ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS 173, 386-388 (1976)

COMMUNICATION

Transport Adenosine Triphosphatase: Absence of ATP: p-Nitrophenol

Phosphotransferase Activity’

It is generally accepted that transport adenosine triphosphatase hydrolyzes both ATP and p-nitrophenyl phosphate, and other authors have shown that the enzyme can be phosphorylated in the same location by either substrate. We could detect no label exchange between ATP and p-nitrophenol. This finding indicates that any common phosphorylated intermediate must be formed from either substrate in a poorly reversible reaction and places constraints on models for the sodium pump.

Current evidence for the mechanism of potassium ions stimulate the p-NPPase activity as (Na++K+)-ATPase2 (i.e., the membrane-bound en- well as the E,-P dephosphorylation; (iii) both Na+ zyme thought to carry out active sodium and potas- and ATP are inhibitors of the K+-dependent phos- sium transport) supports a model in which ATP phatase (4, 5); (iv) ouabain inhibits the p-NPPase phosphorylates a site on the enzyme; this “high- activity (5) as well as the (Na+ + K+)-ATPase activ- energy” intermediate (E, - P) is then converted into ity; and (v) Inturrisi and Titus have shown (6) that a another form (E,-P) which is subsequently dephos- unique enzyme site can be labeled from either 32P- phorylated in the presence of KC (1, 2):

Mg” (PM), Na’ E + ATP \ ’ E,-P + ADP

I Mg” (rnM) (inhibited by oligomycin)

E + P, <K’ E,-P

Apart from its ATPase activity, the (Na++K+)-ATP- ase also possesses ATP:ADP phosphotransferase ac- labeled orthophosphate orp-[32P]NPP and concluded tivity, apparently connected with the initial phos- that this is the same site which is phosphorylated by phorylation step (1). ATP.

Also closely associated with the (Na++K+)-ATP- Evidence has accumulated, however, that the K+- ase is a potassium-stimulated phosphatase which dependent phosphatase is more than a simple mani- can hydrolyze, among other substrates, acetyl phos- festation of the terminal dephosphorylation in the phate and p-nitrophenyl phosphate @-NPP). It is ATPase reaction: Na+ and ATP have a stimulating commonly postulated that this phosphatase activity effect on the phosphatase when added together in resides in the terminal hydrolytic step of the low concentrations (7, 8); in addition, Robinson (9, (Na++K+)-ATPase activity because: (i) the two ac- 10) has found enzyme labeling from p-V*PlNPP tivities do not separate upon purification (3); (ii) which is stimulated by K+ at pH 5 but by Na+ at

neutral pH and has suggested that, in the presence ’ This research was carried out in the Department of Na+, the phosphatase substrates may use the en-

of Biophysics, University of Maryland School of tire pathway of the ATPase reaction as either the Medicine, Baltimore. Supported by grants from the major mechanism of hydrolysis, in the case of acetyl National Institutes of Health. phosphate, or a parallel alternative, in the case of

* Abbreviations used: (Na++K+)-ATPase, so- p-NPP. dium- and potassium-ion activated adenosine tri- Since the transport enzyme can be labeled from phosphatase; p-NP, p-nitrophenol; p-NPP, p-nitro- either [Y-~*P]ATP or p-[32P]NPP, and since enzyme phenyl phosphate; p-NPPase, p-nitrophenyl phos- labeling (E + P) presumably occurs in the por- phatase; E, - P, E,-P, high- and low-energy in- tion of the ATPase reaction associated with the termediate forms of the adenosine triphosphatase. [‘%]ADP-ATP exchange activity, it is not unrea-

386

Copyright 0 1976 by Academic Press, Inc. All right of reproduction in any form reserved.

COMMUNICATION 387

sonable to inquire whether phosphorylated interme- diates formed from p-NPP might participate in such exchanges as, for example, transfer of label from [y-3ZP]ATP into p-[32P]NPP, or from p-13*P]NPP into [32P]ATP. We attempted to detect such 3zP ex- changes under several conditions favoring proposed intermediates of the ATPase and thep-NPPase. The

E, - P intermediate was presumed to exist under conditions supporting sodium stimulated [“CIADP- ATP exchange, i.e., 50 mM Na+, 0.05 mM Mg*+, oligomycin, 10 pglml. Under the conditions de- scribed as favoring E,-P, the medium contained 50 mM Na+ and 3 mM Mg’+.

Rat brain synaptosomes were prepared according to the method of Gray and Whittaker (111, except that prior to density gradient centrifugation, the preparation was osmotically shocked in ice-cold dis- tilled water (15 ml/gram original brain weight). [y-32P]ATP and p[‘%]nitrophenol (p-NP) were ob- tained from ICN, P-[~*P]NPP from AmershamSearle and [‘%]ADP from Schwarz/Mann. All other bio- chemicals were obtained from Sigma. Incubations for isotopic exchange experiments were carried out at 37°C for 30 min; the reaction was stopped by spotting 2-pl aliquots onto thin-layer sheets of poly- ethyleneimine cellulose (Brinkmann) which were then developed in 1 M, Tris-maleate, pH 7, in order to separatep-13*P]NPP (12), or stepwise in 2 and 4 M ammonium formate, pH 3.4, in order to separate [32P]ATP (13). The sheets were allowed to dry at room temperature. The spots were located under uv light, cut out, and glued onto planchettes to be counted in a Beckman Widebeta II gas flow counter. (Na+ + K+)-ATPase activity was measured in a medium containing ATP, 3 mM, and MgCl,, 3 mM, by comparing the release of orthophosphate in the presence and absence of 50 mM NaCl + 10 mM KCl. p-NPPase activity was assayed by measuring the re- lease ofp-NP in a medium containingp-NPP, 4 mM, and MgCl,, 4 mM, in the presence and absence of 8 mM KCl. All solutions were buffered to pH 7.4 at 37°C with 90 mM Tris buffer. In control samples, equivalent amounts of Tris-chloride were substi- tuted for NaCl and KCl.

