FIGURE 2.

Ca²⁺ dependence of accumulation of ADP-insensitive EP in the steady state in mutant Y122A. Microsomes expressing the mutant Y122A were phosphorylated with [γ-³²P]ATP at various Ca²⁺ concentrations and pHs as indicated at 0 °C for 5 min in a mixture containing 20 μg/ml microsomal protein, 50 mm MOPS/Tris, 0.1 m KCl, 7 mm MgCl₂, 0.01 mm EGTA, 3 μm A23187, 10 μm [γ-³²P]ATP, and various concentrations of CaCl₂. For the determination of the accumulated ADP-insensitive EP, an equal volume (50 μl) of a mixture containing 10 mm ADP, 7 mm MgCl₂, 10 mm EGTA, 50 mm MOPS/Tris (pH 6.8, 7.3, or 7.8 as indicated), and 0.1 m KCl was added to the above phosphorylation mixture. At 1 s after this addition, the reaction was quenched with trichloroacetic acid. ADP-sensitive EP disappeared entirely within 1 s after the addition of ADP. The total amounts of EP were nearly the same under all the conditions (data not shown). The amount of ADP-insensitive EP is shown as a percentage of the total amount of EP. Solid lines show the least squares fit to the Hill equation. Apparent Ca²⁺ affinities and Hill coefficients thus obtained were 360 μm and 2.1 (pH 6.8), 160 μm and 1.9 (pH 7.3), and 76 μm and 2.0 (pH 7.8).