Studies of the Ca²⁺ transport mechanism of human erythrocyte inside-out plasma membrane vesicles. I. Regulation of the Ca²⁺ pump by calmodulin

Calcium accumulation by human erythrocyte inside-out vesicles was linear for at least 30 min in the presence of ATP. In untreated inside-out vesicles, 3.76 ± 1.44 nmol of calcium/min/unit of acetylcholinesterase were transported, compared with 10.57 ± 2.05 (± S.D.; n = 11) in those treated with calmodulin. The amount of calmodulin necessary for 50% activation of Ca²⁺ accumulation was 60 ± 22 ng/ml (± S.D.; n = 4). The K(m) (Ca²⁺) for calmodulin-stimulated accumulation was 0.8 ± 0.05 μM (± S.D.; n = 5) using Ca²⁺/ethylene glycol bis(β-aminoethyl ether) N,N,N',N'-tetraacetic acid (EGTA) buffers, or 25 μM with direct addition of unbuffered calcium. In the absence of calmodulin, these values were 0.4 and 60 μM, respectively. K(m) ATP) values of 90 and 60 μM in the presence and absence of calmodulin, respectively, were measured at constant magnesium concentration (3 mM). In the presence of calmodulin, a broad pH profile is exhibited from pH 6.6 to 8.2. Maximal calcium accumulation occurs at pH 7.8. In the absence of calmodulin, the pH profile exhibits a linear upward increase from pH 7.0 to 8.2 The (Ca²⁺-Mg²⁺)-ATPase activity, measured under identical conditions, was 2.40 ± 0.72 nmol of P(i)/min/unit of acetylcholinesterase in the untreated vesicles and 11.29 ± 2.87 nmol of P(i)/min/unit of acetylcholinesterase (± S.D.; n= 4) in calmodulin-treated vesicles. A stoichiometry of 1.6 Ca²⁺/ATP hydrolyzed was determined in the absence of calmodulin; in the presence of calmodulin, this ratio was decreased to 0.94 Ca²⁺/ATP hydrolyzed.