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)

Sulphated glycosaminoglycans have been analysed in cloned bovine aortic endothelial cells cultured on collagen gels after incubation with [3H]glucosamine and Na2(35)SO4. Radioactive products were analysed in the culture medium, in sequential collagenase and trypsin extracts of the cell monolayer and the associated extracellular matrix, and in the remaining viable cells. Heparan sulphate and chondroitin sulphate were found in each compartment: the heparan sulphate had a low degree of sulphation (approximately 0.4 N-sulphate and 0.2 O-sulphate groups per disaccharide unit on average). In the nitrous acid scission products of heparan sulphate, O-sulphated substituents were confined to disaccharide and tetrasaccharide fragments, indicating that local regions of the chain (which might be susceptible to excission by the platelet endoglycosidase) are highly sulphated. Only minor structural differences in heparan sulphate were observed between the various compartments. In contrast the chondroitin sulphate found in the collagenase extract had a higher iduronic acid content than corresponding material in the trypsin extract and the culture medium, indicating that collagenase and trypsin may extract glycosaminoglycans from different regions of the extracellular and pericellular matrix.
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PMID:Synthesis of glycosaminoglycans by cloned bovine endothelial cells cultured on collagen gels. 641 71

The effect of ascorbic acid on the synthesis, distribution and sulphation of glycosaminoglycans by human skin fibroblasts has been examined. Medium was supplemented with ascorbate over several days, and cultures incubated with [3H]glucosamine and Na2(35)SO4 for 48 h, followed by analysis of the glycosaminoglycans in the medium, in collagenase and trypsin extracts, and in cell fractions. Ascorbate feeding resulted in a reduction in hyaluronate synthesis, which was the main 3H-labelled component and was distributed mainly in the medium fractions. Sulphated glycosaminoglycans showed a reduction in incorporation of 3H label, but increased sulphation following ascorbate feeding. In control cultures 53% of 3H-labelled sulphated glycosaminoglycans and 63% of 35S-labelled glycosaminoglycans were present in the medium fraction, while in ascorbate-fed cultures, 41% of 3H label and 38% 35S label were incorporated into medium-sulphated glycosaminoglycans. Ascorbate also caused an increase in cell density and in collagen production and deposition.
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PMID:Changes in the synthesis, distribution and sulphation of glycosaminoglycans of cultured human skin fibroblasts upon ascorbate feeding. 642 Apr 22

The globular domain of collagen IV was solubilized by collagenase digestion from a mouse tumor, human placenta and bovine aorta and was purified by chromatographic methods. The materials show a unique, mainly non-collagenous amino acid composition and contain small amounts of glucosamine and galactosamine. The globular structures with Mr = 170 000 appear as a hexameric assembly originating from two collagen IV molecules. Subunits of this assembly are two different dimers Da and Db (Mr about 56 000) and monomers (Mr = 28 000). Their N-terminal amino acid sequences start with short triple-helical sequences, which overlap with the C-terminal triple helix of the alpha 1(IV) and alpha 2(IV) chain, demonstrating that the globule originates from the C terminus of collagen IV. Dimers arise from monomers by disulfide cross-linking (form Db) and/or formation of non-reducible cross-links (form Da). Reduction under non-denaturing conditions causes partial dissociation of the globule and of collagen IV dimers, indicating that reducible cross-links are formed between monomers of two different collagen IV molecules. Dissociation of the hexamer into the subunits can be achieved with 8 M urea, sodium dodecyl sulfate or in the pH range 2.5-4. The latter indicates that carboxyl groups are essential for association. Mixtures of the subunits (monomers and dimers) or purified dimers reassemble in neutral buffer into hexamers as shown by ultracentrifugation and electron microscopy. Reconstituted hexamers, however, dissociate in a much broader pH range than the native globules. Circular dichroic spectra indicate that the structure is more completely refolded from acid-treated than from urea-treated material. These data suggest that globules originating from monomers (as existing in single collagen IV molecules) are stabilized by the adjacent triple helix. Covalent cross-link formation stabilizes the globular structure and allows reconstitution in stoichiometric proportions.
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PMID:Subunit structure and assembly of the globular domain of basement-membrane collagen type IV. 669 21

Islets of Langerhans were isolated by collagenase digestion from the pancreas of a 39 year-old female renal transplant donor. The islets were subjected to three consecutive periods of tissue culture, after each of which they were incubated in vitro with various agents whose effects on insulin release from islets of laboratory animals have previously been established. After the first culture period, the basal insulin secretion rate of 5.2 microunits/islet/h seen with 2 mmol/l glucose was increased approx. 5-fold on raising the glucose concentration to 20 mmol/l. The islets retained the insulin-secretory response to 20 mmol/l glucose throughout the period of study. Insulin secretion was also stimulated by mannose, leucine, alpha-ketoisocaproate, dihydroxyacetone and 3-hydroxybutyrate, but not by fructose or N-acetyl-glucosamine. Fructose however increased insulin release in the presence of 4 mmol/l glucose. Caffeine elicited insulin release in the absence of glucose and enhanced insulin release in response to 10 mmol/l glucose. Glucose-stimulated insulin release was inhibited by trifluoperazine (25 mumol/l).
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PMID:Insulin release from human pancreatic islets in vitro. 699 14

