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)

Rat ovarian granulosa cells were isolated from immature female rats after stimulation with pregnant mare's serum gonadotropin and maintained in culture. Proteoglycans were labeled with [35S]sulfate, [3H]glucosamine, [3H]serine, or [3H]mannose as precursors. A low buoyant density dermatan sulfate proteoglycan was separated from a larger hydrodynamic size, high buoyant density dermatan sulfate proteoglycan and from a heparan sulfate proteoglycan using DEAE-Sephacel and Sepharose CL-4B chromatography under dissociative conditions in the presence of detergent. This low buoyant density dermatan sulfate proteoglycan, which constituted approximately 30% of the 35S-labeled proteoglycans in the culture medium, has a relatively small hydrodynamic size (Kd = 0.45 on Sepharose CL-4B) and shows a broad buoyant density distribution in CsCl density gradients, primarily due to the heterogeneity in glycosaminoglycan composition. The average molecular weight of the protein coreoligosaccharide complex obtained by chondroitinase ABC digestion of the proteoglycan was estimated to be approximately 230,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After digestion with chondroitinase ABC, the dermatan sulfate chains (average Mr = 33,000) yielded 81% 4-sulfated disaccharides and 17% disulfated disaccharides (sulfate groups on the 6-position of the galNAc and probably on the 2-position of the iduronic acid). Alkaline borohydride treatment of this proteoglycan released three distinct size species of oligosaccharides; a species of N-linked oligosaccharide which contains mannose, glcNAc, and sialic acid, and two species of O-linked oligosaccharides. Trypsin digestion of the proteoglycan generated fragments which contain (a) glycosaminoglycan-peptides with an average of 2 dermatan sulfate chains, (b) clusters of O-linked oligosaccharide-peptides, and (c) N-linked oligosaccharide-peptides.
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PMID:Characterization of low buoyant density dermatan sulfate proteoglycans synthesized by rat ovarian granulosa cells in culture. 641 57

Purified proteoglycan subunits from human articular, bovine articular and nasal cartilages, and a rat chondrosarcoma were phosphorylated in vitro by beef heart cAMP-dependent protein kinase in the presence of gamma 32P-ATP. In these experiments, a maximum of 1.7 moles of 32P were incorporated per mole of proteoglycan from human cartilage. Phosphorylation was dependent on the presence of cAMP. Analysis by autoradiography revealed that serine residues in the core protein of the proteoglycan were the sites of phosphorylation. Treatment of proteoglycan subunits with chondroitinase ABC and alkaline phosphatase prior to reaction with cAMP-dependent protein kinase increased the incorporation of 32P by 12-30% when compared with untreated proteoglycans. These data indicate that proteoglycans in cartilage can be phosphorylated by cAMP-dependent protein kinase.
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PMID:Phosphorylation of proteoglycans from human articular cartilage by a cAMP-dependent protein kinase. 647 53

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

Deglycosylation of bovine skin proteodermatan sulfate with chondroitinase ABC yielded a protein core with an apparent molecular weight of about 45,000. The amino acid sequence of this preparation was determined up to position 24. This region was enriched in acidic amino acids and proline compared with the whole protein core and it was predicted to be highly folded. The amino acid sequence determined in these experiments has a gap at position 4. Results obtained after beta-elimination-sulfite addition showed that residue 4 was an O-substituted hydroxyamino acid. The latter was identified as serine by sequencing the NH2-terminal region of the protein core (Mr approximately 43,000) isolated after a more complete deglycosylation of the proteoglycan with anhydrous HF. Serine 4 may be an attachment site for one of the few dermatan sulfate chains present in the proteoglycan.
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PMID:The NH2-terminal amino acid sequence of bovine skin proteodermatan sulfate. 665 8

Rat chondrosarcoma chondrocytes were cultured in the presence of puromycin to induce premature termination of core protein precursor. The structure and function of intracellular and extracellular proteoglycans were assessed by molecular sieve chromatography and polyacrylamide gel electrophoresis. [3H]Serine incorporation was maximally inhibited by 3 X 10(-4) M puromycin but unaffected by 10(-5) M puromycin. Proteoglycans synthesized in the presence of puromycin exhibited increased monomer size due to increased chondroitin sulfate chain size, typical of proteoglycans synthesized in the presence of protein synthesis inhibitors, but no loss in ability to bind to hyaluronic acid; and no loss in core protein size was observed after treatment with chondroitinase. These data suggest that chondrocytes select only completed or nearly completed core protein molecules to process into proteoglycans.
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PMID:Effect of puromycin on cartilage proteoglycan structure and capacity to bind hyaluronic acid. 671 56

