THE JOURNAL OF BIOLOGICAL CHEMWPRY Vol. 244, No. 24, Issue of December 25, pp. 6596-6604, 1969

Printed in U.S.A.

A Cycle for Ouabain Inhibition of Sodium- and

Potassium-dependent Adenosine

Triphosphatase*

(Received for publication, August 22, 1968)

AMAR IL SEN AND T. TOBIN

From the Department of Pharmacology, Faculty of Medicine, University of Toronto, Toronto 6, Ontario, Canada

R. L. POST

From the Department of Physiology, School of Medicine, Vanderbilt University, Nashville, Tennessee 37203

SUMMARY

Physiological ligands of (sodium-potassium)-dependent adenosine triphosphatase influenced the sensitivity of the enzyme to the inhibitor, ouabain. Phosphorylation of the native enzyme is accelerated by sodium ions, while potas- sium ions accelerate dephosphorylation. Exposure of the phosphoenzyme to ouabain slowed its turnover and rendered it insensitive to potassium ion. This action of ouabain did not require free magnesium ion and was accelerated by high concentrations of sodium ion. The ouabain-treated phos- phoenzyme gradually yielded a dephosphoenzyme which could not be rephosphorylated from adenosine triphosphate but could incorporate inorganic phosphate.

The native dephosphoenzyme on incubation with ouabain lost its ability to be phosphorylated by adenosine triphos- phate. This effect was accelerated by magnesium ion or magnesium ion plus inorganic phosphate and could be pre- vented but not reversed by sodium ion at 0”. At 23” or above it was reversible with or without sodium ion. The inhibited dephosphoenzyme incorporated inorganic phos- phate in the presence of magnesium ion. All phosphorylated forms of the enzyme yielded identical patterns on electro- phoresis and radioautography of proteolytic digests.

A cyclic reaction sequence for inhibition of the enzyme by ouabain is proposed and related to physiological transport of sodium and potassium ions.

An enzymatic pump transports sodium and potassium ions across cell membranes (l-5). The pump mechanism is inhibited by cardiac glycosides such as ouabain, which apparently act

* This work was supported by Grants MA-2485 from the Medi- cal Research Council of Canada. 51201 HE-01974 from the Nationa.1 Heart Institute, and 5POl AM-67462 from the National Institute of Arthritis and Metabolic Diseases. Preliminary reports of this work were presented at the Second International Biophysics Congress, Vienna, 1966; the Federation Meetings, Chicago, 1967; and the Canadian Federation Meetings, Kingston, 1968.

upon a phosphorylated derivative of the enzyme (6-11). It seemed profitable to study further the effects of this specific steroid inhibitor on the kinetics of phosphorylation and dephos-

phorylation, in order to interpret the mechanism of the ouabain effect.

The pump appears to be stoichiometric. In human erythro- cytes three Na+ are transported outward per cycle for every two K+ transported inward, with cleavage of one terminal phosphate group of intracellular ATP in the presence of intracellular Mg++. The ADP and inorganic phosphate are released intracellularly (12-15).

The discovery of (Na+ + K+)-ATPase in crab nerve mem- branes and the demonstration of its participation in the pump action led to consideration of the pump as an enzyme, or enzyme system, embedded in the membrane with sites on both surfaces permitting the binding of the ions to be translocated (1, 4). Sodium and potassium ions compete with each other at both surfaces, Na+ having the higher affinity at the inside and K+ having the higher affinity at the outside (16-18). Futhermore, the (Naf + K+)-ATPase activity of the intact cell requires the presence of ATP and Mg++ on the inner surface of the mem- brane. Since Na+ stimulates formation of the phosphorylated intermediate, and K+ stimulates its hydrolysis, it is a reasonable assumption that phosphorylation of the enzyme on the inner surface of the membrane takes place during ion transport (14).

The actions of ouabain have not previously been explained in sufficient detail to account for the observed effects. Inhibition of the pump by cardiac glycosides is stoichiometric (19) and thought to be reversible (20). In the squid giant axon, ouabain was effective only when applied to the outer, and not the inner, surface of the membrane (21). Ouabaininhibitionof cation trans- port by erythrocytes was augmented by a high extracellular Na+ concentration and was partially and competitively inhibited by extracellular K+ (22). Corresponding effects have been ob- served on the (Na+ + K+)-ATPase of broken membranes from calf heart (23). In this paper we present a study of the effects of ouabain on intermediate phosphorylation steps in the reaction sequence, and the effects of various ligands on the sensitivity of this enzyme to ouabain inhibition. The findings indicate that ouabain reacts primarily with the Kf-sensitive phosphorylated form of the enzyme.

by guest on July 12, 2020http://w

ww

.jbc.org/D

ownloaded from

Issue of December 25, 1969 A. K. Sen, T. Tobin, and R. L. Post 6597

METHODS

Mater&---(Naf + K+)-dependent ATPase from guinea pig or rabbit kidney cortex and (Y-~~P)-ATP were prepared as de- scribed by Post and Sen (24, 25). In most cases fresh kidneys were used, but for a few preparations frozen kidneys were ob- tained from Pel-Freez, Rogers, Arkansas. The enzyme prep- aration contains residual Na+ and Mg+f, probably adsorbed to the phospholipids of the membranes. When a Na+- and Mg++- free enzyme was required, the insoluble preparation was washed three times by alternate resuspension and centrifugation at 39,100 X g in a solution containing 25 mM imidazole, 12.5 mM histidine hydrochloride, and 0.01 mM Hd (EDTA) at pH 7.20 f 0.1. When removal of only 1 ion was necessary, this was facilitated by addition of the other; about 1 mM Na+ in the washing solution accelerated loss of Mgf+ and aice versa. The preparation was stored at 4” in a solution containing 10 mM imidazole and 0.1 mM Hq (EDTA) with the pH adjusted to 6.9 f 0.1 with HCl. The final preparation contained approximately 3 mg of protein per ml. The specific activity of the (Na+ + K+)-ATPase was 1.7 to 2.5 units per mg of protein. One unit splits 1 pmole of ATP per min at 37”. Of the total ATPase activity, 90% or more was (Naf + K+)-dependent. Protein was determined by the method of Lowry et al. (26), and total phosphate by a modifica- tion of the method of Bartlett (27). Ouabain (Strophanthin-G, octahydrate) was obtained from Sigma, and a fresh solution was prepared every 14 days.

