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 proteoglycans of cartilage are complex molecules in which chondroitin sulphate and keratan sulphate chains are covalently linked to a protein core, forming a polydisperse population of proteoglycan monomers. By interaction with hyaluronic acid and link proteins, the monomers form large macromolecular complexes. In vivo the proteoglycans mainly occur in such aggregates. In the electron microsope, the cartilaginous matrix can be seen to be made up of thin collagen fibrils and polygonal granules about 10-50 nm in diameter Addition of the polyvalent cationic dye Ruthenium Red to glutaraldehyde and osmium tetroxide fixatives yields a dense selective staining of the matrix granules. Following a short digestion of cartilage slices with either of the chondroitin sulphate-degrading enzymes hyaluronidase and chondroitinase or with the proteolytic enzyme papain, the matrix granules were few in number or completely absent and the proteoglycan content, measured as hexosamine, decreased by up to 90%. Similarly, extraction of the cartilage with 4 M guanidine-HCl removed all matrix granules and most of the proteoglycans. From these findings, it can be concluded that the matrix granules represent proteoglycans, most probably in aggregate form, and that Ruthenium Red staining may be used to study the distribution of these macromolecules in thin sections. As a complement to chemical studies on proteoglycan structure, it is also possible to observe and measure individual molecules in the electron microscope after spreading them into a monomolecular layer with cytochrome c. This technique has been applied in investigations on proteoglycans isolated from bovine nasal cartilage and other hyaline cartilages. The molecules in the monomer fractions appeared as an extended central core filament to which about 25--30 side-chain filaments were attached at various intervals. The core filament, averaging about 300 nm in length, was interpreted as representing the polysaccharide binding part of the protein core and the side-chain filaments, averaging about 45 nm in length, as representing the clusters of chondroitin sulphate chains. Statistical treatment of the collected data indicated that no distinct subpopulations existed within the monomer fractions. The electron microscopic results correlated well with chemical data for the corresponding fractions and together with recent observations on various aggregate fractions strongly support present concepts of proteoglycan structure.
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PMID:Electron microscopy of cartilage proteoglycans. 6 24

Heparan sulfates were isolated from the urine of normal individuals and patients with genetic mucopolysaccharidoses after exhaustive digestion with chondroitinase ABC. Electrophoresis of these preparations on cellulose acetate membrane revealed one spot corresponding in mobility to reference heparan sulphate in barium acetate buffer, while electrophoresis in 0.1 M HCl resulted in two distinct spots for each case; one corresponded in migration rate to reference heparan sulfate, and the other was faster in mobility than reference heparan sulfate but slightly retarded when compared with reference heparin. On thin-layer gel filtration on Sephadex G-200 (superfine) heparan sulfate from normal urine was polydispersed in character and its molecular size was larger than those of other preparations. Heparan sulfates from Hunter's and Sanfilippo's urine were monodispersed and small in molecular size. The molecular size of heparan sulfate from Sanfilippo's urine was the smallest of all. Heparin sulfate from Hurler's urine appeared to be composed of two populations; one corresponded in molecular size to heparan sulfate from normal urine, and the other corresponded to that of Hunter's urine.
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PMID:Molecular size difference of urinary heparan sulfates from normal individuals and genetic mucopolysaccharidoses. 12 36

Proteoglycans were extracted from bovine articular cartilage with guanidine-HCl and fractionated in cesium chloride density gradients by equilibrium ultracentrifugation. The acidic glycosaminoglycan (AGAG) components were then determined enzymatically with chondroitinase-ABC and streptomyces hyaluronidase. Under associative and dissociative conditions, the distribution of the AGAG components was as follows: the ratio of 4-sulfated disaccharide units to total AGAG increased with decreasing density gradients whereas that of 6-sulfated disaccharide units to total AGAG increased with increasing density gradients. The ratio of disulfated disaccharide units to total AGAG increased somewhat with decreasing density gradients whereas that of non-sulfated disaccharide units tended to decrease. Although the cartilage proteoglycan macromolecules were heterogeneous, a certain regularity was observed with respect to the distribution of sulfate and the degree of sulfation in the chondroitin sulfate chains of the proteoglycans.
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PMID:Constitutional heterogeneity of the glycosaminoglycans in articular cartilage proteoglycans. 14 4

