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)

We have previously shown the development in vitro of tryptase+ human mast cells from fetal liver cells cocultured with murine 3T3 fibroblasts. In this study, recombinant human stem cell factor (rhuSCF), the ligand for the c-kit proto-oncogene product called Kit, stimulated the growth and differentiation primarily of mast cells from dispersed fetal liver cells, whereas recombinant human interleukin-3 (rhuIL-3) stimulated the differentiation of basophils along with other cell types. Cultures of fetal liver cells were initiated and maintained in the presence of rhuSCF or rhuIL-3 for up to 6 weeks. Metachromatic cells in cytospins were identified as mast cells primarily on the basis of tryptase expression, and as MCT or MCTC by immunohistochemistry using monoclonal antibodies against tryptase and chymase, whereas basophils were metachromatic, polymorphonuclear, and lacked these proteases. Levels of tryptase and histamine were measured by radioimmunoassay, tryptase and chymase activities by peptide hydrolysis, and cell surface Kit by flow cytometry with the monoclonal antibody YB5.B8. The predominant presence of mast cells occurred only in the cultures supplemented with rhuSCF. The percentage and total number of mast cells increased over time with increasing concentrations of rhuSCF and reached a plateau at 55 ng/mL. At this concentration of rhuSCF, mast cells first appeared by day 7; by day 42, 106% of the starting number of cells were present and 85% of these were tryptase+, 31% being weakly chymase+. These mast cells appeared immature by ultrastructural criteria; most cells were mononuclear, but some had nuclei with deeply divided lobes. DNA synthesis in tryptase+ mast cells at days 21 and 28 of culture with rhuSCF was demonstrated by incorporation of bromodeoxyuridine. Calculated levels of histamine (1.2 pg/mast cell) and tryptase (0.9 pg/mast cell) were similar to those determined previously in coculture experiments with murine 3T3 fibroblasts. Chymase activity was undetectable in most cell extracts. On day 0, 4% to 20% of fetal liver cells expressed cell surface Kit. In the presence of rhuSCF, the percentages and total numbers of Kit+ cells and the apparent concentration of Kit per cell increased along with the number of tryptase+ cells. In the presence of rhuIL-3, toluidine blue+, tryptase- cells first and maximally appeared at day 14 (11% +/- 2.5%). The percentage of these toluidine blue+ cells then declined to about 6% by days 21 and 35, while the total number of positive cells declined over 10-fold. Kit+ cells in the presence of rhuIL-3 declined from 9% on day 3 to 2% on day 35.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Recombinant human stem cell factor stimulates differentiation of mast cells from dispersed human fetal liver cells. 128 84

This article reviews the ultrastructural and immunohistochemical features of normal human mast cells (MC) at multiple tissue sites. Current literature indicates that granules containing discrete scrolls (scroll-rich morphology) are frequent in MC from bowel mucosa and lung, locations where the majority of MC show only tryptase immunoreactivity (MCT). In contrast, most MC from skin, breast parenchyma, axillary lymph nodes, and bowel submucosa are characterized by scroll-poor morphology (that is, granules are rimmed by incomplete scrolls forming parallel lamellae and containing central, amorphous granular material or grating/lattice-like structures) and show both tryptase and chymase immunoreactivity (MCTC). MC having granules with both scroll-rich and scroll-poor features can occur in all tissue sites, and an occasional MC, especially in lung and bowel, may show only chymase immunoreactivity (MCC). Chymase immunoreactivity in MC also is closely associated with avidin binding and carboxypeptidase reactivity. We conclude that there is ultrastructural and immunophenotypic diversity among normal human MC, although certain forms predominate in specific tissue environments. In skin, breast tissue, axillary lymph nodes, and bowel submucosa MC tend to have scroll-poor granules and stain for avidin, chymase, tryptase, and carboxypeptidase, whereas, in lung and bowel mucosa MC granules tend to be scroll-rich and stain only for tryptase with currently available reagents.
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PMID:Ultrastructural and immunohistochemical characterization of normal mast cells at multiple body sites. 200 49

