Neither barium nor calcium prevents the inhibition by Bacillus thuringiensis δ-endotoxin of sodium- or potassium gradient-dependent amino acid accumulation by tobacco hornworm midgut brush border membrane vesicles

Rapid filtration assays were used to determine the effects of barium, calcium and an insecticidal δ-endotoxin from Bacillus thuringiensis on sodium and potassium ion gradient dependent phenylalanine accumulation by brush border membrane vescles from the larval midgut of the tobacco hornworm (Manduca sexta). Neither barium nor calcium had a significant effect on sodium ion gradient dependent phenylalanine accumulation by the membrane vesicles. Both barium and calcium inhibited potassium ion gradient dependent phenylalanine accumulation by the membrane vesicles. B. thuringiensis δ-endotoxin inhibited both sodium and potassium ion gradient dependent phenylalanine accumulation by the vesicles. Inhibition of both sodium and potassium ion gradient dependent phenylalanine accumulation increased similarly with increasing δ-endotoxin inhibition of either sodium or potassium dependent phenylalanine accumulation by the vesicles.     

Na-dependent uptake of phenylalanine in the midgut of a cockroach (Blabera gigantea)

Summary A net absorption of sodium ions and ofl-phenylalanine, in the absence of chemical gradients, occurs across the isolated midgut of the cockroachBlabera gigantea. Both sodium and amino acid net fluxes were abolished by the haemolymphatic addition of the Na–K ATPase inhibitor ouabain, at a concentration 1 mM. A purified fraction of brush border membranes, prepared from the midgut tissue by Ca-precipitation, was used to investigate the occurrence of a cotransport system in the luminal membrane of the cockroach enterocyte. An inwardly directed Na gradient (100 mM outside the vesicles, 0 mM inside) drives the uphill movement of phenylalanine into the vesicular space, whilst other monovalent cations fail to induce the concentrative uptake of the amino acid. Moreover, the amino acid uptake seems to be dependent on the transmembrane potential, since inwardly directed gradients of different sodium salts determine a decreasing rate of phenylalanine uptake in agreement with the presumptive permeabilities of Na counterions. These data suggest the presence of a Na-phenylalanine cotransport system located on the brush border membrane ofB. gigantea midgut.Abbreviations BBMV brush border membrane vesicles - HEPES N-2-hydroxyethyl-piperazine-N-2-ethanesulfonic acid - PD transepithelial electrical potential difference - TMA tetramethylammonium - Tris tris-(hydroxymethyl) aminomethane     

The receptor for Bacillus thuringiensis CrylA(c) delta-endotoxin in the brush border membrane of the lepidopteran Manduca sexta is aminopeptidase N

A 120 kDa glycoprotein in the larval midgut membrane of the Iepidopteran Manduca sexta, previously identified as a putative receptor for Bacillus thuringiensis CrylA(c) δ-endotoxin, has been purified by a combination of protoxin affinity Chromatography and anion exchange chromatography. In immunoblotting experiments, the purified glycoprotein has the characteristics predicted of the receptor: it binds CrylA(c) toxin In the presence of GlcNAc but not GalNAc; it binds the lectin SBA; but it does not bind CrylB toxin. N-terminal and internal amino acid sequences obtained from the protein show a high degree of similarity with the enzyme aminopeptidase N (EC 3.4.11.2). When assayed for aminopeptidase activity, purified receptor preparations were enriched 5.3-fold compared to M. sexta brush border membrane vesicles. We propose that the receptor for CrylA(c) toxin in the brush border membrane of the lepidopteran M. sexta is the metalloprotease aminopeptidase N.     

Phenylglyoxal suppresses cationic lysine/K+ symport under alkaline conditions in brush border membrane vesicles from larval Manduca sexta midgut

The arginine-specific reagent, phenylglyoxal, decreases the initial rate of lysine/K⁺ symport (cotransport) as well as maximum lysine accumulation at pH 9.2, by brush border membrane vesicles obtained from the larval midgut of the lepidopteran, Manduca sexta. The symport of a neutral amino acid, leucine, remained unaffected. Following exposure to phenylglyoxal, the apparent dissociation constant for lysine increased by a factor of 2.5 whereas the maximum uptake rate decreased by a factor of 0.4. More than one arginine residue appears to react with phenylglyoxal. Apparently phenylgyoxal reacts preferentially with arginine residues on a symporter that is specific for positively charged lysine. Phenylglyoxal shows promise as a specific covalent label for the identification of a cationic amino acid symporter. © 1995 Wiley-Liss, Inc.     

