Sugar digestion in mosquitoes: identification and characterization of three midgut alpha-glucosidases of the neo-tropical malaria vector Anopheles aquasalis (Diptera: Culicidae)

Dietary carbohydrates provide an important source of energy for flight, and contribute to longevity and fecundity of mosquitoes. The most common sugar mosquitoes ingest is sucrose, and digestion of this substance is carried out mainly by alpha-glucosidases. In the current work, we tested the efficiency of sucrose on Anopheles aquasalis female diet. The best longevity (days) was reached when sugar was available in the diet, whereas most only blood fed females were dead 6 days after emergence. Three alpha-glucosidase isoforms were detected in the adult female midgut, named alphaGlu1, alphaGlu2 and alphaGlu3. These are acidic alpha-glucosidases with optima pH around pH 5.5. alphaGlu1 and alphaGlu2 are present in both secreted and membrane-bound forms, whereas alpha-Glu3 only in anchored to membranes. The alpha-glucosidase activity is concentrated mainly in the posterior midgut (70%), both in non-fed or 10% sucrose fed females. The single form of these alpha-glucosidases seemed to be approximately 70 kDa polypeptides, although alphaGlu2 is presented in >or=600 kDa self-aggregates. Km values of alphaGlu1, alphaGlu2 and alphaGlu3 differed significantly from each other, supporting the statement that three alpha-glucosidases are produced in the female midgut. Together, all data suggest that sugar is an essential component of A. aquasalis female diet. In addition, alpha-glucosidases are synthesized in the same place where sucrose is digested and absorbed, the midgut.     

Phylogenetic distribution of cysteine proteinases in beetles: evidence for an evolutionary shift to an alkaline digestive strategy in Cerambycidae

We characterized the digestive proteinases of eight species of beetles to improve our understanding of the phylogenetic distribution of serine and cysteine proteinases. Serine proteinases function optimally under alkaline pH conditions, whereas cysteine proteinases require acidic pH. The phylogenetic distribution of cysteine proteinases suggests that they first appeared in an early cucujiform ancestor, however, data for some groups is patchy, and there has been speculation that they have been lost in at least one group, the long-horned beetles (Cerambycidae). The pattern we found supports the hypothesized origin of the proteinases and extends their distribution to an additional superfamily. In addition, we confirmed the presence of cysteine proteinases in some Curculionoidea. Cysteine proteinases were absent, however, from all three species of cerambycids surveyed, supporting the hypothesis that this group has reverted to the more ancestral serine (alkaline) digestive strategy. In four species we compared the pH optima for total proteolytic activity to the actual pH of the midgut and found the match between optimal and actual pH to be weaker in the cerambycids. These findings suggest that either a close correlation between midgut pH and the proteolytic pH optimum is not needed for adequate digestive efficiency, or that midgut pH is a more constrained digestive feature and there has been insufficient time for it to shift upwards to maximize serine proteinase activity.     

The larval midgut of the cassava hornworm (Erinnyis ello)

Summary Columnar cells of the larval midgut of the cassava hornworm, Erinnyis ello, display microvilli with vesicles pinching off from their tips (anterior and middle midgut) or with a large number of double membrane spheres budding along their length (posterior midgut). Basal infoldings in columnar cells occur in a parallel array with many openings to the underlying space (posterior midgut) or are less organized with few openings (anterior and middle midgut). Goblet cells have a cavity, which is formed by invagination of the apical membrane and which occupies most of the cell (anterior and middle midgut) or only its upper part (posterior midgut). The infolded apical membrane shows modified microvilli, which sometimes (posterior midgut) or always (anterior and middle midgut) contain mitochondria. The cytoplasmic side of the membrane of the microvilli that contain mitochondria are studded with small particles. The results suggest that the anterior and middle region of the midgut absorbs water, whereas the posterior region secretes it. This results in a countercurrent flux of fluid, which is responsible for the enzyme recovery from undigested food before it is expelled. Intermediary and final digestion of food probably occur in the columnar cells under the action of plasma membrane-bound and glycocalix-associated enzymes.     