Recent studies have led to a rapid expansion of knowledge concerning the structure and biology of the two major mast cell proteinases, tryptase and chymase. Tryptase is an abundant, trypsin-like enzyme found in the secretory granules of all human lung mast cells. The subunits of the heparin-associated tryptase tetramer appear to be the products of a multigene family whose intron-exon organization is unique and is not closely related to that of other mast cell or leukocyte serine proteinases. In vitro studies suggest that tryptases may participate in lung and airway responses by regulating airway neuropeptide activity, bronchomotor tone, and fibroblast mitogenesis. Mast cell chymases are chymotrypsin-like proteinases related closely to neutrophil cathepsin G and lymphocyte granzymes. The cDNA-derived structures of tryptase and chymase suggest that the two enzymes may differ in modes of activation from proenzyme forms, although the mature enzymes are packaged and released together. Chymase expression appears to be limited to a subset of human lung mast cells most prevalent in the airway submucosa. Possible roles for chymase include inactivation of sensory neuropeptides, regulation of submucosal gland secretion, and potentiation of histamine-induced vascular permeability.
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PMID:The structure and airway biology of mast cell proteinases. 202 78

Many unexpected biological functions as bioreactants of the intracellular proteases and their endogenous inhibitors have been found recently. Chymase and tryptase in histamine granules of mast cells and basophile cells play an important role in the process of IgE-mediated degranulation and in the formation of an allergic inflammation profile. Furthermore, the relationship between membrane proteases and their endogenous inhibitor has been taken up as a key and key-hole relation which plays an important role for special recognition apparatus of biological information like the relation of peptide hormones (growth factors) and their specific receptors. Amino acid sequences of the active site of trypstatin are homologous with the neutralizing epitope beta of gp120 of AIDS virus (HIV-1). The trypstatin and anti-tryptase M antibody inhibited syncytium formation in HIV infected Molt 4, clone 8 cells. Therefore, the relationship between tryptase M with trypstatin and the recognition site of epitope beta of HIV-1 with the receptor of helper T-cells are the common keys. The precursor of Alzheimer's deposition protein contains a Kunitz-type trypsin inhibitor domain. The A4-precursor proteins are located in axons of pyramidal neurons in brain and secretory granules of chromaffin cells in adrenal medulla. Those may be secreted into the extracellular milieu. We propose that the A4 inhibitor inhibits a special type of tryptase in the brain and disturbs the complete degradation of secreted A4-precursor protein causing amyloid deposition in alzheimer disease by abnormal proteolysis. Human c-Ha-ras p21 shows 58% homology with cystatin beta, an endogenous inhibitor of cathepsin. Actually, p21 inhibits cathepsin L specifically, but not cathepsin H, papain and cathepsin B. However, the metabolic significance of this inhibitory activity is still unknown.
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PMID:New biological functions of intracellular proteases and their endogenous inhibitors as bioreactants. 220 23

Exogenous addition of purified chymase, a rat serosal mast cell (RSMC) chymotryptic enzyme, results in RSMC degranulation at 37 degrees, but not at 1 degree. Chymase can cause an active site-dependent inducing event at 1 degree such that RSMC degranulation occurs if the cells are later incubated at 37 degrees. RSMC exposed to chymase or other stimuli were surface radiolabelled using 125I and Iodo-Gen, solubilized with 1% Nonidet-40, and the resulting 25,000 g supernatants analysed by SDS-PAGE and autoradiography. A 125I-labelled RSMC membrane protein of approximate 90,000 MW decreased upon exposure to either chymase or alpha-chymotrypsin (alpha-CT) for 5 min at 37 degrees or to chymase for 60 min at 1 degree. Exposure of RSMC to the secretagogues ionophore A23187, compound 48/80, and anti-IgE for 5 min at 37 degrees resulted in beta-hexosaminidase (a secretory granule enzyme) release, but did not cause a detectable change in the 90,000 MW surface-labelled protein. Lima bean trypsin inhibitor, which inhibits both the esterase and RSMC degranulation activities of chymase and alpha-CT, prevented the disappearance of the 125I-labelled 90,000 MW band when added with chymase or alpha-CT. Exposure of RSMC to chymase at 1 degree for 0-10 min, prior to addition of LBTI, led to a progressive disappearance of the 90,000 MW band, which corresponded to the kinetics of priming for subsequent RSMC degranulation at 37 degrees. When RSMC were exposed to trypsin (2.5 micrograms/ml) for 0-120 min at 1 degree, a progressive disappearance of the 90,000 MW band occurred, in association with a loss of sensitivity to subsequent activation by chymase at 37 degrees. The disappearance of the 90,000 MW determinant in association with chymase-mediated priming for degranulation and the inability of chymase to mediate degranulation of trypsin-treated RSMC, which lack this membrane protein, suggests that it is involved in chymase-mediated RSMC degranulation.
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PMID:Cleavage of a rat serosal mast cell membrane component during degranulation mediated by chymase, a secretory granule protease. 231 65