Brush border membrane and amino acid transport

Fundamental differences in midgut structure, physiology, brush border proteins, and transporters among Leptinotarsa decemlineata, lepidopteran caterpillars, other insect taxa, and vertebrates are reviewed. The effects of dietary protein concentration on Manduca sexta midgut amino acid transport and brush border membrane proteins are reported. M. sexta fed diet with reduced protein had elevated levels of leucine aminopeptidase in the brush border membrane. No changes in amino acid transport or alkaline phosphatase activity due to dietary differences were detected. Changes in brush border proteins could affect the toxicity and pathogenicity of microbial agents. © 1996 Wiley-Liss, Inc.     

Pore-forming properties of the Bacillus thuringiensis toxin Cry9Ca in Manduca sexta brush border membrane vesicles

The toxicity and pore-forming ability of the Bacillus thuringiensis Cry9Ca insecticidal toxin, its single-site mutants, R164A and R164K, and the 55-kDa fragment resulting from its proteolytic cleavage at residue 164 were investigated using Manduca sexta neonate larvae and fifth-instar larval midgut brush border membrane vesicles, respectively. Neither the mutations nor the proteolytic cleavage altered Cry9Ca toxicity. Compared with Cry1Ac, Cry9Ca and its mutants formed large poorly selective pores in the vesicles. Pore formation was highly dependent on pH, however, especially for wild-type Cry9Ca and both mutants. Increasing pH from 6.5 to 10.5 resulted in an irregular step-wise decrease in membrane permeabilization that was not related to a change in the ionic selectivity of the pores. Pore formation was much slower with Cry9Ca and its derivatives, including the 55-kDa fragment, than with Cry1Ac and its rate was not influenced by the presence of protease inhibitors or a reducing agent.     

Biotinylation of Bacillus thuringiensis Insecticidal Crystal Proteins

Biotinylation of Bacillus thuringiensis insecticidal crystal proteins (ICPs) was evaluated for its potential use in an alternative ICP screening method and in the characterization of ICP receptors. In vivo biological activity of CryIA(b), as inferred from bioassays with Manduca sexta and Ostrinia nubilalis and from histopathological effects on O. nubilalis midgut cells induced by force feeding, was not affected by biotinylation at moderate biotinylation ratios. A competitive radioreceptor assay showed that there was only a minor reduction in binding affinity of biotin-labeled CryIA(b) for M. sexta brush border membrane vesicles. On midgut tissue sections, the binding pattern along the midgut epithelium and the staining intensity of biotinylated ICPs detected with streptavidin-enzyme conjugate were virtually identical to the binding pattern and staining intensity of native CryIA(b) detected with antibodies. The specificity of biotinylated ICP binding to larval midgut tissue was demonstrated by performing homologous competition experiments. The relationship between different ICP receptor types in Plutella xylostella, as inferred from radioligand binding studies, was confirmed by the results of heterologous competition experiments performed with biotinylated and native ICPs.     

Amino acid and divalent ion permeability of the pores formed by the Bacillus thuringiensis toxins Cry1Aa and Cry1Ac in insect midgut brush border membrane vesicles

The pores formed by Bacillus thuringiensis insecticidal toxins have been shown to allow the diffusion of a variety of monovalent cations and anions and neutral solutes. To further characterize their ion selectivity, membrane permeability induced by Cry1Aa and Cry1Ac to amino acids (Asp, Glu, Ser, Leu, His, Lys and Arg) and to divalent cations (Mg²⁺, Ca²⁺ and Ba²⁺) and anions (SO₄²⁻ and phosphate) was analyzed at pH 7.5 and 10.5 with midgut brush border membrane vesicles isolated from Manduca sexta and an osmotic swelling assay. Shifting pH from 7.5 to 10.5 increases the proportion of the more negatively charged species of amino acids and phosphate ions. All amino acids diffused well across the toxin-induced pores, but, except for aspartate and glutamate, amino acid permeability was lower at the higher pH. In the presence of either toxin, membrane permeability was higher for the chloride salts of divalent cations than for the potassium salts of divalent anions. These results clearly indicate that the pores are cation-selective.     

