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)

We have identified and sequenced a cDNA encoding human neutrophil collagenase from a lambda gt11 cDNA library constructed from mRNA extracted from the peripheral leukocytes of a patient with chronic granulocytic leukemia. The library was screened with an oligonucleotide probe constructed from the putative zinc-binding region of fibroblast collagenase. Eleven positive clones were identified, of which the one bearing the largest insert (2.2 kilobases (kb)) was sequenced. From the nucleotide sequence of the 2.2-kb cDNA clone we have deduced a 467-amino acid sequence representing the entire coding sequence of the enzyme. The deduced protein was confirmed as neutrophil collagenase by conformity with the amino-terminal sequence analyses of three tryptic peptides of purified neutrophil collagenase. The cDNA clone hybridizes to a 3.3-kb mRNA present in RNA extracted from human bone marrow but did not hybridize with RNA isolated from U937 cells induced to differentiate with phorbol myristate acetate. Neutrophil collagenase was found to possess 57% identity with the deduced protein sequence for fibroblast collagenase with 72% chemical similarity. Certain regions of the molecule, including the putative zinc-binding region, are highly conserved. When compared with the published sequence for fibroblast collagenase, neutrophil collagenase contains four additional sites for glycosylation. Medium from COS-7 cells transfected with a pcDNA1 eucaryotic expression vector containing cDNA for neutrophil collagenase degraded type I collagen into the three-quarter, one-quarter fragments characteristic of mammalian interstitial collagenase activity. Thus, definitive evidence based on the cDNA sequence confirms the neutrophil collagenase is a distinct gene product and a member of the family of matrix metalloproteinases.
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PMID:Human neutrophil collagenase. A distinct gene product with homology to other matrix metalloproteinases. 216 2

The action of human fibroblast collagenase (HFC) on six substrates of markedly different size, sequence, and conformation, including rat type I collagen, rat alpha 1(I) gelatin, beta-casein, and the three synthetic oligopeptides Gly-Pro-Gln-Gly-Ile-Ala-Gly-Gln, Asp-Val-Ala-Gln-Phe-Val-Leu-Thr-Pro-Gly, and Pro-Val-Gln-Pro-Ile-Gly-Pro-Gln, has been examined. The first peptide is a model for the collagenase cleavage site in the alpha 1(I) chain of type I collagen, while the latter two peptides are models for the autolytic activation and degradation sites in pro-HFC, respectively. The goal of these studies was to assess whether HFC hydrolyzes all of these disparate substrates at the same active site. Individual kinetic parameters for the hydrolysis of all six substrates have been determined. Gel zymography experiments using collagen, gelatin, and casein as substrates show that all three activities are associated solely with HFC rather than impurities. Recombinant HFC expressed in Escherichia coli also exhibits caseinase activity, reinforcing the view that this activity is not due to a contaminating protease from fibroblasts. The ratios of these activities agree within experimental error for several independent HFC preparations and do not change when two additional affinity purification steps are employed. The inhibition of the hydrolysis of these substrates by both 1,10-phenanthroline and Boc-Pro-Leu-Gly-NHOH is identical within experimental error. A series of assays carried out in the presence of pairs of these substrates clearly shows that they compete for the same active site.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Proteolytic activities of human fibroblast collagenase: hydrolysis of a broad range of substrates at a single active site. 216 39

Continuous topical application of epidermal growth factor (EGF) to granulation tissue increases the rate of collagen accumulation. It is believed that the clinical use of growth factors, such as EGF, may become common in the treatment of impaired wound healing in the near future. Impairments in the production and degradation of wound collagens have been demonstrated in diabetes mellitus. We studied the effects of a single, local application of EGF on collagen content, collagenase activity, and the ratio of type III and type I collagens within granulation tissue using polytetrafluoroethylene (PTFE) wound cylinders in 48 streptozotocin-induced diabetic rats in order to determine potential benefits of EGF to wound healing in diabetics. Wound collagen content in EGF-treated diabetic animals was significantly higher than in diabetic controls during the first 10 days of wound healing (236% on day 5, P less than .001; 140% on day 10, P less than .01), but decreased to significantly lower levels by day 15 of healing (71% of diabetic controls, P less than .01; 47% of nondiabetic controls, P less than .01). An 18% increase in diabetic wound protease activity was observed following application of EGF (P less than .001). The ratio of type III collagen to total wound collagen within the granulation tissue was significantly reduced (P less than .001) following EGF application. We demonstrate that a single, topical application of EGF promotes early synthesis of type I collagen, thereby deranging the usual type III/total collagen ratio, and is associated with increased wound protease activity.
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PMID:EGF increases short-term type I collagen accumulation during wound healing in diabetic rats. 216 27

Human fibrosarcoma cells derived from a patient with multiple metastases extensively degrade artificial basement membranes (BM) and secrete interstitial type of collagenase, a proteolytic enzyme responsible for degradation of type I collagen. Exposure of invasive cell line to TGF beta abrogates destruction of BM.TGF beta reduces collagenase activity and stimulates specific metalloproteinase inhibitor (TIMP) in invasive tumor cells. We preassume that TGF beta could play a protective role in tumor invasion.
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PMID:Protective role of transforming growth factor beta (TGF beta) in tumor-induced degradation of basement membranes. 216 65

