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)

1. Homogenates of rat uteri removed 1 and 2 days post partum were centrifuged at 6000 g. Both pellets and supernatants degraded Azocoll, a general proteinase substrate, at pH 7.5. More than 80% of the total activity was in the pellet fraction. 2. Part of the pellet activity was in a latent form. Trypsin and 4-aminophenylmercuric acetate (a thiol-blocking agent) both activated this latent form, indicating that it is an enzyme--inhibitor complex. An endogenous serine proteinase activated part of the latent enzyme during the assay. 3. The enzyme activity was low before parturition and after involution; it was highest during the first 2 days post partum, when the largest losses of uterine wet weight and matrix macromolecules occur. 4. Up to 70% of the enzyme in the pellets was extracted by heating at 60 degrees C for 4 min in 0.1 M-CaCl2/0.05 M-Tris/HCl, pH 7.5. Approx. 30% of the extracted enzyme was still latent. 5. The extracted enzyme was a metalloproteinase, since it was inhibited completely by 1,10-phenanthroline, but not by inhibitors of thiol or serine proteinases. 6. The enzyme was further purified 15--30-fold by gel chromatography and precipitation with (NH4)2SO4. The apparent molecular weight, estimated by gel filtration, was 24000 for the latent form and 12000 for the active form. The pH optimum was 7--7.5. 7. The enzyme also degraded cartilage proteoglycan. This activity was studied by viscometry and the products were analysed by analytical ultracentrifugation. The major product had a mol.wt. of approx. 100000. The sites of cleavage were in the protein core, since no free oligosaccharides were detected. 8. This neutral metalloproteinase is distinct from uterine collagenase and from a uterine metal-dependent endopeptidase that hydrolyses a heptapeptide related to collagen.
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PMID:The extraction of a neutral metalloproteinase from the involuting rat uterus, and its action on cartilage proteoglycan. 701 17

1. Single smooth muscle cells were prepared from the guinea-pig taenia coli and the porcine coronary artery by treatment with collagenase, in order to measure the 45Ca flux with special reference to the effects of external cations. 2. In excess [K]o solution, cell suspensions prepared from both tissues showed an increased 45Ca uptake within 3-5 min. In Na-free solution, the cells prepared from taenia coli, but not from coronary artery showed an increased 45Ca uptake. The Ca uptake of the cells paralleled with the tension increase recorded from tissues of both species. 3. The efflux of 45Ca into Ca-free EGTA containing solution was markedly increased by [Na]o in cells from the taenia coli, but not in cells from the coronary artery. 4. The [Na]o-activated 45Ca efflux from cells of the taenia coli was slightly larger in Ca-free solution than in the Ca-containing (10)-4) M) solution. Depolarization of membranes produced by excess [K]o did not effect the [Na]o-activated 45Ca efflux. 5. Increase in [Na]i by treatment with K-free solution suppressed the [Na]o-activated 45Ca efflux in the taenia coli. Re-addition of [K]o reactivated the [Na]o-activated 45Ca efflux. This re-activation was blocked by ouabain. 6. The efflux of 45Ca was slightly activated by [Ca]o in cells from the taenia coli. This [ca]o-activated 45Ca efflux was larger in Na-free solution than in Na-containing solution, thus suggesting interactions between [Na]o and [Ca]o on the Ca efflux. 7. In cells from the taenia coli, 45Ca efflux could still be observed in nominally Na-and Ca-free solution. This residual 45Ca efflux made a large contribution to the total 45Ca efflux. 8. When 45Ca uptake was measured in Na-free (Tris) solution, the [Na]o-activated, [Ca]o-activated and residual 45Ca effluxes of cells from the taenia coli were accelerated, non-selectively. 9. These results obtained with cells prepared from the guinea-pig taenia coli are comparable to the Ca2+ efflux mechanism seen in the squid axon. However, maintenance of low concentrations of [Ca]i seems to require not only the above three 45Ca efflux mechanisms, but also Ca sequestering mechanisms in the cell.
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PMID:Effects of external cations on calcium efflux from single cells of the guinea-pig taenia coli and porcine coronary artery. 723 38

