Pore formation activity of Cry1Ab toxin from Bacillus thuringiensis in an improved membrane vesicle preparation from Manduca sexta midgut cell microvilli.
The pore formation activity of Cry1Ab toxin is analyzed in an improved membrane preparation from apical microvilli structures of Manduca sexta midgut epithelium cells (MEC). A novel methodology is described to isolate MEC and brush border membrane vesicles (BBMV) from purified microvilli structures. The specific enrichment of apical membrane enzyme markers aminopeptidase (APN) and alkaline phosphatase (APh) were 35- and 22-fold, respectively, as compared to the whole midgut cell homogenate. Ligand-blot and Western-blot experiments showed that Cry1A specific receptors were also enriched. The pore formation activity of Cry1Ab toxin was fourfold higher in the microvilli membrane fraction that showed low intrinsic K+ channels and higher APN and APh activities than in the basal-lateral membrane fraction harboring high intrinsic K+ channels. These data suggest that basal-lateral membrane was separated from apical membrane.This procedure should allow more precise studies of the interaction of Cry toxins with their target membranes, avoiding unspecific interaction with other cellular membranes, as well as the study of the pore formation activity induced by Cry toxins in the absence of endogenous channels from M. sexta midgut cells.
Bravo
Alejandra
A
Departamento de Microbiología, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Cuernavaca, Mexico. bravo@ibt.unam.mx
Miranda
Raúl
R
Gómez
Isabel
I
Soberón
Mario
M
D000469:Alkaline Phosphatase / Q000032:analysis; D000626:Aminopeptidases / Q000032:analysis; D000818:Animals; D001413:Bacillus thuringiensis; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000737:chemistry / Q000494:pharmacology*; D001427:Bacterial Toxins / Q000494:pharmacology*; D015415:Biomarkers / Q000032:analysis; D002458:Cell Fractionation; D004731:Endotoxins / Q000737:chemistry / Q000494:pharmacology*; D006460:Hemolysin Proteins; D007422:Intestines / Q000187:drug effects / Q000201:enzymology; D007814:Larva / Q000187:drug effects; D018402:Manduca / Q000187:drug effects*; D008565:Membrane Proteins / Q000737:chemistry; D010539:Permeability; D010572:Pest Control, Biological; D013347:Subcellular Fractions / Q000201:enzymology
D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D015415:Biomarkers; D004731:Endotoxins; D006460:Hemolysin Proteins; D008565:Membrane Proteins; C030956:insecticidal crystal protein, Bacillus Thuringiensis; D000469:Alkaline Phosphatase; D000626:Aminopeptidases