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)

Metachromatic leukodystrophy and Maroteaux-Lamy syndrome can be diagnosed by assay of leukocyte or fibroblast arylsulfatase A and B activity with the fluorogenic substrate 4-methylumbelliferyl sulfate. The arylsulfatases are extracted into a 27000 x g supernatant by sonication in 0.9% sodium chloride and then separated with CM-32 on columns or in test tubes. In 0.05 M sodium acetate pH 6.0, arylsulfatase A is not absorbed while arylsulfatase B is retained by the resin. The arylsulfatase B is then eluted from the resin with 0.3 M sodium chloride. The arylsulfatase A activity obtained from normal leukocytes and fibroblasts is linear for the initial 10 minutes of the reaction, is stimulated 3-fold by 6 mM lead acetate and inhibited 80% by 0.24 mM silver nitrate. After separation with CM-32, the arylsulfatase B activity is stimulated 3-fold by Triton X-100 (0.1%). Arylsulfatase A but not arylsulfatase B is destroyed by heat (60 degrees). Both leukocyte and fibroblast arylsulfatase A activity was reduced to 11% of control values in metachromatic leukodystrophy. Essentially no arylsulfatase B activity was detected in cells from patients with Maroteaux-Lamy syndrome. Metachromatic leukodystrophy heterozygotes but not Maroteaux-Lamy syndrome heterozygotes can also be distinguished by this method. A heat inactivation technique utilizing the differential thermal stabilities of the two enzymes for diagnosis of patients with Marotezux-Lamy syndrome is also described. The advantages of these 4-methylumbelliferyl sulfate assay procedures over the p-nitrocatechol sulfate method of assay are greater sensitivity, selectivity for the desired enzyme and potential for use in large scale testing.
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PMID:Arylsulfatases A and B in metachromatic leukodystrophy and Maroteaux-Lamy syndrome: studies with 4-methylumelliferyl sulfate. 0 5

The distribution of glycosaminoglycans and glycoproteins has been studied in cytoplasmic and particulate fractions of neurons isolated in bulk from rat cerebrum. Lysis of the neurons in 25 mM sodium phosphate buffer at pH 7.5 released 20% of the protein and over 90% of the lactate dehydrogenase in a soluble form. Eighty-two percent of the chondroitin sulfate was also released, together with 55% of the heparan sulfate and 24-25% of the hyaluronic acid and glycoproteins. The chondroitin sulfate remaining in the membranes was completely depolymerized to disaccharides after treatment with chondroitinase ABC, and treatment of the neuronal membranes with 0.1% trypsin removed 55-63% of the chondroitin sulfate and heparan sulfate but only 25% of the sulfated glycoproteins. The results reported here support our previous conclusion that the soluble chondroitin sulfate proteoglycan of brain is largely a cytoplasmic constitutent of neurons (and astrocytes) and is not primarily present in nervous tissue as an extracellular ground substance.
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PMID:Presence of chondroitin sulfate in the neuronal cytoplasm. 28 11

Chondroitinase B and chondroitinase C were separated from an extract of Flavobacterium heparinum induced with chondroitin 6-sulfate by using column chromatography on hydroxylapatite. Chondroitinase C was eluted together with the activities of hyaluronidase, delta4,5glycosiduronase, and sulfatase. The latter two activities were eliminated exclusively by passing the crude chondroitinase C fraction through a phosphono-cellulose column pre-equilibrated with 0.07M sodium phosphate buffer (pH 6.8). Chondroitinase C was then purified by affinity chromatography using dermatan sulfate-bound AH-Sepharose 4B coated with the same glycosaminoglycan. Purification of the enzyme was achieved 18-fold and in 73% yield. On the other hand, the activities of delta4,5glycosiduronase and sulfatase were decreased to 50 and 60%, respectively, as compared with those in the crude chondroitinase B fraction, after passing the fraction through a column of phosphono-cellulose pre-equilibrated with 0.1M sodium phosphate buffer (pH 6.8). The remaining activities of these two enzymes were then eliminated from chondroitinase B by affinity chromatography with heparin-bound AH-Sepharose 4B coated with dermatan sulfate. In the affinity chromatography used in the present study, non-covalent coating of the glycosaminoglycan-bound (covalently) AH-Sepharose 4B with the same or another glycosaminoglycan was found to be important.
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PMID:Purification of chondroitinase B and chondroitinase C using glycosaminoglycan-bound AH-Sepharose 4B. 42 37

