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)

Serpins represent a diverse class of endogenous protease inhibitors that regulate important biological functions. In consideration of the importance of regulated proteolysis within secretory vesicles for the production of peptide hormones and neurotransmitters, this study revealed the molecular identity of a novel serpin, endopin 1, that is localized to neurosecretory vesicles of neuropeptide-containing chromaffin cells (chromaffin granules). Endopin 1 of 68-70 kDa was present within isolated chromaffin granules. Stimulated cosecretion of endopin 1 with chromaffin granule components, [Met]enkephalin and a cysteine protease known as "prohormone thiol protease," demonstrated localization of endopin 1 to functional secretory vesicles. Punctate, discrete immunofluorescence cellular localization of endopin 1 in chromaffin cells was consistent with its secretory vesicle localization. Endopin 1 contains a unique reactive site loop with Arg as the predicted P1 residue, suggesting inhibition of basic residue-cleaving proteases; indeed, trypsin was potently inhibited (K(i(app)) of 5 nM), and plasmin was moderately inhibited. Although endopin 1 possesses homology with alpha(1)-antichymotrypsin, chymotrypsin was not inhibited. Moreover, endopin 1 inhibited the chromaffin granule prohormone thiol protease (involved in proenkephalin processing). These results suggest a role for the novel serpin, endopin 1, in regulating basic residue-cleaving proteases within neurosecretory vesicles of chromaffin cells.
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PMID:Molecular cloning of endopin 1, a novel serpin localized to neurosecretory vesicles of chromaffin cells. Inhibition of basic residue-cleaving proteases by endopin 1. 1056 88

We investigated the NF-kappaB-like factor induced in the late-passage human oral mucosal fibroblasts stimulated with interleukin-1 (IL-1). Compared with the NF-kappaBs of HeLa cells and early-passage fibroblasts, the NF-kappaB-like factor of late-passage (passage 15) fibroblasts migrated faster in the electrophoretic mobility shift assay (EMSA) and behaved like a 70-80 kDa protein in the gel filtration chromatography. Both antibodies against p50 and p65 subunits of NF-kappaB could supershift the small NF-kappaB-like factor of late-passage cells in the EMSAs. A 47-kDa band was detected in late-passage fibroblasts by immunoblotting against p50. The mobility of the trypsin-degraded NF-kappaB of HeLa cells corresponded to that of the small NF-kappaB-like factor of late-passage fibroblasts in the EMSAs. Furthermore, when the nuclear extracts of the IL-1-stimulated HeLa cells were incubated with those of the IL-1-stimulated old fibroblasts, the p65-p50 NF-kappaB band disappeared, leaving behind a small NF-kappaB-like band. This reduction of NF-kappaB was prevented by the addition of a cysteine protease inhibitor leupeptin. These results suggest that the small NF-kappaB-like factor of late-passage fibroblasts is a part of the NF-kappaB truncated by aging-induced protease(s).
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PMID:Aging-dependent proteolysis of NF-kappaB in human fibroblasts. 1062 89

beta-Lapachone (beta-lap) effectively killed MCF-7 and T47D cell lines via apoptosis in a cell-cycle-independent manner. However, the mechanism by which this compound activated downstream proteolytic execution processes were studied. At low concentrations, beta-lap activated the caspase-mediated pathway, similar to the topoisomerase I poison, topotecan; apoptotic reactions caused by both agents at these doses were inhibited by zVAD-fmk. However at higher doses of beta-lap, a novel non-caspase-mediated "atypical" cleavage of PARP (i.e., an approximately 60-kDa cleavage fragment) was observed. Atypical PARP cleavage directly correlated with apoptosis in MCF-7 cells and was inhibited by the global cysteine protease inhibitors iodoacetamide and N-ethylmaleimide. This cleavage was insensitive to inhibitors of caspases, granzyme B, cathepsins B and L, trypsin, and chymotrypsin-like proteases. The protease responsible appears to be calcium-dependent and the concomitant cleavage of PARP and p53 was consistent with a beta-lap-mediated activation of calpain. beta-Lap exposure also stimulated the cleavage of lamin B, a putative caspase 6 substrate. Reexpression of procaspase-3 into caspase-3-null MCF-7 cells did not affect this atypical PARP proteolytic pathway. These findings demonstrate that beta-lap kills cells through the cell-cycle-independent activation of a noncaspase proteolytic pathway.
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PMID:Activation of a cysteine protease in MCF-7 and T47D breast cancer cells during beta-lapachone-mediated apoptosis. 1069 31

