The pH dependence of the cardiac sarcolemmal Ca2(+)-transporting ATPase: evidence that the Ca2+ translocator bears a doubly negative charge

The pH dependence of the Ca2(+)-transporting ATPase of bovine cardiac sarcolemma was determined in a membrane vesicle preparation. The maximal velocity (Vmax) at saturating external Ca2+ showed a sigmoidal pH dependence with maximal values in the 6.0-6.5 range, a half-maximal value at 7.2 and minimal (less than or equal to 15%) values at pH greater than or equal to 8.0. The apparent affinity for Ca2+ (1/Km) varied over 10(4)-fold for 6.0 less than or equal to pH less than or equal to 8.5, increasing with increasing pH. Plots of log(1/Km) vs. pH were biphasic. In the acid range (6.0 less than or equal to pH less than or equal to 7.2), a slope of 2.6 was observed for the calmodulin-activated form of the pump. For 7.2 less than or equal to pH less than or equal to 8.5, a slope of 0.5 was observed. At pH 7.4, the Km is approx. 48 +/- 19 nM. The Ca2+ pump of cardiac sarcoplasmic reticulum in the same preparation had a Km of 304 +/- 115 nM and showed a similar pH dependence except that the slope in the acid range was 1.7. When calmodulin was removed from the sarcolemmal pump, its Km was raised to approx. 1.0 microM, the slope in the acid range was reduced to 1.7 and the Vmax was markedly reduced. The results are explicable in terms of a model in which each of the two Ca2+ binding sites on the pump contains two buried COO- groups responsible for high affinity. The Km effect is explained by 2 H+ vs. 1 Ca2+ competition for occupation of each of the two cytoplasmically-oriented translocators (4 H+ vs. 2 Ca2+). The Vmax effect is explained by counter-transport of H+. The findings are considered in terms of the published amino acid sequence of the cardiac sarcolemmal pump and recent site-directed mutagenesis vs. function studies identifying the Ca2+ binding site in the skeletal sarcoplasmic reticulum pump. The kinetic data are also applied to pump behavior under conditions of ischemia and acidosis.