FIGURE 7.

Lumenal Ca²⁺- and ADP-induced reverse E2P decay of Y122A in the presence of K⁺. A, microsomes (100 μg/ml) expressing the mutant Y122A were phosphorylated with ³²P_i for 10 min at room temperature in the absence of Ca²⁺ in a medium containing 50 mm MOPS/Tris (pH 7.3), 7 mm MgCl₂, 1 mm EGTA, 15 μm A23187, 0.1 mm ³²P_i, and 20% Me₂SO (that favors extremely the E2P formation (38)), and then the reaction mixture was chilled on ice. Subsequently, the phosphorylated sample was diluted at 0 °C with a 20-fold volume of a chase solution containing 50 mm MOPS/Tris (pH 7.3), 105 mm KCl, 7 mm MgCl₂, 1 mm EGTA, 0.1 mm non-radioactive P_i, and 0.105 mm ADP without or with various concentrations of CaCl₂ to give the final free Ca²⁺ concentrations as indicated. At the indicated periods, the chase reaction was terminated by trichloroacetic acid and the amount of EP was determined. Solid lines show the least squares fit to a single exponential decay. The decay rates thus obtained were plotted versus the Ca²⁺ concentration in panel B.