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 extracted from adult chicken were initially purified by DEAE-chromatography. Digestion of these proteoglycans with chondroitinase ABC generated a single 40-kDa core protein while digestion with keratanase generated a single 52-kDa core protein. Digestion with both enzymes combined, however, increased the amount of 40-kDa core protein produced. This suggested that the 40-kDa core protein exists with chondroitin/dermatan sulfate (C/DS) side chains alone and with both C/DS and keratan sulfate (KS) side chains. The proteoglycan fraction was initially digested with chondroitinase ABC, and the M(r) = 40,000 core protein derived from proteoglycans containing C/DS side chains alone was isolated. Amino-terminal sequencing showed it to be the chick cognate of decorin. The remaining proteoglycans were then digested with keratanase, and both the 40-kDa core protein and the 52-kDa core proteins derived from KS-containing proteoglycans were purified. The M(r) = 40,000 core protein derived from proteoglycans containing both C/DS and KS side chains had the same amino-terminal sequence as decorin and cross-reacted with antibodies to decorin. Sequence from the 52-kDa core protein derived from KS-containing proteoglycans showed it to be lumican. The results of this study suggest that adult chick corneas contain two isoforms of decorin: one containing C/DS side chains and the other, a hybrid, containing both C/DS and KS side chains. Embryonic corneas did not contain the hybrid isoform of decorin. These results suggest that different post-translational modifications occur to the decorin gene product during corneal development and maturation.
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PMID:Isolation and partial characterization of lumican and decorin from adult chicken corneas. A keratan sulfate-containing isoform of decorin is developmentally regulated. 140 Mar 83

Adhesion properties of rat embryo fibroblast cultures and proteoglycans (PGs) produced both in the growth medium and in the cell layer were investigated with increasing passages. Both cell-cell and cell-substrate adhesion increased with increasing subculture number. Cell adhesion properties were improved by cell treatment with chondroitinase ABC. The increase in subculture number was coupled with a constant increase of PG molecular size, which was particularly evident in cell layer extracts. The ratio HS-PGs/DS-PGs increased with increasing passages. PG modifications are likely to represent evidence of changes in extracellular matrix organization and could play a role in the increase of cell adhesion properties.
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PMID:Modifications of adhesion properties and proteoglycan structure in rat embryo fibroblast cultures with increasing passages. 142 2

A sensitive chemiluminescence high-performance liquid chromatographic method has been developed for the determination of hyaluronic acid, chondroitin sulphate and dermatan sulphate as their unsaturated disaccharide-dansylhydrazine derivatives involving an effective sample clean-up system. The dansylhydrazones of the unsaturated disaccharides derived from the hyaluronic acid, chondroitin sulphate and dermatan sulphate by chondroitinase ABC and/or chondroitinase ACII, were separated by reversed-phase chromatography using a mixture of 0.1 M sodium acetate buffer (pH 6.0) and 80% acetonitrile on a column (250 mm x 4.0 mm I.D.) packed with amide-80 silica beads (5 microns diameter). For post-column elution in the chemiluminescence system, 1 mM bis[2-(3,6,9-trioxadecanyloxycarbonyl)-4-nitrophenyl]oxalate and 3mM hydrogen peroxide in acetonitrile were used. The detection limit of each glycosaminoglycan was 100 fmol. The method was applicable to the determination of the levels of hyaluronic acid, chondroitin sulphate and dermatan sulphate in rat peritoneal mast cells.
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PMID:Chemiluminescence high-performance liquid chromatography for the determination of hyaluronic acid, chondroitin sulphate and dermatan sulphate. 142 67

This paper proposes a new method for simultaneous analysis of unsaturated disaccharides derived from glycosaminoglycans by enzymatic digestion with chondroitinase ABC, based on high-performance capillary electrophoresis (HPCE) of their 1-phenyl-3-methyl-5-pyrazolone derivatives. The O-sulphate group is stable in this derivatization, and this method allows reproducible microdetermination of glycosaminoglycans. This paper also demonstrates the applicability of this method to estimation of urinary chondroitin sulphates. Urinary creatinine as an inherent internal standard could also be estimated by HPCE, though in another mode of separation, i.e. ion-exchange electrokinetic chromatography.
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PMID:High-performance capillary electrophoresis of unsaturated oligosaccharides derived from glycosaminoglycans by digestion with chondroitinase ABC as 1-phenyl-3-methyl-5-pyrazolone derivatives. 143 32