The results of these experiments, shown in Table I, revealed no detectable transfer of 32P from 13*P]ATP onto p-NP to produce P-[~~P]NPP, or from P-[~*P]NPP onto ADP to produce 13*P]ATP, under any of the conditions tested. Absence of p-YPINPP production could mean that p-NP cannot be phos- phorylated by either E, - P or E,-P, but it does not rule out the formation of either of these inter- mediates fromp-[32P]NPP. However, lack of produc- tion of [32P]ATP from P-[~~P]NPP + ADP, under conditions which have been shown to support Na+- dependent [‘%]ADP-ATP exchange, makes forma- tionofE, - P from p-NPP seem highly unlikely.

Dudding and Winter (14) failed to find 3ZP-label transfer onto ADP by phosphorylated (Na++K+)-

TABLE I

ACTIVITIES OF (Na++K+)-ATPase AND ASSOCIATED REACTIONS IN A TYPICAL SYNAPTOSOMAL

PREPARATIONS

Activity (nmol min’ (mg of

protein)-‘)

Reaction

(Na++K+)-ATPase Na+-dependent [‘%]ADP-ATP

exchange K+-stimulated p-nitrophenyl

phosphatase in absence of sodium and ATP

K+-stimulated p-nitrophenyl phosphatase, Na+ = 8 mM; ATP = 0.1 mM

[32PlATP + p-[32P]NPP

(E, - P conditionsP P-[~~P]NPP -+ [3zP]ATP

0% - P co.nditionsY 13*P]ATP - P-[~*P]NPP

(E,-P conditions)”

p-l’%lNP-p-NPP exchange’ Mg’+ = 4 mM MgZ+ = 4 mM; Na+ = 20 mM MgZ+ = 4 mM; Na+ = 8 m&r; K+=BmM

230 2 7 14.3 k 1.0

39.8 k 1.0

22.1 ? 3.3

0.056 + 0.105

-0.011 % 0.151

-0.031 * 0.157

-0.145 * 0.504 0.298 T 0.477

-0.186 f 0.692

” 32P and 14C exchange assays were performed in quadruplicate, the other reactions in triplicate. See text footnote 2 for abbreviations.

b NaCl, 50 mM; MgCl,, 0.05 mM; [32P]ATP, 5 mM; p-NP, 20 mM; oligomycin, 10 g/ml.

c NaCl, 50 mM; MgCl,, 0.05 mM; ATP, 0.1 mM; ADP, 2 mM; P-[~~P]NPP, 4 mM; oligomycin, 10 pg/ ml.

d NaCl, 50 mM; MgCl,, 3 mM; [32PlATP, 5 mM; p- NP, 20 mM.

(’ All solutions contained p-NPP, 4 mM, and p- l’%]NP, 2.5 mM.

ATPase made from acetyl phosphate in the presence of Mg*+. They postulated that labeling of the enzyme by acetyl phosphate produced E,-P in an irreversible reaction, since no ‘%-label transfer between acetate and acetyl phosphate could be detected. In an analo- gous experiment, we also found no transfer of label from %-labeled p-NP to p-NPP (bottom of Table Il. It seems likely, therefore, that, under ordinary con- ditions, the (Na++K+)-ATPase and the K+ phospha- tase reactions do not have a common intermediate of high enough energy to rephosphorylate either ADP, p-NP, or acetate. In this regard, it is worth recalling that Inturrisi and Titus (6) found labeling of the (Na++K+)-ATPase from p-NPP only under condi- tions where labeling from inorganic 32P can also occur. It may be, therefore, that under some condi-

388 COMMUNICATION

tions p-NPP behaves as an orthophosphate analog. In conclusion, it appears that no counterpart of

the Na+-dependent [“CIADP-ATP exchange exists between ATP and p-NPP in either direction or be- tween p-NP and p-NPP. Nor does it seem that the E, - P pathway contributes significantly to the hy- drolysis of p-nitrophenyl phosphate. The constraint placed by these findings on models for the (Na++K+)-ATPase is that, if the ATPase and phos- phatase reactions have any phosphorylated inter- mediate in common at all, it must be formed from both substrates in an irreversible reaction.

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4. TOSTESON, D. C., BLAUSTEIN, M. P., AND Mouti TON, R. H. (1961) Fed. PFOC. 20, 138.

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7. NAGAI, K., AND YOSHIDA, H. (1966) Biochim. Biophys. Acta 128, 410-412.

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10. ROBINSON, J. D. (1970) AFC~. Biochem. Biophys. 139, 164-171.

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12. HOBBS, A. S. (1975) Anal. Biochem. 66,620-622. 13. RANDERATH, K. (1966) Thin Layer Chromatogra-

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ANN S. HOBBS Laboratory of Neurochemistry, NINCDS National Institutes of Health Bethesda, Maryland 20014

PAUL DE WEER Department of Physiology and Biophysics Washington University School of Medicine St. Louis, Missouri 63110

Received August 25, 1975