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:3.1.6.12 (chondroitinase)
2,183 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

The ultrastructural identification and characterization of lung proteoglycans was studied using the polycationic dye, ruthenium red. Treating lung parenchyma with the detergent Triton X-100 increased epithelial permeability and allowed the dye to penetrate alveolar walls and stain the alveolar basement membrane and lung collagen. Ruthenium red stained numerous 10- to 40-nm granules concentrated at the lamina surface of basement membrane and attached to the major doublet collagen band. The granules attached to collagen were digested by chondroitinase ABC and papain, indicating that they represent proteoglycan aggregates containing chondroitin or dermatan sulfate. Granules observed on the alveolar basement membrane were resistant to digestion by collagenase and by all glycosidases, suggesting that heparin or heparan sulfate is the predominant glycosaminoglycan in epithelial basement membrane. Ruthenium red in association with tannic acid also stained a fine network of 3- to 10-nm filaments in which collagen was enmeshed, forming the interfibrillar matrix. This network was resistant to collagenase and glycosidase digestion but was removed after papain digestion, suggesting that it was a protein or glycoprotein that did not contain glycosaminoglycans. These methods have allowed visualization of lung proteoglycans and have identified a structure that does not contain glycosaminoglycan that is intimately associated with collagen. This technique can now be applied to explore the potential role of proteoglycans in lung development and in restructuring the lung in various disease states.
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PMID:Ultrastructural localization and characterization of proteoglycans in the pulmonary alveolus. 9 9

Incorporation of sulfate into alcian blue-precipitable glycosaminoglycan of 12-day-old chick embryo sterna is stimulated by addition, separately or together, of normal human serum and physiological concentrations of thyroid hormones (Audhya, T.K., and Gibson, K.D. (1975) Proc. Natl. Acad, Sci. U. S. A. 72, 604--608). We present evidence that this stimulation is due to increased synthesis of at least one proteoglycan, with minor alterations in the size and chemical composition of the glycosaminoglycans. Pulse-chase experiments showed no detectable loss of label during the chase, in control sterna or sterna incubated with serum and L-3,5,3'-triiodothyronine; thus, all incorporation was the result of synthesis of glycosaminoglycans. In double-label experiments, with 35SO4(2-) and [3H]acetate, the molar ratio of 3H and 35S incorporated into glycosaminoglycans was changed little, if at all, by addition of serum or triiodothyronine or both, at concentrations which increased incorporation up to 2-fold. Glycosaminoglycans isolated from these and other incubations gave similar elution patterns from agarose columns, and identical electrophoretic patterns on cellulose acetate. Digestion with chondroitinase ABC (chondroitin ABC lyase; EC 4.2.2.4.) showed that incorporation was into chondroitin sulfate and possibly hyaluronic acid, and that the proportions of non-sulfated, 4-sulfated, and 6-sulfated disaccharide units differed little between stimulated and unstimulated sterna. Incorporation of [3H]serine into glycosaminoglycans from papain digest of sterna paralleled incorporation of 35SO4(2-), and indicated a number average molecular weight between 21,000 and 25,000 for the newly synthesized chondroitin sulfate. This value was confirmed by gel filtration chromatography, which also showed that the average molecular weight of the newly synthesized chondroitin sulfate decreased up to 15% under conditions of 2-fold stimulation. Proteoglycans were extracted from sterna incubated with [3H]serine and 35SO4(2-) and analyzed by isopycinic centrifugation in CsCl and by zone sedimentation in a sucrose gradient. A major proteoglycan fraction could be separated by either method. Incorporation of both isotopes into this proteoglycan fraction, and into glycosaminoglycans isolated after papain digestion, was stimulated in a coordinate manner. Almost identical results were obtained with both separation techniques. The results indicate that the synthesis of the major proteoglycan, and probably also of a minor one, is stimulated by serum and triiodothyronine.
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PMID:Stimulation of proteoglycan synthesis in chick embryo sternum by serum and L-3,5,3'-triiodothyronine. 13 41