Insoluble elastins were isolated from control and aneurytic aortas by a sequential extraction procedure involving the use of purified collagenase. Marked differences in amino acid analyses and susceptibilities to pancreatic elastase were observed between normal and pathological samples. The incorporation of either 14C-lysine or 14C-glucosamine into proteins of the vessel wall was also studied. In addition, high amounts of elastase-type activity was extractable from pathological aorta specimens which may contribute significantly to the loss of elastic tissue evidenced by ultra-structural studies and confirmed by the biochemical technics. We propose therefore that increased elastase-type protease activity in these pathological aortas does significantly contribute to the weakening of the aortic wall and also may well be the main cause of the rupture of aneurysms observed occasionally.
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PMID:Studies on elastic tissue of aorta in aortic dissections and Marfan syndrome. 703 99

1. Glycoproteins of hamster, rat and baboon lung parenchyma were investigated by using [14C]glucosamine incorporation in vitro followed by sequential extraction of the macromolecular components and characterization of the glycoproteins in the extracts. 2. Slices of lung parenchyma maintained in vitro incorporated [U-14C]glucosamine linearly with time into non-diffusible macromolecules for up to 5h. All the macromolecule-associated 14C label was present as [14C]glucosamine. 3. These 14C-labelled macromolecules were extracted from previously delipidated and salt-extracted lung by 5M-guanidinium chloride in the presence of dithiothreitol and proteinase inhibitors before (extract A1) and after (extract A2) hydrolysis of the collagen by collagenase. The [14C]glucosamine-labelled glycoproteins in extracts A1 and A2 contained 55 and 5% respectively of the total [14C]glucosamine incorporated in the lung of all three species studied. 4. The [14C]glucosamine-labelled glycoproteins were analysed by gel-filtration chromatography, sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and isoelectric focusing. The major [14C]glucosamine-labelled glycoproteins of baboon lung parenchyma had apparent mol.wts. of about 400 000, 140 000 and 65 000 with isoelectric points respectively of 4.8, 5.4 and 5.4. The hamster lung glycoproteins with isoelectric points of 4.1 and 5.8 were devoid of hydroxyproline and contained galactose, mannose and N-acetylglucosamine. These experiments indicate that several distinct glycoproteins are synthesized in situ by the cells of pulmonary parenchyma and may well play a role in its structure and function.
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PMID:Isolation and characterization of lung connective-tissue glycoproteins. 711 3

Rat submandibular gland was dissociated by enzymatic digestion with collagenase and hyaluronidase, followed by mild mechanical shearing and filtration through a nylon mesh. The dissociated cell populations contained predominantly groups of acinar cells which maintained their acinar arrangement. The morphological and functional viability of the cells was confirmed by electron microscopic examination and a normal secretory response to beta-adrenergic or cholinergic stimulation was observed. Both isoproterenol (IPR) and carbachol caused the fusion of secretory granules into large vacuoles which were also continuous with the lumen, and into which the secretory product was released. Secretion was assessed quantitatively from the incorporation of 14C-glucosamine into the acinar cells and its subsequent release into the culture medium as labelled glycoprotein. IPR stimulated secretion to 125% of untreated controls in the concentration range t x 10(-5) to 5x 10(-7) M and to 110% of controls at 5 x 10(-8) M, after 40 min incubation. Carbachol stimulated secretion to 131% of controls at 5 x 10(-5) M and to 115% at 5 x 10(-6) M but had no effect at 5 x 10(-7) or 5 x 10(-8) M. The secretory response was blocked by the respective beta-adrenergic and cholinergic antagonists, propranolol and atropine. These findings show that dissociated rat submandibular acinar cells provide a useful in vitro model for the study of mucus synthesis and secretion.
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PMID:The secretory response in dissociated acini from the rat submandibular gland. 737 55

Parenchymal cells, isolated from normal adult rat liver (3 x 10(7) cells/g liver) by collagenase perfusion and maintained in nondividing monolayer culture, were employed to investigate cell surface properties of hepatocytes. Membrane transport systems for asialoorosomucoid (A-OM) and methotrexate (MTX) were lost rapidly in culture, whereas induction of tyrosine aminotransferase and transport of alpha-aminoisobutyrate actually increased during the first 3 days. Alterations in the membrane transport systems for A-OM and MTX reflected more generalized modifications of cell surface components induced during primary culture. Thus, the binding of concanavalin A(Con A) and wheat germ agglutinin (WGA) to cultured hepatocytes increased approximately 2-fold between 24 and 96 hr, and the incorportion of radioactive mannose and glucosamine into trichloroacetic acid-insoluble proteins increased 13-fold and 4-fold, respectively. Plasma membranes were isolated from cultured hepatocytes and the major structural proteins and glycoproteins were analyzed by SDS-polyacrylamide gel electrophoresis. Membrane instability between 24 and 96 hr of culture was characterized by time-dependent alterations in specific polypeptides and extensive changes in Con A- and WGA-binding glycoproteins. Although addition of a complex hormone supplement to the medium increased the number of viable cells and sustained A-OM and MTX transport systems for 24 hr, it had no influence on the altered membrane protein and glycoprotein profiles observed in its absence.
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PMID:Membrane characteristics of adult rat liver parenchymal cells in primary monolayer culture. 741 32

