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

Abstract

Ca²⁺-Mg²⁺-dependent ATPase [EC 3. 6. 1. 3] was prepared from the SR by salt fractionation after solubilization of the SR by treatment with DOC. The affinities of the enzyme for Ca²⁺ and Mg²⁺ ions during the formation and decomposition of a phosphorylated intermediate (EP) were determined at an ionic strength of 0.16 at pH 7.0 and 0°C. The following results were obtained. 1. The formation of EP required Ca²⁺ ions and was inhibited by Mg²⁺ ions which competed with Ca²⁺. The effects of Ca²⁺ and Mg²⁺ions on the rate of EP-formation. V_f, were expressed as The values of K_ca, K_Mg, and V_f were found to be 0.35μM, 10.6mM, and 1.33 mole/10⁶g.sec, respectively. 2. The decomposition of EP required Mg²⁺ ions and was inhibited by Ca²⁺ ions, which competed with Mg²⁺. The effects of Mg²⁺ and Ca²⁺ ions on the rate of EP-decomposition, v_o/[EP], were given by where v_o and [EP] are the rate of ATP-hydrolysis and the concentration of EP at the steady state, respectively. The values of K_mg/K_ca, and K_d were found to be 2.45 and 0.12 sec⁻¹, respectively. These two results show that the affinities of the enzyme for Ca²⁺ and Mg²⁺ ions change dramatically during EP-formation. 3. The Ca²⁺ ions required for EP-formation could be replaced by Sr²⁺ ions, but the Mg²⁺ ions required for EP-decomposition could not be replaced by other cations, such as Mn²⁺, Zn²⁺, La³⁺ and Ce³⁺.