The calmodulin-activated form of the Ca2(+)-pumping ATPase of the cardiac sarcolemmal membrane produces Ca2+ gradients with a thermodynamic efficiency of 100%.
The thermodynamic efficiency of the calmodulin-activated form of the Ca2+-pumping ATPase of the bovine cardiac sarcolemma (SL) was evaluated in sealed vesicles under reversible conditions. The free internal Ca2+ concentration ([Ca2+]i) established in the SL vesicle lumen by action of the ATPase was determined as a function of the [ATP]/([ADP][Pi]) ratio for the following experimental conditions: 250 mM sucrose, 100 mM KCl, 0.1 mM Mg2+, 25 mM HEPES, 25 mM Tris, pH 7.40, at 37 degrees C, [Ca2+]o = 50 nM (1 mM Ca/EGTA buffer), 0.75 mM Mg-ATP, 0.1 mM Pi, variable [ADP]. Under these conditions, with the pump working near its Km of 64 nM, the [Ca2+]i achieved was less than or equal to 18 mM, decreasing with increasing [ADP] for [ADP] greater than or equal to 0.84 mM. A plot of the square of the [Ca2+]i/[Ca2+]o ratio against [ATP]/([ADP][Pi]) gave a straight line with a slope of 1.5 x 10(7) M. This was in agreement, within the experimental error, with the equilibrium constant for ATP hydrolysis under these conditions (1.09 x 10(7) M). These results demonstrate (1) tight coupling between Ca2+ transport and ATP hydrolysis with a stoichiometry of 2 Ca2+ moved per ATP split and (2) a low degree of passive leakage. Analysis at low [ADP] (less than 0.83 mM) showed the unexpected result that ADP increases the rate of the forward reaction of the pump. The maximal effect on the initial rate is a 96 +/- 5% increase, with an EC50 of approximately 0.4 mM (ADP). Similar but lesser stimulation was observed with CDP. The implications of the above results for the energetics of the pump and for its physiological function in the beating heart are discussed.
Dixon
D A
DA
Department of Pharmacology, University of Miami School of Medicine, Florida 33101.
Haynes
D H
DH
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000502:physiology*; D002147:Calmodulin / Q000502:physiology*; D002417:Cattle; D004789:Enzyme Activation / Q000502:physiology; D066298:In Vitro Techniques; D007700:Kinetics; D008954:Models, Biological; D009206:Myocardium / Q000378:metabolism*; D012508:Sarcolemma / Q000378:metabolism*; D013816:Thermodynamics
D002147:Calmodulin; D000252:Calcium-Transporting ATPases; D002118:Calcium