Calcium gradient-dependent and calcium gradient-independent phosphorylation of sarcoplasmic reticulum by orthophosphate. The role of magnesium.
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 degrees 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 Ionophore X-537 A or phospholipase-A-treated sarcoplasmic reticulum vesicles. Maximum phosphoprotein formation independent of calcium gradient at 20 degrees 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 . Pi . Mg) in equilibrium with the phosphoprotein (E-Pi . Mg) and, secondly, an interdependence of both ions in the formation of the ternary complex. The apparent equilibrium constant was 0.6 and the apparent dissociation constants KMg, KMg', KPi and KPi' 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.
D000818:Animals; D002118:Calcium / Q000494:pharmacology*; D000252:Calcium-Transporting ATPases / Q000378:metabolism; D004533:Egtazic Acid / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007832:Lasalocid / Q000494:pharmacology; D008274:Magnesium / Q000494:pharmacology*; D010710:Phosphates / Q000378:metabolism*; D010740:Phospholipases / Q000494:pharmacology; D010766:Phosphorylation; D011189:Potassium Chloride / Q000494:pharmacology; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000378:metabolism*; D013757:Tetraethylammonium Compounds / Q000494:pharmacology
D010710:Phosphates; D013757:Tetraethylammonium Compounds; D004533:Egtazic Acid; D011189:Potassium Chloride; D010740:Phospholipases; D000252:Calcium-Transporting ATPases; D008274:Magnesium; D002118:Calcium; D007832:Lasalocid