Reaction Mechanism of the Ca++-Dependent ATPase of Sarcoplasmic Reticulum from Skeletal Muscle*

We have previously reported the dependencies on the concentration of Ca++ and ATP of the Ca++-dependent ATPase EC 3.6.1.3 activity of sarcoplasmic reticulum from rabbit skeletal muscle. We have also observed the 82P-incorporation into the enzyme, when it was incubated with γ-82P-ATP in the presence of Ca++. The P incorporation increased with increase in the concentration of Ca++. Furthermore, the rate of Ca++-dependent ATPase activity was proportional to the amount of P-incorporated. In the present work, we studied the mechanism of the P-incorporation and obtained the following results. 1. The amount of P incorporated increased with increase in pH and reached a maximum above pH 8.3. The pH-activity curve of the Ca++-dependent ATPase was bell-shaped with a maximum at pH 7.0. The pH dependence of the ratio of the ATPase activity to the amount of 82P-incorporation indicated the participation of a functional group with a pK value of about 7.2 in the decomposition of the enzyme-phosphate complex. 2. When 2mM EGTA was added after formation of the enzyme-phosphate complex, the amount of the complex decreased rapidly. The rate of its decomposition decreased with increase in pH value. The life-time of the complex measured in this way agreed well with the value obtained kinetically, i.e., from the ratio of the ATPase activity to the amount of the complex. 3. When 2 mM of cold ATP were added after the formation of the enzyme-82P complex, the amount of 82P incorporated decreased with time. The rate of its decrease was 3 fold that observed after the addition of EGTA. 4. The rate of decomposition of the enzyme-phosphate complex was found to be unaffected by removal of magnesium ion which was required for the formation of the enzyme-phosphate complex. 5. The enzyme-phosphate complex isolated by TCA-treatment was stable in acidic medium but unstable in alkaline medium. The complex was very unstable in the presence of 0.5 M hydroxylamine.