FIGURE 4.

Ca²⁺ concentration dependence of Ca²⁺ binding in non-phosphorylated Ca²⁺-ATPase from COS-1 cells. A and B, microsomes expressing the wild type (A) or mutant L119A (B) were incubated at 4 °C for 20 s with various concentrations of ⁴⁵Ca²⁺ in a mixture containing 20 μg/ml microsomal protein, 50 mm MOPS/Tris (pH 7.3), 0.1 m KCl, 7 mm MgCl₂, 3 μm A23187, and 10 μm ⁴⁵CaCl₂ with 0–0.09 mm EGTA to give the indicated free Ca²⁺ concentration in the presence (open triangles) or absence (open circles) of 1 μm TG. Then, as described under “Experimental Procedures,” 50 μl of the mixture was spotted on a membrane filter and washed immediately for 3 s with 1 ml of a Ca²⁺ binding assay medium containing 10 mm CaCl₂ and 0.1 mm ATP in 50 mm HEPES/Tris (pH 8.0), 0.1 m KCl, and 7 mm MgCl₂. The values presented are the mean ± S.D. (n = 3–4). The amount of Ca²⁺ specifically bound to the Ca²⁺-ATPase (trapped as occluded Ca²⁺ in E1PCa₂, closed circles) was obtained by subtracting the amount of nonspecific Ca²⁺ binding determined in the presence of TG. The solid lines show the least squares fit to the Hill equation with the fitting parameters K_d and Hill coefficient of 0.22 μm and 1.6 for the wild type and 0.29 μm and 1.6 for the mutant L119A. C, the maximum amount of specific ⁴⁵Ca²⁺ binding in 10 μm ⁴⁵Ca²⁺ was determined as above (Ca) and compared with the maximum amount of EP (EP) determined in the presence of 10 mm Ca²⁺ as in Fig. 2A (i.e. essentially under the same conditions as described for the determination of the catalytic site content in the preparation (45)). It should be mentioned here that, for the comparison, one preparation of microsomes was used throughout for the wild type and the mutant. Actual values of the amount of bound Ca²⁺ and that of EP (pmol/mg of microsomal protein (n = 4)) for the wild type were 287 ± 14 and 136 ± 8, respectively, giving a stoichiometry of Ca/EP = 2.11. Those for the L119A mutant were 95.6 ± 16.2 and 48.6 ± 2.6, respectively, providing a stoichiometry of Ca/EP = 1.97. Note that these maximum Ca²⁺ binding values are slightly different from those in A and B, in which different preparations were used.