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 tectorial membrane is a gel-like, acellular connective tissue overlying the microscopic organ of Corti--the auditory sensory structure. It is instrumental in the sound-synchronous deflection of the stereocilia of the hair cells, a central event in auditory transduction. It is well established that collagen, primarily type II, constitutes the major protein of the tectorial membrane, with smaller amounts of types IX and XI also present. However, conclusive information on the proteoglycans in this structure is lacking. Tectorial membranes were extracted with a 4 M guanidine--HCl solvent, and proteoglycans isolated after ethanol precipitation and collagenase treatment. A colorimetric assay based on the binding of the cationic dye safranin O to glycosaminoglycans, in combination with enzymatic techniques, detected significant amounts of chondroitin sulfate and keratan sulfate (0.29 and 0.17% on a wet weight basis, respectively). Agarose-polyacrylamide electrophoresis of chondroitinase-digested samples revealed a core protein with a similar molecular mass to that of the large cartilage proteoglycan aggrecan. This proteoglycan reacted with the antibody 3-B-3 (recognizing modified chondroitin 6-sulfate linkage region oligosaccharides). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed several low molecular mass proteins which reacted with 5-D-4, specific for keratan sulfate, one of which showed characteristics of fibromodulin. Comparison of the quantitative aspects of various connective tissue components of tectorial membrane with other type II collagen-containing structures revealed that this tissue resembles highly hydrated cartilage.
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PMID:Uronic acid-containing glycosaminoglycans and keratan sulfate are present in the tectorial membrane of the inner ear: functional implications. 827 27

The signals that trigger the cytodifferentiation of oligodendrocytes (OLGs) are largely unknown. Using as a model system cultures of pure OLGs, we have shown that adhesion to a substratum initiates myelinogenesis (Yim SH, Szuchet S, Polak PE, J Biol Chem 261:11808-11815, 1986). It was of interest to investigate whether components such as proteoglycans (PGs) play any role in the biology of OLGs as it pertains to myelinogenesis. We set out to determine first, whether OLGs carry PGs; second, the nature of the association of these components with OLG plasma membrane; and third, if and how these PGs are modulated by OLG-substratum interaction. We compared the expression and characteristics of PGs extracted with different solvents from nonattached (B3.f) and attached (B3.fA) OLGs. B3.f and B3.fA OLG cultures were labeled with carrier-free 35SO4(2-) in serum-free medium. After removing excess label, OLGs were treated with heparin to extract susceptible components. Pellets were then exposed to 1% Triton X-100 plus 0.1 M NaCl and subsequently to 4 M guanidine-HCl plus 0.5 M NaCl. Solutions containing extracted material were characterized by size-exclusion chromatography, SDS-PAGE, and enzymatic degradation. Herein we report that (1) OLGs display [35S]PGs on their surface within 24 hr of substratum adhesion, and (2) these PGs can be operationally classified as peripheral and integral. We further show that the peripheral PGs are of high and intermediate size as assessed by size-exclusion chromatography and are segregated within the plasma membrane in such a way that the species with intermediate mass are extracted while OLGs remain adhered, whereas the high-molecular-weight species are only extracted after OLGs have been detached. Heparin also dislodges a number of sulfated proteins/Gps. Only a single class--high molecular weight--of integral PGs was identified; this PG requires guanidine-HCl for extraction. All PGs belong to the heparan sulfate class as evidenced by their degradation with heparitinase and their lack of susceptibility to chondroitinase ABC. The common theme of our findings is that these macromolecules have basal levels of expression in the nonadhered OLGs but undergo an adhesion-induced enhancement in their syntheses. We postulate that these PGs (1) play a role in OLG-substratum adhesion and hence myelinogenesis, and (2) may be determinants in establishing OLG polarity. Such polarization is the first overt sign of OLG functional differentiation and occurs prior to any morphological differentiation, e.g., extension of processes does not occur until 48 hr later when the plasma membrane is already polarized.
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PMID:Oligodendrocyte proteoglycans: modulation by cell-substratum adhesion. 847 42

