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)

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

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

This study determines whether calcium affects glutathione metabolism and whether glutathione metabolism may influence parathyroid (PTH) secretion in collagenase dispersed bovine parathyroid cells. Reduced glutathione (GSH) and glutathione disulfide (GSSG) were measured fluorometrically and enzymatically while PTH secretion was determined by radioimmunoassay. The total GSH and GSSG content of parathyroid cells was found to range from 1.59 to 1.71 micrograms/mg cell protein, and this did not vary significantly with changes in extracellular calcium. An increase in the medium calcium concentration from 0.5 to 2.0 mM did, however, cause an increase in GSSG from 0.43-0.54 to 1.19-1.20 micrograms/mg protein with a concomitant decrease in GSH. The compound 2-cyclohexen-1-one was used to deplete the cells of GSH at a low-calcium medium (0.5 mM) to levels seen in high-calcium medium (2.0 mM). This treatment was found to inhibit PTH secretion in the low-calcium medium, as if the cells were incubated in high medium calcium. Both 2-cyclohexen-1-one and calcium caused a rapid decrease in reduced GSH levels and in hormone secretion. The ketone was not found to affect cellular protein synthesis, indicating that there was no nonspecific toxic effect of this treatment on the cells. These results suggest that changes in the calcium concentration of the medium affect the GSH/GSSG ratio of dispersed parathyroid cells. Changes in the GSH/GSSG ratio induced by calcium may be related to changes in PTH secretion.
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PMID:Involvement of glutathione oxidation reduction in parathyroid hormone secretion. 396 88

Matrix metalloproteinases (MMPs) and neutrophil elastase (NE) may each contribute to fibrillar collagen degradation in various disease states. Little, however, is known about the activation and localization of MMP in the heart. Accordingly, we extracted MMP and examined mechanisms of proMMP activation in whole tissue extracts of the adult rat myocardium. Incubation of extracts with serine proteases (i.e., trypsin or neutrophil elastase) at 37 degrees C resulted in a time-dependent activation of proMMPs. Based on immunoblot and measurements of MMP activity by zymography, the molecular weight of active MMP was deduced to be 52 kDa. The second-order rate constant for activation of proMMP by serine protease was 5.5 +/- 0.2 x 10(5) M-1min-1 and for oxidized glutathione (GSSG) 1.5 +/- 0.1 M-1min-1. Incubation of the extract with both serine protease and GSSG increased the rate of activation 30-fold. Based on reverse zymographic analysis of collagenase inhibition, tissue inhibitors of metalloproteinases were identified. Indirect immunofluorescence localized proMMPs/MMPs to the endothelium and subendothelial space of the endocardium and throughout the interstitial space found between groups of muscle fibers. These results suggest that the mechanism of activation of MMPs by either a serine protease and by oxidizing, thiol-modifying reagents are mechanistically different and the presence of either a serine protease or GSSG synergistically increase the rate of activation of proMMPs. Our results also suggest that MMPs may be regulated by its own endogenous inhibitors. The contribution of this proteolytic enzyme to tissue remodeling and wound healing responses that occur in various diseases states remains to be established.
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PMID:Myocardial matrix metalloproteinase(s): localization and activation. 810 89

To examine the modification of reactive sulfhydryls of carbonic anhydrase III (CA III), hepatocytes were prepared by collagenase perfusion from Se deficient and Se-adequate male Sprague-Dawley rats. After 24 h in culture, hepatocytes were treated for 15-30 min with one of two oxidative stressors, t-butyl hydroperoxide (t-BuOOH) or menadione. Modification of CA III was measured by isoelectric focusing/immunoblotting. Formation of glutathione disulfide (GSSG) during oxidative stress was markedly less in hepatocytes of Se-deficient rats than in those of Se-adequate rats. During treatment with t-BuOOH, GSSG formation in hepatocytes from Se-adequate rats reached a maximum at 3 min, and then GSSG was gradually reduced to glutathione. After menadione treatment, intracellular GSSG irreversibly increased in hepatocytes of Se-adequate rats but not in those of Se-deficient rats. A modification of CA III that was reversible by dithiothreitol treatment concurred with the formation of GSSG during treatment with either t-BuOOH or menadione. Although modification of CA III occurred in hepatocytes from Se-deficient rats, the extent of modification was significantly less than in Se adequacy, and the modification was less reversible by dithiothreitol than in hepatocytes from Se-adequate rats. Selenium deficiency may be useful in examining the importance of modification of specific proteins subjected to oxidative stress.
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PMID:Selenium deficiency suppresses the S-glutathiolation of carbonic anhydrase III in rat hepatocytes under oxidative stress. 836 Jul 74