Rat ovarian granulosa cells were isolated from immature female rats after stimulation with pregnant mare's serum gonadotropin and then maintained in culture. Proteoglycans were labeled using [35S]sulfate, D-[3h]glucosamine, or L-[3H]serine as precursors. 35S-labeled proteoglycans in the medium increased linearly up to 72 h after a 6- to 8-h lag period, and those in a 4 M guanidine HCl extract of the cell layer increased for about 16 h and then reached a plateau and stayed fairly constant up to 72 h. Two distinct sizes of proteoglycans were observed in the medium. The smaller (Kav = 0.60 on Sepharose CL-2B) had lower buoyant densities in dissociative gradients (rho less than 1.4 g/ml). The larger (Kav = 0.26 on Sepharose CL-2B) had high buoyant densities (recovered mainly in the bottom (D1) fraction of the dissociative gradient). More than 90% of the D1 proteoglycans contained dermatan sulfate chains (average Mr = 38,000) which yielded 84% 4-sulfated and 15% disulfated disaccharides after digestion with chondroitinase ABC. About 8% of the 35S-label in D1 was present as a heparan sulfate proteoglycan. When [3H]-glucosamine was used as a precursor, 28% of the 3H activity in the D1 proteoglycans was located in three major oligosaccharide components, two of which were similar or identical with those observed previously in D1 proteoglycans isolated from porcine follicular fluid. These results plus similar susceptibility of the labeled proteoglycans to proteolytic enzymes, especially plasmin, suggest that the granulosa cells synthesize the predominant follicular fluid proteoglycans.
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PMID:Biosynthesis of proteoglycans by rat granulosa cells cultured in vitro. 50 Jul 20

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

The cytoplasmic sialomucin in Paget cells of both mammary and extramammary Paget's diseases was examined, using a new method proposed by Volz et al. (1987a, b). The staining methods used involved an electrolyte-Alcian Blue (pH = 5.8) and periodic acid Schiff. Oxidation was performed with 0.4 mmol/l periodate in 1 mol/l HCl at 4 degrees C or 50 mmol/l periodate in distilled water at room temperature for 1 h. Methylation, saponification, borohydride reduction, and digestion with diastase, neuraminidase (Vibrio cholerae) or chondroitinase ABC, were also employed. The cytoplasmic mucin was found to exhibit positive reaction for the above staining which were variously altered by the chemical modification procedures and diminished in intensity or abolished by digestion with neuraminidase. These results suggest that the cytoplasmic mucin in Paget cells is sialomucin without side-chain substituent in genital Paget's disease, and that with a substituent at C7 in mammary Paget's disease.
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PMID:Histochemical analysis of sialomucin in Paget cells of mammary and extramammary Paget's disease. 137 8

In this report we describe a system capable of resolving all of the known unsaturated disaccharides derived from the chondroitin sulphates, dermatan sulphate and hyaluronic acid by chondroitinase digestion. This system is superior to others in that the non-sulphated and mono-, di- and tri-sulphated disaccharides can be separated with good resolution in approximately 40 min in an isocratic solvent. The system employs an amino-cyano silica gel column (Whatman Partisil 5 PAC, 25 cm) and is eluted with an isocratic solvent consisting of 48% (v/v) acetonitrile, 14% (v/v) methanol and 38% (v/v) aqueous buffer. This aqueous buffer contains 0.5 M Tris-HCl, 0.1 M boric acid, 23.4 mM sulphuric acid, pH 8.0. UV absorption is monitored at 229 nm and for most disaccharides as little as 150 ng can be reliably determined. The addition of boric acid to the eluent is essential for good resolution of all components and the addition of low concentrations of sulphuric acid is used to control the elution times of various components. The system was applied to the analysis of glycosaminoglycan standards and excellent agreement with previous compositional analyses was obtained.
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PMID:High pressure liquid chromatographic identification of hyaluronic acid and chondroitin sulphate disaccharides. 179 40

