The ADP- and Mg2+-reactive calcium complex of the phosphoenzyme in skeletal sarcoplasmic reticulum Ca2+-ATPase

The effects of ATP on Ca2+ binding in the absence of added Mg2+ to the purified sarcoplasmic reticulum Ca2+-ATPase were studied at pH 7.0 and 0 degrees C. ATP increased the number of Ca2+-binding sites of the enzyme from 2 to 3 mol per mol of phosphorylatable enzyme. The association constant for the ATP-induced Ca2+ binding was 4 X 10(5) M-1, which was not significantly different from that obtained in the absence of ATP. AdoP[CH2]PP had little effect on the Ca2+-binding process. The amount of phosphoenzyme formed was equivalent to the level of ATP-induced Ca2+ binding. ADP decreased the level of ATP-induced Ca2+ binding and phosphoenzyme by the same amount. These results suggest that ATP-induced Ca2+ binding exists in the form of an ADP-reactive phosphoenzyme . Ca complex. In addition, the Ca2+ bound to the enzyme in the presence of ATP was released on the addition of 1 mM MgCl2; after the release of Ca2+, the phosphoenzyme decayed. These observations suggest that Mg2+, added after the ATP-induced Ca2+-binding process, may replace the Ca2+ on the phosphoenzyme and initiate phosphoenzyme decomposition.