The type and distribution of mineral binding and collagenous matrix-associated chondroitin sulphate and dermatan sulphate proteoglycans in rabbit alveolar bone were studied biochemically and immunocytochemically, using three monoclonal antibodies (mAb 2B6, 3B3, and 1B5). The antibodies specifically recognize oligosaccharide stubs that remain attached to the core protein after enzymatic digestion of proteoglycans and identify epitopes in chondroitin 4-sulphate and dermatan sulphate; chondroitin 6-sulphate and unsulphated chondroitin; and unsulphated chondroitin, respectively. In addition, mAb 2B6 detects chondroitin 4-sulphate with chondroitinase ACII pre-treatment, and dermatan sulphate with chondroitinase B pre-treatment. Bone proteins were extracted from fresh specimens with a three-step extraction procedure: 4 M guanidine HCl (G-1 extract), 0.4 M EDTA (E-extract), followed by guanidine HCl (G-2 extract), to characterize mineral binding and collagenous matrix associated proteoglycans in E- and G2-extracts, respectively. Biochemical results using Western blot analysis of SDS-polyacrylamide gel electrophoresis of E- and G2-extracts demonstrated that mineral binding proteoglycans contain chondroitin 4-sulphate, chondroitin 6-sulphate, and dermatan sulphate, whereas collagenous matrix associated proteoglycans showed a predominance of dermatan sulphate with a trace of chondroitin 4-sulphate and no detectable chondroitin 6-sulphate or unsulphated chondroitin. Immunocytochemistry showed that staining associated with the mineral phase was limited to the walls of osteocytic lacunae and bone canaliculi, whereas staining associated with the matrix phase was seen on and between collagen fibrils in the remainder of the bone matrix. These results indicate that mineral binding proteoglycans having chondroitin 4-sulphate, dermatan sulphate, and chondroitin 6-sulphate were localized preferentially in the walls of the lacunocanalicular system, whereas collagenous associated dermatan sulphate proteoglycans were distributed over the remainder of the bone matrix.
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PMID:Nature and distribution of chondroitin sulphate and dermatan sulphate proteoglycans in rabbit alveolar bone. 881 81

The submandibular gland proteoglycans were investigated biochemically and immunohistochemically in male Sprague-Dawley rats. Proteoglycans were extracted with 4 M guanidine-HCl, followed by ultracentrifugation in a CsCl density gradient, and fractionated by ion-exchange chromatography and gel filtration. The molecular weight of PGs was estimated by SDS-PAGE and immunoblot analysis with monoclonal antibodies (HepSS-1 or 6-B-6). The glycosaminoglycan side-chains in the proteoglycan fractions were identified by electrophoresis on cellulose acetate membrane. Three proteoglycan fractions were obtained. One was a heparan sulphate proteoglycan that migrated as a diffuse band of about 210 kDa. The other two fractions contained at least two dermatan sulphate proteoglycans of 70-85 kDa and 40-50 kDa. Digestion of these two proteoglycans with chondroitinase ABC, but not heparitinase, produced two bands of 50 and 21 kDa, which were core proteins. The smaller dermatan sulphate proteoglycan may be a portion of the other, as the core protein of both bound to 6-B-6 antibody, and sugar chains of both were the same (20-30 kDa). Heparan sulphates recognized by antibody HepSS-1 were observed widely in the basement membrane, fibrous connective tissue, and striated and excretory ductal cells, while dermatan sulphate proteoglycans recognized by antibody 6-B-6 were located in the connective tissue surrounding striated and excretory ducts.
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PMID:Characteristics and localization of rat submandibular gland proteoglycans. 903 2

Cell-fibronectin interactions, mediated through several different receptors, have been implicated in a wide variety of cellular properties. Among the cell surface receptors for fibronectin, integrins are the best characterized, particularly the prototype alpha5beta1 integrin. Using [125I]iodine cell surface labeling or metabolic radiolabeling with sodium [35S]sulfate, we identified alpha5beta1 integrin as the only sulfated integrin among beta1 integrin heterodimers expressed by the human melanoma cell line Mel-85. This facultative sulfation was confirmed not only by immunoprecipitation reactions using specific monoclonal antibodies but also by fibronectin affinity chromatography, two-dimensional electrophoresis, and chemical reduction. The covalent nature of alpha5beta1 integrin sulfation was evidenced by its resistance to treatments with high ionic, chaotrophic, and denaturing agents such as 4 M NaCl, 4 M MgCl2, 8 M urea, and 6 M guanidine HCl. Based on deglycosylation procedures as chemical beta-elimination, proteinase K digestion, and susceptibility to glycosaminoglycan lyases (chondroitinase ABC and heparitinases I and II), it was demonstrated that the alpha5beta1 heterodimer and alpha5 and beta1 integrin subunits were proteoglycans. The importance of alpha5beta1 sulfation was strengthened by the finding that this molecule is also sulfated in MG-63 (human osteosarcoma) and HCT-8 (human colon adenocarcinoma) cells.
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PMID:Post-translational modifications of alpha5beta1 integrin by glycosaminoglycan chains. The alpha5beta1 integrin is a facultative proteoglycan. 913 4

