Effect of temperature on phosphorylation and ouabain binding to N-ethylmaleimide-treated (Na+, K+)-ATPase.

Treatment of purified sodium and potassium ion-activated adenosine triphosphatase from sheep kidney with N-ethylmaleimide in the presence of KC1 and ATP reduced hydrolytic activity by 83% while steady state phosphorylation was inhibited only 24%. At 3O”C, in the presence of magnesium plus inorganic phosphate, the ouabain-binding capacity of the N-ethylmaleimidetreated enzyme was only 15% less than the capacity of the native enzyme. The phosphorylated intermediate formed from the N-ethylmaleimide-treatedenzyme was less sensitive to decomposition by KC1 and more sensitive to ADP when measured at either 0” or 22”C, compared to a native enzyme. The ouabain-binding capacity of native enzyme at 0” and 30°C in the presence of either magnesium plus inorganic phosphate or magnesium plus ATP plus sodium was the same. The ouabain-binding capacity of the N-ethylmaleimidetreated enzyme in the presence of magnesium plus inorganic phosphate was the same at 0°C as it was at 30°C. However, in the presence of magnesium plus ATP plus sodium, the treated enzyme bound only half as much ouabain at 0°C as it did at 3O”C, even when the reaction was extended to several hours. The rates of ouabain binding were approximately equal for the native and N-ethylmaleimide-treated enzyme when measured in the presence of magnesium alone or magnesium plus ATP. N-Ethylmaleimide treatment, however, reduced the rates of ouabain binding in the presence of magnesium plus inorganic phosphate, magnesium plus ATP plus sodium, or magnesium plus ATP plus sodium plus potassium. The results indicate that ouabain does interact with N-ethylmaleimide-treated enzyme and that multiple conformers of (Na+,K+)-ATPase, as de-