FIGURE 4.

Effect of various ionic conditions on phosphorylation (from [γ-³²P]ATP) of Drs2p-Bad. A and B, the effect of pH and KCl was explored in the absence (open symbols) or presence (gray symbols) of 1 mm orthovanadate. C, the effect of manipulating the concentrations of a few metal ions was tested by adding either 0.6 or 5 mm EGTA (which reduced the free Ca²⁺ concentrations to 0.1 or 0.01 μm as indicated below the corresponding bars); by adding 4, 7.5, or 15 mm EDTA (which also lowered the free Ca²⁺ concentrations to various extents but in addition reduced the free Mg²⁺ concentrations); or by adding 50 or 500 μm Zn²⁺ or Co²⁺, or 50 μm La³⁺, or 10 μm H⁺ ionophore carbonyl cyanide m-chlorophenylhydrazone (CCCP). Phosphorylation (on ice for 30 s) was triggered by addition of 0.5 μm [γ-³²P]ATP followed by acid quenching. Data are presented as the mean ±SD (error bars) of duplicates.