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)

An inhibitor of neovascularization from the conditioned media of scapular chondrocytes established and maintained in serum-free culture has been isolated and characterized. To determine whether this chondrocyte-derived inhibitor (ChDI) was capable of inhibiting neovascularization in vivo, this protein was assayed in the chick chorioallantoic membrane assay. ChDI was a potent inhibitor of angiogenesis in vivo (4 micrograms = 87% avascular zones). This inhibitor is also an inhibitor of fibroblast growth factor-stimulated capillary endothelial cell (EC) proliferation and migration, as well as being an inhibitor of mammalian collagenase. ChDI significantly suppressed capillary EC proliferation in a dose-dependent, reversible manner with an IC50 (the inhibitory concentration at which 50% inhibition is achieved) of 2.025 micrograms/ml. Inhibition by ChDI of growth factor-stimulated capillary EC migration was also observed using a modified Boyden chamber assay (IC50 = 255 ng/ml). SDS-PAGE analysis followed by silver staining of ChDI purified to apparent homogeneity revealed a single band having an M(r) of 35,550. Gel elution experiments demonstrated that only protein eluting at this molecular weight was anti-angiogenic. These studies are the first demonstration that chondrocytes in culture can produce a highly enriched, potent inhibitor of neovascularization which also inhibits collagenase.
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PMID:Isolation and characterization of an inhibitor of neovascularization from scapular chondrocytes. 138 32

Some patients with hereditary nephritis (HN) who have received a renal transplant have been shown to form antibody with specificity for the NC1 domain of collagen type IV, a major constituent of glomerular basement membranes (GBM). We attempted to duplicate this phenomenon in a family of dogs with X-linked HN, a model for human X-linked HN, by immunizing affected male dogs with normal dog NC1 domain. A collagenase digest was prepared from normal dog GBM, the NC1 domain was separated into dimer (approximately 50 kDa) and monomer (24 kDa and 26 kDa) components by SDS-PAGE, and injected into two affected male dogs. Antisera obtained from both dogs contained antibody which reacted with the NC1 domain of dog and human GBM by a plate-binding radioimmunoassay, bound to the dimer and 26 kDa monomer bands by Western blotting, and staining dog and human GBM by immunofluorescence (IF). The affected male dog antiserum reacted equally by radioimmunoassay with the NC1 domain isolated from GBM of unaffected, affected male, and carrier female dogs in the family with X-linked HN, and bound by Western blotting to dimers and the 26 kDa monomer band of the NC1 domain of GBM in each group of dogs. However, the affected male dog antiserum differentiated these dogs by IF; it produced global staining of GBM of unaffected dogs, failed to stain GBM of affected male dogs, and produced segmental staining of GBM of carrier female dogs. Absorption of the affected male dog antiserum with normal dog NC1 domain eliminated the staining of dog GBM by IF, whereas staining persisted after absorption with affected male dog NC1 domain. The abnormal staining patterns of GBM seen by IF in the affected male and carrier female dogs and the results of the absorption studies imply an abnormality of one or more determinants in the 26 kDa monomer band of the NC1 domain of their GBM. Amino acid sequencing of this band identified the alpha 1(IV) chain of collagen type IV, a finding that has implications for the pathogenesis of canine X-linked HN. Absent and segmental staining respectively were also seen by IF in GBM of a male and female patient with HN, using the affected male dog antiserum. Thus, the results obtained in affected male and carrier female dogs with X-linked HN may also be relevant to patients with this disease.
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PMID:Production of anti-NC1 antibody by affected male dogs with X-linked hereditary nephritis: a probe for assessing the NC1 domain of collagen type IV in dogs and humans with hereditary nephritis. 146 51

