Inhibition of hydrolysis of phosphorylated Ca2+,Mg2+-ATPase of the sarcoplasmic reticulum by Ca2+ inside and outside the vesicles.

Abstract

The effects of intra- and extravesicular calcium and magnesium ions on the hydrolysis of the phosphoenzyme (EP) intermediate formed in the reaction of Ca2+,Mg2+-dependent ATPase of the sarcoplasmic reticulum were investigated. The rate constants of EP hydrolysis were measured under conditions that allowed a single turnover of ATP hydrolysis to minimize the increase in calcium concentration inside the vesicles. The EP formed during a single turnover was hydrolyzed biphasically and could be resolved into fast- and slow-decomposing components. When free Mg2+ outside the vesicles was chelated by adding excess EDTA, EP could also be kinetically resolved into two components; EDTA-sensitive EP, which could be quickly decomposed by adding EDTA, and EDTA-insensitive EP, which could be prevented from decomposing by adding EDTA. The amount of EDTA-sensitive EP decreased rapidly during the initial phase of the reaction, while that of EDTA-insensitive EP decreased slowly with the same rate constant as that of the slow-decomposing EP. These results showed that the biphasic time course of EP hydrolysis was caused by the formation of EDTA-sensitive and -insensitive EP during the reaction. The time course of EP hydrolysis could be quantitatively analyzed in terms of the following reaction mechanism. (formula; see text) The decomposition of EDTA-insensitive EP required Mg2+ outside the vesicles and was competitively inhibited by extravesicular Ca2+. The decomposition of EDTA-sensitive EP was inhibited by Ca2+ inside the vesicles but not by external Ca2+. The linear relationships between the inverse of the rate constants of EP decomposition during the initial phase and the intravesicular CaCl2 concentrations suggested that decomposition of EDTA-sensitive EP was inhibited by the binding of 1 mol of intravesicular Ca2+ to 1 mol of EP. Furthermore, Mg2+ inside the vesicles scarcely affected the inhibition of EP hydrolysis by intravesicular Ca2+. These results suggested that magnesium ions are not counter-transported during the active transport of calcium by SR vesicles.