Transient state kinetic studies of Ca2+-dependent ATPase and calcium transport by cardiac sarcoplasmic reticulum. Effect of cyclic AMP-dependent protein kinase-catalyzed phosphorylation of phospholamban.

Phosphorylation of the 22,000-dalton protein phospholamban of cardiac microsomes (sarcoplasmic reticulum) by CAMP-dependent protein kinase was shown to alter profoundly the intermediary steps of microsomal Ca'+-dependent ATPase.To define the control of ATPase by its putative regulator phospholamban, we examined transient kinetics of the formation of the phosphorylated intermediate EP of ATPase and calcium binding by the membrane through a rapid quenching device, after microsomes were incubated with and without CAMP-dependent protein kinase.When microsomes were suspended with ethylene glycol bis( 8-aminoethyl ether)NJV"tetraacetic acid (EGTA) prior to the assay (Ca'+-free microsomes), the initial rates of EP formation and calcium binding were markedly enhanced in phosphorylated microsomes, whereas those incubated with calcium/EGTA buffer (Ca2+-bound microsomes) exhibited less pronounced enhancement.In Ca'+-free microsomes, these effects were evident within a wide range of ionized Ca2+ between 0.1 and 20 pM.Under these conditions, the initial rates and maximal amounts of EP formation were enhanced significantly by microsomal phosphorylation above 10 p~ ATP, while those were reduced at lower ATP (1 to 6 p~).Latter effects were attributed to the predominance of a protein kinase-induced increase in EP decomposition at lower ATP ranges, where the EP formation is ratedetermining.These results indicate that CAMP-dependent phosphorylation of phospholamban produces marked increases in the rate of a step associated with calcium binding to the ATPase enzyme and the rate at which EP is subsequently formed, resulting in an increased rate at which calcium is translocated across the microsomal membrane.Together with our previous report (Tada, M.,