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)

From the inner shoots of the chive Allium tuberosum, a single-chained protein with a molecular weight of 36 kDa and an N-terminal sequence manifesting resemblance to chitinases but lacking in cysteine residues characteristic of a cysteine-rich domain present in chitinases of other Allium species, was purified. The isolation procedure entailed affinity chromatography on Affi-gel blue gel, ion-exchange chromatography on DEAE-cellulose and Mono S, and gel filtration on Superdex 75. The protein was unadsorbed on DEAE-cellulose and adsorbed on Affi-gel blue gel and Mono S. It exhibited antifungal activity against Rhizoctonia solani, Fusarium oxysporum, Coprinus comatus, Mycosphaerella arachidicola, and Botrytis cinerea. The IC(50) for its antifungal effect against Botrytis cinerea was 0.2 microM. The antifungal activity was stable after 1 h at pH 1.6 and 12.3, and up to 60 degrees C for 5 min. Incubation of the protein with trypsin or chymotrypsin at an enzyme:substrate ratio of 1:100 and pH 7.6 up to 150 min did not affect its antifungal activity. The protein did not exhibit antibacterial activity. The protein inhibited cell-free translation in a rabbit reticulocyte system with an IC(50) of 0.8 microM, but did not affect the proliferation of mouse splenocytes. It exerted some cytotoxic effect on breast cancer cells and was inhibitory toward HIV-1 reverse transcriptase.
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PMID:A robust cysteine-deficient chitinase-like antifungal protein from inner shoots of the edible chive Allium tuberosum. 1111 20

Genomic blot analysis raised the possibility that uncharacterized tryptase genes reside on chromosome 17 at the complex containing the three genes that encode mouse mast cell protease (mMCP) 6, mMCP-7, and transmembrane tryptase (mTMT). Probing of GenBank's expressed sequence tag data base with these three tryptase cDNAs resulted in the identification of an expressed sequence tag that encodes a portion of a novel mouse serine protease (now designated mouse tryptase 4 (mT4) because it is the fourth member of this family). 5'- and 3'-rapid amplification of cDNA ends approaches were carried out to deduce the nucleotide sequence of the full-length mT4 transcript. This information was then used to clone its approximately 5.0-kilobase pair gene. Chromosome mapping analysis of its gene, sequence analysis of its transcript, and comparative protein structure modeling of its translated product revealed that mT4 is a new member of the chromosome 17 family of mouse tryptases. mT4 is 40-44% identical to mMCP-6, mMCP-7, and mTMT, and this new serine protease has all of the structural features of a functional tryptase. Moreover, mT4 is enzymatically active when expressed in insect cells. Due to its 17-mer hydrophobic domain at its C terminus, mT4 is a membrane-anchored tryptase more analogous to mTMT than the other members of its family. As assessed by RNA blot, reverse transcriptase-polymerase chain reaction, and/or in situ hybridization analysis, mT4 is expressed in interleukin-5-dependent mouse eosinophils, as well as in ovaries and testes. The observation that recombinant mT4 is preferentially retained in the endoplasmic reticulum of transiently transfected COS-7 cells suggests a convertase-like role for this integral membrane serine protease.
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PMID:Tryptase 4, a new member of the chromosome 17 family of mouse serine proteases. 1125 27

A novel ribosome-inactivating protein with a molecular weight of 20 kDa was isolated from fruiting bodies of the mushroom Hypsizigus marmoreus. The isolation procedure entailed ion exchange chromatography on CM-cellulose, affinity chromatography on Affi-gel Blue Gel and ion exchange chromatography on Mono Q. The protein designated hypsin demonstrated an inhibitory action against mycelial growth in various fungal species including Mycosphaerella arachidicola, Physalospora piricola, Fusarium oxysporum, and Botrytis cinerea with an IC50 of 2.7, 2.5, 14.2, and 0.06 microM, respectively. Translation in the rabbit reticulocyte lysate system was inhibited with an IC50 of 7 nM and HIV-1 reverse transcriptase activity was inhibited with an IC50 of 8 microM. Antiproliferative activity against mouse leukemia cells and human leukemia and hepatoma cells was observed. About 60% of the translation-inhibitory activity was retained after heating at 100 degrees C for 10 min. No loss of translation-inhibitory activity occurred after brief treatment with trypsin.
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PMID:Hypsin, a novel thermostable ribosome-inactivating protein with antifungal and antiproliferative activities from fruiting bodies of the edible mushroom Hypsizigus marmoreus. 1146 62

