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)

Recent reports have demonstrated that a series of probands with severe osteogenesis imperfecta had single base mutations in one of the two structural genes for type I procollagen that substituted amino acids with bulkier side chains for glycine residues and decreased the melting temperature of the triple helix. Here we demonstrate that the type I procollagen synthesized by cultured fibroblasts from a proband with a severe form of osteogenesis imperfecta consisted of normal molecules and molecules over-modified by post-translational reactions. The thermal stability of the intact type I collagen was normal as assayed by protease digestion under conditions in which a decrease in thermal stability was previously observed with eight other substitutions for glycine in the alpha 1(I) chain. In contrast, the thermal stability of the one-quarter length B fragment generated by digestion with vertebrate collagenase was decreased by 2-3 degrees C under the same conditions. Nucleotide sequencing of cDNAs and genomic DNA established that the proband had a substitution of A for G in one allele of the pro alpha 1(I) gene that converted the codon for alpha 1-glycine 844 to a codon for serine. The results also established that the alpha 1-serine 844 was the only mutation that could account for the decrease in thermal stability of the collagenase B fragment. There are at least two possible explanations for the failure of the alpha 1-serine 844 substitution to decrease the thermal stability of the collagen molecule whereas eight similar mutations decreased the melting temperature. One possibility is that the effects of glycine substitutions are position specific because not all glycine residues make equivalent contributions to cooperative blocks of the triple helix that unfold in the predenaturation range of temperatures. A second possible explanation is that substitutions of glycine by serine have much less effect on the stability of protein than the substitutions by arginine, cysteine, and aspartate previously studied.
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PMID:Substitution of serine for alpha 1(I)-glycine 844 in a severe variant of osteogenesis imperfecta minimally destabilizes the triple helix of type I procollagen. The effects of glycine substitutions on thermal stability are either position of amino acid specific. 251 Nov 92

It has been suggested that the trabecular meshwork may be involved in the pathogenesis of open-angle glaucoma, and some authors have pointed out that disorders of the extracellular matrix components may play a role; nevertheless, nothing is known about the normal metabolism of connective tissue molecules in this particular tissue. We recently initiated some studies in this field and have focused on the in vitro effects of aqueous humor on collagen metabolism. We report the finding of a latent collagenase of low molecular weight in aqueous humor obtained from cataractous patients; the enzyme was identified through several methods, including its in vitro activity against radiolabelled type I collagen and additionally with a zymogram technique. It was partially characterized by high-performance liquid chromatography.
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PMID:A latent collagenase in human aqueous humor. 253 47

Tumour necrosis factor-alpha (TNF-alpha) is secreted by macrophages in response to inflammation, infection and cancer. Sublethal doses of recombinant TNF-alpha to rats causes cachexia, anaemia and inflammation. TNF-alpha plays a major part in tissue inflammation and remodelling by stimulating production of collagenase. Cellular responses to TNF-alpha are initiated by binding to high-affinity cell surface receptors. TNF-alpha then profoundly affects gene regulation, stimulating the fos, myc, interleukin-1 and interleukin-6 genes and inhibiting the type I collagen gene. Here we demonstrate that TNF-alpha also stimulates collagenase gene transcription; this stimulation is mediated by an element of the gene that is responsive to the transcription factor AP-1, the major component of which (jun/AP-1) is encoded by the jun gene; and that TNF-alpha stimulates prolonged activation of jun gene expression. This prolonged induction of jun contrasts with its transient activation by the phorbol ester TPA and provides a physiological example of the ability of jun/AP-1 to stimulate its own transcription. This may be a key mechanism for mediating at least some of the biological effects of TNF-alpha.
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PMID:Prolonged activation of jun and collagenase genes by tumour necrosis factor-alpha. 253 68

A fluorogenic substrate for vertebrate collagenase and gelatinase, Dnp-Pro-Leu-Gly-Leu-Trp-Ala-D-Arg-NH2, was designed using structure-activity data obtained from studies with synthetic inhibitors and other peptide substrates of collagenase. Tryptophan fluorescence was efficiently quenched by the NH2-terminal dinitrophenyl group, presumably through resonance energy transfer. Increased fluorescence accompanied hydrolysis of the peptide by collagenase or gelatinase purified from culture medium of porcine synovial membranes or alkali-treated rabbit corneas. Amino acid analysis of the two product peptides showed that collagenase and gelatinase cleaved at the Gly-Leu bond. The peptide was an efficient substrate for both enzymes, with kcat/Km values of 5.4 microM-1 h-1 and 440 microM-1 h-1 (37 degrees C, pH 7.7) for collagenase and gelatinase, respectively. Under the same conditions, collagenase gave kcat/Km of about 46 microM-1 h-1 for type I collagen from calf skin. Since both enzymes exhibited similar Km values for the synthetic substrate (3 and 7 microM, respectively), the higher catalytic efficiency of gelatinase reflects predominantly an increase in kcat. Both enzymes were inhibited by HSCH2(R,S)CH[CH2CH(CH3)2]CO-L-Phe-L-Ala-NH2 in this assay (50% inhibition at 20 nM and less than 1 nM for collagenase and gelatinase, respectively). Soluble type I collagen was a competitive inhibitor of peptide hydrolysis by collagenase (KI = 0.8 microM) and exhibited mixed inhibition of gelatinase (KI = 0.3 microM).
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PMID:Comparison of vertebrate collagenase and gelatinase using a new fluorogenic substrate peptide. 253 33

