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)

Isolated rabbit glomeruli were incubated in vitro with 35SO4 in order to analyze the proteoglycans synthesized. Proteoglycans extracted with 4 M guanidine HCl from whole isolated glomeruli and from purified glomerular basement membrane (GBM) were analyzed by gel filtration chromatography. Two types of sulfated proteoglycans were found to be synthesized by rabbit glomeruli and these contained either heparan sulfate or chondroitin/dermatan sulfate glycosaminoglycan chains. These glycosaminoglycans were characterized by their sensitivity to selective degradation by nitrous acid or chondroitinase ABC, respectively. The major proteoglycan extracted from the whole glomeruli was a chondroitin/dermatan sulfate species (75%), while purified GBM contained mostly heparan sulfate (70%). The glycosaminoglycan chains were estimated to be about 12,000 molecular weight which is consistent with previous estimates for similar molecules extracted from the rat GBM.
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PMID:Biosynthesis of proteoglycans by isolated rabbit glomeruli. 662 17

Ruthenium red and toluidine blue O precipitates were described associated with lathyritic elastic fibers in aortas of chickens treated with beta-aminopropionitrile fumarate (I. Pasquali-Ronchetti, C. Fornieri, I. Castellani, G. M. Bressan, and D. Volpin (1981). Alterations of the connective tissue components induced by beta-aminopropionitrile. Exp. Mol. Pathol. 35, 42-56). In this report evidence is given that these precipitates reveal the presence of proteoglycans, as they are completely removed by 5 M guanidine-HCl incubation and by specific enzymatic digestions. In particular, proteoglycans associated with the poorly cross-linked lathyritic elastin can be removed by testicular hyaluronidase, chondroitinase ABC, heparitinase, and nitrous acid treatments, whereas they are rather resistant to streptococcal hyaluronidase and chondroitinase AC. On the contrary, proteoglycans of the matrix or associated with collagen fibers are particularly sensitive to these latter enzymatic treatments. The conclusion is reached that glycosaminoglycans associated with beta-aminopropionitrile-induced lathyritic elastin (i) are different from those of the matrix or associated with collagen, and (ii) include mainly dermatan and heparan sulfates.
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PMID:Elastin fiber-associated glycosaminoglycans in beta-aminopropionitrile-induced lathyrism. 670 93

An acidic glycoconjugate containing mannose, galactose and phosphate in approximately equimolar amounts was extracted from Leishmania donovani promastigotes and partially characterized. The glycoconjugate could be metabolically labeled with either [3H]mannose or [3H]galactose and was extractable from a delipidated residue fraction with water/ethanol/diethyl ether/pyridine/concentrated NH4OH (15:15:5:1:0.017) at 25 degrees C. The radioactively labeled glycoconjugate was found to possess the following characteristics: 1) comprised 45-60% of the total [3H]mannose label incorporated into macromolecules; 2) was soluble in alkaline solvents and 0.5% Triton X-100; 3) migrated as a broad band upon electrophoresis on sodium dodecyl sulfate-polyacrylamide gels with an approximate molecular weight of 15,000-30,000; 4) bound to DE52 cellulose and was eluted with a salt gradient of 0-0.1 M NaCl; 5) was insensitive to Pronase, hyaluronidase, chondroitinase, endo-beta-N-acetylglucosaminidase H, and endo-beta-galactosidase; and 6) possessed hydrophobic properties. An unusual feature of the glycoconjugate was its lability to mild acid hydrolysis (0.02 N HCl, 15 min, 60 degrees C). As determined by alkaline phosphatase and glycosidase digestion and paper chromatographic analysis, the major fragment generated by mild acid hydrolysis was found to be a phosphorylated galactosyl-beta-mannose disaccharide. All of these characteristics suggest that the glycoconjugate may be a polysaccharide and, possibly, may be important in parasite-host cell interactions.
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PMID:Expression of an unusual acidic glycoconjugate in Leishmania donovani. 670 85

