Dual origin of gut proteases in Formosan subterranean termites (Coptotermes formosanus Shiraki) (Isoptera: Rhinotermitidae)

Cellulose digestion in lower termites, mediated by carbohydrases originating from both termite and endosymbionts, is well characterized. In contrast, limited information exists on gut proteases of lower termites, their origins and roles in termite nutrition. The objective of this study was to characterize gut proteases of the Formosan subterranean termite (Coptotermes formosanus Shiraki) (Isoptera: Rhinotermitidae). The protease activity of extracts from gut tissues (fore-, mid- and hindgut) and protozoa isolated from hindguts of termite workers was quantified using hide powder azure as a substrate and further characterized by zymography with gelatin SDS-PAGE. Midgut extracts showed the highest protease activity followed by the protozoa extracts. High level of protease activity was also detected in protozoa culture supernatants after 24 h incubation. Incubation of gut and protozoa extracts with class-specific protease inhibitors revealed that most of the proteases were serine proteases. All proteolytic bands identified after gelatin SDS-PAGE were also inhibited by serine protease inhibitors. Finally, incubation with chromogenic substrates indicated that extracts from fore- and hindgut tissues possessed proteases with almost exclusively trypsin-like activity while both midgut and protozoa extracts possessed proteases with trypsin-like and subtilisin/chymotrypsin-like activities. However, protozoa proteases were distinct from midgut proteases (with different molecular mass). Our results suggest that the Formosan subterranean termite not only produces endogenous proteases in its gut tissues, but also possesses proteases originating from its protozoan symbionts.     

Digestive proteolytic activity in larvae and adults of Bactrocera oleae Gmelin (Diptera: Tephritidae)

Digestive proteolytic activity in larvae and adults of Bactrocera oleae was studied using specific substrates and inhibitors. The optimal pH for general proteolytic activity was 4 and 10 for soluble and membrane-bound fractions of larvae, and 9 for the soluble fraction of adults. The highest activities of general proteases were revealed at temperatures of 25 °C and 45 °C for both the soluble and membrane-bound fractions of larvae as well as the soluble fraction of adults. Determination of the specific protease activities demonstrated the presence of serine and cysteine proteases in addition to two exopeptidases in the larvae and adults. However, trypsin-like protease, chymotrypsin-like protease, and two exopeptidases of larvae, and chymotrypsin-like protease as well as cathepsin L of adults had no activity in the soluble fraction. The presence of specific proteases was verified by using specific inhibitors such as PMSF, TLCK, TPCK, E-64, EDTA, phenanthroline, and DTT. Finally, feeding of B. oleae larvae on different olive varieties revealed the highest trypsin-like protease, chymotrypsin-like protease, elastase, cathepsin B, cathepsin L, and cathepsin D on Amigdalifolia, Coratina, Baladi, Mari, Conservalia, Baladi, and Arbequina, respectively. These results showed digestive proteolytic activities in B. oleae for the first time, and could be the basic knowledge required for finding a control procedure to decrease the damage of this destructive pest around the world.     

Digestive proteolytic activity in the pistachio green stink bug,Brachynema germari Kolenati (Hemiptera: Pentatomidae)

Proteolytic activity in the digestive system of the pistachio green stink bug, Brachynema germari, was investigated. The maximum total proteolytic activity in the midgut extract was observed at pH 5, suggesting the presence of cysteine proteases. Hydrolyzing the specific substrates for cysteine proteases revealed the presence of cathepsin B and cathepsin L activities in the midgut extract. The presence of cysteine proteases was confirmed by their noticeable inhibition and activation due to specific inhibitors and activators, respectively. The significant inhibition of chymotryptic activity by the inhibitors showed the presence of chymotrypsin in the midgut. No considerable tryptic activity was observed in the midgut extract. There was no detectable total proteolytic activity in the salivary gland extract. Tryptic activity of the salivary gland extract was also inhibited by the specific inhibitors. The substrates for cysteine proteases were also slightly hydrolyzed by the salivary gland extract. Zymogram analysis showed at least one distinct band due to cysteine protease activity in the midgut extract, and the cysteine protease inhibitor caused almost complete disappearance of the band. Cathepsin B and L activities were mainly detected in midgut divisions m₁ and m₃, respectively, and maximum chymotrypsin and trypsin activities were observed in m₃. In general, the results revealed the significant presence of cathepsin B, cathepsin L, and chymotrypsin proteases in the midgut extract. The major proteolytic activity in the salivary glands seems to be conducted by trypsin-like proteases.     

