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 hamster ovary (CHO) cells. We purified this toxin to homogeneity by sequential ammonium sulfate precipitation, gel filtration with Sephacryl S-200, hydrophobic interaction chromatography with phenyl-Sepharose CL-4B, ion-exchange chromatography with DEAE-Sephadex A-50, and affinity chromatography. The toxin is heat labile and sensitive to proteases, with 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 sulfate-polyacrylamide gel electrophoresis, respectively. The toxin did not 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-cholrea toxin serum. Mixed gangliosides and gangliosides GM1, GD1a, GD1b, Gq1b, GT1b, GD2, GD3, GM2, and GM3 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 accumulation in suckling mice. These results and the lack of homology between V. hollisae DNA and DNA coding for cholera toxin or the heat-labile toxin of Escherichia coli suggest that the V. hollisae toxin is structurally and functionally different from other CHO cell-elongating toxins.
Kothary
M H
MH
Division of Virulence Assessment, Food and Drug Administration, Washington, D.C. 20204, USA.
Claverie
E F
EF
Miliotis
M D
MD
Madden
J M
JM
Richardson
S H
SH
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001427:Bacterial Toxins / Q000737:chemistry / Q000302:isolation & purification* / Q000494:pharmacology; D016466:CHO Cells / Q000187:drug effects*; D048429:Cell Size / Q000187:drug effects; D002772:Cholera Toxin / Q000276:immunology; D006224:Cricetinae; D003429:Cross Reactions; D000242:Cyclic AMP / Q000378:metabolism; D004269:DNA, Bacterial / Q000235:genetics; D005798:Genes, Bacterial; D007526:Isoelectric Point; D008970:Molecular Weight; D014733:Vibrio / Q000737:chemistry / Q000472:pathogenicity*; D014882:Water-Electrolyte Balance / Q000187:drug effects
D000596:Amino Acids; D001427:Bacterial Toxins; D004269:DNA, Bacterial; D002772:Cholera Toxin; D000242:Cyclic AMP