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.     

Effects of Bacillus thuringiensis delta-endotoxin on the permeability of brush border membrane vesicles from tobacco hornworm (Manduca sexta) midgut

1. The effect of two recombinant Bacillus thuringiensis delta-endotoxins on brush border membrane vesicles of Manduca sexta midgut was investigated using an in vitro assay system, based on ion-amino acid cotransport. 2. A CryIA(b)-toxin provoked an increase in the permeability of the vesicles. 3. A CryIB-toxin, not toxic to M. sexta larvae in vivo, had no effect in our assay. 4. In contrast to earlier reports, the increase in permeability was found to be neither selective for K+ nor specifically inhibited by Ca2+ or Ba2+. 5. Our data support the hypothesis that B. thuringiensis delta-endotoxins create non-specific pores.     

Bacterial infection of a model insect: Photorhabdus luminescens and Manduca sexta

Invertebrates, including insects, are being developed as model systems for the study of bacterial virulence. However, we understand little of the interaction between bacteria and specific invertebrate tissues or the immune system. To establish an infection model for Photorhabdus, which is released directly into the insect blood system by its nematode symbiont, we document the number and location of recoverable bacteria found during infection of Manduca sexta. After injection into the insect larva, P. luminescens multiplies in both the midgut and haemolymph, only later colonizing the fat body and the remaining tissues of the cadaver. Bacteria persist by suppressing haemocyte-mediated phagocytosis and culture supernatants grown in vitro, as well as plasma from infected insects, suppress phagocytosis of P. luminescens. Using GFP-labelled bacteria, we show that colonization of the gut begins at the anterior of the midgut and proceeds posteriorly. Within the midgut, P. luminescens occupies a specific niche between the extracellular matrix and basal membrane (lamina) of the folded midgut epithelium. Here, the bacteria express the gut-active Toxin complex A (Tca) and an RTX-like metalloprotease PrtA. This close association of the bacteria with the gut, and the production of toxins and protease, triggers a massive programmed cell death of the midgut epithelium.     

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.     

A Tenebrio molitor GPI-anchored alkaline phosphatase is involved in binding of Bacillus thuringiensis Cry3Aa to brush border membrane vesicles

Bacillus thuringiensis Cry toxins recognizes their target cells in part by the binding to glycosyl–phosphatidyl–inositol (GPI) anchored proteins such as aminopeptidase-N (APN) or alkaline phosphatases (ALP). Treatment of Tenebrio molitor brush border membrane vesicles (BBMV) with phospholipase C that cleaves out GPI-anchored proteins from the membranes, showed that GPI-anchored proteins are involved in binding of Cry3Aa toxin to BBMV. A 68 kDa GPI-anchored ALP was shown to bind Cry3Aa by toxin overlay assays. The 68 kDa GPI-anchored ALP was preferentially expressed in early instar larvae in comparison to late instar larvae. Our work shows for the first time that GPI-anchored ALP is important for Cry3Aa binding to T. molitor BBMV suggesting that the mode of action of Cry toxins is conserved in different insect orders.     

Mercurial perturbation of brush border membrane permeability in rabbit ileum

The sulfhydryl reagents Hg++ and p-chloromercuribenzene sulfonate (PCMBS) at millimolar concentrations reduced the mucosal entry of sugars and amino acids to 80-90% of control levels within several minutes. Based on 50% levels of inhibition, Hg++ proved to be 20 and 10 times as potent as PCMBS in blocking sugar and amino acid transport, respectively; both systems were equally sensitive to Hg++. Concomitant measurements of 203Hg-PCMBS demonstrated a progressive tissue uptake, which, unlike inhibition, did not saturate with increasing times of exposure, thus suggesting appreciable epithelial entry with prolonged exposures (less than 30 min at 1 mM). At similar dose levels, no significant change in mucosal Na+ entry was detected. Inhibition was not reversed by 30-min washes in cholinesalt solutions; however, 10-min exposures to dithiothreitol [10 mM] reversed Hg++ and PCMBS inhibition by 40 and 100%, respectively. Alanine and galactose influx kinetics measured at concentrations of 0-100 mM exhibited a linear or diffusional entry component in addition to the usual saturable component for both control and Hg++-treated ileum. The presence of a diffusional term in the flux equation resulted in two sets of parameters giving nearly equal fits to these measurements. It was shown that this ambiguity could be resolved by determining the change in diffusional entry with Hg++ treatment. A 20-min exposure to 0.5 mM Hg++ caused an increase from 0.050 and 0.045 to 0.064 and 0.070 cm/hr in the coefficient of diffusional entry for alanine and galactose, respectively. On the basis of this increase, it is argued that Hg++ causes a decrease in Jmax and little change in Km for both transport mechanisms. This analysis has a general bearing on kinetic measurements of transport in which passive fluxes are comparable to those mediated by specific pathways. The alanine results are consistent with bimolecular reactions between mercurial and two membrane inhibitory sites, each producing approximately 40% reduction in membrane translocation rate. The estimated reaction rate constants were 5.0 and 0.4 mM min.     

