Summary The insecticidal crystal proteins of Bacillus thuringiensis show a high degree of specificity. In vitro binding studies with several crystal proteins demonstrated a correlation between toxicity and binding to receptors of larval midgut epithelial cells. In order to study the domain‐function relationships of the toxic fragment, hybrid crystal proteins based on CrylA(b) and CrylC were constructed. Two out of 11 hybrid proteins constructed exhibited insecticidal activity. Both displayed an insectidial spectrum similar to that of the parental crystal protein from which the C ‐terminal part of the toxic fragment originated. In addition, in vitro binding studies directly demonstrated the involvement of the C ‐terminal part of the toxic fragment in receptor binding. These results demonstrate that the C ‐terminal part of the toxic fragment determines specific receptor binding, which in turn determines, to a large extent, the insect specificity.

Bacillus thuringiensis

10.1111/j.1365-2958.1991.tb01988.x

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Citations (33)

Tight agonist binding may prevent the correct interpretation of agonist competition binding curves for alpha 2-adrenergic receptors.

Molecular Pharmacology (1987)

André Convents J.-P. De Backer Daniel Convents Georges Vauquelin

alpha 2-Adrenergic receptors in calf retina membranes can be specifically labeled with the tritiated antagonist 3H-RX 781094. Saturation binding occurs to a single class of noncooperative sites. The number of sites amounts to 1070 +/- 243 and 935 +/- 178 fmol/mg of protein, and the equilibrium dissociation constants equal 1.8 +/- 0.4 and 3.8 +/- 0.3 nM at 25 degrees and 37 degrees, respectively. Binding is rapid, equilibrium being reached within 5 min, and is reversible. At both temperatures, (-)-epinephrine competition binding curves are shallow in the presence of magnesium ions. The curves, obtained for incubation periods varying between 5 and 60 min, are superimposable at 37 degrees. Computer-assisted analysis indicates that approximately 75% of the receptors (RH sites) display high agonist affinity for (-)-epinephrine as well as for the other agonists tested: (-)-norepinephrine, clonidine, and UK 14304. However, the (-)-epinephrine competition curves display a time-dependent leftward shift at 25 degrees. This can be attributed to an increase in agonist affinity for the RH sites. Addition of 0.1 mM Gpp(NH)p causes a marked steepening and rightward shift of the curves, at both 25 and 37 degrees. These curves are superimposable for all of the incubation times tested. The nonequilibrium of agonist competition binding at 25 degrees can be attributed to slow dissociation of the agonist (i.e., tight binding) when the receptor is coupled to the regulatory component Ni. This dissociation rate can be measured by preincubation of the membranes with 10 microM (-)-epinephrine, followed by extensive washing and incubation with 3H-RX 781094 for increasing lengths of time. The first order rate of agonist dissociation (i.e., receptor recovery) is appreciably faster at 37 degrees than at 25 degrees: i.e., 0.029 min-1 and 0.0044 min-1, respectively. These findings are confirmed by kinetic experiments using the radiolabeled agonist 3H-UK 14304. Slow agonist dissociating kinetics may prevent the correct evaluation of the agonist binding parameters by computerized analysis of competition binding curves when the incubation time is too short, especially at low temperature.

Dissociation constant

10.1016/s0026-895x(25)13762-0

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Citations (23)

Intramolecular Proteolytic Cleavage ofBacillus thuringiensisCry3A δ-Endotoxin May Facilitate Its Coleopteran Toxicity

Journal of Invertebrate Pathology (1997)

Joe Carroll Daniel Convents Jo Van Damme Annemie Boets Jeroen Van Rie

Leptinotarsa

Bacillus thuringiensis

Colorado potato beetle

Bacillaceae

Cleavage (geology)

10.1006/jipa.1997.4656

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Citations (76)

The Bacillus thuringiensis delta-endotoxin. Evidence for a two domain structure of the minimal toxic fragment.

