scholarly journals Toxicity, Binding, and Permeability Analyses of FourBacillus thuringiensis Cry1 δ-Endotoxins Using Brush Border Membrane Vesicles of Spodoptera exigua and Spodoptera frugiperda

1999 ◽  
Vol 65 (2) ◽  
pp. 457-464 ◽  
Author(s):  
Ke Luo ◽  
David Banks ◽  
Michael J. Adang
Keyword(s):  
Binding Site ◽  
Spodoptera Frugiperda ◽  
Brush Border ◽  
Spodoptera Exigua ◽  
Brush Border Membrane ◽  
Membrane Vesicles ◽  
Brush Border Membrane Vesicles ◽  
Binding Assays ◽  
Competition Binding

ABSTRACT The binding and pore formation properties of four Bacillus thuringiensis Cry1 toxins were analyzed by using brush border membrane vesicles from Spodoptera exigua andSpodoptera frugiperda, and the results were compared to the results of toxicity bioassays. Cry1Fa was highly toxic and Cry1Ac was nontoxic to S. exigua and S. frugiperda larvae, while Cry1Ca was highly toxic to S. exigua and weakly toxic to S. frugiperda. In contrast, Cry1Bb was active against S. frugiperda but only marginally active against S. exigua. Bioassays performed with iodinated Cry1Bb, Cry1Fa, and Cry1Ca showed that the effects of iodination on toxin activity were different. The toxicities of I-labeled Cry1Bb and Cry1Fa against Spodoptera species were significantly less than the toxicities of the unlabeled toxins, while Cry1Ca retained its insecticidal activity when it was labeled with 125I. Binding assays showed that iodination prevented Cry1Fa from binding to Spodoptera brush border membrane vesicles. 125I-labeled Cry1Ac, Cry1Bb, and Cry1Ca bound with high-affinities to brush border membrane vesicles fromS. exigua and S. frugiperda. Competition binding experiments performed with heterologous toxins revealed two major binding sites. Cry1Ac and Cry1Fa have a common binding site, and Cry1Bb, Cry1C, and Cry1Fa have a second common binding site. No obvious relationship between dissociation of bound toxins from brush border membrane vesicles and toxicity was detected. Cry1 toxins were also tested for the ability to alter the permeability of membrane vesicles, as measured by a light scattering assay. Cry1 proteins toxic to Spodoptera larvae permeabilized brush border membrane vesicles, but the extent of permeabilization did not necessarily correlate with in vivo toxicity.

Critical domains in the specific binding of radiolabelled Vip3Af insecticidal protein to brush border membrane vesicles from Spodoptera spp. and cultured insect cells

2021 ◽  
Author(s):  
Yudong Quan ◽  
Maria Lázaro-Berenguer ◽  
Patricia Hernández-Martínez ◽  
Juan Ferré
Keyword(s):  
Brush Border ◽  
Binding Sites ◽  
Brush Border Membrane ◽  
Ex Vivo ◽  
Specific Binding ◽  
Membrane Vesicles ◽  
Brush Border Membrane Vesicles ◽  
Cry Proteins ◽  
Competition Binding

Vegetative insecticidal proteins (Vip3) from Bacillus thuringiensis have been used, in combination with Cry proteins, to better control insect pests and as a strategy to delay the evolution of resistance to Cry proteins in Bt crops (crops protected from insect attack by the expression of proteins from B. thuringiensis ). In this study, we have set up the conditions to analyze the specific binding of 125 I-Vip3Af to Spodoptera frugiperda and Spodoptera exigua brush border membrane vesicles (BBMV). Heterologous competition binding experiments revealed that Vip3Aa shares the same binding sites with Vip3Af, but that Vip3Ca does not recognize all of them. As expected, Cry1Ac and Cry1F did not compete for Vip3Af binding sites. By trypsin treatment of selected alanine-mutants, we were able to generate truncated versions of Vip3Af. Their use as competitors with 125 I-Vip3Af indicated that only those molecules containing domains I to III (DI-III and DI-IV) were able to compete with the trypsin-activated Vip3Af protein for binding, and that molecules only containing either domain IV or domains IV and V (DIV and DIV-V) were unable to compete with Vip3Af. These results were further confirmed with competition binding experiments using 125 I-DI-III. In addition, the truncated protein 125 I-DI-III also bound specifically to Sf21 cells. Cell viability assays showed that the truncated proteins DI-III and DI-IV were as toxic to Sf21 cells as the activated Vip3Af, suggesting that domains IV and V are not necessary for the toxicity to Sf21 cells, in contrast to their requirement in vivo. IMPORTANCE This study shows that Vip3Af binding sites are fully shared with Vip3Aa, only partially shared with Vip3Ca, and not shared with Cry1Ac and Cry1F in two Spodoptera spp. Truncated versions of Vip3Af revealed that only domains I to III were necessary for the specific binding, most likely because they can form the functional tetrameric oligomer and because domain III is supposed to contain the binding epitopes. In contrast to results obtained in vivo (bioassays against larvae), domains IV and V are not necessary for the ex vivo toxicity to Sf21 cells.

