Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.4.21.4 (trypsin)
42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Two digestive trypsin-like proteinases from Sesamia nonagrioides Lef. (Lepidoptera: Noctuidae) larvae were purified by benzamidine-Sepharose affinity chromatography. The purified enzymes showed molecular size of 27 (trypsin-I) and 24 KDa (trypsin-II). Amino acid analysis and N-terminal sequencing confirmed their relationship with other trypsins from lepidopteran larvae. However, trypsin-I presented one lysine at position 11, being the first report of this amino acid in the sequence of a lepidopteran digestive trypsin. Trypsin-I had an isoelectric point of 6.0, and a Km of 2.2 x 10(-4) M and 3.9 x 10(-5) M for BApNa and BAEE, respectively. Trypsin-II presented an isoelectric point of 8.7, and Km values of 1.7 x 10(-4) M (BApNa) and 3.8 x 10(-5) M (BAEE). Both enzymes were differentially inhibited by some proteinase inhibitors. In particular, trypsin-I was inhibited by E-64 (ID50 = 6 microM) but not by lima bean trypsin inhibitor (LBI), whereas trypsin-II was inhibited by LBI (ID50 = 1 microM) and poorly by E-64 (ID50 = 85 microM). Changes in the susceptibility of the trypsin-like activity of midgut extracts from different larval instars to these inhibitors suggest that the relative proportion of these two enzymes varied through larval development, being predominant in early instars trypsin-I and in late instars trypsin-II.
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PMID:Isolation and characterization of two digestive trypsin-like proteinases from larvae of the stalk corn borer, Sesamia nonagrioides. 1019 40

Enteropeptidase [EC 3.4.21.9] is a membrane-bound serine endopeptidase present in the duodenum that converts trypsinogen to trypsin. We previously cloned the cDNA of the porcine enzyme and deduced its entire amino acid sequence [M. Matsushima et al. (1994) J. Biol. Chem. 269, 19976-19982]. In the present study, we purified the porcine enzyme approximately 2,200-fold in a 12% yield from a duodenal mucosal extract to apparent homogeneity by an improved procedure comprising four steps of chromatography including benzamidine-Sepharose affinity chromatography. Lectin blotting analysis suggested that the enzyme is glycosylated mainly with N-linked carbohydrate chains of the tri- and/or tetraantennary complex type. The H and L chains of the enzyme were separated into two major bands upon SDS-PAGE under reducing conditions, suggesting that the enzyme mainly comprises two isoforms, a higher molecular weight form and a lower molecular weight form. The enzyme was also separated by lectin affinity chromatography into two major fractions, named isoforms I and II, which corresponded to the higher and lower molecular weight forms, respectively. These two isoforms appeared to be different only in the carbohydrate moiety, having essentially the same enzymatic properties. The enzyme was optimally active at pH 8.0 toward Gly-Asp-Asp-Asp-Asp-Lys-beta-naphthylamide, and was inhibited strongly by various serine proteinase inhibitors. Furthermore, it was also strongly inhibited by E-64 [L-trans-epoxysuccinyl-leucylamide-(4-guanido)-butane], a cysteine proteinase inhibitor. Substrate specificity studies involving various synthetic peptides indicated that acidic residues at the P2, P3, and/or P4 positions are especially favorable for maximal activity, but are not absolutely necessary, at least in the cases of peptide substrates.
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PMID:Purification and further characterization of enteropeptidase from porcine duodenum. 1022 May 88

