Changes in Affinity for Calcium Ions with the Formation of Two Kinds of Phosphoenzyme in the Ca2+, Mg2+-Dependent ATPase of Sarcoplasmic Reticulum1

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Abstract
The amount of Ca 2+ bound to the Ca 2+ , Mg 2+ -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 Ca 2+ bound cooperatively to 1 mol of active site with high affinity and were removed rapidly by addition of EGTA. AMPPNP did not affect the Ca 2+ binding to the ATPase in the presence of MgCl 2 . Under the conditions where most EP was ADP sensitive at steady state (58 μM Ca 2+ , 50 μM EGTA, and 20 mM M g Cl 2 at pH 7.0 and 0°C), bound Ca 2+ 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 45 Ca 2+ on addition of unlabeled Ca 2+ + EGTA was biphasic, and 70% of bound 45 Ca 2+ 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 Ca 2+ , 30 μm EGTA, and 20 mM M g Cl 2 at pH 8.8 and 0°C), the amount of bound Ca 45 Ca 2+ 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 Ca 45 Ca 2+ , 25 μm EGTA, and 1 mM M g Cl 2 at pH 8.8 and 0°C), the amount of bound Ca 45 Ca 2+ did not change upon addition of ATP. These findings suggest that the Ca 2+ bound to the enzyme becomes unremovable by EGTA upon formation of ADP-sensitive EP and is released upon its conversion to ADP-insensitive EP.