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)

Keratan sulfate-rich peptides were isolated after digestion of proteoglycans from bovine nasal cartilage and bovine nucleus pulposus with chondroitinase ABC, trypsin and chymotrypsin. The keratan sulfate enriched peptides from nucleus pulposus were larger than those from nasal cartilage. Keratan sulfate chains were isolated after treatment of the keratan sulfate-rich peptides under alkaline, reductive conditions. Proteoglycans from nucleus pulposus contain longer keratan sulfate chains, as is shown primarily by gel chromatography of the keratan sulfate-rich peptides and the keratan sulfate chains, but also from end-group analyses of the keratan sulfate chains.
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PMID:Skeletal keratan sulfate from different tissues. Characterization and alkaline degradation. 4 89

After chondroitinase digestion of bovine nasal and tracheal cartilage proteoglycans, subsequent treatment with trypsin or trypsin followed by chymotrypsin yielded two major types of polypeptide-glycosaminoglycan fragments which could be separated by Sepharose 6B chromatography. One fragment, located close to the hyaluronic acid-binding region of the protein core, had a high relative keratan sulfate content. This fragment contained about 60% of the total keratan sulfate, but less than 10% of the total chondroitin sulfate present in the original proteoglycan preparation. The weight average molecular weight of the keratan sulfate-enriched fragment was 122,000, as determined by sedimentation equilibrium centrifugation. The chemical and physical data indicate that this fragment contains an average of 10 to 15 keratan sulfate chains, if the average molecular weight of individual chains is assumed to be about 8,000, and about 5 chondroitin sulfate chains attached to a peptide of about 20,000 daltons. The other population of fragments was derived from the other end of the proteoglycan molecule, the chondroitin sulfate-enriched region, and contained mainly chondroitin sulfate chains. About 90% of the total chondroitin sulfate, but only 20 to 30% of the total keratan sulfate was recovered in these fragments. On the average, approximately 5 chondroitin sulfate chains and 1 keratan sulfate chain could be linked to the same peptide. Another 10 to 20% of the total keratan sulfate, originally found in or near the hyaluronic acid-binding region, was not separated from the chondroitin sulfate-enriched fragments. Hydroxylamine could be used to liberate a large molecular size, chondroitin sulfate-enriched fragment (Kav 0.54 on Sepharose 2B) from the proteoglycan aggregates. The remainder of the protein core, containing the keratan sulfate-enriched region, was bound to hyaluronic acid with the link proteins and recovered in the void volume on the Sepharose 2B column.
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PMID:Distribution of keratan sulfate in cartilage proteoglycans. 13 6

Monomer proteoglycan was isolated from porcine ovarian follicular fluid by isopycnic CsCl centrifugation in the presence of 4 M guanidine HCl and protease inhibitors. The elution profile of the D1 preparation on Sepharose 2B was similar to that of monomer proteoglycan from bovine nasal cartilage, indicating a similar molecular size. Follicular fluid proteoglycans consist of about 20% protein, 50% dermatan sulfate, and 20% oligosaccharides rich in sialic acid, galactose, mannose, glucosamine, and galactosamine. The amino acid composition of this proteoglycan is significantly different from that of cartilage proteoglycans, with a higher proportion of aspartic acid, threonine, and lysine, and lower amounts of proline and glycine. Alkali-released dermatan sulfate chains are larger on Sepharose 6B (average Mr = 56,000) than chondroitin sulfate chains from cartilage proteoglycans (average Mr = 25,000), and iduronic acid accounts for 9% of total hexuronic acid. Disaccharide units released by chondroitinase ABC consists of 67% 4-sulfated, 22% 6-sulfated, 5% non-sulfated, and 5% disulfated disaccharides. After treatment with 0.05 M NaOH, 1 M NaBH4 at 45 degrees C for 24 h, two major sialic acid-containing oligosaccharides were observed on Sephadex G-25, corresponding to penta- and hexasaccharides. The pentasaccharide contained sialic acid, galactose, glucosamine, and galactosamine in the proportions 1:2:1:1. The galactosamine is O-glycosidically linked to the protein core. This oligosaccharide accounts for approximately 77% of all the sialic acid in the follicular fluid proteoglycans. The hexasaccharide fraction contained sialic acid, galactose, mannose, and glucosamine in the proportions 1:2:1:2. It also contained a small amount of fucose and galactosamine. The linkage of these oligosaccharides to the protein core remains to be determined. The follicular fluid proteoglycans, unlike those from cartilage, do not interact with hyaluronic acid. Digestion with trypsin, chymotrypsin, or plasmin released dermatan sulfate-peptides nearly as small as those released by papain or alkali; in contrast, cartilage proteoglycans were resistant to plasmin and released peptides containing an average of more than four chondroitin sulfate chains after trypsin or chymotrypsin digestion.
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PMID:Isolation and characterization of proteoglycans from porcine ovarian follicular fluid. 76

