pouchan & Rega, 1970).

0250-

C 0

- w

'us .0 I I

0 5 10 15 20Time (min)

F~ig. 1. Time course of p-NPP uptake by red cells incubated in solutionsof similar composition than those used for the Na loss experiments and con-taining 17, 35 and 70 mM-p-NPP. The incubation mixture was preparedin an ice bath and at zero time the tubes were transferred to a bath at370 C. After 5 and 20 mmn the tubes were returned to the ice bath and cooledfor 3 miin before centrifugation. The experimental points are the averagefor the three concentrations of p.NPP. The vertical lines are the rangesfor the three conditions. The full line corresponds to the equation (p.NPP)in/(p-NPP) out =0125 (1-exp0231t).

Thefate of intracellular -NAPP. Part of the p-NPP taken up by the cellsis hydlrolysed mainly by the unspecific soluble phosphatase activity of thered cells. As a consequence of this, p-NPP uptake is accompanied by theapparition of p-NP and Pi within the cells. Fig. 2 shows the intracellularbuild up of p-NPP and that of the products of its hydrolysis in cells in-cubated in a medium containing 70 mM-p-NPP. It is clear that after20 min incubation intracellular p-NP is 1-4 m-mole/l. cell water while P1concentration is 4 m-mole/l. cell water higher than that at the start of the

P. J. GARRAHAN AND A. F. REGAexperiment. The larger increase in internal Pi as compared with that ofp-NP probably results from the fact that p-NP rapidly equilibrates withthe external medium (Garrahan et al. 1969).

Table 1 shows the effect of inosine on the intracellular content of Pi incells incubated for 20 min in media containing either 10 or 70 mM-p-NPP.

18

I

p-NPP',

0 10 20Time (min)

Fig. 2. Build up of p-NPP, p-NP and Pi in red cells incubated at 370 C ina medium like that of experiment in Fig. 1, containing 70 mM-p-NPP. Theconcentration of P1 is the difference between the concentration at a giventime and the concentration at the start of the experiment.

TABLE 1. The effects of inosine and iodoacetamide on the intracellularconcentration of P1 in red cells incubated in media containing p-NPP

Intracellular P1Additions (mM) (m-mole/l. cells)

None 2-1p-NPP 10 6-5p-NPP 12+inosine 15 1-6p-NPP 10+inosine 15+iodoacetamide 2 1n5p-NPP 70 6-1p-NPP 70 +i isine 1iodoacetamide 2 2 6

Cells were incubated for 20 min at 370 C in media similar to that in Fig. 1. Forother details see Methods.

It is clear that: (i) the accumulation of internal P1 is not significantlydifferent in cells incubated in 10 or 70 mM externalp-NPP. This is probablydue to the low Km Of the intracellular soluble phosphatase of red cells(M. I. Pouchan, unpublished); (ii) The increase in internal P1 upon addi-tion of p-NPP is almost completely prevented by 15 mM inosine even in

600


the presence of iodoacetamide in sufficient concentration as to com-pletely block the the red cell glycolysis. Control experiments (not shown)demonstrated that, like the (Na + K)-ATPase (Garrahan & Glynn, 1967 c),the K-activated phosphatase of cell membranes is not affected by 2 mMiodoacetamide.

Effects ofp-NPP on active Na and Rb movements

ATP-free cellsFig. 3 shows the result of an experiment in which the uptake of Rb was

measured as a function ofp-NPP concentration in cells freed of ATP by a24 hr-long pre-incubation in a substrate-free medium. The experiment was

50

-cX

@ 25

2E / Control_ ./ * Ouabain

0.

-o:

0 20 40Extracellular p-NPP concentration (mM)

0 5 10Intracellular p-NPP concentration (mM)

Fig. 3. Effects of increasing concentrations of p-NPP on Rb uptake incells pre-incubated during 24 hr in a substrate-free medium. The finalintracellular p-NPP concentration was calculated to be one-fourth theexternal concentration (see Fig. 1). The incubation media contained 15 mminosine and 2 mm iodoacetamide. For other conditions see Methods.

performed in the presence of inosine to avoid the increase in intracellularPi and of iodoacetamide to prevent repletion of ATP by inosine. It isevident that as p-NPP concentration is raised there is a progressive in-crease in Rb uptake, but that this increase is not prevented by ouabain ina concentration sufficient to block K-dependent phosphatase activitycompletely (Garrahan et al. 1969). Essentially similar results were obtainedin three other experiments in which p-NPP concentration was raised to70 mM. The effect ofp-NPP on Na loss in ATP-free cells is shown in Table 2.

P. J. GARRAHAN AND A. F. REGAIt is clear that: (i) in the presence of 70 mm external p-NPP, which givesan internal concentration about 3 times higher than the Km of the mem-brane phosphatase, no ouabain-sensitive Na loss can be detected; (ii) thelack of effect ofp-NPP on Na loss persists even after the adverse gradientsfor transport are cancelled by complete removal of external Na (Expt.no. 2).

