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)

In this report we describe a system capable of resolving all of the known unsaturated disaccharides derived from the chondroitin sulphates, dermatan sulphate and hyaluronic acid by chondroitinase digestion. This system is superior to others in that the non-sulphated and mono-, di- and tri-sulphated disaccharides can be separated with good resolution in approximately 40 min in an isocratic solvent. The system employs an amino-cyano silica gel column (Whatman Partisil 5 PAC, 25 cm) and is eluted with an isocratic solvent consisting of 48% (v/v) acetonitrile, 14% (v/v) methanol and 38% (v/v) aqueous buffer. This aqueous buffer contains 0.5 M Tris-HCl, 0.1 M boric acid, 23.4 mM sulphuric acid, pH 8.0. UV absorption is monitored at 229 nm and for most disaccharides as little as 150 ng can be reliably determined. The addition of boric acid to the eluent is essential for good resolution of all components and the addition of low concentrations of sulphuric acid is used to control the elution times of various components. The system was applied to the analysis of glycosaminoglycan standards and excellent agreement with previous compositional analyses was obtained.
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PMID:High pressure liquid chromatographic identification of hyaluronic acid and chondroitin sulphate disaccharides. 179 40

The temporomandibular joint (TMJ) provides articulation between the jaw and cranium, which associate with jaw movement and growth. The articular disc of TMJ separates the surfaces of the temporal bone and mandibular condyle. An understanding of its biochemical composition is very important, because the TMJ exhibits variety of pathological derangements including anterior displacement of disc. Proteoglycan (PG), major component of the disc, is one of the non-collagenous protein, which relates to the tissue viscoelasticity and physiological stress. This paper describe the isolation and characterization of proteoglycans from bovine articular disc. Articular discs obtained from bovine were cutted into small pieces. They were then extracted with 0.05 M Tris-HCl buffer, pH 7.4, containing 4 M guanidium HCl (Gdm HCl) and protease inhibitors for 12h at 4 degrees C. PGs were isolated by chromatography of Gdm HCl extract. The sequential chromatography steps consisted of ion-exchange chromatography on DEAE-Sephacel in 4 M Urea, rechromatography of FPLC Superose 6 in 4 M Urea. The two forms of PGs (on SDS-PAGE, Mr = 120-130 K and 200 K) were isolated by these steps. The core protein of two forms of PGs liberated by chondroitinase ABC were shown by SDS-PAGE as Mr = 58,000. Also the glycosaminoglycan (GAG) chains of PGs liberated by papain digestion were shown by SDS-PAGE as Mr = 70-80 K. Moreover GAG chains of PGs were consisted of chondroitin sulfate A, C and dermatan sulfate. Antisera raised against bovine periodontal ligament PGs cross-react with core protein of disc PGs (obtained after chondroitinase digestion), but not with bone small PG. These data suggested that two forms of PGs have a identical core protein. However 120-130 K PG might have one GAG chain, and 200 K PG might have two GAG chains. These small PGs were different from bone small PG, especially dermatan sulfate contents, which may be important in disc tissue.
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PMID:[Purification and partial characterization of proteoglycans of bovine articular disc]. 213 66

The monoclonal antibody 75d7, specific for type XII collagen (Sugrue, S.P., Gordon, M.K., Seyer, J., Dublet, B., van der Rest, M., and Olsen, B. R. (1989) J. Cell Biol., in press), was used to characterize the intact form of type XII collagen from chick embryo leg tendons. On an immunoblot of a 6% polyacrylamide gel of tendon extracts, one sharp band is recognized by the antibody at Mr = 220,000, while two fuzzy and poorly resolved bands are seen at Mr = 270,000 and Mr = 290,000. By immunoprecipitation of radiolabeled tendon culture media and electrophoresis of the precipitated material, bands with the same mobilities are observed, indicating that type XII collagen is not proteolytically processed in the extracellular space. Type XII collagen was extracted from tendons with 1 M NaCl in a Tris-HCl buffer and partially purified by concanavalin A-Sepharose and gel permeation chromatographies, using dot immunoblots to monitor the purification. Fractions highly enriched in bacterial collagenase-sensitive proteins with the same electrophoretic properties as type XII collagen were obtained. These fractions did not stain with Alcian blue and neither they nor the immunostained type XII collagen were affected by chondroitinase ABC digestion, indicating that type XII collagen is not a proteoglycan. A disulfide-bonded trimeric CNBr peptide was isolated by affinity chromatography on an antibody column and further purified by gel electrophoresis. Its NH2-terminal amino acid sequence was shown to be unique, demonstrating that type XII collagen is a homotrimer [alpha 1 (XII)]3. After bacterial collagenase digestion, both the immunopurified radiolabeled preparation and the purified tendon extract fraction showed by gel electrophoresis the presence of a large disulfide-bonded, 3 x 190-kDa, collagenase-resistant domain. Rotary shadowing and electron microscopy of the purified type XII fraction demonstrated that the molecule has the structure of a cross consisting of a 75 nm collagenase-sensitive tail, a central globule, and three 60 nm arms each ending in a small globule. After heat denaturation and renaturation, only a very large globule can be seen, attached to the triple helical tail. These results show that type XII collagen has a unique structure and is different from the other matrix constituents described so far.
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PMID:The structure of avian type XII collagen. Alpha 1 (XII) chains contain 190-kDa non-triple helical amino-terminal domains and form homotrimeric molecules. 275 5