One of the more recently discovered collagens, type V (or A-B) collagen, in its native fibillar form mediates human platelet aggregation and the release of serotonin. In agreement with a recent report, it has no detectable effect on human platelets in the soluble or amorphous form. The possibility that the observed results might be due to contaminating interstitial collagens was eliminated by taking advantage of unusual solubility properties of type V collagen. Type V collagen dissolved in 0.1M acetic acid formed native-type fibrils when dialyzed against PBS and amorphous fibrils when dislyzed against 0.05M Tris/0.13M NaCl, pH 7.4, at 4 degrees C. Interstitial collagens remained in solution under both of these conditions. In addition, type V collagen treated with sufficient, purified synovial collagenase to digest all contaminating interstitial collagen retained its platelet-aggregating properties. The purity of type V collagen was confirmed by SDS-PAGE of CNBr digests. These data indicate that the quaternary structure of type V collagen is important in its recognition by platelet membranes.
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PMID:Collagen-platelet interaction. Type V(A-B) collagen induced platelet aggregation. 735 Feb 45

Myocardial cells were isolated after treatment with collagenase (0.05%) and hyaluronidase (0.1%) by discontinuous-gradient centrifugation on 3% Ficoll. Nuclei derived from these myocardial cells were then fractionated on a discontinuous sucrose density gradient with the following steps: (I) 2.0M/2.3M, (II) 2.3M/2.4M, (III) 2.4M/2.5M, (IV) 2.5M/2.6M, and (V) 2.6M/2.85M. The myocardial nuclei were sedimented in the interfaces of gradient fractions (II) and (III). Nuclei from whole ventricles that had been treated with the enzymes before isolation sedimented into five major subsets of nuclei. These findings suggest that nuclei sedimented in the isopycnic gradient at fractions (II) and (III) are most probably derived from myocardial cells. However, this procedure is laborious and lengthy, and the recovery of myocardial-cell nuclei is low. An alternative method was developed to isolate an enriched fraction of myocardial-cell nuclei from whole ventricular tissue without exposing the tissues to enzyme digestion. These ventricular nuclei could be fractionated into five nuclear subsets by using the same discontinuous sucrose density gradient as that described above. The content of DNA, RNA and protein per nucleus for each band was determined. Although the DNA content per nucleus was constant (10pg), that of RNA varied from 1.5 to 4.5pg and that of protein from 16 to 24pg. Nuclei from each band were examined by light-microscopy: large nuclei occurred in the ligher regions whereas smaller nuclei were found in the denser regions of the gradient. From the size distribution pattern of myocardial-cell nuclei compared with that of total ventricular nuclei, it was found that nuclear subsets (II), (III), and (IV) were similar to myocardial nuclei. Electrophoretic analyses of the proteins solubilized in sodium dodecyl sulphate/phenol or Tris/EDTA/2-mercaptoethanol/phenol obtained from each nuclear subset indicate that these fractions are similar, with limited qualitative differences. These findings indicate that isolation of an enriched fraction of myocardial-cell nuclei could be achieved by discontinuous-sucrose-density-gradient centrifugation.
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PMID:Fractionation of rat ventricular nuclei. 739 68

Serum amyloid P component (SAP) is a glycoprotein in human plasma. We recently showed the localization of SAP in human atherosclerotic lesions by immunohistochemical staining. In this study, the presence of SAP in atherosclerotic lesions was confirmed, and the biochemical character of SAP in atherosclerotic intima was investigated and compared with that of native SAP. Atherosclerotic intima was sequentially extracted with 2 mmol/L CaCl2-Tris-buffered saline (TBS), 10 mmol/L EDTA-TBS, 3 mol/L guanidine-TBS, and collagenase digestion. The character of SAP in each extract was studied with double immunodiffusion, electroimmunoassay, crossed immunoelectrophoresis, and Western immunoblotting. The total amount of SAP in atherosclerotic intima was 190 +/- 64 micrograms/g wet tissue with an SAP-albumin ratio of 1:22.7, which is 44 times higher than the relative plasma ratio of 1:1000. This suggests that SAP is specifically localized in atherosclerotic lesions. SAP from the intima was indistinguishable from plasma or purified SAP with respect to immunological character and molecular weight. However, electrophoretic mobility and the binding of SAP to atherosclerotic intima appeared heterogeneous. Of total extractable SAP, about 43% appeared in the CaCl2-TBS fraction, 25% in the EDTA-TBS fraction, and 32% in the collagenase digestion fraction. SAP is one of the two pentraxins in human plasma; the other is C-reactive protein, which has also been reported to locate in atherosclerotic lesions. Our findings suggest a role for SAP in atherogenesis and encourage efforts to determine more precisely the physiological contributions of the pentraxin family to the development of atherosclerosis.
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PMID:Characterization of serum amyloid P component from human aortic atherosclerotic lesions. 774 34

