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)

Monoclonal antibodies against an ovarian tumor cell line, OC-3-VGH, were generated using modified hybridoma technology. Among the seven that were selected for their high specificity and affinity to ovarian cancer cells and low cross-reactivity to most normal human tissues, RP 215 was shown to react specifically with a tumor-associated antigen, COX-1, from certain ovarian/cervical cancer cell lines. By Western blot assay, COX-1 was shown to have a subunit molecular mass of about 60 kDa and exist as an aggregate in the native state. COX-1 could also be detected in the shed medium of certain cultured tumor cells. A solid-phase sandwich enzyme-immunoassay procedure was designed for quantitative determinations of COX-1 in the shed medium or in patients' sera using RP 215 for both well-coating and the signal detection. Highly purified COX-1 was obtained from the shed medium of cultured OC-3-VGH tumor cells mainly by hydroxyapatite and immunoaffinity chromatography with RP 215 as the affinity ligand. At neutral pH, purified COX-1 also exists as an aggregate and is relatively stable at temperatures below 50 degrees C. Its immunoactivity was found to decrease with time in the presence of trypsin. However, the immunoactivity of COX-1 was not affected upon incubation with carbohydrate-digestive enzymes or concanavalin A and only partially inactivated in the presence of NaIO4 or iodoacetamide. Treatments of COX-1 with dithiothreitol and guanidine thiocyanate resulted in a complete loss of activity. Furthermore, rabbit antisera raised against purified COX-1 exhibited similar immunospecificity to that of RP 215. The results of this study suggest that COX-1 is a glycoprotein consisting of a 60 kDa subunit, which is recognized by RP 215 through its peptide determinant. Preliminary retrospective clinical studies were performed to assess the utility of a COX-1 enzyme immunoassay kit for detection and monitoring of patients with ovarian and cervical cancers.
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PMID:Studies of a tumor-associated antigen, COX-1, recognized by a monoclonal antibody. 161 19

Prostaglandin endoperoxide H synthases (PGHS)-1 and -2 are integral membrane proteins of the endoplasmic reticulum (ER). The luminal versus cytoplasmic orientations of several epitopes of PGHS-1 and PGHS-2 were determined by immunocytofluorescent staining of cells following treatment with membrane-selective permeants. With serum-stimulated, murine NIH/3T3 cells expressing PGHS-2, an anti-peptide antibody directed against a domain near the COOH terminus of this isozyme caused staining only after all membranes were permeabilized with 0.2% saponin; no staining occurred with 3T3 cells treated with digitonin to permeabilize only the plasma membrane. Similarly, cos-1 cells expressing ovine PGHS-1 were stained with anti-peptide antibodies directed against (a) the amino terminus (residues 25-35), (b) a domain containing the tryptic cleavage site at Arg277 (residues 272-284), or (c) a region near the carboxyl terminus (residues 583-594) following permeabilization with saponin but not with digitonin or streptolysin O. The results obtained with the antibodies against the Arg277-containing domain of PGHS-1 were surprising because the enzyme is susceptible to tryptic cleavage at Arg277 in microsomal preparations. However, enzymatic and immunochemical analyses of microsomes prepared from ovine vesicular glands and cos-1 cells indicated that these microsomes are not intact. Accordingly, our results indicate that the trypsin cleavage site (Arg277) as well as the NH2 and COOH termini of ovine PGHS-1 are on the luminal side of the ER. The NH2 terminus, the Arg277 domain, and the N-glycosylation sites of ovine PGHS-1 are part of a large soluble, globular structure in crystalline ovine PGHS-1 (Picot, D., Loll, P. J., and Garavito, M. (1994) Nature, 367, 243-249). We conclude that PGHS-1 and, by analogy, the highly homologous PGHS-2 are luminal ER proteins. Assuming that the PGHS-1 and PGHS-2 present in the ER are functional in intact cells, our results indicate that PGH2 synthesis from arachidonate occurs in the lumen of the ER.
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PMID:The orientation of prostaglandin endoperoxide synthases-1 and -2 in the endoplasmic reticulum. 805 Oct 68