We have recently presented biochemical evidence for collagen and gelatin degrading activities associated with plasma membranes of various human cancer cell lines. In this report we describe the localization of interstitial collagenase at the basal plasma membrane of the human pancreatic cancer cell line RWP-I, using immunofluorescence and ultrastructural immunogold labeling techniques. Collagenase was expressed on the extracellular face of the plasma membrane. Furthermore, the immunogold labeling was concentrated on the long, finger-like microvillous projections typically seen on the basal cell surface, while the short, brush-like projections characteristic of the apical cell surface were unlabeled. When the cytoplasmic face of the membrane was made accessible, the number of reactive sites increased markedly, indicating a high concentration of enzyme at the inner surface of the plasma membrane. When plasma membrane fractions of RWP-I cells were prepared by differential centrifugation, high salt washes virtually failed to extract collagenase activity from the membrane, while detergent extraction with n-octyl glucoside, a detergent used in the purification of integral membrane proteins, yielded soluble collagenase activity. When detergent extracted membrane fractions were passed over an anticollagenase immunoaffinity column, collagenase was specifically bound, as demonstrated by the TCA and TCB degradation of type I collagen by the bound material. Gelatinolytic activity did not bind to the column. Furthermore, immunoprecipitation of 125I-labeled detergent extracts of tumor membranes yielded a single Mr 55,000 band consistent with the zymogen form of the connective tissue collagenase. These morphological and biochemical findings suggest that collagenase is a tightly associated component of the basal plasma membrane, where it occupies a strategic location for directional proteolysis during cell migration and invasion.
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PMID:Localization of collagenase at the basal plasma membrane of a human pancreatic carcinoma cell line. 217 14

Conditioned culture medium derived from Interleukin-I alpha-activated human articular chondrocytes contained both collagen- and proteoglycan-degrading activities. Preparations of soluble type I collagen and the cartilage collagens type II, IX, X and XI were all degraded when incubated with the conditioned culture medium at 35 degrees C. Fractionation of the enzymic activities using column chromatography with Ultragel AcA 34 and Heparin-Sepharose allowed the separation and identification of neutral proteinase, collagenolytic and proteoglycan-degrading activities. Eluant fractions which contained type I collagenase activity effectively degraded collagen type II, but these fractions did not correspond precisely with those which degraded collagen types IX, X and XI. These observations indicate that chondrocytes have the potential to produce a conventional interstitial type II collagenase together with other enzymes having some specificity for the minor collagens. Thus IL-1-activated chondrocytes produce a range of collagenolytic and proteoglycan-degrading enzymes which can process most of the structural components of the cartilage matrix.
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PMID:Degradation of cartilage collagens type II, IX, X and XI by enzymes derived from human articular chondrocytes. 217 Aug 28

Inactivation of the plasma serine-proteinase inhibitor alpha 1-antitrypsin (alpha 1-AT) by neutrophil metalloproteinases has been reported [Vissers, George, Bathurst, Brennan & Winterbourn (1987) Fed. Proc. Fed. Am. Soc. Exp. Biol. 46, 1390a; (1988) J. Clin. Invest. 82, 706-711; Desrochers & Weiss (1988) J. Clin. Invest. 81, 1646-1650]. To identify the enzyme responsible, supernatant from neutrophils stimulated with phorbol 12-myristate 13-acetate was subjected to preparative SDS/PAGE, both with and without activation of latent metalloproteinases with HgCl2. The lanes were subsequently sliced into pieces, the slices incubated with equimolar amounts of type I collagen and alpha 1-AT in the presence of HgCl2, and the reaction products separated by SDS/PAGE. With the latent supernatant, the characteristic collagen-cleavage products and cleaved alpha 1-AT were present in the same slices, corresponding to an Mr of 80,000-85,000. On treatment with HgCl2 both degradative activities underwent the same molecular-mass shift to a position corresponding to Mr 60,000-65,000. Western blots of neutrophil supernatants, using a polyclonal antibody to purified collagenase, showed Mr values of 83,000 for the latent enzyme and 63,000 for the HgCl2-activated enzyme. Neutrophil collagenase was purified to homogeneity and shown also to exist in a second latent form with Mr 70,000. When activated to the Mr-63,000 form by HgCl2 and incubated with equimolar amounts of collagen and alpha 1-AT, collagenase cleaved alpha 1-AT at almost twice the rate at which collagen was cleaved. alpha 1-AT cleavage was inhibited by 1,10-phenanthroline and by high concentrations of collagen. That the purified collagenase did not contain a contaminant proteinase such as stromelysin was indicated by inability of the preparation to cleave casein. Taken together these results lead us to conclude that neutrophil collagenase is capable of degrading alpha 1-AT. Neutrophil gelatinase also cleaved alpha 1-AT, but cleavage was slow when compared with its activity against gelatin.
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PMID:Human neutrophil collagenase cleaves alpha 1-antitrypsin. 217 52