scholarly journals Alteration of a Cry1A Shared Binding Site in a Cry1Ab-Selected Colony of Ostrinia furnacalis

Toxins ◽  
2022 ◽  
Vol 14 (1) ◽  
pp. 32
Author(s):  
Daniel Pinos ◽  
Yueqin Wang ◽  
Patricia Hernández-Martínez ◽  
Kanglai He ◽  
Juan Ferré
Keyword(s):  
Binding Site ◽  
Brush Border ◽  
Binding Sites ◽  
Brush Border Membrane ◽  
Membrane Vesicles ◽  
Brush Border Membrane Vesicles ◽  
Membrane Receptors ◽  
Cross Resistance ◽  
Ostrinia Furnacalis ◽  
Binding Assays

The Asian corn borer, Ostrinia furnacalis (Guenée, 1854), is a highly damaging pest in Asia and the Pacific islands, and larvae feed mainly from corn crops. To determine the suitability of Bt-corn technology for the future control of this pest, understanding the potential to develop resistance to Cry1Ab and the basis of cross-resistance to other Cry1 proteins is of great interest. Here, we have explored the binding of Cry1A proteins to brush border membrane vesicles from two O. furnacalis colonies, one susceptible (ACB-BtS) and one laboratory-selected with Cry1Ab (ACB-AbR). The insects developed resistance to Cry1Ab and showed cross-resistance to Cry1Aa, Cry1Ac, and Cry1F. Binding assays with radiolabeled Cry1Ab and brush border membrane vesicles from susceptible insects showed that Cry1A proteins shared binding sites, though the results were not conclusive for Cry1F. The results were confirmed using radiolabeled Cry1Aa. The resistant insects showed a reduction of the specific binding of both Cry1Ab and Cry1Aa, suggesting that part of the binding sites were lost or altered. Competition binding assays showed full competition between Cry1Ab and Cry1Aa proteins in the susceptible colony but only partial competition in resistant insects, confirming the alteration of some, but not all, binding sites for these two proteins. The binding site model for Cry1A proteins in O. furnacalis is in agreement with the occurrence of multiple membrane receptors for these proteins.

Potential differences influence amino acid/Na+ symport rates in larval Manduca sexta midgut brush-border membrane vesicles.

1994 ◽  
Vol 189 (1) ◽  
pp. 55-67
Author(s):  
R Parthasarathy ◽  
W R Harvey
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Manduca Sexta ◽  
Brush Border ◽  
Brush Border Membrane ◽  
Membrane Vesicles ◽  
Brush Border Membrane Vesicles ◽  
Rate Determining Step ◽  
Half Saturation Constant

The time-dependent fluorescence intensity of an intravesicular potential-sensitive dye was used to probe the real-time kinetics of potential difference (PD)-dependent amino acid/Na+ symport at pH9 into brush-border membrane vesicles obtained from larval Manduca sexta midgut. Neutral amino acids (alanine, proline) are symported at higher rates as the vesicles are hyperpolarized. The symport rates of acidic (glutamate) and basic (arginine) amino acids are almost PD-independent. The half-saturation constant of alanine is PD-independent between -108 and -78 mV, although the maximal symport velocity increases by half as the voltage is increased. Amino acid throughput is evidently enhanced as the relatively high transmembrane PDs (> 150 mV, lumen positive) measured in vivo are approached. The half-saturation concentrations of Na+ were in the range 15-40 mmol l-1 for most of the amino acids examined and increased with voltage for alanine. The Vmax observed as a function of cation or amino acid concentration increased as the vesicle was hyperpolarized in the case of leucine and alanine. The data support the hypothesis that carrier and substrates are at equilibrium inasmuch as substrate translocation seems to be the rate-determining step of symport.

Indirect coupling of urate and p-aminohippurate transport to sodium in human brush-border membrane vesicles

1996 ◽  
Vol 270 (1) ◽  
pp. F61-F68 ◽  
Author(s):  
F. Roch-Ramel ◽  
B. Guisan ◽  
L. Schild
Keyword(s):  
Brush Border ◽  
Brush Border Membrane ◽  
Membrane Vesicles ◽  
Brush Border Membrane Vesicles ◽  
Indirect Coupling ◽  
Sodium Gradient ◽  
Beta Hydroxybutyrate ◽  
Sodium Cotransport ◽  
Stimulation Of

