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:3.4.21.37 (neutrophil elastase)
4,078 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have characterized by bioassay and reversed-phase high-performance liquid chromatography analysis (rp-HPLC) the conversion of human big endothelin-1 (bET-1) to endothelin-1 (ET-1) by the cytosolic fraction from human polymorphonuclear leukocytes (PMNs). Either the general serine protease inhibitor 3,4-dichloroisocoumarin (DCI; 50 microM) or the selective elastase inhibitor ONO-5046 (100 microM) blocked the formation of ET-1 from bET-1. Interestingly, human leukocyte elastase formed some of the same products from bET-1 as the PMN cytosol, but generated negligible amounts of ET-1. However, coincubation of the elastase-derived fragments of bET-1 with the PMN cytosol in the presence of ONO-5046 resulted in a 17-fold increase in the formation of ET-1, indicating that an elastase-derived intermediate of bET-1 was subsequently cleaved by a soluble protease(s) to form mature ET-1. We have identified by electrospray-mass spectrometry (ESMS) analysis this intermediate as bET-1(1-22). Analysis of bET-1 digestion by human leukocyte cathepsin G revealed the formation of a biologically active metabolite chromatographically distinct from ET-1, identified as bET-1(1-31) by ESMS. These findings indicate the presence of complex enzyme systems in human PMNs capable of activating bET-1.
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PMID:Characterization of serine protease-derived metabolites of big endothelin in the cytosolic fraction from human polymorphonuclear leukocytes. 128 76

The metabolism of big endothelin 1 (bET) and endothelin 1 (ET-1) by subcellular fractions from human polymorphonuclear leukocytes (PMNs) was investigated by bioassay and reversed-phase high-performance liquid chromatography. More than 80% of endothelin-converting activity was recovered from the cytosolic fraction, which in addition to ET-1 generated other peptides from bET. The processing of bET to all its metabolites including ET-1 was prevented by the serine protease inhibitor 3,4-dichloroisocoumarin (DCI; 50 microM) or the elastase inhibitor ONO-5046 (100 microM) but not by phenylmethylsulfonyl fluoride (PMSF; 143 microM), another serine protease inhibitor. Paradoxically, human leukocyte elastase, despite generating a bET fragmentation pattern similar to that of PMN cytosol, produced very little ET-1. However, subsequent treatment of the elastase-derived metabolites of bET with PMN cytosol in the presence of ONO-5046 dramatically increased the amount of ET-1 formed. The generation of ET-1 following this intervention was inhibited by DCI. The PMN membrane preparation degraded ET-1 to a major metabolite, similar to that produced from ET-1 by elastase, and several minor products, paralleled by a loss of its smooth muscle contracting activity. The degradation of ET-1 by PMN microsomes was prevented by DCI, PMSF, or ONO-5046. Our results suggest that an elastase-initiated serine protease cascade is responsible for the sequential conversion of bET to ET-1 by the PMN cytosol. Elastase also partly accounts for the ET-metabolizing properties of PMN microsomes.
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PMID:The two-step conversion of big endothelin 1 to endothelin 1 and degradation of endothelin 1 by subcellular fractions from human polymorphonuclear leukocytes. 149 79

Antithrombin III (AT-III) is a serine protease inhibitor (serpin) that can be catalytically inactivated by human neutrophil elastase (HNE) without inhibiting HNE activity. As with catalytic inactivation of most serpins, the cleaved form of the inhibitor is difficult to measure in the presence of active inhibitor. One major difference between the cleaved and intact forms of AT-III is that the cleaved form adopts a more stable conformation. Using sodium dodecyl sulfate (SDS), we were able to devise an enzyme-linked immunosorbent assay (ELISA) capable of detecting cleaved AT-III in the presence of intact AT-III. It seems likely that the SDS alters the intact AT-III so that it is not detected in the ELISA. As little as 5 micrograms/ml HNE-cleaved AT-III could be detected when spiked into human plasma; HNE-cleaved AT-III spiked into human plasma at different levels was recovered as expected. Thrombin-cleaved AT-III was also detected using this ELISA. The generation of cleaved AT-III in human plasma by HNE in the presence of heparin could be monitored as well. The cleaved AT-III ELISA is a novel, yet simple way to measure proteolytically inactivated AT-III in the presence of intact AT-III and should be useful for studying the role of proteolytic inactivation of serpins such as AT-III in vivo.
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PMID:Enzyme-linked immunosorbent assay for proteolytically inactivated antithrombin-III: use of sodium dodecyl sulfate to eliminate signal due to intact antithrombin-III. 151 63

