EVIDENCE FOR TWO DIFFERENT Na+‐DEPENDENT [3H]‐OUABAIN BINDING SITES OF A Na+‐K+‐ATPASE OF GUINEA‐PIG HEARTS

Abstract

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, (K_0.5 = 4.5 mM); this type of [³H]-ouabain binding was strongly correlated to the Na⁺ concentration necessary for half maximum phosphorylation (K_0.5 = 1 mM). The other ouabain binding process was predominant at high Na⁺ concentrations (K_0.5 = 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.