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)

The biosynthesis of fibronectin during the in vivo development of matrix-induced endochondral bone was investigated by using [35S]methionine in rats. The dmineralized bone matrix that was implanted subcutaneously to induce local bone formation bound circulating fibronectin. This may be an important initial requirement for cell attachment to the matrix. Fibronectin was present throughout the development of bone but accounted for the largest percentage of total protein synthesized during mesenchymal cell proliferation and hematopoiesis. Fibronectin was identified in tissue extracts by its (i) comigration on electrophoretic NaDodSO4/polyacrylamide gels with human and rat plasma fibronectin, (ii) affinity for denatured collagen, (iii) crossreactivity with purified antibody of rat plasma fibronectin, and (iv) insensitivity to collagenase digestion. Fibronectin was localized by immunofluorescence in the extracellular matrix during the period of mesenchymal cell proliferation. During chondrogenesis, fibronectin was demonstrated in the differentiating chondrocytes. Fibronectin was detectable in the cartilage matrix only after hyaluronidase treatment. During vascular invasion, prior to osteogenesis, fibronectin was localized in association with endothelial cells. These observations demonstrate a possible role of fibronectin in collagenous matrix-mesenchymal cell interaction in vivo.
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PMID:Synthesis and localization of fibronectin during collagenous matrix-mesenchymal cell interaction and differentiation of cartilage and bone in vivo. 699 Apr 20

The penetration of cercariae through the skin initiates infection of the host with the human trematode parasite Schistosoma mansoni. Many larvae fail to migrate into the living epidermal cell layer. In order to determine if chemical as well as mechanical barriers to cercarial skin penetration exist, inhibitory activity of epidermal cell extracts against the proteinase obtained from cercarial secretions was assayed. An inhibitor was purified 50-fold by gel filtration on Sephadex G 75 and cation exchange chromatography at pH 5.8 and 4.9. The inhibitor has a relative molecular mass (Mr) of approx. 40 000-53 000. Oxidation of the inhibitor with N-chlorosuccinimide eliminated its inhibitory activity and thus indicated a critical methionine residue. The inhibitor was active against a wide spectrum of serine proteinases: porcine pancreatic elastase, human granulocyte elastase, bovine trypsin, and bovine alpha-chymotrypsin. However, no inhibition was detected against papain or clostridial collagenase. The inhibitor did not cross react with antiserum to human or rat serum alpha 1-proteinase inhibitor.
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PMID:Partial purification and characterization of an inhibitor from newborn-rat epidermis with activity against the proteinase of Schistosoma mansoni cercariae. 716 4

1. The kinetics of the degradation of the kinins bradykinin and Met-Lys-bradykinin, angiotensins I and II and the tachykinin substance P by PMNL-collagenase (MMP 8), PMNL-gelatinase (MMP 9) and by the recombinant catalytic domain of MMP 8 (rcd-PMNL-c) was examined by RP-HPLC. The resulting fragments were identified by automated Edman degradation or by amino acid analysis. 2. The initial degradation rates of substance P at a substrate concentration of 25 microM were 5 min-1 for MMP 9 and 150 min-1 for MMP 8. The kinetic constants KM and kcat were determined by concentration-dependent measurements. For MMP 8/substance P the constants were KM = 78 +/- 14 microM and kcat = 412 +/- 67 min-1. For MMP 9/substance P the constants were KM = 91 +/- 15 microM and kcat = 25 +/- 4 min-1. Both enzymes cleaved substance P between Gln6 and Phe7 and between Gly9 and Leu10. 3. Under the same conditions, MMP 8 degraded angiotensin I at an initial rate of 20 h-1, resulting mainly in the vasoactive fragments angiotensin II and angiotensin(1-7). At a substrate concentration of 25 microM and an enzyme/substrate ratio of 1:100, angiotensin II was degraded very slowly (19% in 24 h) by MMP 8. Under these conditions, MMP 9 degraded angiotensin I to a lesser extent than MMP 8 (25% in 24 h) and was unable to cleave angiotensin II. 4. Under the same conditions, bradykinin and Met-Lys-bradykinin were cleaved by PMNL-collagenase at a rate of 20% in 24 h, producing BK(1-7) and BK(1-8). PMNL-gelatinase was unable to cleave the kinins under these conditions. 5. In all cases, rcd-PMNL-c produced the same fragments as wild type PMNL-collagenase, but at a significantly lower rate.
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PMID:Degradation of kinins, angiotensins and substance P by polymorphonuclear matrix metalloproteinases MMP 8 and MMP 9. 753 73