Fragmentation of subendothelial matrix-bound fibronectin by proteases released from stimulated leukocytes has been implicated in lung vascular injury. We studied the degradation of fibronectin bound to denatured collagen by inflammatory polymorphonuclear leukocytes (PMNL). Tissue culture wells coated with denatured collagen (gelatin) were pretreated with 125I rat plasma fibronectin to allow for fibronectin binding prior to the addition of rat inflammatory PMNL. The release of both intact and fragmented fibronectin from the 125I-labelled artificial matrix was quantified following the addition of PMNL stimulated by the phagocytosis of opsonized zymosan as well as leukocyte elastase. Stimulated PMNL released three times more radiolabelled fibronectin from the denatured collagen surface during a 4 h incubation as compared with unstimulated PMNL. This pattern of 125I-fibronectin release could also be elicited by the addition of purified leukocyte elastase alone, in the absence of PMNL. The release of radiolabelled fibronectin by stimulated PMNL was blocked in a dose-dependent manner by the addition of both methoxysuccinyl-alanine-alanine-valine chloromethyl ketone (AAPVCK), a leukocyte elastase specific inhibitor as well as phenylmethylsulfonylfluoride (PMSF), a non-specific serine protease inhibitor. Western blot analysis coupled with autoradiography confirmed the presence of fibronectin fragments in the medium after addition of PMNL or leukocyte elastase. The large molecular weight fragments (60-200 kD) were not labelled, but the smaller molecular weight fragments (less than 45 kD), derived from the artificial matrix, were labelled. Thus, fibronectin complexed with denatured collagen is susceptible to proteolytic degradation by stimulated inflammatory PMNL. Such a process may have a role in the pathogenesis of acute vascular injury following microvascular margination of activated blood leukocytes.
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PMID:Proteolysis of gelatin-bound fibronectin by activated leukocytes: a role for leukocyte elastase. 165 37

The role of proteolytic enzymes in the hCG-induced increase in testicular vasopermeability and neutrophil extravasation was studied using protease inhibitors. An intra-testicular injection of hCG together with incubation medium conditioned by polymorphonuclear leucocytes (PMNs) caused a significant increase in vasopermeability and a coincident extravasation of PMN's from the postcapillary venules in the rat testis. When p-aminobenzamidine, a serine protease inhibitor which inhibits urokinase-type plasminogen activator, was administered together with hCG in the incubation medium, both the permeability increase and PMN extravasation were prevented. Aprotinin, another serine protease inhibitor, and Eglin C, a specific neutrophil elastase and cathepsin G inhibitor were, however, without effect. None of these inhibitors caused any non-specific vascular effects in the testis at the concentrations used. These results support the concept that the hCG-induced increase in vasopermeability in the rat testis is related to extravasation of PMNs and suggest that urokinase-type plasminogen activator is involved in migration of these cells through the postcapillary venular walls.
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PMID:Plasminogen activator is involved in the hCG-induced neutrophil extravasation and vasopermeability increase in the rat testis. 169 41