Phosphorylution-The entire procedure was normally carried out in an ice bath, at a temperature of approximately 0”. In a few specific instances, incubation was performed at a higher temperature before termination of the reaction with cold acid (25). The reaction mixture contrained 0.8 to 1.0 mg of protein and 10 pmoles each of imidazole and glycylglycine at pH 7.5 f 0.1 in a 50-ml polycarbonate test tube. Additions of all other reagents were made in volumes of 0.1 ml; the final reaction volume was 1.0 ml. For maximal phosphorylation the usual amounts added were 16 Mmoles of NaCl and 1.0 pmole of MgC12. The reaction was started by addition of 0.04 pmole of (T-~~P)- ATP with a specific activity of 10 to 50 X lo6 cpm per pmole. When K+ was substituted for Naf in the standard mixture, about 5 to 10% of maximal phosphorylation was found; this amount was subtracted from all values as a blank. The maximal value minus the blank was taken as 100% phosphorylation for any given enzyme preparation. This procedure allows ready comparison of results obtained with enzyme preparations of different specific activity. The range of maximal phosphate in- corporation was 125 to 250 ppmoles of a2P per mg of protein depending on the enzyme activity. The reaction mixture was stirred continuously with a magnetic stirrer at the highest speed which did not make bubbles. Additions were made with a hand- operated syringe at times estimated from an electric stopclock. The reaction was stopped, at the times indicated, by pouring in 35 ml of ice-cold 0.25 M trichloracetic acid which contained 0.6 mM Nas AT!? and 0.8 mrvr HaPOd. For estimation of phos- phorylation capacity the reaction was usually stopped within 6 set after addition of (a2P)-ATP.

The denatured membrane suspension was centrifuged immedi- ately at 10,000 X g for 20 min. The precipitate was washed twice by homogenization in 35 ml of 0.3 M trichloracetic acid and centrifugation at 10,000 x g for 10 min. The precipitate was then rehomogenized in 10 ml of 0.3 M trichloracetic acid, trans- ferred to a 12-ml thick-walled graduated centrifuge tube, and

spun at 1,200 x g for 20 min. The supernatant was poured off, the inverted tube was drained for 1 min, and the precipitate was homogenized in 0.1 ml of water. The volume was made up to 5 ml with a fresh mixture of chloroform, methanol, and formic acid (4:2 : 1 ,v/v/v). This procedure yielded a uniform dis- persion of the denatured membranes. A l.O-ml aliquot of the suspension was dried on a planchet for measurement of radio- activity with a thin window gas flow counter. Protein and total

0 PRE-INCUBATION TIME WITH OUABAIN

IN MINUTES

FIG. 1. Influence of magnesium ion and inorganic phosphate on inhibition of the dephospho enzyme by ouabain. Freshly pre- pared enzyme was used. The incubation reaction mixture con- tained 0.25 pmole of ouabain with: 5 pmoles of (Tris)4EDTA (U-O); 0.4 pmole of MgC12 (A-A); or 0.4 Nmole of MgCL and 1 rmole of (Tris) 3PO4 ( l - - -0 ) . At the times indicated the native enzyme was phosphorylated by addition of 0.04 pmole of (azP)-ATP and 16 pmoles of NaCl, with 6 pmoles of MgClz in addi- tion to overcome the EDTA when present (0-O). The reac- tion was stopped with acid 5 set later.

-1 Es l\l-o--o-------------o- )

011

0 a 16 24 32

MINUTES AFTER OUABAIN

FIG. 2. The effect of sodium ion on the time course of ouabain inhibition of phosphorylation. The reaction mixture contained 0.4 pmole of MgC12. Incubation was started with 0.25 pmole of ouabain. (O-O ), 0.04 pmole of (32P)-ATP and 60 pmoles of NaCl were added at times indicated on the abscissa. C---C and A-.-A, 60 pmoles of NaCl were added at 2 or 4 min, respec- tively, as indicated by arrows and 0.04 pmole of (32P)-ATP was added at the times indicated on the abscissa. The reactions were terminated with cold acid 5 set after addition of the (32P)-ATP.

by guest on July 12, 2020http://w

ww

.jbc.org/D

ownloaded from

Ouabain Inhibition of &a+ + K+)-ATPase Vol. 244, No. 24

0” 01 o-4 I.0 2.0 4 0 8.0 16.0

mM NO*

FIG. 3. Influence of the concentration of sodium ion inhibition of the dephospho enzyme by ouabain. The reaction mixture con- tained 0.4 pmole of MgClz and quantities of NaCl to produce the final concentrations in 1 ml as indicated. Incubation with 0.25 Mmole of ouabain was for 16 min. Phosphorylation was started with 0.04 fitmole of (““P) -ATP and 16 pmoles of NaCl; it was stopped with acid 3 set later. Inset: l , replot of these data as percentage of prevention of inhibition, that is, 100 minus the percentage of maximal inhibition; 0, percentage of maximal phosphorylation in the absence of ouabain. The latter data are replotted from Fig. 8 in Reference 6.

phosphate were determined in duplicate on aliquots of 0.2 ml and 0.5 ml, respectively, taken to complete dryness before analy- sis. The radioactivity was divided by the protein or total phos- phate to correct for uneven recovery from the washing procedure.

RESULTS

During the course of these experiments it became clear that un- der appropriate conditions ouabain reacted with the enzyme either before or after phosphorylation by ATP. At low ionic strength and in the absence of monovalent inorganic cations, ouabain reacted with the dephosphoenzyme and prevented phosphoryla- tion by ATP. In the presence of sodium ion, ouabain reacted only after phosphorylation and prevented acceleration of dephos- phorylation by K+. The former results are presented first.

Reaction of Enzyme with Ouabain Prior to Phosphorylation by ATP

E$ects of Mgf+, Pi, and Na+ on Ouabain Inhibition of Phos- phorylation-In the presence of optimal concentrations of Na+, Mg++, and ATP, phosphorylation of the enzyme is complete in less than 3 set (28). In the absence of K+ dephosphorylation is relatively slow. Therefore, changes in incorporation of azP from ATP during incubation for less than 6 set indicate changes in the amount of enzyme available for phosphorylation. By this approach, it was found that when enzyme was incubated at 0” with ouabain in the absence of added Na+ and Mg++, there was a slow reduction in the ability of t,he enzyme to be phos- phorylated on subsequent completion of the reaction mixture. If a small quantity of Mg++ was present during the incubation period, inhibition developed more rapidly. Pi further acceler- ated the inhibition in the presence of Mg++ (Fig. 1) but was ineffective in its absence (not shown). However, ouabain inhibi- tion was prevented by Na+. Addition of 60 mM Na+ to the

TABLE I

Reversal of ouabain inhibition after combination with dephospho- enzyme

Just before use, the enzyme was washed free of Na+ with 1 rnM MgClz in the washing solution given under “Methods.” It was incubated with ouabain for 2 min. Reversal was started with 20 pmoles of HNas-EDTA at zero time. After the intervals indi- cated, phosphorylation was started with 0.04 rmole of (32P)-ATP and 30 pmoles of MgC12. The reaction was stopped with acid 5 set later. The temperature was 23” except that the acid was at 0”. There was no inhibition when the HNas-EDTA was added before the ouabain.