[14C]urate and p-[14C]aminohippurate (PAH) uptake by human brush-border membrane vesicles (BBMV) were measured in the presence of an inwardly oriented sodium gradient. No direct sodium cotransport was observed. Indirect [14C]urate coupling to sodium transport was demonstrated by cis-stimulation of [14C]urate with nicotinate or pyrazinoate (PZA) in the extravesicular medium but not by adding lactate, alpha-ketoglutarate, or beta-hydroxybutyrate. Indirect sodium coupling of [14C]PAH uptake was observed only when alpha-ketoglutarate was added to the extravesicular medium, a mechanism similar to that of basolateral membranes. The ability for PZA (and nicotinate) to cis-stimulate urate uptake was correlated with a high apparent affinity for the urate/anion exchanger. In urate-loaded vesicles, for identical medium concentrations, [14C]PZA uptake via the urateanion exchanger was 10 times higher than [14C]lactate uptake. Such high PZA affinity for the urate exchanger, working in parallel with PZA sodium cotransport can account for the stimulation of urate reabsorption by PZA in vivo.

Cholera toxin facilitates calcium transport in jejunal brush border vesicles

1986 ◽  
Vol 64 (5) ◽  
pp. 568-574 ◽  
Author(s):  
David D. Maenz ◽  
G. W. Forsyth
Keyword(s):  
Cholera Toxin ◽  
Brush Border ◽  
Brush Border Membrane ◽  
Equilibrium Dialysis ◽  
Membrane Vesicles ◽  
Second Messenger ◽  
Brush Border Membrane Vesicles ◽  
Ionophore A23187

Cholera toxin is very well characterized in terms of the activation of adenylate cyclase. In some systems, however, this cyclase activation does not seem to account for all of the physiological responses to the toxin. On the premise that cholera toxin may also exert effects through other second messenger compounds we have studied the effect of cholera toxin on the rate of Ca2+ movement across the membrane of intestinal brush border vesicles. Increasing concentrations of cholera toxin progressively accelerated the passive uptake of Ca2+ into, and the efflux of Ca2+ from, an osmotically active space in brush border membrane vesicles. This effect of cholera toxin was saturable by excess Ca2+ and was relatively specific, as the toxin did not affect vesicle permeability to an uncharged polar solute. The toxin had two high affinity Ca2+ binding sites on the A subunit as measured by equilibrium dialysis. Ca2+ transport facilitated by cholera toxin was temperature dependent, required the holotoxin, and could be inhibited by preincubation of the toxin with excess free ganglioside GM1.This increased rate of Ca2+ influx caused by the in vitro addition of cholera toxin to brush border membrane vesicles may have physiological significance as it was comparable to rates observed with the Ca ionophore A23187. Similar effects occurring in vivo could permit cholera toxin to increase cytoplasmic Ca2+ concentrations and to produce accompanying second messenger effects.

scholarly journals Binding Analyses of Bacillus thuringiensis Cry δ-Endotoxins Using Brush Border Membrane Vesicles of Ostrinia nubilalis

2001 ◽  
Vol 67 (2) ◽  
pp. 872-879 ◽  
Author(s):  
Gang Hua ◽  
Luke Masson ◽  
Juan Luis Jurat-Fuentes ◽  
George Schwab ◽  
Michael J. Adang
Keyword(s):  
Bacillus Thuringiensis ◽  
Binding Site ◽  
Brush Border ◽  
Ostrinia Nubilalis ◽  
European Corn Borer ◽  
Binding Proteins ◽  
Brush Border Membrane ◽  
Membrane Vesicles ◽  
Brush Border Membrane Vesicles ◽  
Cry Toxin

ABSTRACT Transgenic corn expressing the Bacillus thuringiensisCry1Ab gene is highly insecticidal to Ostrinia nubilalis(European corn borer) larvae. We ascertained whether Cry1F, Cry9C, or Cry9E recognizes the Cry1Ab binding site on the O. nubilalis brush border by three approaches. An optical biosensor technology based on surface plasmon resonance measured binding of brush border membrane vesicles (BBMV) injected over a surface of immobilized Cry toxin. Preincubation with Cry1Ab reduced BBMV binding to immobilized Cry1Ab, whereas preincubation with Cry1F, Cry9C, or Cry9E did not inhibit BBMV binding. BBMV binding to a Cry1F-coated surface was reduced when vesicles were preincubated in Cry1F or Cry1Ab but not Cry9C or Cry9E. A radioligand approach measured 125I-Cry1Ab toxin binding to BBMV in the presence of homologous (Cry1Ab) and heterologous (Cry1Ac, Cry1F, Cry9C, or Cry9E) toxins. Unlabeled Cry1Ac effectively competed for 125I-Cry1Ab binding in a manner comparable to Cry1Ab itself. Unlabeled Cry9C and Cry9E toxins did not inhibit 125I-Cry1Ab binding to BBMV. Cry1F inhibited125I-Cry1Ab binding at concentrations greater than 500 nM. Cry1F had low-level affinity for the Cry1Ab binding site. Ligand blot analysis identified Cry1Ab, Cry1Ac, and Cry1F binding proteins in BBMV. The major Cry1Ab signals on ligand blots were at 145 kDa and 154 kDa, but a strong signal was present at 220 kDa and a weak signal was present at 167 kDa. Cry1Ac and Cry1F binding proteins were detected at 220 and 154 kDa. Anti-Manduca sexta aminopeptidase serum recognized proteins of 145, 154, and 167 kDa, and anti-cadherin serum recognized the 220 kDa protein. We speculate that isoforms of aminopeptidase and cadherin in the brush border membrane serve as Cry1Ab, Cry1Ac, and Cry1F binding proteins.

