Fig. 2.

Phosphorimages of SDS/PAGE gels showing the response regulator dephosphorylation time courses. (A) 400 μM CheY₁ was added to 2 μM CheA₃P1-P in the absence (left half of gel) and presence of 2.5 μM CheA₃ (right half of gel). (B) 400 μM CheY₆ was added to 30 μM CheA₃P1-P in the absence (left half of gel) and presence of 2.5 μM CheA₃ (right half of gel). 10-μl reaction samples were taken at the time points indicated and quenched in 20 μl of 1.5× SDS/EDTA loading dye. The quenched samples were analyzed by SDS/PAGE and detected by phosphorimaging. ATP was not present in any of the reactions, so after the phosphotransfer reactions, which were completed before the first-time point, the only reaction occurring was RR-P dephosphorylation. As has been observed for E. coli CheA, a small fraction of CheA₃P1-P (< 4%) failed to transfer phosphoryl groups to the RRs (42). Phosphatase activity is indicated by a reduction in CheY-P levels in the presence of 2.5 μM CheA₃ when compared with those in the absence of CheA₃ (seen in B but not in A).