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Phosphorylation of the calcium transport atpase of cardiac sarcoplasmic reticulum by ortho phosphate



Phosphorylation of the calcium transport atpase of cardiac sarcoplasmic reticulum by ortho phosphate



European Journal of Biochemistry 77(3): 611-620



Membranes of sarcoplasmic reticulum prepared from dog hearts were phosphorylated by [32P]-Pi in the presence of a Ca load and Mg. The [32P]phosphate incorporation into sarcoplasmic reticulum followed Michaelis-Menten kinetics with an apparent Km of 0.5 mM for Pi (pH 7.0). The phosphorylation was pH-dependent, with an optimum pH of 6.0-6.2 (maximum phosphoprotein steady state level 0.6-0.8 nmol/mg protein). The phosphorylation of sarcoplasmic reticulum by [32P]-Pi was strongly inhibited by Ca in the medium. Half-maximum inhibition occurred at an Ca2+ concentration of about 0.8 .mu.M. The phosphoprotein steady-state level was reduced by 85-90% by phospholipase-A treatment or solubilization of Ca-preloaded sarcoplasmic reticulum with Triton X-100. The phosphoprotein of sarcoplasmic reticulum formed from [32P]-Pi was dephosphorylated by ADP with resultant in ATP synthesis. Phosphoprotein formation and ATP synthesis by sarcoplasmic reticulum were unaltered by azide, dinitrophenol, dicyclohexyl carbodiimide and oligomycin. The phosphoprotein was acid-stable. The trichloroacetic-acid-denatured 32P-labeled membrane complex was dephosphorylated by hydroxylamine, indicating that the phosphorylated protein was an acylphosphate. Polyacrylamide gel electrophoresis (performed with phenol/acetic acid/water) of sarcoplasmic reticulum phosphorylated by [32P]-Pi demonstrated that the phosphate incorporation occurred into a protein with a MW of about 100,000, as in the case of phosphoprotein formation from ATP. The data suggested that the phosphoprotein of the Ca-dependent ATPase formed from Pi represented a high-energy intermediate of the reverse reaction of the Ca pump of cardiac sarcoplasmic reticulum.

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