alpha- and beta-tryptase genes encode serine proteases that are abundantly expressed by mast cells. Under physiologic conditions other myeloid cells are virtually tryptase negative. However, tryptases are also expressed in several myeloid leukemia cell lines. In this study, serum total tryptase levels were determined in 150 patients with acute leukemias (de novo acute myeloid leukemia [AML], n = 108; secondary AML, n = 25; acute lymphoid leukemia [ALL], n = 17) by fluoroenzyme immunoassay. In healthy subjects (n = 30), tryptase levels ranged between 2.0 and 12.6 ng/mL. Elevated tryptase levels (> 15) were detected in 42 (39%) of 108 patients with de novo AML and in 11 (44%) of 25 patients with secondary AML. No elevated tryptase levels were found in patients with ALL. In de novo AML, elevated tryptase levels were frequently detected in patients with French-American-British classification M0 (6 of 9), M2 (9 of 14), M3 (4 of 6), and M4eo (7 of 7), and less frequently in M1 (7 of 20), M4 (6 of 26), M5 (2 of 18), M6 (0 of 5), or M7 (1 of 3). The highest tryptase levels were found in M4eo. Immunohistochemical staining of bone marrow sections with anti-tryptase antibody as well as immunoelectron microscopy revealed tryptase expression in the cytoplasm of myeloblasts. As assessed by Northern blotting and reverse transcriptase-polymerase chain reaction, AML cells expressed alpha-tryptase messenger RNA (mRNA) but little or no beta-tryptase mRNA. In AML patients with elevated serum tryptase before chemotherapy, who entered complete remission, tryptase levels returned to normal or near normal values. Blast cell persistence or regrowth was associated with a persistently elevated level or recurrent increase of tryptase. Together, tryptase is expressed in myeloblasts in a group of AML and may serve as a useful disease-related marker.
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PMID:Expression of mast cell tryptase by myeloblasts in a group of patients with acute myeloid leukemia. 1156 8

Trypsin and mast cell tryptase cleave proteinase-activated receptor 2 (PAR2) to induce alterations in contraction of airway smooth muscle that have been implicated in asthma in experimental animals. Although tryptase inhibitors are under development for treatment of asthma, little is known about the localization and function of PAR2 in human airways. We detected PAR2 expression in primary cultures of human airway smooth muscle cells using reverse transcriptase/polymerase chain reaction (RT-PCR) and immunofluorescence. The PAR2 agonists trypsin, tryptase, and an activating peptide (SLIGKV-NH2) stimulated calcium mobilization in these cells. PAR2 agonists strongly desensitized responses to a second challenge of trypsin and SLIGKV-NH2, but not to thrombin, indicating that they activate a receptor distinct from the thrombin receptors. Immunoreactive PAR2 was detected in smooth muscle, epithelium, glands, and endothelium of human bronchi. Trypsin, SLIGKV-NH2, and tryptase stimulated contraction of isolated human bronchi. Contraction was increased by removal of the epithelium and diminished by indomethacin. Thus, PAR2 is expressed by human bronchial smooth muscle where its activation mobilizes intracellular Ca2+ and induces contraction. These results are consistent with the hypothesis that PAR2 agonists, including tryptase, induce bronchoconstriction of human airway by stimulating smooth muscle contraction. PAR2 antagonists may be useful drugs to prevent bronchoconstriction.
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PMID:Expression and function of proteinase-activated receptor 2 in human bronchial smooth muscle. 1167 22