Bacillus thuringiensis subsp. Aizawai δ-endotoxin inhibits the k+/amino acid cotransporters of lepidopteran larval midgut

1. The δ-endotoxin of Bacillus thuringiensis subsp. aizawai inhibits in a dose-dependent manner the K⁺-driven accumulation of histidine and lysine as well as their equilibrative uptake into brush border membrane vesicles from the midgut of Bombyx mori larvae.2. The decrease of uptake in the absence of a K⁺-gradient is neither due to a leakage of the labelled substrate from the vesicles nor to a reduction of the osmotically active space available, as a result of a detergent-like effect of the toxin.3. The toxin acts as a non competitive inhibitor of the K⁺/amino acid cotransporters of the larval midgut of Lepidoptera, with no preference for a specific transport system.     

Sodium-cotransport systems in intestine and kidney of the winter flounder

Summary Brush-border membrane vesicles were isolated from the intestine and kidney of the winter flounder,Pseudopleuronectes americanus, and the transport ofd-glucose,l-alanine and sodium was examined by a rapid filtration technique.d-glucose,l-alanine, and sodium entered the same osmotically reactive space suggesting that uptake into vesicles represents transport across rather than binding to the membrane. d-glucose andl-alanine uptake by intestinal and renal brush-border membrane vesicles was stimulated by sodium as compared to potassium or choline. In the presence of a sodium chloride gradient, overshooting uptake was observed indicating a transient intravesicular accumulation ofd-glucose andl-alanine. The sodium-dependentd-glucose uptake was inhibited by phlorizin andd-galactose while the transport ofl-alanine was inhibited byl-phenylalanine. The sodium-dependent transport ofd-glucose andl-alanine was affected by the electrical potential difference across the vesicle membrane; the addition of valinomycin in the presence of an inwardly directed potassium chloride gradient inhibited sodium-dependent solute uptake, whereas replacing chloride or gluconate with more permeant anions, such as SCN^–, stimulated uptake. Similar results were obtained with intestinal and renal membranes; they document the presence of sodium/d-glucose and sodium/l-alanine cotransport systems in the brush-border membrane of intestine and kidney.Sodium uptake into brush border membrane vesicles from the flounder intestine and kidney was saturable (tracer replacement) and trans-stimulated (tracer coupling), indicating transport via facilitated diffusion systems. Additionally, sodium uptake was only slightly affected by superimposing diffusion potentials demonstrating that the majority of sodium transport was by electroneutral coupled processes. In both the intestinal and kidney brush-border membrane vesicles sodium uptake was inhibited by an inwardly directed proton gradient suggesting the presence of a sodium/proton exchange mechanism. In intestinal, but not in renal membrane preparations, sodium uptake was stimulated by chloride. Chloride stimulation was abolished after preincubation with furosemide indicating the presence of an additional coupled sodium-chloride transport in the intestinal brush-border membranes.The experiments were carried out at the Mount Desert Island Biological Laboratory, Salsbury Cove, Maine 04672, USAAddress effective February 1, 1980: Albert Einstein College of Medicine, Department of Physiology, 1300 Morris Park Avenue, Bronx, New York 10461, USA     

Na+-independent l-arginine transport in rabbit renal brush border membrane vesicles

Na⁺-independent l-arginine uptake was studied in rabbit renal brush border membrane vesicles. The finding that steady-state uptake of l-arginine decreased with increasing extravesicular osmolality and the demonstration of accelerative exchange diffusion after preincubation of vesicles with l-arginine, but not d-arginine, indicated that the uptake of l-arginine in brush border vesicles was reflective of carrier-mediated transport into an intravesicular space. Accelerative exchange diffusion of l-arginine was demonstrated in vesicles preincubated with l-lysine and l-ornithine, but not l-alanine or l-proline, suggesting the presence of a dibasic amino acid transporter in the renal brush border membrane. Partial saturation of initial rates of l-arginine transport was found with extravesicular [arginine] varied from 0.005 to 1.0 mM. l-Arginine uptake was inhibited by extravesicular dibasic amino acids unlike the Na⁺-independent uptake of l-alanine, l-glutamate, glycine or l-proline in the presence of extravesicular amino acids of similar structure. l-Arginine uptake was increased by the imposition of an H⁺ gradient (intravesicular pH<extravesicular pH) and H⁺ gradient stimulated uptake was further increased by FCCP. These findings demonstrate membrane-potential-sensitive, Na⁺-independent transport of l-arginine in brush border membrane vesicles which differs from Na⁺-independent uptake of neutral and acidic amino acids. Na⁺-independent dibasic amino acid transport in membrane vesicles is likely reflective of Na⁺-independent transport of dibasic amino acids across the renal brush border membrane.     