TABLE 2. The effect of p-NPP on Na loss from ATP-free red cells

p-NPP Na loss (m-mole/l. cells.hr)concentration ,

(mM) Control Control + ouabainExpt 1

0 0-54 0-5470 1-26 1-29

Expt 20 0-85 0-8270 2-36 2-41

The cells were depleted by a 24 hr long incubation in a substrate-free medium. InExpt. 2 all the choline chloride and the NaCl in the incubation medium was replacedby an equivalent amount of KC1. All final incubation media contained 15 mMinosine and 2 mM iodoacetamide.

TABLE 3. Effect of K on Na loss from ATP-free reconstituted ghostsincubated in a Na-free medium containing 10 mM-p-NPP

Na loss(m-mole/l.sealed

ghosts . hr)

K-free medium 0-40K-containing medium 0-40

Preparation of the ghosts and determination of the volume of sealed ghosts wasas previously described (Rega et al. 1970). The ghosts were sealed in a solution con-taining (mM): NaCl, 50; Tris-HCl (pH 7-8 at 370C), 100; MgCl2, 5. The incubationmedium contained (mM): MgCl2, 5; Tris-HCl (pH 7-8 at 370 C), 170, p-NPP (Trissalt, pH 7-8), 10. When present 20 mM-KCI replaced an equivalent amount of Tris-HCL. The ghost suspension had a haematocrit of 10% in terms of original cells. Naloss was calculated measuring by flame photometry the extracellular concentrationof Na after 5 and 65 min incubation at 37° C.

The experiments of Fig. 3 and Table 2 suggest that in intact cells,hydrolysis ofp-NPP is unable to replace hydrolysis of ATP in energizingactive movements of cations. This conclusion is valid only if intracellularconditions are such as to allow a reasonable rate of ouabain-sensitivephosphatase activity. Though this seems to be the case (see Discussion) nodirect test was attempted since the very high unspecific phosphatase

602


activity made unfeasible the assay of membrane phosphatase in intact redcells. To overcome this uncertainty the effect ofp-NPP was also tested inATP-free reconstituted ghosts prepared as to insure a negligible retentionof unspecific phosphatase and optimal intracellular concentration ofcofactors of the membrane phosphatase (Rega et al. 1970). Table 3 showsthe results of such an experiment which makes evident that even underconditions which allow a good rate of K-dependent phosphatase activity,addition of K to reconstituted ghosts incubated in a medium containingp-NPP is unable to promote K-dependent Na loss.

ATP-containing cellBFurther insight on the interaction between p-NPP and the active trans-

port system was obtained studying the effects of p-NPP on ouabain-sensitive cation movements in ATP-containing cells. Since ATP decreases

o Na loss1-0 \o Na loss 3 0 Rb uptake

E o~~~~~~~~~~

0 * 00 35 70 0 35 70Extracellular p-NPP concentration (mM) Extracellular p-NPP concentration (mM)0 8-7 17-5Intracellular p-NPP concentration (mM)

Fig. 4a and b. Effects of p-NPP concentration on the ouabain-sensitiveNa loss and Rb uptake in cells pre-incubated for 4 hr in a substrate-freemedium. All final incubation media contained 15 mm inosine and 2 mMiodoacetamide. Haematocrit was 10%. Final intracellular concentrationof p-NPP was calculated to be one fourth the external concentration (seeFig. 1). For other details see Methods.

the apparent affinity of the membrane phosphatase for p-NPP (Garrahanet al. 1970), the experiments were performed in cells whose ATP contentwas reduced by a 4 hr long pre-incubation in a substrate-free medium.

Fig. 4a and b show the results of an experiment in which the ouabain-sensitive Na loss and Rb uptake were measured as a function of p-NPP


concentration. It can be observed that as p-NPP concentration in theincubation media is raised there is a progressive decline in both ouabain-sensitive Na loss and Rb uptake. Fig. 4b demonstrates that when the reci-procals of the ouabain-sensitive Na or Rb movements are plotted againstthe concentration of p-NPP, the experimental points for both cations fallalong straight lines. The Na and Rb fluxes against p-NPP concentrationcurve can therefore be fitted to equations of the form:

flux = 1 + ((p-NPP)IK1)'

where V is the flux in the absence of p-NPP and KI is equal to the p-NPPconcentration which reduces the flux to one half the control rate. Theequation predicts that as p-NPP concentration is raised the ouabain-sensitive cation movements will tend to zero along a rectangular hyperbola.For both Na and Rb, the value of KI obtained from the intercept on theabscissa of the plots in Fig. 4b was 77 mm external p-NPP, that is about19 mm internal p-NPP.

