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)

The purpose of this study was to determine the distribution of mucin glycoprotein 1 (MG1) within submandibular, parotid, labial and palatine salivary tissues. Formalin-fixed and frozen tissue sections were examined histochemically with PAS, Alcian blue and Meyer's mucicarmine, and immunocytochemically with an anti-mucin glycoprotein 1 monoclonal antibody (clone 3/E8). Clone 3/E8 was produced in Balb/c mice using mucin-enriched chromatographic fractions from submandibular-sublingual saliva. The monospecificity of 3/E8 was confirmed by immuno-dot blotting and SDS-PAGE/electrophoretic transfer. Clone 3/E8 (IgG1; kappa) was of moderate affinity, and was directed to a carbohydrate-containing, TPCK-trypsin-insensitive and pronase-insensitive epitope on this mucin, which was not blood-group specific. The location of mucin glycoprotein 1 was determined by both indirect (peroxidase-antiperoxidase) and direct methods. Mucin glycoprotein 1 was localized within all labial acini examined, but was not found within parotid tissues. Histochemical methods stained all submandibular, palatine and labial acini, but immunocytochemistry with monoclonal antibody revealed heterogeneous staining with clone 3/E8 in submandibular and palatine tissues. These findings suggest the presence of mucin glycoprotein 1-specific acinar cell subpopulations within human submandibular and palatine salivary tissues.
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PMID:Immunochemistry of high molecular-weight human salivary mucin. 218 37

The complete amino acid sequence of a major trypsin inhibitor (FMTI-II) from seeds of foxtail millet (Setaria italica) was determined by analysis of peptides derived from the reduced and S-carboxymethylated protein by digestion with TPCK-trypsin and Staphylococcus aureus V8 protease. FMTI-II consists of 67 amino acid residues, including 10 half-cystine residues which are involved in 5 disulfide bridges in the molecule. The established sequence had a high degree of homology to Bowman-Birk type inhibitors from leguminous and gramineous plants. The trypsin reactive-site peptide bond in FMTI-II also appears to be Lys (16)-Ser (17) by comparison with these sequences.
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PMID:The complete amino acid sequence of a major trypsin inhibitor from seeds of foxtail millet (Setaria italica). 229 95

A fluorescence titration assay was used to detect the effects of various modifications of E.coli elongation factor Tu on the formation of the ternary complex with aminoacyl-tRNAs. The treatment of EF-Tu.GDP with TPCK, an analogue of the 3'terminus of aminoacyl-tRNA, was found to have no influence on the conversion of EF-Tu.GDP to 'active' EF-Tu.GTP, but does decrease the affinity of the activated protein for yeast aminoacyl-tRNA by more than three orders of magnitude. Modification of the elongation factor by limited cleavage with trypsin, leading to the excision of amino acid residues 45-58, has only a minor influence on ternary complex formation. The equilibrium dissociation constant of the ternary complex with this trypsin-treated EF-Tu.GTP and E.coli Phe-tRNA(Phe) is only one order of magnitude higher than that of the ternary complex with native EF-Tu. Mutations in the amino acid residues 222 and 375 of EF-Tu also have little effect on ternary complex formation. Compared with TPCK-treated EF-Tu, the affinities of the two mutant species, designated EF-tuAR and EF-TuBO respectively, for [AEDANS-s2C]Tyr-tRNA(Tyr) are only slightly reduced and in the same range as trypsin-cleaved EF-Tu.
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PMID:The influence of different modifications of elongation factor Tu from Escherichia coli on ternary complex formation investigated by fluorescence spectroscopy. 240 11

The possible involvement of cell surface-associated proteolytic enzymes in human NK cell-mediated cytotoxicity and the mechanism by which such enzymes exert their activity have been studied. The treatment of intact cells with 3H-DFP under restricted conditions that predominantly bind surface-associated enzymes resulted in the labeling of five to six enzyme bands. Among these were a 35,000-dalton enzyme, which may be a previously identified trypsin-like proteinase engaged in cytotoxicity, and a 58,000-dalton elastase. The latter seems not to be involved in the reaction, as potent inhibitors of this enzyme have negligible effect on cytotoxicity. Of the membrane-associated enzymes, those engaged in cytotoxicity seem to be concealed from the external environment, as pretreatment of the effector cells with protease inhibitors such as trasylol and PMSF have no effect on the reaction. Immediately upon addition of the target cells and the initiation of cytotoxicity, the reaction becomes highly sensitive to inhibitors for a limited time interval of 2 to 5 min when trasylol is employed and 5 to 10 min when TPCK is the inhibitor, suggesting that target cell binding triggers the exposure of the enzymes to the external environment, rendering them accessible to the inhibitors. This short sensitivity period parallels the interval in which the reaction is sensitive to the microfilament inhibitor cytochalasin B. As the reaction proceeds, it becomes increasingly resistant to inhibitors of both proteolysis and cytoskeleton, at the same time suggesting that microfilament action and the unraveling of proteases may be processes that bear a close linkage with one another. The surface-associated elastase on the other hand maintains a constitutive mode of activity distinctive and unrelated to that of enzymes engaged in cytotoxicity. These findings suggest the existence on the surface of the NK lymphocyte of a mechanism that associates the receptor for target cells with an array of enclaved proteolytic enzymes via microfilaments. The resting cytotoxic structures become activated as the receptor attaches to the target cell, triggers the exposure of the proteolytic moiety, and initiates the lytic phase of the reaction.
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PMID:The mechanism of human NK cell-mediated cytotoxicity. Mode of action of surface-associated proteases in the early stages of the lytic reaction. 240 53

