6533b7d5fe1ef96bd1264616

RESEARCH PRODUCT

Proteolytic Processing ofBacillus thuringiensisCryIIIA Toxin and Specific Binding to Brush-Border Membrane Vesicles ofLeptinotarsa decemlineata(Colorado Potato Beetle)

subject

description

Abstract The mode of action of Bacillus thuringiensis insecticidal proteins in lepidopteran insects is known to involve five steps: ingestion, solubilization, protease activation, binding to midgut membrane receptors, and disruption of the intestinal membrane. Two of these steps, protease activation and binding to midgut membrane receptors, have been analyzed in the major potato pest, the coleoptera Leptinotarsa decemlineata (Colorado potato beetle). Unlike recently proposed, after treatment of the coleopteran-specific B. thuringiensis toxin CryIIIA with gut content from the Colorado potato beetle, a 42-kDa processing polypeptide has been identified. The study of binding to midgut membrane receptors has demonstrated specific and saturable binding of chymotrypsinized CryIIIA to brush-border membrane vesicles from the Colorado potato beetle. The affinity constant and the concentration of binding sites values ( K d = 37.5 ± 8.6 n M, R t = 17 ± 4 pmol/mg of protein) were in the range of the ones previously estimated for low affinity binding sites in lepidopteran insects. Taking into account that CryIIIA can be proteolytically processed by the Colorado potato beetle midgut proteases, along with the fact that, in our hands, binding can be demonstrated only if the toxin is chymotrypsin processed, these results suggest that the mode of action of the coleopteran-specific B. thuringiensis toxin CryIIIA is probably the same as that of lepidopteran-specific toxins.