Chondrons have recently been extracted from adult articular cartilages and techniques developed to study their structure and composition in isolation. This study introduces methods to immobilize isolated canine chondrons in thin layers of agarose gel for immunohistochemistry and future in vitro studies. An antibody to Type VI collagen which stained the chondron in suspension was used to successfully validate the system and its feasibility for immunoelectron microscopy. Monoclonal and polyclonal antibodies to a variety of epitopes on the proteoglycan molecule were tested on fresh and fixed plugs cored from chondron-agarose gels. Plugs were immunolabeled with peroxidase-diaminobenzidine before or after digestion with testicular hyaluronidase or chondroitinase ABC. Trypsin/chymotrypsin were used to challenge epitopes of the core protein. The results indicate that epitopes to keratan sulfate, chondroitin sulfate, hyaluronate binding region, and core protein are localized in the chondron. Consistent staining was found in the tail and interconnecting segments between chondrons, whereas staining of the pericellular matrix and capsule adjacent to the chondrocyte varied according to the enzyme pre-treatment employed. We conclude that isolated chondrons are rich in proteoglycan monomer, which is particularly concentrated in the tail and interconnecting segments of the chondron where it could function to protect and stabilize the chondrocyte.
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PMID:Chondrons from articular cartilage. (IV). Immunolocalization of proteoglycan epitopes in isolated canine tibial chondrons. 171 45

Human promyelocytic cells (HL-60) were labeled with 35S-sulfate and either 3H-glucosamine or 3H-serine as precursors. Accumulation of 35S-labeled macromolecules was approximately linear for up to 96 h, with a mean cell:medium ratio of 5.5:1, although activity/10(5) viable cells reached a plateau level after 24 h. Virtually none of the cell-associated proteoglycan was removed by trypsinization, consistent with a predominantly intracellular localization. Proteoglycan heterogeneity was investigated by DEAE-Sephacel chromatography, isopyknic CsCl gradient centrifugation, and gel filtration chromatography. HL-60 cells appeared to synthesize a single proteoglycan species, Kav = 0.46 on Sepharose CL-4B and Kav = 0.32 on Sepharose CL-6B, recovered primarily from the high-density fractions of a dissociative CsCl gradient (rho greater than 1.40 g/l). Degradation products of lower charge density, lower buoyant density, and lower hydrodynamic size were also present, mainly in the cell pellets. The major proteoglycan was found to contain chondroitin sulfate chains of average Mr = 14.5 kD, yielding virtually 100% 4-sulfated disaccharides on digestion with chondroitinase ABC. The proteoglycan was resistant to trypsin, chymotrypsin, plasmin, and papain, and the core protein Mr was approximately 20 kD by molecular sieve chromatography. Induction of HL-60 cells with 0.15 dimethyl sulfoxide (DMSO) resulted in differentiation to a more mature granulocytic phenotype and was associated with a reduction in 35S-sulfate incorporation to 45% of control values or 32%, expressed as activity/10(5) cells. Proteoglycans synthesized by DMSO-treated cells were identical to those from untreated cells in terms of hydrodynamic size, glycosaminoglycan Mr, and sulfation.
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PMID:Biosynthesis of proteochondroitin sulfate by HL-60 human promyelocytic cells. 291 Oct 20

