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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 Acinetobacter spec collagenase has been almost completely purified. This enzyme is a true collagenase the activity of which is high on collagen. The enzyme is active on insoluble collagen, gelatin and the synthetic Pz-peptide, but has no proteolytic activity on casein or bovine serum-albumin. The collagenase was obtained on a simple medium with gelatin and yeast extract. The enzyme was purified by (NH4)2SO4 precipitation. DEAE cellulose column chromatography, Sephadex G 200 gel-filtration. The molecular weight of the enzyme was found to be 102 000 daltons, and its isoelectric point was found to be 7,7 +/- 0,2. The optimum pH and temperature for insoluble collagen hydrolysis were 7.6 and 37 degrees C, respectively; so, this collagenase corresponds to true collagenase. Hydrolysis of Pz-peptide is activated by Ca2+ and inhibited by metal ions (Cu2+, Fe3+, Zn2+, Pb2+, Hg2+). EDTA and o-phenanthroline induced a very significant reduction in enzyme activity. Iodoacetate and p-CMB induced a slight reduction in enzyme activity only at high concentrations (10-2M). The collagenase is most stable for temperatures less than or equal to 50 degrees C.
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PMID:[Purification and physico-chemical properties of collagenase synthesized by a bacterium of the type Acinetobacter sp]. 4 44

Collagenases (EC 3.4.24.3) from human skin, rat skin and rat uterus were inhibited by the chelating agents EDTA, 1,10-phenanthroline and tetraethylene pentamine in the presence of excess Ca2+, suggesting that a second metal ion participates in the activity of the enzyme. Collagenase inhibition by 1,10-phenanthroline could be both prevented and reversed by a number of transition metal ions, specifically Zn2+, Co2+, Fe2+ and Cu2+. However, Zn2+ is effective in five-fold lower molar concentrations (1-10(-4) M) than the other ions. Furthermore, Zn2+ was the only ion tested able to prevent and reverse the inhibition of collagenase by EDTA in the presence of excess Ca2+. Atomic absorption analysis of purified collagenase for Zn2+ showed that Zn2+ was present in the enzyme preparations, and that the metal co-purifies with collagenase during column chromatography.
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PMID:Evidence for mammalian collagenases as zinc ion metalloenzymes. 19 65

The effect of copper sulfate (CuSO4) on cultured human vascular endothelial (HVE) cells and cultured human fibroblasts (HAIN-55) was investigated. HVE cells were collected from umbilical veins by enzymatic digestion with collagenase. The viability, subsequent growth and DNA synthesis of both cell types were inhibited concentration-dependently by the addition of copper. The cytotoxic effect of copper on the morphology of these cells was also concentration-dependent. However, the cytotoxic effect of copper on the viability, subsequent growth and DNA synthesis was greater in HVE cells than in HAIN-55 cells. These results suggest that HVE cells are more susceptible to concentration-dependent copper cytotoxicity than HAIN-55 cells are, and that copper could induce vascular endothelial injury, which may be involved in the pathogenesis of cardiovascular disease.
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PMID:Injury to cultured human vascular endothelial cells by copper (CuSO4). 128 65

The involvement of leukocytes in corneal neovascularization has been known for a long time. Recent observations suggest that collagenase from leukocytes may be a common mediator for prostaglandin E1 (PGE1)- and copper-induced corneal neovascularization. This study was designed to investigate the effect of copper ion on collagenase activity from leukocytes and other sources and leukocyte infiltration in the corneal angiogenic process induced by PGE1. These results demonstrated that collagenase production from leukocytes was stimulated in a dose-dependent manner by copper ion but not by PGE1. Copper chloride 0.2 mM produced the highest stimulation. Copper ion had no effect on collagenase release from corneal fibroblasts and capillary endothelium. There were more polymorphonuclear leukocytes (PMN) in the prostaglandin E1 treated corneas than in the control. The time-course study showed that the appearance of PMN reached a peak on day 2 and new vessel growth could not be identified until day 4. These results supported an earlier suggestion that leukocytes play a role in corneal neovascularization and further suggested that copper in corneal neovascularization can stimulate the release of collagenase from leukocytes.
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PMID:Effect of copper ion on collagenase release. Its implication in corneal vascularization. 131 69

