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:3.4.24.3 (collagenase)
18,340 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Despite advances in human islet isolations, there remain inconsistencies in human islet yield and viability after collagenase digestion. It has been suggested that trypsin may contribute to the proteolysis of collagenase and the destruction of islet cells, or possibly exert indirect effects on the pancreas by activating other endogenous serine proenzymes. This study evaluated the effects of serine proteases on collagenase activity and profiled the kinetics of serine protease activity throughout human islet isolations with and without addition of Pefabloc, a serine protease inhibitor. Cadaveric pancreases were perfused in the presence (n = 12) and absence of Pefabloc (0.4 mmole; n = 8). Samples were collected before and throughout the digestion process and were assayed for trypsin, chymotrypsin, and elastase activity. A study of the enzyme kinetics of serine proteases throughout human islet isolations showed an increase in activity levels throughout the digestion period. There was a significant difference in the chymotrypsin (1342 +/- 503 and 384 +/- 71 units) and elastase (7.94 +/- 1.1 and 2.761 +/- 0.69 units) levels between the control and Pefabloc-supplemented isolations, respectively. There was no significance difference noted among the trypsin (88 +/- 27 and 54 +/- 18 units) levels between the control and Pefabloc-supplemented isolations, respectively. This demonstrates that serine proteases are effectively inhibited by Pefabloc during the islet isolation process. These data show that the presence of serine proteases may likely damage the islets upon prolonged digestion of the pancreatic tissue.
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PMID:An evaluation of the activation of endogenous pancreatic enzymes during human islet isolations. 1461 84

Inflammatory foci are rich in proteases released by neutrophils (serine proteases) and macrophages (metalloproteases). These enzymes can degrade extracellular matrix proteins and cell membrane bound proteins thus contributing to the development and progression of inflammatory reaction. In this study we have investigated the influence of collagenase (metalloprotease) and trypsin (serine protease) on murine resident and oil-induced peritoneal macrophages (Mf). Short in vitro treatment of Mf, not affecting cell viability, significantly reduced the release of reactive oxygen intermediates (ROIs) and at the same time triggered the increase of IL-6 production and to lesser extent of TNF-alpha production. Both these effects were dependent on enzyme concentration used and were particularly well pronounced in resident macrophages. In addition both enzymes cleaved a number of cell-membrane molecules, including CD23, CD14, CD95L, and Mac-3. We hypothesize that the enzymatic digestion of certain Mf surface receptor proteins in inflammatory foci may be responsible for modification of cell behaviour either by preventing the generation of specific signal or alternatively by delivering a mock substitute signal to the cell interior. In effect inhibition of ROIs production limits their destructive effects and the increase in the secretion of IL-6 stimulates the synthesis of acute phase proteins and triggers other anti-inflammatory mechanisms thus directing Mf present in inflammatory foci into regulatory pathway rather than allowing them to perform solely the effector function.
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PMID:Modulation of macrophage activity by proteolytic enzymes. Differential regulation of IL-6 and reactive oxygen intermediates (ROIs) synthesis as a possible homeostatic mechanism in the control of inflammation. 1476 Sep 41

Despite advances in human islet isolation, islet yield remains inconsistent and unreliable. In recent studies, it has been suggested that serine proteases, in particular trypsin, have been shown to have a damaging effect on islet yield. This study evaluated enzyme activity levels throughout 42 human islet isolation procedures. Trypsin, chymotrypsin, and elastase activity was determined spectrophotometrically using suitable chromophoric substrates. The results of the islet isolations were rated as successful (n = 19) or unsuccessful (n = 23) based on the islet yield and functionality. The enzyme activity profiles of the isolations were compared. No significant differences in donor-related variables were found in this study. However, in the successful isolations, a significantly greater amount (85.6 +/- 1.9%; p = 0.0017) of the pancreas was digested in a significantly shorter digestion time (19.7 +/- 0.6 min; p = 0.0054) compared with 74.8 +/- 2.5% of digested tissue in 22.6 +/- 0.7 min in the poor isolations. This study showed no significant effect of serine protease levels on the outcome of islet isolations, regardless of enzyme inhibitor supplementation. These data suggest that serine protease activity does not sufficiently affect islet yield. However, the data show that the most successful human islet isolations are achieved when the maximum amount of tissue is digested in the shortest amount of time. This suggests that further understanding of the isolation process should focus on the role of the collagenase digestion solution in the dissociation of the endocrine-exocrine tissue connection.
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PMID:Endogenous pancreatic enzyme activity levels show no significant effect on human islet isolation yield. 1512 61