An isolation procedure comprising affinity chromatography on Affi-gel blue gel, ion exchange chromatography on SP-Toyopearl, and fast protein liquid chromatography on Mono S was used to purify a peptide from broad beans which manifested antifungal activity toward Mycosphaerella arachidicola, Fusarium oxysporum, and Botrytis cinerea. The peptide demonstrated a molecular mass of 7.5 kDa. N-terminal sequence analysis disclosed the identity of the antifungal peptide to be a trypsin-chymotrypsin inhibitor. The trypsin-chymotrypsin inhibitor also exerted an inhibitory action on chymotrypsin activity and HIV-1 reverse transcriptase activity. Proliferation of murine splenocytes was stimulated in the presence of the trypsin-chymotrypsin inhibitor. This report constitutes the first observation of antifungal activity of a leguminous peptidic protease inhibitor.
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PMID:A Bowman-Birk-type trypsin-chymotrypsin inhibitor from broad beans. 1170 82

Mast cells have been reported to release not only chemical mediators, but also cytokines upon Fc epsilon receptor I(Fc epsilon RI) cross-linking. Recently, we have established a culture system to derive chymase-rich human mast cells from mononuclear cells in peripheral blood. However, the functional properties of these mast cells have remained unrevealed. In this study, we examined the functions of peripheral blood-derived human cultured mast cells (pHCMCs). pHCMCs expressed functional Fc epsilon RI, and most of them contained tryptase. These pHCMCs sensitized with immunoglobulin E (IgE) and interleukin 4 (IL-4) were activated through cross-linking of Fc epsilon RI. The time-dependent mRNA expression profiles of Fc epsilon RI subunits, cytokines and chemokines in the sensitized pHCMCs upon Fc epsilon RI engagement were examined by reverse transcriptase polymerase chain reaction (RT-PCR). mRNA for most of cytokines and chemokines, which were observed in allergic inflammation, was detected in activated pHCMCs. In addition, gene expression for monocyte chemoattractant protein 3 (MCP-3) in human mast cells, and liver and activation-regulated chemokine (LARC), thymus and activation-regulated chemokine (TARC) and macrophage-derived chemokine (MDC) in mast cells was revealed for the first time in our study. Fc epsilon RI-mediated cytokine and chemokine production at protein level was evaluated using enzyme-linked immunosorbent assay (ELISA). These data suggest that pHCMCs, which are capable of producing a variety of cytokines and chemokines, can be a useful candidate for investigating roles of mast cells as a conductor for allergic inflammation.
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PMID:Gene expression profiles for Fc epsilon RI, cytokines and chemokines upon Fc epsilon RI activation in human cultured mast cells derived from peripheral blood. 1179 24

The purification and unique carbohydrate binding properties, including blood group B-specific agglutination and preferential binding to Galalpha1,3Gal-containing sugar epitopes, of the Marasmius oreades agglutinin (MOA) are reported in an accompanying paper (Winter, H. C., Mostafapour, K., and Goldstein, I. J. (2002) J. Biol. Chem. 277, 14996-15001). Here we describe the cloning, characterization, and expression of MOA. MOA was digested with trypsin and endoproteinase Asp-N, and the peptide fragments were purified by high performance liquid chromatography. Amino acid sequence data were obtained for eight peptides. Using oligonucleotides deduced from the peptide sequences for a reverse transcriptase-PCR, a 41-base pair cDNA was obtained. The 41-base pair fragment allowed the generation a full-length cDNA using 5' and 3' rapid amplification of cDNA ends. MOA cDNA encodes a protein of 293 amino acids that contains a ricin domain. These carbohydrate binding domains were first described in subunits of bacterial toxins and are also commonly found in polysaccharide-degrading enzymes. Whereas these proteins are known to display a variety of sugar binding specificities, none to date are known to share MOA's high affinity for Galalpha1,3Gal and Galalpha1,3Galbeta1,4GlcNAc. Recombinantly expressed and purified MOA retains the specificity and affinity observed with the native protein. This study provides the basis for analyzing the underlying cause for the unusual binding specificity of MOA.
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PMID:Cloning, expression, and characterization of the Galalpha 1,3Gal high affinity lectin from the mushroom Marasmius oreades. 1183 54

