The site of action of a Vero toxin (VT2 or Shiga-like toxin 11) from enterohemorrhagic Escherichia coli and Shiga toxin from Shigella dysenteriae 1 on eukaryotic ribosomes was studied. Treatment of eukaryotic ribosomes with either toxin caused the release of a fragment of 400 nucleotides from 28s ribosomal RNA when the isolated ribosomal RNA was treated with aniline. Release of this fragment with aniline treatment was accompanied by inhibition of protein synthesis and of elongation-factor-1-dependent aminoacyl-tRNA binding to ribosomes. Analysis of the nucleotide sequence of the 3'-terminal fragment of 553 nucleotides of 28s rRNA of rat liver 60s ribosomal subunits suggested that an adenine base at position 4324 (A-4324) was absent in toxin-treated 28s rRNA. Further analysis by thin-layer chromatography demonstrated quantitative release of adenine from rat liver ribosomes on treatment with the toxins. These results indicate that both VT2 and Shiga toxin inactivate 60s ribosomal subunits by cleaving the N-glycosidic bond at A-4324 in 28s ribosomal RNA. (T. Ogasawara et al., unpublished results) were recently shown to inactivate 60s ribosomal subunits and thereby inhibit EF-1-dependent binding of aminoacyl-tRNA to ribosomes, in this study we examined whether these bacterial cytotoxins inactivate 60s ribosomal subunits by the same mechanism as that of other plant and fungal cytotoxins. MATERIALS AND METHODS Purqications of Shiga toxin and VT2Shiga toxin was isolated and purified from the culture supernatant of S. dysenteriae 1 (RIMD 3101010) by ammonium sulfate fractionation, DEAE-cellulose column chromatography and repeated chromatofocusing column chromatographies, as described previously [28, 291. VT2, which has been shown to be immunologically unrelated to the Shiga toxin, but has similar biological activities to the latter, was isolated and purified from an enterohemorrhagic E. cali 0157:H7 strain J-2 as described earlier [12]. Both purified toxins gave a single band staining for protein on conventional polyacrylamide disc gel electrophoresis and polyacrylamide gel isoelectrofocusing. Preparation of anti-toxin sera against Shiga toxin and VT2Anti-toxin sera against Shiga toxin and VT2 were prepared by immunizing rabbits with purified Shiga toxin and purified VT2 as described previously [12, 281. Immunoglobulins

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