Nature of the Transport Adenosine Triphosphatase Digitalis Complex IV. EVIDENCE THAT SODIUM-POTASSIUM COMPETITION MODULATES THE RATE OF OUABATN INTERACTION

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 pM [3H]ouabain (30’). When assays were carried out for longer periods in the presence of at least 0.2 pM [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 lo4 s-r ~-l at 30” and 4.3 X lo4 s-l M-l 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 (&.s) for potassium but had no effect on Ko.s for sodium. Changes in KO.a 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 13H]ouabain binding rates suggested that sodium and potassium compete for a common site; dissociation constants for sodium and potassium at 37” were 13.7 rnr+d and 0.213 mu, respectively. This relationship predicted the sensitivity (i.e. in terms of inhibition of enzyme activity) of (Na+ + K+)-ATPase to ouabsin in the presence