Cytoplasmic granules of basophilic leukocytes stain metachromatically and have been thought to contain sulfated glycosaminoglycans, presumably heparin. To test this hypothesis, we identified the [35S]glycosaminoglycans synthesized by guinea pig blood basophils in culture and in vivo. Basophils isolated from guinea pig blood were cultured for 20 hr in F12 medium--10% guinea pig serum containing sodium [35S]sulfate. Alternatively, basophils were purified from animals receiving repeated i.v. injections of sodium [35S]sulfate. Glycoaminoglycans were isolated from these basophils after pronase digestion and identified by the use of selective glycosaminoglycan-degrading enzymes. Approximately 55% of the [35S]glycosaminoglycans was degraded by chondroitinase AC, indicating the presence of chondroitin sulfate; an additional 30 to 35% could be degraded by chondroitinase ABC, indicating that dermatan sulfate was also present. The 15% glycosaminoglycan remaining after chondroitinase ABC digestion was degraded by purified heparitinase (heparanase), which has no effect on authentic heparin but degrades heparan sulfate. Thus, the glycosaminoglycan content of guinea pig basophils is a mixture of chondroitin sulfate, dermatan sulfate, and smaller amounts of heparan sulfate. No heparin was detected.
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PMID:Sulfated glycosaminoglycans of guinea pig basophilic leukocytes. 68 51

The structurally related type XII-like collagen molecules TL-A and TL-B were recently identified in fetal bovine epiphyseal cartilage and subsequently shown to be collagen types XII and XIV, respectively. By indirect immunofluorescent staining of cartilage using monoclonal antibodies to the NC3 domains of each molecule, it was shown that type XII collagen was present predominantly around cartilage canals, the articular surface, subperichondrial margins, and the perichondrium, was less so in the remaining cartilage matrix, and was absent from the growth plate region. In the permanent cartilage of trachea, type XII stained somewhat more intensely in the margins beneath the loose connective tissue. Type XIV collagen localized more uniformly throughout the articular cartilage and was also absent from the growth plate region, whereas in tracheal cartilage, its distribution was similar to type XII. We have characterized the structure of these cartilage molecules and compared them with those from fetal bovine skin. Extraction of cartilage with 1 M NaCl and differential NaCl precipitation yields a fraction enriched for these two collagens. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting with monoclonal antibodies to the large amino-terminal non-triple-helical domain, NC3, revealed the presence in cartilage of two forms of type XII collagen: type XIIB, the molecule previously identified in chick and bovine tissues, and type XIIA, a much larger form equivalent to the molecule recently identified in WISH-transformed epithelial cell culture medium (Lunstrum, G. P., McDonough, A. M., Marinkovich, M. P., Keene, D. R., Morris, N. P., and Burgeson, R. E. (1992) J. Biol. Chem. 267, 20087-20092). Digestion with bacterial collagenase shows that the increased mass is present in the NC3A domain. Additional purification by velocity sedimentation and observation of rotary-shadowed images demonstrates molecules with extended non-triple-helical arms approximately 80 nm in length analogous to the WISH cell molecules. Electrophoretic mobilities of bands corresponding to type XIIA, but not type XIIB, are sensitive to chondroitinase ABC, indicating that type XIIA is a chondroitin sulfate proteoglycan and that modification occurs predominantly within the NC3A domain distal to NC3B. Neither type XIIB from skin nor type XIIA from WISH cells are chondroitinase-sensitive. By similar analysis, a portion of the type XIV collagen chains in cartilage was also sensitive to chondroitinase digestion. Chondroitin sulfate is apparently not located on its NC3 domain. As in skin, collagen types XII and XIV have subtly different distributions within cartilage and type XII may have a tissue-specific structure.
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PMID:Characterization of collagen types XII and XIV from fetal bovine cartilage. 140 Mar 27

