PURIFICATION AND CHARACTERIZATION OF A CHINESE-HAMSTER OVARY CELL ELONGATION-FACTOR OF VIBRIO-HOLLISAE

The halophilic bacterium Vibrio hollisae, isolated from patients with diarrhea, produces an extracellular toxin which elongates Chinese hams ter ovary (CHO) cells. We purified this toxin to homogeneity by sequen tial ammonium sulfate precipitation, gel filtration with Sephacryl S-2 00, hydrophobic interaction chromatography with phenyl-Sepharose CL-4B , ion-exchange chromatography with DEAE-Sephadex A-50, and affinity ch romatography, The toxin is heat labile and sensitive to proteases, wit h an isoelectric point of about 6.5 and molecular weights of about 83, 000 and 80,000, as estimated by gel filtration and sodium dodecyl sulf ate polyacrylamide gel electrophoresis, respectively, The toxin did no t react with immunoaffinity-purified antibodies to cholera toxin in a plate enzyme-linked immunosorbent assay and in a Western blot, and its activity could not be neutralized by anti-cholera toxin serum, Mixed gangliosides and gangliosides G(M1), G(D1a), G(D1b), G(q1b), G(T1b), G (D2), G(D3), G(M2), and G(M3) failed to block its activity. Elongation of CHO cells induced by the toxin was not accompanied by an increase in the levels of cyclic AMP. The toxin induced intestinal fluid accumu lation in suckling mice. These results and the lack of homology betwee n V. hollisae DNA and DNA coding for cholera toxin or the heat-labile toxin of Escherichia coli suggest that the V. hollisae toxin is struct urally and functionally different from other CHO cell-elongating toxin s.