In physiologic remodeling of bone and other connective tissues, proteinases such as the matrix metalloproteinases (MMPs) which can cleave Type I collagen play a critical role. In bone, MMP-1 is secreted by stromal fibroblasts, osteoblasts, and osteoclasts. Only the collagenases (MMP-1 and MMP-8) cleave native undenatured collagen at neutral pH. The cleavage is site specific at a single locus in the alpha 1(I) chain between Gly775/Ile776. The authors have altered the amino acid sequences around the collagenase cleavage site by site-directed mutagenesis of the murine Col1a-I gene, introducing Pro for Gln774, Pro for Ala777, and Met for Ile776. The mutant Col1a-I gene has been expressed in Mov13 fibroblasts, and secreted Type I collagen molecules have been found to be resistant to cleavage at Gly775/Ile776 by MMP-1 or MMP-8. This subtle mutation was introduced recently into the endogenous Col1a-I gene by homologous recombination in embryonic stem cells to determine the role of collagenase in vivo. Chimaeric mice derived from blastocysts injected with these embryonic stem cells transmitted the mutant Col1a-I gene to their offspring. Surprisingly, homozygous mutant mice reproduce and appear to develop normally. The mechanisms of collagen resorption in remodeling of bone and soft tissues in these mice are being examined currently. Information should be derived that will be useful in interpreting human disorders characterized by increased collagen deposition, such as osteopetrosis and dermal fibrosis.
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PMID:Is collagenase (matrix metalloproteinase-1) necessary for bone and other connective tissue remodeling? 764 97

In the present study, we describe two procedures for isolation and culture of Sertoli cells from the testes of adult Siberian hamsters. In procedure I, collagenase and pancreatin were used for differential enzymatic digestion of the tissue at 32 degrees C, and effects of several attachment factors were examined. Sertoli cells isolated from adult hamsters were not affected by addition of Na selenite, epidermal growth factor, insulin, and transferrin, with or without 5% fetal bovine serum, to Dulbecco's modified Eagle's medium + Ham's F-12. Significant increase in the yield and plating efficiency of Sertoli cells was achieved by developing a novel procedure (procedure II) for Sertoli cell isolation. In this procedure, testicular tissue was exposed to only one enzyme (collagenase I) for two consecutive digestions at 37 degrees C, and the total period of enzymatic exposure was reduced relative to that of procedure I. Another objective of this study was to compare the functions of Sertoli cells isolated from spermatogenetically active and inactive adult testes. Isolated Sertoli cells from 60 +/- 5-day-old hamsters raised in long day conditions (LD, 16L:8D) or in short day conditions (SD, 6L:18D) were stimulated with FSH and testosterone in the presence of 35S-methionine for 24 h on Day 4 of culture. The medium was concentrated, and equal amounts of radioactive proteins from LD and SD hamster Sertoli cell cultures were analyzed by two-dimensional PAGE. Compared to Sertoli cells derived from SD hamsters, Sertoli cells from LD animals produced greater amounts of two secretory proteins and a smaller amount of one.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Isolation and culture of Sertoli cells from the testes of adult Siberian hamsters: analysis of proteins synthesized and secreted by Sertoli cells cultured from hamsters raised in a long or a short photoperiod. 775 59