EF-Tu from Thermus thermophilus was first labelled with N-[14C]tosyl-L-phenylalaninechloromethylketone and then cleaved by the combined action of CNBr and trypsin. The resulting peptides were separated by reversed-phase HPLC. Analysis of the isolated, labelled peptide led to the identification of a sequence which was identical to residues 76-88 in T. thermophilus EF-Tu. The TPCK reactive site is at Cys-82. Kinetic measurements of the incorporation of TPCK into native EF-Tu and EF-Tu nicked at position Arg-59 were performed. The results provide evidence that the cleavage of the peptide bond between Arg-59 and Gly-60 does not lead to a dramatic conformational change of EF-Tu at the aa-tRNA binding site.
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PMID:Identification of the N-tosyl-L-phenylalanyl chloromethylketone modification site in Thermus thermophilus elongation factor Tu. 258 65

Human glandular kallikrein was purified from urine and subjected to detailed structural characterization. The protein was carboxymethylated with iodoacetic acid and digested with TPCK-trypsin, Staphylococcal aureus V-8 protease and endo LysC peptidase. The resulting peptide fragments were separated by reverse-phase HPLC using C-4 columns and acetonitrile-trifluoroacetic acid gradient elution. The complete amino acid sequence of the carboxymethylated derivative was elucidated by sequence analysis and alignment of peptides derived from different proteolytic cleavages. A procedure using in situ CNBr cleavage of a large endo LysC peptidase-derived peptide followed by direct sequencing was carried out to provide overlap for two glycosylation sites at residues 78 and 84. Three Asn-linked glycosylation sites were confirmed by the direct sequence analysis of the isolated glycopeptides. However, the third glycosylation at Asn-144 occurs only in 60% of kallikrein molecules. Reverse-phase HPLC effectively separates two species of HUK which correspond to molecules glycosylated and non-glycosylated at Asn-144, respectively. The human urinary kallikrein contains 238 amino acid residues with Ile and Ser as N- and C-terminal amino acids, respectively. The primary structure is completely identical to that deduced from a human genomic DNA sequence (F.K. Lin et al., manuscript in preparation) and is different in one amino acid (Lys-162 vs. Glu-162) from that deduced from pancreatic or kidney cDNA sequence.
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PMID:Human urinary kallikrein. Complete amino acid sequence and sites of glycosylation. 266 27

We have defined three categories of cultured cell lines on the basis of their permissiveness (susceptibility to initial infection) to mouse hepatitis virus (MHV). Fully permissive L-2 cells gave rise to 100-1000-fold higher numbers of infectious centers than did semi-permissive LM, LM-K or C-1300 cells, whereas non-permissive Vero or C-6 cells were refractory to MHV infection. On an infected cell basis, there was no deficiency on the part of semi-permissive cell lines to replicate total viral RNA, viral polypeptides or progeny virions. Two of the semi-permissive cell lines (LM and LM-K) supported persistent MHV infection, while a third (C-1300) succumbed to lytic infection. LM and LM-K cells, but not C-1300 cells showed resistance to MHV-induced membrane fusion, even when placed in contact with fusion-active MHV-infected L-2 cells. The ability of a given cell to undergo fusion did not correlate with membrane lipid characteristics (unsaturated fatty acid and sterol content) which contribute to membrane "fluidity". In order to more closely study the parameters of MHV-induced cell fusion, membranes were prepared from MHV-infected L-2 cells and monitored for their fusogenic potential with permissive L-2 cells, semi-permissive LM cells and non-permissive vero cells. Fusion was only observed with the permissive L-2 cells, and only when exogenous protease (trypsin or chymotrypsin) was added. When the membranes were prepared from 35S-methionine-labeled MHV-infected L-2 cells and subjected to protease treatment, the radiolabeled 180,000 dalton form of the E2-glycoprotein underwent proteolytic cleavage to yield a major product of approximately 90,000 daltons. Both trypsin and chymotrypsin were effective in this proteolytic cleavage and in activating membrane fusion. In a normally permissive, fusogenic infection of MHV in L-2 cells, the protease inhibitors TPCK and ZPCK, but not TLCK, were found to inhibit cell fusion. In MHV-infected L-2 cells, E2 was found almost exclusively as the 180,000 dalton form but turned over rapidly as shown by pulse-chase studies. TPCK and ZPCK but not TLCK inhibited turnover. The results suggest that L-2 cells contain a protease which cleaves at aromatic amino acids such as phenylalanine, and that this protease cleaves the 180,000 dalton form of the E2 to peptide fragments, one or more of which may activate cell fusion.
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PMID:The role of protease-dependent cell membrane fusion in persistent and lytic infections of murine hepatitis virus. 282 27

