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Phosphorylation of the calcium‐transport ATPase of skeletal muscle sarcoplasmic reticulum by inorganic phosphate was investigated in the presence or absence of a calcium gradient. The maximum phosphoprotein formation in the presence of a calcium gradient at 20 °C and pH 7.0 is approximately 4 nmol/mg sarcoplasmic reticulum protein, but only between 2.4 and 2.8 nmol/mg protein in the absence of a calcium gradient, using lonophore X‐537 A or phospholipase‐A‐treated sarcoplasmic reticulum vesicles. Maximum phosphoprotein formation independent of calcium gradient at 20 °C and pH 6.2 is in the range of 3.6–4 nmol/mg protein. Half‐maximum phosphoprotein formation dependent on calcium gradient was achieved with 0.1–0.2 mM free orthophosphate at 10 mM free magnesium or at 0.1–0.2 mM free magnesium at 10 mM free orthophosphate. Phosphoprotein formation independent of calcium gradient is in accordance with a model which assumes, firstly, the formation of a ternary complex of the ATPase protein with orthophosphate and magnesium (E · P i · Mg) in equilibrium with the phosphoprotein (E‐P i · Mg) and, secondly, and interdependence of both ions in the formation of the ternary complex. The apparent equilibrium constant was 0.6 and the apparent dissociation constants K Mg , K Mg′ , K Pi and K Pi′ were 8.8, 1.9, 7.2 and 1.5 mM respectively, assuming a total concentration of the phosphorylation site per enzyme of 7 nmol/mg protein.
Punzengruber et al. (Fri,) studied this question.