Stable immobilization of lipid vesicles for kinetic studies using surface plasmon resonance.
In order to study the kinetics of binding between membrane vesicle surface receptors to the lepidopteran insecticidal toxins from Bacillus thuringiensis using surface plasmon resonance, we have developed a technique to immobilize membrane vesicles purified from the brush border of dissected guts from the lepidopteran insect pest Choristoneura fumiferana. Two methods using immobilized immunoglobulins against either avidin or biotin were successful in achieving stable immobilization of the vesicles (> 1.5 h). Specificity of the immobilized receptors exposed on the vesicle surface was demonstrated, in part, by the inability of bovine serum albumin to bind to the immobilized brush border membrane vesicles. Homologous and heterologous competition experiments further demonstrated specific binding of trypsin-activated CryIA(c) toxin to the cell-surface receptors on the vesicles. Kinetic rate constants for activated cryIA(b) toxin binding to brush border vesicles were determined, revealing the presence of a high-affinity receptor on the surface of the immobilized brush border membrane vesicles.
Masson
L
L
Biotechnology Research Institute, National Research Council of Canada, Montreal, Quebec.
Mazza
A
A
Brousseau
R
R
D000818:Animals; D001180:Arthropod Venoms / Q000378:metabolism; D001413:Bacillus thuringiensis; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000378:metabolism* / Q000633:toxicity; D001427:Bacterial Toxins / Q000378:metabolism* / Q000633:toxicity; D015374:Biosensing Techniques; D004355:Drug Stability; D004731:Endotoxins / Q000378:metabolism* / Q000633:toxicity; D006460:Hemolysin Proteins; D007700:Kinetics; D050356:Lipid Metabolism; D008055:Lipids / Q000737:chemistry; D008081:Liposomes; D008566:Membranes / Q000378:metabolism; D008871:Microvilli / Q000378:metabolism; D008954:Models, Biological; D010572:Pest Control, Biological; D013379:Substrate Specificity
D001180:Arthropod Venoms; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D004731:Endotoxins; D006460:Hemolysin Proteins; D008055:Lipids; D008081:Liposomes; C030956:insecticidal crystal protein, Bacillus Thuringiensis