Biochemical response between insects and plants: an investigation of enzyme activity in the digestive system of Leucoptera coffeella (Lepidoptera: Lyonetiidae) and leaves of Coffea arabica (Rubiaceae) after herbivory

Plant defence mechanisms can reduce the digestive enzyme activity of insect pests. The aim of this study was to determine the relationship between the production of proteinase inhibitors, lipoxygenase and polyphenol oxidase activity in Coffea arabica (Catuai IAC 15) plants, and the digestive enzyme activity in the pest Leucoptera coffeella (Lepidoptera: Lyonetiidae) after feeding on the plant. The production of proteinase inhibitors was evaluated with L‐BApNA as a substrate. We studied lipoxygenase activity with linoleic acid and polyphenol oxidase activity with catechol substrates, in coffee plants damaged (T1) and not damaged (T2) by L. coffeella. L. coffeella digestive enzyme activity was verified by trypsin‐like (substrate l ‐BApNA and l ‐TAME), chymotrypsin‐like (BTpNA and ATEE), cysteine proteases (l ‐BApNA) and total protease (azocasein). Proteinase inhibitor production and lipoxygenase and polyphenol oxidase activity in C. arabica increases (P ≤ 0.05) with L. coffeella damage. Our results provide important information that these enzymatic activities may play a role in plant defence processes in C. arabica. Trypsin‐like activity increases, whereas chymotrypsin‐like and cysteine protease activity decrease in the midgut of L. coffeella, which acts as a defence mechanism.     

Effect of pH on protease activities in the large intestine

The influence of pH on proteolysis in different fractions of human faeces was studied with a variety of chromogenic substrates. The pH optima of proteases in the washed cell fraction and washed particulate fraction were neutral to alkaline, whereas extra-cellular proteolysis in the cell-free supernatant fraction was relatively insensitive to pH over the range 6·4–8·0. Measurements with p -nitroanilide substrates suggested the presence of more than one elastase-like, trypsin-like and chymotrypsin-like protease in the gut.     

Biochemical characterization of midgut digestive proteases from Mamestra brassicae (cabbage moth; Lepidoptera: Noctuidae) and effect of soybean Kunitz inhibitor (SKTI) in feeding assays

Proteolytic activities in soluble protein extracts from Mamestra brassicae (cabbage moth) larval midgut were analysed using specific peptide substrates and proteinase inhibitors. Serine proteinases were the major activities detected, with chymotrypsin-like and trypsin-like activities being responsible for approximately 62% and 19% of the total proteolytic activity towards a non-specific protein substrate. Only small amounts of elastase-like activities could be detected. The serine proteinases were active across the pH range 7-12.5, with both trypsin-like and chymotrypsin-like activities maximal at pH 11.5. The digestive proteinases were stable to the alkaline environment of the lepidopteran gut over the timescale of passage of food through the gut, with 50% of trypsin and 40% of chymotrypsin activity remaining after 6h at pH 12, 37 degrees C. Soybean Kunitz trypsin inhibitor (SKTI) ingestion by the larvae had a growth-inhibitory effect, and induced inhibitor-insensitive trypsin-like activity. Qualitative and quantitative changes in proteinase activity bands after gel electrophoresis of gut extracts were evident in SKTI-fed larvae when compared with controls, with increases in levels of most bands, appearance of new bands, and a decrease in the major proteinase band present in extracts from control insects.     

