| Abstract: | The aim of the present study was to test simple reaction sequences which describe calcium-independent plus calcium-dependent phosphorylation of sarcoplasmic reticulum transport. ATPase by orthophosphate including the function of magnesium in phosphoenzyme formation. The reaction schemes considered were based on the reaction sequence for calcium-independent phosphorylation proposed previously; namely that the transport enzyme (E) forms a ternary complex (Mg . E . Pi), by random binding of free magnesium and free orthophosphate, which is in equilibrium with the magnesium-phosphoenzyme (Mg . E-P). Phosphorylation, performed at pH 7.0 20 degrees C and a constant free orthophosphate concentration using sarcoplasmic reticulum vesicles either unloaded or loaded passively with calcium in the presence of 5 mM or 40 mM CaCl2, resulted in a gradual decrease in the apparent magnesium half-saturation constant and an increase in maximum phosphoprotein formation with increasing calcium loads. When phosphorylation of sarcoplasmic reticulum vesicles preloaded in the presence of 5 mM CaCl2 was performed at a constant free magnesium 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 calcium inside has been removed by ionophore X-537A plus EGTA treatment; however, both parameters remained unchanged by increasing free magnesium from 20 mM to 30 mM. When phosphorylation of sarcoplasmic reticulum vesicles passively loaded with calcium in the presence of 40 mM CaCl2, at which the saturation of the low-affinity calcium 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 magnesium 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. Firstly, calcium present at the inside of the sarcoplasmic reticulum membrane binds to the free enzyme and in sequential order to E . Mg . Pi or Mg . E-P or to both, but neither to E. Mg nor to E . Pi. Secondly, calcium-independent and calcium-dependent phosphoproteins are magnesium-phosphoenzymes. Calcium-dependent phosphoenzyme is a magnesium-calcium-enzyme phosphate complex with 1 magnesium, 2 calciums and 1 orthophosphate (the last covalently) bound to the enzyme Mg . E-P . (Cai)2], and not a 'calcium-phosphoprotein' without bound magnesium. |
