Degradation of 2-phosphoglycerate by cytotoxin B of Clostridium difficile

Floyd Knoop; Robert Martig; Michaela Owens

doi:10.1016/0014-5793(90)80274-M

Degradation of 2-phosphoglycerate by cytotoxin B of Clostridium difficile

Floyd Knoop, Robert Martig, Michaela Owens

Department of Medical Microbiology and Immunology

Research output: Contribution to journal › Article › peer-review

4 Scopus citations

Abstract

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 × 10⁸ units mg protein and showed a single protein band with an estimated molecular weight of 163000 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.

Original language	English (US)
Pages (from-to)	9-12
Number of pages	4
Journal	FEBS Letters
Volume	267
Issue number	1
DOIs	https://doi.org/10.1016/0014-5793(90)80274-M
State	Published - Jul 2 1990

All Science Journal Classification (ASJC) codes

Biophysics
Structural Biology
Biochemistry
Molecular Biology
Genetics
Cell Biology

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title = "Degradation of 2-phosphoglycerate by cytotoxin B of Clostridium difficile",

abstract = "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 × 108 units mg protein and showed a single protein band with an estimated molecular weight of 163000 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.",

author = "Floyd Knoop and Robert Martig and Michaela Owens",

note = "Funding Information: Health Service Grant RR 05390 from the National Institutes of Health and by the US Army Department of Medical Research.",

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T1 - Degradation of 2-phosphoglycerate by cytotoxin B of Clostridium difficile

AU - Knoop, Floyd

AU - Martig, Robert

AU - Owens, Michaela

N1 - Funding Information: Health Service Grant RR 05390 from the National Institutes of Health and by the US Army Department of Medical Research.

PY - 1990/7/2

Y1 - 1990/7/2

N2 - 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 × 108 units mg protein and showed a single protein band with an estimated molecular weight of 163000 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.

AB - 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 × 108 units mg protein and showed a single protein band with an estimated molecular weight of 163000 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.

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