Proteolytic gut activities in the rice water weevil,Lissorhoptrus brevirostris Suffrian (Coleoptera: Curculionidae)

Digestive endoprotease activities of the rice water weevil, Lissorhoptrus brevirostris Suffrian (Coleoptera: Curculionidae), were characterized based on the ability of gut extracts to hydrolyze specific synthetic substrates, optimal pH, and hydrolysis sensitivity to protease inhibitors. Larvae of this species were found to use a complex proteolytic system that includes cathepsin D-, cathepsin B-, trypsin-, and chymotrypsin-like activities. Trypsin-like activity was evenly distributed among the anterior, middle, and posterior portions of the gut, whereas cathepsin B- and cathepsin D-like activities were mainly located in the anterior and middle sections, and the chymotrypsin-like activity was highest in the middle and posterior sections. Gelatin-containing native-PAGE gels indicated the presence of several aspartyl, cysteine, and serine protease forms and confirmed the spatial organization of the proteolytic digestive process.     

Purification and characterization of a digestive alkaline protease from the larvae of Spilosoma obliqua

A digestive protease from Spilosoma obliqua (Lepidoptera: Arctiidae) fifth instar larval guts was purified and characterized. The protease was purified using ammonium sulfate fractionation, ion-exchange chromatography, and hemoglobin-sepharose affinity chromatography. The purification procedure resulted in a 37-fold increase in the specific activity of the protease. Protease thus obtained was found to be electrophoretically pure under native and denaturing conditions. The purified protease had a molecular mass of 90 kDa as determined by gel filtration, and a pH optimum of 11.0. The purified protease optimally hydrolyzed casein at 50 degrees C. A Km of 2 x10(-6) M was obtained using BApNA as a substrate for the purified alkaline protease. The ability of S. obliqua protease and bovine trypsin to hydrolyze various synthetic substrates (BApNA, BAEE, and BAME), and the inhibition patterns of S. obliqua and bovine trypsin with "classical" trypsin inhibitors are also reported.     

麻蝇幼虫肠道类胰蛋白酶的分离纯化及性质(英文)

麻蝇幼虫肠液经硫铵沉淀, ＤＥＡＥ－Ｓｅｐｈａｄｅｘ Ａ－２５离子交换层析, ＳＢＢＩ－Ｓｅｐｈａｒｏｓｅ ４Ｂ亲和层析,分离纯化出一种分子量为 １６ｋＤ的蛋白酶。底物及抑制剂的特异性表明,该酶为类胰蛋白酶。其能够强烈地降解蛋白酶非专一底物酪蛋白和 Ｈｉｄｅ ｐｏｗｄｅｒ ａｚｕｒｅ,以及类胰蛋白酶专一底物 Ｂｚ－Ｐｈｅ－Ｖａｌ－Ａｒｇ ＮＡ, Ｂｚ－Ｐｒｏ－Ｐｈｅ－Ａｒｇ ＮＡ和Ｂｚ－Ｖａｌ－Ｇｌｙ－Ａｒｇ ＮＡ．该酶又能被丝氨酸蛋白酶抑制剂ＰＭＳＦ,类胰蛋白酶抑制剂 ＳＢ－ＢＩ和Ｌｅｕｐｅｐｔｉｎ强烈地抑制。蛋白酶在酸性环境下极不稳定,在弱碱环境（ｐＨ８．５－９．５）中活性最高。     

Proteolytic activity in Plagiodera versicolora Laicharting (Coleoptera: Chrysomelidae): Characterization of digestive proteases and effect of host plants

