ADP-ribosylation and de-ADP-ribosylation of the rho protein by Clostridium botulinum exoenzyme C3. Regulation by EDTA, guanine nucleotides and pH

Abstract Pretreatment of rho protein purified from pig brain cytosol with EDTA (3 mM) for 10 min at 30°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 GDPβS present during the pretreatment period completely prevented the decrease in ADP-ribosylation with half-maximal and maximal effects at 3 and 300 μM, respectively. GTP or GTPγS were less efficacious in preventing the decrease in ADP-ribosylation, but were more potent (half-maximal and maximal effects at 0.1 and 3 μM, respectively). [ 32 P]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, [ 32 P]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.