FIGURE 7.

Short and long term effects of detergents on phosphorylation (from [γ-³²P]ATP) of Drs2p-Bad. P3 membranes, either D⁺ (open bars or open symbols) or D*⁺ (gray bars or gray symbols), were suspended at 2 mg/ml in SB supplemented with PMSF and a complete protease inhibitor mixture. A, samples were then incubated for 1 min in the presence of various detergents all at 20 mg/ml; the detergents used are indicated (Digit, digitonin; TX-100, Triton X-100). After such incubation, samples were diluted 4-fold into buffer A supplemented with 0.1 mm CaCl₂, and phosphorylation was measured as described earlier using two superimposed A/E filters. Both filters were counted, and the sum of the radioactivities in the two filters was plotted. B, for selected detergents, similar measurements were repeated after incubation on ice for a few hours. The detergents tested were DDM (triangles), C₁₂E₈ (squares), and Triton X-100 (diamonds). C, DDM was tested at various concentrations, resulting in the various detergent (det.)-to-protein (prot.) ratios indicated. Data are presented as the mean ± S.D. (error bars, n = 3).