Induction of oxidative stress in leukocytes by an Enterobacter cloacae toxin able to form oligomers and binding to proteins

A leukotoxic and hemolytic toxin was purified from cultures of Enterobacter cloacae. Stimulation of oxidative stress was observed and the production o f reactive oxidant species was measured in leukocytes treated with toxin by means of nitroblue tetrazolium and chemiluminescence assays. Molecular wei ght of toxin was estimated by chromatography and SDS-PAGE. Two protean peak s with toxic activity were found in Sephadex G-100 (P1, 42.0 kDa; and P2, 1 3.3 kDa), The relative amounts between the peaks (P1/P2 = 0.36) changed whe n g-mercaptoethanol was employed (P1/P2 = 0.59), When Sephadex G-200 chroma tography was performed, a protean peak of Ve = 113 mt (100 kDa) was found; its was dissociated with 3 M urea in toxic proteins of lower mass: 42, 27, and 13.3 kDa, SDS-PAGE (15%) showed a single toxin band of purified monomer (13.3 kDa), but electrophoresis of a 42-kDa toxin with urea presented thre e bands of trimer, dimer, and monomer. An increase of casein hydrolysate an d albumin molecular weight was observed by chromatography after incubation with toxin due to the binding of both proteins with toxin, (C) 2000 Academi c Press.