No substrate for glycolysis was present during the experiment shown in Fig. 4.At 370 C the half-time for ATP decay in the absence of substrate is about 3 hr (Lew,1971), so that the drop in ATP content during the 20 min long flux experiment canbe neglected. However, it has been shown that under certain conditions, addition ofintracellular P, to cells treated with inosine and iodoacetamide can induce a largeincrease in the rate of ATP disappearance (Lew, 1971). It could be argued that aneffect of this kind may account for the concentration-dependent inhibition by p-NPPsince the cells were incubated with inosine and iodoacetamide and p-NPP is addingP, to the internal medium. This interpretation is made unlikely by the fact that thereis no significant difference in the accumulation of Pi in cells treated with 10 or 70mM-p-NPP (see Table 1). However a direct test of this alternative was performedby comparing the effect of 70 mM-p-NPP in partially starved cells incubated with andwithout inosine and iodoacetamide. Results (see Table 4) demonstrated that omissionof inosine and iodoacetamide from the incubation medium had no detectable effecton the inhibitory action of p-NPP.

Effects of p-NPP on the behaviour of the Na pump in the absence ofexternal K. It is known (Garrahan & Glynn, 1967a) that when external Kis removed, the Na pump in red cells, instead of exchanging Na for K,catalyses a ouabain-sensitive 1: 1 exchange ofNa across the cell membrane.This exchange requires but does not consume ATP and thus presumablyis associated with the phosphorylation but not with the dephosphorylationof the transport system (Garrahan & Glynn, 1967c, d). Table 4 summarizesthe results of three experiments in which the effects of 70 mM-p-NPP onthe ouabain-sensitive Na loss was studied in the presence and absence ofexternal K. It is clear that addition of p-NPP inhibits to a similar extentboth the Na:K exchange as judged by the ouabain-sensitive Na loss in the

604


presence of external K, and the Na: Na exchange as judged by the ouabain-sensitive Na loss in the absence of external K.

Comparison of the experiments in Table 4 with that in Fig. 4 shows that raisingfrom 10 to 100 mm the external Na concentration had no detectable effect on theinhibitory action ofp-NPP. This result may seem at first hand rather surprising, sinceit is known that Na, by competing with K, lowers the apparent affinity of the mem-brane phosphatase for p-NPP (Garrahan et al. 1969). Lack of effect of external Naprobably arises from the fact that internal Na and ATP markedly raise the apparentaffinity of the membrane phosphatase for K (Rega et al. 1970) bringing its value closeto that of the (Na +K)-ATPase. If this were the case 100 mM-Na would have noeffect in the presence of 20 mm external K.

TABLE 4. Comparison of the effect ofp-NPP on the ouabain-sensitive Naloss in partially starved cells incubated in media with and without K

Ouabain-sensitive Na loss(m-mole/l. cells. hr)

Additions K-free medium K-medium

None 0-30 0-45(0.32-0.36) (0.31-0.53)

p-NPP 70 mM 0-16 0-22(0.10-0.25) (0.12-0-31)

The cells were pre-incubated for 4 hr in a substrate-free medium. The finalincubation media contained (mM): NaCl, 100; choline chloride, 10; MgCl2, 1; TrisHC1 (pH 7-8 at 370 C), 70; inosine, 15; iodoacetamide, 2. When present 10 mM-KClreplaced the choline chloride and 70 mM-p-NPP (Tris salt, pH 7-8) replaced theTris-HCl. Haematocrit was 2 %. For other details see Methods. The results are themean of three separate experiments, ranges are indicated by the figures in brackets.

The mode of action ofp-NPPThe experiments reported up to now demonstrate that addition of p-

NPP to the incubation media of ATP-containing cells leads to inhibitionof ouabain-sensitive ion movements. For the inhibitory effect ofp-NPP tobe of physiological significance it is necessary to show that it is due to thecombination ofp-NPP with the active centre ofthe membrane phosphatase.If this were the case it could be predicted that: (i) the observed effectshould be due to p-NPP and not to the products of its hydrolysis, (ii) itshould be reduced as internal ATP is raised, and (iii) it should be exertedonly when p-NPP is at the inner surface of the cell membrane.

Effects of PI and p-NP. Data in Fig. 4 and Table 1 show that inhibitionof active transport depends on p-NPP concentration whereas, for the samerange of p-NPP concentration accumulation of Pi does not, suggestingrather strongly that inhibition by p-NPP cannot be attributed to accumu-lation of Pi. Furthermore, the red cells being highly permeable to p-NP,p-NP concentration will depend not only on the rate ofp-NPP hydrolysis


but also on the haematocrit used. Comparison of results in Fig. 4 andTable 4 shows that a fivefold reduction in haematocrit, and thus in intra-cellular p-NP has no detectable effect on the inhibitory action of 70mM-p-NPP.

