Isolation and characterization of brush border membrane vesicles from whole Aedes aegypti larvae

Studies of the binding interactions of dipteran-specific Bacillus thuringiensis delta-endotoxins are hindered by the lengthy midgut dissection procedure needed for preparation of brush border membrane vesicles. In an attempt to resolve this problem, brush border membrane vesicles were isolated from homogenates of whole Aedes aegypti larvae by a modification of the method of Macintosh ct al. (1994). These preparations were found to resolve well on SDS-PAGE and appeared as spherical vesicles of various sizes under electron microscopic examination. Specific activities of the brush border membrane marker enzymes alkaline phosphatase and leucine amino acid arylamidase were enriched 10.9- and 10.7-fold, respectively. Direct binding experiments using S-35-labeled B. thuringiensis CryIC toxin revealed a single class of high-affinity binding sites with a dissociation constant (K-d) of 27 +/- 0.6 nM and a maximum binding capacity (B-max) of approximately 27 +/- 1.2 pmol/mg BBMV protein. These binding parameters are similar to those of vesicles prepared from isolated midguts, indicating that whole larval brush border membrane vesicles are suitable for in vitro membrane binding studies. (C) 1999 Academic Press.