Pore formation activity of Cry1Ab toxin from Bacillus thuringiensis in an improved membrane vesicle preparation from Manduca sexta midgut cell microvilli

The pore formation activity of Cry1Ab toxin is analyzed in an improved membrane preparation from apical microvilli structures of Manduca sexta midgut epithelium cells (MEC). A novel methodology is described to isolate MEC and brush border membrane vesicles (BBMV) from purified microvilli structures. The specific enrichment of apical membrane enzyme markers aminopeptidase (APN) and alkaline phosphatase (APh) were 35- and 22-fold, respectively, as compared to the whole midgut cell homogenate. Ligand-blot and Western-blot experiments showed that Cry1A specific receptors were also enriched. The pore formation activity of Cry1Ab toxin was fourfold higher in the microvilli membrane fraction that showed low intrinsic K+ channels and higher APN and APh activities than in the basal-lateral membrane fraction harboring high intrinsic K+ channels. These data suggest that basal-lateral membrane was separated from apical membrane.This procedure should allow more precise studies of the interaction of Cry toxins with their target membranes, avoiding unspecific interaction with other cellular membranes, as well as the study of the pore formation activity induced by Cry toxins in the absence of endogenous channels from M. sexta midgut cells.     

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.     

Kinetics of leucine transport in brush border membrane vesicles from lepidopteran larvae midgut.

The kinetics of K(+)-leucine cotransport in the midgut of lepidopteran larvae was investigated using brush border membrane vesicles. Initial rate (3 s) of leucine uptake was determined under experimental conditions similar to those occurring in vivo, i.e. in the presence of delta psi much greater than 0 (inside negative) and a delta pH of 1.4 units (7.4in/8.8out). Leucine and K+ bind to the carrier according to a sequential mechanism, and the binding of one substrate changed the dissociation constant for the other substrate by a factor of 0.15. Both trans-K+ and trans-leucine were mixed-type inhibitors of leucine uptake. Moreover, a portion of total leucine uptake was K+ independent, and it was competitively inhibited by trans-leucine. We interpret the trans inhibitory effects to mean that the partially loaded K+ only form is virtually unable to translocate across the membrane, whereas the binary complex carrier, leucine, can isomerize from the trans to the cis side of the membrane. However, the K(+)-independent leucine uptake occurs with a Keq greater than 1, i.e. the efflux route through the partially loaded leucine only form is slower than the rate of isomerization of the unloaded carrier from trans to cis side. Taken together, these results suggest a model in which transport occurs by an iso-random Bi Bi system. Since K+ does not act as a pure competitive activator, this model is different from that proposed for most of the Na(+)-linked solutes transport agencies and may be related to the broadening of the cation specificity of the amino acid transporters in lepidopteran larvae.     

A Novel Insecticidal Toxin from Photorhabdus luminescens, Toxin Complex a (Tca), and Its Histopathological Effects on the Midgut of Manduca sexta

Photorhabdus luminescens is a bacterium which is mutualistic with entomophagous nematodes and which secretes high-molecular-weight toxin complexes following its release into the insect hemocoel upon nematode invasion. Thus, unlike other protein toxins from Bacillus thuringiensis (δ-endotoxins and Vip’s), P. luminescens toxin (Pht) normally acts from within the insect hemocoel. Unexpectedly, therefore, the toxin complex has both oral and injectable activities against a wide range of insects. We have recently fractionated the protein toxin and shown it to consist of several native complexes, the most abundant of which we have termed Toxin complex a (Tca). This complex is highly active against the lepidopteran Manduca sexta. In view of the difference in the normal mode of delivery of P. luminescens toxin and the apparent communality in the histopathological effects of other gut-active toxins from B. thuringiensis, as well as cholesterol oxidase, we were interested in investigating the effects of purified Tca protein on larvae of M. sexta. Here we report that the histopathology of the M. sexta midgut is similar to that for other novel midgut-active toxins. Following oral ingestion of Tca by M. sexta, we observed an acceleration in the blebbing of the midgut epithelium into the gut lumen and eventual lysis of the epithelium. The midgut shows a similar histopathology following injection of Tca into the insect hemocoel. These results not only show that Tca is a highly active oral insecticide but also confirm the similar histopathologies of a range of very different gut-active toxins, despite presumed differences in modes of action and/or delivery. The implications for the mode of action of Tca are discussed.     