Effect of serine protease inhibitor Camostat Mesilate (Foipan) on primary glomerulonephritis and it's mechanism were evaluated. Forty-two patients having primary glomerulonephritis (13 cases of IgA nephropathy, 11 cases of membranous nephropathy and others), aged 18 to 81 years were selected for this study. At the start of our study, twenty-one patients had received other drugs (13 cases of dipyridamole and 13 cases of prednisolone). A control period of four weeks was established to confirm that the levels of proteinuria and renal functions were stable. Patients were orally administered with 600 mg of Camostat Mesilate per day for four weeks. Effect of Camostat Mesilate was judged by urinary protein excretion, hematuria, serum total protein, albumin, Ccr, creatinine and BUN. In order to reveal the mechanism of the effect laboratory data such as granulocyte elastase, CH50, C3, C4, fibrinogen, platelate factor 4, beta-thromboglobulin, thromboxane B2 and prostaglandin F1 alpha were evaluated before and after the treatment. Parameters were analyzed by using paired t-test. Mean (+/- SEM) urinary protein excretion reduced from 4.31 +/- 0.91 to 2.80 +/- 0.43 g/day (p less than 0.05), and score of hematuria decreased from 1.8 +/- 0.16 to 1.5 +/- 0.15. A significant decrease in urinary protein excretion was seen in membranous nephropathy and a significant decrease in hematuria was seen in IgA nephropathy. In combination therapy (dipyridamole, prednisolone) urinary protein excretion markedly decreased (p less than 0.05) and in Camostat Mesilate therapy score of hematuria markedly decreased (p less than 0.05). Camostat Mesilate had no effects on renal function assessed by Ccr, creatinine and BUN.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:[Effect of protease inhibitor on primary glomerulonephritis and the mechanism of the effect]. 177 Jun 35

We have shown that platelet-activating factor (PAF) primes neutrophil (PMN) responses and enhances their ability to damage endothelial cells. Furthermore, thrombin-stimulated endothelial cells which produce PAF can augment and prime PMN superoxide production, elastase release and adhesion to endothelium. We wondered whether these marginated neutrophils (PMN) themselves, or their release products, might feedback and further amplify endothelial cell activation. To measure cellular activation, we assessed changes in endothelial cell intracellular calcium [( Ca2+]i) in endothelial monolayers loaded with Fura-2, and PAF production by [3H]acetate incorporation into phospholipid. Resting PMNs induced no change in [Ca2+]i, while N-formyl-L-methionine-L-leucyl-L-phenylalanine stimulated PMN and their lysosomal products caused a 25% increase in endothelial cell calcium. Sonicates of PMN produced a much larger increase in [Ca2+]i than activated PMN; the effect of PMN sonicates could in part be reduced by the serine protease inhibitor, alpha 1 antitrypsin. In contrast, purified neutrophil elastase did not alter endothelial cell [Ca2+]i. Despite hydrogen peroxide's ability to increase [Ca2+]i, catalase failed to inhibit the PMN-induced rise in [Ca2+]i. Since polyanionic heparin inhibited the PMN sonicate rise in calcium, a cationic protein released by PMN was thought to be responsible. The cationic primary granule enzyme, cathepsin G, duplicates the rise in [Ca2+]i seen with PMN sonicates. Furthermore, PAF production increased threefold in response to neutrophil sonicates. Thus, during inflammation, when coagulation and inflammatory cells are activated the endothelium responds by priming PMNs and promoting margination.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Endothelial cell platelet-activating factor primes neutrophil responses: amplification of endothelial activation by neutrophil products. 196 17

Alpha 1-Antitrypsin (alpha 1-AT) is similar to other members of the serine protease inhibitor (serpin) supergene family in that it undergoes structural rearrangement during the formation of a covalently stabilized inhibitory complex with its cognate enzyme, neutrophil elastase. We have recently demonstrated an abundant, high-affinity cell surface receptor on human hepatoma cells and human mononuclear phagocytes which recognizes a conformation-specific domain of the alpha 1-AT-elastase complex as well as of other serpin-enzyme complexes (Perlmutter, D. H., Glover, G. I., Rivetna, M., Schasteen, C. S., and Fallon, R. J. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 3753-3757). Binding to this serpin-enzyme complex (SEC) receptor activates a signal transduction pathway for increased expression of the alpha 1-AT gene and may be responsible for clearance of serpin-enzyme complexes. In this study, we show that there is time-dependent and saturable internalization of alpha 1-AT-elastase and alpha 1-AT-trypsin complexes in human hepatoma HepG2 cells. Internalization is mediated by the SEC receptor as defined by inhibition by synthetic peptides corresponding to residues 359-374 of alpha 1-AT. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of intracellular radioactivity demonstrated that intact 75- and 66-kDa alpha 1-AT-trypsin complexes were internalized. Kinetic analysis of internalization at 37 degrees C showed that a single cohort of 125I-alpha 1-AT-trypsin complexes, prebound to cells at 4 degrees C, disappeared from the cell surface and accumulated intracellularly within 5-15 min at 37 degrees C. The intracellular concentration of radiolabeled complexes then decreased rapidly coincident with appearance of acid-soluble degradation products in the extracellular culture fluid. Intracellular degradation was inhibited by internalization at 18 degrees C or by internalization at 37 degrees C in the presence of weak bases ammonium chloride, primaquine, and chloroquine, indicating that degradation is lysosomal. These results indicate that in addition to its role in signal transduction the SEC receptor participates in internalization and delivery of alpha 1-AT-protease complexes to lysosome for degradation.
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PMID:Endocytosis and degradation of alpha 1-antitrypsin-protease complexes is mediated by the serpin-enzyme complex (SEC) receptor. 221 87