13C nmr spectral parameters were measured for intact bovine nasal cartilage tissue, the purified proteoglycan aggregate, and chondroitin 4-sulfate. A comparison of integrated intensities obtained for four different samples of fresh tissue with an ethylene glycol standard indicated that at least 80% of the total glycosaminoglycan carbons in the tissue contributed to the spectrum. This result was confirmed by intensity measurements obtained at 56 degrees on fresh tissue and at 37 degrees after extensive papain digestion of fresh tissue. Spin lattice relaxation times and nuclear Overhauser enhancements were analyzed in terms of the following models of molecular motion: (a) single correlation time; (b) log X2 distribution of correlation times; and (c) anisotropic motion. The analysis indicates that the segmental motions of glycosaminoglycan chains are characterized by a broad distribution of correlation times centered at about 50 ns. Slow motion contributions to glycosaminoglycan line widths were reduced by dipolar decoupling (gammaH2/2pi = 65 kHz). Collagen intensity was observed in dipolar decoupled spectra, but not in scalar decoupled spectra of intact tissue, showing that the type II collagen in cartilage undergoes anisotropic motion like the type I collagen in tendon. Only glycosaminoglycan resonances were observed in spectra of a solution of proteoglycan aggregate before and after chondroitinase digestion. After subsequent digestion with papain, protein resonances were observed. These results suggest that the protein portions of the proteoglycan aggregate structure, in contrast with the glycosaminoglycan chains, have restricted backbone mobility and consequently a defined backbone structure.
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PMID:Investigation of molecular motion of proteoglycans in cartilage by 13C magnetic resonance. 14 Aug 75

Rat liver cells grown in primary cultures in the presence of [(35)S]sulphate synthesize a labelled heparan sulphate-like glycosaminoglycan. The characterization of the polysaccharide as heparan sulphate is based on its resistance to digestion with chondroitinase ABC or hyaluronidase and its susceptibility to HNO(2) treatment. The sulphate groups (including sulphamino and ester sulphate groups) are distributed along the polymer in the characteristic block fashion. In (3)H-labelled heparan sulphate, isolated after incubation of the cells with [(3)H]galactose, 40% of the radioactive uronic acid units are l-iduronic acid, the remainder being d-glucuronic acid. The location of heparan sulphate at the rat liver cell surface is demonstrated; part of the labelled polysaccharide can be removed from the cells by mild treatment with trypsin or heparitinase. Further, a purified plasma-membrane fraction isolated from rats previously injected with [(35)S]sulphate contains radioactively labelled heparan sulphate. A proteoglycan macromolecule composed of heparan sulphate chains attached to a protein core can be solubilized from the membrane fraction by extraction with 6m-guanidinium chloride. The proteoglycan structure is degraded by treatment with papain, Pronase or alkali. The production of heparan [(35)S]sulphate by rat liver cells incubated in the presence of [(35)S]sulphate was followed. Initially the amount of labelled polysaccharide increased with increasing incubation time. However, after 10h of incubation a steady state was reached where biosynthetic and degradative processes were in balance.
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PMID:Structure and metabolism of rat liver heparan sulphate. 14 28

Glycosaminoglycans were isolated from purified fractions of glomerular basement membranes and partially characterized by chemical analysis and cellulose acetate electrophoresis. Basement membranes were prepared by detergent treatment of rat glomeruli and subjected to digestion with papain and Pronase. Glycosaminoglycans were isolated from the digests by precipitation with cetyl pyridinium chloride and ethanol. Results of cellulose acetate electrophoresis of the isolated glycosaminoglycan fraction revealed the presence of one major and one minor spot. The major spot was identified as heparan sulfate because it comigrated with the heparan sulfate standard and was sensitive to heparinase and to nitrous acid oxidation but insensitive to chondroitinase ABC and to testicular or leech hyaluronidase. The minor spot was tentatively identified as hyaluronic acid based on its migratory behavior and sensitivity to leech and testicular hyaluronidase. The chemical composition of the isolated glycosaminoglycan was typical of that of heparan sulfate (high carbazole/orcinol ratio, high sulfate content, absence of galactosamine). The data support and confirm the cytochemical data obtained previously [Kanwar, Y. S. & Farquhar, M. G. (1979) Proc. Natl. Acad. Sci. USA 76, 1303-1307] demonstrating that heparan sulfate is the only sulfated glycosaminoglycan detectable in the glomerular basement membrane. The present results suggest that in addition to sulfated glycosaminoglycan some nonsulfated glycosaminoglycan (hyaluronic acid) may also be present in the glomerular basement membrane.
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PMID:Isolation of glycosaminoglycans (heparan sulfate) from glomerular basement membranes. 15 57