Definite evidence on the lack of effect of both Pi and p-NP is presentedin Table 5. In Expt 1 the effect of 70 mMi-p-NPP was compared in cellsincubated with and without inosine and iodoacetamide. It is evident thatreduction in the intracellular P, by inosine had no effect on inhibition byp-NPP, suggesting not only that P, is unable to inhibit Na loss but also that

TABLE 5. Comparison of the inhibitory effect of p-NPP withthat of the products of its hydrolysis


0 mM p-NPP 70 mm p-NPPConditions medium medium

Expt 1Control 0 63 0 27Control + 15 mm inosine + 2 mM iodoacetamide 0-66 0.29

Expt 2Control 0-88 0-42Cells loaded with Pi and p-NP 0-85

All cells were pre-incubated for 4 hr in a substrate-free medium. Loading with Piwas achieved by pre-incubating the cells in a medium containing (mM): KCl, 100;NaCl, 40; orthophosphoric acid titrated with Tris base to pH 7-4 at 370 C, 25. Thecells loaded with Pi were incubated, for measuring Na loss, in a medium containing1 mM-p-NP. Intracellular Pi at the end of the final incubation was 5-4 m-mole/l.cells for the cells loaded with P, and 6-0 m-mole/l. cells for the cells incubated withp-NPP. Haematocrit was 10%.

under the experimental conditions used there is no significant competitionbetween Pi and p-NPP (cf. Nagai, Izumi & Yoshida, 1966). In Expt 2 theouabain-sensitive Na loss was measured in control cells, in cells incubatedin 70 mM-p-NPP and in cells loaded with P, and p-NP. It is clear that P1and p-NP have no significant effect on Na loss even when their concen-tration during the whole experiment is similar to the final concentration inthe p-NPP treated cells.

Effects of ATP. It is known that as ATP concentration is raised there isa progressive decline in K-dependent phosphatase activity. This effect isdue to an increase in the Km of the phosphatase for its substrate (Garrahanet al. 1970). If inhibition by p-NPP were due to its combination with theactive centre of the membrane phosphatase, it can be predicted that theeffectiveness of p-NPP should depend on the intracellular concentration

606


of ATP. The results of an experiment designed to test this prediction aregiven in Table 6, which compares the effect of 70 mM-p-NPP in cells pre-incubated for 4 hr in media with and without glucose. The experiment wasperformed under identical conditions and on the same batch of cells. It canbe seen that the inhibitory effect ofp-NPP on ouabain-sensitive Na loss isstrongly enhanced in the cells pre-incubated in the substrate-free medium,suggesting that inhibition by p-NPP depends on the intracellular con-centration of ATP.

TABLE 6. The effects of substrate depletion on the inhibition byp-NPP of the ouabain-sensitive Na loss


Substrate in A Inhibitionpre-incubation 0 mM-p-NPP 70 mM-p-NPP by p-NPP

medium medium medium %Glucose 10 mm 1-4 1.1 22None 0 5 0.22 66

Cells were pre-incubated during 4 hr. Haematocrit was 10 %. For other conditionssee Methods.

TABLE 7. The effects of ethacrynic acid (ETHA) on the inhibition by p-NPP ofouabain-sensitive Na loss and on the intracellular concentration of p-NPP

Ouabain-sensitive Na loss Final(m-mole/l. cells. hr) intracellular

Inhibition p-NPP0 mM-p-NPP 70 miu-p-NPP by p-NPP (m-mole/l.

Additions medium medium (%) cell H20)

None 066 0.29 56 17.3ETHA 1 mM 0 68 0 58 15 4.6

The cells were pre-incubated for 4 hr in a substrate-free medium. Haematocritwas 10% . For other conditions see Methods.

Effects sf intracellular p-NPP. The large permeability of the red cellmembrane to p-NPP makes it very difficult to determine on which surfaceofthe cell membranep-NPP is effective as an inhibitor. A tool for analysingthe asymmetrical requirements of p-NPP action was provided by ourfinding that ethacrynic acid significantly reduces the rate of penetrationofp-NPP into red cells. The effects of 1 mM ethacrynic acid on both, theinhibition of ouabain-sensitive Na loss by p-NPP and on the intracellularconcentration of p-NPP are compared in Table 7. It can be seen that:(i) under our experimental conditions ethacrynic acid has little effect onouabain-sensitive Na loss in the absence ofp-NPP, (ii) at constant extra-cellular p-NPP concentration, the reduction in intracellular p-NPP by


ethacrynic acid is associated with a proportional decrease in the inhibitoryeffect ofp-NPP on the ouabain-sensitive Na loss. The fact that the decreasein inhibition by p-NPP upon addition of ethacrynic acid can be fullyaccounted by the drop in intracellular p-NPP is consistent with the ideathat the site for the inhibitory effect of p-NPP is located, like the activecentre of the membrane phosphatase, at the inner surface of the cellmembrane.

The lack of effect of ethacrynic acid on ouabain-sensitive Na loss in the absenceof p-NPP was confirmed in three other experiments using two different batches ofthe compound. Although this was not studied in detail, the failure of ethacrynicacid to block Na efflux may have resulted from the experimental conditions used.

Accepting that inhibition of active transport is due to intracellularp-NPP, the curves of Fig. 4a and b can now be represented as a functionof the intracellular concentration of p-NPP. If this is done, rectangularhyperbolae with K, values of around 19 mm are obtained. This sort ofbehaviour is expected from the interaction of p-NPP with the mem-brane phosphatase, since in the presence of 0-25 mM-ATP the velocity vs.substrate concentration curve for the phosphatase follows a Michaelis-likecurve with a Km of about 20 mm (Pouchan, Garrahan & Rega. 1969).

