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)

Sheep mast cell proteinase-1 (sMCP-1), a serine proteinase with dual chymase/tryptase activity, is expressed in gastrointestinal mast cells, and released systemically and on to the mucosal surface during gastrointestinal nematode infection. The potential for native plasma proteinase inhibitors to control sMCP-1 activity was investigated. Sheep alpha1-proteinase inhibitor (alpha1PI) inhibited sMCP-1 slowly, with second-order association rate constant (kass) 1. 1x10(3) M-1.s-1, whereas sheep contrapsin inhibited trypsin (kass 2.2x10(6) M-1.s-1) but not sMCP-1. Western-blot analysis and gel filtration showed that when added to serum or plasma, sMCP-1 was partitioned between alpha1PI and alpha2-macroglobulin. The possibility that significant cleavage of plasma proteins could occur before sMCP-1 was inhibited was investigated using gel filtration and SDS/PAGE after adding sMCP-1 to plasma. Cleavage of ovine fibrinogen occurred in the presence of excess alpha1PI and alpha2-macroglobulin, the alpha-chain being cleaved C-terminally and the beta-chain at the putative Lys-27. In addition, sMCP-1 was found to be mitogenic for bovine pulmonary artery fibroblasts, but was not mitogenic in the presence of soya-bean trypsin inhibitor. In terms of fibrinogen cleavage and fibroblast stimulation, sMCP-1 shows functional similarities to mast cell tryptase.
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PMID:Sheep mast cell proteinase-1, a serine proteinase with both tryptase- and chymase-like properties, is inhibited by plasma proteinase inhibitors and is mitogenic for bovine pulmonary artery fibroblasts. 916 5

We have investigated the potential of tryptase to stimulate an increase in microvascular permeability following injection into the skin of guinea pigs. Tryptase was isolated from high salt extracts of human lung tissue by octyl-agarose and heparin-agarose chromatography. Injection of purified tryptase (2.5 ng-2.5 microg/site) into the skin of guinea pigs which had been injected intravenously with Evans blue dye provoked a dose-dependent increase in microvascular permeability. The skin reactions elicited by tryptase were apparent up to 80 min following injection, while histamine-induced microvascular leakage resolved completely by 40 min. Heat-inactivation of tryptase, or preincubating the proteinase with certain proteinase inhibitors, significantly reduced the extent of microvascular leakage, suggesting dependency on an intact catalytic site. No evidence was found for a synergistic or antagonistic interaction between tryptase (2.5 ng-2.5 microg/site) and histamine (1-10 microg/site) when these mast cell products were injected together. Addition of heparin to tryptase (10:1; w/w) prior to injection was without effect on tryptase-induced microvascular leakage. Pretreatment of guinea pigs with a combination of the histamine H1 receptor antagonist pyrilamine and the histamine H2 receptor antagonist cimetidine (both 10 mg/kg), partially abolished tryptase-induced microvascular leakage as well as attenuating the reaction to histamine. Reasoning that the microvascular leakage induced by tryptase is likely to involve the release of histamine, we investigated the ability of tryptase to stimulate histamine release from dispersed guinea-pig skin and lung cells in vitro. Tryptase was found to induce concentration-dependent histamine release from both sources of tissue. Mast cell activation stimulated by tryptase in vitro was inhibited by heat treating the enzyme or by addition of proteinase inhibitors, suggesting a requirement for an intact catalytic site. Histamine release was inhibited also by preincubating cells with the metabolic inhibitors antimycin A and 2-deoxy-D-glucose indicating that the mechanism was energy-requiring and non-cytotoxic. We conclude that human mast cell tryptase may be a potent stimulus of microvascular leakage. The activation of mast cells by this proteinase may represent an amplification process in allergic inflammation.
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PMID:Human mast cell tryptase: a stimulus of microvascular leakage and mast cell activation. 920 74