Secretion of beta-glycosidase by middle midgut cells and its recycling in the midgut of Tenebrio molitor larvae

There are four β-glycosidases (βgly1, βgly2, βgly3, and βgly4) in Tenebrio molitor midgut larvae. βgly1 and βgly2 have identical kinetic properties, and differ in a few amino acid residues. Purified βgly1 was used to raise antibodies in a rabbit. The resulting antiserum recognizes in a Western blot only βgly1 and βgly2 in midgut tissue homogenates and contents. An immunocytochemical study carried out using confocal fluorescence and immunogold techniques showed that βgly1+βgly2 are secreted by exocytosis mainly from the distal part of the second third of T. molitor midguts. This is the first immunocytochemical study of an insect digestive enzyme that does not have polymers as substrates. Enzyme assays with 0.3 mM amygdalin, a condition that detects only βgly1+βgly2, revealed that most of those β-glycosidases are found in the lumen of anterior and middle midgut. This supports the hypothesis that a countercurrent flux of fluid occurs in T. molitor midgut that is able to carry βgly1 and βgly2 to anterior midgut, in agreement with the enzyme recycling mechanism thought to occur in most insects.     

Interaction of intestinal brush border membrane vesicles with small unilamellar phospholipid vesicles. Exchange of lipids between membranes is mediated by collisional contact

The kinetics of lipid transfer from small unilamellar vesicles as the donor to brush border vesicles as the acceptor have been investigated by following the transfer of radiolabeled or spin-labeled lipid molecules in the absence of exchange protein. The labeled lipid molecules studied were various radiolabeled and spin-labeled phosphatidylcholines, radiolabeled cholesteryl oleate, and a spin-labeled cholestane. At a given temperature and brush border vesicle concentration similar pseudo-first-order rate constants (half-lifetimes) were observed for different lipid labels used. The lipid transfer is shown to be an exchange reaction leading to an equal distribution of label in donor and acceptor vesicles at equilibrium (time t----infinity). The lipid exchange is a second-order reaction with rate constants being directly proportional to the brush border vesicle concentration. The results are only consistent with a collision-induced exchange of lipid molecules between small unilamellar phospholipid vesicles and brush border vesicles. Other mechanisms such as collision-induced fusion or diffusion of lipid monomers through the aqueous phase are negligible at least under our experimental conditions.     

Renal brush border glutamine transport: comparison between in situ and isolate membrane vesicle uptake

Glutamine uptake by renal cortical brush-border vesicles was compared to transport expressed by the functioning isolated kidney. Comparisons were made with regard to sodium dependency and the adaptive increase induced by chronic metabolic acidosis in the rat. The results show an absolute dependency upon a sodium gradient; sodium-independent glutamine uptake has no counterpart in situ. In addition, acidosis-induced adaptive increase in vesicle glutamine uptake has no counterpart in situ. Rather, the apparent adaptation reflects extravesicular gamma-glutamyltransferase-mediated conversion to glutamate and subsequent accumulation; acidosis-induced adaptation of this enzyme largely explains the apparent adaptation in glutamine uptake. Consequently the role of membrane transport in glutamine flux regulation can be assessed providing metabolic conversion is controlled.     

Ecdysone oxidase and 3-oxoecdysteroid reductases in Manduca sexta midgut: Kinetic parameters

Ecdysone and 20-hydroxyecdysone are converted to their 3-epimers by enzymes in the midgut cytosol of Manduca sexta larvae. A partially purified cytosol preparation has been used to analyze the nature of and the interaction between these enzymes. The cytosol was shown to contain ecdysone oxidase, one or more 3-oxoecdysteroid 3α-reductase(s), and one or more 3-oxoecdysteroid 3β-reductase(s). The reductases reacted at different velocities with NADH and NADPH. With NADH, 3α-reduction was the major reaction; with NADPH, 3β-reduction was the major reaction. The apparent kinetic parameters for the enzymes support the assumed two-step mechanism for the 3-epimerization with a 3-oxoecdysteroid as intermediate.     