To explore possible mechanisms responsible for the absence of cell re-colonization of mural thrombi in aneurysms, we analyzed the release and storage of leukocyte proteases in the most luminal layer versus intermediate and abluminal layers of 10 mural thrombi of human abdominal aortic aneurysms. The luminal layer contained many polymorphonuclear leukocytes (PMNs), which released pro-matrix metalloproteinase (MMP)-9 and MMP-8. Leukocyte elastase was also stored and released by the luminal layer (immunohistochemistry, activity on synthetic substrates, and casein zymography). Acid buffer allowed extraction of leukocyte elastase from the luminal layer, which was inhibited by elastase inhibitors. Casein zymography of luminal extracts and conditioned medium from formyl-methionyl-leucyl-phenylalanine (fMLP)-stimulated PMNs exhibited a similar lysis pattern, corresponding to elastase activity. Smooth muscle cell (SMC) seeding resulted in colonization of the intermediate thrombus layer ex vivo but not of the luminal layer. Extracts of the luminal layer induced loss of anchorage of both cultured human smooth muscle cells and stromal cells of bone marrow origin (anoikis). This anoikis was prevented by preincubation of the extracts with serine protease inhibitors. Moreover, adhesion of human SMCs and stromal bone marrow cells on fibrin gels was strongly inhibited when the gel was preincubated with pure elastase, medium of fMLP-stimulated PMNs, or extracts of luminal layers of mural thrombi. This loss of cell anchorage was prevented by the preincubation of the medium or extracts with alpha(1)-antitrypsin, but not when alpha(1)-antitrypsin was added after binding of elastase to the fibrin gel. In conclusion, elastase released by PMNs trapped within the mural thrombus impairs the spontaneous anchorage of mesenchymal cells to a fibrin matrix. This phenomenon could be one mechanism by which cellular healing of the mural thrombus in aneurysms is prevented.
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PMID:Role of leukocyte elastase in preventing cellular re-colonization of the mural thrombus. 1516 42

Extracellular enzymes, including serine protease, chitinase and collagenase, corresponding to the main chemical constituents of the nematode cuticle and eggshell, have been reported to be involved in the infectious process as virulence factors. This review will focus on the categories, characterization, purification, cloning and potential function of these virulence enzymes and will attempt to provide new insights into the mechanisms of fungal pathogenesis in nematodes.
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PMID:Extracellular enzymes serving as virulence factors in nematophagous fungi involved in infection of the host. 1556 74

Previous work has shown that endothelial cell (EC)-derived matrix metalloproteinases (MMPs) regulate regression of capillary tubes in vitro in a plasmin- and MMP-1 dependent manner. Here we report that a number of serine proteases can activate MMP-1 and cause capillary tube regression; namely plasma kallikrein, trypsin, neutrophil elastase, cathepsin G, tryptase and chymase. Plasma prekallikrein failed to induce regression without coactivators such as high molecular weight kininogen (HMWK) or coagulation Factor XII. The addition of trypsin, the neutrophil serine proteases (neutrophil elastase and cathepsin G) and the mast cell serine proteases (tryptase and chymase) each caused MMP-1 activation and collagen type I proteolysis, capillary tubular network collapse, regression and EC apoptosis. Capillary tube collapse is accompanied by collagen gel contraction, which is strongly related to the wound contraction that occurs during regression of granulation tissue in vivo. We also report that proMMP-10 protein expression is markedly induced in ECs undergoing capillary tube morphogenesis. Addition of each of the serine proteases described above led to activation of proMMP-10, which also correlated with MMP-1 activation and capillary tube regression. Treatment of ECs with MMP-1 or MMP-10 siRNA markedly delayed capillary tube regression, whereas gelatinase A (MMP-2), gelatinase B (MMP-9) and stromelysin-1 (MMP-3) siRNA-treated cells behaved in a similar manner to controls and regressed normally. Increased expression of MMP-1 or MMP-10 in ECs using recombinant adenoviral delivery markedly accelerated serine protease-induced capillary tube regression. ECs expressing increased levels of MMP-10 activated MMP-1 to a greater degree than control ECs. Thus, MMP-10-induced activation of MMP-1 correlated with tube regression and gel contraction. In summary, our work demonstrates that MMP-1 zymogen activation is mediated by multiple serine proteases and MMP-10, and that these events are central to EC-mediated collagen degradation and capillary tube regression in 3D collagen matrices.
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PMID:MMP-1 activation by serine proteases and MMP-10 induces human capillary tubular network collapse and regression in 3D collagen matrices. 1587 Jan 7

A thermophilic actinomycete strain Thermoactinomyces sp. 21E producing a highly thermostable serine collagenase was isolated from Bulgarian soil. The collagenase, produced extracellular by Thermoactinomyces sp. 21E, was purified to homogeneity by heat treatment, ultrafiltration, saturation with ammonium sulfate and gel filtration chromatography with a 101-fold increase in specific activity and 58% recovery. The collagenase has a relative molecular mass of 50000 by SDS-PAGE. The optimum temperature for the enzyme activity was 60-65 degrees C in the absence of Ca(2+) and 70-75 degrees C in the presence of Ca(2+). About 40% of the original activity remaining after incubation at 85 degrees C for 30 min in the presence of Ca(2+). The optimum pH for the enzyme activity was 9.0-9.5 and the enzyme was stable for 1h at 70 degrees C in the pH range from 7.5 to 12.5. The collagenase was strongly inhibited by active-site inhibitors of serine protease PMSF and DFP, which indicated that the enzyme is serine protease. The enzyme activity was completely inhibited by Hg(2+), Cu(2+) and Fe(2+). However, Ca(2+ )strongly activated the collagenase activity. The collagenase from Thermoactinomyces sp. 21E showed high activity toward type I collagen, acid-soluble collagen, gelatin and Pz-PLGPR. However, elastin for collagenase was inert as substrate. The properties of the collagenase from strain 21E suggest that this enzyme is a new collagenolytic protease that differs from the collagenases and serine proteases reported so far.
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PMID:Novel thermostable serine collagenase from Thermoactinomyces sp. 21E: purification and some properties. 1684 31

