Watkins P A, Moss J, Vaughan M
J Biol Chem. 1981 May 25;256(10):4895-9.
Major labeled bands of Mr = 42,000 and 47,000 were observed on sodium dodecyl sulfate polyacrylamide gel electrophoresis of human skin fibroblast membranes incubated with choleragen and [32P]NAD. Prior incubation of intact fibroblasts with choleragen blocked specifically the subsequent in vitro labeling of these two proteins. The effect of choleragen was dependent on time, temperature, and toxin concentration. Neither the choleragen A subunit nor the B subunit nor the A1 peptide could replace the holotoxin in cell incubations. Inhibition of subsequent in vitro labeling by prior exposure of cells to choleragen was correlated with increased cellular cAMP. Incubation of fibroblasts with prostaglandin E1 and isoproterenol, which activate adenylate cyclase by different mechanisms, did not block subsequent labeling with choleragen and [32P]NAD. The results suggest that proteins of Mr = 42,000 and 47,000 may be in vivo substrates for choleragen in human fibroblasts.
在用霍乱毒素和[32P]NAD孵育的人皮肤成纤维细胞膜的十二烷基硫酸钠聚丙烯酰胺凝胶电泳上，观察到分子量为42,000和47,000的主要标记条带。用霍乱毒素预先孵育完整的成纤维细胞可特异性地阻断这两种蛋白质随后的体外标记。霍乱毒素的作用取决于时间、温度和毒素浓度。在细胞孵育中，霍乱毒素A亚基、B亚基或A1肽均不能替代全毒素。细胞预先暴露于霍乱毒素对随后体外标记的抑制作用与细胞内cAMP增加相关。用前列腺素E1和异丙肾上腺素孵育成纤维细胞，它们通过不同机制激活腺苷酸环化酶，但并未阻断随后用霍乱毒素和[32P]NAD进行的标记。结果表明，分子量为42,000和47,000的蛋白质可能是人成纤维细胞中霍乱毒素的体内底物。