Immunohistochemical staining for mast cell tryptase and chymase was used to examine the distribution, activation, and tryptase/chymase phenotype of mast cells (MCs) in 250 samples of atherosclerotic lesions (type I to VI) of human carotid arteries. Dual immunolocalization and histochemical techniques were used to identify the associations of MCs with macrophages, smooth muscle cells, and extracellular matrix components. Whereas normal carotid arteries contained very few MCs within the intima, atherosclerotic lesions showed increased MC numbers with variable focal accumulations. MCs were identifiable from the earliest stages of atherosclerosis, and especially at the shoulder regions of the fully formed atheroma. They were observed in close association with macrophages (HAM56 positive) and extracellular lipid, as well as at sites of foam cell formation. MCs and diffuse tryptase staining were also evident within sites of new calcification and around small calcified deposits. Extensive MC activation/degranulation, as judged by diffuse extracellular tryptase staining, was a common feature of the advanced atherosclerotic plaques complicated by fissure, haemorrhage, and thrombus formation. Moreover, such sites of extracellular MC tryptase were often associated with localized oedema and disruption of the stromal matrix. MCs which contained both tryptase and chymase (the MCTC phenotype) represented approximately 80-95 per cent of all MCs. These studies are the first to demonstrate significant numbers and focal accumulations of MCs in all developmental stages of atherosclerotic carotid arteries. Since MCs contain or express a variety of potent mediators, their release could profoundly influence the development and pathological complications of atherosclerotic plaques.
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PMID:Mast cell distribution, activation, and phenotype in atherosclerotic lesions of human carotid arteries. 922 50

Proteinase-activated receptor-2 (PAR-2) is a G protein-coupled receptor that is cleaved and activated by trypsin-like enzymes. PAR-2 is highly expressed by small intestinal enterocytes where it is activated by luminal trypsin. The location, mechanism of activation, and biological functions of PAR-2 in the colon, however, are unknown. We localized PAR-2 to the muscularis externa of the rat colon by immunofluorescence. Myocytes in primary culture also expressed PAR-2, assessed by immunofluorescence and RT-PCR. Trypsin, SLIGRL-NH2 (corresponding to the PAR-2 tethered ligand), mast cell tryptase, and a filtrate of degranulated mast cells stimulated a prompt increase in [Ca2+]i in myocytes. The response to tryptase and the mast cell filtrate was inhibited by the tryptase inhibitor BABIM, and abolished by desensitization of PAR-2 with trypsin. PAR-2 activation inhibited the amplitude of rhythmic contractions of strips of rat colon. This response was unaffected by indomethacin, l-NG-nitroarginine methyl ester, a bradykinin B2 receptor antagonist and tetrodotoxin. Thus, PAR-2 is highly expressed by colonic myocytes where it may be cleaved and activated by mast cell tryptase. This may contribute to motility disturbances of the colon during conditions associated with mast cell degranulation.
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PMID:Mast cell tryptase regulates rat colonic myocytes through proteinase-activated receptor 2. 929 3

Mast cells are the principal effector cells in IgE-dependent hypersensitivity reactions. Despite reports that rodent mast cells proliferate in the presence of nerve growth factor (NGF), human mast cells reportedly do not respond to this factor. To determine if human mast cells express the NGF receptors, TrkA tyrosine receptor and the low affinity NGF receptor (LNGFR), we first analyzed the mRNA expression by RT-PCR of TrkA and LNGFR in a human mast cell line (HMC-1) and in human mast cells cultured in the presence of stem cell factor. Both HMC-1 and cultured human mast cells were found to express TrkA but not LNGFR. TrkA protein was demonstrated by Western blot analysis of HMC-1 lysates. Using flow cytometric analysis and mast cell tryptase as a mast cell marker, both HMC-1 cells and cultured human mast cells were shown to coexpress tryptase and TrkA. Treatment of mast cells with NGF resulted in phosphorylation of TrkA on tyrosine residues as detected by immunoblotting with an antiphosphotyrosine antibody. Furthermore, NGF induced the immediate early gene c-fos in HMC-1 cells. HMC-1 cells and cultured human mast cells were also found to express NGF mRNA, and conditioned medium from HMC-1 cells stimulated neurite outgrowth from chicken embryonic sensory ganglia in culture. This effect was blocked by anti-NGF. Thus, mast cells express functional TrkA and synthesize NGF, suggesting a mechanism by which NGF may act as an autocrine factor for human mast cells, and by which mast cells and nerves may interact.
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PMID:Human mast cells express functional TrkA and are a source of nerve growth factor. 934 72

