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 glycopeptide fraction containing glucuronic acid as a component sugar was extracted and purified from squid cartilage to give a single band migrating much slower than hyaluronic acid in cellulose acetate electrophoresis. The molecular weight of the glycopeptide was fairly large since its Kav value in Sephadex G-200 chromatography was 0.18; however, it was soluble in 66% ethanol. This glycopeptide contained glucuronic acid, glucosamine, galactosamine, galactose, and fucose. The total amino acid content was 1.87 mumol of amino acid per mg of the glycopeptide. Threonine, serine and proline represented 80% of the amino acids. Digestion with chondroitinase ABC or reaction with nitrous acid did not result in degradation of the glycopeptide; however, it was completely degraded by reaction with 0.5 M KOH at 37 degrees C. Two hexasaccharides were separated from the alkaline degradation products, and they both contained glucuronic acid, fucose, galactosamine, and reducing terminal glucosamine in the molar ratio, 2:1:2:1. These results indicated that the glycopeptide contains glucuronic acid-containing sugar chains that are distinct from any known glycosaminoglycan.
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PMID:Glucuronic acid-containing glycopeptide from squid cartilage. 662 77

Rat 13762NF mammary adenocarcinoma cell surface glycoproteins from s.c. tumor- or lung metastases-derived cell clones of differing spontaneous metastatic potentials were examined for their relationship to metastasis. After treatment with neuraminidase, lectin-binding assays showed that highly metastatic clone MTLn3 cells express approximately twice the quantity of peanut agglutinin (PNA) binding sites (approximately 2.3 X 10(8) sites/cell) than clones of lower metastatic potential. However, the number of wheat germ agglutinin (WGA)-binding sites on the various cell clones decreased slightly as the metastatic potential of the clones increased. The quantities of concanavalin A (conA)-binding sites were similar (approximately 1.7 X 10(8) sites/cell) in all cell clones and growth conditions. Glycoprotein analysis was performed by electrophoresis on polyacrylamide gels in the presence of sodium dodecyl sulfate (SDS-PAGE) and subsequent staining with 125I-labeled lectins. SDS-PAGE gels stained with 125I-labeled conA revealed mainly one glycoprotein (Mr approximately 150 kD), and the amounts of this glycoprotein did not correlate with metastasis. Differences in WGA-binding glycoproteins were detected between s.c. tumor- and lung metastases-derived cell clones. Several desialylated glycoproteins were detected with 125I-labeled PNA after SDS-PAGE, and the labeling intensity of one (Mr approximately 580 kD) correlated with the metastatic potentials of the various cell clones. This high Mr galactoprotein was further analyzed by [3H]glucosamine metabolic labeling, solubilization, sequential gel filtration, and chondroitinase ABC treatment prior to SDS-PAGE. The 580 kD galactoprotein was expressed in increased amounts on the more highly metastatic clones. Chemical labeling of cell surface sialic acid residues using periodate treatment followed by [3H]borohydride reduction showed an additional change in a major sialoglycoprotein (Mr approximately 80 kD), which decreased in labeling intensity on clones of increasing metastatic potential. The results suggest quantitative changes in cell surface glycoproteins rather than major qualitative alterations are associated with differences in the metastatic behavior of 13762NF tumor cell clones.
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PMID:Cell surface glycoproteins of 13762NF mammary adenocarcinoma clones of differing metastatic potentials. 668 89

