title: Purification and properties of a 28-kilodalton hemolytic and mosquitocidal protein toxin of Bacillus thuringiensis subsp. darmstadiensis 73-E10-2.
abstract: The mosquitocidal crystal of Bacillus thuringiensis subsp. darmstadiensis 73-E10-2 was purified, bioassayed against third-instar Aedes aegypti larvae (50% lethal concentration, 7.5 micrograms/ml), and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, revealing polypeptides of 125, 50, 47, and 28 kilodaltons (kDa). When solubilized and proteolytically activated by insect gut proteases or proteinase K, the crystal was cytotoxic to insect and mammalian cells in vitro and was hemolytic. By using nondenaturing polyacrylamide gel electrophoresis, a polypeptide of 23 kDa, derived from the 28-kDa protoxin, was identified which was hemolytic and cytotoxic to Aedes albopictus, A. aegypti, and Choristoneura fumiferana CF1 insect cell lines. The 23-kDa polypeptide was purified by ion-exchange chromatography and gave 50% lethal dose values of 3.8, 3.3, and 6.9 micrograms/ml against A. albopictus, A. aegypti, and C. fumiferana CF1 cells lines, respectively. Cytotoxicity in vitro was both dose and temperature dependent, with a sigmoidal dose-response curve. The cytotoxicity of the 23-kDa toxin and the solubilized and proteolytically activated delta-endotoxin was inhibited by a range of phospholipids containing unsaturated fatty acids and by triglyceride and diglyceride dispersions. An interaction with membrane phospholipids appears important for toxicity. Polyclonal antisera prepared against the 23-kDa polypeptide did not cross-react with polypeptides in the native crystals of four other mosquitocidal strains.
authors.affiliation: Department of Biochemistry, University of Cambridge, United Kingdom.
authors.forename: F A
authors.initials: FA
authors.lastname: Drobniewski
authors.forename: D J
authors.initials: DJ
authors.lastname: Ellar
mesh_terms: D000818:Animals; D001413:Bacillus thuringiensis / Q000032:analysis*; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000276:immunology / Q000302:isolation & purification* / Q000494:pharmacology; D001427:Bacterial Toxins; D015153:Blotting, Western; D003429:Cross Reactions; D009033:Culicidae / Q000187:drug effects*; D046508:Culture Techniques; D004731:Endotoxins; D004789:Enzyme Activation / Q000187:drug effects; D005231:Fatty Acids, Unsaturated; D006460:Hemolysin Proteins; D007422:Intestines / Q000201:enzymology; D007814:Larva; D008081:Liposomes; D008563:Membrane Lipids / Q000378:metabolism; D008970:Molecular Weight; D010447:Peptide Hydrolases / Q000378:metabolism; D013329:Structure-Activity Relationship
chemical_list: D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D004731:Endotoxins; D005231:Fatty Acids, Unsaturated; D006460:Hemolysin Proteins; D008081:Liposomes; D008563:Membrane Lipids; C030956:insecticidal crystal protein, Bacillus Thuringiensis; D010447:Peptide Hydrolases