Protonation of the sarcoplasmic reticulum Ca-ATPase during ATP hydrolysis.

pH changes of the reaction medium (pH 6.35) following addition of MgATP were determined at 4 degrees C with sarcoplasmic reticulum vesicles in the presence of 0.1 mM CaCl2 and 5 mM MgCl2. With intact vesicles, a pronounced acidification following a slight alkalinization was induced by MgATP, indicating H+ ejection from vesicles. This agrees with previous observations by several investigators that suggested H+ counter-transport coupled to Ca2+ uptake. When vesicles were made leaky, a markedly enhanced alkalinization occurred after addition of MgATP, and then the pH returned to the level before the MgATP addition. The decay of this alkalinization coincided with the disappearance of phosphoenzyme (EP) which was formed from MgATP and the Ca-ATPase of sarcoplasmic reticulum. The CaCl2 concentration dependence of the alkalinization agreed well with that of EP formation. Furthermore, the alkalinization was prevented by modification of a specific SH group of the enzyme essential for EP decomposition. These findings give evidence that the alkalinization with leaky vesicles represents protonation of the enzyme during ATP hydrolysis. The enhanced alkalinization with leaky vesicles suggests protonation occurring inside vesicles. When CaATP in place of MgATP was added in the absence of MgCl2, neither the H+ ejection from intact vesicles nor the enhanced alkalinization with leaky vesicles occurred. Under these conditions, CaATP did not efficiently support active Ca2+ uptake. These results are compatible with the view that the observed protonation could represent an initial event occurring inside vesicles in the H+ countertransport coupled to Ca2+ uptake.