The ADP- and Mg 2+-reactive calcium complex of the phosphoenzyme in skeletal sarcoplasmic reticulum Ca 2+-ATPase

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The effects of ATP on Ca 2+ binding in the absence of added Mg 2+ to the purified sarcoplasmic reticulum Ca 2+-ATPase were studied at pH 7.0 and 0°C. ATP increased the number of Ca 2+-binding sites of the enzyme from 2 to 3 mol per mol of phosphorylatable enzyme. The association constant for the ATP-induced Ca 2+ binding was 4·10 5 M −1, which was not significantly different from that obtained in the absence of ATP. Ado P[CH 2] PP has little effect on the Ca 2+-binding process. The amount of phosphoenzyme formed was equivalent to the level of ATP-induced Ca 2+ binding. ADP decreased the level of ATP-induced Ca 2+ binding and phosphoenzyme by the same amount. These results suggest that ATP-induced Ca 2+ binding exists in the form of an ADP-reactive phosphoenzyme·Ca complex. In addition, the Ca 2+ bound to the enzyme in the presence of ATP was released on the addition of 1 mM MgCl 2; after the release of Ca 2+, the phosphoenzyme decayed. These observations suggest that Mg 2+, added after the ATP-induced Ca 2+-binding process, may replace the Ca 2+ on the phosphoenzyme and initiate phosphoenzyme decomposition.