Comparison of the localization of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> Cry1A δ-endotoxins and their binding proteins in larval midgut of tobacco hornworm, <Emphasis Type="Italic">Manduca sexta</Emphasis>

Tobacco hornworm, Manduca sexta, is a model insect for studying the action of Bacillus thuringiensis (Bt) Cry toxins on lepidopterans. The proteins, which bind Bt toxins to midgut epithelial cells, are key factors involved in the insecticidal functions of the toxins. Three Cry1A-binding proteins, viz., aminopeptidase N (APN), the cadherin-like Bt-R₁, and membrane-type alkaline phosphatase (m-ALP), were localized, by immunohistochemistry, in sections from the anterior, middle, and posterior regions of the midgut from second instar M. sexta larvae. Both APN and m-ALP were distributed predominantly along microvilli in the posterior region and to a lesser extent on the apical tip of microvilli in the anterior and middle regions. Bt-R₁ was localized at the base of microvilli in the anterior region, over the entire microvilli in the middle region, and at both the apex and base of microvilli in the posterior region. The localization of rhodamine-labeled Cry1Aa, Cry1Ab, and Cry1Ac binding was determined on sections from the same midgut regions. Cry1Aa and Cry1Ab bound to the apical tip of microvilli almost equally in all midgut regions. Binding of Cry1Ac was much stronger in the posterior region than in the anterior and middle regions. Thus, binding sites for Bt proteins and Cry1A toxins are co-localized on the microvilli of M. sexta midgut epithelial cells.     

The digestive system of the “stick bug” Cladomorphus phyllinus (Phasmida,Phasmatidae): A morphological,physiological and biochemical analysis

This work presents a detailed morphofunctional study of the digestive system of a phasmid representative, Cladomorphus phyllinus. Cells from anterior midgut exhibit a merocrine secretion, whereas posterior midgut cells show a microapocrine secretion. A complex system of midgut tubules is observed in the posterior midgut which is probably related to the luminal alkalization of this region. Amaranth dye injection into the haemolymph and orally feeding insects with dye indicated that the anterior midgut is water-absorbing, whereas the Malpighian tubules are the main site of water secretion. Thus, a putative counter-current flux of fluid from posterior to anterior midgut may propel enzyme digestive recycling, confirmed by the low rate of enzyme excretion. The foregut and anterior midgut present an acidic pH (5.3 and 5.6, respectively), whereas the posterior midgut is highly alkaline (9.1) which may be related to the digestion of hemicelluloses. Most amylase, trypsin and chymotrypsin activities occur in the foregut and anterior midgut. Maltase is found along the midgut associated with the microvillar glycocalix, while aminopeptidase occurs in the middle and posterior midgut in membrane bound forms. Both amylase and trypsin are secreted mainly by the anterior midgut through an exocytic process as revealed by immunocytochemical data.     

Inhibition of cysteine and aspartyl proteinases in the alfalfa weevil midgut with biochemical and plant-derived proteinase inhibitors

Proteolytic activities in alfalfa weevil (Hypera postica) larval midguts have been characterized. Effects of pH, thiol activators, low-molecular weight inhibitors, and proteinase inhibitors (PIs) on general substrate hydrolysis by midgut extracts were determined. Hemoglobinolytic activity was highest in the acidic to mildly acidic pH range, but was maximal at pH 3.5. Addition of thiol-activators dithiothreitol (DTT), 2-mercaptoethanol (2-ME), or L-cysteine had little effect on hemoglobin hydrolysis at pH 3.5, but enhanced azocaseinolytic activity two to three-fold at pH 5.0. The broad cysteine PI E-64 reduced azocaseinolytic activity by 64% or 42% at pH 5 in the presence or absence of 5 mM L-cysteine, respectively. Inhibition by diazomethyl ketones, Z-Phe-Phe-CHN(2) and Z-Phe-Ala-CHN(2), suggest that cathepsins L and B are present and comprise approximately 70% and 30% of the cysteine proteolytic activity, respectively. An aspartyl proteinase component was identified using pepstatin A, which inhibited 32% (pH 3.5, hemoglobin) and 50% (pH 5, azocasein) of total proteolytic activity. This activity was completely inhibited by an aspartyl proteinase inhibitor from potato (API), and is consistent with the action of a cathepsin D-like enzyme. Hence, genes encoding PIs with specificity toward cathepsins L, B and D could potentially be effective for control of alfalfa weevil using transgenic plants.     

