Moss J, Manganiello V C, Vaughan M
Proc Natl Acad Sci U S A. 1976 Dec;73(12):4424-7. doi: 10.1073/pnas.73.12.4424.
Choleragen and the isolated A protomer catalyzed the hydrolysis of NAD to ADP-ribose and nicotinamide. The protein with NADase activity (NAD nucleosidase; NAD glycohydrolase, EC 3-2-2-5) migrated on polyacrylamide gels with choleragen, and chromatographed on Bio-Gel P-60 columns with the A protomer. The NADase activity of choleragen and of the A protomer was increased markedly in acetate and phosphate buffers, and enhanced over 10-fold by dithiothreitol in high concentration. NAD hydrolysis was proportional to choleragen concentration; the Michaelis constant for NAD was about 4 mM with both choleragen and the A protomer. The demonstration that the A protomer of choleragen catalyzes an enzymatic reaction involving activation of the ribosyl-nicotinamide bond of NAD, a reaction analogols to those catalyzed by diphtheria toxin, supports the hypothesis that activation of adenylate cyclase by choleragen involves the ADP-ribosylation of an appropriate acceptor protein.
霍乱毒素和分离出的A亚基催化NAD水解为ADP-核糖和烟酰胺。具有NAD酶活性的蛋白质（NAD核苷酶；NAD糖水解酶，EC 3-2-2-5）在聚丙烯酰胺凝胶上与霍乱毒素一起迁移，并在Bio-Gel P-60柱上与A亚基一起进行层析。霍乱毒素和A亚基的NAD酶活性在醋酸盐和磷酸盐缓冲液中显著增加，在高浓度二硫苏糖醇作用下增强超过10倍。NAD水解与霍乱毒素浓度成正比；霍乱毒素和A亚基对NAD的米氏常数约为4 mM。霍乱毒素的A亚基催化涉及NAD核糖基-烟酰胺键激活的酶促反应，该反应类似于白喉毒素催化的反应，这一证明支持了霍乱毒素激活腺苷酸环化酶涉及适当受体蛋白的ADP-核糖基化的假说。