A quantitative collagenase assay detecting soluble collagen fragments is described in this paper. Using the reagent N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP) type I collagen was conjugated with horseradish peroxidase (POD) which was employed as a reporter enzyme. POD was preferentially linked to the TC B fragment in a ratio of 1.4 mol POD/mol collagen. The conjugation product was immobilized on AH-Sepharose via carbodiimide coupling to form the final collagenase substrate used in the assay. POD activity in the supernatants caused by liberated TC B fragments exhibited a linear relationship for collagenase concentrations up to 100 micrograms/ml bacterial collagenase. Over an incubation period of 4 h the lowest detection limits found were 20 ng/100 microliters for bacterial collagenase and 60 ng/100 microliters for human leukocyte collagenase. Incubation of the assay mixture with 5 micrograms trypsin resulted in 3.8% of the activity released by the equivalent amount of leukocyte collagenase. The assay developed here has been shown to be sensitive and specific for collagenase, with the additional advantage that this method is suited for simple and economic handling.
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PMID:A spectroscopic collagenase assay using peroxidase-labeled collagen. 254 Jun 72

The specific role of proteolytic enzymes in the degradation by live cells of fibrillar model matrices (fibrin, collagen) was studied using monoclonal and polyclonal inhibitory (anti-catalytic) antibodies. Dissolution of fibrin by plasminogen-supplemented human HT-1080 cells was blocked by (1) omission of plasminogen, (2) inhibitory anti-plasmin antibody, and (3) inhibitory anti-u-PA antibody but not by non-inhibitory control antibodies. Using a similar approach, it was shown that the dissolution of reconstituted type I collagen fibrils by trypsin-supplemented live human skin fibroblasts was blocked by inhibitory antibodies to fibroblast-type procollagenase but not by noninhibitory control antibodies. These findings permit us to deduce that, at least in culture, the dissolution of fibrin by plasminogen-supplemented HT-1080 cells was mediated by plasminogen-assisted proteolysis which entailed the extracellular conversion of plasminogen to plasmin by cell-derived u-PA, and that the dissolution of collagen fibrils by trypsin-supplemented skin fibroblasts was mediated by a collagenase-dependent pathway.
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PMID:Use of inhibitory (anti-catalytic) antibodies to study extracellular proteolysis. 254 25

The role of human neutrophil proteases in the further degradation of the native triple-helical characteristic cleavage products 3/4- and 1/4-collagen fragments generated by neutrophil interstitial collagenase from native type I collagen was studied. Purified human neutrophil collagenase did not further degrade the characteristic collagen fragments whether they were in triple-helical (native collagen) or random-coil (gelatin) conformation. Neutrophil extract treated with 1 mM phenylmercuric chloride (PMC) degraded native type I collagen at +37 degrees C producing multiple protein bands. Neutrophil extract at +18 degrees C in the presence of the serine protease inhibitors phenylmethylsulfonyl fluoride and banzamidine did not degrade native type I collagen. Inclusion of PMC to active latent collagenase caused neutrophil extract to degrade native type I collagen to 3/4- and 1/4-fragments. In addition, native 3/4- and 1/4-fragments were further degraded in a time-dependent manner by PMC-treated neutrophil extract. Both native 3/4- and 1/4-collagen fragments were degraded by specific rather than by multiple cleavage. Further fragmentation was inhibited by divalent cation chelators EDTA and 1,10-phenanthroline. The results indicate the presence of latent metalloprotease(s), as distinct from collagenase, gelatinase and serine proteases, that are capable of further degrading by specific cleavage both native 3/4- and 1/4-collagen fragments generated by collagenase in human neutrophils. The enzyme(s) may augment the action of collagenase and other neutral proteases in connective tissue destruction associated with the etiopathogenesis of periodontal diseases.
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PMID:Identification of protease(s) capable of further degrading native 3/4- and 1/4-collagen fragments generated by collagenase from native type I collagen in human neutrophils. 254 68