Identification and characterization of Aedes aegypti aminopeptidase N as a putative receptor of Bacillus thuringiensis Cry11A toxin

Bacillus thuringiensis subsp. israelensis, which is used worldwide to control Aedes aegypti larvae, produces Cry11Aa and other toxins during sporulation. In this study, pull-down assays were performed using biotinylated Cry11Aa toxin and solubilized brush border membrane vesicles prepared from midguts of Aedes larvae. Three of the eluted proteins were identified as aminopeptidease N (APN), one of which was a 140 kDa protein, named AaeAPN1 (AAEL012778 in VectorBase). This protein localizes to the apical side of posterior midgut epithelial cells of larva. The full-length AaeAPN1 was cloned and expressed in Eschericia coli and in Sf21 cells. AaeAPN1 protein expressed in Sf21 cells was enzymatically active, had a GPI-anchor but did not bind Cry11Aa. A truncated AaeAPN1, however, binds Cry11Aa with high affinity, and also Cry11Ba but with lower affinity. BBMV but not Sf21 expressed AaeAPN1 can be detected by wheat germ agglutinin suggesting the native but Sf21 cell-expressed APN1 contains N-acetylglucosamine moieties.     

Diversity of digestive proteinases in Tenebrio molitor (Coleoptera: Tenebrionidae) larvae

The spectrum of Tenebrio molitor larval digestive proteinases was studied in the context of the spatial organization of protein digestion in the midgut. The pH of midgut contents increased from 5.2-5.6 to 7.8-8.2 from the anterior to the posterior. This pH gradient was reflected in the pH optima of the total proteolytic activity, 5.2 in the anterior and 9.0 in the posterior midgut. When measured at the pH and reducing conditions characteristic of each midgut section, 64% of the total proteolytic activity was in the anterior and 36% in the posterior midgut. In the anterior midgut, two-thirds of the total activity was due to cysteine proteinases, whereas the rest was from serine proteinases. In contrast, most (76%) of the proteolytic activity in the posterior midgut was from serine proteinases. Cysteine proteinases from the anterior were represented by a group of anionic fractions with similar electrophoretic mobility. Trypsin-like activity was predominant in the posterior midgut and was due to one cationic and three anionic proteinases. Chymotrypsin-like proteinases also were prominent in the posterior midgut and consisted of one cationic and four anionic proteinases, four with an extended binding site. Latent proteinase activity was detected in each midgut section. These data support a complex system of protein digestion, and the correlation of proteinase activity and pH indicates a physiological mechanism of enzyme regulation in the gut.     

Compartmentalization of proteinases and amylases in Nauphoeta cinerea midgut.

Compartmentalization of proteinases, amylases, and pH in the midgut of Nauphoeta cinerea Oliv. (Blattoptera:Blaberidae) was studied in order to understand the organization of protein and starch digestion. Total proteolytic activity measured with azocasein was maximal at pH 11.5 both in anterior (AM) and posterior (PM) halves of the midgut, but the bulk of activity (67%) was found in PM. Total AM and PM preparations were fractionated on a Sephadex G-50 column and further analysed by means of activity electrophoresis and specific inhibitors and activators. The major activity in PM was classified as an unusual SH-dependent proteinase with M(r) 24,000 and pH optimum with synthetic substrate BApNA at 10.0. The enzyme was 43-fold activated in the presence of 1 mM DTT, insensitive to synthetic inhibitors of serine (PMSF, TLCK, TPCK) and cysteine (IAA, E-64) proteinases, strongly inhibited by STI, and displayed four active bands on zymograms. In PM, activities of trypsin-like, chymotrypsin-like, subtilisin-like, and cysteine proteinases were observed. Aspartic and metalloproteinases were not detected. In AM, activity of unusual SH-dependent proteinase also dominated and activity of chymotrypsin-like proteinase was observed, but their levels were much lower than in PM. Distribution of amylase activity, exhibiting an optimum at pH 6.0, was quite the opposite. The major part of it (67%) was located in AM. Treatment of amylase preparation with proteinases from AM and PM reduced amylase activity twofold. pH of the midgut contents was 6.0-7.2 in AM, 6.4-7.6 in the first and 8.8-9.3 in the second halves of PM. Thus, pH in AM is in good agreement with the optimal pH of amylase, located in this compartment, but the activity of proteinases, including the ability to degrade amylase, in such an environment is low. Active proteolysis takes place in the second half of PM, where pH of the gut is close to the optimal pH of proteinases.     

