Regulation of Ca2+-pumping ATPase of heart sarcolemma by a phosphorylation-dephosphorylation Process

The Ca2+-ATPase of dog heart sarcolemma (1, 2) is affected by phosphorylation. As normally prepared, sarcolemmal vesicles are phosphorylated to a high degree, resulting in a relatively low additional incorporation of hydroxylamine resistant [32P]phosphate from [gamma-32P]ATP. The 32P incorporation is increased up to 20-fold by pretreating the vesicles with phosphorylase phosphatase and is inhibited by an inhibitor of cAMP-dependent protein kinases. The phosphatase treatment inhibits markedly the Ca2+-ATPase and the ATP-dependent Ca2+ uptake. The inhibition is more evident at relatively higher levels of free Ca2+ and is reversed by preincubation with ATP. The Ca2+-pumping activity is stimulated markedly by phosphorylase b kinase and inhibited by the (cAMP-dependent) protein kinase inhibitor. Both the protein kinase inhibitor and ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid prevent the rephosphorylation of sarcolemmal vesicles, but the effects are not additive. The Ca2+ dependence curve of the Ca2+ uptake in phospho- and dephosphorylated vesicles suggests that the phosphorylation might affect the efficiency of the enzyme (turnover rate) rather than its affinity for Ca2+.