Dorner F, Hughes C, Nahler G, Högenauer G
Proc Natl Acad Sci U S A. 1979 Oct;76(10):4832-6. doi: 10.1073/pnas.76.10.4832.
Escherichia coli heat-labile enterotoxin was synthesized in a cell-free system directed by DNA of the plasmid P307. Synthesis of the toxin, assayed by the elongation induced in Chinese hamster ovary cells, was strongly stimulated by cyclic AMP and occurred at physiological levels of Mg2+ only when the polyamine spermidine was present. Activity was abolished by heat and antisera prepared against the enterotoxins of both E. coli P263 and Vibrio cholera. Tritium-labeled enterotoxin was purified by immunoprecipitation and examined by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. When gel slices were assayed for the ability to stimulate adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] activity in erythrocyte ghosts, two peaks were found, one at Mr 26,000 and frequently, but not always, another at Mr 23,000. Detection of radiolabeled protein by fluorography and scintillation counting of gel slices revealed three prominent polypeptides, two corresponding to the peaks having adenylate cyclase-stimulating activity and a further one of Mr 11,500, identical to that of the cholera subunit B. The data suggest that the E. coli heat-labile enterotoxin synthesized in the cell-free system has a subunit structure.
大肠杆菌不耐热肠毒素是在由质粒P307的DNA指导的无细胞系统中合成的。通过中国仓鼠卵巢细胞中诱导的伸长来测定毒素的合成，其受到环磷酸腺苷的强烈刺激，并且仅当存在多胺亚精胺时才在生理水平的Mg2+条件下发生。加热以及用针对大肠杆菌P263和霍乱弧菌的肠毒素制备的抗血清可使活性丧失。通过免疫沉淀纯化氚标记的肠毒素，并通过十二烷基硫酸钠/聚丙烯酰胺凝胶电泳进行检测。当对凝胶切片检测刺激红细胞血影中腺苷酸环化酶[ATP焦磷酸裂解酶（环化），EC 4.6.1.1]活性的能力时，发现了两个峰，一个在Mr 26,000处，并且经常（但不总是）另一个在Mr 23,000处。通过凝胶切片的荧光自显影和闪烁计数检测放射性标记的蛋白质，发现了三种突出的多肽，其中两种对应于具有腺苷酸环化酶刺激活性的峰，另一种Mr 11,500的多肽与霍乱毒素B亚基相同。数据表明在无细胞系统中合成的大肠杆菌不耐热肠毒素具有亚基结构。
