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)

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

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

A series of experiments has been carried out to characterize 58-kDa human neutrophil collagenase (HNC) and compare it with human fibroblast collagenase (HFC). N-Terminal sequencing of latent and spontaneously activated HNC shows that it is a distinct collagenase that is homologous to HFC and other members of the matrix metalloproteinase gene family. Activation occurs autolytically by hydrolysis of an M-L bond at a locus homologous to the Q80-F81-V82-L83 autolytic activation site of HFC. This releases a 16-residue propeptide believed to contain the "cysteine switch" residue required for latency. Polyclonal antibody raised against HNC cross-reacts with HFC but with none of the other major human matrix metalloproteinases examined. Treatment of HNC with endoglycosidase F or N-glycosidase F indicates that it is glycosylated at multiple sites. The deglycosylated latent and spontaneously activated enzymes have molecular weights of approximately 44K and 42K, respectively. Differences in the carbohydrate processing of HFC and HNC may determine why HFC is a secreted protein while HNC is stored in intracellular granules. The kinetic parameters kcat and KM for the hydrolysis of the interstitial collagen types I, II, and III in solution by both collagenases have been determined. The strong preferences of HNC for type I collagen and of HFC for type III collagen found in earlier studies have been confirmed. The preference of HNC for type I over type III collagen is almost abolished when fibrillar collagens are used as substrates, but the preference for HFC for type III over type I collagen is only partially decreased.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Characterization of 58-kilodalton human neutrophil collagenase: comparison with human fibroblast collagenase. 217 76

An understanding of the immunologic relationships between collagenases of various cellular origins is necessary to define the roles of various cell types in the pathologic tissue destruction seen in chronic inflammatory diseases, such as rheumatoid arthritis. We compared the immunologic cross-reactivity of human neutrophil and skin fibroblast collagenases, utilizing polyclonal antisera prepared to purified enzymes. Polyclonal antisera from rabbits immunized with neutrophil collagenase recognized fibroblast collagenase, as well as the neutrophil enzyme, when analyzed by immunoblot techniques. The cross-reactive epitopes constituted a major proportion of the antibody population, as shown by competitive inhibition enzyme-linked immunosorbent assay; 50% of the antibody to neutrophil collagenase was inhibited by skin collagenase. Paradoxically, antisera to fibroblast collagenase failed to recognize the neutrophil enzyme, either by immunoblot techniques or competitive inhibition enzyme-linked immunosorbent assay, an observation which supports the notion that there are unique immunodominant epitopes. The cross-reactivity with skin fibroblast collagenase shown by the neutrophil antibody suggests a conservation of epitopes between collagenases of different cellular origins. The presence of epitopes unique for each enzyme, however, could lead to a feasible approach for investigating the differential contribution of various cell types to collagenolytic activity in inflamed tissues.
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PMID:The immunologic relationship of human neutrophil and skin collagenases. 244 Apr 53

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

Collagenase is secreted from neutrophils as a latent or proenzyme. In an effort to understand the mechanism of collagenase activation in inflammation, human peripheral neutrophils (PMNs) were isolated and incubated with the tumor promotor, phorbol myristate acetate (PMA), which induces the neutrophils to degranulate and secrete proteinases. Neutrophil media were then treated with various activators or inhibitors of collagenase and other proteinases, and the collagenase activity was measured. A serine proteinase secreted from neutrophils, cathepsin G, was found to activate latent collagenase, but it was also found to require activation itself. Both hypochlorous acid (HOCl) and oxidized glutathione (GSSG) were tested for their collagenase-activating ability and were found to be successful only in the presence of active cathepsin G. A specific cathepsin G inhibitor (0.5 mM Z-Gly-Leu-Phe-CH2Cl) prevented the activation of latent collagenase by HOCl. To confirm these results, purified neutrophil cathepsin G was incubated with a neutrophil proteinase mixture which contained latent collagenase. The collagenase was shown to be activated upon incubation with purified cathepsin G. These results indicate that cathepsin G is a key mediator in neutrophil collagenase activation.
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PMID:Activation of neutrophil collagenase by cathepsin G. 254 91

In an effort to understand the mechanism of collagenase activation in inflammation, human peripheral neutrophils were isolated and incubated with the tumor promoter, phorbol myristate acetate (PMA), which induces the neutrophils to degranulate and secrete proteinases. Neutrophil media were then treated with HOCl with or without various proteinase inhibitors then collagenase activity was measured. Added HOCl was able to activate latent collagenase. However, a serine proteinase, cathepsin G, was found to be necessary for collagenase activation to occur by HOCl. The results indicate that cathepsin G is a key mediator in neutrophil collagenase activation and that HOCl under certain conditions leads to the activation of cathepsin G or the stimulation of cathepsin G's ability to activate neutrophil collagenase.
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PMID:Hypochlorous acid (HOCl) activation of neutrophil collagenase requires cathepsin G. 255 76

