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:3.1.6.4 (chondroitinase)
2,039 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A dermatan sulfate proteoglycan has been isolated from a murine parietal yolk sac cell line, which in culture synthesizes basement membrane components. The proteoglycan has a molecular weight of 200,000-300,000 with 10-15 dermatan sulfate chains of Mr = 14,000-16,000. The glycosaminoglycan chains carry sulfate residues predominantly attached to C-4 of the galactosamine unit; less than 10% of the sulfate groups occur as 6-sulfated galactosamine units. About 60% of the uronic acid residues are of the glucuronic configuration, the rest being iduronic acid. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of chondroitinase ABC-treated 125I-labeled proteoglycan reveals two polypeptides with molecular weights of 34,000 and 27,000. Results from papain digestion of the proteoglycan suggest that most of the polysaccharide chains are clustered at a papain-resistant segment of the core protein (Mr = 8,000). This proteoglycan is distinctly different from the large cartilage proteoglycan in the smaller size of its core protein, and its relationship to other small chondroitin and dermatan sulfate proteoglycans and to the chondroitin sulfate proteoglycan recently located in rat tissue basement membranes will be discussed.
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PMID:Characterization of a dermatan sulfate proteoglycan synthesized by murine parietal yolk sac (PYS-2) cells. 405 55

An explant culture of 15 cynomolgus monkey corneas was incubated with [35S]sulfate and [2-3H]mannose as labeling precursors. A 4 M guanidine HCl extract of the corneal stromas was prepared and combined with a 4 M guanidine HCl extract of stromas from 300 unlabeled corneas. The keratan sulfate proteoglycans in the combined extracts were purified by a combination of DEAE-cellulose chromatography, chondroitinase ABC digestion to remove chondroitin-dermatan sulfate proteoglycans, and elution from immobilized concanavalin A. The purified keratan sulfate proteoglycan was digested with papain and the digest was eluted on DEAE-Sephacel. The unbound fraction contained 59% of the 3H activity and consisted of intact oligosaccharide-peptides. The bound fraction, consisting of keratan sulfate chains linked to peptides, eluted during a linear 0-0.75 M NaCl gradient as a peak centered at approximately 0.6 M NaCl and contained 41% of the 3H and all of the 35S activity in the original proteoglycan. The chains were digested with endo-beta-galactosidase, and the digest was eluted on DEAE-Sephacel with a linear 0-0.75 M NaCl gradient. Most of the sulfated digestion fragments from the chains eluted as several distinct peaks during the gradient. All the 3H activity eluted in the unbound volume along with a small proportion of 35S activity. This unbound fraction was eluted on Bio-Gel P-10 to give a 3H peak (Kav = 0.46) well resolved from the remaining 35S activity which eluted near the total volume. This 3H peak contained the oligosaccharide-peptides derived from the linkage region between the keratan sulfate chains and the core protein. Structural analyses of the linkage region oligosaccharides and the intact oligosaccharides (Nilsson, B., Nakazawa, K., Hassell, J.R., Newsome, D.A., and Hascall, V.C. (1983) J. Biol. Chem. 258, 6056-6063) in combination with the 3H-labeling data suggest that the intact keratan sulfate proteoglycans contain an average of about one intact oligosaccharide per keratan sulfate linkage site.
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PMID:Purification of keratan sulfate proteoglycan from monkey cornea. 622 39

Fibroblasts in culture were incubated with [35S]sulfate/[3H]glucosamine or [35S]sulfate/[3H]leucine. Proteoglycans were isolated from the medium and a 4 M guanidinium chloride extract of the cell layer or from a trypsin digest of the cells and an extract of the cell residue. Proteoglycans were isolated by density gradient centrifugation, gel permeation, and ion exchange chromatography after digesting contaminating proteogalactosaminoglycans with chondroitinase ABC. Gel chromatography suggests that the cell-derived protoheparan sulfate had an Mr = 350,000 whereas the trypsin-released and the medium-derived counterparts both had an Mr = 140,000. Reduction and alkylation of the cell-derived proteoglycan gave rise to a component with Mr = 140,000, whilst the medium-derived form was not affected. Degradation of cell-associated proteoheparan sulfate by trypsin followed by papain or alkali suggest that the core protein consists of three types of regions, heparan sulfate-containing regions of Mr = 140,000, oligosaccharide-containing regions, and nonglycosylated peptide regions containing most of the [3H]leucine. The heparan sulfates of the cell- and medium-derived proteoglycans were similar in size distribution and charge density and with regard to the proportions and arrangements of various building blocks.
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PMID:Proteoheparan sulfate from human skin fibroblasts. Isolation and structural characterization. 622 77

