Binding of the delta endotoxin from Bacillus thuringiensis to brush‐border membrane vesicles of the cabbage butterfly (Pieris brassicae)

Abstract

The insecticidal delta endotoxin of Bacillus thuringiensis was labeled with iodine-125. Brush-border membrane vesicles, prepared from the midgut epithelium of Pieris brassicae larvae, known to be highly susceptible to the toxin, and from a non-target tissue: the small intestine of rat, were examined for binding of '251-toxin. The toxin was bound specifically only to insect vesicles. Its binding to the insect membrane system was competitively inhibited by '271-toxin and non-iodinated toxin, whereas the binding of the 1251-toxin to the mammalian membrane system was not affected by unlabeled toxin. Vesicles of P. brassicae possess two individual bindingsite populations for iodinated toxin with dissociation constants of 46 nM and 490 nM. The Hill coefficients of both sites were approximately 1 and the binding capacities were 0.2 pmol and 30 pmol/mg vesicle protein for the high and the low-affinity sites respectively. The estimation of the dissociation constant for non-iodinated toxin, using a competition experiment, revealed only one binding-site population which possessed a dissociation constant of 235 nM. It is concluded that this is the binding site for the native toxin. This site was sensitive towards treatment with proteases or mixed glycosidases. It is suggested that it is a protein or a glycoprotein.The delta endotoxin of Bacillus thuringiensis is a proteinaceous, crystalline insecticide, toxic to the larval stages of several important pest insects in agriculture and forestry. The crystals are composed of subunits (protoxin) with a molecular mass of about 130 kDa [l -31. The toxic moiety is located on the N-terminal half of the protein [4-61 and has a molecular mass of approximately 60 -68 kDa. The molecular mass of the protoxin and that of the activated toxin vary depending on the strain and on the proteolytic procedure used for the activation. In the case of the strain studied here, activation of the protoxin (136 kDa) by trypsin leads to toxic fragments with molecular masses of 55 kDa and 70 kDa. In vitro the protoxin needs proteolytic activation to exhibit toxicity against insect cell cultures [7, 81. In vivo the protoxin is enzymatically activated within the digestive tract. Furthermore, the toxins of various strains differ in their host spectrum [9, 101. The delta endotoxin destroys the gut epithelia of susceptible insect larvae [ll]; the results of several studies have shown that the permeability of the gut membrane undergoes changes (for review, see [12]).

An analysis of Bacillus thuringiensisδ‐endotoxin action on insect‐midgut‐membrane permeability using a light‐scattering assay

“…The slow activation of CryIB to a 55-kDa polypeptide by l? brussicue gut extract was similar to the action of trypsin on this protein (Hofmann et al, 1988b).…”

Binding of Bacillus sphaericus binary toxin to a specific receptor on midgut brush‐border membranes from mosquito larvae

“…Dissociation constants and binding site concentrations for other insecticidal crystal proteins such as B. thuringiensis aendotoxins have been reported for either susceptible or resistant Lepidoptera larvae (Hofmann, 1988a;1988b;Van Rie et al, 1989;Ferrk et al, 1991). For susceptible insects, the Kd for most 8-endotoxins (0.5 -10 nM) were in the same range as those for B. sphaericus binary toxin, and too high to be determined for field or laboratory resistant species (Garczynski et al, 1991 ;E'erri: et al, 1991).…”

Safety Evaluation of Bt Plants

“…No Cry toxin receptors have been identified on the membrane of mammalian cells 27,46 . Tsuda et al succeeded in making an unsusceptible mammalian cell susceptible to Cry toxin by transferring the receptor gene derived from silkworm (Bombyx mori) to the cell 56 .…”