The expression of high-molecular-weight and low-molecular-weight kininogen mRNAs in the whale liver was examined by reverse transcription-polymerase chain reaction. The nucleotide sequences of the high-molecular-weight and low-molecular-weight kininogen cDNAs were analyzed and deduced to the amino acid sequences. The high-molecular-weight kininogen composed of 609 amino acid residues with 18 signal peptides possessed the consensus sequences of the cysteine protease inhibitor domains I and II, the bradykinin domain, the histidine-rich region, and the prekallikrein-binding region. Except for the histidine-rich region, the overall homologies with bovine, human, and rat high-molecular-weight kininogens were 81%, 76%, and 62%, respectively. The low-molecular-weight kininogen is composed of 408 amino acid residues. The nucleotide sequence down to C(1200) as well as the amino acid sequence till Ile(382) is identical to that of the high-molecular-weight kininogen. The remaining low-molecular-weight kininogen-specific carboxy-terminal portion possessed an amino acid sequence similar to that of the land mammals. The overall homologies with bovine, human, and rat low-molecular-weight kininogens were 82%, 79%, and 64%, respectively. The amino acid sequences of both whale high-molecular-weight and low-molecular-weight kininogens are most similar to those of the bovine among the land mammals analyzed so far. An incubation of dolphin/whale plasma with human plasma kallikrein, or with bovine trypsin, in the presence of carboxypeptidase inhibitors generated bradykinin antigen as well as the spasmogenic activity to the estrous rat uterus. The amount of bradykinin released by the latter enzyme was almost double of the former, indicating that the dolphin/whale plasma contained similar concentrations of low-molecular-weight and high-molecular-weight kininogens.
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PMID:Whale high-molecular-weight and low-molecular-weight kininogens. 1070 58

The metabolism of three opioid tetrapeptides, Tyr-D-Arg-Phe-Nva-NH2, Tyr-D-Arg-Phe-Phe-NH2 and Tyr-D-Ala-Phe-Phe-NH2, was investigated in the presence of pure pancreatic enzymes (trypsin, chymotrypsin, elastase, carboxypeptidase A and carboxypeptidase B), as well as in the presence of pure carboxylesterase and aminopeptidase N. The cleavage patterns of the pure pancreatic enzymes were then compared with those found in rat and human jejunal fluid. Metabolism was also studied in homogenates from different intestinal regions (duodenum, jejunum, ileum and colon) and in enterocyte cytosol from rats. The effect of various protease inhibitors was investigated in the jejunal homogenate. The parent peptides were assayed by high-performance liquid chromatography and metabolites were identified by means of liquid chromatography-mass spectrometry. Of the pure enzymes, the quickest hydrolysis of the peptides was observed for the pancreatic enzymes chymotrypsin, trypsin and carboxypeptidase A. In most cases they formed the corresponding deamidated tetrapeptides (chymotrypsin and trypsin) or tripeptides with a missing C-terminal amino acid (carboxypeptidase A). Regional differences in intestinal metabolism rates were found for all three peptides (P < 0.001), with the highest rates observed in jejunal and/or colonic homogenates. The deamidated tetrapeptides were formed both in rat intestinal homogenates and in enterocyte cytosol. Metabolism in the jejunal homogenate was markedly inhibited by some serine and combined serine and cysteine protease inhibitors. In conclusion, the C-terminal amide of these tetrapeptides did not fully stabilise them against intestinal deamidase and carboxypeptidase activities. The significant hydrolysis of the peptides by pure chymotrypsin, trypsin and carboxypeptidase A showed that lumenal pancreatic proteases might be a clear metabolic obstacle in oral delivery even for small peptides such as these tetrapeptides.
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PMID:Investigations of the in-vitro metabolism of three opioid tetrapeptides by pancreatic and intestinal enzymes. 1093 29

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

Protease inhibitors from potato juice of cv. Elkana were purified and quantified. The protease inhibitors represent ca. 50% of the total soluble proteins in potato juice. The protease inhibitors were classified into seven different families: potato inhibitor I (PI-1), potato inhibitor II (PI-2), potato cysteine protease inhibitor (PCPI), potato aspartate protease inhibitor (PAPI), potato Kunitz-type protease inhibitor (PKPI), potato carboxypeptidase inhibitor (PCI), and "other serine protease inhibitors". The most abundant families were the PI-2 and PCPI families, representing 22 and 12% of all proteins in potato juice, respectively. Potato protease inhibitors show a broad spectrum of enzyme inhibition. All the families (except PCI) inhibited trypsin and/or chymotrypsin. PI-2 isoforms exhibit 82 and 50% of the total trypsin and chymotrypsin inhibiting activity, respectively. A strong variation within the latter activities was shown within one family and between protease inhibitor families.
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PMID:Relative abundance and inhibitory distribution of protease inhibitors in potato juice from cv. Elkana. 1140 80

