Reaction Mechanism of the Ca2+-dependent ATPase of Sarcoplasmic Reticulum from Skeletal Muscle

The SR was loaded with Ca2+ ions by simply pre-incubating it in solutions containing various concentrations of CaCl2, 0.1 M KCl, and 50 mM Tris-maleate at pH 7.0 and 0°C for several hours. When the Ca2+-loaded reticulum was put into a solution containing 10 mM EGTA, 20 mM MgCl2, 5 mM 32P1, 90 mM KCl, and 50 mM Tris-maleate at pH 7.0 and 20°C, P was rapidly incorporation into the SR. The maximum amount of P-incorporation reached 4 moles/105 g SR protein. The time-course of P-incorporation showed a lag phase, and a remarkably large dependence on temperature. The dependence of the amount of P incorporated ([EP]) in the steady state on the Ca2+-gradient across the membrane and the concentrations of Mg2+ ions and P1 in the phosphorylation medium was given by where ɛ is the total concentration of phosphorylation site, and [Cai] and [Ca°] are the electrochemical activities of Ca2+ ions inside and outside the membrane, respectively. When [EP]/ɛ was 0.5 in the presence of 20 mM MgCl2 and 5 mM Pi, the free energy obtained by transporting 2 moles of Ca2+ ions from inside to outside the membrane was estimated to be about 12 kcal. The phosphorylation of the Ca2+-loaded SR with P1 was competitively inhibited by ATP. At various pH values the stability of the acid-denatured phosphorylated intermediate formed by the reaction with P1 was equal to that of the intermediate formed by the reaction with ATP. Decomposition of the acid-denatured phosphorylated intermediate produced by the reaction with P1 was accelerated by adding hydroxylamine in the same way as that of the intermediate produced by the reaction with ATP. Furthermore, the SDS-gel electrophoretogram of the phosphorylated intermediate showed that P was incorporated in the protein moiety of the ATPase [EC 3. 6. 1. 3]. When ADP was added to the Ca2+-loaded SR 30 sec after starting the phosphorylation experiment, the amount of P incorporated decreased markedly and rapidly, and ATP was formed. The amount of ATP synthesized was exactly half the amount of Ca2+ ions loaded into the SR during the pre-incubation.