Kidneys were perfused with [35S]sulfate at 4 h in vitro to radiolabel sulfated proteoglycans. Glomeruli were isolated from the labeled kidneys, and purified fractions of glomerular basement membrane (GBM) were prepared therefrom. Proteoglycans were extracted from GBM fractions by use of 4 M guanidine-HCl at 4 degrees C in the presence of protease inhibitors. The efficiency of extraction was approximately 55% based on 35S radioactivity. The extracted proteoglycans were characterized by gel-filtration chromatography (before and after degradative treatments) and by their behavior in dissociative CsCl gradients. A single peak of proteoglycans with an Mr of 130,000 (based on cartilage proteoglycan standards) was obtained on Sepharose CL-4B or CL-6B. Approximately 85% of the total proteoglycans were susceptible to nitrous acid oxidation (which degrades heparan sulfates), and approximately 15% were susceptible to digestion with chondroitinase ABC (degrades chondroitin-4 and -6 sulfates and dermatan sulfate). The released glycosaminoglycan (GAG) chains had an Mr of approximately 26,000. Density gradient centrifugation resulted in the partial separation of the extracted proteoglycans into two types with different densities: a heparan sulfate proteoglycan that was enriched in the heavier fraction (p greater than 1.43 g/ml), and a chondroitin sulfate proteoglycan that was concentrated in the lighter fractions (p less than 1.41). The results indicate that two types of proteoglycans are synthesized and incorporated into the GBM that are similar in size and consist of four to five GAG chains (based on cartilage proteoglycan standards). The chromatographic behavior of the extracted proteoglycans and the derived GAG, together with the fact that the two types of proteoglycans can be partially separated into the density gradient, suggest that the heparan sulfate and chondroitin sulfate(s) are located on different core proteins.
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PMID:Partial characterization of newly synthesized proteoglycans isolated from the glomerular basement membrane. 679 99

The proteoglycans of the cynomolgus monkey corneal stroma were isolated and characterized by using a combination of physiochemical and biochemical methods. Proteoglycans were biosynthetically radiolabeled by incubating whole corneas in medium containing [35S]sulfate and either [3H]serine or [3H]mannose as precursors. Macromolecules were extracted from the corneal stromas with 4 M guanidine-HCl. After dialysis into 8 M urea, proteoglycans in the extracts were initially purified by DEAE-cellulose chromatography. A portion of the proteoglycan fraction was digested with chondroitinase ABC, and the keratan sulfate proteoglycans were then isolated by rechromatography of the digest on DEAE-cellulose. Another portion of the proteoglycan fraction was digested with endo-beta-galactosidase and the dermatan sulfate-proteoglycans were then isolated by chromatography of the digest on Sepharose CL-4B. Each proteoglycan population was further fractionated by chromatography on concanavalin A-Sepharose and by CsCl density gradient centrifugation. Four subpopulations for both the keratan sulfate proteoglycans and the dermatan sulfate proteoglycans were isolated. Based on differences in binding to concanavalin A-Sepharose, buoyant densities, and glycosaminoglycan content, subpopulations of each proteoglycan differ by the number and properties of both the glycosaminoglycan chains and the mannose-containing oligosaccharides attached to their protein core.
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PMID:Heterogeneity of proteoglycans in monkey corneal stroma. 683 14

The synthesis of hyaluronic acid by cultured chondrocytes from the Swarm rat chondrosarcoma was examined using [3H]glucosamine as a precursor. [3H]Hyaluronate in samples was estimated as the specific unsaturated disaccharide released by incubation with chondroitinase ABC under conditions where all [3H]hyaluronate was converted to disaccharide even in the presence of excess chondroitin sulfate. The products of digestion of chondroitin sulfate and hyaluronate were well separated by cellulose thin layer chromatography. A second method involving quantitation of the 3H-oligosaccharides released by digestion with Streptomyces hyaluronidase gave nearly identical results. [3H]Hyaluronate formed about 12% of the [3H]glycosaminoglycans synthesized by the chondrocytes, although levels as high as 20% were found in one set of cultures. The incorporation of [3H]glucosamine into chondroitin sulfate and hyaluronate was linear and at a constant ratio over a 24-h period. The distributions of [35S]proteoglycans, [3H]chondroitin sulfate, and [3H]hyaluronate between the medium, a 4.0 M guanidine HCl extract of the cell layer, and the cell residue, were investigated over 24 h. Both hyaluronate and proteoglycan accumulated in the medium with little retention in the cell layer. Pulse-chase experiments indicated that a greater proportion of the [3H]hyaluronate than the [3H]chondroitin sulfate was initially retained in the cells, but that subsequently [3H]hyaluronate accumulated more rapidly in the medium, suggesting differences in the transit time through the extracellular matrix for the two molecules once secreted from the cell. In cultures labeled for 6 h, 4.0 M guanidine HCl extracted 66% of the hyaluronate and 58% of the chondroitin sulfate associated with the cell layer, while 1% Zwittergent, a zwitterionic detergent, extracted 59% and 29%, respectively. The selective extraction with detergent suggests that there is a pool of hyaluronate in the cell layer which is not associated with proteoglycan aggregates. Equilibrium density gradient centrifugation of cell layer extracts and culture media, under dissociative conditions, showed that 89% of the [3H]hyaluronate banded at densities between 1.43-1.55 g/ml with a peak at 1.47 g/ml. Disaccharide analyses of the gradient fractions showed that the main proteoglycan component contained primarily chondroitin 4-sulfate but also revealed a minor proteoglycan at lower densities which was considerably enriched in chondroitin 6-sulfate.
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PMID:Biosynthesis of hyaluronic acid in cultures of chondrocytes from the Swarm rat chondrosarcoma. 706 20