The regulatory effect of endogenously synthesized eicosanoid metabolites on the expression of tissue inhibitor of metalloproteinases (TIMP), interstitial collagenase, and 92-kDa gelatinase by human macrophages was examined. TIMP and metalloproteinase production were stimulated with three agonists that produce distinct patterns of eicosanoid synthesis: lipopolysaccharide (10 micrograms/ml), denatured collagen (10 micrograms/ml), or zymosan (1 mg/ml). Indomethacin (3 micrograms/ml) or MK886 (3 microM), a specific inhibitor of 5-lipoxygenase, was used to examine the role of endogenous metabolites of arachidonic acid. Regardless of the agonist used, TIMP production by macrophages was inhibited 65% by indomethacin, synthesis of interstitial collagenase was reduced 70%, and expression of 92-kDa gelatinase was decreased 40%. In contrast, inhibition of leukotriene synthesis had no effect on metalloproteinase or TIMP production. The agonist-stimulated increase in TIMP and collagenase production was directly correlated to the cumulative prostaglandin E2 level induced by the agonist used. However, if response to an agonist was poor, the exogenous addition of prostaglandin E2 could not increase TIMP or collagenase production more than twofold, indicating an important permissive effect of the agonist on the regulation of each protein's expression. The mechanism of indomethacin inhibition of TIMP and collagenase production was studied by labeling the cells with [35S]-methionine and performing immunoprecipitation using specific antiserum. Indomethacin markedly inhibited the lipopolysaccharide-induced biosynthesis of both TIMP and collagenase. Northern analysis revealed parallel suppression of TIMP and collagenase steady-state mRNA levels by indomethacin, indicating pretranslational control. The regulation of inflammatory-cell TIMP and interstitial collagenase expression by prostaglandin E2 suggests that therapy inhibiting the cellular response to prostaglandins may be useful in cutaneous and systemic disease states involving macrophage-mediated connective-tissue destruction.
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PMID:Agonist-induced expression of tissue inhibitor of metalloproteinases and metalloproteinases by human macrophages is regulated by endogenous prostaglandin E2 synthesis. 779 41

During the past decade, strains of Bacteroides fragilis that produce an enterotoxin have been implicated in diarrheal disease in animals and humans. The extracellular enterotoxin has been purified and characterized as a single polypeptide (M(r), approximately 20,000). Single specific primer-PCR was used to clone a portion of the B. fragilis enterotoxin gene. The recombinant protein expressed by the cloned gene fragment reacted with monospecific antibodies to B. fragilis enterotoxin by enzyme-linked immunosorbent assay and immunoblot analysis. The deduced amino acid sequence revealed a signature zinc-binding consensus motif (HEXXHXXGXXH/Met-turn) characteristic of metalloproteases termed metzincins. Sequence comparisons showed close identity to matrix metalloproteases (e.g., human fibroblast collagenase) within the zinc-binding and Met-turn region. Purified enterotoxin contained 1 g-atom of Zn2+ per molecule and hydrolyzed gelatin, azocoll, actin, tropomyosin, and fibrinogen. The enterotoxin also underwent autodigestion. The N-terminal amino acid sequences of two autodigestion products were identical to the deduced amino acid sequence of the recombinant enterotoxin and revealed cleavage at Cys-Leu and Ser-Leu peptide bonds. Gelatinase (type IV collagenase) activity comigrated with the toxin when analyzed by gel fractionation and zymography, indicating that protease activity is due to the enterotoxin and not to a contaminating protease(s). Optimal proteolytic activity occurred at 37 degrees C and pH 6.5. Primary proteolytic cleavage sites in actin were identified, revealing cleavage at Gly-Met and Thr-Leu peptide bonds. Enzymatic activity was inhibited by metal chelators but not by inhibitors of other classes of proteases. Additionally, cytotoxic activity of the enterotoxin on human carcinoma HT-29 cells was inhibited by acetoxymethyl ester EDTA. The metalloprotease activity of the enterotoxin suggests a possible mechanism for enterotoxicity and may have additional implications in the study of disease caused by B. fragilis.
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PMID:The enterotoxin of Bacteroides fragilis is a metalloprotease. 780 55