N-(Carboxyalkyl)maleimides are rapid as well as time-dependent inhibitors of prostaglandin endoperoxide synthase (PGHS). The corresponding N-alkylmaleimides were only time-dependent inactivators of PGHS, suggesting that the carboxylate is critical for rapid inhibition. Several N-substituted maleimide analogs containing structural features similar to those of the nonsteroidal anti-inflammatory drug aspirin were synthesized and evaluated as inhibitors of PGHS. Most of the aspirin-like maleimides inactivated the cyclooxygenase activity of purified ovine PGHS-1 in a time- and concentration-dependent manner similar to that of aspirin. The peroxidase activity of PGHS was also inactivated by the maleimide analogs. The cyclooxygenase activity of the inducible isozyme, i.e., PGHS-2, was also inhibited by these compounds. The corresponding succinimide analog of N-5-maleimido-2-acetoxy-1-benzoic acid did not inhibit either enzyme activity, suggesting that inactivation was due to covalent modification of the protein. The mechanism of inhibition of PGHS-1 by N-(carboxyheptyl)maleimide was investigated. Incubation of apoPGHS-1 with 2 equiv of N-(carboxyheptyl)[3,4-14C]maleimide led to the incorporation of radioactivity in the protein, but no adduct was detected by reversed-phase HPLC, suggesting that it was unstable to the chromatographic conditions. Furthermore, hematin-reconstituted PGHS-1, which was rapidly inhibited by N-(carboxyheptyl)maleimide, displayed spontaneous regeneration of about 50% of the cyclooxygenase and peroxidase activities, suggesting that the adduct responsible for the inhibition breaks down to regenerate active enzyme. ApoPGHS-1, inhibited by N-(carboxyheptyl)maleimide, did not display regeneration of enzyme activity, but addition of hematin to the inhibited apoenzyme led to spontaneous recovery of about 50% of cyclooxygenase activity. These results suggest that addition of heme leads to a conformational change in the protein which increases the susceptibility of the adduct toward hydrolytic cleavage. ApoPGHS-1, pretreated with N-(carboxyheptyl)maleimide, was resistant to trypsin cleavage, suggesting that the carboxylate functionality of the maleimide binds in the cyclooxygenase channel. A model for the interaction of N-(carboxyheptyl)maleimide in the cyclooxygenase active site is proposed.
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PMID:Design, synthesis, and biochemical evaluation of N-substituted maleimides as inhibitors of prostaglandin endoperoxide synthases. 864 9

Many nonsteroidal antiinflammatory agents (NSAIDs) bind to prostaglandin endoperoxide synthase (PGHS) and induce a conformational change in the PGHS apoprotein that renders it resistant to cleavage by trypsin at Arg277. In the present study, the trypsin protection assay was modified to permit detection of conformational changes at times as short as 5 s after the addition of inhibitor. The kinetics of the induction and reversal of trypsin resistance in apoPGHS-1 by a series of NSAIDs and isozyme-specific PGHS-1 and PGHS-2 inhibitors were determined. All compounds induced resistance to trypsin cleavage at a rapid rate. The conformational change induced by competitive inhibitors was reversed on prolonged incubation with trypsin (approximately 5 min). In contrast, the resistance induced by irreversible inhibitors was not lost during a 5 min incubation with trypsin. All of the selective PGHS-2 inhibitors protected against tryptic cleavage of apoPGHS-1 but did not inhibit the protein's cyclooxygenase activity. The results suggest that induction of trypsin resistance is a reflection of the initial association of reversible as well as irreversible inhibitors with the apoprotein.
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PMID:Kinetics of the interaction of nonsteroidal antiinflammatory drugs with prostaglandin endoperoxide synthase-1 studied by limited proteolysis. 870 11

Prostaglandin H synthase (PGHS) catalyzes a key step in the biosynthesis of a variety of bioactive lipid mediators. The two known isoforms (PGHS-1 and -2) share about 60% amino acid identity, but exhibit distinct interactions with substrates, activators, and inhibitors. Ovine PGHS-1 has previously been shown to have a distinctive protease-sensitive site near Arg277; cleavage by trypsin, chymotrypsin, or proteinase K produces fragments of 33 and 38 kDa and loss of activity. The ovine PGHS-1 crystal structure shows Arg277 located in an exposed loop structure; homology modeling predicts similar loop structures for both human isoforms (hPGHS-1 and -2). We have used limited proteolytic digestion of recombinant hPGHS-1 and hPGHS-2 to probe their structures. Incubation of hPGHS-1 with either trypsin or proteinase K produced 33- and 38-kDa fragments and loss of activity. In contrast, incubation of hPGHS-2 with the same proteases led to cleavage of only a 2- to 3-kDa fragment, with no decrease in activity. Immunoblotting with site-specific antibodies demonstrated that the cleaved fragment originated from the hPGHS-2 C-terminus. Similar immunoblotting experiments indicated that trypsin did not attack the ovine PGHS-1 C-terminus. Mutagenesis was used to replace Pro263 of hPGHS-2 (corresponds to Arg277 of ovine PGHS-1) with arginine, inserting a potential trypsin site. Incubation of this P263R hPGHS-2 mutant with either trypsin or proteinase K resulted in cleavage near the C-terminus and retention of activity, just as with wild-type hPGHS-2. A peptide containing residues 259-268 of the P263R mutant was cleaved by trypsin at the same rate as a peptide corresponding to hPGHS-1 residues 272-281, demonstrating that the sequence differences were not responsible for the lack of tryptic cleavage at residue 263 in the hPGHS-2 mutant. Preincubation of hPGHS-2 with graded levels of guanidinium HCl before incubation with proteinase K did not produce large proteolytic fragments, indicating that the hPGHS-2 loop region was not selectively unfolding. The results point to two regions of significant structural difference between PGHS-1 and -2: the Arg277 loop, which is protease-sensitive in PGHS-1 but protease-resistant in PGHS-2, and the C-terminus, which is protease-sensitive in PGHS-2 but not in PGHS-1.
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PMID:Comparison of prostaglandin H synthase isoform structures using limited proteolytic digestion. 924 92

