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)

Periportal and perivenous hepatocytes were isolated by the digitonin-collagenase perfusion technique. The activity of the cytosolic glutathione S-transferase was higher in perivenous cells, but the cytosolic glutathione reductase and the microsomal glutathione S-transferase activities were evenly distributed. In contrast, both the Se-dependent and the microsomal Se-independent glutathione peroxidase activity and the glucose-6-phosphate dehydrogenase activity was much lower in perivenous hepatocytes, suggesting that these cells have a lowered detoxification capacity, which may contribute to their greater susceptibility to damage by xenobiotics. The mechanism of the ethanol-induced GSH depletion in vivo was studied by incubating conventionally isolated hepatocytes. In the absence of glutathione precursors, ethanol (80 mM) did not influence the GSH content, despite accumulation of acetaldehyde (10-100 MicroM). L-Methionine or L-cysteine stimulated GSH replenishment to in vivo rates. Ethanol oxidation resulted in acetaldehyde accumulation, but did not inhibit GSH replenishment from L-methionine and even stimulated that from L-cysteine. This seems to exclude conjugation of GSH with acetaldehyde as a mechanism by which ethanol suppresses GSH levels in vivo.
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PMID:Glutathione metabolism in isolated rat hepatocytes: acinar heterogeneity of detoxifying enzymes and effects of ethanol. 342 86

The acinar distribution of glutathione S-transferase (GST), glutathione peroxidase (GPx), glutathione reductase (GR), and glucose-6-phosphate dehydrogenase (G-6-PDH) was examined by analyzing periportal (p.p.) and perivenous (p.v.) rat hepatocytes selectively isolated by the digitonin-collagenase perfusion. The cytosolic GST activity was higher in p.v. cells, but the microsomal GST and cytosolic GR were found to be evenly distributed in the acinus. In contrast, the activity of both the Se-dependent GPx and the microsomal (Se-independent) GPx, as well as G-6-PDH, was much lower in the p.v. than in the p.p. cells. The heterogeneous distribution of GST, GPx and G-6-PDH was confirmed by analyzing liver perfusion effluents collected after ante- or retrograde digitonin infusion. The relatively low activities of GPx and G-6-PDH in the p.v. cells could partly explain the susceptibility of this region to chemical injury.
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PMID:Acinar distribution of glutathione-dependent detoxifying enzymes. Low glutathione peroxidase activity in perivenous hepatocytes. 359 5

A method for the isolation of intact viable rainbow trout liver cells in high numbers is described. The technique involves perfusion of collagenase through the liver. A major part of the cytochrome P-450 in isolated liver cells was present in the oxidized non-substrate bound form. It was observed that 7-ethoxycoumarin was rapidly taken up by the liver cells and bound to cellular cytochrome P-450. The substrate binding spectrum for isolated trout liver cells was slightly modified compared with that obtained with trout liver microsomes. The microsomal affinity of 7-ethoxycoumarin, calculated as the apparent spectral dissociation constant (ks), was elevated 11-fold after fish were treated with beta-naphthoflavone, indicating a qualitative alteration in the nature of the constitutive cytochrome P-450. The metabolism of 7-ethoxycoumarin in isolated liver cells was found to be of a comparable rate to that obtained in liver microsomes. Pretreatment of fish with Clophen A50 or beta-naphthoflavone significantly increased the content of cytochrome P-450 and elevated the rate of 7-ethoxycoumarin deethylation in isolated liver cells. Furthermore, the rate of conjugation of 7-hydroxycoumarin was significantly elevated in liver cells isolated from beta-naphthoflavone treated fish when compared with the control rate. In isolated liver cells, 90% of the 7-hydroxycoumarin formed from deethylation of 7-ethoxycoumarin was further metabolized to conjugated products. However, in beta-naphthoflavone of Clophen A50 treated fish the fraction of conjugated metabolites was markedly decreased, indicating a changed balance between cytochrome P-450 dependent reactions and conjugation reactions in the cell.
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PMID:Spectral properties of substrate-cytochrome P-450 interaction and catalytic activity of xenobiotic metabolizing enzymes in isolated rainbow trout liver cells. 399 55

