Permeability of plasma membrane vesicle to ouabain and Mg2+ as a factor determining rate of binding of ouabain to Na+ and K+ dependent ATPase.

Na+, K+-ATPase of the plasma membrane isolated from sheep kidney medulla exhibits functional asymmetry for the cardiac glycoside ouabain. In this vesicular membrane preparation the rate of binding of ouabain was slow (time constant greater than 60 min) when the vesicles were incubated in the presence of isotonic sucrose. Upon treatment of the preparation with hypoosmotic shock or phospholipase A the initial rate of ouabain binding was enhanced at least 3 fold. In equilibrium a concentration of the ouabain-enzyme-complex was obtained which was about twofold that of the untreated vesicles. This result suggests two types of ouabain binding sites with an approximate stoichiometry of 1 to 1. The stoichiometry seems to be maintained at high concentrations of ouabain where binding curves show a biphasic time course. Additional information about heterogeneity of binding sites comes through experiments in which the vesicles were treated with Mg2+ prior to the addition of ouabain. A minor fraction of the binding sites were occupied by ouabain only after longtime incubation with Mg2+.