ADP-RIBOSYLATION AND DE-ADP-RIBOSYLATION OF THE RHO-PROTEIN BY CLOSTRIDIUM-BOTULINUM EXOENZYME-C3 - REGULATION BY EDTA, GUANINE-NUCLEOTIDES AND PH

Pretreatment of rho protein purified from pig brain cytosol with EDTA (3 mM) for 10 min at 30-degrees-C inhibited its ADP-ribosylation by Clostridium botulinum C3 ADP-ribosyltransferase by more than 90%. The EDTA effect was not caused by alteration of C3. GDP or GDP-beta-S present during the pretreatment period completely prevented the decrease in ADP-ribosylation with half-maximal and maximal effects at 3 and 300-mu-M, respectively. GTP or GTP-gamma-S were less efficacious in preventing the decrease in ADP-ribosylation, but were more potent (half-maximal and maximal effects at 0.1 and 3-mu-M, respectively). [P-32]ADP-ribose incorporated in pig brain rho by C3 was de-ADP-ribosylated by the enzyme in the presence of nicotinamide and at low pH. Concomitantly, [P-32]NAD was formed. The pH optima for ADP-ribosylation and de-ADP-ribosylation were pH 7.5 and 5.5, respectively. De-ADP-ribosylation was most efficient with nicotinamide, less effective with 3-acetylpyridine and not observed with 3-aminopyridine, 4-aminopyridine, 4-acetylpyridine and isonicotinic acid. As observed for the ADP-ribosylation, the de-ADP-ribosylation by C3 was maximal with the GDP-bound form of rho and blocked after EDTA treatment.