Mature (average patient age = 29.5 yr, closed apical foramen) and immature (average patient age = 17.5 yr, open apical foramen) root shards were placed in dialysis tubing and demineralized to completion using either 10% disodium EDTA plus protease inhibitors or 0.6 N HCl. The demineralized shards were re-extracted (five times) with 0.05 M tris-HCl, 1.0 M NaCl and then collagenase digested. No major differences were observed in chromatograms of extracts, re-extracts or collagenase digests from root shards demineralized in either way. In contrast, chromatograms of immature and mature roots showed qualitative differences. Chromatograms of mature roots demineralized in either way showed broader protein peaks and less organic phosphorus than those from immature tooth roots. A distinct band amid degraded phosphoprotein (150 K) was found in SDS-PAGE gels (7.5%) from EDTA-extracted immature tooth roots but not from mature tooth roots. Electroelution of this band revealed a typical phosphoprotein amino-acid profile containing increased aspartic acid and serine residues. Comparison of the total phosphoprotein and amino acid composition of extracts, re-extracts and collagenase digests revealed that phosphoprotein, serine and to a lesser extent aspartic acid were recovered in greater quantities from immature roots than mature tooth roots. These data suggest that the degree of maturation is crucial to the isolation of an intact phosphoprotein and provides additional evidence that human dentine phosphoprotein undergoes amino acid compositional changes during maturation.
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PMID:Comparison of phosphoprotein isolated from mature and immature human tooth roots. 147 54

Procollagenase M(r) 85,000 (SDS-PAGE) was purified from buffy coat to homogeneity and represents a stable single polypeptide chain forming the entire proenzyme. The procollagenase can be activated by various proteinases, e.g. trypsin, chymotrypsin, cathepsin G, kallikrein and stromelysin and by different mercurial compounds. Proteolytic conversion of the latent enzyme to the active form by chymotrypsin is accompanied by a molecular weight reduction to an apparent M(r) 64,000. This active enzyme lacks the first 79 N-terminal residues. Activation by trypsin leads to a latent intermediate of apparent M(r) 70,000, lacking 48 N-terminal residues. The active enzyme is therefore generated upon prolonged incubation with trypsin by further cleavage of 22 N-terminal residues. Another latent intermediate form with apparent M(r) 69,000 is generated from the proenzyme upon incubation with leukocyte elastase by N-terminal cleavage of 53 or 64 residues, respectively. However, latent collagenase cannot be activated by plasmin. Activation by different mercurial compounds finally results in the formation of active collagenase with apparent M(r) 64,000. In contrast to the proenzyme, active collagenase can autolyse to give active M(r) 57,000 and 45,000 intermediates and two M(r) 28,000 fragments. Purification of latent leukocyte gelatinase yields three final products with apparent M(r) 98,000, 125,000 and 220,000 (SDS-PAGE; non reduced). Upon reduction, only the M(r) 98,000 form can be detected. The latent gelatinase can be activated in a similar manner as collagenase. Proteolytic activation by trypsin leads after N-terminal cleavage to an active gelatinase with sequence homology to leukocyte collagenase.
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PMID:Latent collagenase and gelatinase from human neutrophils and their activation. 148 34

The tissue inhibitor of metalloproteinases (TIMP, M(r) 30,000) is secreted by many cell and tissue types and has been shown to inhibit most secreted mammalian metalloproteinases. In matrix and tissue invasion assays, the inactivation or removal of TIMP enhances invasiveness. However, many of the cells that secrete TIMP also secrete other metalloproteinase inhibitors. By analysis of medium conditioned by various endothelial, mesenchymal, and neural cells on SDS-.substrate-polyacrylamide-inhibitor gels (reverse zymograms), we have detected at least three other distinct inhibitors of metalloproteinases (IMPs). Some or all of these IMPs have been detected in secretions of mouse, rabbit, sheep, and human cells and are all smaller in apparent molecular size than TIMP (IMP-1, M(r) 26,000; IMP-2, M(r) 21,000; IMP-3, M(r) 18,000). These IMPs are not proteolytic degradation products of TIMP nor do they represent nonglycosylated TIMP. The IMPs do not cross-react in the native or denatured state with any of several anti-TIMP antibodies. The IMPs appear to be regulated independently of each other and of TIMP. In vitro, the complex consisting of one of the IMPs, or TIMP, and a metalloproteinase can be dissociated into functional inhibitor and metalloproteinase. Whether this characteristic is significant in vivo is not known. IMP-2 has been purified from several sources and shares sequence homology with TIMP, suggesting that the IMPs and TIMP may constitute a gene family. The most significant characteristic of IMP-2 is that it appears to preferentially inhibit, on a mole:mole basis, the M(r) 68,000 gelatinase rather than collagenase or stromelysin.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Secreted inhibitors of metalloproteinases (IMPs) that are distinct from TIMP. 148 40