The type and distribution of sulphated proteoglycans (PGs) in the midshaft subperiosteal bone of 15-18-day embryonic chick femurs were studied immunocytochemically and biochemically, using four monoclonal antibodies (MAb 2B6, 3B3, 1B5, and 5D4). These MAb specifically recognize epitopes in chondroitin 4-sulphate (C4-S) and dermatan sulphate (DS); chondroitin 6-sulphate (C6-S) and unsulphated chondroitin (C0-S); C0-S; and keratan sulphate (KS) respectively. Immunohistochemistry showed that staining of C4-S, DS, and KS, but not of C6-S and C0-S, was limited to osteoid, the cell surface of osteocytes, and to the walls of osteocytic lacunae and bone canaliculi in 15-18-day embryonic specimens. However, no significant difference in the distribution and intensity of immunostaining was observed in these specimens. Bone proteins were extracted from fresh 18-day embryonic specimens with a three extraction procedure, 4 M guanidine HCl (GdnCl, G-1 extract), 0.4 M EDTA (E-extract), followed by GdnCl (G-2 extract), to characterize mineral binding and collagenous matrix associated PGs in E- and G2-extracts respectively. Western blot analysis of E- and G2-extracts demonstrated that chondroitinase ABC-digested PGs with a molecular weight (Mr) approximately of 45,000 containing GAGs predominantly corresponding to C4-S and/or DS, with no detectable C6-S or C0-S present in the mineral and matrix phase, whereas KSPGs having an Mr of approximately 72,000 are only present in the mineral phase. These results indicate that embryonic chick bone contains small PGs having C4-S, DS, and KS chains with preferential localization to osteoid, the cell surface of osteocytes, and to the walls of osteocytic lacunae and bone canaliculi.
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PMID:Immunohistochemical and biochemical characterization of sulphated proteoglycans in embryonic chick bone. 935 33

The eggshell of the chicken is a useful model to study matrix components which affect biomineralization. As an extension of our previous immunohistochemical work which suggested the presence of dermatan sulfate proteoglycans in the mineralized region of the eggshell, a study was undertaken to characterize these molecules biochemically. After demineralization with HCl and extraction with 4 M guanidinium chloride containing protease inhibitors, the extract was partitioned by anion exchange chromatography. Step elution with 0.25 M and 1.0 M sodium chloride resulted in the generation of two fractions, both of which contain chondroitinase-sensitive proteoglycans with molecular weights estimated at 200,000 by gel electrophoresis. The proteoglycans in each fraction have core proteins with molecular weights of approximately 120,000 and glycosaminoglycans with average molecular weights of 22,000. Based on differential sensitivity to chondroitinase ABC and AC II, these glycosaminoglycans contain a small proportion of dermatan sulfate. The disaccharide compositions of these glycosaminoglycans differ for the proteoglycans eluted with 0.25 M and 1.0 M sodium chloride. Those eluted with lower sodium chloride are enriched in unsulfated chondroitin and have much more 4-sulfated than 6-sulfated disaccharides; those eluted with 1.0 M sodium chloride contain primarily 4-sulfated disaccharides, a small amount of 6-sulfated disaccharides, and less unsulfated disaccharides than the proteoglycans eluted with 0.25 M sodium chloride. The large difference in the proportions of unsulfated chondroitin may be the reason for the elution at different sodium chloride concentrations. Both of the anion exchange column fractions contain other proteins in addition to the proteoglycans. These proteins are not separated from the proteoglycans by a second anion exchange column or by molecular sieve chromatography under dissociative conditions. Of particular interest is the observation that the eggshell proteoglycans and their core proteins are recognized by a monoclonal antibody which recognizes an epitope on the core protein of avian versican. This suggests that, in spite of the large differences in the sizes of the core proteins of versican and the eggshell proteoglycans, these core proteins share some homology. Because anionic molecules are thought to be important regulators of biomineralization, and because preparations like those analyzed in this study have been shown to influence in vitro calcium carbonate crystallization, the eggshell proteoglycans may play a role in eggshell mineralization.
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PMID:Dermatan sulfate proteoglycans from the mineralized matrix of the avian eggshell. 951 87