We examined biochemically and immunocytochemically the type and distribution of mineral binding proteoglycans (PGs) in rat mid-shaft subperiosteal bone using three monoclonal antibodies (MAb 1-B-5, 9-A-2, and 3-B-3) which specifically recognize unsulfated chondroitin, chondroitin 4-sulfate (C4-S) and dermatan sulfate (DS), and chondroitin 6-sulfate. Bone proteins were extracted from fresh specimens with a three-step technique: 4 M guanidine HCl (GdnCl), aqueous EDTA without GdnCl (E-extract), followed by GdnCl. Western blot analysis of SDS-polyacrylamide gel electrophoresis revealed that E-extract after chondroitinase ABC digestion reacted strongly with MAb 9-A-2 but not with MAb 1-B-5 or 3-B-3. After adehyde fixation, ethanolic trimethylammonium EDTA was used as a demineralizing agent for light and electron immunocytochemistry. This provided good retention of water-soluble PGs in the specimens. After chondroitinase ABC pre-treatment of tissue sections, MAb 9-A-2 specifically stained C4-S and/or DS in the walls of osteocyte lacunae and bone canaliculi in the mineralized matrix as well as in the unmineralized matrix such as pre-bone, vascular canals, and pericellular matrix surrounding osteocytes; the remainder of the mineralized matrix lacked staining. These results indicate that mineral binding PGs contain C4-S and/or DS and are exclusively localized in the walls of the bone lacuna and canaliculus.
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PMID:Biochemical and immunocytochemical characterization of mineral binding proteoglycans in rat bone. 189 98

Proteoglycans (PGs) synthesized by the epidermis during stages crucial to the subepidermal migration of neural crest cells in the trunk of the axolotl (Ambystoma mexicanum, Urodela, Amphibia) embryo were studied. The glycosaminoglycan chains were biosynthetically labeled with [35S]sulfate in vitro during a period corresponding to the onset of migration. After extraction with guanidine HCl, the radiolabeled PGs were separated according to size by molecular-sieve chromatography on Sepharose CL-2B under dissociative conditions. This resulted in the separation of high-molecular-weight PGs, which eluted in the void volume, and low-molecular-weight PGs, eluting in a broad peak with a mean Kav of 0.7. The large PGs were also found to elute in the void volume when chromatographed on a Sephacryl S-1000 column. The low-molecular-weight PGs contained heparan sulfate and chondroitin sulfate (CS) and were not further characterized. The glycosaminoglycan component of the high-molecular-weight PG was completely degraded by chondroitinase ABC, while a large portion was resistant to chondroitinase AC, indicating the presence of dermatan sulfate (DS). These CS/DS chains were of unusually large size (Mr approximately 150,000) as estimated by chromatography on Sepharose CL-4B, relating the elution position to hyaluronan standards. Moreover, the chains were found to have a lower surface charge density than standard CS, and may therefore be undersulfated. After reduction and alkylation the high-molecular-weight PGs were included on both Sepharose CL-2B and Sephacryl S-1000 columns, eluting at Kav 0.2 and 0.4, respectively. Hence, the high-molecular-weight material appears to consist of large PG complexes, stabilized by intermolecular disulfide bonds. A CS/DSPG of similar size as the reduced monomeric form of the high-molecular-weight PG was found in small amounts in the total extract of 35S-labeled material.
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PMID:Large disulfide-stabilized proteoglycan complexes are synthesized by the epidermis of axolotl embryos. 192 7

1. Isomeric chondroitin sulfate proteoglycans were extracted from human, bovine, swine and rabbit aortas by 4 M guanidine-HCl and were fractionated and purified by CsCl isopycnic centrifugation, Sepharose CL-4B gel filtration, DEAE-Sepharose ion-exchange chromatography and octyl-Sepharose hydrophobic interaction chromatography. 2. The molecular size and the composition of isomeric chondroitin sulfate proteoglycans varied among species. Variations were also noted in the composition and molecular weight of constituent glycosaminoglycan chains. 3. Observations made on chondroitinase ABC and chondroitinase AC digests of proteoglycans indicate that dermatan sulfate is linked to the core proteins through chondroitin sulfates.
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PMID:Variations in the composition of arterial wall isomeric chondroitin sulfate proteoglycans among different animal species. 212 68


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