Degradation of 2-phosphoglycerate by cytotoxin B of Clostridium difficile.
Cytotoxin B of C. difficile was highly purified by selective ammonium sulfate precipitation, Biogel A5m chromatography, phenyl boronate hydrophobic interaction chromatography and ultracentrifugation. The final cytotoxic product had a specific activity of 7.8 X 10(8) units/mg protein and showed a single protein band with an estimated molecular weight of 163,000 when subjected to SDS-PAGE. Immunoelectrophoresis of the final product showed a single precipitin arc. The addition of cytotoxin B to imidazole-HCl buffer (pH 7.4) containing MgSO4, KCl and the substrate 2-phosphoglycerate resulted in the formation of phosphoenolpyruvate as demonstrated by spectrophotometric analysis. Phosphoglycerate conversion was absent when the cytotoxin was heat-inactivated of reacted with specific antitoxin prior to assay.
Knoop
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F
Department of Medical Microbiology, Creighton University School of Medicine, Omaha, NE 68178.
Martig
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R
Owens
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M
D000311:Adrenal Glands / Q000187:drug effects / Q000378:metabolism; D000645:Ammonium Sulfate; D000818:Animals; D001426:Bacterial Proteins; D001427:Bacterial Toxins / Q000378:metabolism*; D002499:Centrifugation, Density Gradient; D003013:Clostridium / Q000378:metabolism; D003603:Cytotoxins / Q000378:metabolism*; D005988:Glyceric Acids / Q000378:metabolism*; D006868:Hydrolysis; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D051379:Mice; D010728:Phosphoenolpyruvate / Q000378:metabolism; D010751:Phosphopyruvate Hydratase / Q000494:pharmacology; D011770:Pyruvate Kinase / Q000378:metabolism
D001426:Bacterial Proteins; D001427:Bacterial Toxins; D003603:Cytotoxins; D005988:Glyceric Acids; C031815:toxB protein, Clostridium difficile; C008885:2-phosphoglycerate; D010728:Phosphoenolpyruvate; D007770:L-Lactate Dehydrogenase; D011770:Pyruvate Kinase; D010751:Phosphopyruvate Hydratase; D000645:Ammonium Sulfate