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)

The amount of glycosaminoglycan (GAG) in dry costal cartilage tissue of rats decreased with aging, while the GAG content in mg DNA (unit cartilage cell) remained the same with aging. These results can be explained by the finding that the total number of cartilage cells decreased with aging. Electrophoretic analysis showed that chondroitin 4-sulfate was the major GAG in rat costal cartilage of various ages. Rat costal cartilage of different ages was incubated with radioactive precursors, and newly synthesized GAG was prepared and the radioactivity analyzed to determine the biosynthetic activity. As to changes in the radioactivity uptake with aging per mg dry cartilage tissue, aging influenced [35S]sulfate incorporation into GAG more significantly than [3H]glucosamine incorporation into GAG. There was a significant decrease in the specific radioactivity of [35S]sulfate per mg DNA (unit cartilage cell), whereas the specific radioactivity of [3H]glucosamine per mg DNA did not change significantly with aging. Both the total sulfotransferase activity and the specific activity per mg DNA decreased significantly with aging. Analysis of disaccharide units formed after chondroitinase ABC digestion of labeled GAG isolated from young and old cartilage showed that the percentage of incorporation of [3H]glucosamine into deltaDi-OS increased significantly with aging. These results suggested that the appearance of nonsulfated positions in the structure of the chondroitin sulfate chain increased with aging. On the basis of gel chromatography on Bio-Gel A-1.5 m no significant difference in the approximate molecular size of chondroitin sulfate was observed between the young and old GAG samples. The present study indicated that the sulfation of chondroitin sulfate chains from rat costal cartilage decreased with the process of aging.
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PMID:The effect of aging on the synthesis of hexosamine-containing substances from rat costal cartilage. A decrease in sulfation of chondroitin sulfate with aging. 42 44

Confluent cultures of human endothelial cells deposit into extracellular matrix (ECM) distinct heparan sulfate proteoglycans (HSPG) which modulate acidic fibroblast growth factor's (aFGF) ability to stimulate human endothelial cell mitogenic capacity. Extracellular matrix 35S-HSPG were isolated from cultures metabolically labelled with Na235SO4 by DEAE-Sepharose, Sepharose CL-4B, and aFGF-Affi-Gel 15 column chromatography and identified by resistance to chondroitinase ABC and sensitivity to nitrous acid. Fifty to sixty percent of the 35S-HSPG deposited into ECM do not bind aFGF. The bound 35S-HSGP (40-50% of the total counts applied) eluted from the aFGF-Affi-Gel column after the addition of buffer containing 2 M NaCl. aFGF-binding and aFGF-nonbinding 35S-HSPG were individually pooled and further purified by Sepharose CL-4B column chromatography. 35S-HSPG which bind aFGF, designated HSPGP, were 100-fold superior to heparin in augmenting the mitogenic efficacy of aFGF in sparse proliferating cultures. In contrast, however, 35S-HSPG, which did not bind aFGF, designated HSPG1, inhibited aFGF-stimulated proliferation in both sparse and subconfluent endothelial cell cultures. The majority of the biological activity of both aFGF-potentiating HSPGP and aFGF-inhibitory HSPG1 was contained in the glycosaminoglycan chains released by alkaline borohydride treatment of intact HSPGP or HSPG1, respectively. 3H-Core protein derived from HSPGP or HSPG1 contained only minor biological activity. The ability of heparitinase or heparinase (Flavobacterium heparinum) to abolish biological activity differed, depending upon the HSPG tested, also suggested that these are two distinct HSPGs.
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PMID:Extracellular matrix heparan sulfate proteoglycans modulate the mitogenic capacity of acidic fibroblast growth factor. 252 52