An alternative explanation for the observed inhibition is that it results from theability of p-NPP to chelate Mg, thereby lowering the concentration of the ATP-Mg complex in the cells. If this were the case an ATP-dependent and asymmetricalinhibition of ouabain-sensitive cation movements would also be observed. In cellscontaining 2 mM-P,, 5 mm phosphate esters and 0-25 mM-ATP, it can be calculated(see Methods) that addition of 50mm-p-NPP to the intracellular medium would reducethe concentration of the Mg-ATP complex from 0-24 to 0-23 mm. It is therefore clearthat even at the highest intracellular concentration reached - that is, 17 mM-p-NPPwould have a negligible effect on the concentration of the Mg-ATP complex.

Effects ofp-NPP on ouabain-insensitive cation movementsApart from the effects already shown addition ofp-NPP was also found

to increase the ouabain-resistant uptake and loss of both Na and Rb. Thiseffect already mentioned in connexion with the experiment in Fig. 1 wasapparent under all the conditions tested. Comparison of the effects ofp-NPP with that of P, and p-NP in Table 8 makes clear that stimulationof ouabain-resistant Na loss is due to p-NPP rather than to the productsof its hydrolysis.

Fig. 5 shows an experiment in which the p-NPP dependent ouabain-resistant Na and Rb uptakes were measured as a function of the extra-cellular Na or Rb concentrations. It is clear that though the effect ofp-NPP is significantly larger for Rb than for Na, the uptake of both cationsis linearly related to their external concentration. This result stronglysuggests that the observed effect of p-NPP is due to an increase in the

608


passive permeability of the cell membrane. Further evidence favouringthis view comes from the experiment in Table 9 which shows that p-NPPincreases the Na movements leaving unaltered the ratio of Na uptake toNa loss as it is to be expected if p-NPP increased the membrane per-meability without altering the driving force for Na movements.

TABLE 8. The effect of p-NPP, P. and p-NP on ouabain-resistant Na loss

Type of cell

ControlCells loaded with Pi and p-NP

Ouabain-resistant Na loss(m-mole/l. cells.hr)

0 mM-p-NPP 70 mM-p-NPPmedium medium

3*13 2

5.3

The cells were pre-incubated for 4 hr in a substrate-free medium. Other conditionsand the intracellular composition of the red cells are those mentioned in Table 5.

30 -

Rb

E

-e 15_/

CL

CL

z

0 50 100Cation concentration (mm)

Fig. 5. The effects of the external cation concentration on the p-N-PP-dependent Na and Rb uptake in the presence of ouabain. p-NPP-dependentcation uptake is the difference between the uptakes in the presence andabsence of p-NPP. External p-NPP concentration was 10 mmi in the Rbuptake experiment and 20 mmi in the Na uptake experiment. Na or Rbreplaced an equivalent amount of choline chloride in the incubation media.Haematocrit was 10%. For other details see Methods.


Effects ofp-NPP concentration on passive permeability to cations. Fig. 6shows the effect of increasing concentrations ofp-NPP on the Na loss andRb uptake in the presence of ouabain. The experiment was performedunder the same conditions and on the same batch ofred cells. It is clear that:(i) the effect of p-NPP is about 4 times larger on Rb uptake than on Na

TABLE 9. Effects ofp-NPP on the uptake and loss of Na in the presenceof ouabain

Concentration of p-NPPin the incubationmedium (mM)

010

Ouabain-resistant Na movements(m-mole/l. cell. hr)

Na uptake

0-210-34

Na loss

1-131-72

The cells were pre-incubated for 4 hr in a substrate-free medium. Haematocrit was10 %. For other conditions see Methods.

5-0r

"' 2-50

E

0

* Rb uptakeo Na loss

0 35 70Extracellular p-NPP concentration (mM)

0 8-8 17-5

Intracellular p-NPP concentration (mM)

Fig. 6. The effects ofp-NPP on ouabain-resistant Na loss and Rb uptake.Cells were pre-incubated for 4 hr in a substrate-free medium. Haematocritwas 10%. For other details see Methods.

loss. This result confirms the observation reported in Fig. 5 and shows thatp-NPP not only increases cation permeability but also makes the red cellmembrane more selective to Rb than that of the control cells; (ii) theincrease in passive cation movements follows a curve that tends to level

Na uptakeNa loss

0-190-20

610


off as p-NPP concentration is raised, suggesting that the effect ofp-NPPmay follow saturation kinetics.The latter point was examined in more detail in the experiment in

Fig. 7 which shows a Lineweaver-Burk plot of the p-NPP dependent in-crease in ouabain-resistant Rb uptake as a function of externalp-NPP con-centration in fresh, partially starved and fuilly starved red cells. The results

3.75

-c4.