Oral submucous fibrosis (OSF) is characterized by an abnormal accumulation of collagen fiber in oral submucosa. OSF is a collagen disease and is also regarded as a precancerous lesion. In the previous study, we discovered that the collagen content in oral mucosa of OSF is statistically higher than in normal mucosa. This research examined the relationship between the fibrosis and collagenase activity. Collagenase activity was determined by using soluble 14C-glycine-labeled collagen (9813 cpm/200 micrograms/tube) as a substrate in a solution incubated for 30 hours at 35 degrees C. The results showed that the collagenase activity of the OSF was much lower than that of normal oral mucosa (65.23 +/- 19.49 units/g tissue in normal mucosa vs. 29.48 +/- 5.69 units/g tissue in OSF). Furthermore, the cleavage pattern revealed by SDS-Polyacrylamid gel electrophoresis (SDS-PAGE) confirmed that the partially purified OSF collagenase was likely to be typical mammalian collagenase, with a molecular weight of about 68.0 kDa.
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PMID:Collagenase activity in oral submucous fibrosis. 149 4

Matrix proteins of bone, dentin and cementum have been shown to play a role in bone induction during the mineralization process, and in regulating the activities of several types of mesenchymal cells. Whether these biological functions are mediated through the same mechanism or whether there is specific modulation in each biological process is still open to speculation. The purpose of this pilot investigation was to compare the non-collagenous proteins among these tissues. Bone and teeth, obtained from clinically healthy subjects, were sectioned into 1 mm thick pieces. With the aid of a dissecting microscope, cementum and dentin were separated and collected. Tissue specimens were extracted sequentially in three steps by solutions containing 0.5 M acetic acid, 4 M Guanidine/0.5 M EDTA, and 250 units/ml bacterial collagenase, respectively. Proteins extracted were dialyzed, lyophilized and then further analyzed by both 10% SDS-PAGE (1-D) and two-dimensional (2-D) SDS-PAGE. Comparison showed that the three extraction buffers had relatively different extraction capacities within and among each tissue. The components extracted by acetic acid and Guanidine/EDTA were similar, but seemed different from that extracted by bacterial collagenase as shown by 10% SDS-PAGE. Two-dimensional SDS-PAGE further characterized numerous distinct protein spots from bone (MW of 61, 55, 40, 35, 34, 33 kD and eleven distinct spots which showed MW between 10 and 29 kD, pI range of 5.6-6.4), dentin (MW of 59, 54, 35, 28, 25, 24, 21 kD), and cementum (MW of 71, 64-65, 58, 55, 52, 50, 47, 43, 40, 31, 19 kD).(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Biochemical comparisons of matrix proteins extracted from healthy human alveolar bone, dentin and cementum. 149 6

The Alport antigen, a component of normal glomerular basement membranes (GBM) which is absent in Alport familial nephritis, is characterized as a 26 kD non-collagenous (NC1) peptide identified by a monoclonal antibody (Mab A7) and an Alport alloantibody. Both antibodies discriminate X-linkage of the Alport defect using indirect immunofluorescence of hemizygous and heterozygous Alport GBM and epidermal basement membrane (EBM). Immunoblotting of SDS-PAGE gels of collagenase-digested Alport renal BM shows absence of monomeric and dimeric components of the Alport antigen, alpha 3(IV) NC1, and alpha 4(IV) NC1. By immunoprecipitation experiments with specific antibodies, the Alport antigen is distinct from the 26 kD NC1 peptide derived from alpha 1(IV). The monoclonal antibody to the Alport antigen and rabbit antiserum to a non-consensus sequence of alpha 5(IV) NC1 react similarly by immunofluorescence with normal kidney and both fail to bind to Alport renal BM. Two dimension Western blots of collagenase-digested BM show that the anti-Alport antigen and the ant-alpha 5(IV) NC1 react similarly with monomeric and dimeric components of BM collagen. These studies are consistent with the likelihood that the Alport antigen and alpha 5(IV) NC1 are the same or are highly homologous molecules. The precise relationship will require characterization of alpha 5(IV) NC1 protein and determination of the nucleotide sequence of the Alport antigen. The associated absence of alpha 3(IV) NC1 and alpha 4(IV) (NC1) from Alport BM is consistent with other observations for a molecular association of these chains in a novel collagen network.
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PMID:Immunochemical studies of the Alport antigen. 150 19