A technique for preparing viable and functional isolated hepatocytes from cattle liver is described. The basic procedure, which was adapted from published methods established for laboratory species, employed a two-step in vitro vascular perfusion of the caudate lobe: (1) perfusion with a calcium-free buffer containing ethylene bis(oxyethylenenitrilo)tetraacetic acid (EGTA) for removal of blood cells and extracellular calcium and (2) perfusion with calcium-fortified buffer containing collagenase for cell dissociation. Hepatocyte suspensions prepared from the caudate lobes of 20 cattle possessed a mean viability of 81.3% as determined by trypan blue exclusion. Mean yield was 2.2 X 10(7) viable hepatocytes/g of liver (wet wt). Viable hepatocytes utilized O2 at a rate 2.82 times greater than nonviable hepatocytes. Biochemical function of the hepatocyte suspensions was assessed by rates of gluconeogenesis and fatty acid oxidation. Glucose production from added lactate ranged from .88 to 1.47 mumol X min-1 X g-1 of liver tissue (dry wt). Both gluconeogenic and fatty acid oxidation rates were substantially greater in isolated hepatocytes when compared with liver slices. Isolated hepatocyte contained .398 +/- .033 (SE) nmol cytochromes P-450/mg microsomal protein and .285 +/- .025 nmol cytochrome bs/mg microsomal protein, which was comparable with amounts in liver tissue from the same animals (.568 +/- .056 and .298 +/- .033 nmol/mg protein, respectively). No significant decline of either cytochrome was detectable for isolated hepatocytes for up to 5.5 h after euthanasia. The potential usefulness of isolated bovine hepatocytes in xenobiotic metabolism studies is illustrated by the epoxidation of aldrin.
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PMID:A technique for isolation of bovine hepatocytes. 401 47

Parenchymal cells from adult rat liver have been established in primary monolayer culture. Donor animals are subjected to a partial hepatectomy and, 4 days later, cells are prepared by collagenase perfusion of the regenerated liver. The hepatic parenchymal cells, separated from nonparenchymal material and suspended in serum-free medium, are placed in plastic tissue culture dishes, where they form a monolayer within 24 h. The monolayer cells exhibit minimal mitotic activity and demonstrate several major metabolic functions characteristic of liver in vivo; these include albumin synthesis and secretion, gluconeogenesis from 3-carbon precursors, responsiveness to insulin and glucagon, glycogen synthesis, and activity of two microsomal enzymes. These functions are present in the monolayer cells for several days at activities similar to those observed in the liver in vivo. The findings indicate that hepatic parenchymal cells in this monolayer system are viable and behave in many respects like normal adult rat liver.
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PMID:Parenchymal cells from adult rat liver in nonproliferating monolayer culture. I. Functional studies. 435 60

The first method for the qualitative and quantitative evaluation of extracellular and intracellular protease activities responsible for degradation of newly synthesized collagen is described. In a double incubation method, underhydroxylated collagen chains (protocollagen) serve as substrate for protease extract and then for the indicator enzyme, 4 prolyl hydroxylase. It was possible to characterize at least four types of protocollagen sites sensible to these proteases. The microsomal fraction of chick embryo liver contained a protease active on protocollagen and whose activity was similar to that of purified human synovial collagenase.
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PMID:A method for the measurement and characterization of protease activities responsible for extracellular or intracellular degradation of collagen precursors. 609 44

