FIG. 1.

VieS autophosphorylation is stimulated by Ca²⁺ and Mn²⁺ and exhibits second-order kinetics. (A) Autophosphorylation of MBP-′VieS occurs in the presence of Mn²⁺ or Ca²⁺ but not Mg²⁺. The cytoplasmic portion of VieS was fused to Escherichia coli MBP and purified. MBP-′VieS was incubated for 30 min at 30°C in phosphorylation buffer in the presence of 30 μM [γ-³²P]ATP and the divalent cations indicated. The samples were subjected to SDS-PAGE and subsequent autoradiography for 12 h. (B) Autophosphorylation of MBP-VieS is not inhibited by Mg²⁺. Autophosphorylation of MBP-′VieS was carried out as described above in the presence of 5 mM Mn²⁺ and increasing concentrations of Mg²⁺ to determine the effect that the presence of both cations would have on the kinase activity of MBP-′VieS. (C) Phosphorylation of MBP-′VieS at twofold serial dilutions. Autophosphorylation was carried out as described above in the presence of 10 mM Ca²⁺. (D) The results from panel B were quantitated and plotted (solid circles) compared with theoretical values obtained if MBP-′VieS followed perfect second-order kinetics (open squares) or first-order kinetics (dashed line).