REGULATION SYSTEM OF ADP-RIBOSYLTRANSFERASE ACTIVITY OF CHOLERA TOXING BY ARE AND ARI :

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Cholera toxin, a secretory product of Vibrio cholerae, causes the diarrheal syndrome characteristic of cholera by activating the adenylate cyclase of intestinal mucosal cells resulting in the elevatin of cell cAMP content that is responsible for effects on fluid and electroyte transport. Cholera toxin, by transferring ADP-ribose from NAD to a critical amino acid in the alpha subunit of Gs, enhances the ability of Gs to activate the cyclase catalytic unit. Toxin-catalyzed ADP-ribosylation of Gschi can be enhanced by several membrance or soluble factors. A 20 kDa protein termed ARF, ADP-ribosylation factor, that enhances the toxin-catalyzed modification in a reconstituted system containing purified Gsalpha was a guanine nucleotide-binding protein. It appears that ARF GTP, by interacting directly with A subunit of cholera toxin, alters the allosteric properties of the toxin resulting in increased catalytic activity with subsaturating substrates. A 20 kDa membrane protein termed ARI, ADP-ribosylation inhibitor, that inhibits cholera toxin-catalyzed modification was purified and the mechanism of inhibition of cholera toxin-catalyzed ADP-ribosylation by ARI was studied. To determine the mechanism of inhibitory effect of ARI on cholera toxin-catalyzed ADP-ribosylation, a Lineweaver-Burk analysis was performed specifically for observing NAD : agmatine ADP-ribosyltransferase activity of cholera toxin A subunit in the presence and absence of ARI.ARI had no effect on the Km for NAD, but significantly reduced the maximal velocity of the reaction. In addition to examining the effect of ARI on NAD, the kinetics of ADP-ribosylation using agmatine as a variable substrate was studied in the presence and absence of ARI.These was a significant increase in the Km for agmatine and a decrease in the maximal velocity of the reaction. These data show that ARI decreases reaction rate and substrate affinity of cholera toxin A subunit for agmatine and that ARI has no effect on the substrate