We previously suggested that periodontal pathogens might mediate connective tissue degradation in periodontal diseases through the ability of antigens from their cell walls to stimulate cytokine production by circulating mononuclear cells. Such cytokines would then induce metalloproteinase (MP) synthesis by resident gingival cells and thus initiate matrix degradation. In the present investigation human gingival fibroblasts (HGFs) were grown on [14C]-labelled type I collagen films and stimulated with either tumor necrosis factor (TNF) or interleukin-1 (IL-1) for 48 h. Collagenolysis occurred in a dose-dependent manner; the optimal dose for human rTNF alpha was 100 ng/ml and for rIL-1 alpha and rIL-1 beta, 1 ng/ml. Collagen degradation was accompanied by increased synthesis and release of the MPs collagenase, gelatinase and stromelysin, and there was a reduction in free TIMP (tissue inhibitor of metalloproteinases): collagenase and stromelysin were detected in both active and latent forms. Cytokine-stimulated collagenolysis was abolished by the addition of exogenous human rTIMP (5 units/ml). We also measured collagenase and TIMP by ELISAs which recognize all forms of collagenase (latent, active or complexed) and TIMP (free or complexed). These showed that while collagenase activity (0.6-1.2 microgram/ml) correlated with lysis, total TIMP levels remained unchanged at approximately 0.2 microgram/ml. These results demonstrate important roles for MPs and TIMP in regulating type I collagen degradation by HGFs, and support the hypothesis that connective tissue destruction during inflammatory diseases may be initiated, at least in part, by TNF and IL-1.
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PMID:Gingival fibroblasts degrade type I collagen films when stimulated with tumor necrosis factor and interleukin 1: evidence that breakdown is mediated by metalloproteinases. 255 Jun 4

The regeneration of connective tissue attachment is a major goal of clinical periodontics. Recent investigations on biochemically mediated periodontal regeneration have attempted to define the various biological response modifiers which may provide a mechanism for periodontal regeneration. Fibronectin and endothelial cell growth factor have been shown to selectively enhance periodontal ligament (PDL) cell adhesion, migration, and proliferation. In addition, dentin preconditioned with tetracycline HCl (TTC) or citric acid (CA) supports PDL cell adhesion, presumably by exposing collagen fibers. We have now extended these studies to include basic fibroblast growth factor (b-FGF) as a potential meditor of periodontal regeneration. Using AFSCM (assays for specific cell migration), b-FGF in concentrations as low as 10 ng per dentin block significantly stimulated PDL cell chemotaxis, while the antibody against b-FGF inhibited both the chemotactic and proliferative characteristics of the mitogen. We also found that 5 ng and above of b-FGF per dentin block significantly stimulated human endothelial cell migration and proliferation. Using 125I-b-FGF, we demonstrated that the factor binds to native dentin. This binding was increased when the dentin blocks were preconditioned by TTC or CA and reduced when the dentin was subsequently treated with collagenase. 125I-b-FGF also bound with moderate affinity to a type I collagen affinity column whereas the binding to a hydroxylapatite affinity column was negligible. The combination of FN and b-FGF was a marginally more potent chemo-attractant than b-FGF alone for PDL cells.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Repopulation of dentin surfaces by periodontal ligament cells and endothelial cells. Effect of basic fibroblast growth factor. 255 Jun 5

Bovine aortic medial tissue and medial smooth muscle cells were demonstrated for the first time to synthesize a latent collagenase together with collagenase inhibitor in culture. Molecular weights of the latent collagenase and its inhibitor derived from aortic medial tissue explant were estimated to be about 52 K by gel filtration and 26.5 K by electrophoresis, respectively. Activated aortic collagenases cleaved type I collagen in solution into 3/4 (alpha A) and 1/4 (alpha B) length cleavage fragments and were inhibited by EDTA, o-phenanthroline, dithiothreitol, bovine serum, and highly purified dental pulp and aortic collagenase inhibitors. The aortic inhibitors showed inhibitory activity against all the animal collagenases tested, except for bacterial collagenase. Double-immunodiffusion analysis using a monospecific antiserum prepared against dental pulp inhibitor showed that the aortic inhibitors are immunologically identical to the pulp inhibitor. Using the same antiserum, we found immunoreactive collagenase inhibitor protein to be localized along the collagen fibers between elastic membranes in aortic medial tissue.
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PMID:Synthesis of latent collagenase and collagenase inhibitor by bovine aortic medial explants and cultured medial smooth muscle cells. 255 72


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