Regulation of the nucleotide dependence of the cardiac sarcolemma Ca(2+)-ATPase.
The nucleotide dependence of the Ca(2+)-ATPase purified from cardiac sarcolemma by calmodulin-affinity chromatography was investigated for preparations either in the basal state or activated by three procedures: (i) addition of calmodulin; (ii) addition of phosphatidylserine and (iii) controlled proteolysis. Upon activation, the maximal velocity of ATP hydrolysis increases by a factor of 4-5, while the curves of ATP dependence of ATP hydrolysis change from hyperbolic to biphasic, revealing the presence of two Kmapp for ATP. A tight coupling between Ca2+ and ATP binding sites was also observed. At high ATP concentration, the ATPase activity of the basal state shows a complex dependence on Ca2+ concentration, increasing sharply at millimolar Ca2+. Our results indicate that this increase in ATPase activity is paralleled by the appearance of a second, low affinity Kmapp for ATP. When only the high affinity site for ATP is occupied the ATPase activity of the basal state displays a simple, hyperbolic dependence on the Ca2+ concentration. In addition, increasing Ca2+ concentration appears to decrease the ATP binding at the low affinity site of the enzyme. The effect of ADP on ATP hydrolysis was also examined. The finding that ADP is a potent inhibitor of the purified Ca(2+)-ATPase from heart suggests that the stimulatory action of ADP observed in cardiac sarcolemmal vesicles is not an intrinsic property of the enzyme.
Pasa
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TC
Departamento de Bioquímica, ICB, C.C.S. Universidade Federal do Rio de Janeiro, Brasil.
Otero
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AS
Barrabin
H
H
Scofano
H M
HM
D000244:Adenosine Diphosphate / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000378:metabolism / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000378:metabolism; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D002417:Cattle; D006868:Hydrolysis; D066298:In Vitro Techniques; D007700:Kinetics; D009206:Myocardium / Q000201:enzymology*; D012508:Sarcolemma / Q000201:enzymology
D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D000252:Calcium-Transporting ATPases; D002118:Calcium