Interleukin-1 beta (IL-1 beta)-converting enzyme (ICE, caspase-1) processes the IL-1 beta precursor to mature inflammatory cytokine IL-1 beta. ICE has been identified as a unique cysteine protease, which cleaves Asp-X bonds, shows resistance to E-64 (an inhibitor of most cysteine proteases) and has a primary structure that is homologous to CED-3, a protein required for apoptosis (programmed cell death) in the nematode Caenorhabditis elegans, and to mammalian cysteine proteases that initiate and execute apoptosis, e.g., apopain/CPP32/caspase-3. The inhibitors of the ICE/CED-3 family or caspases, as they are called recently, may constitute therapeutic agents for amelioration of inflammatory and apoptosis-associated diseases. The most efficient ICE inhibitors are peptide aldehydes and peptidyl chloro or (acyloxy)methanes. A recent study revealed that both D- and L-Asp are accepted by ICE at the P1 of such inhibitors, and the peptidyl (acyloxy)methane analogues having the beta-homo-aspartyl residue [-NH-CH(CH2COOH)-CH2CO-] are inactive. These findings we reexamined in terms of two issues. (a) ICE's resistance to E-64. Since it was thought to be caused by the enzyme's unique substrate specificity, we prepared substrate-based analogues, which were not inhibitory suggesting significant structural difference between the active centers of ICE and papain-like enzymes. (b) Tolerance for D-stereochemistry at the P1 of these inhibitors. In view of the mechanism of cysteine protease inhibition by peptidyl X-methanes, we thought that this phenomenon should be a general characteristic of cysteine proteases and the hAsp-containing analogues should behave as reversible inhibitors. Here, we analyzed the inhibition of ICE and apopain in comparison with that of papain, thrombin, and trypsin by peptide L/D-alpha-aldehydes and their L-beta-homo-aldehyde [-NH-CH(R)-CH2-CHO] analogues. The following results were found. (1) The peptidyl L-beta-homo-aspartals are potent inhibitors for caspases. (2) The L-beta-homo analogues of peptide aldehyde inhibitors designed for other proteases are not inhibitory. (3) Unlike trypsin and thrombin (serine proteases), papain (cysteine protease) shows tolerance for D-stereochemistry at the P1 site of peptide aldehydes in proportion to the lability of the alpha-hydrogen of the P1-D-residue. The complete tolerance of ICE for P1-D-Asp may arise from this residue's high tendency to epimerization. (4) Reaction of cysteine proteases with peptide aldehyde or peptidyl X-methane inhibitors containing P1-D-residues may include alpha-proton abstraction followed by asymmetric induction leading to P1-L-residue-containing products.
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PMID:Peptidyl beta-homo-aspartals (3-amino-4-carboxybutyraldehydes): new specific inhibitors of caspases. 1038 Mar 58

Recently, it has emerged that extracellular proteases have specific regulatory roles in modulating immune responses. Proteases may act as signaling molecules to activate the Raf-1/extracellular regulated kinase (ERK)-2 pathway to participate in mitogenesis, apoptosis, and cytokine production. Most reports on the role of protease-mediated cell signaling, however, focus on their stimulatory effects. In this study, we show for the first time that extracellular proteases may also block signal transduction. We show that bromelain, a mixture of cysteine proteases from pineapple stems, blocks activation of ERK-2 in Th0 cells stimulated via the TCR with anti-CD3epsilon mAb, or stimulated with combined PMA and calcium ionophore. The inhibitory activity of bromelain was dependent on its proteolytic activity, as ERK-2 inhibition was abrogated by E-64, a selective cysteine protease inhibitor. However, inhibitory effects were not caused by nonspecific proteolysis, as the protease trypsin had no effect on ERK activation. Bromelain also inhibited PMA-induced IL-2, IFN-gamma, and IL-4 mRNA accumulation, but had no effect on TCR-induced cytokine mRNA production. This data suggests a critical requirement for ERK-2 in PMA-induced cytokine production, but not TCR-induced cytokine production. Bromelain did not act on ERK-2 directly, as it also inhibited p21ras activation, an effector molecule upstream from ERK-2 in the Raf-1/MEK/ERK-2 kinase signaling cascade. The results indicate that bromelain is a novel inhibitor of T cell signal transduction and suggests a novel role for extracellular proteases as inhibitors of intracellular signal transduction pathways.
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PMID:Bromelain, from pineapple stems, proteolytically blocks activation of extracellular regulated kinase-2 in T cells. 1045 95

