Mechanism of the stimulation of calcium ion dependent ATPase of cardiac sarcoplasmic reticulum by adenosine 3',5'-monophosphate dependent protein kinase

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Abstract
Canine cardiac sarcoplasmic reticulum (SR) is known to be phosphorylated by cAMP-dependent protein kinase on a 22,000 dalton protein. Phosphorylation enhances the initial rate of Ca2+ uptake and Ca2+-ATPase activity. To determine the molecular mechanism by which phosphorylation regulates the Ca2+ pump in SR, the effect of cAMP-dependent protein kinase on the individual steps of the Ca2+-ATPase reaction sequence was examined. Cardiac SR was preincubated with cAMP and cAMP-dependent protein kinase in the presence (phosphorylated SR) and absence (control) of ATP. Control and phosphorylated SR were subsequently assayed for formation (4-200 ms) and decomposition (0-73 ms) of the acid-stable phosphorylated enzyme (E .apprx. P) of Ca2+-ATPase in media containing 100 .mu.M [ATP] and various free [Ca2+]. cAMP-dependent phosphorylation of SR resulted in pronounced stimulation of initial rates and levels of E .apprx. P formed at low free [Ca2+] (.ltoreq. 7 .mu.M), but the effect was less at high free Ca2+ (.gtoreq. 10 .mu.M). This stimulation was associated with a decrease in the Kd for Ca2+ binding and a possible increase in Ca2+ sites. The observed rate constant for E .apprx. P formation of Ca2+-preincubated SR was not significantly altered by phosphorylation. Phosphorylation also increased the initial rate of E .apprx. P decomposition. Phosphorylation of cardiac SR by cAMP-dependent protein kinase apparently regulates several steps in the Ca2+-ATPase reaction sequence which result in an overall stimulation of the Ca2+ pump observed at steady state.

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