Preparation and partial characterization of amino acid transporting brush border membrane vesicles from the larval midgut of the cabbage butterfly (Pieris brassicae)

Wolfersberger, Michael G.; Luethy, Peter; Maurer, Andreas; Parenti, Paolo; Sacchi, F.; Giordana, B.; Hanozet, Giorgio M.

doi:10.1016/0300-9629(87)90334-3

Cited by 575 publications

(324 citation statements)

References 31 publications

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“…This fraction is referred to as the crude membrane. BBMV from B. mori midguts was prepared by the method of Wolfersberger et al 27 ) BBMV from rabbit kidney cortexes and small intestines were prepared by the method of Kessler et al 28 ) Binding assay. Membrane fractions were incubated with 125I-Iabeled toxins in the presence or absence of excess unlabeled toxin in PBS-BSA at room temperature (23°C).…”

Section: Methodsmentioning

confidence: 99%

Specific Toxicity of δ-Endotoxins fromBacillus thuringiensistoBombyx mori

Ihara

Kuroda

Wadano

et al. 1993

Bioscience, Biotechnology, and Biochemistry

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Section: Methodsmentioning

confidence: 99%

Specific Toxicity of δ-Endotoxins fromBacillus thuringiensistoBombyx mori

Ihara

Kuroda

Wadano

et al. 1993

Bioscience, Biotechnology, and Biochemistry

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“…Midgut tissue was dissected from fifth instars, washed in ice-cold MET buffer (0.3 M mannitol, 5 mM ethyleneglycoltetraacetic acid (EGTA, Sigma), 17 mM Tris/HCl, pH 7.5), snap-frozen in liquid nitrogen, and stored at −80°C. Brush border membrane vesicles (BBMV) were prepared from the stored tissue by the method of Wolfersberger et al (1987), and subsequently solubilised with CHAPS following the protocol of Liao et al (2005), except that two of the protease inhibitors, pepstatin and 1,10-phenanthroline, were not included. The affinity protocol was essentially as described by Liao et al (2005).…”

Section: Purification Of Cry1ac-binding Proteinsmentioning

confidence: 99%

Diversity of aminopeptidases, derived from four lepidopteran gene duplications, and polycalins expressed in the midgut of Helicoverpa armigera: Identification of proteins binding the δ-endotoxin, Cry1Ac of Bacillus thuringiensis

Angelucci

Barrett-Wilt²,

Hunt

et al. 2008

Insect Biochemistry and Molecular Biology

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Helicoverpa armigera midgut proteins that bind the Bacillus thuringiensis (Bt) δ-endotoxin Cry1Ac were purified by affinity chromatography. SDS-PAGE showed that several proteins were eluted with N-acetylgalactosamine and no further proteins were detected after elution with urea. Tandem mass spectral data for tryptic peptides initially indicated that the proteins resembled aminopeptidases (APNs) from other lepidopterans and cDNA sequences for seven APNs were isolated from H. armigera through a combination of cloning with primers derived from predicted peptide sequences and established EST libraries. Phylogenetic analysis showed lepidopteran APN genes in nine clades of which five were part of a lepidopteran-specific radiation. The Cry1Ac-binding proteins were then identified with four of the seven HaAPN genes. Three of those four APNs are likely orthologs of APNs characterised as Cry1Ac-binding proteins in other lepidopterans. The fourth Cry1Ac-binding APN has orthologs not previously identified as Cry1Ac-binding partners. The HaAPN genes were expressed predominantly in the midgut through larval development. Each showed consistent expression along the length of the midgut but five of the genes were expressed at levels about two orders of magnitude greater than the remaining two. The remaining mass spectral data identified sequences encoding polycalin proteins with multiple lipocalin-like domains. A polycalin has only been previously reported in another lepidopteran, Bombyx mori, but polycalins in both species are now linked with binding of Bt Cry toxins. This is the first report of hybrid, lipocalin-like domains in shorter polycalin sequences that are not present in the longest sequence. We propose that these hybrid domains are generated by alternative splicing of the mRNA.

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“…BBMV to be used in binding assays were prepared according to the method described by Wolfersberger [20]. The same protocol was used for the preparation of the BBMV used in oligomerization assays, except that EGTA was excluded from the buffers.…”

Section: Bbmv Preparationmentioning

confidence: 99%

Mutations in the Bacillus thuringiensis Cry1Ca toxin demonstrate the role of domains II and III in specificity towards Spodoptera exigua larvae

Herrero

González‐Cabrera

Ferré

et al. 2004

Biochemical Journal

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Several mutants of the Bacillus thuringiensis Cry1Ca toxin affected with regard to specific activity towards Spodoptera exigua were studied. Alanine was used to replace single residues in loops 2 and 3 of domain II (mutant pPB19) and to replace residues 541-544 in domain III (mutant pPB20). Additionally, a Cry1Ca mutant combining all mutations was constructed (mutant pPB21). Toxicity assays showed a marked decrease in toxicity against S. exigua for all mutants, while they retained their activity against Manduca sexta, confirming the importance of these residues in determining insect specificity. Parameters for binding to the specific receptors in BBMV (brush border membrane vesicles) of S. exigua were determined for all toxins. Compared with Cry1Ca, the affinity of mutant pPB19 was slightly affected (2-fold lower), whereas the affinity of the mutants with an altered domain III (pPB20 and pPB21) was approx. 8-fold lower. Activation of Cry1Ca protoxin by incubation with S. exigua or M. sexta BBMV revealed the transient formation of an oligomeric form of Cry1Ca. The presence of this oligomeric form was tested in the activation of the different Cry1Ca mutants, and we found that those mutated in domain II (pPB19 and pPB21) could not generate the oligomeric form when activated by S. exigua BBMV. In contrast, when oligomerization was tested using BBMV prepared from M. sexta, all of the Cry1Ca mutants showed the formation of a similar oligomeric form as did the wild-type toxin. Our results show how modification of insect specificity can be achieved by manipulation of different parts of the toxin structure involved in different steps of the mode of action of B. thuringiensis toxins.

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Preparation and partial characterization of amino acid transporting brush border membrane vesicles from the larval midgut of the cabbage butterfly (Pieris brassicae)

Cited by 575 publications

References 31 publications

Specific Toxicity of δ-Endotoxins fromBacillus thuringiensistoBombyx mori

Specific Toxicity of δ-Endotoxins fromBacillus thuringiensistoBombyx mori

Diversity of aminopeptidases, derived from four lepidopteran gene duplications, and polycalins expressed in the midgut of Helicoverpa armigera: Identification of proteins binding the δ-endotoxin, Cry1Ac of Bacillus thuringiensis

Mutations in the Bacillus thuringiensis Cry1Ca toxin demonstrate the role of domains II and III in specificity towards Spodoptera exigua larvae

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