Dietary copper deficiency has been shown to reduce copper-dependent superoxide dismutase (SOD) activity and to increase lipid peroxidation in rats. Circulating reduced glutathione (GSH) concentrations are elevated in copper-deficient (CuD) rats, which suggests an increased GSH synthesis or decreased degradation, perhaps as an adaptation to the oxidative stress of copper deficiency. GSH synthesis was examined in isolated hepatocytes from CuD rats. Isolated hepatocytes were prepared by collagenase perfusion and incubated in Krebs-Henseleit bicarbonate buffer, pH 7.4, 10 mM glucose, 2.5 mM Ca2+ in the presence and absence of 1.0 mM buthionine sulfoximine (BSO), a specific inhibitor of GSH synthesis. Cell viability was assessed by trypan blue exclusion. GSH and oxidized glutathione (GSSG) were measured by the glutathione reductase recycling assay. Copper deficiency depressed hepatocyte Cu by greater than 90% and increased intracellular GSH by 41-117% over the 3-h incubation, with a two- to threefold increase in the rate of intracellular GSH synthesis. Intracellular GSSG values were minimally influenced by CuD, with a constant mol% GSSG. Extracellular total glutathione (GSH + 2GSSG) synthesis was increased by approximately 33%. Both intracellular GSH and extracellular total glutathione synthesis were inhibited by BSO. The pattern of food consumption in CuD rats, meal fed versus ad libitum fed, had no effect on glutathione synthesis. The results indicate an increased hepatic GSH synthesis as a response to dietary copper deficiency and suggest an interrelationship between the essential nutrients involved in oxyradical metabolism.
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PMID:Glutathione production in copper-deficient isolated rat hepatocytes. 155 18

Cationic glucose oxidase, prepared by amidation of its free carboxylic groups, has prolonged retention in tissues, resulting in sustained release of hydrogen peroxide generated during oxidation of endogenous glucose. Increased levels of hydrogen peroxide can inhibit superoxide dismutase activity, thereby promoting reduction of transition metal ions, particularly iron and copper, by superoxide anions. Therefore, hydrogen peroxide can generate highly reactive hydroxyl radicals through a superoxide-driven Fenton reaction. Amidated glucose oxidase injected into rabbit cornea produces corneal opacification within 3-4 days and severe corneal damage by 7 days. Ultrastructural studies revealed typical tissue lesions observed in corneal melting. Heat-inactivated amidated glucose oxidase had no effect during the first 3-4 days. However, a gradual opacification occurred thereafter, resulting in some cases, in a severe opacity by 7 days. These results are consistent with an oxidative attack on corneal glycoconjugates by radicals derived from glucose oxidase-generated hydrogen peroxide during the first 3-4 days. Invading phagocytic cells are responsible for lesions observed with the inactive enzyme and for the progression of the initial lesions caused by the active enzyme. Stimulated phagocytic cells not only produce active oxygen species during the respiratory burst, but also release neutral collagenase and acid lysosomal hydrolases that contribute to and amplify the degradation of the extracellular matrix.
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PMID:Role of active oxygen species in corneal ulceration. Effect of hydrogen peroxide generated in situ. 215 13

Synapsin I is believed to play an important role in the regulation of neurotransmitter release, since it is able to bind to synaptic vesicles, to the cytoskeleton and to membrane proteins; in addition, it bundles F-actin and microtubules. These properties, which are controlled by phosphorylation, could be explained if synapsin has different and multiple binding sites or if synapsin I is able to form polymers by self-association. In this study we present experimental evidence that synapsin I at low concentration forms self-associated dimers, as revealed after mild treatments with cross-linking agents. We have especially studied here the effects of copper/o-phenanthroline, a zero-length cross-linking agent which forms covalent links by oxidative formation of S-S bridges between adjacent cysteines. The time course and concentration-dependence of synapsin-dimer formation are studied; interestingly, these experiments could suggest a different behaviour of the two polypeptides. Limited proteolysis of phosphorylated synapsin I by V8 protease, alpha-chymotrypsin or collagenase, performed on the isolated dimer and monomer, allows us to localize tentatively in the central hydrophobic core of the molecule the cysteine residues the oxidation of which by copper/o-phenanthroline gives rise to synapsin dimers.
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PMID:Detection by chemical cross-linking of bovine brain synapsin I self-association. 251 53

