Binding of adenosine triphosphate to sodium and potassium ion-stimulated adenosine triphosphatase.

In order to characterize a step in the reaction sequence of sodium and potassium ion-dependent adenosine triphosphatase (EC 3.6.1.3), the binding of radioactive adenosine triphosphate to kidney cell membranes was estimated by flow dialysis in the presence of a chelator of divalent cations, which prevented hydrolysis of the adenosine triphosphate. Adenosine triphosphate was bound to a single site on sodium and potassium ion-stimulated adenosine triphosphatase (a) because binding capacity of the membranes was equal, one for one, to the phosphorylation capacity of this enzyme, (b) because binding was inhibited by pretreatment of the membranes with the spec%c inhibitor, ouabain, and (c) because binding was inhibited by potassium ion with antagonism of this inhibition by sodium ion, which had no direct effect. The dissociation constant was 0.22 pM. Other nucleotides displaced ATP including, in order of decreasing effectiveness: deoxyadenosine triphosphate, p, y-methylene adenosine triphosphate, cytidine triphosphate, and inosine triphosphate. Monovalent cations reduced the binding affinity at progressively higher cation concentrations, as follows: potassium, rubidium, thallous, ammonium, and cesium ions. Lithium ion had no effect. Binding was stable between pH 5.6 and 7.6, but declined sharply above pH 8.0. In the presence of potassium ion alone, there appeared to be one or more binding sites on this enzyme with a much lower affinity for adenosine triphosphate. These may be regulatory sites.