Reversal time

- I.

Ouabain, Ouabain, Ouabain, 2.5 X 2.5 x 10-s M 2.5 x 10-4 M 10e4 &I; Pi, 0.3 lTlP

set % % % 5 59 8 6

60 65 15 11 120 74 25 18 240 79 43 30 480 94 47 41

=P-intermediate

medium during the incubation period abruptly blocked inhibi- tion but did not reverse it (Fig. 2). When the concentration of Na+ present during incubation was less than 16 mu, the rate of development of the ouabain effect was correspondingly increased. This prevention may be due to combination of Na+ with its active sites for (Na+ + K+)-ATPase activity. The half-maxi- mal concentration of Nat for phosphorylation is about 1.6 mM.

The concentration of Na+ for half-maximal prevention of inhibi- tion was about the same (Fig. 3). Apparently at least two func- tionally different states of the dephosphoenzyme could be dis- tinguished. When combined with Mg++ at low ionic strength, it was sensitive to ouabain; when combined with Na+, it was resistant.

Reversibility of Ouabain Inhibition-Reversibility of ouabain inhibition of this enzyme is controversial (20, 29). In our ex- periments reversal of inhibition did not appear at 0” within 30 min (e.g. Fig. 2) with or without EDTA. At 23” reversal was 35% after 8 min in the presence of Naf and EDTA (Table I). At 37” reversal was 77 ‘% after 5 min, with cooling to 0” before phos- phorylation. In all reversal experiments ouabain remained in the solution with the enzyme during the whole period.1

Optimal Conditions for Ouabain Inhibition-Development of inhibition appeared to require a low ionic strength. The optimal concentration of Mg++ in t,he absence of Pi was between 0.1 and 0.4 mM. In 10 mM MgClz there was & as much inhibition; and in 10 mM choline chloride, Tris-sulfate, or Tris-phosphate there was 4 as much inhibition. At the optimal concentration of Mg++ and an incubation time of 16 min, the ouabain concentra- tion for half-maximal inhibition was 5 X 1O-5 M.

Phosphorylation of Inhibited Enzyme by Pi--It has previously been shown that Pi will not phosphorylate the native enzyme (6). However, the ouabain-inhibited enzyme was phosphorylated by Pi.

The enzyme was first incubated with 0.04 mM 32Pi at 37” for 30 min, to label any residual inorganic phosphate in the prep-

1 HZ-Ouabain studies in progress show that the enzyme-ouabain complex is essentially irreversible at O”, while at 37” binding is readily reversible.

by guest on July 12, 2020http://w

ww

.jbc.org/D

ownloaded from

Issue of December 25, 1969 A. K. Sen, T. T&n, and R. L. Post 6599

y //---- ,’ ,/-\

/’ I

if I 0 A 0 CONTROL

4

ti 60 I E40-

1’

‘Ok 0 6 12 18 24

SECONDS AFTER 32P-ATP

I I ,OMIT ATP

I I I I I I

0 IO 20 30 40 50

SECONDS AFTER EXCESS COLD ATP

FIG. 4 (upper). Time dependence of ouabain inhibition of en- zyme turnover following phosphorylation from ATP. The reac- tion mixture contained 16.0pmoles of NaCl and 0.4 pmole of MgClz with (solid symbols) or without (open symbols) 0.25 pmole of oua- bain. The NaCl was added before the ouabain. The reaction was started with 0.04 rmole of unlabeled (Tris)sATP 48 set before (0, n ), 5 set before (A, A), or simultaneously with (0, 0) a tracer quantity of (32P)-ATP added at zero time. The reaction was stopped with acid at the times indicated.

FIQ. 5 (lower). Breakdown of 32P-intermediate in the presence and absence of ouabain. The reaction mixture contained 16 pmoles of NaCl and 1 pmole of MgC12. Phosphorylation was effected by addition of 0.04 pmole of (32P)-ATP 12 set before indi- cated zero time. Where present, 0.25 pmole of ouabain, was added 6 set before zero time. At zero time, 2 pmoles of unlabeled (Tris)tMgATP were added. q , disappearance of the 32P-inter- mediate in the presence of ouabain and absence of unlabeled (Tris)sMg-ATP; 0, the disappearance of the S2P-intermediate in the presence of ATP without ouabain; 0, the disappearance of the azP-intermediate with ouabain and ATP present.

aration. It was washed free of soluble radioactivity and then incubated with 0.25 pmole of ouabain and 0.2 pmole of MgC& at 0”. After 180 set, an aliquot was phosphorylated with 0.04 pmole of C2P)-ATP and 16 pmoles of NaCI. There was 50% incorporation of 32P from ATP as compared with the same prep-

aration before exposure to ouabain. Another portion was phos- phorylated with (32P)-ATP together with 0.4 pmole of 32Pi of the same specific activity as the (a2P)-ATP. Phosphorylation now amounted to 85 ‘%. The presence of 32P i thus led to phosphoryla- tion of the inhibit.ed enzyme (compare also References 29-31).

n ::

\ \ I

‘1, w \ \ I l \

6( I- \

i

\ I ’ \

\ A- I ‘A--

\ ---A---- I --A \ ---_ \ I ---_

I --.

L-4, ---_ I 20

-- --- I ------A I I

r

Y

I A

I I I I 0 10 20 30 40 50

Seconds

’ A I I f---A-

I

I I

OUABAIN ABSENT o A I

1 0’ 20 60 100 140

Seconds after EDTA

FIG. 6 (upper). Formation of a K+-resistant phosphorylated intermediate in the presence of ouabain. The reaction mixture contained 16 rmoles of Na+ and 1.0 pmole of Mg++. Two minutes after addition of 0.25 pmole of ouabain the reaction was started with 0.04 pmole of (32P) -ATP. l , rapid formation and slow disap- pearance of phosphorylated enzyme. After 3, 12, and 36 set, 16 pmoles of KC1 were added. The reaction was stopped at the times indicated by A. In the absence of ouabain (0) there was rapid formation and persistence of the intermediate. After 32 set, 16 pmoles of KC1 with 1 pmole of unlabeled ATP were added. The reaction was stopped with cold acid at 36 set (A).