Journal of Biological Chemistry (1990)

Daniel Convents C Houssier Ignace Lasters Marc Lauwereys

The conformational characteristics of the minimal toxic fragment of the delta-endotoxin from Bacillus thuringiensis berliner 1715 were examined by fluorescence and circular dichroism spectroscopy. This insecticidal protein, specifically toxic to lepidopteran species, was found to consist of two structural domains. Experimental evidence for this conclusion was provided by biphasic guanidine hydrochloride unfolding curves at different pH values and electrophoretic patterns of protease digests. Two stable fragments of comparable molecular weight were obtained using four different broad specificity proteolytic enzymes. A secondary structure model was constructed using seven B. thuringiensis toxin sequences. These toxins were selected on the basis of their limited sequence homology and represent all known insecticidal specificities. Despite this divergence, a consensus secondary structure pattern was obtained, confirming the structural homology among the toxins. The N-terminal halves of all toxins are predicted to be relatively rich in alpha-helix structure and the C-terminal parts to contain alternating beta-strand and coil structures. The latter seems characteristic for a beta-sheet conformation. Comparing this model to the unfolding data obtained by circular dichroism, whose far UV signal gives a measure of the alpha-helix content, allowed us to delineate the structural domains into the primary structure.

Random coil

Bacillus thuringiensis

Guanidine

Homology

Helix (gastropod)

10.1016/s0021-9258(19)40023-9

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Citations (42)

Structure-function relationship of the Bacillus thuringiensis d-endotoxin

Daniel Convents

Bacillus thuringiensis

Source

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Two structural domains as a general fold of the toxic fragment of the Bacillus thuringiensisδ‐endotoxins

European Journal of Biochemistry (1991)

Daniel Convents Marc Cherlet J. Van Damme Ignace Lasters Marc Lauwereys

The unfolding by guanidine hydrochloride of the toxic fragment of a Bacillus thuringiensis toxin belonging to the CryIC class reveals a two‐step denaturation under both acid and alkaline conditions. This demonstrates the existence of two structural domains as building blocks for this toxin. Protease digests performed on a CryIA(b) and CryIC B. thuringiensis toxin, under native and partially denatured conditions, confirm this conclusion. Whereas the native CryIC toxin is completely protease resistant, the CryIA(b) toxin, earlier described as consisting of two structural domains [Convents, D., Houssier, C., Lasters, I. & Lauwereys, M. (1990) J. Biol. Chem. 265 , 1369–1375], is cleaved by three proteases, resulting in at least two common fragments. This suggests that this toxin is built up of two globular units linked by a protease‐susceptible linker. The detection of a stable intermediate along the denaturation curve allows us to study and compare the consecutive unfolding of the structural domains for both toxins. By addition of a protease, under conditions where such an unfolding intermediate exists, a single denaturation phase can be assigned to a specific part of the protein. These experiments lead to the conclusion that the domain whose stability is highly dependent on pH corresponds to the N‐terminal half of both toxins.

Bacillus thuringiensis

Guanidine

Denaturation (fissile materials)

Linker

Bacillaceae

10.1111/j.1432-1033.1991.tb15747.x

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Citations (31)

Glycoprotein nature of α₂‐adrenergic receptors labeled with p‐azido[³H] clonidine in calf retina membranes

FEBS Letters (1988)

André Convents Jean‐Paul De Backer Edilbert Van Driessche Daniel Convents Sonia Beeckmans

α 2 ,‐Adrenergic receptors in calf retina membranes can be specifically labeled with the tritiated agonist p ‐azido[ 3 H]clonidine. Saturation binding in the dark occurs with high affinity (1.3 ± 0.3 nM) to a single class of sites (1122 ± 67 fmol/mg protein). Irradiation of the membrane‐bound radioligand results in the labeling of a peptide band with an apparent size of 65 kDa and a characteristic pharmacological profile for an α 2 ‐adrenergic receptor. The carbohydrate moieties of the α 2 ‐receptor are characterized by lectin affinity chromatography and glycosidase treatment. The Nonidet P‐40‐solubilized, p ‐azido[ 3 H]clonidine‐labeled receptors are completely retained by Con A‐ as well as WGA‐Sepharose columns. Neuraminidase, α‐mannosidase and TFMS do not affect the electrophoretic mobility of the receptor on SDS‐PAGE whereas endoglycosidase F reduces the apparent size to 45 kDa.

Endoglycosidase H

Photoaffinity labeling

10.1016/0014-5793(88)80142-x

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Citations (5)

p-アジド[ 3 H]クロニジンで標識したウシ網膜中のアドレナリンα 2 受容体の糖蛋白質としての性質

FEBS Letters (1988)

André Convents De Backer J-P Van Driessche E Daniel Convents S Beeckmans

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