FIGURE 3.

Stabilization of E1PCa₂ formed by reverse conversion from E2P. Wild-type Ca²⁺-ATPase in microsomes was phosphorylated at 25 °C with 0.1 mm ³²P_i in a mixture containing 200 μg/ml microsomal protein, 50 mm MOPS/Tris (pH 7.3), 7 mm MgCl₂, 1 mm EGTA, 30 μm A23187, 7 mm MgCl₂, and 20% (v/v) Me₂SO that strongly favors E2P formation. The mixture was chilled at 4 °C and then, at zero time, diluted with a 19-fold volume of a solution containing 50 mm HEPES/Tris (pH 8.0), 0.105 m KCl, 7 mm MgCl₂, and 10.5 mm CaCl₂ (open and closed circles) or 1 mm EGTA (open triangles) in the absence (open symbols) or presence (closed circles) of 10.5 mm ADP, and the amount of EP was determined at the indicated times.