Protein digestion in the lesser grain borer, Rhyzopertha dominica (F.) (Coleoptera: Bostrichidae), results from the action of a complex of serine proteinases present in the midgut. In this study we partially characterized trypsin-like enzyme activity against N-alpha-benzoyl-L-arginine p-nitroanilide (BApNA) in midgut preparations and cloned and sequenced three cDNAs for trypsinogen-like proteins. BApNAase activity in R. dominica midgut was significantly reduced by serine proteinase inhibitors and specific inhibitors of trypsin, whereas BApNAase activity was not sensitive to specific inhibitors of chymotrypsin or aspartic proteinases. However, trans-epoxysuccinyl-L-leucylamido-(4-guanidino) butane (E-64) inhibited BApNAase activity by about 30%. BApNAase was most active in a broad pH range from about pH 7 to 9.5. The gut of R. dominica is a tubular tract approximately 2.5 mm in length. BApNAase activity was primarily located in the midgut region with about 1.5-fold more BApNAase activity in the anterior region compared to that in the posterior region. Proteinases with apparent molecular masses of 23-24 kDa that were visualized on casein zymograms following electrophoresis were inhibited by TLCK. Three cDNAs for trypsinogen-like proteins were cloned and sequenced from mRNA of R. dominica midgut. The full cDNA sequences consisted of open reading frames encoding 249, 293, and 255 amino acid residues for RdoT1, RdoT2, and RdoT3, respectively. cDNAs RdoT1, RdoT2, and RdoT3 shared 77-81% sequence identity. The three encoded trypsinogens shared 54-62% identity in their amino acid sequences and had 16-18 residues of signal peptides and 12-15 residues of activation peptides. The three predicted mature trypsin-like enzymes had molecular masses of 23.1, 28, and 23.8 kDa for RdoT1, RdoT2, and RdoT3, respectively. Typical features of these trypsin-like enzymes included the conserved N-terminal residues IVGG62-65, the catalytic amino acid triad of serine proteinase active sites (His109, Asp156, Ser257), three pairs of conserved cysteine residues for disulfide bridges, and the three residues (Asp251, Gly274, Gly284) that determine specificity in trypsin-like enzymes. In addition, RdoT2 has both a PEST-like sequence at the C-terminus and a free Cys158 near the active site, suggesting instability of this enzyme and/or sensitivity to thiol reagents. The sequences have been deposited in GenBank database (accession numbers AF130840 for RdoT1, AF130841 for RdoT2, and AF130842 for RdoT3).
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PMID:Characterization of midgut trypsin-like enzymes and three trypsinogen cDNAs from the lesser grain borer, Rhyzopertha dominica (Coleoptera: Bostrichidae). 1061 40

A serine protease from mite faecal extract, Dermatophagoides farinae, was purified using DEAE-Sephacel anion exchange chromatography and Superdex 75 pg gel chromatography. The molecular weight of this protease was 34 kD on SDS-PAGE under reducing conditions. The optimal pH and temperature of the protease were 8.0 and 47 degrees C, respectively. In addition, this protease cleaved arginyl or lysyl residue containing substrates selectively and was only inhibited by aprotinin, FUT-175, and soy bean trypsin inhibitor and not by chymostatin, E-64 and iodoacetic acid. These results show that our purified serine protease belongs to the trypsin-type. Purified trypsin-like protease was shown to be allergenic by enzyme-linked immunosorbent assay. Antigenicity of trypsin-like protease was completely different from those of Der f I and Der f II. Both, 20 N-terminal amino acid sequence and amino acid compositions of the purified protease were very similar to those of Der f III. Good similarities were found between trypsin-like protease and Der f III concerning physicochemical properties such as molecular weight on SDS-PAGE and ammonium sulphate solubility. Summarizing the above data, it can be concluded that a trypsin-like protease from mite faecal extract is actually the Der f III allergen and that it may be involved in the digestive process of the mite as it was found not in mite body but in mite faeces.
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PMID:Trypsin-like protease of mites: purification and characterization of trypsin-like protease from mite faecal extract Dermatophagoides farinae. Relationship between trypsin-like protease and Der f III. 1077 9