gamma-Glutamyl transpeptidase (gamma GTP) is an enzyme found in cerebral capillary endothelial cells, the presumed site of the blood-brain barrier, but not in endothelial cells lining blood vessels in other parts of the body. Using a line of mouse cerebral microvessel endothelial cells (ME-ly cells) and a sensitive colorimetric assay to measure gamma GTP levels we demonstrated that primary cultures of mouse astrocytes and a line of rat C6 glioma cells released a soluble product(s) that induced the production of gamma GTP in cultured endothelial cells by 34% and 39%, respectively, over control levels. Cerebrovascular smooth muscle cells had no significant effect on gamma GTP levels in ME-ly cells, and the astrocyte product(s) had no effect on rabbit aortic endothelial cells. The induction of gamma GTP levels in ME-ly cells was apparent after one day of exposure to the astrocyte product(s) and increased in magnitude with increasing time of exposure of the ME-ly cells to the product(s). Removal of the product(s) from the ME-ly cells resulted in a return to control levels of gamma GTP in the ME-ly cells within 2 days. The presence of a protein synthesis inhibitor during incubation with the product(s) blocked the induction of gamma GTP in ME-ly cells, and treatment of the product(s) with 200 U/ml TPCK-trypsin destroyed its inductive properties.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Induction of gamma-glutamyl transpeptidase in cultured cerebral endothelial cells by a product released by astrocytes. 288 71

Protein-mixed disulfides (PSSG) were formed by interaction of glutathione disulfide (GSSG) with lens crystallins. Total water-soluble crystallins and alpha-crystallin purified on a Sephacryl S-200 column were separately incubated with 0, 2, 4, and 8 mM (final concentrations) GSSG overnight and then dialyzed to remove unbound GSSG and GSH. Either TPCK-treated trypsin or TLCK-treated alpha-chymotrypsin were added to about 200 micrograms crystallin samples and incubated for 20 min at room temperature. Reactions were terminated by boiling in SDS-mercaptoethanol-Tris (pH 6.8) solution and subjected to electrophoresis on 10% polyacrylamide slab gels. Comparison of SDS-PAGE patterns of proteolysis with or without GSSG treatment showed that GSSG at a concentration of 2 mM or higher reduced or abolished proteolysis of alpha-crystallin by trypsin but not by alpha-chymotrypsin. The protective effect of GSSG was greater with alpha-crystallin than with beta-crystallins. Addition of alpha-crystallin-mixed-disulfide to an assay system in which trypsin was hydrolyzing N-alpha-benzoyl-DL-arginine-P-anilide (BAPNA) inhibited the tryptic activity. Direct addition of GSSG or native alpha-crystallin had no significant inhibitory effect on trypsin. Based on these results, it is speculated that alpha-crystallin glutathione mixed-disulfide appears to become resistant to trypsin probably by non-competetive inhibition of the enzyme.
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PMID:Resistance of alpha-crystallin-glutathione mixed-disulfide to tryptic digestion. 301 92

The ability of synthetic inhibitors of trypsin-like (TLCK) and chymotrypsin-like (TPCK) proteinases and natural antiproteinase oligopeptides of animal (aprotinin) and microbial (enzistatin) origin to suppress multicycle replication of different alpha viruses (Semliki, Sindbis, Venezuelan equine encephalomyelitis viruses) in cultured cells was studied. Antiviral activity was found to be induced by TPCK and aprotinin (Gordox). These compounds were shown to reduce virus yield 100-fold and to prevent the involvement of cells into infection process. The mechanisms of antiviral activity and chemotherapeutic possibilities of antiproteinase compounds are discussed.
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PMID:[Antiviral activity of proteinase inhibitors in cultured cells infected with alpha-viruses]. 302 88


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