Administration of (D+) catechin (100 mg/kg body wt) to rats resulted in an increase in the amount of total sulphated glycosaminoglycans (GAG) in liver. The increase was more pronounced in the case of heparan sulphate than chondroitin sulphate and dermatan sulphate. The liver slices prepared from catechin-treated rats showed a significant increase in the rate of incorporation of 35S-sulphate into GAG. Similarly there was a concentration-dependent increase in the rate of 35S-sulphate incorporation into GAG by normal liver slices in presence of catechin in vitro. Susceptibility to nitrous acid degradation and chondroitinase ABC digestion showed that more than 80% of the GAG labelled in vivo with 35S-sulphate, was heparan sulphate and about 10% chondroitin sulphate and dermatan sulphate. Gel filtration of the 35S-labelled material isolated from livers of normal and catechin-treated animals over sephacryl S-300 did not show any difference probably excluding the possibility of free GAG chains initiated on catechin or any of its metabolites in vivo. These results indicate that catechin stimulates the synthesis of sulphated GAG, particularly heparan sulphate in liver.
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PMID:D(+)catechin enhances heparan sulphate content in rat liver. 253 88

Two discrete peptido-keratan sulphate fragments were isolated via chondroitinase ABC and trypsin digestion of a proteoglycan aggregate fraction prepared from bovine femoral head cartilage (six year old animals). The larger fragments (K(av) = 0.07, CL-6B) contained peptides substituted with several keratan sulphate (KS) chains from the KS-rich region of the proteoglycan and the smaller fragments (K(av) = 0.5, CL-6B) contained peptides with, perhaps, only one KS chain and the stubs of post-chondroitinase-treated chondroitin sulphate chains. The two peptido-KS samples and the KS chains derived from these by alkaline borohydride reduction were characterised by 13C-NMR spectroscopy. The two populations of KS chains were also examined by chromatography (Sephadex G-75), and keratanase digestion followed by chromatography on Bio-Gel P-10. From the results it was concluded that the KS chains from the two major trypsin-derived peptido-KS fragments had similar sulphation levels, distributions of hydrodynamic sizes and susceptibilities to keratanase.
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PMID:Structural studies of two populations of keratan sulphate chains from mature bovine articular cartilage. 253 85

In a previous study we described a family of monoclonal antibodies directed against tracheal antigens having a variety of cellular and subcellular distributions. In the present study, we have extended our findings on four representative antibodies to determine the periodate sensitivity, glycosidase sensitivity, and apparent molecular weight of the corresponding antigens. Since mild periodate oxidation selectively cleaves carbohydrate moiety leaving amino acids intact, loss of antigenicity following this treatment suggests the involvement of sugar residues in the antigenic determinant. This can be confirmed by testing the sensitivity of the antigens to specific glycosidases. By enzyme-linked immunosorbent assay (ELISA), all four antibodies were found to have highest affinity for void volume components isolated by Bio-Gel A15m chromatography of the total tracheal secretion. Further analysis of this void volume material by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions followed by immunoblot analysis revealed that all antigens were carried by high-molecular-weight species (greater than 200,000) which were periodate-Schiff positive but reacted poorly with Coomassie blue. In parallel experiments using immunofluorescence and ELISA, antibody binding was compared under control conditions and following periodate treatment of antigens under varying intensities (10 mM IO4-, 10 min, 4 degrees C; 50 mM IO4-, 1 h, 4 degrees C; 100 mM IO4-, 12 h, 20 degrees C). Similar results were obtained with the two methods, indicating a partial loss of antigenicity for one of the four antigens following the mildest periodate treatment, and total loss of antigenicity for all four antigens following each of the two prolonged treatments. All four antigens showed marked sensitivity to digestion with mixed exoglycosidases and three antigens were also susceptible to endo-beta-galactosidase digestion. Antigenicity was not decreased during incubation with chondroitinase ABC, heparitinase, or heparinase. Immunofluorescence analysis of tracheal tissue sections showed that the four antibodies recognized determinants in different locations, including gland and goblet cell cytoplasmic granules and the apical epithelial membrane. The characteristic immunofluorescence patterns of all antibodies were abolished by periodate incubation of the tracheal sections. Thus, the four antibodies appear to recognize carbohydrate antigens carried by high-molecular-weight glycoproteins, each with different cellular origins.
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PMID:Tracheal carbohydrate antigens identified by monoclonal antibodies. 301 42