1. Although mast cell hyperplasia is a feature of rheumatoid arthritis and osteoarthritis, the extent and nature of mast cell activation in joint disease have not been clearly established. 2. We have investigated the levels of mast cell tryptase and histamine and also of eosinophil cationic protein in synovial fluid collected from 31 patients with rheumatoid arthritis, 14 with seronegative spondyloarthritis and nine with osteoarthritis. Two RIAs for tryptase were employed: one with monoclonal antibody AA5, which was found to bind equally well to both alpha and beta isoforms on Western blots of the recombinant enzyme, and the other with antibody G5, which recognizes predominantly beta-tryptase. 3. alpha-Tryptase, which is likely to be released constitutively from mast cells, appeared to be the major form in synovial fluid, as the assay with antibody AA5 detected appreciably more tryptase than that with antibody G5. beta-Tryptase, which is released on anaphylactic activation of mast cells, was detected in 14 out of 45 synovial fluid samples studied, with concentrations of up to 12 micrograms/l measured by the G5 assay. The apparent levels of beta-tryptase, but not of alpha-tryptase, were closely correlated with those of histamine in the synovial fluid. Patients with osteoarthritis appeared to have a greater proportion of beta-tryptase in the synovial fluid than those with rheumatoid arthritis, as well as higher concentrations of histamine. Eosinophil cationic protein was present at high levels in the synovial fluid, although eosinophil numbers were low, and its concentrations were not correlated with the concentrations of the mast cell products. 4. These data suggest that anaphylactic degranulation of mast cells may have occurred to a greater extent in osteoarthritis than in rheumatoid arthritis, despite the relative lack of synovial inflammation in osteoarthritis. Although the eosinophil cationic protein detected may not reflect eosinophilic inflammation in the joint, the presence in synovial fluid of tryptase of both major forms, and of histamine, appears to indicate that mast cell products are secreted constitutively, as well as by processes of anaphylactic degranulation in rheumatoid arthritis, seronegative spondyloarthritis and osteoarthritis.
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PMID:Mast cell activation in arthritis: detection of alpha- and beta-tryptase, histamine and eosinophil cationic protein in synovial fluid. 940 29

There is an accumulation of evidence to suggest that mast cells may play a key role in gastrointestinal inflammation. We have investigated the numbers and heterogeneity in staining properties of mast cells in biopsies of the duodenum of normal subjects (n = 10), and of normal duodenum from patients with Crohn's disease of the ileum and/or colon (n = 7) or with Helicobacter-associated gastritis of the antrum/corpus (n = 6). In normal donors, two subsets of mast cells, one located in the duodenal mucosa and the other in the submucosa, were clearly distinguished by their morphology and dye-binding properties. Whereas submucosal mast cells stained metachromatically with Toluidine Blue after neutral formalin fixation and emitted a yellow fluorescence after staining with Berberine sulphate, those in the mucosa were invisible using these stains. In patients with gastritis or Crohn's disease, there were marked changes in the numbers of mucosal mast cells compared with control subjects even though the duodenal biopsies were from apparently uninvolved tissue. Gastritis was associated with increased mucosal mast cell numbers (controls: 187 +/- 23 cells mm-2; gastritis: 413 +/- 139 cells mm-2; p = 0.0004), but mean mucosal mast cell counts in the uninvolved duodenum of Crohn's patients were actually decreased (34 +/- 30 cells mm-2, p = 0.0147). The clear differentiation between mucosal and submucosal mast cells on the basis of metachromasia with Toluidine Blue was not seen in biopsies from the patients with gastritis or Crohn's disease. Previous studies which have suggested that there are no distinct mucosal and submucosal mast cell subsets in the human intestine may, therefore, have been affected by the use of tissue from diseased subjects. Heterogeneity in the expression of mast cell tryptase and chymase was seen by immunohistochemistry using specific antibodies, but the relative numbers of mast cell subsets were critically dependent on the methods used. Using a sensitive staining procedure, the majority of mucosal mast cells stained positively for chymase as well as for tryptase, an observation confirmed by immunoelectron microscopy and immunoabsorption studies. Our findings suggest that early stages in intestinal inflammation may be reflected in changes in mast cell numbers and in their staining properties, and call for a reappraisal of mast cell heterogeneity in the human intestinal tract.
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PMID:Number, fixation properties, dye-binding and protease expression of duodenal mast cells: comparisons between healthy subjects and patients with gastritis or Crohn's disease. 942 79