We have evaluated the prostaglandin (PG) production and PG biosynthetic gene expression in a choriodecidual dispersed cell culture system. Cells dispersed from human choriodecidual membranes by dispase and trypsin digestion were evaluated after 1,3,5 and 7 days of culture for basal and tumour necrosis factor alpha (F-alpha) stimulated PGE2 production. The highest rates of production (P < 0.05) were obtained with cells treated after 3 days of culture, (3.7 +/- 1) x 10(2) pg PGE2 per 16 h per microg total cellular protein (mean +/- SEM), which was 3.9 times basal rate after 3 days culture. In choriodecidual cells treated after 3 days in culture, expression of prostaglandin endoperoxide H synthase-2 (PGHS-2) mRNAwas similarly responsive toTNF-alpha (3.9 times basal within 3 h of 30 ng/ml TNF-alpha) while there was little effect on PGHS-1 or cytosolic phospholipase A2 expression. Hence, the dispersed choriodecidual cell culture system described retainsTNF-alpha responsive PG biosynthetic capacity which is at least in part upregulated via increased expression of PGHS-2 mRNA.
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PMID:Regulation of prostaglandin biosynthesis in dispersed choriodecidual cells in culture. 1199 16

The underlying respiratory disease is activated by unknown mechanism and results in an intense infiltration of mast cells and eosinophils into the entire respiratory mucosa. These cells synthesize leukotrienes (LTs) at a very high rate and mast cells also release histamine and tryptase and synthesize PGD(2) a vasodilator and bronchoconstrictor. Furthermore, AERD patients under synthesize from arachidonic acid (AA) a peculiar product called lipoxins, which opposes inflammation generated by leukotrienes. Finally, cysLT1 receptors are over expressed and highly responsive to LTE(4), further augmenting the underlying inflammatory disease. This inflammatory condition is partly inhibited by synthesis of PGE(2) through COX-1. PGE(2) partially inhibits 5-lipogygenase conversion of AA to LTA(4) and blocks release of histamine and tryptase from mast cells. When COX-l is inhibited by ASA or NSAIDs, PGE(2) synthesis stops and an enormous release of histamine and synthesis of LTs occurs. The upper respiratory reaction is mediated by both histamine and LTs but the bronchospastic reaction is mediated by LTs. The systemic effects of flush, gastric pain and hives are mediated by histamine. Aspirin desensitization can not be explained by disappearance of LT synthesis since urine LTE(4) levels are still elevated at acute ASA desensitization. However, mast cell products such as histamine, tryptase and PGD(2) are no longer released or synthesized at acute desensitization. It is more likely that a diminution in number or function of cysLT receptors accounts for the diminished inflammatory response found in ASA desensitization.
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PMID:Pathogenesis of aspirin-exacerbated respiratory disease. 1266 97

