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The effects of an adenosine 3': 5'-monophosphate (cyclic AMP) -dependent protein kinase on Ca2+-activated adenosine triphosphatase (ATPase) activity and calcium uptake of canine cardiac microsomes were examined. The cardiac microsomes, which represent an enriched preparation of fragmented sarcoplasmic reticulum, were preincubated with varying concentrations of cyclic AMP-dependent protein kinase or cyclic AMP or both in the presence of Mg2+ and ATP. Ca2+-activated ATPase activity and calcium uptake were determined in the presence of oxalate and various concentrations of Ca2+. Ionized Ca2+ concentrations were maintained with Ca2+ buffers containing CaC and ethylene glycol bis (P-aminoethyl ether) -N, N'-tetraacetic acid. The initial rates of both Ca"+-activated ATPase and calcium uptake were increased 2 to 3 times following lo-min preincubation with protein kinase and 1 pM cyclic AMP. Pretreatment with protein kinase in the absence of cyclic AMP caused a lesser degree of stimulation, whereas pretreatment with cyclic AMP alone had no detectable effect. Stimulation of the Ca2+-activated ATPase and calcium uptake by protein kinase was dependent on cyclic AMP concentration: maximal stimulation was seen at approximately 10e6 M with apparent half-maximal stimulation at approximately 1OV M. Marked stimulation of both Ca2+-activated A'IPase activity and calcium uptake by cyclic AMP and protein kinase was seen at a Ca2+ concentration of approximately 1 pM. The stoichiometric coupling of Ca2+ -activated ATPase and calcium uptake was maintained at 2 moles of calcium taken up per mole of ATP hydrolyzed, following stimulation by protein kinase and cyclic AMP. The stimulatory effects on Ca2+-activated
Tada et al. (Tue,) studied this question.