Botulinum ADP-ribosyltransferase C3

Purification of the enzyme and characterization of the ADP-ribosylation reaction in platelet membranes


Correspondence to K. Aktories, Rudolf-Buchheim-Institut für Pharmakologie, Frankfurter Straße 107, D-6300 Gießen, Federal Republic of Germany


A novel ADP-ribosyltransferase C3 was purified to homogeneity from filtrates of certain strains of Clostridium botulinum type C by ammonium sulfate precipitation, gel filtration, ion-exchange chromatography and heat treatment. The molecular mass of botulinum ADP-ribosyltransferase C3 was found to be 25 kDa. In the presence of [32P]NAD but not with [carbonyl-14C]NAD, C3 labelled 21–24-kDa protein(s) in membranes of human platelets and other tissues. The Km value of the ADP-ribosylation reaction for NAD was about 2 μM. Labelling of the 21–24-kDa protein(s) by C3 was largely reduced by addition of nicotinamide. Snake venom phosphodiesterase cleaved the ADP-ribose attached to the 21–24-kDa protein(s) by C3 and released 5'AMP. C3 catalyzed hydrolysis of [carbonyl-14C]NAD and released [carbonyl-14C]nicotinamide. ADP-ribosylation of 21–24-kDa platelet membrane protein(s) was biphasically regulated by Mg2+, Mn2+ and Ca2+. In the absence of free divalent cations GTP, GTP [γS] and GDP but not GDP[βS], GMP, ATP or ATP[γS] increased labelling by C3. In the presence of Mg2+, GTP[γS] was inhibitory. Guanine nucleotides prevented heat inactivation of the substrate protein(s) with the rank order GTP[γS] = GTP = GDP > GDP[βSL] > GMP ≫ ATP = ATP[γS]. The data support the view that the novel ADP-ribosyltransferase C3 modifies eukaryotic 21–24-kDa GTP-binding protein(s).


guanosine 5′-[γ-thio]triphosphate


guanosine 5′-[β-thio]diphosphate


adenesine 5′-[γ-thio]triphosphate


the inhibitory guanyl-nucleotide-binding regulatory protein of adenylate cyclase


the stimulatory guanyl-nucleotide-binding regulatory protein of adenylate cyclase


novel botulinum ADP-ribosyltransferase


NADase (EC


phosphodiesterase I (EC