FIG. 7 (lower). Possible precursor-product relationship be- tween inhibited phosphoenzyme and inhibited dephospho enzyme. The reaction mixture contained 16 bmoles of NaCl and 0.1 pmole of MgC12. The reaction was started 40 set before zero time with the addition of 0.04 rmole of (32P)-ATP. When present, 0.25 rmole of ouabain was added 30 set before zero time. Data with ouabain are shown by solid symbols and data without ouabain are shown by open symbols. At zero time, 10 pmoles of (Tris)rEDTA and 0.04 rmole of KC1 were added in order to prevent further for- mation of phospho enzyme and to dephosphorylate any native phospho enzyme that might be present. At 20 or 110 set (indi- cated by arrows) 15 rmoles of MgClz and 200 pmoles of NaCl were added in order to phosphorylate again the native enzyme and expose the inhibited dephospho enzyme. Data with this addition are shown by A and A, and data without this addition are shown by l and 0. The reaction was stopped with acid at the times indicated.

Reaction of Enzyme with Ouabain after Phosphorylation by ATP

Effect on Rephosphorylation-Addition of ouabain to the phos- phoenzyme produced a slow decline in the amount of the phos- phorylated intermediate after more than 6 sec. Similarly, when

by guest on July 12, 2020http://w

ww

.jbc.org/D

ownloaded from

Ouabain Inhibition of (Naf + K+)-ATPase Vol. 244, No. 24

TABLE II

Reversal of ouabain inhibition of dephosphoenzyme formed from inhibited phosphoenzyme

The reaction mixture contained 1 Fmole of MgC12 and 16 pmoles of NaCl. The reaction was started with 0.04 pmole of (3”P) -ATP. After 10 see 0.25 rmole of ouabain was added. After 60 set phos- phorylation was stopped, and any native phosphoenzyme present was dephosphorylated with 10 rmoles of (Tris)c-EDTA and 0.04 pmole of KCl. After 180 set the reaction vessels were placed in a water bath at 37” to start reversal (80 set of warming brought the temperature of the reaction mixture only to 23’). After thewarm- ing intervals indicated below, t,he reaction vessels were placed in ice water for 180 set for the shorter interval warming and for 360 set for the longer. Then phosphorylation was started again with 15 wmoles of MgCla and 200 pmoles of N&l. The reaction was stopped with acid 5 set later.

The Without warming With warmine

/ Before (Mg++ After (I@++ Before (MS++ After @Q++

+ Na+) 1 + Na+) 1 + Na+) j + Na+)

JCG % % % %

180 12 21 440 1 15 1 28 660 1 14 1 40

- a The effective warming periods were 80 and 120 set with re-

phosphorylations at 440 and 660 set, respectively.

ouabain inhibition was blocked by Na+ (e.g. Fig. 3) and the enzyme was subsequently phosphorylated by ATP, the amount of phosphoenzyme slowly declined as incubation continued for longer than 6 set (Figs. 4 and 5). To investigate these kinetics, we examined the rate and extent of phosphorylation of enzyme which had been turning over in the presence of ouabain. The enzyme was protected with Na+, ouabain was added, and the reaction was started with unlabeled ATP; tracer (32P)-ATP was

added subsequently. In the absence of ouabain the incorpora- tion of a2P corresponded to the normal slow turnover of the intermediate in the absence of K+, as no exchange between E-32P and ATP occurs under these conditions (6). In an enzyme which had been turning over in the presence of ouabain the final level of 32P incorporation was progressively reduced and the rate of incorporation was also proportionately less the longer the exposure to ouabain (Fig. 4). This experiment showed that the phosphorylated enzyme, unlike the native enzyme in the presence of Na+, was ouabain-sensitive, and it suggests that the gradual decline in steady state level of the intermediate was due to an inhibition of rephosphorylation of the enzyme as it turned over.

E$ect on Dephosphorylation-The action of ouabain on the stability of the phosphoenzyme was tested. The enzyme was phosphorylated, ouabain was added, and 6 see later further for- mation of radioactive intermediate was prevented by addition of excess unlabeled ATP. The rate of disappearance of radioac- tivity from the intermediate indicated its stability. It was clear that the phosphoenzyme became more stable in the presence of

ouabain than it was in its absence (Fig. 5). Ouabain also ap- peared to react more rapidly with the phosphoenzyme than with the dephosphoenzyme.

Effect of Ouabain on K+ Xensitivity of Phosphorylated Enzyme- As ouabain appeared to reduce the rate of dephosphorylation, which is normally accelerated by K+, it was appropriate to test

ADD OUABAIN

AT 4 SECONDS

0 8 16 24 32

SECONDS AFTER CHELATOR LOG ho+? MM

FIG. 8 (left). Ouabain inhibition of the phospho enzyme in the absence of Mg++. The reaction mixture contained 16 pmoles of NaCl and 0.4 rmole of MgCl,. The reaction was started at 10 set before zero time with 0.04 pmole of (32P)-ATP. At zero time, 4 pmoles of (Tris)4(1,2-cyclohexylenediamino)tetraacetate were added to chelate the free Mg++ and block further phosphorylation. At 4 set (shown by the arrow) 0.25 rmole of ouabain was (O-O ) or was not (O- - -0) added. The reaction was stopped with acid at the times indicated.

FIG. 9 (right). The effect of sodium ion concentration on the rate of conversion of phospho enzyme to inhibited phospho enzyme by ouabain. The reaction mixture contained 1 pmole of MgClz and 2 to 200 rmoles of NaCl to provide the concentrations of Na+ indicated on the abscissa in the final volume of 1 ml. The reaction was started with 0.04 pmole of (32P)-ATP. At 10 set, 0.25 rmole of ouabain was added. At 14 or 18 set, 1 pmole of unlabeled ATP and 16 pmoles of KC1 were added. The reaction was stopped with acid 4 set later. These samples provided data for estimation of the amount of the K+-resistant inhibited phospho enzyme. For each concentration of Na+, the initial quantity of the phospho enzyme was obtained by stopping the reaction at 10 set and the background level of phosphorylation was obtained by omitting ouabain from a sample otherwise identical with that to which ATP and KC1 were added at 18 sec. The background level was sub- tracted from the other three. The rate constant, k, was estimated from the fraction of the initial phospho enzyme, j, remaining after exposure to ouabain for 4 or 8 set of time, t, in the following for- mula: j = e+t. The value of k was 5yo lower at 8 set than at 4 set on the average (range, -14% to +lO%) perhaps because of failure to estimate formation of inhibited dephospho enzyme. The values of k at 4 and 8 set were averaged. 0, 0, or A, a different enzyme preparation.