The biosynthesis of proteoglycans in short term organ culture of human colon and colon carcinoma was studied. Proteoglycans, labeled with [35S]sulfate and [3H]serine, were extracted with either 4 M or 0.5 M guanidine HCl in the presence of protease inhibitors and sequentially purified by associative and dissociative CsCl density gradient ultracentrifugation. Normal colon synthesized two polydisperse classes of proteoglycans: a large heparan sulfate-containing monomer, with a Kav of 0.48 on Sepharose CL-2B and a small dermatan sulfate-containing monomer with a Kav of 0.65. A portion (25%) of the proteoglycans was found as aggregate when chromatographed under associative conditions, and the larger monomers interacted with hyaluronic acid to an extent greater than the smaller proteoglycans. Following papain or alkali treatment, the free glycosaminoglycan side chains of both monomers eluted as a single broad peak (Kav = 0.5) from Sepharose CL-6B, with an estimated Mr of 20 X 10(3). In contrast, colon carcinoma synthesized only one proteoglycan monomer, which aggregated to a limited extent (12%). This proteoglycan population, with a Kav of 0.7 on CL-2B, contained chondroitin sulfate as the major glycosaminoglycan (greater than 81%), with small amounts of dermatan sulfate. The glycosaminoglycans had an estimated Mr of 9 X 10(3), and the disaccharides released by chondroitinase ABC consisted of 32% 4-sulfate and 68% 6-sulfate. Electron microscopy of mixed proteoglycancytochrome c monolayers from the associative fractions of normal and neoplastic colon revealed aggregated complexes which were similar in over structure, although smaller than the proteoglycan aggregates from cartilage.
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PMID:Isolation and characterization of proteoglycans synthesized by human colon and colon carcinoma. 710 48

The ultrastructural characteristics of alveolar (ABM) and capillary (CBM) basement membranes in the adult rat lung have been defined using tannic acid fixation, ruthenium red staining, or incubation in guanidine HCl. ABM is dense and amorphous, has 3- to 5-nm filaments in the lamina rara externa (facing the alveolus) that run between the lamina densa and the basal cell surface of the epithelium, has an orderly array of ruthenium red-positive anionic sites that appear predominantly (79%) on the lamina rara externa, and has discontinuities beneath alveolar type II cells but not type I cells that allow penetration of type II cytoplasmic processes into the interstitium of the alveolar wall. The CBM is fibrillar and less compact than ABM, has no lamina rara filaments, and has one fifth the number of ruthenium red-positive anionic sites of ABM that appear predominantly (64%) overlying the lamina densa. Incubation of lung tissue with Flavobacterium heparinum enzyme or with chondroitinase has shown that ABM anionic sites represent heparan sulfate proteoglycans, whereas CBM anionic sites contain this and other sulfated proteoglycans. The CBM fuses in a local fashion with ABM, compartmentalizing the alveolar wall into a thick and thin side and establishing a thin, single, basement-membrane gas-exchange surface between alveolar air, and capillary blood. The potential implications of ABM and CBM ultrastructure for permeability, cell differentiation, and repair and morphogenesis of the lung are discussed.
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PMID:Structural features of alveolar wall basement membrane in the adult rat lung. 719 26

