THE PHOSPHORYLATION OF ESCHERICHIA-COLI ISOCITRATE DEHYDROGENASE IN INTACT-CELLS

The isocitrate dehydrogenase of Escherichia coli ML308 can be reversibly activated by addition of pyruvate to cells growing on acetate. Cells pulse-labeled with [32P]Pi were used to show that the activation and inactivation of the enzyme in these conditions correlate with its dephosphorylation and rephosphorylation, respectively. Incubation of cell extracts prepared during an activation/inactivation cycle with purified isocitrate dehydrogenase phosphatase confirmed that the pyruvate-induced activation of the dehydrogenase goes essentially to completion. The reversible changes in the activity of the dehydrogenase in cells grown on acetate are solely due to phosphorylation/dephosphorylation. Inactive 32P-labeled isocitrate dehydrogenase was isolated from cells incubated with [32P]Pi in the presence of acetate. Both this material and purified enzyme phosphorylated in vitro were digested with chymotrypsin and the phosphopeptides were isolated and analyzed. Only one phosphopeptide was observed in each case; the residue phosphorylated in vivo is identical with that phosphorylated by purified isocitrate dehydrogenase kinase in vitro.