To demonstrate the intra- and extracellular localization of hyaluronic acid (HA) in articular cartilage of the rabbit tibia, biotinylated HA binding region, which specifically binds to the HA molecule, was applied to the tissue. In comparison with the localization of HA, that of chondroitin sulfate (CS), keratan sulfate (KS), and the protein core (PC) of the proteoglycan was examined by immunohistochemistry. Strong positive staining for HA was detected in chondrocytes located in the transition between the superficial and middle zones of the tissue. Pre-treatment with chondroitinase ABC, keratanase II, or trypsin enhanced the stainability for HA in peri- and intercellular matrices. Immunohistochemistry with or without enzymatic pre-treatment demonstrated that immunoreactivity for CS, KS, and PC was distinctly discerned in chondrocytes and in the extracellular matrix located in the middle and deep zones. In particular, the immunoreactivity for KS and PC was augmented by pre-treatment with chondroitinase ABC not only in chondrocytes but in the extracellular matrix located in the middle and deep zones. Microbiochemical analysis corresponded well with histochemical and immunohistochemical results. These results suggest that HA is abundantly synthesized and secreted in chondrocytes located in the transition between the superficial and middle zones.
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PMID:Intra- and extracellular localization of hyaluronic acid and proteoglycan constituents (chondroitin sulfate, keratan sulfate, and protein core) in articular cartilage of rabbit tibia. 143 Oct 58

Adult human epidermis was cultured in whole skin organ culture under serum-free conditions in the presence of 35SO4. Proteoglycans (PG) comprised about 25% of the total (35SO4)-labeled material produced by epidermis. The rest of the incorporated activity displayed solubility characteristics typical of lipids. The molecular mass and the composition of the 35SO4-labeled epidermal PG and glycosaminoglycans (GAG) were studied using gel filtrations and agarose gel electrophoresis. The 35SO4-label of the epidermal PG was located in heparan sulfate (HS, approximately 75%) and chondroitin/dermatan sulfate (CS/DS, 25%), but not in keratan sulfate as determined by nitrous acid, chondroitinase AC II, chondroitinase ABC, and keratanase digestions, respectively. The molecular mass of the GAG chains was 10-40 kDa. The 35SO4-labeled PG were distributed between 60 and 600 kDa in agarose gel electrophoresis, with the highest activity at 350 kDa. Smaller activity peaks occurred at 150 and 60 kDa. Digestion of the PG with heparitinase removed most of the activity at 350 and 150 kDa, whereas chondroitinase ABC removed that at 60 kDa. A small amount of activity migrating between 600 and 1000 kDa was not affected by any of the GAG-degrading enzymes. Pulse chase experiments showed that the epidermal PG had an average half life of 24 h. The results thus demonstrate that human epidermis produces at least three different, rapidly metabolized PG. The PGs from 150 to 350 kDa contained heparan sulfate chains, whereas those at 60 kDa were chondroitin/dermatan sulfate PG.
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PMID:Proteoglycans synthesized by adult human epidermis in whole skin organ culture. 143 Dec 25

Isolated, purified small chondroitin (dermatan) sulphate proteoglycans from corneas of cow and rabbit and cow sclera were stained with Cupromeronic blue in 'model' experiments. The lengths and thicknesses of the images were compared with those of the same proteoglycans stained in the tissue, using the critical electrolyte concentration principle to give specificity for sulphated proteoglycans, and keratanase 1 or chondroitinase ABC digestion to distinguish between chondroitin and keratan sulphate. Corrections for orientation of the stained glycan filaments within the section plane were made to convert the observed lengths to true average lengths. Observed lengths of stained chondroitin (dermatan) sulphate were greater than those of keratan sulphate, both in models and tissues, in agreement with published data from biochemical and rotary-shadowing studies, in both species. Corrected (true) average lengths of stained isolated chondroitin (dermatan) sulphate proteoglycans were slightly, but not significantly, longer than expected from rotary shadowing or biochemical measurements. Keratan sulphate lengths were similarly somewhat longer. The data support the idea that Cupromeronic blue acts as a scaffold that helps maintain polyanion shape against distortion on staining. Stained filaments in tissues were sometimes over twice the length of isolated stained proteoglycans, suggesting that 2 glycan chains were aligned end-to-end. Thicknesses of proteoglycan filaments suggested that at least 2 glycan chains were aligned side-by-side, both in models and in tissues. A scheme for proteoglycan tertiary structure in cornea is proposed, in which glycan chains may bridge collagen fibrils in duplexed forms similar to those observed in rotary shadowed preparations.
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PMID:Morphometry of cupromeronic blue-stained proteoglycan molecules in animal corneas, versus that of purified proteoglycans stained in vitro, implies that tertiary structures contribute to corneal ultrastructure. 145 71