U

-50E

125

4,4,

z

i'I I0 0-2 0-4

1/Extracellular p-NPP concentration (mM)-

Fig. 7. A Lineweaver-Burk plot of p-NPP dependent Rb uptake in thepresence of ouabain as a function ofp-NPP concentration in fresh (*-*)partially starved (0-0) and fully starved (A-A) red cells. The experi-ment was performed on cells from the same batch. Haematocrit was 10 %.For other conditions see Methods.

make evident that regardless of the metabolic conditions of the cells, allexperimental points fall along a single straight line, showing that: (i) theeffect of p-NPP on ouabain-resistant Rb uptake follows a Michaelis-likeequation with a Km of 60 mN external p-NPP, i.e. 15 mm internal p-NPPand a Vm of 8 m-mole/l. cell. hr, and (ii) in contrast with the effect onouabain-sensitive movements, the effect of p-NPP on ouabain-resistantmovements is independent of the intracellular concentration of ATP,providing additional evidence that it is due to an increase in the passivepermeability of the cell membrane.

Effect of intracellular p-NPP. In Table 1O the effects of 1 mm ethacrynicacid on the p-NPP-dependent passive cation movements and on the intra-

23 PH Y 223


cellular concentration ofp-NPP are compared. Results make clear that atconstant external p-NPP, ethacrynic acid partially inhibits the p-NPP-dependent passive cation movements and that this inhibition can be fullyaccounted for by the reduction in intracellular p-NPP concentration.Though alternative explanations cannot be discarded, these results suggestthat the site with which p-NPP has to combine to increase cation per-meability is accessible only from the inner surface of the cell membrane.

TABLE 10. Effects of ethacrynic acid (ETHA) on the p-NPP-dependent Na loss andRb uptake in the presence of ouabain and on the intracellular concentration ofp-NPP

p-NPP-dependent Intracellular p-NPPion movement concentration

m-mole/l. m-mole/l.cells. hr % cell H20 %

Na lossControl 0.80 100 16.3 100ETHA 1 mm 0 40 50 6.7 41

Rb uptakeControl 3-6 100 17.3 100ETHA nmm 0-98 27 4-6 26

The cells were pre-incubated in a substrate-free medium for 4 hr. p-NPP-dependention movements are the difference between the fluxes in the presence and in theabsence of 70 mm external p-NPP. Haematocrit was 10 %. For other conditions seeMethods.

In contrast with the lack of effect on ouabain-sensitive cation movements men-tioned before, ethacrynic acid inhibited ouabain-resistant cation movements in theabsence of p-NPP. The inhibitory effect was however considerably less than thatreported by other authors (Hoffman & Kragenow, 1966) using different experimentalconditions. In our experiments inhibition of control fluxes by ethacrynic acid rarelyexceeded 20% of the total ouabain-insensitive flux.

DISCUSSION

The first conclusion to be drawn from the experiments reported in thispaper is thatp-NPP hydrolysis by the K-activated membrane phosphataseseems to be unable to drive cation movements through the active transportsystem. Since this assertion is based on the inability of p-NPP to induceouabain-sensitive Na or Rb movements in ATP-free red cells, it is neces-sary to show that under the conditions used the rate of ouabain-sensitivep-NPP hydrolysis was large enough to induce measurable ion fluxes. Thecomposition of the media in which the cells were incubated (see Methods)was near that which allows optimal rate ofp-NPP hydrolysis. But, since nodirect test of membrane phosphatase is feasible in intact cells, some un-

612


certainties concerning the adequacy ofthe intracellular medium of red cellsfor phosphatase activity have to be considered. The first of these refersto the concentration ofMg ions in intracellular water. We have shown pre-viously that in order to activate the membrane phosphatase, Mg ions haveto be present internally (Rega et al. 1970) and that for p-NPP concen-trations up to 50 mm maximum activity is reached at about 3 mM-Mg(Garrahan et al. 1969). The total intracellular concentration of Mg in redcells is about 5 m-mole/l. cell water (Harrison, Long & Sidle, 1968), butthere are no data about the fraction of intracellular Mg that is ionized.In ATP-free cells containing 2 mM-Pi, and 5mM phosphate esters, andassuming that there is no binding of Mg by haemoglobin (see Schatzman& Vincenzi, 1969) it can be calculated (see Methods) that 5 mm total Mgwill yield 2-2 mM-Mg ions. This value seems to be reasonable as it corre-sponds to the intracellular Mg concentration in Gibbs-Donnan equilibriumwith plasma Mg.Another source of uncertainty in predicting the membrane phosphatase

activity of intact cells lies in the presence of 2 mM-Pi in the intracellularmedium. It has been shown in other preparations that Pi is a competitiveinhibitor of membrane phosphatase activity (Nagai et al. 1966). In theexperiments reported in this paper no detectable stimulation of ouabain-sensitive fluxes in starved cells was observed even when p-NPP concen-tration exceeded several fold the Michaelis constant of the membranephosphatase. Under these conditions any significant inhibition of themembrane phosphatase due to competition between Pi and p-NPP shouldhave been largely overcome. The uncertainties on the adequacy of intra-cellular composition for phosphatase activity disappear when recon-stituted ghosts are used. Experiments shown in this paper clearly demon-strate that addition of p-NPP is unable to drive active Na loss in ATP-free reconstituted ghosts with internal composition adjusted as to giveoptimal rate of phosphatase activity.Though it seems likely that intracellular composition of red cells would