Dermatan sulfate proteoglycans (DS-PGs) isolated from bovine articular cartilage have been examined for their effects on the adhesive responses of BALB/c 3T3 cells and bovine dermal fibroblasts on plasma fibronectin (pFN) and/or type I collagen matrices, and compared to the effects of the chondroitin sulfate/keratan sulfate proteoglycan monomers (CS/KS-PGs) from cartilage. DS-PGs inhibited the attachment and spreading of 3T3 cells on pFN-coated tissue culture substrata much more effectively than the cartilage CS/KS-PGs reported previously; in contrast, dermal fibroblasts were much less sensitive to either proteoglycan class unless they were pretreated with cycloheximide. Both cell types failed to adhere to substrata coated only with the proteoglycans; binding of the proteoglycans to various substrata has also been quantitated. While a strong inhibitory effect was obtained with the native intact DS-PGs, little inhibitory effect was obtained with isolated DS chains (liberated by alkaline-borohydride cleavage) or with core protein preparations (liberated by chondroitinase ABC digestion). In marked contrast, DS-PGs did not inhibit attachment or spreading responses of either 3T3 or dermal fibroblasts on type I collagen-coated substrata when the collagen was absorbed with pFN alone, DS-PGs alone, or the two in combination. These results support evidence for (a) collagen-dependent, fibronectin-independent mechanisms of adhesion of fibroblasts, and (b) different sites on the collagen fibrils where DS-PGs bind and where cell surface "receptors" for collagen bind. Experiments were developed to determine the mechanism(s) of inhibition. All evidence indicated that the mechanism using the intact pFN molecule involved the binding of the DS-PGs to the glycosaminoglycan (GAG)-binding sites of substratum-bound pFN, thereby inhibiting the interaction of the fibronectin with receptors on the cell surface. This was supported by affinity chromatography studies demonstrating that DS-PGs bind completely and effectively to pFN-Sepharose columns whereas only a subset of the cartilage CS/KS-PG binds weakly to these columns. In contrast, when a 120-kD chymotrypsin-generated cell-binding fragment of pFN (CBF which has no detectable GAG-binding activity as a soluble ligand) was tested in adhesion assays, DS-PGs inhibited 3T3 adherence on CBF more effectively than on intact pFN. A variety of experiments indicated that the mechanism of this inhibition also involved the binding of DS-PGs to only substratum-bound CBF due to the presence of a cryptic GAG-binding domain not observed in the soluble CBF.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Fibronectin-mediated adhesion of fibroblasts: inhibition by dermatan sulfate proteoglycan and evidence for a cryptic glycosaminoglycan-binding domain. 295 85

Three fluorescein-labeled lectins were shown to bind differentially to cell surfaces in different epithelial layers of rat oral mucosa regardless of the age or the site of origin of the tissue. Griffonia simplicifolia (GS-1-B4), specific for alpha-D-galactosyl end groups, labeled basal cells only; Ulex europeus (Ulex 1) specific for alpha-L-fucosyl groups labeled spinous cells; and Bandeiraea simplicifolia (BSII), specific for N-acetyl-D-glucosamine, labeled cornified cells. Pretreatment of sections with alpha-galactosidase completely abolished the staining of basal cells by GS-1-B4, but had no effect on the staining of spinous cells by Ulex 1. In contrast, alpha-fucosidase abolished the staining of spinous cells by Ulex 1 and caused staining of both basal and spinous cells by GS-1-B4. Neuraminidase and chondroitinase ABC produced results similar to one another, with staining of basal cells by GS-1-B4 and labeling of both basal and spinous cells with Ulex 1. beta-galactosidase, beta-glucuronidase, and testicular hyaluronidase did not affect the staining pattern of GS-1-B4 or Ulex 1, whereas chymotrypsin completely abolished any staining with either lectin. The results demonstrate a complex arrangement of cell surface carbohydrates in the epithelium of rat oral mucosa. The findings indicate a possible simplification in the spatial arrangements of cell surface carbohydrates during the differentiation of basal to spinous cells.
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PMID:Preferential lectin binding to specific layers of rat oral epithelium and modification by enzyme pretreatment. 299 51

