FIGURE 9.

Ca²⁺ affinity determined with EP formation. The microsomes expressing wild type or mutant were preincubated for 20 min at 25 °C in 45 μl of a mixture containing 1–5 μg of microsomal protein, 2 mm EGTA, 1 μm A23187, 0.1 m KCl, 7 mm MgCl₂, 50 mm MOPS/Tris (pH 7.0), and various concentrations of CaCl₂ to give the desired Ca²⁺ concentrations. After cooling, the Ca²⁺-ATPase was phosphorylated with 10 μm [γ-³²P]ATP for 15 s at 0 °C, and the amount of EP formed was determined. In the inset, the typical Ca²⁺ dependence of EP formation was shown for the wild type and the two mutants including 5Gi-110/111, which gave the highest K_d value. The K_d value and Hill coefficient were estimated by least squares fit to the Hill equation (solid line). The Hill coefficient obtained was ∼2 (actually 1.6–2) in the wild type and all of the mutants. Statistical significance compared with the wild type is shown: *, p < 0.05; ‡, p < 0.01. Error bars, S.D.