Calcium inhibition of the ATPase and phosphatase activities of (Na ++K +)-ATPase

Abstract

In experiments performed at 37°C, Ca 2+ reversibly inhibits the Na +- and (Na ++K +)-ATPase activities and the K +-dependent phosphatase activity of (Na + + K +)-ATPase. With 3 mM ATP, the Na +-ATPase was less sensitive to CaCl 2 than the (Na ++K +)-ATPase activity. With 0.02 mM ATP, the Na +-ATPase and the (Na ++K +)-ATPase activities were similarly inhibited by CaCl 2. The K 0.5 for Ca 2+ as (Na ++K +)-ATPase inhibitor depended on the total MgCl 2 and ATP concentrations. This Ca 2+ inhibition could be a consequence of Ca 2+Mg 2+ competition, Ca·ATP-Mg·ATP competition or a combination of both mechanisms. In the presence of Na + and Mg 2+, Ca 2+ inhibited the K +-dependent dephosphorylation of the phosphoenzyme formed from ATP, had no effect on the dephosphorylation in the absence of K + and inhibited the rephosphorylation of the enzyme. In addition, the steady-state levels of phosphoenzyme were reduced in the presence both of NaCl and of NaCl plus KCl. With 3 mM ATP, Ca 2+ alone sustained no more than 2% of the (Na ++K +)-ATPase activity and about 23% of the Na +-ATPase activity observed with Mg 2+ and no Ca 2+. With 0.003 mM ATP, Ca 2+ was able to maintain about 40% of the (Na + + K +)-ATPase activity and 27% of the Na +-ATPase activity seen in the presence of Mg 2+ alone. However, the E 2(K)-E 1K conformational change did not seem to be affected. Ca 2+ inhibition of the K +-dependent p- nitrophenylphosphatase activity of the (Na ++K +)-ATPase followed competition kinetics between Ca 2+ and Mg 2+. In the presence of 10 mM NaCl and 0.75 mM KCl, the fractional inhibition of the K +-dependent p- nitrophenylphosphatase activity as a function of Ca 2+ concentration was the same with and without ATP, suggesting that Ca 2+ indeed plays the important role in this process. In the absence of Mg 2+, Ca 2+ was unable to sustain any detectable ouabain-sensitive phosphatase activity, either with p- nitrophenylphosphate or with acetyl phosphate as substrate.