the K+ sensitivity of the intermediate formed in the presence of ouabain. In the absence of ouabain, K+ produced a rapid and almost complete dephosphorylation (Fig. 6). When phospho- rylation was carried out in the presence of ouabain and K+ was added after different intervals, dephosphorylation was biphasic, a rapid phase being followed by a slow one. The extent of the rapid phase decreased progressively, and the slow phase increased in magnitude the longer the interval before addition of K+. Addition of excess unlabeled ATP (to block formation of the radioactive intermediate) together with the K+ did not modify the results. In other experiments, a reduction of the concentra- tion of Na+ or ouabain in the incubation medium reduced the rate and extent of formation of this K+-insensitive form of the phosphorylated enzyme.

Dephosphorylation of K+-insensitive Intermediate- Aa G;,=arent paradox noted above was that while ouabain consistently pro- duced a slow appearance of the dephosphorylated form of the enzyme (Figs. 4 and 5) it also stabilized the phosphorylated inter-

by guest on July 12, 2020http://w

ww

.jbc.org/D

ownloaded from

Issue of December 25, 1969 A. K. Ben, T. Tobin, and R. L. Post 6601

TABLE III

Radioactivity in peptic fragments of various derivatives of phos- phorylated (Na+ + K+)-ATPase

The reaction mixture contained 2.5 mg of membrane protein from rabbit kidney cortex, 10 pmoles of imidazole glycylglycine, and 0.1 Imole of MgCla in a final volume of 1.0 ml. Samples 1 to 3

also contained 16 rmoles of NaCl and were incubated at 0”. At zero time 40 mrmoles of (““P) -ATP and 1 pmole of (Tris)s-PO4 were added to these samples. At 5 set 0.25 rmole of ouabain was added to Sample 3. At 25 set 1 pmole each of KC1 and (Tris),-ATP (unlabeled) was added to Samples 2 and 3. At 30 set the reaction was stopped with cold acid. Samples 4 to 6 were incubated at

23”. In Sample 6 the MgCll was replaced by 1 pmole of the chelat- ing agent (Tris)J-cyclohexylenediaminotetraacetic acid. At zero time 40 mpmoles of a2Pr were added to Samples 4 to 6. The spe- cific activity of the “Pi was the same as that of the (32P)-ATP. At 5 set 0.25 rmole of ouabain was added to Samples 5 and 6. At 15 set the reaction was stopped with cold acid. All the radio- active denatured precipitates were washed with cold acid and digested with 1 mg of pepsin each. After centrifugation, aliquots of the supernatant were subjected to electrophoresis on paper at pH 2 and radioautography. Radioactive spots appeared only at positions corresponding to Pi and peptic fragments P4, P5, and P6 (31). The radioactive spots were cut out of the paper and counted in a low background gas flow counter. Counts for all three peptic fragments were summed. 32P activities (counts per min) were converted to micromicromoles incorporated. The quantities given in the table correspond to the entire supernatant after peptic digestion.

Source of radioactivity Additions

(“2P) -ATP (3”P) -ATP (““P) -ATP

32p. 3zp: 1 ‘*Pi

Control Kf with ATP Ouabain with

Kf and ATP Control Ouabain Chelator and

ouabain

/q.wnoles

300.0 20.0 5.3 0.6

160.0 10.0

3.0 31.0 20.0 34.0

0.5 25.0

mediate with respect to K+ (Fig. 6). The relatively slow ap- pearance of inhibited dephosphoenzyme, the correspondingly

high stability of the inhibited phosphoenzyme (Figs. 4 and 5),

and the irreversibility of ouabain inhibition at 0”, the temperature

of these experiments, all suggested that the K+-insensitive phos- phorylated intermediate might be converted slowly to a dephos- phorylated form which is resistant to rephosphorylation. An experiment was designed to test this possibility. Addition of EDTA and K+ rapidly broke down the normal phosphorylated

intermediate and prevented its further formation. However, the K+-resistant phospho-form produced in the presence of Na+, Mg++, radioactive ATP, and ouabain disappeared very slowly

on addition of EDTA and K+ (Fig. 7). At 20 or 110 set during this disappearance the enzyme was tested for its ability to be rephosphorylated by addition of excess Mg++ and Na+. The fraction of enzyme remaining unphosphorylated increased from

41% at 25 set to 66% at 115 sec. During this time the K+- resistant phosphorylated intermediate (indicated by the solid

circles) decreased from 48 to 13%. This decrease (35%) was only a little more than the simultaneous increase (25%) in the

nonphosphorylatable dephospho-form (indicated by the difference between solid and open triangles at corresponding t’imes). The result is consistent with a precursor-product relationship. When it was warmed, the inhibited dephosphoenzyme produced from the inhibited phosphoenzyme lost part of its inhibition (Table II), as did the inhibited dephosphoenzyme formed in the absence of ATP (Table I).

InJEuence of Na+ and Mg++ on Rate of Inhibition of Phospho- enzyme-Mg+f accelerated inhibition of the dephosphoenzyme (Fig. l), and Na+ prevented inhibition (Fig. 2). It was appro- priate to test the effects of Mg++ and Na+ on the sensitivity of the phosphoenzyme to ouabain.

A requirement for free Mg++ was tested by first adding excess cyclohexylenediaminetetraacetic acid to remove free Mg++ from the native phosphoenzyme and then adding ouabain. Ouabain reacted with and stabilized the phosphoenzyme at a normal rate (Fig. 8, compare Fig. 5). In contrast t.o the native dephospho- enzyme, the native phosphoenzyme did not require free Mg++ for rapid reaction with ouabain.

The effect of Na+ was tested by adding excess Na+. Excess Na+ not only failed to inhibit but slightly accelerated the ap- pearance of the K+-resistant phosphoenzyme (Fig. 9). The influences of Mg++ and Na+ on ouabain inhibition of the phos- phoenzyme were the opposite of their influences on inhibition of the dephosphoenzyme.

