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 | |
| State | Published - Jul 2 1990 |
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All Science Journal Classification (ASJC) codes
- Biochemistry
- Biophysics
- Molecular Biology
Cite this
Degradation of 2-phosphoglycerate by cytotoxin B of Clostridium difficile. / Knoop, Floyd C.; Martig, Robert; Owens, Michaela.
In: FEBS Letters, Vol. 267, No. 1, 02.07.1990, p. 9-12.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Degradation of 2-phosphoglycerate by cytotoxin B of Clostridium difficile
AU - Knoop, Floyd C.
AU - Martig, Robert
AU - Owens, Michaela
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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UR - http://www.scopus.com/inward/citedby.url?scp=0025299768&partnerID=8YFLogxK
U2 - 10.1016/0014-5793(90)80274-M
DO - 10.1016/0014-5793(90)80274-M
M3 - Article
VL - 267
SP - 9
EP - 12
JO - FEBS Letters
JF - FEBS Letters
SN - 0014-5793
IS - 1
ER -