It has been suggested that IL-1 produces cartilage matrix degradation by metalloproteinases such as collagenase and that such degradation is regulated by metalloproteinase inhibitors. In the present study, the effects of IL-6 and oxygen radical scavengers on cartilage matrix degradation were studied. Superoxide dismutase, catalase, or methionine all significantly inhibited cartilage matrix degradation both in IL-1 beta-stimulated and unstimulated experimental conditions. Both 10 mM EDTA and 100 nM tissue inhibitor of metalloproteinase (TIMP) significantly inhibited cartilage matrix degradation. The addition of methionine significantly inhibited collagenase activity produced in the culture supernatants of chondrocytes stimulated with IL-1 beta. IL-6 significantly suppressed cartilage matrix degradation produced spontaneously or by IL-1 beta stimulation in chondrocytes. IL-6 inhibited superoxide production by chondrocytes both in IL-1 beta-stimulated or unstimulated conditions. These results suggest that oxygen radicals are involved in cartilage matrix degradation mediated by both paracrine and autocrine IL-1 mechanisms and that oxygen radical-mediated activation of collagenase in chondrocytes may explain the mechanisms of how oxygen radicals are involved in cartilage matrix degradation. IL-6 inhibited superoxide production in chondrocytes and thus inhibited cartilage matrix degradation.
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PMID:Role of oxygen radicals and IL-6 in IL-1-dependent cartilage matrix degradation. 784 4

Active digestive enzymes are involved in the pathophysiology of acute pancreatitis. Previous studies have mainly focused on the role of trypsin in the autodigestive process. The present study compares the noxious potential of different pancreatic enzymes to damage acinar cells. Acinar cells were isolated from rat pancreas by collagenase digestion. Cell viability was studied by (1) exclusion of trypan blue, (2) release of lactate dehydrogenase, and (3) release of newly synthesized proteins identified with methionine labeled with sulfur 35. Cells were then incubated in oxygenated N-2-hydroxyethylpiperazine-N-'-2-ethanesulfonic acid-Ringer solution containing different concentrations of various active digestive enzymes. Uptake of trypan blue was the most sensitive and reliable test of cell damage when compared with release of lactate dehydrogenase or radiolabeled newly synthesized proteins. All active digestive enzymes studied caused dose-dependent cell damage. The noxious potential, however, was strikingly different for the various enzymes. Pancreatic elastase in nanomolar concentrations caused marked cell damage after 45 to 90 minutes of incubation. Lipase and chymotrypsin caused a similar damage only at micromolar concentrations, whereas even millimolar concentrations of trypsin failed to cause significant damage. The present results confirmed recent work showing that lipase and phospholipase A2 probably cause cell damage through release of free fatty acids and lysolecithin. Although activation of trypsin might be the trigger to start the activation cascade in acute pancreatitis, trypsin itself is markedly less noxious to acinar cells when compared with other digestive enzymes. Elastase by far had the greatest noxious potential of all enzymes evaluated. Studies analyzing therapeutic effects of protease inhibitors should evaluate not only the inhibitory potential against trypsin but also that against other digestive enzymes, particularly elastase.
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PMID:Active pancreatic digestive enzymes show striking differences in their potential to damage isolated rat pancreatic acinar cells. 784 75

Nearly homogeneous preparations of stromal cells derived from human proliferative endometrium can be obtained by treating endometrial fragments with collagenase in order to disperse stromal elements, filtering the mixture a 25 microns opening sieve to separate them from gland, and incubating the dispersed cells to culture dishes. Exposure of stromal cell cultures to db-cAMP, 8-Br-cAMP or forskolin in RPMI 1640 medium containing 2% ct-FCS and 0.1 U/ml insulin induces the expression of prolactin (PRL), evident from 1) its secretion to the culture medium, measured by radioimmunoassay and by Western blot analysis, 2) the incorporation of 35S-methionine in a protein precipitated with a PRL antibody and co-migrating with authentic PRL during electrophoresis, and 3) the synthesis of PRL mRNA determined by Northern blot analysis. The cAMP effect on PRL production is enhanced by progestins, which by themselves are weak PRL inducers under similar experimental conditions. As expected from previous findings in our laboratory, showing that addition of PRL to the culture medium induces decidualization of endometrial stromal cells, cAMP derivatives not only induce PRL but also provoke differentiation of the fibroblast-like stromal cells to the decidual phenotype, as evident from morphologic changes and by the expression of products characteristic of decidual cells, e.g. IGFBP-1, desmin, hsp 27 and laminin. These findings suggest a PRL-mediated, progesterone-enhanced decidualization mechanism initiated by physiologic agents increasing cAMP levels in stromal cells.
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PMID:Mechanisms involved in the decidualization of human endometrial stromal cells. 798 67


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