Ouabain-Enzyme-Phosphate Derivatives: Similarity of Enzyme-Phosphate Bonds

As demonstrated above, the 32P-intermediate formed from (a2P)-ATP became more stable and K+-insensitive in the presence of ouabain (Fig. 6). Also the ouabain-treated enzyme incor- porated 32P i as an acid-stable derivative in the presence of Mg+f (text). To compare t.he covalent structure of these ouabain- enzyme-phosphate derivatives, we prepared both and subjected them to peptic digestion. For comparison, the Na+- and Mg++- dependent phosphorylated intermediat,e from (32P)-ATP was prepared in the absence of ouabain and similarly digested. Table III shows the distribution of radioactivity in electropherograms of the peptic digests of these phosphorylated derivatives.

In the presence of Na+ and Mg++, 32P-ATP gave a high incor- poration into the peptic fragments. Following the addition of K+ and unlabeled ATP, the 32Pi activity in the fragments was negligible. In the presence of ouabain the fraction of 32P in the K+-resistant intermediate was the same as that in the denatured precipitate in Fig. 6. Samples 4 to 6 (Table III) show that only in the presence of Mg++ and ouabain was there a significant incorporation from 32P i. The poor yield was probably due to the low concentration of Pi. Aliquots of Samples 1, 3, and 5 were treated with performic acid or hydroxylamine and examined by paper electrophoresis, together with untreated aliquots of the same samples. As reported previously, the relative mobilities of the spots derived from all three samples were identical (30, 31). All three were altered in the same manner by treatment with performic acid or hydroxylamine. Other aliquots of the same three reaction mixtures were treated with pronase and subjected to performic oxidation. Paper electrophoresis again showed iden- tical patterns (see Reference 31, Figs. 19, 20).

In other experiments the turnover times of the ouabain- enzyme-phosphate complexes formed from ATP or from P; were similar. Following the ouabain-dependent incorporation of 32P i into the enzyme, further formation of this phospho-form was

by guest on July 12, 2020http://w

ww

.jbc.org/D

ownloaded from

6602 Ouabain Inhibition of (iVa+ + K+)-ATPase

TABLE IV

Vol. 244, No. 24

Phosphorylattin by Pi of inhibited enzyme generated by ouabain and unlabeled ATP

The reaction mixture contained 0.3 pmole of MgCla. The reaction was started with 0.25 pmole of ouabain at zero time. When present, 10 pmoles of unlabeled (Tris)k-ATP were added at 10 see; 32 pmoles of NaCl or KC1 were added beforehand or at 180 set, as indicated; 0.4 pmole of (32P)-(Tris)s-PO1 was added at 190 sec. The a,P in the denatured precipitate is expressed as a percentage of the maximal amount of the phosphorylated intermediate, which was 178 pprnoles per pmole of total P. The experiment was performed at 0”.

Experiment Salt added beforehand

1 NaCl 2 KC1 3 NaCl 4 KC1 5 -

6 i

-

-

-.

-

Ouabain added ATP added at 0 set at 10 see

- -

+ + - -

sak&dee at 8% added at Acid added at 190 set 200 set

-

-

-

-

-

NaCl KC1

+ +

+ +

+ +

+ +

+ + + +

- I f

__

-

=P in denatured precipitate

%tlpl&

% 7

13 37 7

37 33

Omit ouabain 1ifference

% %

4 3 8 5

7 30 7 0

15 22

10 23

‘ooFr==--l 0s 75

W” t 1

PRE-INCUBATION TIME WITH OUABAIN IN MINUTES

I,;;,

2 4 6 6 IO 12 DAYS OF STORAGE AT 4O

FIG. 10. Effect of storage of the enzyme preparation of 4” on ouabain inhibition of phosphorylation at 0”. Upper panel, en- zyme preparations either 1 day (0) or 9 days (m) old. They were incubated with 0.25 pmole of ouabain and 5 pmoles (Tris)dEDTA. At the times indicated, the enzyme was phosphorylated by the addition of 16 pmoles of NaCl, 6 rmoles of MgC12, and 0.04 rmole of (32P)-ATP. The reaction was stopped with acid 5 set later. Lower panel, the results obtained when enzyme preparations dif- fering in age were incubated for 16 min and phosphorylated as in the upper panel. The composition of the storage solution is given under “Methods.”

blocked with the chelating agent, 1,2-cyclohexylenediaminetetra- acetic acid. Under these conditions, the turnover time of the ouabain-enzyme-phosphate complex was 55 set, close to the 60- set turnover time of the ouabain-enzyme-phosphate complex formed from (32P)-ATP in the presence of Na+ and Mg++ (Fig.7). Warming reversed inhibition of both types of dephosphoenzyme (Tables I and II). Furthermore, inhibited dephosphoenzyme formed either directly (text and Table IV) or from inhibited phosphoenzyme (Table IV) could be phosphorylated by Pi. In Experiment 3 of Table IV Na+ blocked inhibition until the enzyme was phosphorylated by ATP. Time was allowed for dephosphorylation (compare Fig. 7), and then the enzyme was phosphorylated by azPi. Without ATP (Experiments 1 and 2) or with K+ in place of Na+ (Experiment 4), ouabain was ineffec-

tive. In the absence of ATP, Na+ or K+ did not block phos- phorylation when added after ouabain (Experiments 5 and 6).

Thus, inhibited phospho- and dephosphoenzyme appeared to be the same regardless of the pathway of formation.

Effect of Aging on Ouabain Inhibition of Enzyme

Early experiments showed little inhibition of phosphorylation when the native enzyme was incubated with ouabain in the presence of EDTA. However, aged enzyme preparations were relatively rapidly inhibited by ouabain even in the presence of EDTA (upper panel, Fig. 10). The amount of ouabain inhibition occurring in 16 min in the presence of EDTA increased with the age of the enzyme in days (Fig. 10, lower panel).

DISCUSSION

There is good evidence for stoichiometric, noncovalent binding of ouabain to inhibited (Na+ + K+)-ATPase (20,29,33). Mat- sui and Schwartz (19) showed specific binding of tritiated digoxin in the presence of Na+, ATP, and Mg++ and suggested that digoxin binds with the phosphoenzyme. Schwartz, Matsui, and Laughter (34) showed binding also in the presence of Mg++ and Pi; this binding was antagonized by Na+. They thought that “the conformational state of the enzyme is probably of primary significance in glgcoside binding.” Albers, Koval, and Siegal (29) confirmed these findings with tritiated ouabainand correlated binding with inhibition of activity. In other work in progress we have also observed binding of tritiated ouabain to the dephospho- or the phosphoenzyme under the conditions employed in the present work. The correspondence of conditions for binding and for alterations of phosphorylation indicates that ouabain is proba- bly bound to the inhibited enzyme.

