Quantitative aspects of the interaction between ouabain and (Na + + K +)-activated ATPase in vitro

Abstract

The inhibitory effect of ouabain on (Na + + K +)-activated ATPase (Mg 2+-dependent, (Na + + K +)-activated ATP phosphohydrolase, EC 3.6.1.3) obtained from rat brain microsomal fraction was re-examined using a modified method to estimate the inhibited reaction velocity. This method involves a preincubation of a ouabain-enzyme mixture in the presence of Na +, Mg 2+ and ATP to bring the ouabain-enzyme reaction to near equilibrium. The (Na + + K +)-activated ATPase reaction was subsequently started by the addition of a KCl solution. When the ouabain-enzyme reaction was brought to near equilibrium prior to the estimation of the ATPase activity, it was kinetically reversible, although overwhelmingly in favor of the ouabain-enzyme complex. This method resulted in a significant shift of the log concentration-response curve to the left. The concentration of ouabain to inhibit 50 % of the (Na + + K +)-activated ATPase activity was 0.12 μM, whereas it was 0.52 μM with the conventional method for the ATPase assay. Hence, the specific binding of ouabain to the (Na + + K +)-activated ATPase molecule was a slow process. This modified method was not suitable for the study of the effect of p-chloromercuribenzoate on (Na + + K +)-activated ATPase since the presence of ATP during the preincubation period protected the enzyme from the SH-blocking reagent. With the modified method, the effect of K + to antagonize ouabain inhibition of (Na + + K +)-activated ATPase was markedly reduced, indicating that the well-documented effect of K + was on the velocity of the ouabain-enzyme complex formation rather than on that of the ouabain-inhibited ATPase reaction. The ouabain-enzyme reaction was competitive with respect to K + at KCl concentrations below 5 mM, although the competition by K + was not remarkable. Above this concentration, the reaction was non-competitive with respect to K +. Ouabain released from the ouabain-enzyme complex was rebound to the enzyme during an incubation in the presence of Na +, Mg 2+ and ATP more easily than ouabain added to the incubation mixture.