Pothoulakis C, Barone L M, Ely R, Faris B, Clark M E, Franzblau C, LaMont J T
J Biol Chem. 1986 Jan 25;261(3):1316-21.
Toxin B, a potent cytotoxin produced by Clostridium difficile, was purified to homogeneity from 6-day broth cultures of a toxigenic isolate. Cytotoxin was purified approximately 4000-fold by sequential ammonium sulfate precipitation, DEAE-Sepharose chromatography, and high performance liquid chromatography on a Mono Q anion-exchange column. The molecular weight of reduced purified toxin was 50,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, compared to 150,000 for unreduced toxin. Dose-response studies indicated that subpicogram concentrations of purified toxin caused rounding of approximately 20,000 IMR-90 fibroblasts. The phenomenon of cell rounding caused by toxin B was correlated with the ratio of globular to filamentous actin in fibroblasts as measured by two techniques. The toxin caused a significant increase in the ratio of globular to filamentous actin which was nearly completed prior to the onset of rounding. We conclude that cell rounding of fibroblasts exposed to toxin B is related to an increase in the ratio of globular to filamentous actin which is produced by small numbers of toxin molecules/cell.
毒素B是由艰难梭菌产生的一种强效细胞毒素，从一株产毒分离株的6天肉汤培养物中纯化至同质。通过硫酸铵分级沉淀、DEAE-琼脂糖层析以及在Mono Q阴离子交换柱上进行高效液相色谱，细胞毒素被纯化了约4000倍。在十二烷基硫酸钠-聚丙烯酰胺凝胶电泳上，还原的纯化毒素的分子量为50,000，而未还原毒素的分子量为150,000。剂量反应研究表明，亚皮克级浓度的纯化毒素会导致约20,000个IMR-90成纤维细胞变圆。毒素B引起的细胞变圆现象与通过两种技术测量的成纤维细胞中球状肌动蛋白与丝状肌动蛋白的比例相关。该毒素导致球状肌动蛋白与丝状肌动蛋白的比例显著增加，这一增加在细胞变圆开始之前几乎就已完成。我们得出结论，暴露于毒素B的成纤维细胞的细胞变圆与由少量毒素分子/细胞产生的球状肌动蛋白与丝状肌动蛋白比例的增加有关。