Collagenases are known to be associated with tissue destruction in chronic inflammatory diseases such as periodontal diseases and rheumatoid arthritis. Collagenases are secreted by circulating inflammatory cells (polymorphonuclear leukocytes and monocytes), resident mesenchymal cells and epithelial cells in latent forms, which can be activated by proteases and compounds reacting with protein thiol groups. We have studied here the effects of oxygen-derived free radicals (ODFR) on latent human neutrophil collagenase. Also, in order to elucidate the cellular sources of collagenases, the ability of human gingival crevicular fluid (GCF) collagenases both from adult periodontitis (AP) and localized juvenile periodontitis (LJP) patients to degrade soluble interstitial collagen types I and II was studied. ODFR generated by the xanthine oxidase/hypoxanthine system in the presence of trace amounts of iron and EDTA activated latent neutrophil collagenase to an equal extent as the known activators phenylmercuric chloride and gold thioglucose. ODFR activation was inhibited by desferoxamine and mannitol as well as by superoxide dismutase and catalase. Clear differences in the susceptibility of collagen types I and II to AP and LJP GCF collagenases were observed. AP GCF collagenase degraded type I and II collagens at equal rates, resembling the substrate-specificity of human neutrophil collagenase. LJP GCF collagenase degraded type I collagen considerably faster than type II collagen, which was only negligibly degraded. This corresponds to the substrate specificity of fibroblast collagenase. Zymographic evaluation of gelatinolytic proteases showed the presence of 90 and 68 kD gelatinases in both AP and LJP GCF. Non-proteolytic means apparently provide a potent activation pathway of neutrophil collagenase in vivo and the hydroxyl radical was identified to be one of the potent activating oxidants.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Non-proteolytic activation of latent human neutrophil collagenase and its role in matrix destruction in periodontal diseases. 256 61

Collagenase in human neutrophils is found within intracellular granules which can be stimulated to be secreted with phorbol myristic acetate. This extracellular secreted form of neutrophil collagenase was isolated by immunoaffinity chromatography using a monoclonal antibody previously shown to specifically recognize neutrophil collagenase. The enzyme efficiently bound to this column and was eluted with NaSCN as three major species of 75, 57, and 22 kDa, respectively. These proteins were closely related immunologically since, after radiolabeling and separation by gel filtration, each of the three proteins was precipitated by the monoclonal antibody. Also, the 75- and 57-kDa proteins exhibited collagenase activity after elution from polyacrylamide gels run under nondenaturing conditions. Further, the 57-kDa protein autodegraded into a 22-kDa protein with time. Polyclonal antibody, prepared to the 57-kDa enzyme, also recognized the 75- and 22-kDa proteins using an immunoblot technique. When crude neutrophil supernatants containing latent collagenase were immunoblotted, both the 75- and the 57-kDa enzymes were present. Our immunoaffinity purified active enzymes, although activated during the course of purification, resemble the latent enzymes in crude neutrophil supernatants. The multiple forms of secreted collagenase from degranulated leukocytes may resemble more closely that seen in inflammation.
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PMID:Secreted forms of human neutrophil collagenase. 300 18

Human monocytic tumor cells of the U937 cell line contain substantial quantities of two neutrophil neutral proteinases, elastase and cathepsin G, raising the question of whether their presence reflects an expression of transformation or whether normal monocytes undergo a developmental stage in which they produce certain neutrophil proteinases. To address this issue, we examined U937 cells for production of collagenase, since human alveolar macrophages release fibroblast-like collagenase, an enzyme that is distinct from neutrophil collagenase. Using an immunoassay that utilized antibody to skin fibroblast collagenase, we found that U937 cells secreted barely detectable quantities of enzyme, 10-12 ng/10(6) cells per 24 h, under basal conditions. Upon incubation with 10 nM 12-o-tetradecanoyl-phorbol-13-acetate (TPA), however, collagenase release increased 200-fold, comparable to the amount secreted by phorbol-stimulated human fibroblasts. Metabolic labeling and immunoprecipitation confirmed the enhanced synthesis of U937 cell collagenase upon TPA exposure. This enzyme activity further resembled fibroblast collagenase and differed from neutrophil collagenase by exhibiting preferential cleavage of monomeric type III collagen relative to type I. As previously observed with human alveolar macrophages, U937 cells also released a protein identical to the collagenase inhibitor produced by human skin fibroblasts, a molecule not associated with neutrophils. Release of this inhibitor increased 10-fold with TPA exposure. In contrast to collagenase and collagense inhibitor, TPA-treated U937 cells contained only 10-15% as much elastase and cathepsin G activities as control cells. Thus, TPA-induced differentiation modified the presence of these enzymes in the direction of their content in normal monocytes. Since the neutral proteinase profile of undifferentiated U937 cells resembles that of neutrophils and changes markedly after cellular differentiation to one that is characteristic of monocytes, these data suggest that neutrophilic proteinases may be produced by normal monocytes during the early stages of their differentiation.
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PMID:12-o-Tetradecanoyl-phorbol-13-acetate-differentiated U937 cells express a macrophage-like profile of neutral proteinases. High levels of secreted collagenase and collagenase inhibitor accompany low levels of intracellular elastase and cathepsin G. 300 52


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