Bacillus thuringiensis Cry1Ac Toxin-Binding and Pore-Forming Activity in Brush Border Membrane Vesicles Prepared from Anterior and Posterior Midgut Regions of Lepidopteran Larvae

It is generally accepted that Bacillus thuringiensis Cry toxins insert into the apical membrane of the larval midgut after binding to specific receptors, and there is evidence that the distribution of binding molecules along the midgut is not uniform. By use of the voltage-sensitive dye DiSC₃(5) and ¹²⁵I-labeled Cry1Ac, we have measured the effect of Cry1Ac in terms of permeabilization capacity and of binding parameters on brush border membrane vesicles (BBMV) prepared from the anterior and the posterior regions of the larval midgut from two insect species, Manduca sexta and Helicoverpa armigera. The permeabilizing activity was significantly higher with BBMV from the posterior region than with the one observed in the anterior region in both insect species. Instead, ¹²⁵I-Cry1Ac bound specifically to BBMV from the two midgut regions, with no significant differences in the binding parameters between the anterior and posterior regions within an insect species. N-acetylgalactosamine inhibition patterns on pore formation and binding differed between anterior and posterior midgut regions and between species, providing evidence of a multifaceted involvement of the sugar in the Cry1Ac mode of action. The analysis of binding and pore formation in different midgut regions could be an effective method to study differences in the mode of action of Cry1Ac toxin in different species.     

Analysis of the Properties of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> Insecticidal Toxins Using a Potential-sensitive Fluorescent Probe

A potential-sensitive fluorescent probe, 3,3-dipropylthiadicarbocyanine iodide, was used to analyze, at pH 7.5 and 10.5, the effects of Bacillus thuringiensis toxins on the membrane potential generated by the efflux of K⁺ ions from brush border membrane vesicles purified from the midgut of the tobacco hornworm, Manduca sexta. Fluorescence levels were strongly influenced by the pH and ionic strength of the media. Therefore, characterization of the effects of the toxins was conducted at constant pH and ionic strength. Under these conditions, the toxins had little effect on the fluorescence levels measured in the presence or absence of ionic gradients, indicating that the ionic selectivity of their pores is similar to that of the intact membrane. Valinomycin greatly increased the potential generated by the diffusion of K⁺ ions although membrane permeability to the other ions used to maintain the ionic strength constant also influenced fluorescence levels. In the presence of valinomycin, active toxins (Cry1Aa, Cry1Ab, Cry1Ac, Cry1C and Cry1E) efficiently depolarized the membrane at pH 7.5 and 10.5.     

l- andd-alanine transport in brush border membrane vesicles from lepidopteran midgut: Evidence for two transport systems

Summary In brush border membrane vesicles from the midgut ofPhilosamia cynthia larvae (Lepidoptera) thel- andd-alanine uptake is dependent on a potassium gradient and on transmembrane electrical potential difference. Each isomer inhibits the uptake of the other form: inhibition ofl-alanine uptake byd-alanine is competitive, whereas inhibition ofd-alanine uptake byl-alanine is noncompetitive. Transstimulation experiments as well as the different pattern of specificity to cations suggest the existence of two transport systems. Kinetic parameters for the two transporters have been calculated both when K_out>K_in and K_out=K_in.d-alanine is actively transported also by the whole midgut, but it is not metabolized by the intestinal tissue.     

Fluorometric Assay of Potential Change of Bombyx mori Midgut Brush Border Membrane Induced by δ-Endotoxin from Bacillus thuringiensis

Bacillus thuringiensis δ-endotoxin, CryIA(a), increased ion permeability of brush border membrane vesicles isolated from midgut epithelia of Bombyx mori larvae. This ion permeability change was measured with a potential-sensitive fluorescent dye, 3,3′-dipropylthiadicarbocyanine iodide. This effect was observed at concentrations of the toxin that correspond to normal effective doses in vivo. CryIA(b) and heat-treat CryIA(a) did not show this effect. CryIA(a) did not show this effect on rat renal brush border membranes. The effects depended on the toxin dose in saturable manner. These suggest that this assay system reflects the mode of action of δ-endotoxin in vivo. The toxin increased various ion permeabilities, suggesting that δ-endotoxin forms non-selective cation pores on brush border membranes.     