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

When slices of adult rabbit articular cartilage were incubated in culture medium, the rate of incorporation of [35S]sulphate or [3H]acetate into glycosaminoglycans increased 4-8 fold during the first 5 days of incubation. Similar changes in biosynthetic activity were observed during culture of adult bovine cartilage. The activation of synthesis was not serum-dependent, but appeared to be a result of the depletion of tissue proteoglycan that occurs under these incubation conditions [Sandy, Brown & Lowther (1978) Biochim. Biophys. Acta 543, 536--544]. Thus, although complete activation was observed in serum-free medium, it was not observed if the cartilage was cultured inside dialysis tubing or in medium containing added proteoglycan subunit. The average molecular size of the proteoglycans synthesized by activated tissue was slightly larger than normal, as determined by chromatography on Sepharose CL-2B, and the average molecular size of the glycosaminoglycans synthesized by activated tissue was markedly increased over the normal. The increase in chain size was accompanied by an increase in the proportion of the chains degraded by chondroitinase ABC; these results are consistent with the preferential synthesis by activated chondrocytes of chondroitin sulphate-rich proteoglycans. The increase in glycosaminoglycan chain size was observed whether the chains were formed on endogenous core protein or on exogenous benzyl-beta-D-zyloside. An approximate 4-fold activation in culture of glycosaminoglycan synthesis on protein core was accompanied by a 1.54-fold increase in the rate of incorporation of [3H]serine into the chondroitin sulphate-linkage region of the proteoglycans. A 2.8-fold activation in culture of glycosaminoglycan synthesis on benzyl-beta-D-zyloside was accompanied by a 1.7-fold increase in the rate of incorporation of [3H]benzyl-beta-D-zyloside into glycosaminoglycans. The activation of glycosaminoglycan synthesis was, however, accompanied by no detectable change in the activity of xylosyltransferase (EC 2.4.2.26) in cell-free extracts. These results are discussed in relation to current ideas on the control of proteoglycan synthesis in cartilage.
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PMID:Control of proteoglycan synthesis. Studies on the activation of synthesis observed during culture of articular cartilages. 677 23

13C NMR was used to study the molecular dynamics of the chick limb bud proteoglycan core protein. Because only about 10% of the proteoglycan is protein, [2-13C]glycine and [3-13C]serine were incorporated into the core protein of the chick limb bud proteoglycan monomer using a chondrocyte culture system. The purified labeled monomer was studied in solution (50 mg/ml, 0.05 M sodium acetate/0.05 M sodium phosphate, pH 7.4) at 37 degrees C. Spin-lattice relaxation times, line widths, and nuclear Overhauser enhancements were measured for the labeled carbons in the protein and for the natural abundance carbons in the glycosaminoglycan chains. Analyses of these data show that correlation times for backbone reorientation of protein and polysaccharide chains in the intact monomer are predominantly in the range of 0.5-5.0 ns. These correlation times are 10(2)-10(3) times smaller than the minimum correlation time calculated for a rigid monomer, indicating that the core protein and polysaccharide backbones are segmentally flexible. Signal intensity data show that at least 80% of the protein backbone is flexible but do not exclude the possibility that 20% of the protein backbone has ordered structure. We observe both broad and narrow signal components in the spectrum of the intact monomer, showing that backbone motion is heterogeneous. The broad signal component is not observed after the monomer is digested with chondroitinase. This result and the strong concentration dependence of the 13C line width observed in solutions of chondroitin 4-sulfate suggest an assignment of the broad signal component to residues near the protein-polysaccharide linkage region. The difference in NMR parameters observed for free and substituted serine carbons confirms that motion of the substituted serine side chain is restricted.
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PMID:13C nuclear magnetic resonance suggests a flexible proteoglycan core protein. 678 60

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

A differentiated population of cells with metachromatically staining granules and surface IgE receptors was obtained from mouse bone marrow cultured for 2 weeks in the presence of conditioned medium derived from concanavalin A-stimulated splenocytes. The cells were found to incorporate large amounts of [35S]sulfate into an intracellular 35S-labeled proteoglycan of Mr approximately 200,000 containing a maximum of seven glycosaminoglycan side chains (Mr = 25,000). After chondroitinase ABC treatment of density gradient-purified [3H] serine-labeled proteoglycan, the resulting core was Mr approximately 26,000 as assessed by gel filtration. Two-dimensional cellulose acetate electrophoresis of beta-eliminated 35S-labeled glycosaminoglycan revealed a single type of glycosaminoglycan that migrated at the position of oversulfated chondroitin sulfate E from squid cartilage. Chondroitinase ABC degradation of the 35S-labeled glycosaminoglycan yielded two cleavage products in approximately equal molar amounts which co-migrated in both descending paper chromatography and high voltage paper electrophoresis with a monosulfated disaccharide, 2-acetamido-2-deoxy-3-O-(beta-D-gluco-4-enepyranosyluronic acid)-4-O-sulfo-D-galactose, and a disulfated disaccharide, 2-acetamido-2-deoxy-3-O-(beta-D-gluco-4-enepyranosyluronic acid)-4-6-di-O-sulfo-D-galactose. The release of some free [35S]sulfate from the oversulfated disaccharide with either chondro-4-sulfatase or chondro-6-sulfatase and the complete desulfation by their combined action established that the oversulfated disaccharide contained N-acetylgalactosamine-4,6-disulfate. The 35S]labeled proteoglycan of these unique IgE receptor-bearing and histamine-containing cells, therefore, is composed of chondroitin sulfate E rather than heparin glycosaminoglycan, and thus is the first identification of such an intracellular localized proteoglycan in a mammalian cell.
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PMID:Culture from mouse bone marrow of a subclass of mast cells possessing a distinct chondroitin sulfate proteoglycan with glycosaminoglycans rich in N-acetylgalactosamine-4,6-disulfate. 680 69


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