FIGURE 7.

E2P hydrolysis and luminal Ca²⁺-induced inhibition. A, microsomes expressing wild type or mutant were phosphorylated with ³²P_i at 25 °C for 10 min in 5 μl of a mixture containing 1–5 μg of microsomal protein, 0.1 mm ³²P_i, 1 μm A23187, 0.2 mm EGTA, 7 mm MgCl₂, 50 mm MOPS/Tris (pH 7.0), and 35% (v/v) Me₂SO. The mixture was then cooled and diluted at 0 °C by the addition of 95 μl of a mixture containing 2.1 mm non-radioactive P_i, 105 mm KCl, 7 mm MgCl₂, 50 mm MOPS/Tris (pH 7.0), and 3 mm EGTA (open bar) or 3.2 mm (gray bar) and 21.1 mm (closed bar) CaCl₂ in place of EGTA, and E2P hydrolysis was followed (see typical examples with wild type and 5Gi-106/107 in the inset). The amounts of E2P formed with ³²P_i at zero time are normalized to 100%. Solid lines show the least squares fit to a single exponential, and the rates thus obtained are shown in the main panel. In the absence of Ca²⁺, statistical significance of the mutant rate compared with the wild-type rate is shown above an open bar: *, p < 0.05; ‡, p < 0.01. In each of the mutants and the wild type, statistical significance of the rate in 3 and 20 mm Ca²⁺ compared with that in the absence of Ca²⁺ is shown above a gray bar and closed bar: V, p < 0.05; ↓, p < 0.01. B, the marked retardation and acceleration in the absence of Ca²⁺ (open bars in A) are visualized with α-carbon coloring, red and purple, respectively. Error bars, S.D.