Properties of Brush Border Vesicles Isolated from Rat Kidney Cortex by Calcium Precipitation

Brush border membrane vesicles were isolated from rat kidney cortex by differential centrifugation in the presence of 10 mM calcium. Their properties were compared to brush border vesicles isolated by free-flow electrophoresis. By the calcium precipitation method membrane vesicles were obtained in a shorter time with a similar enrichment of brush border marker enzymes (11- to 12-fold for alkaline phosphatase and maltase), with a similarly reduced activity of the marker enzyme for basal-lateral plasma membranes and an almost identical protein composition as revealed by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. The transport properties of the two membrane preparations for D-glucose, L-phenylalanine, and phosphate are essentially the same; there is some indication for a lower sodium permeability of the vesicles prepared by the calcium precipitation method. The latter vesicles were also shown to exhibit sodium gradient stimulated uptake of L-glutamate.     

A novel regulatory mechanism for amino acid absorption in lepidopteran larval midgut

A number of methyl and ethyl esters of naturally occurring amino acids exert a potent stimulatory effect on the cotransport system responsible for the absorption of most essential amino acids along the midgut of the silkworm Bombyx mori. L-Leucine methyl ester (Leu-OMe), one of the most effective activators, induces a large increase of the initial rate of leucine uptake in midgut brush border membrane vesicles (BBMV) from the anterior-middle (AM) region, and a small effect in BBMV from the posterior (P) region. Nonetheless, the methyl ester causes in both regions a relevant K(+)-, Deltapsi- and pH-independent increase of the intravesicular accumulation of the amino acid. The activation by Leu-OMe proves that amino acid absorption can be modulated all along the B. mori larval midgut and that the AM region, where the ability to transport and concentrate the substrate is very low, is more susceptible than the P region. Leucine uptake in AM-BMMV can be activated by amino acid methyl esters with definite structural requisites, with the following order of potency: L-leucine>L-phenylglycine>L-methionine>L-phenylalanine>L-norleucinez.Gt;L-isoleucine. The activation is stereospecific and occurs also with some ethyl esters (e.g. leucine and phenylalanine). No activation was observed with esters of amino acids with short hydrophobic or polar side-chains. The activation mechanism here described plays a fundamental role in larval growth since silkworms reared on artificial diets supplemented with leucine or methionine methyl esters reach maximum body weight 12-18 h before control larvae and spin cocoons with a larger shell weight. This novel regulatory mechanism of an amino acid transport protein appears to be widespread among lepidopteran larvae.     

Comparison of brush border membrane vesicles prepared by three methods from larval Manduca sexta midgut

Brush border membrane vesicles (BBMV) were prepared from freshly isolated posterior larval Manduca sexta midguts by differential calcium precipitation, differential magnesium precipitation and differential ultrasonication. BBMV were also prepared from frozen posterior larval M. sexta midguts by differential calcium precipitaion and differential magnesium precipitation. The yield of BBMV by both differential precipitation methods was 5–6 times greater than that by the differential ultrasonification method. Enrichments of the brush border membrane marker enzymes alkaline phosphatase, γ-glutamyl transferase, and aminopeptidase were similar in all preparations. The polypeptide composition of all preparations was also similar. The specific activity of mitochondrial and microsomal marker enzymes was higher in BBMV prepared from freshly isolated midguts by the differential precipitation methods than in BBMV prepared by the ultrasonication method. The specific activity of cytochrome-c oxidase was 2.5–7 times higher in BBMV prepared from frozen midguts than in BBMV prepared from fresh tissue.     

Ion requirements for taurocholate transport by ileal brush border membrane vesicles.

The ion requirements for intestinal taurocholate transport were studied using vesicles prepared from the brush borders of guinea pig small intestines. For each experimental electrolyte, parallel uptake experiments were performed with vesicles from jejunal and ileal brush border membranes to differentiate between uptake by passive fluxes and non-specific binding and uptake by the ileal bile salt active transport system. Uptake of taurocholate prior to the addition of electrolyte was the same for vesicles prepared from jejunal and ileal tissue. During the presence of a sodium gradient (extravesicular concentration greater than intravesicular), only ileal vesicles displayed the enhanced uptake which is characteristic of the overshoot phenomenon. When NaCl was replaced by KCl or LiCl, the overshoot was not observed. Replacement of NaCl with NaCNS, Na₂SO₄, or NaSO₃C₂H₄OH, however, resulted in no significant difference in the initial uptake values observed in either the jejunal or ileal vesicles. This pattern of taurocholate transport independence of relative anion permeability differs from the pattern observed by others for the Na⁺ dependent transport of D-glucose by intestinal brush border membrane vesicles. This difference may be attributed in part to the fact that, unlike the situation with glucose, the binding of a taurocholate anion and a sodium cation by the hypothetical carrier would result in an electroneutral addition.