Purification and characterization of heat-labile toxin from Bordetella bronchiseptica.
The heat-labile toxin (HLT) of Bordetella bronchiseptica was purified successively from sonic extracts of phase I organisms grown in Stainer-Scholte medium, by partition in hydrophobic interaction, sucrose density gradient centrifugation, gel filtration through Sepharose 4B and 6B, isoelectric precipitation and isoelectric focusing. The purified HLT was homogeneous by disc polyacrylamide gel electrophoresis and the gel diffusion-test, and free of detectable hemagglutinin and endotoxin activity. A 386-fold purification over the crude extract was obtained at a yield of about 28%, and a minimum dose of 0.9 ng was dermonecrotizing with a lesion 5 mm in diameter in guinea pigs and induced splenoatrophy. The mouse LD50 was 200 ng (intraperitoneal) or 70 ng (intravenous). The HLT was found to be a simple protein with an isoelectric point of pI 6.9. It has a molecular weight of 102,000 estimated by Sepharose 6B gel filtration and was found to consist of two different types of polypeptide by SDS-polyacrylamide gel electrophoresis, their molecular weights being 30,000 and 20,000. Amino acid analysis showed 15 common amino acid residues, and methionine, cysteine and tryptophan were undetectable. The HLT crystallized by methylpentanediol showed a block form. The HLT was inactivated at 56 C when heated for 10 min, and at above pH 9 and below pH 4.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001427:Bacterial Toxins / Q000302:isolation & purification* / Q000633:toxicity; D001884:Bordetella / Q000302:isolation & purification / Q000472:pathogenicity*; D001885:Bordetella Infections / Q000662:veterinary; D003460:Crystallization; D005260:Female; D006168:Guinea Pigs; D051379:Mice; D008813:Mice, Inbred ICR; D008970:Molecular Weight; D009336:Necrosis; D012867:Skin / Q000187:drug effects / Q000473:pathology; D013552:Swine; D013553:Swine Diseases / Q000382:microbiology; D011503:Transglutaminases; D010566:Virulence Factors, Bordetella
D000596:Amino Acids; D001427:Bacterial Toxins; D010566:Virulence Factors, Bordetella; C060969:dermonecrotic toxin, Bordetella; D011503:Transglutaminases