STUDIES ON THE TWO PHOSPHOENZYME CONFORMATIONS OF Na⁺+ K⁺-ATPase (original) (raw)

Many proposed reaction mechanisms for Na+ t K+-ATPase postulate the existence of a phosphoenzyme that reacts readily with ADP and poorly with K+, generally called the E l Q P form of the phosphoenzyme.' The direct identification of this postulated intermediate in native Na+ + K+-ATPase has proved difficult, although it is relatively easy t o demonstrate such an intermediate in enzymes irreversibly inhibited with NEM (N-ethylmaleimide).2 In this communication we present evidence for the direct identification of this intermediate in native Na+ + K+-ATPase and its role in the reaction mechanisms of this enzyme. Methods Rat-brain Na+ + K+-ATPase prepared by the method of Akera and Brody3 or the guinea pig kidney enzyme prepared as described by Post and Sen4 were used throughout. Phosphorylation, [ HI ouabain binding, and Na+ t K+-ATPase activity were measured as described by Tobin and coworkers.' Because of the ease with which El % P may be demonstrated in rat-brain Na+ + K+-ATPase this preparation was used in the phosphoenzyme experiments. In experiments involving [ HI ouabain binding equilibria the guinea pig kidney Na+ + K+-ATPase was used because of the rapidity with which glycoside binding t o this particular enzyme equilibrates. Results One of the ligands of Na++K+-ATPase used to monitor changes in the reactivity of the enzyme in these experiments is [ HI ouabain, and the initial series of experiments deal with the effects of bound ATP on the interaction of this enzyme with [ 3H] ouabain. FIGURE 1 shows the actions of Na+ and ATP on the rate of dissociation of [ HI ouabain from rat-brain Na+ t K+-ATPase. For this experiment [ 3H] ouabain was bound t o the enzyme in the presence of Mg++ and Pi, and the enzyme then washed free of ligands and unbound [ 3 H ]ouabain. The preformed enzyme-ouabain complex was then allowed t o dissociate into a buffered solution containing 1 mM EDTA and unlabeled ouabain. As shown in FIGURE 1 , the rate of dissociation of [3H] ouabain was slow under all