Sequence and function of lysosomal and digestive cathepsin D-like proteinases of Musca domestica midgut

Musca domestica larvae display in anterior and middle midgut contents, a proteolytic activity with pH optimum of 3.0–3.5 and kinetic properties like cathepsin D. Three cDNAs coding for preprocathepsin D-like proteinases (ppCAD 1, ppCAD 2, ppCAD 3) were cloned from a M. domestica midgut cDNA library. The coded protein sequences included the signal peptide, propeptide and mature enzyme that has all conserved catalytic and substrate binding residues found in bovine lysosomal cathepsin D. Nevertheless, ppCAD 2 and ppCAD 3 lack the characteristic proline loop and glycosylation sites. A comparison among the sequences of cathepsin D-like enzymes from some vertebrates and those found in M. domestica and in the genomes of Aedes aegypti, Drosophila melanogaster, Tribolium castaneum, and Bombyx mori showed that only flies have enzymes lacking the proline loop (as defined by the motif: DxPxPx(G/A)P), thus resembling vertebrate pepsin. ppCAD 3 should correspond to the digestive cathepsin D-like proteinase (CAD) found in enzyme assays because: (1) it seems to be the most expressed CAD, based on the frequency of ESTs found. (2) The mRNA for CAD 3 is expressed only in the anterior and proximal middle midgut. (3) Recombinant procathepsin D-like proteinase (pCAD 3), after auto-activation has a pH optimum of 2.5–3.0 that is close to the luminal pH of M. domestica midgut. (4) Immunoblots of proteins from different tissues revealed with anti-pCAD 3 serum were positive only in samples of anterior and middle midgut tissue and contents. (5) CAD 3 is localized with immunogold inside secretory vesicles and around microvilli in anterior and middle midgut cells. The data support the view that on adapting to deal with a bacteria-rich food in an acid midgut region, M. domestica digestive CAD resulted from the same archetypical gene as the intracellular cathepsin D, paralleling what happened with vertebrates. The lack of the proline loop may be somehow associated with the extracellular role of both pepsin and digestive CAD 3.     

Fractionation of digestive proteinases from Tenebrio molitor (Coleoptera: Tenebrionidae) larvae and role in protein digestion

Tenebrio molitor larval digestive proteinases were purified and characterized by gel filtration chromatography combined with activity electrophoresis. Cysteine proteinases, consisting of at least six distinct activities, were found in three chromatographic peaks in anterior and posterior midgut chromatographies. The major activity in the anterior midgut, peak cys II, consisted of cysteine proteinases with Mm of 23 kDa. The predominant peak in the posterior, cys I, was represented by 38 kDa proteinases. The activities of all cysteine proteinases were maximal in buffers from pH 5.0 to 7.0, with 80% stability at pH values from 4.0 to 7.0. In the conditions of the last third of the midgut, the activity and stability of cysteine proteinases was sharply decreased. Trypsin-like activity included a minor peak of "heavy" trypsins with Mm 59 kDa, located mainly in the anterior midgut. An in vitro study of the initial stages of digestion of the main dietary protein, oat 12S globulin, by anterior midgut proteinases revealed that hydrolysis occurred through the formation of intermediate high-Mm products, similar to those formed during oat seed germination. Cysteine proteinases from the cys III peak and heavy trypsins were capable of only limited proteolysis of the protein, whereas incubation with cys II proteinases resulted in substantial hydrolysis of the globulin.     