In the presence of concentrations of Na+ or K+ above 16 mM the dephosphoenzyme did not react with ouabain (Figs. 2 and 3 and Table IV). After phosphorylation by ATP in the presence of Na+ and Mg++ the enzyme was rapidly converted to an in- hibited phospho-form, which later lost its phosphate group but not the inhibition (Figs. 4 to 7). This form was stable at O”, but, warming restored the reactivity of the native enzyme (Table II). This cycle could operate continually under physiological condi- tions where the monovalent cation concentration is above 16 ml\l, Na+, Mg++, and ATP are present inside the cell, and the tem- perature is usually above 23”. Under other conditions this cycle

by guest on July 12, 2020http://w

ww

.jbc.org/D

ownloaded from

Issue of December 25, 1969 A. K. Sen, T. Tobin, and R. L. Post 6603

appeared to be pttrtially reversible. The dephosphoenzyme re- acted with ouabain in the absence of monovalent cations and in the presence of Mg+f. The reaction was accelerated by Pi, and Pi phosphorylated the inhibited enzyme (Fig. 1 and Tables III and IV). Inhibited enzyme appeared to be the same after formation by either the forward or backward direction of the cycle. In both cases inhibition of the dephosphoenzyme was relieved by warming (Tables I and II), and in both cases the inhibited enzyme was phosphorylated by Pi (Table IV). In both cases the turnover of the inhibited phosphoenzyme was about, 60 set (Fig. 7 and text), and the active site of phosphoryl- ation appeared to be the same (text and References 30 and 31). In both cases the phosphate bond was resistant to splitting in the presence of K+ (Fig. 6 and Table IV).

The data suggest the following cycle for ion transport and ouabain inhibition of the enzyme.

f Ouabain +P, EP .- I E).Ou, ’ E,mP . Ou

II Pi ? -pi T

f Mg++ /I + ouabain

il Na+ Mg++ I

7 ~ E,.Na E1-P.Na - E,-P i- NaC

I

-Pi E;-P. K

On the basis of its reactivity the native enzyme is designated E1, the intracellularly reacting form. Ouabain binding by this form is negligible at 37” (text) and extremely slow at 0” (Fig. 1). It is, however, immediately available to Na+ (Fig. 2), for which it has a high affinity (Fig. 3). Naf or ATP directs the reaction to the ATP-dependent pathway. In the presence of Mg+f the enzyme is phosphorylated. Because of its high reactivity with ouabain and K+, it is designated Ez-P, the extracellularly reactive form. K+ splits the enzyme-phosphate bond, and the enzyme returns to the El form through transient Ez-P.K and E1. K forms. Ouabain binds to the phosphoenzyme to give EI-P.Ou. It slows hydrolysis of the enzyme phosphate bond (Fig. 5), renders it resistant to dephosphorylation by K+ (Fig. 6), and afterwards remains bound to the dephosphoenzyme (Ez. Ou) (Figs. 4 and 7). The stability of Ez. Ou at 0” and 23” (Fig. 7 and Table II) suggests that formation of this complex is the basis of ouabain inhibition of the enzyme. Dissociation of the Ez. Ou form allows regeneration of the native enzyme (Fig. 7).

The slow reaction of the native enzyme with ouabain is acceler- ated by 1\Ig++ alone or with Pi (Figs. 1 and 4 and Tables III and IV). The function of Mgff may be to generate a form of the enzyme which reacts more readily with Pi or ouabain or both. The reaction sequence of this pathway is at present unclear. Under physiological conditions with high monovalent cation con- centrations this reaction sequence is presumably of little signif- icance. In contrast to the native enzyme, the ouabain-treated enzyme in t,he presence of Mg +f (Ez. Ou) rapidly incorporates 32P i as an acyl phosphate (Tables III and IV). The phospho- enzyme formed in the presence of ouabain is high energy in that it is an acyl phosphate bond (31) but it failed to react with ADP.2 As suggested by Albers, Koval, and Siegel (29), the free energy change induced by ouabain is probably due to a change in the conformational potential of the system.

* Unpublished experiment.

These phosphorylation experiments have shown a marked difference between the sensitivity of the phosphoenzyme and that of the dephosphoenzyme to ouabain in the presence of sodium ion. Binding of ouabain is clearly stimulated by ATP in the presence of Na+ and Mg++ (19, 29, 33), but other nucleotides are also effective (29, 33). These nucleotides arc much more slowly split by (Naf + K+)-ATPase. However, the ouabain- enzyme complex can be so stable that a very low rate of phos- phorylation could stimulate binding. Furthermore, the influence of ligands on the rate of inhibition was reversed by phosphoryl- ation. Before phosphorylation, Mg++ accelerated (Fig. 1) and Na+ retarded inhibition (Figs. 2 and 3), but after phos- phorylation Mg+f was not required (Fig. 8) and Na+ slightly accelerated inhibition (Fig. 9). Because of the speci- ficity of the structure of cardioactive steroids for function (32) and because of stoichiometric binding to the inhibited enzyme (19, 20, 29, 33), it seems reasonable to think of a specific active site for binding. Because of the complex act,ion of ligands on the sensitivity to inhibition, it seems reasonable, for the present, to explain the conversion of sensitivity by a change in confor- mation of the enzyme.

Following Monod, Wyman, and Changeux (35) and Jardetzky (36) it is proposed that changes in ligand affinity and presentation of the ion carrier sites represent changes in conformat,ion of a pumping enzyme. It is proposed that there are at least two maior conformations of the enzyme which link changes in function as follows.

Conformation

Presentation of carriers L&and affinities Catalyst for transphosphoryl-

ation Associated transient forms Reactivity of phospho-forms

Ollabain alfinity

El

Inward Naf, ATP Na+ and Mg++

331-P With AL)P

Low

EZ

Olltward K+, Olmbain K+

J&P With I-I20 or

ouabain High

Phosphorylation of El gives rise to El-P, which is a transient form (28, 31, 37). The phosphoenzyme then stabilizes as a new conformation, Es-P. On hydrolysis of the enzyme-phos- phate bond a transient E2 form exists. Owing to its conformn- tional potential, it restabilizes as El. Ouabain stabilizes the transient Et form and allows it to react with Pi. It is suggested that these conformational changes produce the changes in car- rier orientation and ligand affinity which allow translocation of the cations (36).

Albers, Koval, and Siegel (29, 30) have emphasized “a drug- enzyme interaction which is essentially irreversible under physio- logical conditions” in their studies on electroplax (Na+ + I<+)- ATPase. There appears to be a species difference between electroplax and guinea pig enzymes in this respect.