Glycosaminoglycan (GAG) was extracted from the porcine thyroid gland with a buffer containing 5.3 M guanidine-HCl and proteolytic enzyme inhibitors and was fractionated by subsequent isodensity CsCl centrifugation. 60% of uronic acid positive materials was accumulated in the bottom one-fourth fraction with high buoyant density. More than 90% of this uronic acid positive material in the thyroid tissue was heparin or heparan sulfate (sensitive to nitrous acid treatment) and the rest was chondroitin sulfate or dermatan sulfate (sensitive to chondroitinase ABC treatment). When the accumulated high buoyant density GAG was analyzed on a Sepharose CL-6-B column, approximately 14% of the heparin sulfate were in the macromolecular portion as a form of proteoglycan because it was destroyed by the papain digestion or alkaline borohydride treatment which extensively digests protein or releases GAG from protein by the elimination reaction, respectively. This study demonstrates the existence of heparin sulfate proteoglycan in thyroid tissue for the first time.
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PMID:Presence of heparan sulfate proteoglycan in thyroid tissue. 623 Nov 80

To investigate the chemical nature of the cationic ferritin (CF)-binding sites of the differentiated microdomains of the capillary endothelium, the vasculature of the mouse pancreas and intestinal mucosa was perfused in situ with neuraminidase, hyaluronidase, chondroitinase ABC, heparinase, and three proteases: trypsin, papain, and pronase. Proteases of broad specificity removed all anionic sites, suggesting that the latter are contributed by acid glycoproteins or proteoglycans. Neuraminidase, hyaluronidase, and chondroitinase ABC reduced the density of CF-binding sites on the plasmalemma proper, but had no effect on either coated pits or fenestral diaphragms. Heparinase removed CF-binding sites from fenestral diaphragms and had no effect on coated pits. Taken together, these results indicate that the anionic sites of the fenestral diaphragms are contributed primarily by heparan sulfate and/or heparin, whereas those of the plasmalemma proper are of mixed chemical nature. The membranes and diaphragms of plasmalemmal vesicles and transendothelial channels do not bind CF in control specimens; this condition is not affected by the enzymic treatments mentioned above.
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PMID:Differentiated microdomains on the luminal surface of the capillary endothelium. II. Partial characterization of their anionic sites. 645 53

A proteoglycan was isolated from ascites fluid produced by a rat yolk sac tumor. The glycosaminoglycan chains of the proteoglycan are all sensitive to digestion with chondroitinase ABC and about 90% are sensitive to chondroitinase AC. The proteoglycan contains 5% protein. Amino acid analysis revealed a high content of serine and glycine which together constitute 37% of the amino acids. Glutamic acid (glutamine) and aspartic acid (asparagine) are also abundant. Galactosamine accounts for 97% of the hexosamine and the remainder is glucosamine. These characteristics indicate that the glycosaminoglycan side chains of this proteoglycan are predominantly chondroitin sulfate with a smaller amount of dermatan sulfate. Antibodies to the proteoglycan were prepared by immunization of a rabbit with purified alkali-treated proteoglycan. Affinity-purified antibodies from the antiserum immunoprecipitated (35S)sulfate-labeled radioactivity from culture media of the yolk sac tumor cells known to contain chondroitin sulfate proteoglycan. This binding was inhibited by the intact purified proteoglycan but not by proteoglycan treated with papain, suggesting dependence of the reactivity of the antibodies on integrity of the protein part of the proteoglycan. Immunofluorescence of the cultured yolk sac tumor cells revealed localization of immune reactive proteoglycans at the cell surface.
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PMID:Isolation of a chondroitin sulfate proteoglycan from a rat yolk sac tumor and immunochemical demonstration of its cell surface localization. 679 88

Colon wall from pig, stripped of most of the mucosal layer to leave material largely composed of muscle, basement membrane, and extracellular matrix, was subjected to procedures for isolation of glycosaminoglycans. A total ethanol precipitate from a papain digest was fractionated by selective ethanol precipitation in the presence of Ca2+. Glycosaminoglycan fractions, freed proteolytically from a high molecular weight glycoprotein component, were further purified by Sepharose CL-6B gel-filtration or DE-52 anion-exchange chromatography. Glycosaminoglycans were identified by chemical composition, 13C-NMR spectroscopy and response to chondroitinase and nitrous acid degradations. The content of glycosaminoglycan in the tissue is low (0.05% dry weight) being comprised of dermatan sulphate (38%), heparin (34%), heparan sulphate (18%) and chondroitin sulphates (10%) as a percentage of total glycosaminoglycan content. Hyaluronic acid and keratan sulphate have not been detected. The composition is generally typical of a high muscle content tissue.
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PMID:The glycosaminoglycans of pig colonic wall connective tissue. 684 74