This study used biochemical and light and electron microscopic immunohistochemical methods to localize and characterize large hyaluronate-binding proteoglycans in the developing mandible of fetal rats at embryonic day 15 (Day 15) to Day 18 using a monoclonal antibody (MAb) 5D5. This antibody is derived from bovine sclera and specifically recognizes the core protein of large proteoglycan such as versican, neurocan and brevican, but not that of aggrecan. At the light microscopic level, MAb 5D5 moderately stained the extracellular matrices among osteoblasts at the centers of ossification in Day 15 mandible specimens. Weaker staining was observed in osteoblasts, whereas Meckel's cartilage lacked staining. Ultrastructural immunocytochemistry showed the presence of immunogold particles over unmineralized matrices among osteoblasts and their intracellular organelles. In Day 16 to 18 specimens, bone nodules were recognized in LR gold sections before immunostaining, but, after immunostaining, consistently appeared devoid of mineral crystals and were seen as a demineralized structure that had an electron dense periphery within which fine filamentous and granular material were present. The appearance of these structures was created by the demineralization of thin sections on grids during immunostaining. Specific immunogold staining was clearly seen over the demineralized structures corresponding to bone nodules. The majority of immunogold particles tended to localize inside of the structures. Bone proteins were extracted from fresh, Day 18 specimens with a three-step technique: 4 M guanidine HCl (GdnCl,-extract), aqueous EDTA without GdnCl (E-extract), followed by GdnCl. Western blot analysis of SDS-polyacrylamide gel electrophoresis after chondroitinase ABC digestion, showed that G1-extract gave a 5D5 reactive band greater than 400 kDa, whereas E-extract produced two major reactive populations of small molecular size with core proteins approximately 63 and 74 kDa. These results indicate that the large proteoglycan having smaller molecular weight is preferentially localized to bone nodules and may correlate with bone matrix mineralization.
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PMID:Immunocytochemical localization and biochemical characterization of large proteoglycans in developing rat bone. 968 Jul 65

A little is known about proteoglycan (PG) changes, occuring in the course of scarring of tissues another than skin. The aim of present study was biochemical characterization of glycosaminoglycans (GAGs) and proteoglycans (PGs) of normal and scarred fascia. Samples of normal fascia lata were taken at autopsy from 23 individuals and samples of scarred fascia lata were removed from 23 patients at reoperations for femoral fracture. The obtained tissues were divided into two samples: first of them was submitted to GAG isolation and the second one to PG isolation. GAGs were extracted by extensive papain digestion followed by the fractionation using cetylpyridinium chloride. In order to qualitative and quantitative characterization GAGs were submitted to electrophoresis on cellulose acetate before and after treatment with enzymes, specifically depolymerizing some kinds of GAGs. PGs were extracted using 4 M guanidine HCl followed by purification by forming complexes with Alcian blue. PGs were submitted to gel permeation chromatography on Sepharose 4B. In order to obtain core proteins PGs were depolymerized with chondroitinase ABC. The purified PGs and their core proteins were separated with sodium dodecyl sulphate/polyacrylamide gel electrophoresis (SDS/PAGE). It was found that total GAGs content was significantly elevated in scarred fascia. Both types of fascia contained chondroitin-, dermatan- and heparan sulphates and hyaluronic acid. Dermatan sulphates (DS) were the predominant GAGs of normal and scarred fascia. The contents of all GAG types were increased in scarred fascia. Both types of fascia contained two kinds of dermatan sulphate proteoglycans (DSPGs); first being similar to biglycan and the second one similar to decorin, as it was judged by molecular weight of their native molecules and core proteins as well as type of GAG components. Densitometric analysis showed that decorin is a predominant DSPG in both fascia types, but in scarred tissue the ratio of biglycan to decorin is considerably higher. Moreover, in scarred fascia a large chondroitin sulphate proteoglycan (CSPG) was also observed. The obtained results have shown that the scar formation is accompanied by quantitative and qualitative alterations in GAGs/PGs resembling those observed in hypertrophic skin scars. The biochemical modification of the scarred fascia lata may partly explain the clinically manifested damage to biomechanical properties of this tissue.
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PMID:An accumulation of proteoglycans in scarred fascia. 1072 38

A method for the determination of hyaluronic acid in shark fin by high performance liquid chromatography (HPLC) is described. At 37 degrees C, with 0.2 mol/L Tris-HCl buffer solution, hyaluronic acid was converted to the hyaluronic acid disaccharide by zymohydrolysis with chondroitinase ABC. The chromatographic conditions were as follows: ZORBAX carbohydrate analysis column (4.6 mm i.d. x 250 mm, 5 microns); room temperature; UV-VIS detector set at 226 nm; mobile phase V(acetonitrile): V(0.5% phosphoric acid) = 2:98; 0.45 micron filter membrane, pumping filter; injection volume 10 microL; flow rate 1 mL/min. The calibration curve for the hyaluronic acid disaccharide was linear over the range of 25 g/L-600 g/L. This method was applied to the analysis of shark fin with satisfactory results. The hyaluronic acid contents in different shark fins were from 0.86% to 1.96%.
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PMID:[Zymohydrolysis with chondroitinase ABC and high performance liquid chromatography used for the determination of hyaluronic acid in shark fin]. 1268 8


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