Isocoumarins are potent mechanism-based heterocyclic irreversible inhibitors for a variety of serine proteases. Most serine proteases are inhibited by the general serine protease inhibitor 3,4-dichloroisocoumarin, whereas isocoumarins containing hydrophobic 7-acylamino groups are potent inhibitors for human leukocyte elastase and those containing 7-alkylureidogroups are inhibitors for procine pancreatic elastase. Isocoumarins containing basic side chains that resemble arginine are potent inhibitors for trypsin-like enzymes. A number of 3-alkoxy-4-chloro-7-guanidinoisocoumarins are potent inhibitors of bovine thrombin, human factor Xa, human factor XIa, human factor XIIa, human plasma kallikrein, porcine pancreatic kallikrein, and bovine trypsin. Another cathionic derivative, 4-chloro-3-(2-isothiureidoethoxy) isocoumarin, is less reactive toward many of these enzymes but is an extremely potent inhibitor of human plasma kallikrein. Several guanidinoisocoumarins have been tested as anticoagulants in human plasma and are effective at prolonging the prothrombin time. The mechanism of inhibition by this class of heterocyclic inactivators involves formation of an acyl enzyme by reaction of the active site serine with the isocoumarin carbonyl group. Isocoumarins with 7-amino or 7-guanidino groups will then decompose further to quinone imine methide intermediates, which react further with an active site residue (probably His-57) to form stable inhibited enzyme derivatives. Isocoumarins should be useful in further investigations of the physiological function of serine proteases and may have future therapeutic utility for the treatment of emphysema and coagulation disorders.
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PMID:Mechanism-based isocoumarin inhibitors for serine proteases: use of active site structure and substrate specificity in inhibitor design. 265 46

The major physiological role of the serine protease inhibitor alpha 1-antitrypsin (alpha 1-AT) is to protect elastic fibers in the lung from excessive hydrolysis by neutrophil elastase. Genetic deficiency of alpha 1-AT predisposes individuals toward the development of emphysema. We have cloned and characterized a mutant alpha 1-AT gene from an individual exhibiting a total absence of immunoreactive alpha 1-AT in serum. Nucleotide sequence analysis of this "null" allele has demonstrated a TC dinucleotide deletion within the codon for Leu318 in exon IV. This frame-shift mutation results in the generation of a premature termination codon at residue 334, which is upstream of the active inhibitory site. To determine the biochemical basis of the null phenotype, the mutant and normal genes were transferred into mouse hepatoma cells for expression analysis. Pulse-chase experiments demonstrated that the mutant gene is expressed into a truncated protein of 45 kDa, which is retained within the rough endoplasmic reticulum. The complete lack of secretion of the truncated protein is consistent with the absence of immunoreactive alpha 1-AT in the patient's serum. In addition, a G to A transition was identified in exon II of the mutant gene, changing the codon for Arg101 to His101. Finally, an A to C transversion was identified in exon V changing the codon for Glu376 to Asp376. Since the latter conservative amino acid substitution has previously been identified in the common PiM2 variant, the frame-shift mutation might have occurred on a PiM2 background chromosome. Using the birthplace of this index case, this mutant alpha 1-AT allele has been designated "nullHong Kong."
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PMID:A frameshift mutation results in a truncated alpha 1-antitrypsin that is retained within the rough endoplasmic reticulum. 325 32


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