Conditions were established to stimulate human gingival fibroblast explant cultures to synthesize milligram quantities of the metalloproteinase proenzymes, prostromelysin and procollagenase. To stimulate enzyme production, cells were treated with 1 nM recombinant human IL-1 beta for approximately 7 days under serum free conditions. Using a combination of rapid column chromatography steps, approximately 10 milligrams of prostromelysin and 5 milligrams of procollagenase were purified from 1 liter of conditioned media. Prostromelysin electrophoresed as a doublet with molecular weights of 55,57 kD, whereas, procollagenase migrated with slightly lower molecular weights of 52, 54 kD. Both proenzymes were treated with trypsin or aminophenylmercuric acetate to generate active species. The molecular weights of the active enzymes were approximately 10 kD smaller than the proenzymes. Active enzymes were inhibited by metal chelators and the natural metalloproteinase inhibitor, tissue inhibitor of metalloproteinase (TIMP), but not by the serine protease inhibitor, phenylmethylsulfonyl fluoride (PMSF). Activated stromelysin degraded a number of substrates including transferrin, proteoglycan monomer, proteoglycan aggregated with hyaluronic acid, and substance P. By contrast, collagenase degraded interstitial type I collagen and the peptide thioester, Ac-Pro-Leu-Gly-SCH(iBu)Co-Leu-GlyOEt. Identity of both enzymes were confirmed by amino-terminal protein sequence analysis as well as by immunoblot analysis using monoclonal antibodies.
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PMID:Production and purification of prostromelysin and procollagenase from IL-1 beta-stimulated human gingival fibroblasts. 217 90

The chymotrypsinlike protease gene (prtA) from Treponema denticola ATCC 35405 was isolated from a lambda gt11 clone bank as one of several clones expressing protease activity. The DNA from one positive clone capable of hydrolyzing type IV collagen was subcloned into plasmid vector pUC119 for further analysis. Deletion analysis of subclone pXQ27.2 revealed the approximate location of the prtA gene on the DNA insert. DEAE-Sephadex chromatography of crude cell extracts of the subclone revealed two distinct T. denticola enzymes, one hydrolyzing SAAPNA (succinyl-L-alanyl-L-alanyl-L-prolyl-L-phenylalanine-p-nitroanilide [chymotrypsin substrate]) and the other hydrolyzing PZ-PLGPA (phenylazobenzyl-oxycarbonyl-L-leucylglycyl-L-prolyl-D -arginine [collagenase substrate]). Each activity was purified to near homogeneity and exhibited by sodium dodecyl sulfate-polyacrylamide gel electrophoresis estimated molecular sizes of 67 and 36 kDa, respectively. Western blot (immunoblot) analysis demonstrated that only the 67-kDa SAAPNA-hydrolyzing enzyme reacted with antibody against the T. denticola chymotrypsinlike protease. The purified SAAPNA-hydrolyzing enzyme degraded type IV collagen, laminin, and fibronectin, but not type I collagen. These results indicate that the prtA gene coding for the chymotrypsinlike protease from T. denticola has been isolated. Another distinct gene encoding an enzyme hydrolyzing PZ-PLGPA appears to be adjacent to the prtA gene.
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PMID:Isolation and characterization of the Treponema denticola prtA gene coding for chymotrypsinlike protease activity and detection of a closely linked gene encoding PZ-PLGPA-hydrolyzing activity. 217 32

Latent and active 58-kDa forms of human neutrophil collagenase (HNC) have been purified to homogeneity. Buffy coats were extracted in the presence and absence of phenylmethanesulfonyl fluoride to generate crude starting preparations that contained latent and active HNC, respectively. The buffers used in preparing these extracts and for all subsequent chromatographic steps contained NaCl at a concentration of 0.5 M or greater, 0.05% Brij-35, concentrations of CaCl2 of 5 mM or greater, and (when feasible) 50 microM ZnSO4 to stabilize the HNC. The collagenase activity in the buffy coat extracts was adsorbed to a Reactive Red 120-agarose column at pH 7.5 in 0.5 M NaCl and was eluted when the NaCl concentration was increased to 1 M. The active and p-(chloromercuri)benzoate-activated latent enzymes were next adsorbed to a Sepharose-CH-Pro-Leu-Gly-NHOH affinity resin in 1 M NaCl at pH 7.5 and desorbed at pH 9 to give a fraction containing only HNC and a small amount of neutrophil gelatinase. The latter enzyme was removed by passage over a gelatin-Sepharose column in 1 M NaCl at pH 7.5. The purified samples of active and latent HNC were obtained with typical cumulative yields of 32 and 82% and specific activities toward soluble rat type I collagen at 30 degrees C of 7200 and 12,000 micrograms min-1 mg-1, respectively. These specific activities are markedly higher than previously reported for HNC. Both active and latent HNC exhibit a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis both in the presence and in the absence of 2-mercaptoethanol. The mobility of latent HNC is consistent with a molecular weight of approximately 58K, with the active form exhibiting a slightly lower (less than 1-2K) molecular weight.
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PMID:Purification to homogeneity of latent and active 58-kilodalton forms of human neutrophil collagenase. 217 75


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