Evidence for two different Na+-dependent [3H]-ouabain binding sites of a Na+-K+-ATPase of guinea-pig hearts.

1. The influence of various Na+ concentrations on [3H]-ouabain binding was studied in experiments on a microsomal Na+-K+-adenosine triphosphatase (ATPase) from guinea-pig hearts. 2. The ATP-independent cardiac glycoside binding was not influenced by increasing Na+ concentrations. However, a good correlation was found between the ATP-dependent [3H]-ouabain binding and Na+ concentration. 3. A more detailed analysis of these results according to Hofstee (1952) revealed two distinct processes involved in this interaction: one ouabain binding process was activated at rather low Na+ concentrations, (K0.5 = 4.5 mM); this type of [3H]-ouabain binding was strongly correlated to the Na+ concentration necessary for half maximum phosphorylation (K0.5 = 1 mM). The other ouabain binding process was predominant at high Na+ concentrations (K0.5 = 69 mM). 4. On the basis of the commonly accepted ATPase reaction cycle a model for the interaction of cardiac glycosides with the Na+-K+-ATPase is proposed, assuming two different binding sites for cardiac glycosides (E2-P and E1-P) and involving a translocation of these drugs from an outer to an inner compartment of the cell membrane.