allow near optimal phosphatase activity, hydrolysis rates considerablybelow the optimal would have given detectable fluxes had p-NPP hydro-lysis been coupled to ion movements. In fact the maximum rate of K-dependent phosphatase activity is about 0x6 m-mole/l. cells. hr (Garrahanet al. 1969); under these conditions, ifp-NPP hydrolysis were coupled toNa and Rb movements with the usual stoicheiometry of the Na pump, anextra oubain-sensitive Na loss of about 1-8 m-mole/I cells. hr and an extraRb uptake of about 1-2 m-mole/l. cells. hr would have been observed. (Cf.Fig. 3 and Table 2).

Results in this paper also show that p-NPP increases the ouabain-resistant Na and Rb movements. It could be argued that the postulated

23-2

P. J. GARRAHAN AND A. F. REGAineffectiveness ofp-NPP actually represents the loss of ouabain-sensitivityof a p-NPP stimulated Na plump. However, the kinetic analysis of thep-NPP-dependent ouabain-resistant cation movements makes thishypothesis untenable since it clearly shows that they are caused by anincrease in the passive permeability of the red cell membrane to Na andRb.

Inability of p-NPP to induce active transport in ATP-free red cells orreconstituted ghosts under the conditions used by us, contrasts with earlierfindings by Askari & Rao (1971) who observed ouabain-sensitive Na lossinduced by p-NPP in red cell ghosts. There is no ready explanation for thisdiscrepancy since both Askari & Rao's and our preparations are able togive ATP-dependent ion transport and K-dependent phosphatase activity.The only conclusion that seems to be possible at this stage is that, if thefluxes observed by Askari & Rao are in fact driven by the Na pump, someother and yet unspecified conditions apart from the existence of K-activated phosphatase activity are necessary to couple this activity toion movements.We also found that p-NPP inhibited ouabain-sensitive Na and Rb

movements when added to ATP-containing cells. A similar observationhas been reported by Tosteson (1962) using sheep and pig red cells. Threemain features of the inhibitory effect ofp-NPP are worth pointing out.

(i) The inhibitory effect ofp-NPP seems to result from the combinationof p-NPP with the active centre of the membrane phosphatase. Thisassertion is based on the fact that the effect of p-NPP depends on theintracellular level of ATP and seems to be exerted at the inner surface ofthe cell membrane with an apparent affinity similar to that of the mem-brane phosphatase.

(ii) Kinetic studies suggest that with excess p-NPP, active transport iscompletely inhibited, i.e. that when all the sites for p-NPP are occupiedno active transport takes place.

(iii) p-NPP inhibits the Na:Na exchange to the same extent as itinhibits the Na:K exchange catalysed by the Na pump, although only thelatter seems to require hydrolysis of the phosphorylated form of the Napump and hence K-dependent phosphatase activity.The effects ofp-NPP on active transport discussed up to now may be

analysed in the light of the recent views on the intermediate stages ofhydrolysis of phosphatase substrates by the Na pump. Bond et al. (1971)and Robinson (1971) have suggested that hydrolysis of phosphatase sub-strates may follow the whole pathway normally used by ATP. If this werethe casep-NPP would block phosphorylation of the transport system fromATP, as has been shown for acetylphosphate (Bond et al. 1971). Sincephosphorylation seems to be required for both Na:Na and Na:K ex-

614


changes, the above-mentioned scheme provides a ready explanation forthe parallel inhibition of both phenomena found upon addition ofp-NPP.However, it is difficult to understand why if phosphatase substratesundergo the same fate as ATP they are unable to energize ion movements.This point has already been raised by Bond et al. (1971) in connexion withthe inability of acetylphosphate to drive ion movements in perfused axons(Brinley & Mullins, 1968). Bond et al. (1971) suggested that in order toinduce ion translocation, a requirement for ATP at a second non-phos-,phorylating site has to be fulfilled. This explanation does not seem to besubstantiated by our experiments since, in the presence of ATP, excessp-NPP seems to lead to complete inhibition of active transport. It may beargued that p-NPP is also able to displace ATP from its postulated secondsite. However, kinetic studies on the effects of ATP on p-NPP hydrolysisby red cell ghosts do not seem to support this view, since some of theseeffects, that require an enzymatically active phosphatase-ATP complex,are apparent even when p-NPP concentration is extrapolated to infinity(see Garrahan et at. 1970, Fig. 9).An alternative view on the mechanism of hydrolysis of phosphatase

substrates by the transport system has been put forward by Dudding &Winter (1971). In their scheme phosphatase substrates phosphorylate theE2 conformation of the Na pump (see Post, Kume, Tobin, Orcutt & Sen,1969) to form E2-P which then reacts with water in the presence ofpotassium to yield P1. Ifthe role played by E2-P in the over-all behaviourof the Na pump is that suggested by Post et al. (1969), there is no reason toexpect that phosphorylation at the E2-P stage will suffice to drive aNa:K or a Na:Na exchange through the Na pump. Moreover, if E2-Pis one of the stages through which the transport system cyclically passesduring ion translocation and, if p-NPP selectively reacts with E2 to giveE2-P, it can be expected that as p-NPP concentration is raised thesystem will progressively be trapped in the E2-P form. If this were thecase, ATP-dependent Na:K and Na:Na exchanges would be progressivelyinhibited.