Proteolytic profiles in the midgut of Plagiodera versicolora were studied using biochemical approaches, and the effects of host plants on possible changes in their activity were determined. Morphology of the alimentary canal revealed several areas of sections, namely bucca, pharynx, esophagus, crop, midgut, ileum, rectum and anus. A pH of 6 and 11 was found to be optimal for soluble and membrane-bound fractions, by using azocasein 2% as a substrate. Determination of specific proteases demonstrates the presence of trypsin-like, chymotrypsin-like, elastase, cathepsin B, cathepsin L and cathepsin D, as well as two exopeptidases. Regarding site of activity for each specific protease, it was found that the major activity of cathepsin B and cathepsin L was in the soluble fraction, chymotrypsin, cathepsin D and two exopeptidases in membrane-bound fraction. Additionally, trypsin-like and elastase activities had no significant differences between fractions. The presence of the above mentioned specific proteases was verified using the specific inhibitors PMSF, TLCK, TPCK, cystatin, phenanthroline and DTT. Feeding of the beetle on four host plants: including Salix aegyptica, S. alba, Populus alba and P. caspica, from the 1st larval instar to adult, revealed the highest trypsin-, chymotrypsin-like and elastase activities in the individuals fed on S. aegyptica and S. alba, respectively. Regarding cathepsins B and L, the highest activities were observed on S. alba and S. aegypticum but cathepsin D was higher in S. Alba and P. alba. Feeding on S. alba and S. aegypticum showed the highest activities of amino- and carboxy-peptidases, respectively.     

PURIFICATION AND PARTIAL CHARACTERIZATION OF A GUT TRYPSIN-LIKE PROTEASE FROM LARVAE OF FLESH FLY BOETTCHERISCA PEREGRIN A (DIPTERA: SARCOPHAGAE)

Abstract A 16kD protease was purified from the gut extract of larvae of Boettcherisca peregrina , after ammonium sulfate precipitation, DEAE-Sephadex A-25 ion-exchange chromatography and SBBI-Sepharose 4B affinity chromatography. The results of substrate and inhibitor specificity indicated that the protease behaved as a trypsin-like protease. It possesses high activity against non-specific substrate casein and Hide powder azure, and against trypsin-specific substrates Bz-Phe-Val-Arg NA, Bz-Pro-Phe-Arg NA and Bz-Val-Gly-Arg NA. It can be strongly inhibited by PMSF, phenymethysulfonyl fluoride (serine protease inhibitor), SBBI, soybean Bowman-Birk inhibitor and Leupeptin (trypsin-specific inhibitor). Activity of this protease was found to be maximal at the alkaline range of pH 8. 5–9. 5.     

Putative protein digestion in a sap-sucking homopteran plant pest (rice brown plant hopper; Nilaparvata lugens: Delphacidae)--identification of trypsin-like and cathepsin B-like proteases

Sap-sucking phytophagous insect species of the order Hemiptera have been assumed not to carry out digestive proteolysis, but instead to rely on free amino acids in the phloem and xylem saps for their nutritional requirements. Extracts prepared from isolated guts of rice brown planthopper (Nilaparvata lugens), a homopteran crop pest, were shown to contain protease activity, with hydrolysis of both protein and synthetic peptide substrates being observed. Assays with specific inhibitors suggested that a trypsin-like serine protease was responsible for most of hydrolytic activity against synthetic substrates. A cDNA library was prepared from RNA extracted from N. lugens gut tissue, and screened for protease-encoding sequences. cDNAs for a cathepsin B-like protease and a trypsin-like protease were isolated and fully characterised; the latter exhibits a novel C-terminal region and an unusual activation mechanism, and represents a small gene family. Soya bean Kunitz trypsin inhibitor (SKTI) is an effective inhibitor of protein hydrolysis by N. lugens gut extracts in vitro, explaining why transgenic rice plants expressing this protein are partially resistant to the insect (Mol. Breed. 5 (1999) 1). It is suggested that digestive proteolysis may be widespread in sap-sucking homoptera, and can make a significant contribution to nutrition.     