Ahmed and Judah (38) observed a reduction in the inhibitory effect of ouabain when the incubation temperature was lowered. The greater stability of the inhibited enzyme at lower t,empera- tures suggests that the rate of formation of the ouabain-sensi- tive form has a higher temperature coefficient than the rate of its disappearance. However, direct experimental proof of this suggestion is not yet available.

Acknowledgments-We are indebted to Prof. H. Kalant for extensive help and criticism in the preparation of this manuscript,

by guest on July 12, 2020http://w

ww

.jbc.org/D

ownloaded from

6604 Ouabain Inhibition of (Naf + K+)-ATPase Vol. 244, No. 24

and to Prof. W. T. Thompson for helpful criticism of it. The 20. GLYNN, I. M., J. Physiol., 136, 148 (1957).

authors are grateful to Mrs. Betty Orcutt, Mrs. Frances Noah, 21. CALDWELL, P. C., AND KEYNES, R. D., J. Physiol., 148, 8P

Mrs. Jean Sorrels, Mr. R. Luben, Mrs. Mila Kopecky, Mrs. Shir- (1959).

19 Wong, and Mr. M. Bhatnagar for expert technical assistance. 22. SCHATZMANN, H. J., Biochim. Biophys. Acta, 94, 89 (1965). 23. MATSUI, H., AND SCHWARTZ, A., Biochem. Biophys. Res. Com-

mun.. 26. 147 (1966). REFERENCES

1. SKOU, J. C., Physiol. Rev., 46, 596 (1965). 2. ALTERS, R. W., Annu. Rev. Biochem., 36, 727 (1967). 3. HEINZ, E., Annu. Rev. Physiol., 29, 21 (1967). 4. POST, R. L., Biochim. Biophys. Acta, 11, 163 (1968). 5. GLY~N, I. %I., Brit. Med. Bull., 24, i65 (1968). 6. POST. R. L.. SEN. A. K.. AND ROSENTHAL. A. S.. J. Biol. Chem..

7. 246, 1437 ‘(1965). ’

I I

FAHN, S., KOVAL, G. J., AND ALBERS, R. W., J. Biol. Chem., 243, 1993 (1968).

8. GIBBS, R., RODDY, P. M., AND TITUS, E., J. Biol. Chem., 240, 2181 (1965).

9. RODNIGHT, R., HEMS, D. A., AND LAVIN, B. E., Biochem. J., 101, 502 (1966).

24. POST, k. i., AND SE&, A. K., in S. P. COLOWICK AND N. 0. KAPLAN (Editors), Methods in enzymology, Vol. 10, Academic Press, New York, 1967, p. 762.

25. POST, R. L., AND SEN, A. K., in S. P. COLOWICK AND N. 0. KAPLAN (Editors), Methods in enzymology, Vol. 10, Academic Press, New York, 1967, p. 773.

26. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J., J. Biol. Chem., 193, 265 (1951).

27. BARTLETT, G. R., J. BioZ. Chem., 234, 466 (1959). 28. SEN, A. K., AND POST, R. L., in Abstracts of the Biophysical

Society, 10th Annual Meeting, 1966, p. 152. 29. ALBERS, R. W., KOVAL, G. J., AND SIEGEL, G. J., Mol. Phar-

macol., 4, 324 (1968).

10. WHITT~M, It., WHEELER, K. P., AND BLAKE, A., Nature, 203, 720 (1964).

30. SIEGEL, G. J., KOVAL, G. J., AND ALBERS, R. W., J. BioZ. Chem., 244, 3264 (1969).

31. POST, R. L., KUME, S., TOBIN, T., ORCUTT, B., AND SEN, A.

11.

12.

NAGANO, K., MIZUNO, N., FUJITA, M., TASHIMA, Y., NAKAO, T., AND NAKAO, M., Biochim. Biophys. Acta, 143, 239 (1967).

GARRAHAN, P. J., AND GLYNN, I. M., J. Physiol., 192, 217 (1967).

13. MURPHY, J. R., J. Lab. Clin. Med., 61, 567 (1963). 14. SEN, A. K., AND POST, R. L., J. Biol. Chem., 239, 345 (1964). 15. WHITTAM, B., AND AGER, M. E., Biochem. J., 97, 214 (1965). 16. MAIZELS, M., J. Physiol., 196, 657 (1968). 17. POST, R. L., MERRITT, C. R., KINSOLVING, C. R., Z\~~ AL-

K.; J. Gen. PhysihZ.,‘64, 306s (1969). 32. GLYNN. I. M.. Pharmacol. Rev.. 16. 381 (1964). 33. HOFF&N, J. P., AND INGRAM, k. j., in k. D&JTSCH, E. GER-

LACH AND K. MOSEN (Editors), Metabolism and membrane permeability of erythrocyte and thrombocytes, Georg Thieme Verlag, Stuttgart, 1968, p. 420.

34. SCHWARTZ, A., MATSUI, H., AND LAUGHTER, A., Science, 169, 323 (1968).

35. MONOD, J., WYMAN, J., AND CHANGEUX, J., J. Mol. Biol., 12, 88 (1965).

BRIGHT. C. D.. J. Biol. Chem.. 236, 1796 (1960). 18. 19.

WHITTAM; R., AAD AGER, M. E., B&hem. k., 93, 337 (1964). MATSUI, H., AND SCHWARTZ, A., Biochim. Biophys. Acta, 167,

655 (1968).

36. JARDETZKY, O., Nature, 211, 969 (1966). 37. FAHN, S., KOVAL, G. J., AND ALDERS, R. W., J. BioZ. Chem.,

241, 1882 (1966). 38. AHMED, K.; AND JUDAH, J. D., Can. J. Biochem., 43,877 (1965).

by guest on July 12, 2020http://w

ww

.jbc.org/D

ownloaded from

Amar K. Sen, T. Tobin and R. L. PostAdenosine Triphosphatase

A Cycle for Ouabain Inhibition of Sodium- and Potassium-dependent

1969, 244:6596-6604.J. Biol. Chem. 

  http://www.jbc.org/content/244/24/6596Access the most updated version of this article at

 Alerts:

  When a correction for this article is posted• 

When this article is cited• 

to choose from all of JBC's e-mail alertsClick here

  http://www.jbc.org/content/244/24/6596.full.html#ref-list-1

This article cites 0 references, 0 of which can be accessed free at

by guest on July 12, 2020http://w

ww

.jbc.org/D

ownloaded from