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 biosynthesis of glycosaminoglycans (GAG) and glycopeptides was studied in rat kidney cortex, glomeruli, and isolated glomerular basement membranes (GBM). Rats were given four intraperitoneal injections of [(35)S]sulfate and [(3)H]glucosamine (over 10 hr) and sacrificed 14 hr after the last injection. Fractions of kidney glomeruli and purified GBM were prepared. The percent of the label incorporated into specific GAG or into glycopeptides was determined by selective degradative techniques in conjunction with gel filtration chromatography using the methods of Hart [Hart, G. W. (1976) J. Biol. Chem. 251, 6513-6521; Hart, G. W. (1978) Dev. Biol. 62, 78-98]. After digestion with Pronase and chromatography on Sephadex G-50, approximately 68% of the total (35)S radioactivity and 10-15% of the total (3)H radioactivity incorporated into cortex, glomeruli, or GBM was found in the GAG fraction, and the remainder ( approximately 32% of (35)S radioactivity and 85-90% of the (3)H radioactivity) was found in glycopeptide fractions. Treatment of GAG fractions isolated from the three sources (cortex, glomeruli, and GBM) with nitrous acid (which degrades heparan sulfates) indicated that the majority (85%, 65%, and 87%) of the (35)S radioactivity as well as the majority (60%, 50%, and 91%) of the (3)H radioactivity from all three sources was degraded by this treatment. When nitrous acid-resistant GAG from GBM were subjected to digestion with Streptomyces hyaluronidase (which degrades hyaluronic acid), approximately 6% of the (3)H-labeled material was sensitive to this treatment. The remaining (35)S- and (3)H-labeled GAG isolated from GBM were digested with chondroitinase ABC (which degrades chondroitin sulfates A and C and dermatan sulfate). Although the ratios of the types of GAG synthesized by all three sources were similar, in GBM the ratios of (35)S- to (3)H-labeled GAG and of (3)H-labeled glycopeptides to (3)H-labeled GAG were higher (2.5 times) than those found for glomeruli. The data demonstrate the synthesis of both sulfated and nonsulfated GAG by rat kidney cortex and glomeruli and their transport to and incorporation into the GBM. Heparan sulfate is the major GAG synthesized by glomeruli, but the glomeruli also synthesize smaller amounts of hyaluronic acid and chondroitin sulfates, which are in part incorporated into GBM. In addition, the renal cortex and the glomeruli synthesize glycopeptides, some of which are sulfated, and incorporate them into GBM.
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PMID:Sulfated and nonsulfated glycosaminoglycans and glycopeptides are synthesized by kidney in vivo and incorporated into glomerular basement membranes. 701 44

Rat serosal mast cells cultured in the presence of heat-inactivated fetal calf serum incorporated (35S) sulfate into heparin proteoglycan of approximately Mr = 750,000 after a 3-h pulse and a 2-h chase. beta-D-Xyloside (0.1 mM) treatments of cultures of rat mast cells resulted in an insignificant increase in total (35S) sulfate incorporation and the appearance of free glycosaminoglycans without a change in proteoglycan size. At higher beta-D-xyloside concentrations, total (35S)sulfate incorporation was inhibited and an increase in the relative glycosaminoglycan content was observed concomitant with a reduction in proteoglycan amount and size. As assessed by susceptibility to digestion by chondroitinase ABC, hydrolysis by nitrous acid, [3H]hexosamine content, and electrophoretic mobility, only heparin chains were polymerized onto the proteoglycan core in all cultures. In contrast, individual glycosaminoglycans which appeared only after beta-D-xyloside treatment were predominantly chondroitin sulfate rather than heparin, indicating that the beta-D-xyloside acceptor supported polymerization of chondroitin sulfate but not of heparin glycosaminoglycan. Thus, the peptide core is an important determinant for the synthesis of heparin glycosaminoglycan by rat peritoneal mast cells.
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PMID:Effect of p-nitrophenyl-beta-D-xyloside on proteoglycan and glycosaminoglycan biosynthesis in rat serosal mast cell cultures. 705 69

Host-derived sulfated components that copurify and are physically associated with the envelope of Rauscher murine leukemia virions grown in JLS-V9 cells were characterized by digestion with chondroitinase ABC and chondroitinase AC II, as well as nitrous acid degradation. A dermatan-sulfate-chondroitin-sulfate copolymer and heparin or heparan sulfate wee shown to be associated with the virions. Competitive binding studies indicated a specificity of the virions for association with heparan sulfate. The physiological importance of the association is discussed.
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PMID:Characterization of glycosaminoglycans associated with Rauscher murine leukemia virions. 707 63