The subcellular route of incorporation of complex carbohydrates into rabbit heterophil primary granules and their subsequent intragranular distribution during granule maturation were studied with ultrastructural, cytochemical, and radioautographic methods. High iron diamine (HID) staining of sulfated glycoconjugates in primary granules was partially diminished after treatment with chondroitinase ABC or after removal of N-sulfate groups with nitrous acid, but was not altered by exposure to hyaluronidase, trypsin, or HCl. Subsequent thiocarbohydrazide-silver proteinate (TCH-SP) straining of thin sections increased the density of the HID reaction product. Golgi-derived spherules and very immature morular granules stained weakly with HID-TCH-SP and labeled intensely after a 10 min incubation with 35SO4. After a 60 min 35SO4 pulse and a 60 min chase, an increase in radiolabeling was observed in granules with HID stained, fused morular material, and some labeling was present in more mature rim stained granules. Fully mature granules lacked HID or HID-TCH-SP staining, but contained most of the 35SO4 labels after a 60 min pulse and 18 hr chase in vitro. Periodate-thiocarbohydrazide-silver proteinate (PA-TCH-SP) staining of unosmicated thin sections localized vicinal glycol-containing complex carbohydrates in Golgi-associated small vesicles. These vesicles lacked HID-TCH-SP staining and apparently contained neutral glycoprotein. They frequently bordered, in a rosette arrangement, the immature morular granules, but not the more mature primary granules. The PA-TCH-SP method localized complex carbohydrates in the rim of granules precursors and enclosed a spherule or morula, but failed to stain the sulfate-containing material in the morulas or spherules. PA-TCH-SP reactivity was diffusely distributed in moderately mature granules and was decreased in fully mature granules. These results indicate that heterophil primary granule contain several complex carbohydrates including O-sulfated and N-sulfated glycosaminoglycans, as well as vicinal glycol-containing glycoproteins. These complex carbohydrates are transported to immature primary granules by different Golgi-derived organelles. The complex carbohydrates are subsequently distributed differently within primary granules and become masked to staining as the granule matures.
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PMID:Ultrastructural cytochemistry and radioautography of complex carbohydrates in heterophil granulocytes from rabbit bone marrow. 741 99

In an earlier analysis of the retinal biosynthesis of proteoglycan, we noted that, following photoreceptor degeneration in the rd (retinal degeneration) mouse, the remaining inner retina exhibited a marked elevation in synthesis of heparan sulfate proteoglycan (HSPG), well above the level observed in the normal (nondegenerate) retina, as well as a pronounced increase in sulfation of protein substrates. Biochemical and autoradiographic results of 35S-amino acid utilization reported here confirm that the 35SO4(2-) differences seen previously are accompanied by increased protein synthesis in the rd retina. An intact photoreceptor cell layer is neither a barrier to nor a sink for the amino acid precursor. Further, we have examined sulfate utilization in four other rodent strains with photoreceptor degenerations. In each of the models examined, an increase in retinal synthesis of 35SO4(2-)-labeled HSPG and glycoproteins occurs following photoreceptor degeneration. We have metabolically labeled with Na2(35)SO4 isolated retinal cultures from the following: (a) mice with light-induced photoreceptor degeneration; (b) rd mice; (c) transgenic mice with photoreceptor degeneration; (d) RCS rats; and (e) rats with light-induced photoreceptor degeneration. Comparisons were made with concurrent cultures of control nondegenerate retinal tissues. Protein and proteoglycan-enriched fractions were prepared from the incubation media and guanidine HCl/detergent extracts of the retinas by ion-exchange chromatography. The 35SO4(2-)-proteoglycans were identified by chondroitinase ABC and nitrous acid treatments. Retinas lacking photoreceptors produced at least five times the amount of 35SO4(2-)-HSPG found in control incubations. The RCS and light-damaged rats also showed increased synthesis of 35SO4(2-)-chondroitin sulfate proteoglycan relative to the control, through the increase was of lesser magnitude than the HSPG effect. 35SO4(2-)-protein in degenerate and light-damaged retinas always contained at least twice the radioactivity found in comparable control preparations. The bulk of the increased radiolabeling was found in N-linked oligosaccharides, including several recognized by the HNK-1 antibody. These data suggest that a sustained increase in HSPG and HNK-1 glycoprotein synthesis is a consistent response of inner retinal cells following loss of photoreceptors and is independent of the cause of photoreceptor degeneration.
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PMID:Increased retinal synthesis of heparan sulfate proteoglycan and HNK-1 glycoproteins following photoreceptor degeneration. 803 98


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