The effect of leukoregulin, a 50-kD lymphokine with unique antitumor properties, was studied in vitro on several fibroblast functions. Leukoregulin did not inhibit fibroblast proliferation, as measured by cell enumeration and [3H]thymidine incorporation, and had no cytotoxic effect in terms of increased membrane permeability detected by trypan blue exclusion, two of the major leukoregulin actions on tumor cells. Leukoregulin induced a dose-dependent decrease in collagen synthesis, demonstrated by decreased [3H]proline incorporation into collagenase-digestible protein, as early as 6 h after the addition of the lymphokine to human fibroblasts. Leukoregulin inhibited the synthesis of both type I and type III collagen, as measured by SDS-PAGE and by specific radioimmunoassay. Neutralizing antibodies to interleukin-1 alpha, interleukin-1 beta, tumor necrosis factor-alpha, and interferon-gamma failed to alter the effect of leukoregulin on collagen synthesis, attesting that leukoregulin action was not due to contamination by these cytokines. Inhibition of collagen synthesis occurred concomitantly with increased secretion of prostaglandin E2 and a transient rise in intracellular cyclic AMP content, peaking at 6 h. However, blocking prostaglandin synthesis with indomethacin did not counteract inhibition of collagen synthesis by leukoregulin, demonstrating independence of this action of leukoregulin from cyclooxygenase metabolites. Leukoregulin also stimulated glycosaminoglycan production in a dose-dependent manner, affecting the synthesis of hyaluronic acid as the major fibroblast-derived extracellular glycosaminoglycan. In addition, secretion of neutral proteases (collagenase, elastase, caseinase) was increased. These observations indicate that leukoregulin is able to regulate synthesis of molecules critical to the deposition of the extracellular matrix by nontransformed nonmalignant fibroblasts.
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PMID:Modulation of human dermal fibroblast extracellular matrix metabolism by the lymphokine leukoregulin. 164 5

A metal-dependent peptidase was isolated from the homogenate of human uterus by standard chromatographic techniques and purified to apparent homogeneity. The peptidase hydrolysed the synthetic vertebrate collagenase substrate 2,4-dinitrophenyl-Pro-Gln-Gly-Ile-Ala-Gly-Gln-D-Arg (Dnp-peptide), the synthetic bacterial collagenase substrate 4-phenylazobenzyloxycarbonyl-Pro-Leu-Gly-Pro-D-Arg (PZ-peptide) and gelatinolytic peptides of gelatin, but was inactive against collagen type I, gelatin and casein. The cleavage site for the Dnp-peptide was the Gly-Ile bond. The enzyme was not only inhibited by metal chelators, such as EDTA, 1,10-phenantroline and dithiothreitol but also by thiol reagents, such as mersalylic acid and N-ethylmaleimid. However, E-64, an inhibitor for thiolproteinases, and leupeptin, an inhibitor for thiol- and serine proteases, did not exhibit any inhibitory activity. Pepstatin, an inhibitor for aspartate proteinases, and inhibitors for serine proteinases like phenylmethanesulfonyl fluoride and Trasylol were ineffective as well. The purified peptidase displayed a single band in the SDS-PAGE with an apparent molecular mass of 65 kDa. Employing isoelectric focusing an IP of 5.0 could be determined. The enzyme's properties are discussed in relation to the proteinase EC 3.4.24.11 and to proteinases of the collagenase family as well as the possibility to discriminate these three metalloproteinase classes by employing the Dnp-peptide.
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PMID:Isolation and properties of a metal-dependent endopeptidase from human uterus hydrolysing synthetic collagenase substrates. 165 Feb 34


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