title: The RNA-N-glycosidase activity of Shiga-like toxin I: kinetic parameters of the native and activated toxin.
abstract: Shiga toxin and Shiga-like toxins are ribosome-inactivating proteins with RNA-N-glycosidase activity which remove a specific adenine from 28S RNA. The toxins are composed of an A subunit non-covalently associated to a multimer of receptor-binding B subunits. Near the COOH-terminus of the A subunit, a disulfide-bonded loop contains two trypsin-sensitive arginine residues. Proteolytic nicking at these sites, followed by reduction, removes from the A subunit the C-terminal end together with the associated B subunits. The requirement of such cleavage for biological activity of Shiga toxin and Shiga-like toxins has been recently questioned. The present paper reports the kinetic constants of the adenine release from highly purified Artemia salina ribosomes catalysed by Shiga-like toxin I and by its A subunit before and after treatment with trypsin, urea and dithiothreitol or urea and dithiothreitol alone. All reactions had approximately the same Km (1 microM). The Kcat was 0.6 min-1 for the untreated holotoxin and 6 min-1 for the isolated A subunit, respectively. The trypsin treatment increased 1000-fold the Kcat of the holotoxin (770 min-1) and 100-fold the Kcat of the A subunit (640 min-1). The same Kcat (693 min -1) was also observed when the A subunit was treated only with urea and dithiothreitol. Thus the full activity of Shiga-like toxin I required not only removal of the B subunits but also activation of the A subunit itself. Such activation could be largely induced in vitro by drastic loosening of the molecule induced by urea and dithiothreitol, but in vivo would probably require a proteolytic cleavage of the toxin. Inactivation of ribosomes by Shiga-like toxin I did not require sensitization of ribosomes by ATP and macromolecular cofactors present in postribosomal supernatants.
authors.affiliation: Dipartimento di Patologia sperimentale dell'Università di Bologna, Italy.
authors.forename: M
authors.initials: M
authors.lastname: Brigotti
authors.forename: D
authors.initials: D
authors.lastname: Carnicelli
authors.forename: P
authors.initials: P
authors.lastname: Alvergna
authors.forename: R
authors.initials: R
authors.lastname: Mazzaracchio
authors.forename: S
authors.initials: S
authors.lastname: Sperti
authors.forename: L
authors.initials: L
authors.lastname: Montanaro
mesh_terms: D000818:Animals; D001427:Bacterial Toxins / Q000378:metabolism*; D004229:Dithiothreitol / Q000494:pharmacology; D007700:Kinetics; D009699:N-Glycosyl Hydrolases / Q000378:metabolism; D011817:Rabbits; D054788:Ribosome Inactivating Proteins; D022622:Shiga Toxin 1; D014357:Trypsin / Q000494:pharmacology; D014508:Urea / Q000494:pharmacology
chemical_list: D001427:Bacterial Toxins; D022622:Shiga Toxin 1; D014508:Urea; D009699:N-Glycosyl Hydrolases; D054788:Ribosome Inactivating Proteins; D014357:Trypsin; D004229:Dithiothreitol