Irreversible Binding Kinetics of Bacillus thuringiensis CryIA δ-Endotoxins to Gypsy Moth Brush Border Membrane Vesicles Is Directly Correlated to Toxicity

Abstract

To examine the binding of Bacillus thuringiensis ␦-endotoxins, CryIAa, CryIAb, and CryIAc, to Lymantria dispar (gypsy moth) brush border membrane vesicles (BBMV), saturation kinetic analyses were conducted according to a two-step interaction schemefor ␦-endotoxin binding to BBMV, rather than the onestep reversible binding presented in prior reports.The order of toxicity of the ␦-endotoxins, as measured by the dose required for a 50% inhibition of weight gain (ID 50 ), was CryIAa (77.3 ng) > CryIAb (157 ng) > CryIAc (187 ng). While both the maximum extent of binding, B max , and the half-maximum insertion rate concentration, K1 ⁄2 , was observed to be indirectly related to toxicity, the rate constant of irreversible binding, k 2 , was found to be directly correlated to toxicity.

Binding Kinetics of Bacillus Sphaericus Binary Toxin to Midgut Brush‐Border Membranes of Anopheles and Culex sp. Mosquito Larvae

“…In contrast, studies conducted with B. thuringiensis toxins and BBMF from Lepidoptera larvae showed that toxicity strongly correlated with irreversible toxin binding. Binding of B. thuringiensis crystals CrylB, CrylAb, CrylC to BBMF of different species of Lepidoptera was irreversible (Hofmann et al, 1988;Van Rie et al, 1989;Liang et al, 1995). Although in some cases, toxin binding was partially reversible.…”

Specificity determinants for Cry insecticidal proteins: Insights from their mode of action

“…For instance, Cry toxins do bind to mammalian proteins in vitro (Shimada et al, 2006b;Vazquez-Padron et al, 2000), but this interaction is mostly non-specific (Hofmann et al, 1988) and thus is not conducive to formation of toxin pores (Shimada et al, 2006a). More difficult to explain are reports of high affinity toxin binding, which suggests toxin insertion in the membrane (Liang et al, 1995), associated with low susceptibility, as in the case of Cry1Ac and Spodoptera frugiperda (Garczynski et al, 1991) or Lymantria dispar (Wolfersberger, 1990). While these observations may be explained by pre-binding specificity determinants, it is also plausible that high affinity binding (i.e.…”

Bacillus thuringiensis Cry1Ac toxin interaction with Manduca sexta aminopeptidase N in a model membrane environment

“…However, very strong binding to target membranes, presumably resulting from toxin insertion into the bilayer, was shown to follow the initial binding event [22] and to be an important determinant of activity in i o [23]. Liang et al [24] began to analyse Cry toxin binding quantitatively in terms of a two-step model, the first step being a low-affinity reversible interaction with the receptor, quickly followed by a higher-affinity, effectively irreversible, association with the target membrane. This approach demonstrated that the activity of different Cry1A toxins against Lymantria dispar was correlated with the highaffinity binding rate constant.…”

“…The fact that Cry toxin binding to BBMV from susceptible insects is quickly irreversible [22], indicating toxin insertion into the membrane, could account for the higher affinity of Cry1Ac for intact BBMV. Indeed, in a number of studies it is the irreversible binding step that has been shown to be correlated with toxin activity in i o [23,24].…”