INTERACTION OF OUABAIN WITH (NA++K+)ATPASE FROM HUMAN-HEART AND FROM GUINEA-PIG HEART

(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 Pi · mg-1 · h-1 and of 25-30 μmol Pi · mg-1 · h-1. Residual Mg2+-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+, Mg2+ 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 kd 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 kd 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. ka for the two classes of binding sites in man were therefore similar, respectively equal to 3.4 × 106 min-1 · M-1 and to 3.7 × 106 min-1 · M-1 · ka for guinea-pig heart was equal to 2.3.106 min-1 · M-1. (6) In guinea-pig heart, KD c from Scatchard plot and from kd/ka ratio were equal to the inhibition constant Ki calculated from Hunter-Downs plot indicating that the binding sites were closely related to (Na+ + K+)ATPase inhibition. (7) In human heart, KD of the low affinity binding sites was close to Ki, whereas KD 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. © 1979.