Influence of PKAi5–24 and ADP on the aspartylphosphate levels obtained with the phosphomimetic S258D mutant. A, upper and lower autoradiograms correspond to representative experiments demonstrating aspartylphosphate levels of Ccc2 wt and S258D at 1 min respectively, in the presence of various concentrations of PKAi5–24. The panel demonstrates the densitometric representation of the autoradiograms obtained with Ccc2 wt (circles; n = 2) and S258D (squares; mean ± S.E., n = 4). Data were normalized and aspartylphosphate levels of Ccc2 wt at 1 min without inhibitor were regarded as 100%. The function described in the legend of Fig. 8 was adjusted to the Ccc2 wt data by non-linear regression. B, representative autoradiograms obtained with membranes of Sf9 cells expressing S258D that were phosphorylated by [γ-32P]ATP for 1, 2, or 3 min (arrows) and supplied with ADP for 10, 20, or 30 s. The figure demonstrates the densitometric representation of three assays (mean ± S.E.). The decrease in phosphoenzyme (% of the corresponding level before ADP addition) corresponds to the ADP-sensitive CuE∼P.