FIGURE 2.

Time course of EP formation and its decay in the wild type. A, microsomes expressing wild-type Ca²⁺-ATPase prepared from COS-1 cells were incubated with 10 μm Ca²⁺ in a mixture containing 20 μg/ml microsomal protein, 20 mm MOPS/Tris (pH 7.3), 0.1 m KCl, 7 mm MgCl₂, 10 μm CaCl₂, and 3 μm A23187 at 4 °C. Then EP formation was initiated at zero time by mixing with an equal volume of a solution containing 20 μm [γ-³²P]ATP, 0.1 m HEPES/Tris (pH 8.0), 0.1 m KCl, and 7 mm MgCl₂ with 10 mm CaCl₂ (closed circles) or 2 mm EGTA (open circles). B, EP was formed in 20 mm MOPS/Tris (pH 7.3), 0.1 m KCl, 7 mm MgCl₂, 10 μm CaCl₂, 3 μm A23187, and 10 μm [γ-³²P]ATP at 4 °C for 10 s. Then the reaction was chased at zero time by mixing with an equal volume of a solution containing non-radioactive 0.2 mm ATP and various concentrations of CaCl₂ to give the indicated final Ca²⁺ concentrations (0.01, 1, and 10 mm) or 2 mm EGTA to remove free Ca²⁺ in 0.1 m HEPES/Tris (pH 8.0), 0.1 m KCl, 7 mm MgCl₂, and 3 μm A23187. The amount of EP was normalized to the value at zero time of the chase.