Formation of Magnesium‐Phosphoenzyme and Magnesium‐Calcium‐Phosphoenzyme in the Phosphorylation of Adenosine Triphosphatase by Orthophosphate in Sarcoplasmic Reticulum

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Abstract
Simple reaction sequences which describe Ca-independent plus Ca-dependent phosphorylation of sarcoplasmic reticulum transport ATPase by orthophosphate including the function of Mg in phosphoenzyme formation were tested. The reaction schemes considered were based on the reaction sequence for Ca-independent phosphorylation proposed previously; namely that the transport enzyme (E) forms a ternary complex (Mg .cntdot. E .cntdot. Pi), by random binding of free Mg and free orthophosphate, which is in equilibrium with the Mg-phosphoenzyme (Mg .cntdot. E-P). Phosphorylation, performed at pH 7.0 20.degree. C and a constant free orthophosphate concentration using sarcoplasmic reticulum vesicles from rabbit skeletal muscle either unloaded or loaded passively with Ca in the presence of 5 mM or 40 mM CaCl2, resulted in a gradual decrease in the apparent Mg half-saturation constant and an increase in maximum phosphoprotein formation with increasing Ca loads. When phosphorylation of sarcoplasmic reticulum vesicles preloaded in the presence of 5 mM CaCl2 was performed at a constant free Mg concentration, a decrease in the apparent orthophosphate half-saturation constant and an increase in maximum phosphoprotein formation was observed as compared with vesicles from which Ca inside was removed by ionophore X-537A [lasalocid] plus EGTA [ethyleneglycol-bis(.beta.-aminoethyl ether)N,N,N'',N''-tetraacetic acid] treatment; both parameters remained unchanged by increasing free magnesium from 20 mM to 30 mM. When phosphorylation of sarcoplasmic reticulum vesicles passively loaded with Ca in the presence of 40 mM CaCl2, at which the saturation of the low-affinity Ca binding sites of the ATPase is presumably near maximum, was performed at increasing concentrations of free orthophosphate, there was a parallel shift of phosphoprotein formation as a function of free Mg and vice versa, with no change in the maximum phosphoenzyme formation. Comparison of the experimental data with the pattern of phosphoprotein formation predicted from model equations for various theoretical possible reaction sequences suggests that phosphoenzyme formation from orthophosphate possesses the following features. Ca present at the inside of the sarcoplasmic reticulum membrane binds to the free enzyme and in sequential order to E .cntdot. Mg .cntdot. Pi or Mg .cntdot. E-P or to both, but neither to E .cntdot. Mg nor to E .cntdot. Pi. Ca-independent and Ca-dependent phosphoproteins are Mg-phosphoenzymes. Ca-dependent phosphoenzyme is a Mg-Ca-enzyme phosphate complex with 1 Mg, 2 Ca and 1 orthophosphate (the last covalently) bound to the enzyme [Mg .cntdot. E-P .cntdot. (Cai)2], and not a Ca-phosphoprotein without bound Mg.
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