Serpins are responsible for regulating a variety of proteolytic processes through a unique irreversible suicide substrate mechanism. To discover novel genes regulated by transforming growth factor-beta1 (TGF-beta 1), we performed differential display reverse transcriptase-PCR analysis of NRP-152 rat prostatic epithelial cells and cloned a novel rat serpin that is transcriptionally down-regulated by TGF-beta and hence named trespin (TGF-beta-repressible serine proteinase inhibitor (trespin). Trespin is a 397-amino acid member of the ov-serpin clade with a calculated molecular mass of 45.2 kDa and 72% amino acid sequence homology to human bomapin; however, trespin exhibits different tissue expression, cellular localization, and proteinase specificity compared with bomapin. Trespin mRNA is expressed in many tissues, including brain, heart, kidney, liver, lung, prostate, skin, spleen, and stomach. FLAG-trespin expressed in HEK293 cells is localized predominantly in the cytoplasm and is not constitutively secreted. The presence of an arginine at the P1 position of trespin's reactive site loop suggests that trespin inhibits trypsin-like proteinases. Accordingly, in vitro transcribed and translated trespin forms detergent-stable and thermostable complexes with plasmin and elastase but not subtilisin A, trypsin, chymotrypsin, thrombin, or papain. Trespin interacts with plasmin at a near 1:1 stoichiometry, and immunopurified mammal-expressed trespin inhibits plasmin in a dose-dependent manner. These data suggest that trespin is a novel and functional member of the rat ov-serpin family.
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PMID:Identification and characterization of a novel rat ov-serpin family member, trespin. 1198 14

Injury to motor neurons associated with mutant Cu,Zn-superoxide dismutase (SOD1)-related familial amyotrophic lateral sclerosis (FALS) results from a toxic gain-of-function of the enzyme. The mechanisms by which alterations to SOD1 elicit neuronal death remain uncertain despite intensive research effort. Analysis of the cellular proteins that are differentially expressed in the presence of mutant SOD1 represents a novel approach to investigate further this toxic gain-of-function. By using the motor neuron-like cell line NSC34 stably transfected with wild-type, G93A, or G37R mutant human SOD1, we investigated the effects of mutant human SOD1 on protein expression using proteomic approaches. Seven up-regulated proteins were identified as argininosuccinate synthase, argininosuccinate lyase, neuronal nitric-oxide synthase, RNA-binding motif protein 3, peroxiredoxin I, proteasome subunit beta 5 (X), and glutathione S-transferase (GST) Alpha 2. Seven down-regulated proteins were identified as GST Mu 1, GST Mu 2, GST Mu 5, a hypothetical GST Mu, GST Pi B, leukotriene B(4) 12-hydroxydehydrogenase, and proteasome subunit beta5i (LMP7). GST assays demonstrated a significant reduction in the total GST activity of cells expressing mutant human SOD1. Proteasome assays demonstrated significant reductions in chymotrypsin-like, trypsin-like, and post-glutamylhydrolase proteasome activities. Laser capture microdissection of spinal cord motor neurons from human FALS cases, in conjunction with reverse transcriptase-PCR, demonstrated decreased levels of mRNA encoding GST Mu 1, leukotriene B(4) 12-hydroxydehydrogenase, and LMP7. These combined approaches provide further evidence for involvement of alterations in antioxidant defenses, proteasome function, and nitric oxide metabolism in the pathophysiology of FALS.
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PMID:Analysis of the cytosolic proteome in a cell culture model of familial amyotrophic lateral sclerosis reveals alterations to the proteasome, antioxidant defenses, and nitric oxide synthetic pathways. 1247 80


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