ADP-Sensitive and -Insensitive Phosphorylated Intermediates of Solubilized Ca2+, Mg2+-Dependent ATPase of the Sarcoplasmic Reticulum from Skeletal Muscle1

Solubilized Ca2+, Mg2+-dependent ATPase [EC 3.6.1.3] with stable activity was prepared from sarcoplasmic reticulum of skeletal muscle by a modification of the method of le Maire et al. (1976) (Biochemistry 15, 2336–2342). The dependence on ATP concentration of the ATPase activity (v0), the amount of EP in the steady state and the initial rate of EP formation followed the Michaelis-Menten equation. ADP inhibited the ATPase activity non-competitively and the value of v0/[EP] was constant over a wide range of ADP concentrations. These results indicate the existence of EP which decreased in amount on addition of ADP. The amounts of two kinds of EP, one of which could react with ADP to form ATP and the other of which could not, were measured at alkaline pH where the EP decomposition was very slow. The following results were obtained. In the presence of a sufficient amount of Ca2+, almost all the EP formed reacted with ADP to form ATP (ADP-sensitive), while in the absence of Ca2+, none of the EP formed could react with ADP (ADP-insensitive). When the reaction was started by adding ATP to the ATPase, ADP-sensitive EP was formed first, then converted into ADP-insensitive EP. 3. The dependence on Mg2+and Ca2+concentrations of the amounts of the two kinds of EP indicated that ADP-sensitive EP binds two mol of Ca2+(CazEP) and ADP-insensitive EP binds two mol of Mg2+per mol of EP (Mg2Ep). Furthermore, the affinities of the enzyme for Ca2+and Mg2+changed markedly with phosphorylation. 4. The decomposition of ADP-insensitive EP was accelerated by the addition of a low concentration of Mg2+and 2inhibited by a high concentration of Mg2+. It was unaffected by Ca2+ions. 5. The conversion of ADP-sensitive EP into -insensitive EP followed first-order kinetics (k=0.5 s-1 at pH 9.0 and 10°C), and was unaffected by the addition of Mg2+. Both the time course of the conversion of ADP-insensitive EP into -sensitive EP and that of formation of the E-ATP complex from ADP-sensitive EP and ADP were biphasic. These results indicate that both ADP-sensitive and -insensitive EP have at least two different conformational states. 6. The conversion of ADP-sensitive EP into -insensitive EP was inhibited by the modification of ATPase with MaINEt.