Rate constants for calmodulin binding to Ca 2+-ATPase in erythrocyte membranes

Abstract

The Ca 2+ -ATPase (ATP phosphohydrolase, EC 3.6.1.3) in human erythrocyte membranes, which is part of the Ca 2+ pump, can be activated by binding of calmodulin. Rate constants ( k 1) for association of calmodulin and enzyme, which depends on the Ca 2+ concentration, have been determined by the aid of an enzyme model. k 1 increased from 0.25 · 10 6 to 17.3 · 10 6 M −1 · min −1 (70 times) when the free Ca 2+ concentration was raised from 0.7 to 20 μM. The binding of calmodulin to the Ca 2+ -ATPase is reversible. The rate constants ( k −1) for dissociation of enzyme-calmodulin complex decreased from 6.0 to 0.044 min −1 (135 times) when the free Ca 2+ concentration was increased from 0.1 to 2–20 μM. The apparent dissociation constant K d = k −1 k 1 accordingly increased from 2.5 nM to 25 μM (or higher) when the Ca 2+ concentration was reduced from 20 to 0.1 μM. Therefore, at 10 −7 M free Ca 2+ most of the Ca 2+ -pump enzyme will not bind calmodulin. For the intact cell the time dependences of activation and deactivation of the Ca 2+ -pump enzyme have been estimated from the rate constants above. The results suggest that the Ca 2+ pump is well suited to maintain a cytosolic concentration of 10 −7 M free Ca 2+ (or lower) in the unstimulated cell and, when the cell is stimulated, to allow transient Ca 2+ signals up to approx. 10 −5 M in the cytosol.