The biosynthesis of proteoglycans in short term organ culture of human colon and colon carcinoma was studied. Proteoglycans, labeled with [35S]sulfate and [3H]serine, were extracted with either 4 M or 0.5 M guanidine HCl in the presence of protease inhibitors and sequentially purified by associative and dissociative CsCl density gradient ultracentrifugation. Normal colon synthesized two polydisperse classes of proteoglycans: a large heparan sulfate-containing monomer, with a Kav of 0.48 on Sepharose CL-2B and a small dermatan sulfate-containing monomer with a Kav of 0.65. A portion (25%) of the proteoglycans was found as aggregate when chromatographed under associative conditions, and the larger monomers interacted with hyaluronic acid to an extent greater than the smaller proteoglycans. Following papain or alkali treatment, the free glycosaminoglycan side chains of both monomers eluted as a single broad peak (Kav = 0.5) from Sepharose CL-6B, with an estimated Mr of 20 X 10(3). In contrast, colon carcinoma synthesized only one proteoglycan monomer, which aggregated to a limited extent (12%). This proteoglycan population, with a Kav of 0.7 on CL-2B, contained chondroitin sulfate as the major glycosaminoglycan (greater than 81%), with small amounts of dermatan sulfate. The glycosaminoglycans had an estimated Mr of 9 X 10(3), and the disaccharides released by chondroitinase ABC consisted of 32% 4-sulfate and 68% 6-sulfate. Electron microscopy of mixed proteoglycancytochrome c monolayers from the associative fractions of normal and neoplastic colon revealed aggregated complexes which were similar in over structure, although smaller than the proteoglycan aggregates from cartilage.
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PMID:Isolation and characterization of proteoglycans synthesized by human colon and colon carcinoma. 710 48

The aim of this study was to investigate the nature and distribution of sulphated macromolecules of the extracellular matrix in rat gastric mucosa. This was achieved by developing an in vivo labelling system. An intraperitoneal injection of 1 mCi [35S]-sulphate was given for either 4 h (0.01% incorporation into macromolecular fraction) or 8 h (0.13% incorporation). At the end of the labelling period the stomach was removed and the mucosa and submucosa was either taken as a single combined sample or separated into four layers by blunt dissection. Each sample was papain digested and analysed by ion-exchange chromatography. This analysis revealed sulphated species of differing charge existing in differing proportions throughout the mucosa. These sulphated species eluted at NaCl concentrations of approximately 0 (A), 0.19 (B), 0.34 (C) and 0.78 mol/L (D) from a Q-Sepharose ion exchange column. Further analysis by size exclusion chromatography and chemical and enzymatic digestion showed that peaks B and C had molecular weights of 2.4 x 10(5) and 2.8 x 10(5), respectively and were resistant to chondroitinase ABC, heparitinase and nitrous acid digestion. Peak D was found to contain a polydisperse population of molecules with a molecular weight range of approximately 1 x 10(4) to 6 x 10(4). This sample was susceptible to nitrous acid and chondroitinase ABC digestion and was found predominantly in the sample isolated from deeper in the tissue. We have thus developed an in vivo labelling technique for sulphated macromolecules that can be used in the further study of injury to the gastric mucosa.
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PMID:Sulphated macromolecules produced by in vivo labelling in the rat gastric mucosa. 778 70

1. The proteoglycan peak from anion exchange chromatography of an extract of bovine aorta was digested with chondroitinase ABC. The residual heparan sulphate proteoglycans were further purified by chromatography on Sepharose CL4B and DEAE-Sephacel to yield two species, of high and low charge density. 2. Higher molecular weight material had a higher proportion of high charge density proteoglycan, while the lower molecular weight species had a higher proportion of low charge density heparan sulphate proteoglycan. 3. The two species shared epitopes as they both reacted with an antibody to heparan sulphate proteoglycan from bovine glomerular basement membrane. 4. On electron microscopy, both high and low charge density proteoglycans were visualized as 'tadpole-like' molecules, which showed a tendency to aggregate via their globular heads. 5. Bovine aortic smooth muscle cells were cultured in the presence of [35S]sulphate and [3H]glucosamine. Proteoglycans were isolated from medium and cell layer extract by the methods outlined above. 6. The major HSPG species isolated from medium were significantly larger than those from cell layer and displayed substantial heterogeneity in both size of HS chain after papain digestion and size of protein core after heparitinase digestion. 7. The major cell layer species yielded two HS species of widely differing mol. wt after papain digestion, and a very small protein core after heparitinase digestion. Therefore cell layer-associated HSPGs show a good deal more homogeneity than those found in the medium. 8. Further ion-exchange chromatography after digestion with chondroitinase ABC revealed HSPG species of lower charge density, possibly derived from a hybrid chondroitin sulphate-dermatan sulphate proteoglycan (CS/DSPG) after removal of the CS/DS chains.
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PMID:Bovine aorta contains at least two related forms of heparan sulphate proteoglycan. 813 21


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