The incorporation of [35S]sulphate into macromolecules by rabbit peritoneal polymorphonuclear neutrophils (PMN) in vitro revealed that two major groups of 35S-labelled macromolecules were synthesized by these cells. The first group did not bind to anion-exchange columns at pH 6.0 and contained 60-80% of the total incorporated radiolabel. The second group did bind to anion-exchange columns at pH 6.0 and eluted as a single peak of radioactivity at an ionic strength characteristic of sulphated proteoglycans; it accounted for the remaining incorporated radiolabel. Analysis of this material on Sepharose CL-6B demonstrated that 35S-labelled macromolecules isolated from the cell extract migrated with Kav. of 0.36, while corresponding material isolated from the medium migrated with Kav. of 0.51. When subjected to electrophoresis on SDS/polyacrylamide gels the intact proteoglycan had a molecular mass of approx. 90 kDa and yielded two core proteins of molecular mass 31 kDa and 28 kDa after digestion with chondroitinase ABC. The peak of labelled macromolecules which did not bind to the anion-exchange column was found, by SDS/PAGE, to comprise 35S-labelled proteins of various molecular masses. The 35S label was displaced from this fraction by treatment with 0.1 M-sodium sulphite, suggesting that the radiolabel was in the form of an S-sulpho sulphite derivative. Using the sulphite-trapping agents N-2,4-dinitroanilinomaleimide and cyst(e)ine, [35S]sulphite was detected in the incubation medium of PMN, indicating that these cells were able to synthesize [35S]sulphite from [35S]sulphate. The release of [35S]sulphite from neutrophil cultures was calculated to be 78 pmol/h per 10(6) cells. When exogenous proteins were included in the incubation medium of cell cultures, the [35S]sulphite reacted with these proteins to form a stable 35S-labelled conjugate.
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PMID:Synthesis of 35S-labelled macromolecules by polymorphonuclear neutrophils. Evidence for the production of [35S]sulphite which can modify both endogenous and exogenous proteins. 146 61

Fixed fragments of bovine nasal septum cartilage were digested for six hours either with testicular hyaluronidase or streptomyces hyaluronidase or flavobacter chondroitinase ABC, and observed with a transmission electron microscope. Collagen fibril diameters (D) were measured to evaluate the effect of enzymatic digestion on the fibril size. This resulted in an increased frequency (17% to 47%) of "thin" fibrils (80 to 32 nm), followed by a decrease (65% to 31%) of the frequency of "mid" fibrils (32 to 64 nm). The frequency of "thick" fibrils (over 64 nm) showed a moderate increase (18% to 22%). Considering the relationship between fibril diameter, fibril volume and collagen content, the apparently relevant increase in number of the "thin" fibrils corresponds to an alteration of only 4% of the total collagen. On the other hand the increase of the "thick" fibrils implies a conspicuous alteration of 20% of the total collagen. The observed fibril rearrangement after digestion may be explained in terms of the wrap of matrix proteoglycans around each fibril. The enzymatic removal of the proteoglycans could make "mid" collagen fibrils free to regress into "thin" as well as to merge together into "thick" fibrils.
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PMID:Collagen fibril ultrastructure alters after glycanolytic digestion. 147 56

The localization of proteoglycans in rat epiphyseal growth plate cartilage was investigated immunoelectron microscopically by the post-embedding method, using mouse monoclonal antibody (2-B-6) which specifically recognizes 4-sulphated chondroitin or dermatan sulphate after digestion of proteoglycans with chondroitinase ABC. Fixation with ruthenium hexamine trichloride (RHT) and embedding in LR White served to preserve chondrocytes in the expanded state and matrix proteoglycans were observed as a reticular network of filaments. Immunoelectron microscopy revealed gold labelling of the secondary antibodies for the demonstration of proteoglycans on these filamentous structures and in elements of the Golgi apparatus. Filaments associated with matrix vesicles were also labelled. After fixation in the presence of RHT, it was clearly demonstrated that cartilage matrix proteoglycans are retained approximately in their original spatial distribution and their antigenicity is well preserved.
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PMID:Immunoelectron microscopic study of proteoglycans in rat epiphyseal growth plate cartilage after fixation with ruthenium hexamine trichloride (RHT). 148 31


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