Characterisation of cysteine proteinases responsible for digestive proteolysis in guts of larval western corn rootworm (Diabrotica virgifera) by expression in the yeast Pichia pastoris

Cysteine proteinases are the major class of enzymes responsible for digestive proteolysis in western corn rootworm (Diabrotica virgifera), a serious pest of maize. A larval gut extract hydrolysed typical cathepsin substrates, such as Z-phe-arg-AMC and Z-arg-arg-AMC, and hydrolysis was inhibited by Z-phe-tyr-DMK, specific for cathepsin L. A cDNA library representing larval gut tissue mRNA contained cysteine proteinase-encoding clones at high frequency. Sequence analysis of 11 cysteine proteinase cDNAs showed that 9 encoded cathepsin L-like enzymes, and 2 encoded cathepsin B-like enzymes. Three enzymes (two cathepsin L-like, DvRS5 and DvRS30, and one cathepsin B-like, DvRS40) were expressed as recombinant proteins in culture supernatants of the yeast Pichia pastoris. The cathepsin L-like enzymes were active proteinases, whereas the cathepsin B-like enzyme was inactive until treated with bovine trypsin. The amino acid residue in the S2 binding pocket, the major determinant of substrate specificity in cathepsin cysteine proteinases, predicted that the two cathepsin L-like enzymes, DvRS5 and DvRS30, should differ in substrate specificity, with the latter resembling cathepsin B in hydrolysing substrates with a positively charged residue at P2. This prediction was confirmed; DvRS5 only hydrolysed Z-phe-arg-AMC and not Z-arg-arg-AMC, whereas DvRS30 hydrolysed both substrates. The enzymes showed similar proteolytic activity towards peptide substrates.     

Cloning and characterization of chymotrypsin- and trypsin-like cDNAs from the gut of the Hessian fly [Mayetiola destructor (Say)

Fifteen unique cDNA clones encoding trypsin- or chymotrypsin-like proteins were cloned and characterized from a gut cDNA library derived from Hessian fly [Mayetiola destructor (Say)] larvae. Based on sequence similarities, the cDNAs were sorted into five gene groups, which were named MDP1 to MDP5. Two of the gene groups, MDP1 and MDP2, encoded chymotrypsin-like proteins; the other three encoded putative trypsins. All deduced proteins have conserved His(87), Asp(136), and Ser(241) residues for the catalytic triad and three pairs of cysteine residues for disulfide bridge configurations. The substrate specificity determination residue at position 235 was also conserved in the putative trypsins and chymotrypsins. In addition, all the deduced protein precursors had a typical secretion signal peptide and activation peptide. Northern blot analysis revealed that all these gene groups were exclusively expressed in the larval stage. The expression profiles for each gene group differed significantly in different ages of the larva, as well as in different tissues. Protease activity analysis of gut extract, using specific inhibitors, demonstrated that serine proteases were the major digestive enzymes in the gut of M. destructor larvae. Serine protease inhibitors inhibited as much as 90% proteolytic activities of gut extract, whereas inhibitors specific to other proteases, including cysteine proteases, aspartic proteases, and metallo-proteases, inhibited only 10-24% of gut protease activity.     

12a-羟基鱼藤酮对斜纹夜蛾生殖力的影响及其作用机理

为探讨12a-羟基鱼藤酮对斜纹夜蛾Prodenia litura (Fabricius)生殖力的影响及其作用机理, 本文测定了该虫取食12a-羟基鱼藤酮后的产卵量。在用高效液相色谱仪确定其卵巢组织中存在12a-羟基鱼藤酮后, 通过MTT法测定该化合物对斜纹夜蛾卵巢细胞（PL细胞）的细胞毒性,并利用流式细胞仪检测该化合物对PL细胞细胞周期、膜电位、胞内钙离子浓度、线粒体膜电位的影响。结果表明:斜纹夜蛾幼虫取食12a-羟基鱼藤酮后产卵量下降80％以上, 并从卵巢组织中检测出该化合物。12a-羟基鱼藤酮对PL细胞增殖具有抑制活性, 其IC₅₀为6.6 mg/L。该化合物将PL细胞周期阻滞于S期, 导致PL细胞膜电位、线粒体膜电位和细胞内游离钙离子浓度均显著升高。该化合物导致斜纹夜蛾繁殖力降低。12a-羟基鱼藤酮将PL细胞增殖阻滞于细胞周期的S期。同时, 该化合物对卵巢细胞具有较弱的毒杀活性, 导致一些卵巢细胞死亡。由于上述原因, 卵巢细胞数量逐渐减少而导致卵巢管萎缩。卵巢管的萎缩使卵巢管内的卵母细胞不能发育成卵细胞, 故其生殖力下降。     