The peptides substance P (SP) and vasoactive intestinal peptide (VIP) released from peptidergic neurons have potent effects on gland secretion and on smooth muscle tone. Because mast cells release proteases during degranulation, and are located in many of the same tissue microenvironments into which SP and VIP are released, we wished to examine whether mast cell proteases, by cleaving and thus inactivating these peptides, could modulate their effects. We used active site-titrated preparations of the two major neutral proteases of mast cell granules, tryptase and chymase, to determine the sites and rates of cleavage of SP and VIP. The proteases were purified from dog mastocytomas. Tryptase cleaved VIP rapidly at two sites with a kcat/Km of 2.2 X 10(5) sec-1 M-1, but had no effect on SP. Chymase cleaved both SP and VIP at primarily a single site with kcat/Km of 3.9 X 10(4) and 5.4 X 10(4) sec-1 M-1, respectively. Thus, these data show that mast cell proteases degrade SP and VIP. The differences in peptidase activity between tryptase and chymase suggest that the consequences of protease release could vary according to mast cell protease phenotype and location in various tissues and species. Tryptase, by cleaving the bronchodilator VIP but not the bronchoconstrictor SP, might promote bronchial hyper-responsiveness in asthma by decreasing the nonadrenergic neural inhibitory influence mediated by VIP. In skin and other tissues, chymase might interrupt axon reflex-mediated neurogenic inflammation by cleaving SP.
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PMID:Substance P and vasoactive intestinal peptide degradation by mast cell tryptase and chymase. 244 73

Human pulmonary mast cells contain the serine proteases tryptase and chymase. Chymase is present in much smaller quantities than tryptase. The definite physiological role of both enzymes remains to be elucidated, angiotensin processing has been proposed as one possible function of chymase. A dose-dependent inhibition of A 23187-induced histamine release from dispersed human lung mast cells was observed after pretreatment with diisopropylfluorophosphate (DFP) or 1-1-tosyamide-2-phenylethyl chloromethyl ketone (TPCK) but not with N-2-p-tosyl-1-lysine chloromethyl ketone (TLCK). In contrast, no inhibition was observed under the same conditions with isolated rat peritoneal mast cells. These results indicate that a chymase is probably an important factor in a late phase of human lung mast cell activation. Current work focuses on the isolation of human lung chymase to further investigate this topic.
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PMID:The role of chymase in ionophore-induced histamine release from human pulmonary mast cells. 246 1

Submucosal glands are the major sources of airway secretions in most mammals. Mast cells are abundant in the environment of airway submucosal glands and are rich sources of secreted proteases. To investigate the hypothesis that mast cell proteases stimulate airway gland secretion, we studied the ability of the two major mast cell granule proteases, chymase and tryptase, to cause secretion of 35S-labeled macromolecules from a line of cultured bovine airway gland serous cells. Mast cell chymase and tryptase were purified from dog mastocytoma cells. Chymase markedly stimulated serous cell secretion in a concentration-dependent fashion with a threshold of 10(-10) M, whereas tryptase had no effect. The response to 10(-8) M chymase (1530 +/- 80% over base line) was approximately 10-fold higher than that evoked by other agonists such as histamine and isoproterenol. The predominant 35S-labeled macromolecule released by chymase was chondroitin sulfate proteoglycan, the glycoconjugate present in serous cell secretory granules. The response to chymase was non-cytotoxic and was blocked by active site inhibitors of chymase (soybean trypsin inhibitor and chymostatin) and by inhibitors of cellular energy metabolism (azide,2,4-dinitrophenol, dicumarol). Supernatant obtained by degranulation of mastocytoma cells caused a secretory response of comparable magnitude to that caused by chymase. These findings demonstrate that chymase, but not tryptase, is a potent secretagogue for airway gland serous cells, and they suggest a possible role for chymase-containing mast cells in the pathogenesis of airway hypersecretion.
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PMID:Mast cell chymase. A potent secretagogue for airway gland serous cells. 249 59