The aim of this study was to detect biologic factors in the structural deterioration of bioprosthetic heart valves. Prostheses were removed from patients after 4-8 years of implantation and submitted to biochemical and morphologic assays. Successive staining of biologic sections revealed colocalization of lipids and glycosaminoglycans underneath calcifications in the disintegrated extracellular matrix. On biochemical assays, the amidolysis of synthetic peptide substrates indicated thrombin, plasmin, and tissue plasminogen activator activities in the nonhemocompatible leaflets; 0.15 mol NaCl, 0.05 mol Tris, and 5 mmol CaCl2 extracts from the prostheses cleaved the peptide substrate for collagenase and lysed gelatin gels. Polyacrylamide gel electrophoresis in sodium dodecyl sulfate disclosed the presence of low molecular mass polypeptides in extracts of the deteriorated prostheses. The detection of plasmin and collagenolytic enzyme(s), and the known broad proteolytic activity of plasmin, may point to the role of activation of the fibrinolytic system in the proteolytic degradation of bioprosthetic valves.
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PMID:Deterioration of bioprosthetic heart valves. 855 4

We evaluated the mechanism of increased serum concentrations of the 7S fragment of the N-terminal domain of type IV collagen (7S collagen) in chronic liver disease. We measured the concentrations of hepatic-free and deposited 7S collagens after extraction with Tris-HCl buffer and bacterial collagenase, then compared them with the serum levels in 8 normal controls and 48 patients with chronic liver disease. The hepatic 7S collagen levels extracted with Tris-HCl buffer and collagenase accounted for 7% and 93%, respectively, of the total 7S collagen levels in normal controls. Both hepatic 7S collagen levels as well as serum levels increased in accordance with the progress of liver disease. Serum levels of 7S collagen showed a closer correlation with the hepatic 7S collagen levels extracted with Tris-HCl buffer (r = .822), compared with those extracted with collagenase (r = .382). On the other hand, the histological degrees of liver fibrosis were highly correlated with the hepatic collagenase-extracted 7S collagen levels (r = .822), compared with serum and the hepatic Tris-HCl buffer-extracted levels (r = .478 and r = .537, respectively). Although there was no difference in serum and hepatic 7S collagen levels between B and C viral patients, the serum and hepatic Tris-HCl buffer-extracted 7S collagen levels were higher in patients with alcoholic cirrhosis than patients with viral cirrhosis. However, the hepatic collagenase-extracted levels were similar in both groups. Gel filtration demonstrated that the serum and hepatic Tris-HCl buffer-extracted 7S collagens were mainly eluted in the macromolecular 7S collagen-reactive fraction in cirrhosis, whereas the hepatic collagenase-extracted 7S collagen was eluted in the authentic 7S collagen-reactive fraction. The results suggest that serum 7S collagen levels are not a particularly reliable measure of hepatic fibrosis but reflect the enhanced metabolism, especially synthesis of type IV collagen in the liver.
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PMID:Relationship between serum and hepatic 7S fragments of type IV collagen in chronic liver disease. 862 Nov 48

