Nature of the Transport Adenosine Triphosphatase Digitalis Complex: IV. EVIDENCE THAT SODIUM-POTASSIUM COMPETITION MODULATES THE RATE OF OUABAIN INTERACTION WITH (Na+ + K+) ADENOSINE TRIPHOSPHATASE DURING ENZYME CATALYSIS

Abstract

Abstract Estimates of initial binding rates for [3H]ouabain interaction with (Na+ + K+)-ATPase were obtained in the presence of ATP, magnesium, sodium, and potassium by using short assay times at 30°. The reactions were terminated by the rapid addition of excess unlabeled ouabain; this did not appear to displace bound [3H]ouabain during subsequent treatment of the suspensions (prior to assay). Initial binding rates were directly proportional to drug concentrations between 0.2 and 2.0 µm [3H]ouabain (30°). When assays were carried out for longer periods in the presence of at least 0.2 µm [3H]ouabain, the rates adhered to pseudo-first order kinetics. Maximal (i.e. at infinite sodium concentration) second order rate constants were 2.2 x 104 s-1 m-1 at 30° and 4.3 x 104 s-1 m-1 at 37°. Hill analyses (plots) for sodium (constant potassium), potassium (constant sodium) and sodium plus potassium (sodium/potassium = 10) activation of (Na+ + K+)-ATPase suggested that ouabain increased the dissociation constant (K0.5) for potassium but had no effect on K0.5 for sodium. Changes in K0.5 for sodium plus potassium activation were less than for potassium, which suggested that sodium modulates ouabain-potassium antagonism during turnover of the enzyme. In the presence of ATP and magnesium, sodium increased and potassium decreased the rate of [3H]ouabain interaction. Kinetic analyses of [3H]ouabain binding rates suggested that sodium and potassium compete for a common site; dissociation constants for sodium and potassium at 37° were 13.7 mm and 0.213 mm, respectively. This relationship predicted the sensitivity (i.e. in terms of inhibition of enzyme activity) of (Na+ + K+)-ATPase to ouabain in the presence of MgCl2, Tris-ATP, and various sodium and potassium concentrations. The relationship did not apply at sodium concentrations less than 10 mm in the presence of 10 mm potassium, which suggested that the validity of the model requires interaction of other cation-binding sites with sodium. The competitive relationship between sodium and potassium may modulate cardiac glycoside interaction with (Na+ + K+)-ATPase.