We characterized the tracheal and bronchial relaxation caused by proteinase-activated receptor-2 (PAR-2) activation in ddY mice and/or in wild-type and PAR-2-knockout mice of C57BL/6 background. Ser-Leu-Ile-Gly-Arg-Leu-amide (SLIGRL-NH(2)) and Thr-Phe-Leu-Leu-Arg-amide, PAR-2- and PAR-1-activating peptides, respectively, caused relaxation in the isolated ddY mouse trachea and main bronchus. The relaxation was abolished by specific inhibitors of cyclooxygenase (COX)-1, COX-2, mitogen-activated protein kinase kinase (MEK), and p38 MAP kinase. The MEK and p38 MAP kinase inhibitors did not affect prostaglandin E(2)-induced relaxation. Inhibitors of cytosolic Ca(2+)-dependent phospholipase A(2) (PLA), Ca(2+)-independent PLA(2), diacylglycerol lipase, tyrosine kinase, and protein kinase C exhibited no or only minor inhibitory effects on the PAR-mediated relaxation. Trypsin, a PAR-2 activator, and 2-furoyl-Leu-Ile-Gly-Arg-Leu-amide, a potent PAR-2-activating peptide, in addition to SLIGRL-NH(2), caused airway relaxation in wild-type C57BL/6 mice, as in ddY mice. In PAR-2-knockout mice, the peptide effects were absent and the potency of trypsin decreased. Desensitization of PAR-2 and/or PAR-1 greatly suppressed the relaxant effect of trypsin. The bronchial and tracheal tissues displayed distinct sensitivities toward trypsin and the PAR-2-activating peptides. Our data indicate an involvement of both COX-1 and COX-2, and the MEK-extracellular signal-regulated kinase and p38 MAP kinase signaling pathways in the PAR-2- and PAR-1-triggered relaxation of mouse airway tissue, and substantiate a role for PAR-2 in regulating both the trachea and bronchial responsiveness in the mouse lung.
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PMID:Proteinase-activated receptor-2-mediated relaxation in mouse tracheal and bronchial smooth muscle: signal transduction mechanisms and distinct agonist sensitivity. 1519 93

The non-specific background reaction produced in avidin-biotin-based immunohistochemistry, particularly after harsh antigen retrieval procedures, has promoted the use of non-avidin-biotin systems, yet there are few reports comparing the performance of non-avidin-biotin, polymer-based methods. In this study we compare two of these methods, ENVISION+trade mark and ImmPRESS, in animal tissues. We examined the immunoreactivity of 18 antigens in formalin-fixed, paraffin-embedded tissues. Antigens were located in the cytoplasmic membrane (CD11d, CD18 and CD79a), cytoplasm (calretinin, COX-1, COX-2, Glut-1, HepPar 1, KIT, Melan A, tryptase and uroplakin III) or nucleus (MUM-1, PGP 9.5 and thyroid transcription factor 1). We also evaluated three infectious agents (Aspergillus, calicivirus and West Nile virus). The staining with ENVISION+ or ImmPRESS was performed simultaneously for each antigen. The intensity of the reaction and background staining were scored. ImmPRESS yielded similar or higher reaction intensity than ENVISION+trade mark in 16/18 antigens. ImmPRESS produced abundant background with the other two antigens (calretinin and COX-2), which hindered interpretation of the specific reaction. The cost of ImmPRESS was 25% lower than for ENVISION+trade mark. Based on these results, ImmPRESS is a good polymer-based detection system for routine immunohistochemistry.
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PMID:Comparison of two polymer-based immunohistochemical detection systems: ENVISION+ and ImmPRESS. 1720 59

Human small airway epithelial cells (HSAEC) form the boundary between the external environmental allergens and the internal lung milieu. Mast cells are present in human lung tissue interspersed within the pulmonary epithelium and can secrete a host of pre- and newly formed mediators from their granules, which may propagate small airway inflammation. In this study, tryptase stimulation of HSAEC increased membrane-associated, calcium-independent phospholipase A(2)gamma (iPLA(2)gamma) activity, resulting in increased arachidonic acid and PGE(2) release. These responses were inhibited by pretreating HSAEC with the iPLA(2)-selective inhibitor bromoenol lactone. The tryptase-stimulated PGE(2) production was inhibited by treating HSAEC with the cyclooxygenase (COX)-1-selective inhibitor SC-560 and the nonselective COX inhibitor aspirin but not by the COX-2-selective inhibitor CAY10404, indicating that the early release of arachidonic acid is metabolized by constitutive COX-1 to form PGE(2) in tryptase-stimulated HSAEC. Additionally, platelet-activating factor production and neutrophil adherence to tryptase-stimulated HSAEC was also increased. This complex response can set up a cascade of inflammatory mediator production in small airways. We speculate that selective inhibition of iPLA(2)gamma-mediated phospholipid hydrolysis may prove beneficial in inflammatory airway diseases.
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PMID:Tryptase activates calcium-independent phospholipase A2 and releases PGE2 in airway epithelial cells. 1879 Sep 94


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