Characterization of a membrane-associated serine protease in Escherichia coli.

Three membrane-associated proteolytic activities in Escherichia coli were resolved by DEAE-cellulose chromatography from detergent extracts of the total envelope fraction. On the basis of substrate specificity for the hydrolysis of chromogenic amino acid ester substrates, the first two eluting activities were determined previously to be protease V and protease IV, respectively (M. Pacaud, J. Bacteriol. 149:6-14, 1982). The third proteolytic activity eluting from the DEAE-cellulose column was further purified by affinity chromatography on benzamidine-Sepharose 6B. We termed this enzyme protease VI. Protease VI did not hydrolyze any of the chromogenic substrates used in the detection of protease IV and protease V. However, all three enzymes generated acid-soluble fragments from a mixture of E. coli membrane proteins which were biosynthetically labeled with radioactive amino acids. The activity of protease VI was sensitive to serine protease inhibitors. Using [3H]diisopropylfluorophosphate as an active-site labeling reagent, we determined that protease VI has an apparent molecular weight of 43,000 in polyacrylamide gels. All three membrane-associated serine proteases were insensitive to inhibition by Ecotin, and endogenous, periplasmic inhibitor of trypsin.     

Molecular cloning of trypsin-like cDNAs and comparison of proteinase activities in the salivary glands and gut of the tarnished plant bug Lygus lineolaris (Heteroptera: Miridae)

Using specific proteinase inhibitors, we demonstrated that serine proteinases in the tarnished plant bug, Lygus lineolaris, are major proteinases in both salivary glands and gut tissues. Gut proteinases were less sensitive to inhibition than proteinases from the salivary glands. Up to 80% azocaseinase and 90% of BApNAse activities in the salivary glands were inhibited by aprotinin, benzamidine, and PMSF, whereas only 46% azocaseinase and 60% BApNAse activities in the gut were suppressed by benzamidine, leupeptin, and TLCK. The pH optima for azocaseinase activity in salivary glands ranged from 6.2 to 10.6, whereas the pH optima for gut proteinases was acidic for general and alkaline for tryptic proteinases. Zymogram analysis demonstrated that approximately 26-kDa proteinases from salivary glands were active against both gelatin and casein substrates. Three trypsin-like cDNAs, LlSgP2-4, and one trypsin-like cDNA, L1GtP1, were cloned from salivary glands and gut, respectively. Putative trypsin precursors from all cloned cDNAs contained a signal peptide, activation peptide, and conserved N-termini (IVGG). Other structural features included His, Asp, and Ser residues for the catalytic amino acid triad of serine proteinase active sites, residues for the binding pocket, and four pairs of cysteine residues for disulfide bridges. Deduced trypsin-like proteins from LlSgP2, LlSgP3, and LlGtP1 cDNAs shared 98-99% sequence identity with a previously reported trypsin-like precursor, whereas the trypsin-like protein of LlSgP4 shared only 44% sequence identity with all other trypsin-like proteins, indicating multi-trypsin forms are present in L. lineolaris.     

