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.