Phosphorylation of the Calcium‐Transport Adenosine Triphosphatase of Cardiac Sarcoplasmic Reticulum by Orthophosphate

Membranes of sacroplasmic reticulum prepared from dog hearts are phosphorylated by [ 32 P]‐orthophosphate in the presence of a calcium load and magnesium. The [ 32 P]phosphate incorporation into sarcoplasmic reticulum follows Michaelis‐Menten kinetics with an apparent Michaelis Constant of 0.5 mM for orthophosphate (pH 7.0). The phosphorylation is 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 [ 32 P]orthophosphate is strongly inhibited by calcium in the medium. Half‐maximum inhibition occurs at an ionized calcium concentration of about 0.8 μM. The phosphoprotein steady‐state level is reduced by 85–90% by phospholipase‐A treatment or solubilization of calcium‐preloaded sarcoplasmic reticulum with Triton X‐100. The phosphoprotein of sarcoplasmic reticulum formed from [ 32 P]orthophosphate is dephosphorylated by ADP with resultant in ATP synthesis. Phosphoprotein formation and ATP synthesis by sarcoplasmic reticulum are unaltered by azide, dinitrophenol, dicyclohexyl carbodiimide and oligomycin. The phosphoprotein is acid‐stable. The trichloroacetic‐acid‐denaturated 32 P‐labelled membrane complex is dephosphorylated by hydroxylamine, indicating that the phosphorylated protein is an acylphosphate. Polyacrylamide gel electrophoresis (performed with phenol/acetic acid/water) of sarcoplasmic reticulum phosphorylated by [ 32 P]orthophosphate demonstrates that the phosphate incorporation occurs into a protein with a molecular weight of about 100 000, as in the case of phosphoprotein formation from ATP. The data suggest that the phosphoprotein of the calcium‐dependent ATPase formed from inorganic phosphate represents a high‐energy intermediate of the reverse reaction of the calcium pump of cardiac sarcoplasmic reticulum.