Changes in Affinity for Calcium Ions with the Formation of Two Kinds of Phosphoenzyme in the Ca²⁺, Mg²⁺-Dependent ATPase of Sarcoplasmic Reticulum

Abstract

The amount of Ca²⁺ bound to the Ca²⁺, Mg²⁺-dependent ATPase of deoxycholic acid-treated sarcoplasmic reticulum was measured during ATP hydrolysis by the double-membrane filtration method [Yamaguchi, M. & Tonomura, Y. (1979), J. Biochem. 86, 509-523]. The maximal amount of phosphorylated intermediate (EP) was adopted as the amount of active site of the ATPase.
In the absence of ATP, 2 mol of Ca²⁺ bound cooperatively to 1 mol of active site with high affinity and were removed rapidly by addition of EGTA. AMPPNP did not affect the Ca²⁺ binding to the ATPase in the presence of MgCl₂.
Under the conditions where most EP was ADP sensitive at steady state (58 μM Ca²⁺, 50 μM EGTA, and 20mM MgCl₂ at pH 7.0 and 0°C), bound Ca²⁺ increased by 0.6-0.7 mol per mol active site upon addition of ATP. The time course of decrease in the amount of bound ⁴⁵Ca²⁺ on addition of unlabeled Ca²⁺+EGTA was biphasic, and 70% of bound ⁴⁵Ca²⁺ was slowly displaced with a rate constant similar to that of EP decomposition. Similar results were obtained for the enzyme treated with N-ethylmaleimide, which inhibits the step of conversion of ADP-sensitive EP to the ADP-insensitive one.
Under the conditions where most EP was ADP insensitive at steady state (58 μM Ca²⁺, 30 μM EGTA, and 20mM MgCl₂ at pH 8.8 and 0°C), the amount of bound Ca²⁺ increased slightly, then decreased slowly by 1 mol per mol of EP formed after addition of ATP. Under the conditions where about a half of EP was ADP sensitive (58 μM Ca²⁺, 25 μM EGTA, and 1mM MgCl₂, at pH 8.8 and 0°C), the amount of bound Ca²⁺ did not change upon addition of ATP.
These findings suggest that the Ca²⁺ bound to the enzyme becomes unremovable by EGTA upon formation of ADP-sensitive EP and is released upon its conversion to ADP-insensitive EP.