Nuclei pulposi were dissected from lumbar discs of radiologically normal human spines of cadavers aged 17, 20 and 21 years. Proteoglycans were extracted with 4 M guanidine hydrochloride (dissociative conditions) with proteinase inhibitors and isolated as A1 fractions by associative density-gradient centrifugation. Aggregating and non-aggregating proteoglycans were separated by Sepharose 2B chromatography. Both aggregating and non-aggregating proteoglycans contained a keratan sulphate-rich region as isolated by chondroitinase/trypsin/chymotrypsin digestion and Sepharose CL-6B chromatography. Agarose/acrylamide-gel electrophoresis of individual fractions of a Bio-Gel A-50m dissociative-column separation of the aggregating proteoglycans revealed two, well-separated bands: S and F, the slower and faster migrating bands respectively. The non-aggregating proteoglycan fractions were eluted under associative conditions (0.5 M-sodium acetate, pH 6.8) and migrated as a single band in the electrophoretic system. The gel-electrophoretic heterogeneity of the aggregating proteoglycans was still evident after hydroxylamine fragmentation and removal of the hyaluronate-binding portion of the molecule. Dissociative density-gradient centrifugation of the aggregating proteoglycans partially separated the Band-S proteoglycans from the Band-F population. Subsequent dissociative chromatography of the high-buoyant-density Band F proteoglycans permitted discrimination of this band into two gel-electrophoresis-distinguishable populations (Bands F-1 and F-2). Enzyme-linked immunosorbent assays with a monoclonal antibody that recognized keratan sulphate demonstrated that the D1 fraction containing the Band F-1 proteoglycans was enriched in keratan sulphate compared with the total aggregating or non-aggregating pool of proteoglycans. The proteoglycans of young adult nucleus pulposus could then be ascribed to one of four structurally and/or electrophoretically distinct populations: (1) the non-aggregating population, which comprised about 70% of the total extractable proteoglycans; (2) the aggregating pool, comprising: (a) Band F-1 proteoglycans, which had a relatively large hydrodynamic size, uronate/protein weight ratio, were enriched in keratan sulphate and had a high buoyant density; (b) Band S proteoglycans, which migrated slower in agarose/acrylamide gels, had a smaller hydrodynamic size, lower buoyant density and a lower uronate/protein ratio than the Band F-1 population; (c) Band F-2 proteoglycans, which were lower in buoyant density, smaller in hydrodynamic size and slightly faster in electrophoretic mobility than the Band F-1 proteoglycans.
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PMID:Proteoglycans of the human intervertebral disc. Electrophoretic heterogeneity of the aggregating proteoglycans of the nucleus pulposus. 304 61

The glycosaminoglycans that exist in rabbit bone marrow were analyzed chemically, and their in situ localization was studied immunohistochemically. Femoral bone marrow of 3-month-old rabbits was defatted with organic solvents. Glycosaminoglycans were prepared from the defatted tissue after its digestion with pronase, treatment with mild alkali, and then digestion with DNase-I. The tissue contained glycosaminoglycans equivalent to 195 mg of hexosamine per femur, which accounted for 27.3% of the total hexosamine in the tissue. Studies with hyaluronidase from Streptomyces hyalurolyticus and chondroitinase ABC showed that the glycosaminoglycans were composed of hyaluronic acid (16% of the total glycosaminoglycan) and chondroitin 6-sulfate (79%). The chondroitin 6-sulfate was separated on Bio-Gel A-0.5m gel into two molecular species with mol wt of greater than 12,000 (Kd greater than 0.2) and approximately 8,000 (Kd = 0.47). Bone marrow digested with chondroitinase ABC and then treated with three monoclonal antibodies 4/8/9-A-2, 5/6/3-B-3, and 5/6/1-B-5, which were specific for unsaturated 4-sulfated, 6-sulfated, and nonsulfated disaccharide structures, respectively, at the nonreducing end of chondroitin sulfate chains, reacted with only 5/6/3-B-3. This result indicated that the chondroitin sulfate, isomer in the bone marrow is chondroitin 6-sulfate, consistent with the biochemical results. The chondroitin 6-sulfate was localized mainly in the extracellular compartment and was considered to be involved in construction of the hemopoietic microenvironment in the bone marrow.
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PMID:Isolation, characterization, and localization of glycosaminoglycans in rabbit bone marrow. 311 61