Midgut proteinase activities in larvae of Anoplophora glabripennis (Coleoptera: Cerambycidae) and their interaction with proteinase inhibitors

The major proteinase activities in the larval midgut of a common poplar tree borer, Anoplophora glabripennis, were characterised. Overall digestive capacity, as measured by casein hydrolysis, showed a pH optimum between 10 and 11.5, suggestive of serine endopeptidase activity. Trypsin, chymotrypsin, and chymotrypsin-like activities were detected using specific p-nitroanilide synthetic substrates and by use of specific serine endopeptidase inhibitors. These activities also showed pH optima in the extreme alkaline range. The absence of cysteine, aspartic, and metallo-endopeptidases were confirmed using class specific proteinase inhibitors. The dominant exopeptidase in the midgut is leucine aminopeptidase with a pH optimum of 7–9. Carboxypeptidase a and b activity were barely detectable. A large range of serine endopeptidase inhibitors were screened and were found to vary widely in their ability to inhibit casein hydrolysis. Potato proteinase inhibitor 1 (a chymotrypsin inhibitor) and wheat-germ trypsin inhibitor 1 inhibited particularly effectively in tandem and represent possible candidates for gene transformation to produce plants tolerant to this pest. © 1996 Wiley-Liss, Inc.     

Digestive physiology and characterization of digestive cathepsin L-like proteinase from the sugarcane weevil Sphenophorus levis

Sugarcane is an important crop that has recently become subject to attacks from the weevil Sphenophorus levis, which is not efficiently controlled with chemical insecticides. This demands the development of new control devices for which digestive physiology data are needed. In the present study, ion-exchange chromatography of S. levis whole midgut homogenates, together with enzyme assays with natural and synthetic substrates and specific inhibitors, demonstrated that a cysteine proteinase is a major proteinase, trypsin is a minor one and chymotrypsin is probably negligible. Amylase, maltase and the cysteine proteinase occur in the gut contents and decrease throughout the midgut; trypsin is constant in the entire midgut, whereas a membrane-bound aminopeptidase predominates in the posterior midgut. The cysteine proteinase was purified to homogeneity through ion-exchange chromatography. The purified enzyme had a mass of 37 kDa and was able to hydrolyze Z-Phe-Arg-MCA and Z-Leu-Arg-MCA with k_cat/K_m values of 20.0 ± 1.1 μM⁻¹ s⁻¹ and 30.0 ± 0.5 μM⁻¹ s⁻¹, respectively, but not Z-Arg-Arg-MCA. The combined results suggest that protein digestion starts in the anterior midgut under the action of a cathepsin L-like proteinase and ends on the surface of posterior midgut cells. All starch digestion takes place in anterior midgut. These data will be instrumental to developing S. levis-resistant sugarcane.     

Molecular cloning and characterization of a midgut chymotrypsin-like enzyme from the lesser grain borer, Rhyzopertha dominica

A cDNA encoding a chymotrypsinogen-like protein in midguts of the lesser grain borer, Rhyzopertha dominica (F.) (Coleoptera: Bostrichidae) was cloned and sequenced. The 901 bp cDNA contains an 816-nucleotide open reading frame encoding 272-amino acids. The predicted molecular mass and pI of the mature enzyme are 23.7 kDa and 4.64, respectively. The encoded protein includes amino acid sequence motifs that are conserved with 5 homologous chymotrypsinogen proteins from other insects. Features of the putative chymotrypsin-like protein from R. dominica include the serine proteinase active site (His(90), Asp(133), Ser(226)), conserved cysteine residues for disulfide bridges, the residues (Gly(220), Gly(243), Asp(252)) that determine chymotrypsin specificity, and both zymogen activation and signal peptides. A TPCK-sensitive caseinolytic protein (P6) with an estimated molecular mass of 24 kDa is present in midgut extracts of R. dominica and can be resolved by electrophoresis on 4-16% polyacrylamide gels. The molecular mass of this caseinolytic enzyme is similar to that of the chymotrypsin deduced from cDNA. Midgut extracts of R. dominica readily hydrolyzed azocasein and N-succinyl-alanine-alanine-proline-phenylalanine-p- nitroanilide (SAAPFpNA), a chymotrypsin-specific substrate. Properties of the enzymes responsible for these activities were partially characterized with respect to distribution in the gut, optimum pH, and sensitivity toward selected proteinase inhibitors. Optimal activity against both azocasein and SAAPFpNA occurs in a broad pH range from about 7 to 10. Both azocasein and SAAPFpNA activities, located primarily in the anterior midgut region, are inhibited by aprotinin, phenylmethyl sulphonylfluoride (PMSF), and soybean trypsin inhibitor (STI). TPCK (N-alpha-tosyl-L-phenylalanine chloromethyl ketone) and chymostatin inhibited more than 60% of SAAPFpNA but only about 10-20% of azocasein activity. These results provide additional evidence for the presence of serine proteinases, including chymotrypsin, in midguts of R. dominica. Arch. Insect Biochem. Physiol. 43:173-184, 2000.Published 2000 Wiley-Liss, Inc.     

