FIGURE 6.

Time course of aspartylphosphate formation of Ccc2 wt and S258A. A, autoradiogram of the 110-kDa band from a representative gel after resolution of the proteins by acidic gel electrophoresis. Membranes of Sf9 cells expressing Ccc2 or S258A were phosphorylated with [γ-³²P]ATP for the indicated times. B, Coomassie Blue staining of the same gels. C, densitometric representation of the time-course of phosphorylation obtained from autoradiograms corrected for the corresponding amount of protein loaded in each lane and for the background signal simultaneously measured with the non-phosphorylating D627A mutant. The phosphoenzyme levels were normalized, taking phosphorylation of Ccc2 wt at 1 min as the reference (see “Experimental Procedures”). Data are mean ± S.E. of 5–7 time course runs with different time intervals. When not shown, the S.E. was smaller than the symbol size. The smooth lines were adjusted to the mean experimental points with the use of Equation 1 (see text), using the mean values (Table 1) of the rate constants of phosphorylation, burst, and EP_max calculated for each individual experiment.