Purification and characterization of the soluble hemagglutinin (cholera lectin)( produced by Vibrio cholerae.
The soluble hemagglutinin (HA) (cholera lectin) produced by Vibrio cholerae strain CA401 was purified to apparent homogeneity by a sequence of ammonium sulfate fractionation, gel filtration, and preparative isoelectric focusing. Soluble HA activity was found to focus at three different pIs, 6.3, 5.3, and 4.7. Each of the factors migrated as a large-molecular-weight protein in sodium dodecyl sulfate-polyacrylamide gel electrophoresis under normal conditions, and each, upon heating in sodium dodecyl sulfate was found to dissociate into 32,000-molecular-weight subunits. Treating the samples with a reducing agent did not affect their mobility. Each gave a reaction of immunological identity with antiserum prepared against the others. Thus, there are apparently three distinct pH isotypes of soluble HA which exist as noncovalently associated polymers of 32,000-molecular-weight subunits. Electron microscopy of purified preparations revealed long filamentous polymers. The molecule does not stain as a glycoprotein; it is hydrophobic; it is inactivated during incubation at 25, 37, or 60 degrees C; and it has significant protease activity. The protease activity likewise focused at pH values of 6.3 and 5.3 to 4.7, and it was inhibited by antiserum against the HA. However, whereas the HA is active at 4 degrees C, the protease is not. The soluble HA is, therefore, a bifunctional molecule capable of mediating hemagglutination and proteolysis. Its amino acid composition is reported. Fab fragments of antibody against the purified HA inhibited attachment of heterologous serotype-biotype V. cholerae to infant rabbit small bowel.
D001426:Bacterial Proteins / Q000302:isolation & purification*; D006388:Hemagglutinins / Q000302:isolation & purification*; D007526:Isoelectric Point; D008854:Microscopy, Electron; D008970:Molecular Weight; D010447:Peptide Hydrolases / Q000378:metabolism; D012995:Solubility; D013696:Temperature; D014734:Vibrio cholerae / Q000032:analysis*
D001426:Bacterial Proteins; D006388:Hemagglutinins; C034971:cholera lectin; D010447:Peptide Hydrolases