Fibroblast activation protein (FAP), as described so far, is a type II cell surface serine protease expressed by fibroblastic cells in areas of active tissue remodelling such as tumour stroma or healing wounds. We investigated the expression of FAP by fibroblast-like synoviocytes (FLSs) and compared the synovial expression pattern in rheumatoid arthritis (RA) and osteoarthritis (OA) patients. Synovial tissue from diseased joints of 20 patients, 10 patients with refractory RA and 10 patients with end-stage OA, was collected during routine surgery. As a result, FLSs from intensively inflamed synovial tissues of refractory RA expressed FAP at high density. Moreover, FAP expression was co-localised with matrix metalloproteinases (MMP-1 and MMP-13) and CD44 splice variants v3 and v7/8 known to play a major role in the concert of extracellular matrix degradation. The pattern of signals appeared to constitute a characteristic feature of FLSs involved in rheumatoid arthritic joint-destructive processes. These FAP-expressing FLSs with a phenotype of smooth muscle actin-positive myofibroblasts were located in the lining layer of the synovium and differ distinctly from Thy-1-expressing and non-proliferating fibroblasts of the articular matrix. The intensity of FAP-specific staining in synovial tissue from patients with RA was found to be different when compared with end-stage OA. Because expression of FAP by RA FLSs has not been described before, the findings of this study highlight a novel element in cartilage and bone destruction of arthritic joints. Moreover, the specific expression pattern qualifies FAP as a therapeutic target for inhibiting the destructive potential of fibroblast-like synovial cells.
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PMID:Fibroblast activation protein is expressed by rheumatoid myofibroblast-like synoviocytes. 1710 46

Lc-WT, the wild-type light chain of antibody, and Lc-Triad, its double mutant with E1D and T27aS designing for the construction of catalytic triad within Asp1, Ser27a, and original His93 residues, were displayed on the cell surface of the protease-deficient yeast strain BJ2168. When each cell suspension was reacted with BODIPY FL casein and seven kinds of peptide-MCA substrates, respectively, a remarkable difference in hydrolytic activities toward Suc-GPLGP-MCA (succinyl-Gly-Pro-Leu-Gly-Pro-MCA), a substrate toward collagenase-like peptidase, was observed between the constructs: Lc-Triad-displaying cells showed higher catalytic activity than Lc-WT-displaying cells. The difference disappeared in the presence of the serine protease inhibitor diisopropylfluorophosphate, suggesting that the three amino acid residues, Ser27a, His93, and Asp1, functioned as a catalytic triad responsible for the proteolytic activity in a similar way to the anti-vasoactive intestinal peptide (VIP) antibody light chain. A serine protease-like catalytic triad (Ser, His, and Asp) is considered to be directly involved in the catalytic mechanism of the anti-VIP antibody light chain, which moderately catalyzes the hydrolysis of VIP. These results suggest the possibility of new approach for the creation of tailor-made proteases beyond limitations of the traditional immunization approach.
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PMID:Design of a serine protease-like catalytic triad on an antibody light chain displayed on the yeast cell surface. 1789 65

Pefabloc and Trasylol are serine protease inhibitors that have been used during islet isolation to inhibit endogenous proteases (trypsin, chymotrypsin, and elastase) and improve islet yields. Using in vitro studies, we evaluated the effects of Pefabloc and Trasylol on the activities of these proteases and the effect of Pefabloc on islet function. In addition, it has been reported that Protector Solution (PS) enhances the efficiency of Pefabloc. Thus, we evaluated the efficacy of Pefabloc in the presence or absence of PS. EnzCheck protease assay was used to measure the activities of trypsin, chymotrypsin, elastase, liberase, and thermolysin in the presence or absence of 0.4 mmol/L Pefabloc (with or without PS) or 0.43 micromol/L Trasylol. We also tested switch samples containing the highest concentration of enzymes. Pefabloc significantly inhibited trypsin, chymotrypsin, elastase, and switch, but not liberase or thermolysin. Trasylol significantly inhibited all enzymes except for elastase and switch sample. Unexpectedly, the potency of Pefabloc was abrogated when diluted first in PS. Insulin release was diminished when islets were incubated or perifused with Pefabloc. In conclusion, Pefabloc when added appropriately significantly blocked in vitro protease activity. Unfortunately, Pefabloc also decreased islet insulin secretion, making it unsuitable for islet isolation. Trasylol cannot be used with collagenase because it impaired both liberase and thermolysin.
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PMID:Endogenous pancreatic protease activity and methods for impeding their function. 1837 65


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