Abstract

The effect of potassium on the binding of ouabain by (Na⁺ + K⁺)-activated adenosine triphosphatase (ATPase) [Mg⁺⁺-dependent, (Na⁺ + K⁺)-activated adenosine triphosphate (ATP) phosphohydrolase, EC 3.6.1.3] and on the release of ouabain from the ouabain-enzyme complex was studied in vitro with dog heart and rat brain enzyme preparations. The bnding was observed in the presence of Mg, Na and ATP or Mg and orthophosphate (P₁). It was a relatively slow process requiring several minutes to complete. The velocity of the (Mg + Na + ATP)-dependent binding was reduced by 1 mM KCl whereas that of (Mg + P₁)-dependent binding was not affected. The ouabain-enzyme complex prepared in the presence of Na, Mg and ATP dissociated according to first-order kinetics. The half-life of the ouabain-enzyme complex at 37°C and pH 7.5 was approximately 7.3 and 4.6 minutes for dog heart and rat brain enzyme, respectively. The release of ouabain from the brain ouabain-enzyme complex was accompanied by reversal of the inhibition of enzyme activity. KCl inhibited the dissociation of the ouabain-enzyme complex significantly with a concomitant prevention of the reversal of the enzyme inhibition. When the ouabain-enzyme complex was prepared by incubation in Mg and P₁, without Na⁺, the rate of dissociation was much slower with a half-life of approximately 30 minutes. KCl had no effect on the dissociation of this complex. The effects of monovalent cations such as NH₄⁺, Rb⁺, K⁺, Cs⁴, Li⁺ and Li⁺ to inhibit the dissociation of the ouabain-enzyme complex could be roughly correlated with the reported ability of these cations to stimulate the ATPase activity in the presence of Na⁺ and Mg⁺, except for Tl. KCl antagonized the inhibitory action of ouabain on (Na⁺ + K⁺)-activated ATPase only when the inhibitor-enzyme reaction was started by the addition of ATP in the presence of ouabain, enzyme, Na⁺, Mg⁺⁺ and K⁺. When the inhibitor-enzyme reaction was started by adding the preformed ouabain-enzyme complex to the incubation mixture, an antagonism between KCl and ouabain was not observed. These effects of KCl are consistent with a conformational change in the enzyme such that the binding sites for ouabain become less accessible.