Tissue homogenates from mouse ear skin exposed to sulfur mustard (HD, which is a military designation and probably originated from a World War I slang term 'Hun Stuff') were assayed for serine and cysteine protease activities. Enzyme activity was measured using synthetic chromogenic thioester and fluorogenic 7-amino-4-methylcoumarin (AMC) substrates. The tissue samples were obtained from animals (n = 6) at 3, 6, 12 and 24 h post-exposure from the right ear (HD exposed), whereas control samples were obtained from the left ear (treated only with dichloromethane vehicle). The samples of naive control (left and right ear) were obtained from animals that received no HD treatment (n = 3). Elastase activity was assayed with t-butyloxycarbonyl-Ala-Ala-Ala-thiobenzylester, tryptase activity with benzyloxycarbonyl-Arg-AMC and benzyloxycarbonyl-Arg-thiobenzylester, chymase activity with succinylAla-Ala-Pro-Phe-thiobenzylester and succinyl-Ala-Ala-Pro-Phe-AMC, cathepsin B activity with benzyloxycarbonyl-Arg-Arg-AMC, cathepsin H activity with Arg-AMC and calpain activity with succinyl-Leu-Tyr-AMC. The HD-exposed skin homogenates obtained at 12 and 24 h post-exposure had higher elastase activity (670% and 1900% increase) than control samples. For tryptase and calpain activities, only HD-exposed skin homogenates at 24h post-exposure showed higher activities (220% and 170% increase) when compared to the control. No differences from control were observed for HD-exposed skin obtained at 3 and 6 h post-exposure for elastase, tryptase and calpain activities. Generally, both unexposed and HD-exposed skin had distinct cathepsin B and cathepsin H enzyme activities and small chymase activity. Enzymatic assays were also performed for other serine, cysteine and metalloproteases. These data document that proteases are involved in HD skin injury and continued assessment of proteolytic activity should be useful for identifying effective antiproteases with therapeutic use in reducing or eliminating tissue injury caused by HD cutaneous exposure.
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PMID:Cutaneous protease activity in the mouse ear vesicant model. 1142 32

Serpins are irreversible covalent 'suicide' protease inhibitors. In the past two years, important advances in the structural biology of serpins have been forthcoming with the crystal structures of a covalent complex between trypsin and alpha1-antitrypsin, and of a Michaelis encounter complex between trypsin S195A and serpin 1B from Manduca sexta. These structures have helped elucidate many aspects of the mechanism of action of serpins. Also, the crystal structure of the cysteine protease caspase-8 in complex with the inhibitor p35 has revealed a new family of suicide protease inhibitors.
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PMID:Serpins and other covalent protease inhibitors. 1175 Oct 56

A premature and intracellular activation of digestive zymogens is thought to be responsible for the onset of pancreatitis. Because trypsin has a critical role in initiating the activation cascade of digestive enzymes in the gut, it has been assumed that trypsin also initiates intracellular zymogen activation in the pancreas. We have tested this hypothesis in isolated acini and lobules from rat pancreas. Intracellular trypsinogen activation was induced by supramaximal secretagogue stimulation and measured using either specific trypsin substrates or immunoreactivity of the trypsinogen activation peptide (TAP). To prevent a trypsin-induced trypsinogen activation, we used the cell-permeant, highly specific, and reversible inhibitor Nalpha-(2-naphthylsulfonyl)-3-amidinophenylalanine-carboxymethylpiperazide (S124), and to prevent cathepsin-induced trypsinogen activation, we used the cysteine protease inhibitor E-64d. Incubation of acini or lobules in the presence of S124 completely prevented the generation of trypsin activity in response to supramaximal caerulein but had no effect whatsoever on the generation of TAP. Conversely, when trypsin activity was recovered at the end of the experiment by either washout of S124 from acini or extensive dilution of lobule homogenates, it was up to 400% higher than after caerulein alone and corresponded, in molar terms, to the generation of TAP. Both trypsin activity and TAP release were inhibited in parallel by E-64d. We conclude that caerulein-induced trypsinogen activation in the pancreas is caused by an E-64d-inhibitable mechanism such as cathepsin-induced trypsinogen activation, and neither involves nor requires intracellular trypsin activity. Specific trypsin inhibition, on the other hand, prevents 80% of trypsin inactivation or autodegradation in the pancreas.
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PMID:Trypsin activity is not involved in premature, intrapancreatic trypsinogen activation. 1180 59


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