Latent matrix metalloproteinases (MMPs) in normal myocardium are activated in end-stage heart failure. In vitro oxidized glutathione (GSSG) activates myocardial MMPs which contains a cysteine residue. In vivo GSSG induce the collagen lysis and cardiac dilatation. To assess whether thiol and non-thiol reducing agents have direct effect on the interstitial human heart fibroblast (HHF) proliferation and MMP expression, HHF and polyoma virus transformed fibroblast cells were cultured with or without the thiol-containing reduced (GSH) or oxidized (GSSG) glutathiones, pyrrolidine dithiocarbamate (PDTC) and N-acetylcysteine (NAC), and non-thiol ascorbic acid. After 100 micrograms/ml (approximately 0.3 mM) GSH or PDTC treatment the proliferative (synthetic) phenotype of transformed fibroblast cells was changed to quiescent (contractile) phenotype. Also, after GSH, PDTC, and ascorbic acid treatment the medium was then analyzed for MMP activity by zymography. The results indicate reduction in MMP expression in transformed fibroblast cells after GSH and PDTC treatments and no effect after ascorbic acid treatment. Based on reverse zymography, we observed the level of tissue inhibitor of metalloproteinase (TIMP) at a decreased level in transformed cells. The effect of the reducing agent at the gene transcription was measured by estimating mRNA (Northern blot analysis) of MMP and of TIMP in the cells that were cultured in medium in the presence and absence of GSH. These results indicate that GSH induces MMP-2 and MMP-1 expression in normal HHF and that GSH reduces MMP-2 and MMP-1 in transformed fibroblast cells. After the treatment, the TIMP-2 level was repressed in normal HHF and TIMP-2 level increased in transformed fibroblast cells. These events are dependent on the nuclear transcription factor activity on the collagenase promoter in normal HHF cells. On the other hand, in polyoma transform fibroblast cells these events are not dependent on this collagenase promoter. These results suggest that oxidative environment induces normal HHF cell proliferation, and the reducing agent decreases normal HHF cell proliferation by inducing MMP and repressing TIMP gene transcription. In transformed cells reducing agents inhibit MMP expression and increase TIMP levels, which suggests a role of antioxidants in preventing tumorigenesis.
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PMID:Reduction-oxidation (redox) state regulation of extracellular matrix metalloproteinases and tissue inhibitors in cardiac normal and transformed fibroblast cells. 872 63

Paraquat (PQ) induces lung, liver and kidney damage. Since PQ mainly is eliminated by the kidney, the kidney damage is of particular importance to the outcome of PQ poisoning. The exact toxic mechanism of PQ is still unclear but it is assumed to involve redox cycling and formation of reactive oxygen species. In this study, further investigations on the toxic mechanism and metabolic effects of PQ were performed using isolated renal proximal tubules from rabbits. Proximal tubules were isolated using a combined iron perfusion and collagenase method. Suspended tubules were incubated for varying periods and concentrations of PQ at 25 or 37 degrees C in Krebs-Ringer phosphate buffer or HCO3-/CO2 buffer. The cytotoxic effect of PQ was evaluated by (1) markers of oxidative stress: status of glutathione (GSH/GSSG) and formation of malondialdehyde (MDA); and (2) markers of tubular metabolism: oxygen consumption (QO2), transport of 14C-p-aminohippuric acid (PAH) and 14C-tetraethylammonium (TEA). Using 0.5 and 5 mM PQ, the GSH/GSSG ratio decreased whereas formation of MDA increased indicating oxidative stress. PQ reduced the accumulation of PAH and TEA, the basal QO2 and the ouabain sensitive QO2 indicating inhibition of the Na/K-ATPase. Nystatin-stimulated QO2 was reduced by PQ, excluding inhibition of Na+ entry as a possible cytotoxic mechanism and suggesting mitochondrial injury. This was confirmed by measuring FCCP-uncoupled QO2. Thus high concentrations of PQ appear to disrupt mitochondrial electron chain transfer resulting in reduction of metabolic functions.
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PMID:The cytotoxic effect of paraquat to isolated renal proximal tubular segments from rabbits. 927 8

