Purification and some properties of cytotoxin produced by Clostridium difficile.
The cytotoxin produced by Clostridium difficile was highly purified by using ammonium sulfate fractionation and successive column chromatographies of DEAE-Sephadex A-25, hydroxyapatite, Bio-Gel A-0.5m, Phenyl-Sepharose CL-4B, and Mono Q. The purified cytotoxin gave a single band on conventional and sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the presence of dithiothreitol. Its molecular weight was estimated to be 260,000 and 50,000 by gel filtration and sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis in the presence of dithiothreitol, respectively. Thus it was supposed that the toxin consists of 5 subunits having molecular weight of approximately 50,000. It had an isoelectric point of 6.6. The toxin was heat-labile (60 C for 10 min) and inactivated by treatment with trypsin and pronase, or at pH below 4 or over 10. The minimum cytotoxic dose of the cytotoxin against Chinese hamster ovary cells was 3 ng. It was also demonstrated that the toxin is antigenically different from enterotoxin of C. difficile.
Katoh
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Department of Bacteriology and Serology, Osaka University.
Honda
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Miwatani
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D000818:Animals; D001426:Bacterial Proteins; D001427:Bacterial Toxins / Q000276:immunology / Q000302:isolation & purification* / Q000633:toxicity; D002478:Cells, Cultured; D003013:Clostridium / Q000032:analysis*; D003603:Cytotoxins / Q000276:immunology / Q000302:isolation & purification* / Q000633:toxicity; D007120:Immunochemistry; D007526:Isoelectric Point; D008970:Molecular Weight
D001426:Bacterial Proteins; D001427:Bacterial Toxins; D003603:Cytotoxins; C031815:toxB protein, Clostridium difficile