The cell-associated proteoglycans synthesized by three dog mastocytoma cell lines were isolated and their structural features compared. The lines were propagated as subcutaneous tumors in athymic mice for over 25 generations. In primary cell culture, all three lines incorporated [35S]sulfate into high molecular weight proteoglycans which were heterogeneous in size and glycosaminoglycan content. Two lines, BR and G, synthesized both a heparin proteoglycan (HPG) and a chondroitin sulfate proteoglycan (ChSPG) in different proportions. The third line, C2, synthesized predominantly a ChSPG with little or no detectable heparin. Gel filtration of the 35S-labeled HPG and ChSPG from the BR line on Sepharose CL-4B in dissociative conditions (4 M guanidine, Triton X-100) yielded a major polydisperse peak (Kav = 0.22) accounting for 70% of 35S activity. Under aggregating conditions (0.1 M sodium acetate) on Sepharose CL-4B, the BR proteoglycans eluted in the excluded volume. Proteoglycans from lines G and C2 also eluted in the void volume under nondissociative conditions, however the C2 line yielded additional fractions of smaller hydrodynamic size (Kav = 0.81) suggesting the presence of intracellular proteoglycan cleavage products or incompletely processed proteoglycans. As assessed by dissociative chromatography on Sepharose CL-4B, proteoglycans from the BR line were resistant to proteinase cleavage under conditions which degraded a rat chondrosarcoma proteoglycan. For all lines, glycosaminoglycans released by pronase/alkaline-borohydride had molecular weights ranging from 20,000 to 50,000 on gel filtration. For line BR, 75% of 35S-labeled glycosaminoglycans were degraded to oligosaccharides by nitrous acid, and the remaining 25% were degraded by chondroitinase ABC. Corresponding percentages for line G were 89% and 11%, and for line C2, 2% and 98%. Paper chromatography of the chondroitinase digestion products from lines BR and C2 showed products corresponding to unsaturated standards delta Di-diSB and delta Di-diSE, derived from the disaccharides IdoUA-2-SO4----GalNAc-4-SO4 and GlcUA----GalNAc-4,6-diSO4 respectively, in addition to smaller amounts of monosulfated disaccharides. Glycans from lines C2 and BR contained small quantities of a trisulfated disaccharide which was degraded to delta Di-diSB upon incubation with chondro-6-sulfatase. The results demonstrate the simultaneous presence of heparin and polysulfated chondroitin sulfate in dog mast cells of clonal origin.
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PMID:Dog mastocytoma proteoglycans: occurrence of heparin and oversulfated chondroitin sulfates, containing trisulfated disaccharides, in three cell lines. 314 22

35S-labelled heparins were recovered from adipose tissue, hearts, lungs, peritoneal cavities and skins of rats given H2(35)SO4. Their purification involved incubation with Pronase, precipitation with cetylpyridinium chloride in 1.0 M-NaCl, gradient elution from DEAE-Sephacel and incubation with chondroitinase ABC. Each product was divided into proteoglycan and "depolymerization products' fractions by gel filtration on Bio-Gel A-15m. Heparin chains were released from a portion of each proteoglycan fraction by beta-elimination with NaOH. Proteoglycans, chains and depolymerization products were separated by gradient elution from a column of antithrombin-agarose into fractions with no affinity, low affinity and high affinity for antithrombin. The relative sizes of the products were determined by gel filtration on columns of Bio-Gel A-50m, A-15m, A-1.5m and A-0.5m. Skin was the major source of heparin and contained the largest proteoglycans and the lowest proportion of depolymerization products. Lungs contained the smallest proteoglycans, the smallest depolymerization products and the highest proportion of depolymerization products. The highest proportions of proteoglycans, chains and depolymerization products with high affinity for antithrombin were found in adipose tissue. The lowest proportions of each of these fractions were found in the peritoneal cavity. The data suggest that there was relatively little biosynthesis of sites with high affinity for antithrombin in peritoneal-cavity mast cells and that heparin catabolism was most active in lungs. Each source of heparin was unique with respect to both biosynthesis and subsequent breakdown of its proteoglycans.
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PMID:Rat heparins. A study of the relative sizes and antithrombin-binding characteristics of heparin proteoglycans, chains and depolymerization products from rat adipose tissue, heart, lungs, peritoneal cavity and skin. 382 37

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


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