Mekalanos J J, Collier R J, Romig W R
J Biol Chem. 1979 Jul 10;254(13):5849-54.
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.
我们使用ADP-核糖基团的人工受体测试了多种测定霍乱毒素ADP-核糖基转移酶活性的方法。几种蛋白质或聚（L-精氨酸）中的任何一种都可以与[腺嘌呤-14C]NAD⁺作为ADP-核糖供体一起使用，但由于潜在受体基团的异质性以及使用昂贵的标记NAD⁺的必要性，这种方法并不理想。因此，我们开发了一种替代测定方法，使用合成的低分子量受体125I-N-鸟苷酪胺（125I-GT）。在β-NAD存在下，125I-GT被巯基处理的霍乱毒素或其A1肽特异性地ADP-核糖基化。通过将未反应的125I-GT分批吸附到阳离子交换树脂上，将ADP-核糖基-125I-GT与未反应的125I-GT分离后进行定量。对125I-GT的ADP-核糖基化动力学分析表明，该反应通过顺序机制而非乒乓机制进行。NAD⁺和125I-GT的Km值分别为3.6 mM和44 μM。L-精氨酸是125I-GT的竞争性抑制剂（KI = 75 mM），但作为ADP-核糖受体，其活性比125I-GT至少低1000倍。