Canatoxin is a protein isolated from jackbean (Canavalia ensiformis), seeds. Injected intraperitoneally, the toxin is lethal to mice but it is inactive if given orally. Canatoxin is also lethal when fed to insects with cathepsin-based digestion while insects with trypsin-based digestion are not affected. The hypothesis that canatoxin is proteolytically activated by cathepsins was investigated. Experiments were performed with 4(th) instar and adult Rhodnius prolixus fed meals containing canatoxin (2.5 microg/mg weight body). While 100% of nymphs died, no effect was observed in adults. Hemolymph taken from nymphs and adults showed the presence of canatoxin's proteolytic fragments. Reduced lethality was seen in R. prolixus 4(th) instars fed meals containing canatoxin and inhibitors of cathepsin enzymes, E-64 (2.0 microM) or Pepstatin-A (2. 0 microM). In another approach, canatoxin was digested in vitro with enzymes from the bruchid, Callosobruchus maculatus, and the resulting peptides were tested in R. prolixus. Three groups of toxic peptides (8,000-12,000 kD range) were separated by gel-filtration. When these peptides were fed to the insects simultaneously with the cathepsin inhibitors, no protective effect was seen. These results confirm the proteolytic activation of canatoxin by insect cathepsin-like enzymes to produce entomotoxic peptide(s). Furthermore, our data point towards overlooked differences in the digestive physiology of distinct life stages of R. prolixus. Arch.
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PMID:Proteolytic activation of canatoxin, a plant toxic protein, by insect cathepsin-like enzymes. 1091 11

The source and nature of zona lytic factors during zona escape of hamster blastocyts were investigated. When cultured in hamster embryo culture medium (HECM)-2h, all 8-cell embryos (n = 135) developed to zona escaped-blastocysts with complete zona lysis. In addition, 2-cell embryos, when co-cultured with zona escaping-blastocysts (at a ratio of 1:10), exhibited zona lysis. Various other embryos at the 1-8-cell stages also showed zona lysis when cultured with zona-escaping blastocysts. However, zonae from mice, rats, sheep and humans were resistant to lysis under these conditions. Pronase treatment resulted in rapid zona lysis in hamsters (7 +/- 1 s), whereas in other species zona lysis was much slower: mouse (662 +/- 27 s), rat (532 +/- 16 s), sheep (120 +/- 12 s) and human (104 +/- 8 s). When cysteine protease inhibitors (antipain, leupeptin, E-64 and p-hydromercuricbenzoate) were tested, they completely inhibited zona escape, while trypsin inhibitors (TLCK and SBTI) did not. Uterine zona lysin contribution in zona escape was discounted since: (i) uterine luminal flushing and endometrial extract from day 4 (the time of zona escape in vivo) pregnant females failed to lyse zonae and (ii) endogenous oocytes and transferred 2-cell embryos (to day 3 pseudopregnant recipients) were all zona-intact, while 71% of transferred blastocysts exhibited zona escape, following their recovery after 24 h. These observations suggest that a species-specific, embryonic proteolytic factor, with a cysteine protease-like activity, is involved in the zona escape of blastocysts in hamsters.
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PMID:Evidence for the involvement of a species-specific embryonic protease in zona escape of hamster blastocysts. 1104 63

A novel type of protease (mantis egg fibrinolytic enzyme, MEF-2) was isolated from the egg cases of Tenodera sinensis. The protease was homogeneous by SDS-PAGE and its apparent molecular mass was 32,900 Da. The amino acids in the N-terminal region were Ile-Val-Gly-Gly-Glu-Glu-Ala-Val-Ala-Gly-Asp-Phe-Pro-Ile-Val-Ser-Leu-Gln-Glu. The enzyme was inhibited by PMSF, TLCK, aprotinin, benzamidine, soybean trypsin inhibitor and also slightly by elastatinal, EDTA, EGTA, cysteine and beta-mercaptoethanol, but TPCK, iodoacetate and E-64 did not affect the activity. MEF-2 was not sensitive to alpha(1)-antitrypsin but antithrombin III and alpha(2)-antiplasmin inhibited the enzyme. MEF-2 preferentially cleaved the oxidized B-chain of insulin between Arg(22) and Gly(23). Among chromogenic protease substrates, the most susceptible to MEF-2 hydrolysis was benzoyl-Phe-Val-Arg-p-nitroanilide with maximal activity at 30 degrees C and pH 5.0. These results indicate that MEF-2 belongs to the trypsin family. Upon incubation of crosslinked fibrin with MEF-2, a steady increase of D-dimer suggests that the enzyme has a strong fibrinolytic activity. In conclusion, MEF-2 is a new type of proteolytic enzyme and has some potential for practical application in fibrinolysis.
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PMID:Purification and characterization of a plasmin-like protease from Tenodera sinensis (Chinese mantis). 1126 96

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.
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PMID:Compartmentalization of proteinases and amylases in Nauphoeta cinerea midgut. 1174 65


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