title: Mechanism of the stimulation of calcium ion dependent adenosine triphosphatase of cardiac sarcoplasmic reticulum by adenosine 3',5'-monophosphate dependent protein kinase.
abstract: Canine cardiac sarcoplasmic reticulum (SR) is known to be phosphorylated by adenosine 3',5'-monophosphate (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 calcium pump in SR, we examined the effect of cAMP-dependent protein kinase on the individual steps of the Ca2+-ATPase reaction sequence. Cardiac sarcoplasmic reticulum was preincubated with cAMP and cAMP-dependent protein kinse in the presence (phosphorylated SR) and absence (control) of adenosine 5'-triphosphate (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 approximately P) of Ca2+-ATPase in media containing 100 microM [ATP] and various free [Ca2+]. cAMP-dependent phosphorylation of SR resulted in pronounced stimulation of initial rates and levels of E approximately P formed at low free [Ca2+] (less than or equal to 7 microM), but the effect was less at high free Ca2+ (greater than or equal to 10 microM). This stimulation was associated with a decrease in the dissociation constant for Ca2+ binding and a possible increase in Ca2+ sites. The observed rate constant for E approximately P formation of calcium-preincubated SR was not significantly altered by phosphorylation. Phosphorylation also increased the initial rate of E approximately P decomposition. These findings indicate that phosphorylation of cardiac SR by cAMP-dependent protein kinase regulates several steps in the Ca2+-ATPase reaction sequence which result in an overall stimulation of the calcium pump observed at steady state.
mesh_terms: D000818:Animals; D000252:Calcium-Transporting ATPases / Q000378:metabolism*; D000242:Cyclic AMP / Q000502:physiology; D004285:Dogs; D004789:Enzyme Activation; D066298:In Vitro Techniques; D007700:Kinetics; D009206:Myocardium / Q000201:enzymology*; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D012519:Sarcoplasmic Reticulum / Q000201:enzymology
chemical_list: D000242:Cyclic AMP; D011494:Protein Kinases; D000252:Calcium-Transporting ATPases