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.     

Prey digestion in the midgut of the predatory bug Podisus nigrispinus (Hemiptera: Pentatomidae)

Pre-oral digestion is described as the liquefaction of the solid tissues of the prey by secretions of the predator. It is uncertain if pre-oral digestion means pre-oral dispersion of food or true digestion in the sense of the stepwise bond breaking of food polymers to release monomers to be absorbed. Collagenase is the only salivary proteinase, which activity is significant (10%) in relation to Podisus nigrispinus midgut activities. This suggests that pre-oral digestion in P. nigrispinus consists in prey tissue dispersion. This was confirmed by the finding of prey muscles fibers inside P. nigrispinus midguts. Soluble midgut hydrolases from P. nigrispinus were partially purified by ion-exchange chromatography, followed by gel filtration. Two cathepsin L-like proteinases (CAL1 and CAL2) were isolated with the properties: CAL1 (14.7 kDa, pH optimum (pHo) 5.5, km with carbobenzoxy-Phe-Arg-methylcoumarin, Z-FR-MCA, 32 μM); CAL2 (17 kDa, pHo 5.5, km 11 μM Z-FR-MCA). Only a single molecular species was found for the other enzymes with the following properties are: amylase (43 kDa, pHo 5.5, km 0.1% starch), aminopeptidase (125 kDa, pHo 5.5, km 0.11 mM l-Leucine-p-nitroanilide), α-glucosidase (90 kDa, pHo 5.0, km 5mM with p-nitrophenyl α-d-glucoside). CAL molecular masses are probably underestimated due to interaction with the column. Taking into account the distribution of hydrolases along P. nigrispinus midguts, carbohydrate digestion takes place mainly at the anterior midgut, whereas protein digestion occurs mostly in middle and posterior midgut, as previously described in seed- sucker and blood-feeder hemipterans.     

Alterations of tight and gap junctions in mouse hepatocytes following administration of colchicine

Summary Mature columnar cells of the midgut of Cubitermes contain a prominent secretion product observed at light- and electron-microscopic levels. At the ultrastructural level the product is resolved as an electron dense material contained in vesicles up to 1 m diameter that accumulate in the apical cytoplasm. The vesicles are composite, apparently formed by coalescence of at least two types of precursor vesicle both of which originate from the Golgi apparatus. Discharge of the product takes place by exocytosis into intercellular space at or in the vicinity of the apical septate junction complex. Augmentation of apical surface area by microvilli is less prominent in Cubitermes than in other termites for which data are available. This and other evidence suggests that absorptive functions are reduced in the midgut of this insect.     