p-NPP and ouabain-resistant cation movementsAs already mentioned we also found that addition ofp-NPP results in an

increase in the passive permeability of the cell membrane to Na and Rb.This effect is due to p-NPP and not to the products of its hydrolysis.The increase in permeability induced by p-NPP is greater for Rb than

for Na. This, together with the fact that the effect of p-NPP -saturatesalong a rectangular hyperbola, contrasts with the linear or second-orderdependence between fluxes and concentration and lack of cation specificityfound by Wieth (1970) for the increases in cation permeability induced by

66P. J. GARRAHAN AND A. F. REGAanions of the lyotropic series. It seems therefore that the mode of actionofp-NPP may be different than that of the anions employed by Weith.The effect ofp-NPP seems to depend on the occupation of a site which

shares some properties with the active centre of the ouabain-insensitivemembrane phosphatase, viz. both face the inward surface of the cell mem-brane, both are' unaffected by ATP and both have similar apparentaffinities forp-NPP (Garrahan et al. 1969, 1970). Therefore, though furtherevidence is necessary, the possibility of some connexion between the in-crease in permeability induced by p-NPP and the ouabain-insensitivemembrane phosphatase cannot be discarded.

This work was supported by grants from the Consejo Nacional de InvestigacionesCientificas y T6cnicas, Argentina, the Universidad de Buenos Aires and the Facultadde Farmacia y Bioquimica, Argentina. We are grateful to C. 0. Poijak from theCentro de Computo de la Facultad de Medicina (Universidad de Buenos Aires) forthe computer solutions of the equations for calculating free Mg and ATP-Mg con-centration and to the Instituto de Transfusiones Luis Agote (Universidad de BuenosAires) for providing the blood used.Most of the preliminary experiments were performed in our laboratory by Dr

Maria-I. Pouchan who also suggested the use of ethacrynic acid. We are deeplyindebted for her collaboration.

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FuNDER, J. & WrITH, J. 0. (1966). Chloride and hydrogen ion distribution betweenhuman red cells and plasma. Acta phyeiol. WCand. 68, 234-245.

GARRAHAN, P. J. & GLYNN, I. M. (1967a). The behaviour of the sodium pump inred cells in the absence of external potassium. J. Physiol. 192, 159-174.

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GARRAISN, P. J. & GLYNN, I. M. (1967d). Factors affecting the relative magnitudesof the sodium:potassium and sodium: sodium exchanges catalyzed by the sodiumpump. J. Phy8iol. 192, 189-216.

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GARRAHAN, P. J., PoucHAN, M. I. & REGA, A. F. (1970). Potassium activatedphosphatase from human red blood cells. The effects of adenosinetriphosphate.J. memb. Biol. 3, 26-42.

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GLYNN, I. M., HOFFMAN, J. F. & LEW, V. L. (1971). Some 'partial reactions' of thesodium pump. Phil. Trans. R. Soc. B 262, 91-102.

HARRISON, D. G., LONG, C. & SIDLE, A. D. (1968). The calcium content of humanerythrocytes. Biochem. J. 108, 40P.

HOFFMAN, J. F. & KREGENow, F. M. (1966). The characterization of new energy-dependent cation transport processes in red blood cells. Ann. N. Y. Acad. Sci. 137,566-576.

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POST, R. L., KuME, S., TOBIN, P., ORcuTr, B. & SEN, A. K. (1969). Flexibility of anactive center in a sodium-plus-potassium adenosinetriphosphatase. J. gen. Physiol.54, 306-3268.

PoucHAN, M. I., GARRAHAN, P. J. & REGA, A. F. (1969). Effects of ATP and Ca2+on a K+-activated phosphatase from red blood cell membranes. Biochim. biophy8.Acta 173, 151-154.

REGA, A. F., GARRAHAN, P. J. & PoucaAN, M. I. (1970). Potassium activatedphosphatase from human red blood cells. The asymmetrical effects of K+, Na+,Mg2+ and adenosinetriphosphate. J. memb. Biol. 3, 14-25.

ROBINSON, J. D. (1971). K+-stimulated incorporation of 32p from nitrophenylphosphate into a (Na++K+)-activated ATPase preparation. Biochem. biophys.Res. Commun. 42, 880-885.

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from the departamento de quimica biol6gica, facultad de

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