Glycosaminoglycans synthesized in polymorphonuclear (PMN) leucocytes isolated from blood (peripheral PMN leucocytes) and in those induced intraperitoneally by the injection of caseinate (peritoneal PMN leucocytes) were compared. Both peripheral and peritoneal PMN leucocytes were incubated in medium containing [35S]sulphate and [3H]glucosamine. Each sample obtained after incubation was separated into cell, cell-surface and medium fractions by trypsin digestion and centrifugation. The glycosaminoglycans secreted from peripheral and peritoneal PMN leucocytes were decreased in size by alkali treatment, indicating that they existed in the form of proteoglycans. Descending paper chromatography of the unsaturated disaccharides obtained by the digestion of glycosaminoglycans with chondroitinase AC and chondroitinase ABC identified the labelled glycosaminoglycans of both the cell and the medium fractions in peripheral PMN leucocytes as 55-58% chondroitin 4-sulphate, 16-19% chondroitin 6-sulphate, 16-19% dermatan sulphate and 6-8% heparan sulphate. Oversulphated chondroitin sulphate and oversulphated dermatan sulphate were found only in the medium fraction. In peritoneal PMN leucocytes there is a difference in the composition of glycosaminoglycans between the cell and the medium fractions; the cell fraction was composed of 60% chondroitin 4-sulphate, 5.5% chondroitin 6-sulphate, 16.8% dermatan sulphate and 13.9% heparan sulphate, whereas the medium fraction consisted of 24.5% chondroitin 4-sulphate, 28.2% chondroitin 6-sulphate, 33.7% dermatan sulphate and 10% heparan sulphate. Oversulphated chondroitin sulphate and oversulphated dermatan sulphate were found in the cell, cell-surface and medium fractions. On the basis of enzymic assays with chondro-4-sulphatase and chondro-6-sulphatase, the positions of sulphation in the disulphated disaccharides were identified as 4- and 6-positions of N-acetylgalactosamine. Most of the 35S-labelled glycosaminoglycans synthesized in peripheral PMN leucocytes were retained within cells, whereas those in peritoneal PMN leucocytes were secreted into the culture medium. Moreover, the amount of glycosaminoglycans in peritoneal PMN leucocytes was significantly less than that in peripheral PMN leucocytes. Assay of lysosomal enzymes showed that these activities in peritoneal PMN leucocytes were 2-fold higher than those in peripheral PMN leucocytes.
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PMID:Comparative study on glycosaminoglycans synthesized in peripheral and peritoneal polymorphonuclear leucocytes from guinea pigs. 669 21

Arterial basement membrane-like material was prepared by a sonication-differential centrifugation technique from cultures of rabbit aortic myomedial cells after metabolic labelling with [35S]sulphate and [3H]glucosamine. Labelled glycosaminoglycans were obtained from isolated basement membrane-like material by proteinase digestion and gel filtration. Glycosaminoglycans were identified by a combination of Sephadex G-50 chromatography and sequential degradation with nitrous acid, Streptomyces hyaluronidase, testicular hyaluronidase and chondroitinase ABC. The data showed that heparan sulphate and chondroitin sulphate were the predominant glycosaminoglycans of myomedial basement membrane-like material. Heparan sulphate accounted for about 55% of [3H]glucosamine-labelled glycosaminoglycans. In addition small amounts of hyaluronic acid was present. Only trace amounts of dermatan sulphate was found. The glycosaminoglycans were analysed by DEAE-cellulose chromatography. Two major peaks were found in the chromatogram consistent with the predominance of heparan sulphate and chondroitin sulphate.
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PMID:Glycosaminoglycans of arterial basement membrane-like material from cultured rabbit aortic myomedial cells. 671 91

In order to study the temporal and topological events involved in the processing and assembly of chondroitin sulfate proteoglycan, a fractionation scheme involving differential centrifugation and discontinuous sucrose density gradient centrifugation was developed for homogenates of chick embryo sternal chondrocytes. The precursors in these subcellular fractions were examined by a series of pulse, pulse-chase, and continuous labeling experiments. When chondrocytes were pulsed for 20 min with [35S]methionine, an immunoprecipitable core protein precursor with an approximate molecular size of 376,000 Da was localized to the rough endoplasmic reticulum fractions. Further incubation under chase conditions showed the presence of the 376,000-Da species as well as two additional polypeptides of higher molecular masses in the smooth membrane-enriched fractions within the next 2 h. This translocation did not occur in the presence of the energy transfer inhibitor carbonyl cyanide-m-chlorophenylhydrazone. The labeling pattern of the newly synthesized core protein precursor with either [3H] mannose or [3H]glucosamine showed that N-linked oligosaccharide addition was found on the earliest synthesized product in the rough endoplasmic reticulum, and the addition of this oligosaccharide was inhibited by co-incubation with tunicamycin. Furthermore, the high mannose oligosaccharide was susceptible to cleavage by endo-beta-N-acetylglucosaminidase H, while upon chase approximately 56 and 31% of the glucosamine- and mannose-labeled oligosaccharides, respectively, were processed to resistant forms, presumably in the Golgi complex. Both direct assay of glycosyl- and sulfotransferases requisite for addition of chondroitin sulfate chains and sensitivity of intracellular precursors to chondroitinase, keratanase , and endoglycosidase H suggest that only the N-linked oligosaccharides are added in the rough endoplasmic reticulum and glycosaminoglycan chain addition occurs predominantly in smooth membranes.
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PMID:Subcellular localization of the synthesis and glycosylation of chondroitin sulfate proteoglycan core protein. 672 88

