Purification and characterization of alpha-toxin produced by Clostridium novyi type A.
Our study describes the production, purification, and properties of alpha-toxin from Clostridium novyi type A 19402. The bacterium produced maximal amounts of alpha-toxin when grown at 37 degrees C for 72 h in dialysis flask cultures containing brain heart infusion supplemented with 0.75% Tween 80 and 2% glycerol. The alpha-toxin was purified by precipitation with polyethylene glycol 6000, followed by chromatography on Q-Sepharose, phenyl-agarose, and Mono-Q. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the toxin exhibited a single band with an M(r) of 200,000. The toxin also exhibited a single immunoprecipitin arc by crossed immunoelectrophoresis with antiserum against crude toxin. It was stable when stored at 4 degrees C and also following exposure to buffers with pHs in the range of 4 to 7. The toxin had a minimum lethal dose in mice of 5 to 10 ng, caused rounding of a variety of cells in tissue culture, and was negative in the rabbit ileal loop assay. The cytotoxic activity was inhibited by agents that affect receptor-mediated processes, and the toxin was less active on a CHO mutant cell line that is defective in endosomal acidification. The analysis of the amino acid composition revealed an unusually high proline content. The N-terminal sequence is Met-Leu-Ile-Thr-Arg-Glu-Gln-Leu-Met-Lys.
Department of Anaerobic Microbiology, Virginia Polytechnic Institute and State University, Blacksburg 24061.
D W
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Ball
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Van Tassell
M D
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Roberts
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Hahn
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Lyerly
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Wilkins
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001427:Bacterial Toxins / Q000737:chemistry / Q000302:isolation & purification* / Q000633:toxicity; D016466:CHO Cells; D002417:Cattle; D002460:Cell Line; D003013:Clostridium / Q000472:pathogenicity; D006224:Cricetinae; D006863:Hydrogen-Ion Concentration; D051379:Mice; D008969:Molecular Sequence Data; D011817:Rabbits; D013696:Temperature
D000596:Amino Acids; D001427:Bacterial Toxins; C075216:Clostridium novyi alpha-toxin