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We tested various methods of assaying the ADP-ribosyltransferase activity of cholera toxin using artificial acceptors of the ADP-ribosyl group. Any of several proteins or poly(L-arginine) could be used with [adenine-14C]NAD+ as ADP-ribosyl donor, but this method was not ideal because of the heterogeneity of potential acceptor groups and the necessity of using costly labeled NAD+. We, therefore, developed an alternative assay using a synthetic low molecular weight acceptor, 125I-N-guanyltyramine (125I-GT). 125I-GT was specifically ADP-ribosylated by thiol-treated cholera toxin or its A1 peptide in the presence of beta-NAD. ADP-ribosyl-125I-GT was quantified after separation from unreacted 125I-GT by batch absorption of the latter to cation exchange resins. Analysis of the kinetics of ADP-ribosylation of 125I-GT indicated that the reaction proceeds by a sequential rather than a ping-pong mechanism. The Km values for NAD+ and 125I-GT were 3.6 mM and 44 microM, respectively. L-Arginine was a competitive inhibitor of 125I-GT (KI = 75 mM), but was at least 1000-fold less active than 125I-GT as an ADP-ribose acceptor.
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