Analysis of the relationship between ARF and cholera toxin and LT

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We examined the relationship between cholera toxin (CT) or heat-labile enterotoxin (LT) and ADP-ribosylation factor. At first, the gene encoding ADP-ribosylation factor II (ARF II) from Saccharomyces cerevisiae was amplified by the polymerase chain reaciton and a fusion gene was constructed with the promoter region and a short stretch of 5'-coding sequence of the E.coli glutathione S-transferase gene. Recombinant ARF II was purified. Its activation of toxin ADP-ribosyltransferase was fully induced by the presence of GTP.Its enzymatic activation of CT was higher than that of porcine or human LT from enterotoxigenic E.coli. A mutant toxin (E112K) of its A subunit inhibited activaiton of LT similar to CT.Moreover, we prepared mutant toxins about the amino acids formed the cavity around Glu112 and analyzed whether these mutant toxin would be activated by rARFII.The mutant at position 110 has enzymatic activity, but its activity was not activated by rARFII.Then, the Glu110 might be involved in the allosteric affect of toxin by rARFII.