The monovalent ionophore, monensin, inhibits secretion of many different proteins from a wide variety of cells. The site of blockage is at the golgi complex. We have exposed chick embryo chondrocytes in suspension culture to monensin, at concentrations ranging from 10(-8) to 10(-6) M. At the higher concentrations, between 10(-7) and 10(-6) M, monensin inhibited secretion of type II procollagen, which accumulated in the chondrocytes. At these concentrations of the ionophore, proteoglycan synthesis was inhibited, as measured by radioactive serine incorporation into core proteins and by radioactive glucosamine or SO4 incorporation into glycosaminoglycans. However, at a monensin concentration of 3 x 10(-8) M, the incorporations of serine and glucosamine were close to normal while SO4 incorporation was at 30% of control values. The ratio of glucosamine to serine in pronase-released glycosaminoglycans from culture media was unaffected by 3 x 10(-8) M monensin but the sulfate to serine ratio decreased to 29% of control values. Examination of the glycosaminoglycans by gel filtration showed a progressive increase in Kav values as sulfation decreased. Undersulfation was demonstrated by radiochromatographic analysis of the digestion products following incubation with chondroitinase ABC. The composite results show that monensin interferes with sulfation of newly synthesized proteoglycans.
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PMID:Undersulfated proteoglycans are secreted by cultured chondrocytes in the presence of the ionophore monensin. 677 Dec 62

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

Proteoglycans deposited in the basal lamina of [14C] glucosamine-labeled normal and [3H]glucosamine-labeled transformed mouse mammary epithelial cells grown on type I-collagen gels, were extracted in 4 M guanidinium chloride and cofractionated over Sepharose CL 4B. The heparan sulfate chains carried by these proteoglycans were isolated by treatment with alkaline borohydride, protease K, chondroitinase ABC, and cetylpyridinium chloride precipitation. Heparan sulfate isolated from transformed cell cultures consistently eluted from DEAE-cellulose at lower salt concentrations and was of smaller apparent Mr when chromatographed over Sepharose CL 6B, than heparan sulfate of normal cell cultures. Experiments using doubly labeled cultures ([3H]glucosamine and [35S]sulfate) demonstrated an approximately 30% reduction in the sulfate/hexosamine ratio in heparan sulfate derived from transformed cultures. Both N- and O-sulfate were decreased. The decreased Mr and decreased sulfation of heparan sulfate upon transformation appear sufficient to explain the altered heparan sulfate/chondroitin sulfate ratios previously observed in these cells. These changes may have implications for the molecular interactions in which these proteoglycans are normally engaged during basal lamina assembly, and cause the poor basal lamina formation displayed by these transformed cells.
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PMID:Transformed mouse mammary epithelial cells synthesize undersulfated basement membrane proteoglycan. 686 47