Two Fischer 344 rat hepatoma cell strains, JM1 and JM2, have been isolated from a primary hepatocellular carcinoma. Primary tumor formation was induced in a two-thirds partially hepatectomized rat by a single low dose (70 mg/kg of diethylnitrosamine followed by chronic phenobarbital administration (0.1 g/100 ml drinking water). The primary tumors were passed three times by subcutaneous implantation of tumor fragments into the inguinal region of syngeneic recipients. The fourth pass was by injection of tumor cells directly into the livers of recipient rats. Several weeks later, the tumor-containing rat livers were subjected to collagenase perfusion. Two cell lines emerged from tissue culture of the cells isolated by perfusion. Each cell line was cloned by serial dilution. Cells JM1 and JM2 were tumorigenic when injected into syngeneic rats. The tumors, which arose from injected cell strains, exhibited several characteristics of hepatocellular carcinoma. Morphology was examined by light and electron microscopy. Histochemical studies of JM1 and JM2 cells grown in vitro and in vivo were done. The levels of tyrosine aminotransferase and three microsomal enzymes of importance to drug and carcinogen metabolism were investigated. To our knowledge, this is the first report of cell strains derived from an initiation promotion protocol in rats.
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PMID:Establishment of two rat hepatoma cell strains produced by a carcinogen initiation, phenobarbital promotion protocol. 613 63

Hepatocytes from rat liver were prepared by perfusion with collagenase, and rough and smooth microsomes and mitochondria were prepared after cell disruption. By applying 1000 lb/in2 (1 lb/in2 = 6.9 kPa) in a nitrogen bomb followed by decompression, 75% of the cells were disrupted after four consecutive treatments. Intact mitochondria, and rough and smooth microsomes with little contamination were prepared from the homogenate. A more rapid disruption was attained by a short sonication with a low output, thus increasing the efficiency of homogenization. The microsomal subfractions prepared from this homogenate were comparable to those obtained after decompression. Sonication resulted in smooth microsomes, which exhibited a higher contamination with non-microsomal membranes. These, however, were partly removed by additional centrifugation with a discontinuous sucrose gradient containing divalent cations.
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PMID:Fractionation of isolated liver cells after disruption with a nitrogen bomb and sonication. 618 14

After intracardial injection of [1,2-3H]dehydroepiandrosterone ([3H]DHA) into female rats, [3H]DHA was found to accumulate and was metabolized in the preputial gland, but not in the diaphragm. The identified metabolites of [3H]DHA in the preputial gland were delta 4-androstenedione-3 alpha, 17 beta-diol. Cells were isolated from the preputial gland after treatment with trypsin and collagenase III, and centrifugation in Ficoll gradients. Activity of the enzyme complex delta 5-3 beta-hydroxysteroid dehydrogenase delta 4-5-isomerase (3 beta-HSD) responsible for transforming DHA into delta 4-androstenedione was found mainly in the 105,000 g pellet (microsomal fraction) of homogenates of the isolated cells. It used preferentially NAD over NADP as a coenzyme, with a pH optimum at 8.5. The apparent Km for DHA was 5.5 X 10(-5) M, and the Vmax was 1.72 nmol/min/mg microsomal protein. These findings indicate that DHA is preferentially taken up by the preputial gland where it undergoes metabolism to form more potent androgens, and suggest that DHA may have important androgenic influence on the preputial gland.
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PMID:delta 5-3 beta-Hydroxysteroid dehydrogenase delta 4-5-isomerase activity and metabolism of dehydroepiandrosterone in rat preputial gland. 623 67

In order to facilitate the homogenization of lung tissue it was previously incubated with collagenase during 30 minutes. Morphological observations were performed in order to ascertain the cell integrity. The enzymatically digested tissue was homogenized in a 0.25 M sucrose solution containing 1 mM EDTA, 3 mM imidazole (pH.7.3) and supplemented with 1 mM imipramine in order to stabilize the mitochondria, which otherwise might contaminate the microsomal fraction. The homogenate was then centrifuged and subdivided into four fractions which were analyzed for their content in protein and for the activities of so-called marker enzymes. The cytochrome P450 level was measured in both control and 3-methylcholanthrene preparations. The activities and the kinetic parameters of lung benzpyrene hydroxylase and aldrin epoxidase were measured using the lung microsomal fractions from control and previously 3-methylcholanthrene treated rats; 3-methylcholanthrene pretreatment modified the catalytiac properties of both enzymes.
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PMID:Preparation and analysis of a lung microsomal fraction from control and 3-methylcholanthrene treated rats. 626 52


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