The RNA-N-glycosidase activity of Shiga-like toxin I: kinetic parameters of the native and activated toxin
| Autor: | Simonetta Sperti, Maurizio Brigotti, Lucio Montanaro, Domenica Carnicelli, Raffaella Mazzaracchio, Paola Alvergna |
|---|---|
| Rok vydání: | 1997 |
| Předmět: |
Protein subunit
Bacterial Toxins Ribosome Inactivating Proteins Toxicology medicine.disease_cause Shiga Toxin 1 Ribosome Dithiothreitol chemistry.chemical_compound medicine Protein biosynthesis Animals Urea Trypsin N-Glycosyl Hydrolases chemistry.chemical_classification biology Toxin Shiga toxin Molecular biology Kinetics Enzyme Biochemistry chemistry biology.protein Rabbits medicine.drug |
| Zdroj: | Toxicon : official journal of the International Society on Toxinology. 35(9) |
| ISSN: | 0041-0101 |
| Popis: | 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 K m (1 μ M). The K cat 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 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 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. |
| Databáze: | OpenAIRE |
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