Previous studies with trans-4-(guanidinomethyl)cyclohexanecarboxylic acid 4-tert-butylphenyl ester (GMCHA-OPhBut), a trypsin inhibitor, strongly suggested the involvement of a trypsin-like protease in histamine release from mast cells induced by various secretagogues (Takei, M., Matumoto, T., Endo, K. & Muramatu, M. (1988) Agents and Actions, in press; Takei, M., Matumoto, T., Ito, T., Endo, K. & Muramatu, M.; Takei, M., Matumoto, T., Endo, K. & Muramatu, M. and Takei, M., Matumoto, T., Urashima, H., Endo, K. & Muramatu, M., unpublished results). Two serine proteases, chymase (Benditt, E.F. & Arase, M. (1959) J. Exp. Med. 110, 451-460) and tryptase Kido, H., Fukusen, N. & Katunuma, N. (1985) Arch. Biochem. Biophys. 239, 436-443) were demonstrated in rat peritoneal mast cells. Both enzymes were purified and the effects of inhibitors for trypsin and chymotrypsin on these proteases were examined. The trypsin-like protease was found in saline extract and purified by successive chromatographies on Sephadex G-100 and DEAE-cellulose columns. The molecular mass of this protease was apparently 120,000 Da. This protease showed maximal activity at pH 7.1 and was named pH 7 tryptase. Chymase was obtained from 1.5M NaCl extract. pH 7 Tryptase markedly hydrolysed Boc-Phe-Ser-Arg-NH-Mec and Boc-Val-Pro-Arg-NH-Mec among the various substrates containing arginyl and lysyl bonds but did not cleave Tos-Arg-OMe. Tos-Lys-CH2Cl and diisopropylfluorophosphate strongly inhibited this protease. Various inhibitors for trypsin inhibited pH 7 tryptase, and those for chymotrypsin inhibited chymase. Among the esters of GMCHA examined, GMCHA-OPhBut most strongly and competitively inhibited pH 7 tryptase but it had no effect on chymase.
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PMID:Tryptase in rat mast cells: properties and inhibition by various inhibitors in comparison with chymase. 306 68

Two of the major enzymes present in and released from rat mast cells are chymotrypsin-type serine protease (chymase) and trypsin-type serine protease (tryptase), and these have been postulated to be important in the inflammatory reactions. There have been no clear data regarding the trypsin-type protease in rat mast cells. Tryptase was recently purified from rat peritoneal mast cells with an associated protein (trypstatin) that inhibited the protease activity above pH 7.5. Chymase was also purified from rat peritoneal cells by employing a one-step method involving hydrophobic chromatography on octyl-Sepharose 4B or arginine-Sepharose 4B. The properties of chymase and tryptase were described in relation to substrate specificity and their relative sensitivity to inhibitors. It was found that proteolytic activities of these enzymes were modulated by naturally occurring substances, such as phosphoglycerides, long-chain fatty acids, and trypstatin. There is as yet little evidence for the physiological roles of these enzymes in the inflammatory reaction. It has been found that the specific, low-molecular-weight inhibitor of chymase, chymostatin, and that of tryptase, leupeptin, inhibit histamine release induced by addition of anti-rat IgE to mast cells. However, the inhibitors with molecular weights of more than 6000 were found to have no effect in this process. The data suggest that chymase and tryptase in mast cell granules play a crucial or significant role in the process of degranulation.
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PMID:Chymotrypsin- and trypsin-type serine proteases in rat mast cells: properties and functions. 389 Jul 54


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