Midgut proteinases of the cockroachNauphoeta cinerea

The study of properties of proteolytic enzymes in midgut of imago of the cockroachNauphoeta cinerea Oliv. Has been carried out. It is shown that the total proteolytic activity of digestive proteases, measured with azocasein as substrate, is maximal at pH 11.5 both in the anterior and in the posterior parts of the midgut. The predominant part of this activity (67%) was present in the posterior part. Fractionation of preparation from the posterior part on a column with Sephadex G-50 and subsequent analysis of the activity in the obtained fractions using specificp-nitroanilide substrates and effects of activators and inhibitors of active center have allowed revealing three types of activity of serine proteinases and one cysteine proteinase. No activity of aspartic and metalloproteinases were detected. Among serine proteinases, one trypsin-like, one unusual SHdependent serine, one chymotrypsin-like, and not less than two enzymes hydrolyzing specific substrate of subtilisin were established. The fractionation of the preparation from the anterior part has allowed revealing only three proteinases that were similar by their properties to cysteine, SHdependent serine, and chymotrypsin-like ones in the posterior part of midgut. Their activity was lower in the anterior, than in the posterior part of the midgut. The probable causes of the low proteolytic activity in the anterior part of the midgut are discussed.     

Nature of the anchors of membrane-bound aminopeptidase,amylase, and trypsin and secretory mechanisms in Spodoptera frugiperda (Lepidoptera) midgut cells

Spodoptera frugiperda larvae have a microvillar aminopeptidase and both soluble and membrane-bound forms of amylase and trypsin. Membrane-bound aminopeptidase is solubilized by glycosyl phosphatidylinositol-specific phospholipase C (GPI-PLC) and detergents, suggesting it has a GPI anchor. Membrane-bound trypsin is not affected by GPI-PLC, although it is solubilized by papain and by different detergents. Membrane-bound amylase is similar to trypsin, although once solubilized in detergent it behaves as a hydrophilic protein. Musca domestica trypsin antiserum cross-reacts with only one polypeptide from S. frugiperda midgut. With this antiserum, trypsin was immunolocalized in the anterior midgut cells at the microvillar surface and on the membranes of secretory vesicles found in the apical cytoplasm and inside the microvilli. The data suggest that in this region trypsin is bound to the secretory vesicle membrane by a hydrophobic anchor. Vesicles migrate through the microvilli and are discharged into the lumen by a pinching-off process. Trypsin is then partly processed to a soluble form and partly, still bound to vesicle membranes, incorporated into the peritrophic membrane. In posterior midgut cells, trypsin immunolabelling is randomly distributed inside the secretory vesicles and at the microvilli surface, suggesting exocytosis. Amylase probably follows a route similar to that described for trypsin in anterior midgut, although membrane-bound forms (peptide anchor) solubilize apparently as a consequence of a pH increase inside the vesicles.     

Differences in midgut serine proteinases from larvae of the bruchid beetles Callosobruchus maculatus and Zabrotes subfasciatus

Proteinase activities in the larval midguts of the bruchids Callosobruchus maculatus and Zabrotes subfasciatus were investigated. Both midgut homogenates showed a slightly acidic to neutral pH optima for the hydrolysis of fluorogenic substrates. Proteolysis of epsilon-aminocaproil-Leu-Cys(SBzl)-MCA was totally inhibited by the cysteine proteinase inhibitors E-64 and leupeptin, and was activated by 1.5 mM DTT in both insects, while hydrolysis of the substrate Z-ArgArg-MCA was inhibited by aprotinin and E-64, which suggests that it is being hydrolysed by serine and cysteine proteinases. Gel assays showed that the proteolytic activity in larval midgut of C. maculatus was due to five major cysteine proteinases. However, based on the pattern of E-64 and aprotinin inhibition, proteolytic activity in larval midgut of Z. subfasciatus was not due only to cysteine proteinases. Fractionation of the larval midgut homogenates of both bruchids through ion-exchange chromatography (DEAE-Sepharose) revealed two peaks of activity against Z-ArgArg-MCA for both bruchid species. The fractions from C. maculatus have characteristics of cysteine proteinases, while Z. subfasciatus has one non-retained peak of activity containing cysteine proteinases and another eluted in a gradient of 250-350 mM NaCl. The proteolytic activity of the retained peak is higher at pH 8.8 than at pH 6.0 and corresponds with a single peak that is active against N-p-tosyl-GlyGlyArg-MCA, and sensitive to 250 microM aprotinin (90% inhibition). The peak contains a serine proteinase which hydrolyzes alpha-amylase inhibitor 1 from the common bean (Phaseolus vulgaris). Arch.