Matrix vesicles (MV) isolated from chicken growth plate by collagenase digestion and incubated in 45Ca-labelled synthetic cartilage lymph (SCL) rapidly induce mineral formation. 45Ca uptake occurs in three distinct stages: (1) an initial lag period of limited accumulation, (2) a period of rapid ion uptake and (3) an extended period of slower uptake. Treatment of MV with buffered aqueous 1,10-phenanthroline (OP), a metal ion chelator, eliminated the lag period, promoting immediate, enhanced Ca2+ uptake. Analysis of MV for trace metals showed them to contain relatively high concentrations of Zn (1.58 mumol/g MV) and lesser amounts of Cu (0.07 mumol/g MV). At least 30-40% of the Zn was readily extractable in isosmotic buffers. Addition of Zn to SCL at levels as low as 5 microM completely inhibited MV mineralization; addition of OP to Zn-inhibited MV restored their ability to mineralize. The findings suggest that Zn2+ ions act as an endogenous regulator of MV Ca2+ uptake and that the normal lag period results from a competition between Zn2+ and Ca2+ for high affinity Ca2+ binding sites in the MV membrane or within the MV lumen. Other metals tested included Cu2+, Pb2+ and Cd2+ which had little or no effect on MV mineralization, Mn2+, which had an intermediate effect, and Al3+, which was found to be almost as inhibitory as Zn2+. This finding may have implications for aluminum-associated osteomalacia.
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PMID:Regulatory effect of endogenous zinc and inhibitory action of toxic metal ions on calcium accumulation by matrix vesicles in vitro. 261 45

A. niger LCF 9 synthesizes a new aspergillopeptidase of potential interest in therapeutics. The properties and operating range of the enzyme were determined. It is a semi-alkaline aspergillopeptidase (EC 3.4.23.4) with one endopeptidase activity. Its pI is 4.10, its molecular weight is 21000 Da and its A1%(1 cm) at 280 nm is 9.75. It rapidly hydrolyzes casein and hemoglobin. Its optimal pH is 7.8 and optimal temperature is 45 degrees C. It is thermally labile above 40 degrees C but can be stabilized by adding calcium ions. It is inhibited by phenylmethylsulfonyl fluoride (PMSF), by ethylenediaminetetraacetic acid (EDTA) and by certain metals ions, e.g. copper, manganese and cobalt ions. It has no dipeptidase or tripeptidase activity and its esterase activity is weak. It has a high collagenase activity and is to our knowledge the only aspergillopeptidase that is active towards benzoyl-arginine p-nitroanilide (BAPNA).
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PMID:Properties of a new alkaline proteinase from Aspergillus niger. 269 84

Active site metal substitutions for both gamma- and zeta-collagenases from Clostridium histolyticum have been made by direct metal exchange. The incubation of Co(II), Cu(II), Ni(II), Cd(II), and Hg(II) with these native collagenases results in changes in activity that parallel those observed for the reconstitution of the respective apoenzymes with these metal ions. For both collagenases, the exchange reactions with Co(II) and Cu(II) are complete within 1 min. However, the changes in activity observed on addition of Ni(II), Cd(II), and Hg(II) to gamma-collagenase and Cd(II) and Hg(II) to zeta-collagenase are time dependent. The kinetic parameters Kcat and KM have been determined for each of the active metallospecies. The substitution of the active-site metal ion in gamma-collagenase results in changes in both kcat and KM, while the effect observed in zeta-collagenase is primarily on KM. This suggests that there are differences in the mechanisms of these two collagenases, at least with respect to the role of the zinc ion in catalysis.
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PMID:Preparation by direct metal exchange and kinetic study of active site metal substituted class I and class II Clostridium histolyticum collagenases. 284 92


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