Previously we isolated a trypsin-like enzyme designated human airway trypsin-like protease from the sputum of patients with chronic airway diseases. This paper describes the cDNA cloning, characterization of the primary protein structure deduced from the cDNA, and gene expression of this enzyme in various human tissues. We obtained an entire 1517-base pair sequence of cDNA with an open reading frame encoding a polypeptide with 418-amino acid residues. The polypeptide consisted of a 232-residue catalytic region and a 186-residue noncatalytic region with a hydrophobic putative transmembrane domain near the NH2 terminus. The polypeptide was suggested to be a type II integral membrane protein in which the COOH-terminal catalytic region is extracellular. Therefore, this protein is thought to be synthesized as a membrane-bound precursor and to mature to a soluble and active protease by limited proteolysis. It showed 29-38% identity in the sequence of the catalytic region with human hepsin, enteropeptidase, acrosin, and mast cell tryptase. The noncatalytic region had little similarity to other known proteins. In Northern blot analysis a transcript of 1.9 kilobases was detectable most prominently in the trachea among 17 human tissues examined.
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PMID:Cloning and characterization of the cDNA for human airway trypsin-like protease. 956 16

A unique property of human mast cell tryptase is its spontaneous inactivation, which may be relevant to the regulation of the activity of this enzyme in vivo. We have found, using size-exclusion chromatography, that the dissociation of the tetrameric active enzyme into the inactive monomer occurred immediately from the beginning of the inactivation process and at a rate significantly faster than that of the appearance of the inactive, tetrameric form. Eventually, a relatively long-lived state of apparent equilibrium between all three forms (active tetramer, inactive monomer, inactive tetramer) was reached. When tryptase was extensively cross-linked with several heterobifunctional photoactivatable reagents, this modified enzyme exhibited a long-term stability in low-ionic-strength buffer and at elevated temperature, unlike that of the native enzyme. Its is suggested that cross-linking prevents the spontaneous inactivation and dissociation of tryptase by 'freezing' the normal association state of the enzyme and supports the hypothesis that the dissociation of native tetrameric tryptase into inactive monomer is the primary event for the entire process of spontaneous inactivation of this enzyme.
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PMID:Spontaneous inactivation of human lung tryptase as probed by size-exclusion chromatography and chemical cross-linking: dissociation of active tetrameric enzyme into inactive monomers is the primary event of the entire process. 963 May 76

Thirty-six new amino acid and peptidyl diphenyl phosphonate esters were synthesized and evaluated to identify potent and selective inhibitors for four trypsin-like proteases: lymphocyte granzymes A and K, human mast cell tryptase, and pancreatic trypsin. Among five Cbz derivatives of Lys and Arg homologues, Z-(4-AmPhe)P(OPh)2 is the most potent inhibitor for granzyme A, and Z-LysP(OPh)2 is the best inhibitor for granzyme K, mast tryptase, and trypsin. The amidino P1 residue D,L-(4-AmPhGly)P(OPh)2 was utilized in a series of compounds with several different N-protecting groups and systematic substitutions at P2 in Cbz-AA derivatives and at P3 in Cbz-AA-Ala derivatives. Generally, these phosphonates inhibit granzyme A and trypsin more potently than granzyme K and tryptase. The P2 Thr and Ala dipeptide phosphonates, Cbz-AA-(4-AmPhGly)P(OPh)2, are the most potent inhibitors for granzyme A, and Cbz-Thr-(4-AmPhGly)P(OPh)2 (kobs/[I] = 2220 M-1 s-1) was quite specific with much lower inhibition rates for granzyme K and trypsin (kobs/[I] = 3 and 97 M-1 s-1, respectively) and no inhibition with tryptase. The most effective inhibitor of granzyme A was Ph-SO2-Gly-Pro-(4-AmPhGly)P(OPh)2 with a second-order rate constant of 3650 M-1 s-1. The most potent inhibitor for granzyme K was 3, 3-diphenylpropanoyl-Pro-(4-AmPhGly)P(OPh)2 with a kobs/[I] = 1830 M-1 s-1; all other phosphonates inhibited granzyme K weakly (kobs/[I] < 60 M-1 s-1). Human mast cell tryptase was inhibited slowly by these phosphonates with Cbz-LysP(OPh)2 as the best inhibitor (kobs/[I] = 89 M-1 s-1). The overall results suggest that scaffolds of Phe-Thr-(4-AmPhe) and Phe-Pro-Lys will be useful to create selective phosphonate inhibitors for granzymes A and K, respectively, and that P4 substituents offer opportunities to further enhance selectivity and reactivity.
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PMID:Synthesis and evaluation of diphenyl phosphonate esters as inhibitors of the trypsin-like granzymes A and K and mast cell tryptase. 963 62


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