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

An inhibitory component that diminishes estrogen receptor (ER) binding to nuclei in vitro is present in cytosol prepared from calf uterus. The inhibitor is heat stable and resistant to enzymatic treatment with trypsin, chymotrypsin, proteinase K, deoxyribonuclease I, or ribonucleases A, T1, and U2. Results of chromatography on DEAE-cellulose and Sephadex G-150 indicate that the factor is a negatively charged macromolecule. Inhibitory activity is sensitive to sequential digestion with chondroitinase ABC, hyaluronidase, and heparinase. Approximately 70% of the inhibitory activity is destroyed by treatment with heparinase alone. Heparitinase destroys only 30% of this activity. Furthermore, the addition of pure hyaluronic acid or chondroitin sulfate to the ER-nuclei binding assay results in little inhibition, whereas addition of heparin inhibits 75% of receptor binding. Overall, these results indicate that glycosaminoglycans, present in bovine uterine cytosol, are capable of inhibiting ER-nuclei interactions. The most potent inhibitory glycosaminoglycan displays heparin-like characteristics.
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PMID:Characterization of a cytosolic inhibitor of calf estrogen receptor binding to nuclei. 330 79

Chondroitin sulfate E proteoglycan was extracted in the presence of protease inhibitors from 6 X 10(9) mouse bone marrow-derived, interleukin 3-dependent mast cells, of which 3 X 10(7) had been biosynthetically labeled with [35S]sulfate or [3H]glycine. Chondroitin sulfate E proteoglycan was purified to apparent homogeneity by density-gradient centrifugation, differential molecular weight dialysis, DEAE-52 ion exchange chromatography, and Sepharose CL-4B gel filtration chromatography. Chondroitin sulfate E proteoglycan, radiolabeled with [3H]glycine or [35S]sulfate, filtered as a single peak of radioactivity on Sepharose CL-4B with a Kav of 0.41. When purified [3H]glycine-labeled proteoglycan was digested with chondroitinase ABC and subjected to gel filtration, all of the radioactivity was shifted to a lower molecular weight. As assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis the Mr of the peptide core obtained by chondroitinase ABC treatment was approximately 10,000. The purified proteoglycan was resistant to degradation by collagenase, clostripain, trypsin, chymotrypsin, elastase, chymopapain, V8 protease, proteinase K, and Pronase, as assessed by gel filtration chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Analysis of the core peptide of the intact proteoglycan revealed that glycine, serine, and glutamic acid/glutamine accounted for 70% of the total amino acids and were present in a molar ratio of 4.3/1.6/1.0. When analyzed for neutral hexose content by gas-liquid chromatography, the proteoglycan contained approximately 2% of its weight as mannose, fucose, galactose, and other sugars, indicating that oligosaccharides were linked to the peptide core. The mouse bone marrow-derived mast cell chondroitin sulfate E proteoglycan, like the rat serosal mast cell heparin proteoglycan, is markedly protease resistant, has highly sulfated glycosaminoglycans, and contains a peptide core that is rich in serine and glycine. These characteristics of the mast cell class of intracellular proteoglycans may contribute to their function in stimulus-induced granule secretion as well as in mediator storage, including retention of cationic neutral proteases.
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PMID:Purification and analysis of the core protein of the protease-resistant intracellular chondroitin sulfate E proteoglycan from the interleukin 3-dependent mouse mast cell. 393 50


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