Structure, processing and midgut secretion of putative peritrophic membrane ancillary protein (PMAP) from Tenebrio molitor larvae

A cDNA coding for a Tenebrio molitor midgut protein named peritrophic membrane ancillary protein (PMAP) was cloned and sequenced. The complete cDNA codes for a protein of 595 amino acids with six insect-allergen-related-repeats that may be grouped in A (predicted globular)- and B (predicted nonglobular)-types forming an ABABAB structure. The PMAP-cDNA was expressed in Pichia pastoris and the recombinant protein (64kDa) was purified to homogeneity and used to raise antibodies in rabbits. The specific antibody detected PMAP peptides (22kDa) in the anterior and middle midgut tissue, luminal contents, peritrophic membrane and feces. These peptides derive from PMAP, as supported by mass spectrometry, and resemble those formed by the in vitro action of trypsin on recombinant PMAP. Both in vitro and in vivo PMAP processing seem to occur by attack of trypsin to susceptible bonds in the coils predicted to link AB pairs, thus releasing the putative functional AB structures. The AB-domain structure of PMAP is found in homologous proteins from several insect orders, except lepidopterans that have the apparently derived protein known as nitrile-specifier protein. Immunocytolocalization shows that PMAP is secreted by exocytosis and becomes entrapped in the glycocalyx, before being released into midgut contents. Circumstantial evidence suggests that PMAP-like proteins have a role in peritrophic membrane type 2 formation.     

Effects of different brush border membrane vesicle isolation protocols on proteomic analysis of Cry1Ac binding proteins from the midgut of Helicoverpa armigera

Brush border membrane vesicles （BBMV） isolated from insect midguts have been widely used to study CrylA binding proteins. Sample preparation is important in two- dimensional electrophoresis （2-DE）, so to determine a suitable BBMV preparation method in Helicoverpa armigera for 2-DE, we compared three published BBMV preparation methods mostly used in sodium dodecyl sulfate-polyacrylamide gel electrophoresis （SDS- PAGE）. All methods yielded similar types and numbers of binding proteins, but in different quantities. The Abdul-Rauf and Ellar protocol was the best of the three, but had limitations. Sufficient protein quantity is important for research involving limited numbers of insects, such as studies of insect resistance to Bacillus thuringiensis in the field. Consequently, we integrated the three BBMV isolation methods into a single protocol that yielded high quantities of BBMV proteins from H. armigera larval midguts, which proved suitable for 2- DE analysis.     

Beta-cyclodextrin facilitates cholesterol efflux from larval Manduca sexta fat body and midgut in vitro

The ability of 2-hydroxypropyl-β-cyclodextrin (HPβCD) and methyl-β-cyclodextrin (MβCD) to promote cholesterol efflux from [³H]cholesterol-labeled larval Manduca sexta fat body and midgut was tested. In fat body, both β-cyclodextrins induced a two-phase efflux of cholesterol. The first rapid phase depended on cyclodextrin concentration and was more rapid for MβCD than for HPβCD. The second, slower, phase was independent of cyclodextrin concentration and type. In midgut, only the concentration-dependent phase was observed; the rate constants are approximately 85% slower than for fat body. In both cases, a low activation energy for transfer was observed, consistent with a collision mechanism where cyclodextrin interacts directly with cholesterol in plasma membrane to affect transfer. In fat body, the second slower phase is suggestive of a second pool of exchangeable cholesterol and most likely represents transfer of cholesterol from internal membranes or different lateral domains of the plasma membrane. The lack of this second phase in midgut suggests that midgut has only a single pool of exchangeable cholesterol. Although the rates are somewhat different, the overall kinetic pattern for cyclodextrin-mediated cholesterol transfer in insect fat body closely resembles that for vertebrate cells, while the single pool behavior of the midgut is not found in vertebrate cells.     

Voltage-current relation and K+ transport in tobacco hornworm (Manduca sexta) midgut

Summary Voltage-current curves for the isolated midgut of the tobacco hornworm were determined by transient and steady voltage clamping over the range of 200 to –200 mV. Over this range the transient method yields a linear relation while the steady method usually yields a curve consisting of two lines of differing slope which intersect at zero voltage. The difference between the results of the methods is due to a slow decline in total conductance which accompanies steady voltage clamping.Holding the midgut at short circuit increases the total conductance of the tissue in a manner consistent with increasing shunt conductance; this effect was seen in both diet-reared and leaf-reared animals.When potassium transport is inhibited by substitution of choline or sodium for potassium in bathing solution the total conductance decreases and the voltage-current curve intersects the normal curve in the hyperpolarizing region. Applying a simple equivalent circuit analysis to the results from partial or total potassium replacement suggests that the electromotive force of the potassium transport system is of the order of 140–190 mV. The conductance decrease during inhibition of potassium transport by transient anoxia is of similar magnitude, suggesting that a major effect of metabolic inhibition is to decrease the active conductance of the potassium transport pathway.