Quantifying cathepsin S activity in antigen presenting cells using a novel specific substrate

Cathepsin S (CatS) is a lysosomal cysteine protease belonging to the papain superfamily. Because of the relatively broad substrate specificity of this family, a specific substrate for CatS is not yet known. Based on a detailed study of the CatS endopeptidase specificity, using six series of internally quenched fluorescent peptides, we were able to design a specific substrate for CatS. The peptide series was based on the sequence GRWHTVGLRWE-Lys(Dnp)-DArg-NH2, which shows only one single cleavage site between Gly and Leu and where every substrate position between P-3 and P-3' was substituted with up to 15 different amino acids. The endopeptidase specificity of CatS was mainly determined by the P-2, P-1', and the P-3' substrate positions. Based on this result, systematically modified substrates were synthesized. Two of these modified substrates, Mca-GRWPPMGLPWE-Lys(Dnp)-DArg-NH2 and Mca-GRWHPMGAPWE-Lys(Dnp)-DArg-NH2, did not react with the purified cysteine proteases cathepsin B (CatB) and cathepsin L (CatL). Using a specific CatS inhibitor, we could further show that these two peptides were not cleaved by endosomal fractions of antigen presenting cells (APCs), when CatS was inhibited and related cysteine proteases cathepsin B, H, L and X were still active. Although aspartic proteases like cathepsin E and cathepsin D were also present, our substrates were suitable to quantify cathepsin S activity specifically in APCs, including B cells, macrophages, and dendritic cells without the use of any protease inhibitor. We find that CatS activity differs significantly not only between the three types of professional APCs but also between endosomal and lysosomal compartments.     

Spectrophotometric assay for protease activity in ionic liquids using chromogenic substrates

A new spectrophotometric assay has been developed to evaluate protease activity in ionic liquids (ILs). The assay consists of two strategies to enable real-time spectrometric analysis of enzymatic reaction in ILs. First, enzymes are modified with a comb-shaped poly(ethylene glycol), PM₁₃, to obtain a transparent enzyme solution in IL. Second, a chromogenic substrate is used to follow the enzymatic reaction in IL. p-Nitroaniline-derivatized substrates are subjected to protease-catalyzed alcoholysis to release chromogenic p-nitroaniline that can be quantitatively detected by a UV-Vis spectrophotometer. By using this method, we can evaluate protease activity in ILs quite easily without separation of products from the reaction mixture. The availability of the novel assay system was demonstrated in a kinetic analysis of subtilisin-catalyzed reaction in the IL 1-ethyl-3-methylimidazolium bis(trifluoromethanesulfonyl)imide ([Emim][Tf₂N]) under different reaction conditions. Because two different serine proteases, subtilisin and α-chymotrypsin, substantially retained its original substrate specificity in the IL, the assay can be extended to other enzymes by using suitable chromogenic substrates.     

Digestive proteolytic activity in larvae of tomato moth,Lacanobia oleracea; effects of plant protease inhibitors in vitro and in vivo

Three distinct digestive protease activities, with strongly alkaline pH optima, were identified in the gut of tomato moth (Lacanobia oleracea) larvae, and characterised using specific synthetic substrates and inhibitors. These were; a trypsin-like activity, a chymotrypsin-like activity specific for substrates and inhibitors containing more than one amino acid residue, and an elastase-like activity, accounting for 40%, 30% and 20% of overall proteolysis respectively. The protease activities differed in their sensitivities to inhibition by different plant protein protease inhibitors (PIs), as estimated by I(50) values. Soya bean Kunitz trypsin inhibitor (SKTI) was the only plant PI tested to inhibit all three digestive protease activities at concentrations <40 &mgr;g/ml (approx. 5x10(-6)M). Incorporation of SKTI into a potato leaf-based artificial diet at 2% of total protein, decreased larval survival and growth (by approx. 33% and 40% respectively after 21 days) and retarded development (by approx. 2 days). However, when SKTI was expressed in transgenic potato plants at approx. 0.5% of total protein, only marginal effects on L. oleracea larvae were observed, which decreased with time. Whilst the presence of SKTI in artificial diet increased endogenous larval trypsin-like activity by up to four-fold, no effects on this activity were observed in larvae feeding on transgenic plants.