The purpose of this study was to determine if exacerbation of apoptosis precedes liver injury during chronic exposure of rats to alcohol. After 7 weeks of feeding an alcohol- or dextrin-containing liquid diet, the animals were treated with gram-negative bacterial lipopolysaccharide (1 mg x kg(-1) body weight, intravenously) or sterile saline and sacrificed 3 hr after the treatment. Alanine:2-oxoglutarate aminotransferase (ALT) and lactate:NAD oxidoreductase [lactate dehydrogenase (LDH)] were measured in plasma. The caudate lobe of the liver was resected for histology, while the rest of the organ was perfused with collagenase to isolate hepatocytes, Kupffer cells (KCs), and sinusoidal endothelial cells (SECs) by centrifugal elutriation. Hepatocyte mitochondria were isolated by differential centrifugation of the cell homogenate. Reduced and oxidized glutathione (GSH and GSSG) in isolated hepatocytes and hepatocyte mitochondria, and malondialdehyde in hepatocytes were assayed. Caspase-3 activity and Fas ligand mRNA expression were determined in hepatocytes, KCs, and SECs. Plasma ALT and LDH activity, liver histology, GSH, GSSG and their ratio, and malondialdehyde content were not affected by alcohol treatment Caspase-3 activity was significantly increased in alcohol-treated rats in all three cell types, with the lowest response observed in hepatocytes and the highest in KCs. Fas ligand mRNA expression, which had the highest level in SECs, followed by KCs and hepatocytes, was not affected by alcohol administration. Lipopolysaccharide had the following effects: an increase in ALT in both pair- and alcohol-fed rats, and LDH only in alcohol-fed rats, a decrease in GSH + GSSG levels in both mitochondria and hepatocytes, an elevation of malondialdehyde content in hepatocytes, a raise in caspase-3 activity in all groups and cell types, and an augmentation of Fas ligand expression in hepatocytes and KCs, but not in SECs. These data suggest that, during chronic alcohol consumption, an exacerbated apoptosis precedes alcohol-induced liver injury.
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PMID:Modulation of caspase-3 activity and Fas ligand mRNA expression in rat liver cells in vivo by alcohol and lipopolysaccharide. 1006 67

Pulmonary fibrosis (PF) is a major side effect of radiotherapy and chemotherapy. Recent clinical trials, unfortunately, have failed to identify any therapeutic agent which has the potential to reduce the consequences of this devastating condition. Reactive oxygen species and tissue remodeling regulators, such as metalloproteinases (MMPs) and their inhibitors (TIMPs), are thought to be involved in the development of PF. We investigated these factors to determine the protective effects of antioxidant alpha-lipoic acid (LA) against antineoplastic agent bleomycin (BLM)-induced oxidant lung toxicity in Sprague-Dawley rats. At different time intervals after BLM administration, pathological changes of the lung were analyzed with the measurement of total protein in bronchoalveolar lavage fluid (BALF), hydroxyproline (HYP) content and the level of three oxidative stress markers, i.e. malondialdehyde (MDA), the GSH/GSSG ratio, and total antioxidative capability (T-AOC). Also, the expression changes of MMP-1 and TIMP-1 were measured. At day 14 or 28 after BLM administration, protein content in BALF, and HYP, MDA and T-AOC contents of the lung increased significantly with a decreased GSH/GSSG ratio, implicating an increased efflux of GSSG from the lung and consumption of GSH. In contrast, treatment with LA protected BLM-induced pulmonary injury by suppressing oxidative stress with the reduction of MDA, and the enhancement of the GSH/GSSG ratio and T-AOC. The BLM-stimulated symptoms of PF were relieved with significant reduction of HYP and total proteins in LA-treated rats. LA also ameliorated the MMP-1/TIMP-1 ratio. These results suggest that LA inhibits BLM-induced lung toxicity associated with oxidative damage. Therefore, antioxidant LA has a potential therapeutic effect in the prevention and alleviation of PF.
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PMID:Therapeutic effects of alpha-lipoic acid on bleomycin-induced pulmonary fibrosis in rats. 1748 18

Pulmonary fibrosis (PF) leads to chronic inflammation and accumulation of macrophages, neutrophils, and lymphocytes in the alveoli. The factors involved in the development of PF include reactive oxygen species and tissue remodelling regulators. The present study demonstrates the effect of andrographolide on bleomycin (BLM)-induced PF in Sprague-Dawley rats. We investigated the total bronchoalveolar lavage fluid protein (BALF) and hydroxyproline (HYP) content along with the level of oxidative stress markers like malondialdehyde (MDA) and GSH/GSSG ratio. In addition, the levels of MMP-1 and TIMP-1 were also analysed. The results revealed an increase in BALF protein, HYP, and MDA contents and decrease in GSH/GSSG ratio of the lungs in animals treated with BLM. However, andrographolide treatment caused a reversal of the BLM induced changes after 20 or 40 days. Treatment with andrographolide suppressed oxidative stress with the decrease of MDA and the increase of the GSH/GSSG ratio. Andrographolide also improved the BLM mediated changes in the MMP-1/TIMP-1 ratio. Therefore, andrographolide has a potential therapeutic effect in the prevention of PF.
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PMID:Andrographolide plays an important role in bleomycin-induced pulmonary fibrosis treatment. 2655 Jan 47


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