A comparison has been made of the synthesis of glycosaminoglycans by human skin fibroblasts cultured on plastic or collagen gel substrata. Confluent cultures were incubated with [3H]glucosamine and Na235SO4 for 48h. Radiolabelled glycosaminoglycans were then analysed in the spent media and trypsin extracts from cells on plastic and in the medium, trypsin and collagenase extracts from cells on collagen gels. All enzyme extracts and spent media contained hyaluronic acid, heparan sulphate and dermatan sulphate. Hyaluronic acid was the main 3H-labelled component in media and enzyme extracts from cells on both substrata, although it was distributed mainly to the media fractions. Heparan sulphate was the major [35S]sulphated glycosaminoglycan in trypsin extracts of cells on plastic, and dermatan sulphate was the minor component. In contrast, dermatan sulphate was the principal [35S]sulphated glycosaminoglycan in trypsin and collagenase extracts of cells on collagen gels. The culture substratum also influenced the amounts of [35S]sulphated glycosaminoglycans in media and enzyme extracts. With cells on plastic, the medium contained most of the heparan sulphate (75%) and dermatan sulphate (> 90%), whereas the collagenase extract was the main source of heparan sulphate (60%) and dermatan sulphate (80%) from cells on collagen gels; when cells were grown on collagen, the medium contained only 5-20% of the total [35S]sulphated glycosaminoglycans. Depletion of the medium pool was probably caused by binding of [35S]sulphated glycosaminoglycans to the network of native collagen fibres that formed the insoluble fraction of the collagen gel. Furthermore, cells on collagen showed a 3-fold increase in dermatan sulphate synthesis, which could be due to a positive-feedback mechanism activated by the accumulation of dermatan sulphate in the microenvironment of the cultured cells. For comparative structural analyses of glycosaminoglycans synthesized on different substrata labelling experiments were carried out by incubating cells on plastic with [3H]glucosamine, and cells on collagen gels with [14C]glucosamine. Co-chromatography on DEAE-cellulose of mixed media and enzyme extracts showed that heparan sulphate from cells on collagen gels eluted at a lower salt concentration than did heparan sulphate from cells on plastic, whereas with dermatan sulphate the opposite result was obtained, with dermatan sulphate from cells on collagen eluting at a higher salt concentration than dermatan sulphate from cells on plastic. These differences did not correspond to changes in the molecular size of the glycosaminoglycan chains, but they may be caused by alterations in polymer sulphation.
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PMID:Synthesis of glycosaminoglycans by human skin fibroblasts cultured on collagen gels. 747 51

Resistance of gastric mucosa to damage is increased after exposure to mild irritants such as bile salts (adaptive cytoprotection). Mucus secretion also contributes to gastric cytoprotection. We investigated whether bile salts stimulate mucous glycoprotein secretion from cultured rabbit gastric mucosal cells. Because prostaglandins (PGs) stimulate mucus secretion, we assessed the role of endogenous PG release in bile salt-stimulated mucus secretion. Because Ca2+ plays a role in PGE2 release, the role of extracellular Ca2+ on PGE2 release and mucus secretion by bile salts was also studied. Rabbit gastric mucosal cells were prepared with collagenase and ethyl-enediaminetetraacetic acid. These cells were cultured as described previously. Cytotoxicity of bile salts was quantified by measuring chromium 51 release from prelabeled cells. PGE2 was measured by radioimmunoassay. Mucous glycoprotein secretion was assessed by tritiated glucosamine release assay. Deoxycholate (DC) and glycodeoxycholate (GDC) stimulated tritiated glucosamine release in doses that were not cytotoxic to the cultured cells. DC stimulated PGE2 release that was blocked by deprivation of extracellular Ca2+. GDC did not stimulate PGE2 release. Neither DC-stimulated nor GDC-stimulated mucus secretion was affected by indomethacin. Deprivation of extracellular Ca2+ did not affect DC-stimulated or GDC-stimulated mucus secretion. Bile salts stimulated mucous glycoprotein secretion from cultured rabbit gastric mucosal cells. This effect occurred independently of changes in endogenous PGE2 or extracellular Ca2+ concentrations.
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PMID:Bile salts stimulate mucous glycoprotein secretion from cultured rabbit gastric mucosal cells. 808 82


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