Extraction of rat glomerular basement membrane, purified by osmotic lysis and sequential detergent treatment, with 8 M urea containing protease inhibitors solubilizes protein that is devoid of hydroxyproline and hydroxylysine. This material represents 8-12% of total membrane protein, elutes mainly as two high molecular weight peaks on agarose gel filtration, and is associated with glycosaminoglycans. Isolated rat renal glomeruli incorporate [35S]sulfate into basement membrane from which this non-collagenous 35S-labeled fraction can be subsequently solubilized. The radioactivity incorporated into urea-soluble glomerular basement membrane eluted primarily with the higher molecular weight peak (Mr greater than 250 000). Cellulose acetate electrophoresis after pronase digestion of the urea-soluble fraction revealed glycosaminoglycan that was resistant to digestion with Streptomyces hyaluronidase and chondroitinase ABC, sensitive to nitrous acid treatment, and contained [35S]sulfate. The findings indicate that one of the non-collagenous components of glomerular basement membrane is a proteoglycan containing heparan sulfate.
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PMID:Non-collagen protein and proteoglycan in renal glomerular basement membrane. 731 55

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

Madin-Darby canine kidney (MDCK) cells were cultured on polycarbonate filters to study the synthesis and sorting of proteoglycans in polarized epithelial cells. Two strains of MDCK cells were used. MDCK I cells resemble distal tubule epithelial cells, and MDCK II cells share some characteristics with proximal tubule cells. Both strains were grown to confluency and labelled with [35S]sulphate for 24 h. The apical and basolateral media and the cell fractions were harvested and analysed by DEAE ion-exchange chromatography. A large portion of the [35S]sulphate-labelled macromolecules bound strongly to the ion-exchange columns, and could be eluted in three distinct peaks. The latest eluting peak was demonstrated to contain almost exclusively chondroitin sulphate, whereas peak 2 contained mostly heparan sulphate, demonstrated by using chondroitinase ABC and nitrous acid (pH 1.5) respectively to depolymerize the [35S]glycosaminoglycan chains. Peak 1 contained negligible amounts of proteoglycans. Large differences could be observed in proteoglycan sorting in MDCK I and II cells. Strain I secreted approx. 67% of the proteoglycans to the apical side and 17% to the basolateral side. The cell fraction contained 17% of the proteoglycans after 24 h of labelling. In contrast, 19% of the proteoglycans were sorted to the apical side of MDCK II cells and 61% to the basolateral side, whereas the cell fraction contained 20%. Furthermore, the level of [35S]proteoglycan biosynthesis (apical and basolateral media and cell fraction total) was higher in MDCK I cells than in strain II. Based on the amount of material degraded by chondroitinase ABC and nitrous acid respectively, and the total amounts of [35S]proteoglycans recovered from the cells, it was calculated that the MDCK I strain synthesized approx. 56% chondroitin sulphate and 44% heparan sulphate. In contrast, the MDCK II strain synthesized 69% heparan sulphate and 31% chondroitin sulphate. To further identify the [35S]proteoglycans synthesized by MDCK I and II cells, antibodies against perlecan, versican and syndecan were used. The antibody against mouse syndecan did not cross-react with any of the proteoglycans produced in MDCK I or II cells. Both MDCK I and II cells expressed perlecan; 57-61% could be recovered from the basolateral fractions and 18-34% from the apical medium. Versican was also found in both MDCK I and II cells. Compared with perlecan, a larger percentage of versican (43-53%) was found in the cell fractions.
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PMID:Proteoglycans in polarized epithelial Madin-Darby canine kidney cells. 748 45