The biosynthesis of glycosaminoglycans (GAG) and glycopeptides was studied in rat kidney cortex, glomeruli, and isolated glomerular basement membranes (GBM). Rats were given four intraperitoneal injections of [(35)S]sulfate and [(3)H]glucosamine (over 10 hr) and sacrificed 14 hr after the last injection. Fractions of kidney glomeruli and purified GBM were prepared. The percent of the label incorporated into specific GAG or into glycopeptides was determined by selective degradative techniques in conjunction with gel filtration chromatography using the methods of Hart [Hart, G. W. (1976) J. Biol. Chem. 251, 6513-6521; Hart, G. W. (1978) Dev. Biol. 62, 78-98]. After digestion with Pronase and chromatography on Sephadex G-50, approximately 68% of the total (35)S radioactivity and 10-15% of the total (3)H radioactivity incorporated into cortex, glomeruli, or GBM was found in the GAG fraction, and the remainder ( approximately 32% of (35)S radioactivity and 85-90% of the (3)H radioactivity) was found in glycopeptide fractions. Treatment of GAG fractions isolated from the three sources (cortex, glomeruli, and GBM) with nitrous acid (which degrades heparan sulfates) indicated that the majority (85%, 65%, and 87%) of the (35)S radioactivity as well as the majority (60%, 50%, and 91%) of the (3)H radioactivity from all three sources was degraded by this treatment. When nitrous acid-resistant GAG from GBM were subjected to digestion with Streptomyces hyaluronidase (which degrades hyaluronic acid), approximately 6% of the (3)H-labeled material was sensitive to this treatment. The remaining (35)S- and (3)H-labeled GAG isolated from GBM were digested with chondroitinase ABC (which degrades chondroitin sulfates A and C and dermatan sulfate). Although the ratios of the types of GAG synthesized by all three sources were similar, in GBM the ratios of (35)S- to (3)H-labeled GAG and of (3)H-labeled glycopeptides to (3)H-labeled GAG were higher (2.5 times) than those found for glomeruli. The data demonstrate the synthesis of both sulfated and nonsulfated GAG by rat kidney cortex and glomeruli and their transport to and incorporation into the GBM. Heparan sulfate is the major GAG synthesized by glomeruli, but the glomeruli also synthesize smaller amounts of hyaluronic acid and chondroitin sulfates, which are in part incorporated into GBM. In addition, the renal cortex and the glomeruli synthesize glycopeptides, some of which are sulfated, and incorporate them into GBM.
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PMID:Sulfated and nonsulfated glycosaminoglycans and glycopeptides are synthesized by kidney in vivo and incorporated into glomerular basement membranes. 701 44

The synthesis of hyaluronic acid by cultured chondrocytes from the Swarm rat chondrosarcoma was examined using [3H]glucosamine as a precursor. [3H]Hyaluronate in samples was estimated as the specific unsaturated disaccharide released by incubation with chondroitinase ABC under conditions where all [3H]hyaluronate was converted to disaccharide even in the presence of excess chondroitin sulfate. The products of digestion of chondroitin sulfate and hyaluronate were well separated by cellulose thin layer chromatography. A second method involving quantitation of the 3H-oligosaccharides released by digestion with Streptomyces hyaluronidase gave nearly identical results. [3H]Hyaluronate formed about 12% of the [3H]glycosaminoglycans synthesized by the chondrocytes, although levels as high as 20% were found in one set of cultures. The incorporation of [3H]glucosamine into chondroitin sulfate and hyaluronate was linear and at a constant ratio over a 24-h period. The distributions of [35S]proteoglycans, [3H]chondroitin sulfate, and [3H]hyaluronate between the medium, a 4.0 M guanidine HCl extract of the cell layer, and the cell residue, were investigated over 24 h. Both hyaluronate and proteoglycan accumulated in the medium with little retention in the cell layer. Pulse-chase experiments indicated that a greater proportion of the [3H]hyaluronate than the [3H]chondroitin sulfate was initially retained in the cells, but that subsequently [3H]hyaluronate accumulated more rapidly in the medium, suggesting differences in the transit time through the extracellular matrix for the two molecules once secreted from the cell. In cultures labeled for 6 h, 4.0 M guanidine HCl extracted 66% of the hyaluronate and 58% of the chondroitin sulfate associated with the cell layer, while 1% Zwittergent, a zwitterionic detergent, extracted 59% and 29%, respectively. The selective extraction with detergent suggests that there is a pool of hyaluronate in the cell layer which is not associated with proteoglycan aggregates. Equilibrium density gradient centrifugation of cell layer extracts and culture media, under dissociative conditions, showed that 89% of the [3H]hyaluronate banded at densities between 1.43-1.55 g/ml with a peak at 1.47 g/ml. Disaccharide analyses of the gradient fractions showed that the main proteoglycan component contained primarily chondroitin 4-sulfate but also revealed a minor proteoglycan at lower densities which was considerably enriched in chondroitin 6-sulfate.
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PMID:Biosynthesis of hyaluronic acid in cultures of chondrocytes from the Swarm rat chondrosarcoma. 706 20


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