Interaction of ouabain with (Na + + K +)ATPase from human heart and from guinea-pig heart

Abstract

(Na + + K +)ATPase (ATP phosphohydrolase, EC 3.6.1.3.) has been prepared from human heart and guinea-pig heart, with respective specific activities of 10–15 μmol P i · mg −1 · h −1 and of 25–30 μmol P i · mg −1 · h −1. Residual Mg 2+—ATPase activities were about 5 per cent. The parameters of (Na + + K +)ATPase activity and of ouabain-interaction have been compared: (1) Half-maximal activity concentrations and Hill coefficients of Na +, K +, Mg 2+ and ATP were similar for the two species. The apparent activation energies calculated from Arrhenius plots were also similar. A transition was observed at about 23°C. (2) Human heart (Na + + K +)ATPase was 10 times more sensitive to ouabain-inhibition than that of guinea-pig. Hunter-Downs plots showed a competitive inhibition for K + at low K + concentration and noncompetitive inhibition at high concentration. (3) The Scatchard plot for [ 3H]ouabain binding was upward-concave with human heart and linear with guinea-pig heart. (4) The dissociation kinetics of [ 3H]ouabain from human preparations studied by an isotopic dilution technique indicated two classes of binding sites with k d of 0.058 min −1 and 0.0092 min −1. The dissociation kinetics with guinea-pig heart indicated one single class of binding sites with a k d of 0.43 min −1. (5) The time-course of 0.2 μM [ 3H]ouabain binding showed pseudo-first order association kinetics in man and in guinea-pig. k a for the two classes of binding sites in man were therefore similar, respectively equal to 3.4 × 10 6 min −1 · M −1 and to 3.7 × 10 6 min −1 · M −1 · k a for guinea-pig heart was equal to 2.3.10 6 min −1 · M −1. (6) In guinea-pig heart, K D c from Scatchard plot and from k d / k a ratio were equal to the inhibition constant K i calculated from Hunter-Downs plot indicating that the binding sites were closely related to (Na + + K +)ATPase inhibition. (7) In human heart, K D of the low affinity binding sites was close to K i , whereas K D of the high affinity binding sites was several times lower. This suggests that only low affinity binding sites might be involved in (Na + + K +)ATPase inhibition by ouabain.