Selective purification by thiol-disulfide interchange chromatography of alveolysin, a sulfhydryl-activated toxin of Bacillus alvei. Toxin properties and interaction with cholesterol and liposomes.
Alveolysin has been purified in milligram quantities to apparent immunochemical and electrophoretic homogeneity from the supernatant culture fluid of Bacillus alvei. Purification involved ultrafiltration with concomitant molecular sieving on Amicon hollow fibers, thiol-disulfide interchange chromatography on thiopropyl-Sepharose 6B, and gel filtration on Sephacryl S-200 and Bio-Gel P-100. The purified toxin was found to be a single polypeptide chain of 582 amino acids (Mr = 63,000) free of carbohydrate, with alanine as NH2-terminal amino acid. Nonpolar amino acids accounted for 40% of the total residues. Immunochemical analysis of the precipitation curve of the toxin with anti-alveolysin immune sera indicated the presence of approximately 13 epitopes. The toxin exhibited a specific activity of 10(6) hemolytic units/mg of protein and behaved as an amphiphilic protein as inferred from charge-shift electrophoresis in a three-detergent system, suggesting the presence of substantial hydrophobic region(s). The inhibitory effects of various thiol reagents and that of cholesterol and related 3 beta-hydroxysterols with aliphatic side chains at carbon 17 indicated that alveolysin is a typical member of the group of bacterial--SH-activated toxins. The stoichiometry of cholesterol-toxin interaction was apparently equimolar. The complexes between alveolysin and [3H]cholesterol were characterized by sucrose gradient ultracentrifugation. Cholesterol-containing liposomes were disrupted by the toxin.
D000596:Amino Acids / Q000032:analysis; D001407:Bacillus / Q000032:analysis*; D002784:Cholesterol / Q000378:metabolism*; D002850:Chromatography, Gel / Q000379:methods; D006460:Hemolysin Proteins / Q000302:isolation & purification* / Q000378:metabolism; D005779:Immunodiffusion; D008081:Liposomes / Q000378:metabolism*; D009930:Organic Chemicals
D000596:Amino Acids; D006460:Hemolysin Proteins; D008081:Liposomes; D009930:Organic Chemicals; C014963:alveolysin; D002784:Cholesterol