scholarly journals Identification of a Newcry1I-Type Gene as a Candidate for Gene Pyramiding in Corn To Control Ostrinia Species Larvae

2015 ◽  
Vol 81 (11) ◽  
pp. 3699-3705 ◽  
Author(s):  
Can Zhao ◽  
Juan Luis Jurat-Fuentes ◽  
Heba M. Abdelgaffar ◽  
Hongyu Pan ◽  
Fuping Song ◽  
...  
Keyword(s):  
Brush Border ◽  
Binding Sites ◽  
European Corn Borer ◽  
Brush Border Membrane ◽  
Gene Pyramiding ◽  
Membrane Vesicles ◽  
Brush Border Membrane Vesicles ◽  
Toxin Gene ◽  
Binding Assays ◽  
Content Type

ABSTRACTPyramiding of diversecrytoxin genes fromBacillus thuringiensiswith different modes of action is a desirable strategy to delay the evolution of resistance in the European corn borer (Ostrinia nubilalis). Considering the dependency of susceptibility to Cry toxins on toxin binding to receptors in the midgut of target pests, a diverse mode of action is commonly defined as recognition of unique binding sites in the target insect. In this study, we present a novelcry1Ietoxin gene (cry1Ie2) as a candidate for pyramiding with Cry1Ab or Cry1Fa in corn to controlOstriniaspecies larvae. The new toxin gene encodes an 81-kDa protein that is processed to a protease-resistant core form of approximately 55 kDa by trypsin digestion. The purified protoxin displayed high toxicity toOstrinia furnacalisandO. nubilalislarvae but low to no activity againstSpodopteraor heliothine species or the coleopteranTenebrio molitor. Results of binding assays with125I-labeled Cry1Ab toxin and brush border membrane vesicles fromO. nubilalislarvae demonstrated that Cry1Ie2 does not recognize the Cry1Ab binding sites in that insect. Reciprocal competition binding assays with biotin-labeled Cry1Ie2 confirmed the lack of shared sites with Cry1Ab or Cry1Fa inO. nubilalisbrush border membrane vesicles. These data support Cry1Ie2 as a good candidate for pyramiding with Cry1Ab or Cry1Fa in corn to increase the control ofO. nubilalisand reduce the risk of resistance evolution.

LEUCINE UPTAKE IN BRUSH-BORDER MEMBRANE VESICLES FROM THE MIDGUT OF A LEPIDOPTERAN LARVA, PHILOSAMIA CYNTHIA

1990 ◽  
Vol 149 (1) ◽  
pp. 207-221
Author(s):  
V. FRANCA SACCHI ◽  
BARBARA GIORDANA ◽  
FLAVIA CAMPANINI ◽  
PATRIZIA BONFANTI ◽  
GIORGIO M. HANOZET
Keyword(s):  
Brush Border ◽  
Brush Border Membrane ◽  
Electrical Potential ◽  
Membrane Vesicles ◽  
Brush Border Membrane Vesicles ◽  
Electrical Potential Difference ◽  
Experimental Conditions ◽  
Leucine Uptake ◽  
Leucine Transport

A potassium- or sodium-activated cotransport of leucine occurs in brush-border membrane vesicles prepared from the midgut of larvae of Philosamia cynthia Drury). The potassium chemical gradient can drive a twofold accumulation of leucine, which is greatly increased under experimental conditions that presumably provide an electrical potential difference (δψ) Kinetic parameters show that leucine transport is improved by these conditions and by a pH gradient similar to that occurring in vivo. However, these gradients cannot drive an intravesicular accumulation of leucine in the absence of potassium. The potassium-dependence of leucine uptake shows that 20% of the transport is potassium-independent and that K50 and Vmax are 30.3± 3.2mmoll−1 and 2584±148pmol 7 s−1mg−1 protein, respectively. The potassium-independent component of leucine transport is also carrier-mediated and some evidence is reported suggesting that the same carrier can cross the membrane as binary carrier and leucine) or ternary (carrier, leucine and potassium) complexes, each having a different mobility

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