Phosphorylation by ATP of sarcoplasmic reticulum ATPase in the absence of calcium.
(Ca2+ + Mg2+)-dependent ATPase purified from sarcoplasmic reticulum was phosphorylated by [gamma-32P]ATP in the presence of ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid, dimethyl sulfoxide, and magnesium up to levels of 1.0 mumol of phosphoenzyme/g of protein. In the presence of 30% (v/v) dimethyl sulfoxide, phosphorylation by ATP measured in the absence of calcium was abolished by 100 mM NaCl or KCl. Optimal pH for phosphoenzyme formation was 5.5 to 6.0. The phosphoenzyme levels were not significantly affected by varying the temperature of the reaction medium in the range from 0 to 20 degrees C. Increasing dimethyl sulfoxide concentrations lead to a progressive increase of the steady state level of phosphoenzyme and to a decrease of both the velocities of formation and breakdown of phosphoenzyme. The observed decrease of phosphoenzyme hydrolysis was greater than that of formation, accounting for the higher phosphoenzyme levels observed with increasing dimethyl sulfoxide concentrations. Phosphoenzyme formed from ATP in the absence of calcium is an acylphosphate-type compound, as demonstrated by its pH and hydroxylamine sensitivity. The phosphate radioactivity was incorporated into the (Ca2+ + Mg2+)-ATPase as shown by polyacrylamide gel electrophoresis. Controls were performed that eliminate both the possibility of calcium bound to the enzyme and the possibility of enzyme phosphorylation by contaminant 32Pi.
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D002118:Calcium / Q000494:pharmacology*; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009994:Osmolar Concentration; D010766:Phosphorylation; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D013696:Temperature
D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D002118:Calcium; D004121:Dimethyl Sulfoxide