FIGURE 5.

Orthovanadate or fluoride compound inhibition of phosphorylation (from [γ-³²P]ATP) of Drs2p-Bad in P3 membranes. D⁺ P3 membranes were incubated at 0.5 mg of protein/ml in buffer A supplemented with 0.1 mm CaCl₂, and phosphorylation was subsequently measured 25 s after addition of 0.5 μm [γ-³²P]ATP (on ice). A and B, incubation lasted 1 h and was performed either at 24 °C (open symbols) or on ice (gray symbols) followed in both cases by an additional 20–40 min on ice. A, incubation was performed in the presence of various concentrations of orthovanadate. B, incubation was performed in the absence or presence of KF (1 mm when present) and in the additional presence of either 50 μm BeCl₂ (squares) or 50 μm AlCl3 (triangles) or in the presence of 5 mm MgCl₂ only (diamonds). C, time dependence on ice of the inhibition by 100 μm VO₄ (gray circles) or by 1 mm KF in the absence (diamonds) or presence of either 50 μm BeCl₂ (squares) or 50 μm AlCl₃ (triangles). Open circles, control incubation in the absence of any added compound. D, D⁺ P3 membranes were diluted on ice to 0.5 mg/ml at time 0 (open circles, dotted line), 50 μm AlCl₃ and 1 mm KF were added at t = 20 min, and the residual phosphorylation level was assayed after various periods. To trigger recovery from inhibition, free aluminum was subsequently chelated at t = 95 min by adding either hydroxyl ions (Tris base) to bring the pH up to 8 (squares), 10 mm EGTA (upside down triangles), or 0.2 mm desferrioxamine (diamonds). For a control, Tris, EGTA, and desferrioxamine were also added to the membranes together with AlCl₃ and KF at t = 20 min (open squares, triangles, or diamonds).