We describe here the purification and partial characterization of a 200 kDa keratan sulphate proteoglycan found in the pericellular matrix of human embryonal carcinoma cells. Previously we have shown that this molecule is recognized by a monoclonal antibody (GCTM-2). The antigen was isolated using ion-exchange chromatography and gel filtration, purification being monitored by e.l.i.s.a. using GCTM-2. Metabolic labelling of GCT 27 C-4 embryonal carcinoma cells with sodium [35S]sulphate resulted in the incorporation of [35S]sulphate into the purified molecule. Throughout the purification procedure, the peaks of 35S radioactivity were coincident with the peaks of immunoreactivity, and this label was released both by digestion with keratanase and chondroitinase, confirming the proteoglycan nature of the antigen. The intact molecule ran as a single broad band of 200 kDa, which has been identified by silver staining and immunoblotting following gel electrophoresis. Amino acid analysis of the purified antigen indicated a high content of serine, glycine and aspartic acid/asparagine residues. Visualization by rotary-shadowing electron microscopy suggests that the purified material forms large aggregates, even under denaturing conditions. Deglycosylation of this preparation with trifluoromethanesulphonic acid yielded a major band of 55 kDa and a minor band of 48 kDa. The biochemical nature of the molecule described here, along with tissue distribution studies using GCTM-2, indicates that the antigen is not related to previously described keratan sulphate proteoglycans.
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PMID:A novel keratan sulphate proteoglycan from a human embryonal carcinoma cell line. 141 56

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

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

The purpose of this study was to investigate the pattern of sulphated glycosaminoglycan synthesis during morphogenesis and cytodifferentiation in mouse tooth rudiments and to compare the results with those obtained in another study for salivary gland, a branched organ. Sulphated glycosaminoglycan was labelled by incubating molar rudiments from day 15 of gestation to day 1 post partum in medium containing [35S]-sodium sulphate. The rudiments were washed, homogenized and digested in pronase and then were sequentially digested by chondroitinase ABC and chemically degraded by nitrous acid oxidation. The fractions from each of these procedures were analysed by chromatography on Sephadex G-50 columns. The analysis revealed that, during morphogenesis, levels of chondroitin sulphate increased to a peak of 91% at day 18 and levels of heparan sulphate diminished to 8% during this period. As cytodifferentiation occurred, the level of chondroitin sulphate dropped to 64% and that of heparan sulphate increased to 35%. These results are similar to those reported for rat submaxillary gland, a branching organ. It appears that this pattern of sulphated glycosaminoglycan synthesis is not a unique feature of branching morphogenesis but may be one which marks the transition between morphogenesis and cytodifferentiation in non-branching rudiments as well.
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PMID:The correlation of temporal regulation of glycosaminoglycan synthesis with morphogenetic events in mouse tooth development. 151 35

Chinese hamster ovary cells transfected with a 4.0-kilobase macrophage colony-stimulating factor (M-CSF) cDNA express two different M-CSF species; one has an apparent molecular weight of 85,000 and is identified as a homodimer of a 43-kDa subunit, and the other has an indeterminate structure greater than 200 kDa. In this study, we investigated the structure of the high molecular weight M-CSF by immunochemical procedures. The high molecular weight M-CSF was easily purified, since it bound tightly to DEAE-Sephacel and eluted at a characteristically high salt concentration. The high molecular weight M-CSF migrated as a diffuse band of over than 200,000 on nonreducing sodium dodecyl sulfate-polyacrylamide gels. Analysis of the same samples under reducing conditions revealed that the larger species consisted of a heteromer of the 43- and 150-200-kDa M-CSF subunits. Digestion of the 150-200-kDa M-CSF subunit with chondroitinase, which degrades the chondroitin sulfate glycosaminoglycan chain, yielded a 100 kDa band. This species was secreted instead of 150-200-kDa species when the cells were cultured in the presence of beta-D-xyloside, which inhibits the elongation of the chondroitin sulfate glycosaminoglycan chain in proteoglycans, providing additional evidence for the existence of a chondroitin sulfate chain in the 150-200-kDa M-CSF subunit. Removal of O- and N-linked carbohydrate from the 150-200-kDa subunit yielded a polypeptide chain with a larger molecular mass (approximately 45 kDa) than that of the 43-kDa subunit (approximately 25 kDa). Collectively, these results indicate that the 150-200-kDa M-CSF subunit is a proteoglycan with a core protein that may be an alternatively processed form of M-CSF.
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PMID:Identification of a high molecular weight macrophage colony-stimulating factor as a glycosaminoglycan-containing species. 153 50


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