The serum protein, alpha 1-proteinase inhibitor (alpha 1-PI), defends the host against serine proteinases, e.g. PMN elastase. Using a rabbit anti-serum against human alpha 1-PI, this protein in GCF was quantified from a standard curve constructed from dot-blot analysis and characterized by Western blot. GCF was collected on filter paper strips from healthy (H), gingivitis (G) and adult periodontitis (AP) patients, then extracted with Tris/NaCl/CaCl2 buffer, pH 7.6. alpha 1-PI concentration increased with G and was highest in AP subjects. H sites only showed intact alpha 1-PI (52 kDa); no degradation fragments (48 kDa) were detected. In G and AP subjects, alpha 1-PI degradation fragments were seen in 17% and 71% of GCF samples, respectively. Both collagenase and alpha 1-PI-degrading activities in GCF increased with severity of inflammation (GCF flow). Moreover, the alpha 1-PI degrading (or serpinolytic) activity was characterized as a matrix metalloproteinase, probably collagenase, based on its in vitro response to a panel of different proteinase inhibitors including doxycycline. We propose: (1) that collagenase promotes periodontal breakdown not only by degrading collagen, but also by depleting alpha 1-PI regulation of elastase and other serine-proteinases, thereby favoring a broader attack on extracellular matrix (ECM) constituents, and (2) based on a recent longitudinal double-blind study using the techniques described above for alpha 1-PI analysis, that low-dose doxycycline administration to humans with adult periodontitis can inhibit this broad cascade of ECM degradation.
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PMID:alpha 1-Proteinase inhibitor in gingival crevicular fluid of humans with adult periodontitis: serpinolytic inhibition by doxycycline. 908 38

Ficolin was initially identified from porcine uterus as a TGF-beta 1 binding protein and is considered to have an overall structure similar to that of the complement protein C1q and the collectins. Recent studies have shown that human ficolin is synthesized mainly by monocytes in peripheral blood and that it could potentially bind to sugar structures on microorganisms. The aim of the present investigations was to isolate ficolin from human plasma by affinity chromatography on immobilized sugars. A human serum protein was identified in the GlcNAc eluate from GlcNAc-Sepharose which migrated as a polypeptide of approx. 40 kDa on SDS-PAGE under reducing conditions and was, after further purification by FPLC on a mono-Q column, shown to have an identical N-terminal sequence, over the first 14 residues, to P35, a plasma protein having similar sequence and domain organisation to ficolin. This protein, named the ficolin-like protein, was shown to be sensitive to collagenase and similar to P35 in that it was also disulphide-linked into an oligomer of approx. 320 kDa. However, unlike P35, its binding to GlcNAc was independent of Ca2+. Gel-filtration studies showed that this ficolin-like protein also had a molecular weight of approx. 320 kDa under non-dissociating conditions. During the course of this study this ficolin-like protein was found to simply bind to CNBr-activated Sepharose which had been inactivated with Tris, and from which it could be eluted with GlcNAc. This ficolin-like protein was also shown to bind to GlcNAc, but not to mannose and maltose. The functional significance of the unusual binding property of this ficolin-like protein is not clear, but it has facilitated the development of a simple method for its purification.
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PMID:Purification and binding properties of a human ficolin-like protein. 920 8

Bioprosthetic heart valves fail as the result of two simultaneous processes: structural deterioration and calcification. Leaflet deterioration and perforation have been correlated with regions of highest stress in the tissue. The failures have long been assumed to be due to simple mechanical fatigue of the collagen fibre architecture; however, we have hypothesized that local stresses-and particularly dynamic stresses-accelerate local proteolysis, leading to tissue failure. This study addresses that hypothesis. Using a novel, custom-built microtensile culture system, strips of bovine pericardium were subjected to static and dynamic loads while being exposed to solutions of microbial collagenase or trypsin (a non-specific proteolytic enzyme). The time to extend to 30% strain (defined here as time to failure) was recorded. After failure, the percentage of collagen solubilized was calculated based on the amount of hydroxyproline present in solution. All data were analyzed by analysis of variance (ANOVA). In collagenase, exposure to static load significantly decreased the time to failure (P < 0.002) due to increased mean rate of collagen solubilization. Importantly, specimens exposed to collagenase and dynamic load failed faster than those exposed to collagenase under the same average static load (P = 0.02). In trypsin, by contrast, static load never led to failure and produced only minimal degradation. Under dynamic load, however, specimens exposed to collagenase, trypsin, and even Tris/CaCl2 buffer solution, all failed. Only samples exposed to Hanks' physiological solution did not fail. Failure of the specimens exposed to trypsin and Tris/CaCl2 suggests that the non-collagenous components and the calcium-dependent proteolytic enzymes present in pericardial tissue may play roles in the pathogenesis of bioprosthetic heart valve degeneration.
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PMID:Mechanical loading of bovine pericardium accelerates enzymatic degradation. 1038 30


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