1. Proteoglycan aggregates from bovine nasal cartilage were studied by using electron microscopy of proteoglycan/cytochrome c monolayers. 2. The aggregates contained a variably long central filament of hyaluronic acid with an average length of 1037nm. The proteoglycan monomers attached to the hyaluronic acid appeared as side chain filaments varying in length (averaging 249nm). They were distributed along the central filament at an average distance of about 36nm. 3. Chondroitin sulphate side chains were removed from the proteoglycan monomers of the aggregates by partial chondroitinase digestion. The molecules obtained had the same general appearance as intact aggregates. 4. Proteoglycan aggregates were treated with trypsin and the largest fragment, which contains the hyaluronic acid, link protein and hyaluronic acid-binding region, was recovered and studied with electron microscopy. Filaments that lacked the side chain extensions and had the same length as the central filament in the intact aggregate were observed. 5. Hyaluronic acid isolated after papain digestion of cartilage extracts gave filaments with similar length and size distribution as observed for the central filament both in the intact aggregate and in the trypsin digests. 6. Umbilical-cord hyaluronic acid was also studied and gave electron micrographs similar to those described for hyaluronic acid from cartilage. However, the length of the filament was somewhat shorter. 7. The electron micrographs of both intact and selectively degraded proteoglycans corroborate the current model of cartilage proteoglycan structure.
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PMID:Cartilage proteoglycan aggregates. Electron-microscopic studies of native and fragmented molecules. 21 57

Proteoglycan subunits (PGS) were isolated from bovine articular cartilage of calves and from cows, 18 months and 8 years old respectively. From the latter cartilage of osteoarthrotic and of non-osteoarthrotic sites was taken. PGS were characterized by gel-chromatography on Sepharose 2B columns and subjected to digestion with chondroitinase ABC and with papain. The isolated keratan sulphate-protein cores obtained from chondroitinase digestion were characterized on Sepharose 4B and the chondroitin sulphate chains on Sephadex G-200 gels. A larger molecular size of PGS was found in calf cartilage than in the other samples. This was attributed to the larger molecular size of chondroitin, whereas no change was observed in the keratan sulphate-protein cores. No change was observed in molecular size of PGS, isolated chondroitin sulphates or keratan sulphate-protein cores in osteoarthrosis in comparison with non-osteoarthrotic cartilage from the same joint or from younger adult animals.
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PMID:Proteoglycan structure of bovine articular cartilage. Variation with age and in osteoarthrosis. 53 16

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

Dermatan sulfate-chondroitin sulfate copolymers have been isolated from human umbilical cord as a major galactosaminoglycan component of this tissue. The galactosaminoglycan fraction was obtained from this tissue by papain [EC 3.4.22.2] digestion followed by precipitation with cetylpyridinium chloride in a yield of 700 mg per 100 g of dry tissue. Ethanol fractionation resolved 4-5 subfractions differing in relative content of L-iduronic acid and D-glucuronic acid. No galactosaminoglycan containing either solely L-iduronic acid or D-glucuronic acid was obtained. The copolymeric structure of the material in each subfraction was demonstrated by analysis of oligosaccharide fragments obtained by chondroitinase-AC [EC 4.2.2.5] digestion. All the polymers contained repeating disaccharide units, D-glucuronosyl-N-acetylgalactosamine, D-glucuronosyl-N-acetylgalactosamine 4-sulfate, D-glucuronosyl-N-acetyl-galactosamine 6-sulfate, and L-iduronosyl-N-acetylgalactosamine 4-sulfate, of which D-glucuronosyl-N-acetylgalactosamine 6-sulfate and L-iduronosyl-N-acetylgalactosamine 4-sulfate were predominant. Both iduronic acid- and glucuronic acid-containing units were arranged in clusters. The presence of a considerable amount of nonsulfated disaccharide units was noted. The copolymers show extensive polydispersity in electrophoresis on cellulose acetate and gel chromatography on Sephadex G-200.
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PMID:Dermatan sulfate-chondroitin sulfate copolymers from ambilical cord. Isolation and characterization. 97 51


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