Proteoglycans metabolically labelled with [35S]sulphate and [3H]glucosamine or [3H]leucine were isolated from the incubation medium and cell layer of human adult mesangial cells and glomerular visceral epithelial cells using sequential DEAE chromatography purification steps followed by gel-filtration chromatography. The proteoglycan composition of each peak was analysed by treatment with HNO2, chondroitinase ABC or chondroitinase AC followed by chromatography on Sephadex G-50 columns. Heparan sulphate proteoglycan (HSPG) and dermatan sulphate proteoglycan were detected in both the culture medium and cell layer of mesangial cells. Culture medium of glomerular visceral epithelial cells contained HSPG and a second proteoglycan with the properties of a hybrid molecule containing HS and chondroitin sulphate (CS). The cell layer contained HSPG and CSPG. Detailed analysis of the hybrid molecule revealed that it had an apparent molecular mass of 400 kDa. SDS/PAGE of hybrid molecules, after treatment with heparitinase and chondroitinase ABC, revealed a core protein of 80 kDa. Using 1.8% polyacrylamide/0.6% agarose-gel electrophoresis, we deduced that the HS and CS were independently attached to one core protein. Because glomerular-basement-membrane HSPG is thought to be derived from mesangial cells and glomerular visceral epithelial cells and this molecule is involved in several kidney diseases, we investigated its synthesis in more detail. Anti-(rat glomerular-basement-membrane HSPG) monoclonal antibodies (JM403) and anti-(human glomerular-basement-membrane HSPG) polyclonal antibodies (both antibodies known to react with the large basement-membrane HSPG, perlecan) reacted strongly with HSPG obtained from both mesangial cells and glomerular visceral epithelial cells. However, the hybrid molecule did not react with these antibodies, suggesting that the HS side chain and the core protein were different from glomerular-basement-membrane HSPG. To quantify HS we performed an inhibition ELISA using mouse antibodies specific for glomerular-basement-membrane HS glycosaminoglycan side chains. Glomerular visceral epithelial cells produced significantly higher levels of HS (between 197.56 and 269.40 micrograms/72 h per 10(6) cells) than mesangial cells (between 29.8 and 45.5 micrograms/72 h per 10(6) cells) (three different cell lines; n = 3; P < 0.001). HS production by these cells was inhibited by cycloheximide, revealing that it was synthesized de novo. Expression of perlecan mRNA, demonstrated using reverse transcriptase PCR, was different in the two cell types. We conclude that glomerular visceral epithelial cells and mesangial cells have characteristic patterns of proteoglycan production. Glomerular visceral epithelial cells produced a hybrid proteoglycan containing CS and HS independently attached to its core protein.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Proteoglycan production by human glomerular visceral epithelial cells and mesangial cells in vitro. 753 59

Histological and histochemical studies were carried out in Jcl:ICR mice to determine the changes in microscopic structures and glycosaminoglycan molecular species in the tissues involved in normal or faulty closure of the embryonic fissure. The purpose of this study was to elucidate the mechanism underlying the faulty closure of the embryonic fissure and to identify the key substances involved in normal and faulty closure. To obtain mice with an appropriate faulty closure of the embryonic fissure, ochratoxin A was employed as a teratogenic agent. Serial sections from tissues undergoing normal and faulty closure of the embryonic fissure were cut at a right angle to the fissure. As the staining procedures, a hematoxylin-eosin procedure and a sensitized high iron diamine method were used. A chemical modification (nitrous acid treatment) or an enzyme digestion procedure (chondroitinase ABC digestion procedure) was employed in combination with the sensitized high iron diamine method to identify the glycosaminoglycan molecular species in the tissues. The results obtained in the present study have substantiated the histophysiological importance of glycosaminoglycan molecular species during the course of histogenesis in the normal and the faulty closure of the embryonic fissure of developing murine eyes.
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PMID:Histological and histochemical studies of the normal and faulty closure of the embryonic fissure in the eye of ICR mouse. 764 79

The aim of this study was to investigate the nature and distribution of sulphated macromolecules of the extracellular matrix in rat gastric mucosa. This was achieved by developing an in vivo labelling system. An intraperitoneal injection of 1 mCi [35S]-sulphate was given for either 4 h (0.01% incorporation into macromolecular fraction) or 8 h (0.13% incorporation). At the end of the labelling period the stomach was removed and the mucosa and submucosa was either taken as a single combined sample or separated into four layers by blunt dissection. Each sample was papain digested and analysed by ion-exchange chromatography. This analysis revealed sulphated species of differing charge existing in differing proportions throughout the mucosa. These sulphated species eluted at NaCl concentrations of approximately 0 (A), 0.19 (B), 0.34 (C) and 0.78 mol/L (D) from a Q-Sepharose ion exchange column. Further analysis by size exclusion chromatography and chemical and enzymatic digestion showed that peaks B and C had molecular weights of 2.4 x 10(5) and 2.8 x 10(5), respectively and were resistant to chondroitinase ABC, heparitinase and nitrous acid digestion. Peak D was found to contain a polydisperse population of molecules with a molecular weight range of approximately 1 x 10(4) to 6 x 10(4). This sample was susceptible to nitrous acid and chondroitinase ABC digestion and was found predominantly in the sample isolated from deeper in the tissue. We have thus developed an in vivo labelling technique for sulphated macromolecules that can be used in the further study of injury to the gastric mucosa.
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PMID:Sulphated macromolecules produced by in vivo labelling in the rat gastric mucosa. 778 70

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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