THE RNA-N-GLYCOSIDASE ACTIVITY OF SHIGA-LIKE TOXIN-I - KINETIC-PARAMETERS OF THE NATIVE AND ACTIVATED TOXIN

Shiga toxin and Shiga-like toxins are ribosome-inactivating proteins w ith RNA-N-glycosidase activity which remove a specific adenine from 28 S RNA. The toxins are composed of an A subunit non-covalently associat ed to a multimer of receptor-binding B subunits, Neat-the COOH-terminu s of the A subunit, a disulfide-bonded loop contains two trypsin-sensi tive arginine residues. Proteolytic nicking at these sites, followed b y reduction, removes from the A subunit the C-terminal end together wi th the associated B subunits, The requirement of such cleavage for bio logical activity of Shiga toxin and Shiga-like toxins has been recentl y questioned. The present paper reports the kinetic constants of the a denine 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 alon e. All reactions had approximately the same K-m (1 mu M). The K-cat wa s 0.6 min(-1) for the untreated holotoxin and 6 min(-1) for the isolat ed A subunit, respectively. The trypsin treatment increased 1000-fold the K-cat of the holotoxin (770 min(-1)) and 100-fold the K-cat of the A subunit (640 min(-1)). The same K-cat (693 min(-1)) was also observ ed when the A subunit was treated only with urea and dithiothreitol. T hus 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 ac tivation could be largely induced in vitro by drastic loosening of the molecule induced by urea and dithiothreitol, but in vivo would probab ly require a proteolytic cleavage of the toxin. Inactivation of riboso mes by Shiga-like toxin I did not require sensitization of ribosomes b y ATP and macromolecular cofactors present in postribosomal supernatan ts. (C) 1997 Elsevier Science Ltd.