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

A novel analytical method for determination of total amount of chondroitin sulfate (CS) based on its conversion to desulfated chondro-disaccharide via an enzyme-catalyzed reaction, was developed. Using the in-capillary enzyme reaction, the method was also applied to the successful construction of an on-line analytical system. Within this system, electrophoretic migration was used to mix zones containing the enzyme mixture (chondroitinase ABC, chondro-4-sulfatase, chondro-6-sulfatase and 2-o-sulfatase) and the substrate (CS). The reaction was then allowed to proceed in the presence of a weak electric field and, finally, the product (desulfated chondro-disaccharide) of enzyme reaction migrated to the detector under the influence of an applied electric field. A polyvinyl alcohol-coated capillary was used to reduce protein adsorption. Desulfated chondro-disaccharide was successfully migrated toward the anode in 10 mM Tris-acetate buffer (pH 7.0) under reversed polarity and detected at 232 nm. The established method was validated and demonstrated to be applicable in the determination of total amount of CS in a commercial ophthalmic solution. No interference from the formulation excipients was observed. Good linearity was obtained, with correlation coefficients above 0.999. Recoveries and precisions ranged from 100.0 to 100.5%, and from 0.2 to 0.6% of the relative standard deviation, respectively. Good agreement was obtained between the established method and traditional photometric method based on carbazole reaction. In this study, application of the method to disaccharide compositional analysis was also performed.
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PMID:Development of a novel analytical method for determination of chondroitin sulfate using an in-capillary enzyme reaction. 1511 83

CE conditions for monitoring the unsaturated disaccharides of hyaluronic acid (di-HA) and chondroitin sulfate (di-CS) using an alkaline tetraborate buffer, electrokinetic sample injection, and UV absorption detection at 232 nm are reported. Separations were performed in an uncoated fused-silica capillary having reversed polarity and reversed electroosmosis generated with the addition of CTAB to the buffer. The influence of various separation parameters, including the concentration of CTAB, buffer pH, concentration of tetraborate, and applied voltage, on the resolution of the two disaccharides was investigated. Baseline separation was obtained with 25 mM tetraborate at pH 10.0 and having 0.05 mM CTAB. Chloride and phosphate in the sample are beneficial for the stacking of the disaccharides, with di-HA forming a much sharper peak than di-CS. Using samples prepared in 25 mM Tris-HCl (pH 7.5) and electrokinetic injection at the cathode at -10 kV for 40 s, linear relationships between the corrected peak area and the concentration of the disaccharides have been found in the ranges of 1.0-400.0 and 0.1-1.0 microg/mL (0.2-1.0 microg/mL for di-CS), with correlation coefficients being >0.9933 in all cases. The RSDs of detection times and corrected peak areas were between 1.13-1.24 and 1.57-2.13%, respectively. Applied to human serum samples that were prepared by ethanol precipitation and depolymerization of the two polysaccharides with chondroitinase ABC reveals comigration of endogenous compounds with di-HA and a sample-dependent detection time. The di-HA content in the serum sample can be estimated via subtraction of the blank peak that is obtained without enzymatic hydrolysis.
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PMID:Analysis of the disaccharides derived from hyaluronic acid and chondroitin sulfate by capillary electrophoresis with sample stacking. 1634 6

We developed a method to extract differentially chondroitin sulfate proteoglycans (CSPGs) that are diffusely present in the central nervous system (CNS) matrix and CSPGs that are present in the condensed matrix of perineuronal nets (PNNs). Adult rat brain was sequentially extracted with Tris-buffered saline (TBS), TBS-containing detergent, 1 m NaCl, and 6 m urea. Extracting tissue sections with these buffers showed that the diffuse and membrane-bound CSPGs were extracted in the first three buffers, but PNN-associated CSPGs remained and were only removed by 6 m urea. Most of the CSPGs were extracted to some degree with all the buffers, with neurocan, brevican, aggrecan, and versican particularly associated with the stable urea-extractable PNNs. The CSPGs in stable complexes only extractable in urea buffer are found from postnatal day 7-14 coinciding with PNN formation. Disaccharide composition analysis indicated a different glycosaminoglycan (GAG) composition for PGs strongly associated with extracellular matrix (ECM). For CS/dermatan sulfate (DS)-GAG the content of nonsulfated, 6-O-sulfated, 2,6-O-disulfated, and 4,6-O-disulfated disaccharides were higher and for heparan sulfate (HS)-GAG, the content of 6-O-sulfated, 2-N-, 6-O-disulfated, 2-O-, 2-N-disulfated, and 2-O-, 2-N-, 6-O-trisulfated disaccharides were higher in urea extract compared with other buffer extracts. Digestions with chondroitinase ABC and hyaluronidase indicated that aggrecan, versican, neurocan, brevican, and phosphacan are retained in PNNs through binding to hyaluronan (HA). A comparison of the brain and spinal cord ECM with respect to CSPGs indicated that the PNNs in both parts of the CNS have the same composition.
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PMID:Composition of perineuronal net extracellular matrix in rat brain: a different disaccharide composition for the net-associated proteoglycans. 1664 27

Macromolecular prodrugs of three non-steroidal anti-inflammatory drugs (NSAIDs), ibuprofen, ketoprofen, and naproxen, were prepared by the covalent attachment of the drugs onto chondroitin sulfate (ChS) using PEG 1000 as a spacer. Drug-PEG adducts were synthesized using 1,1'-carbonyl diimidazole as a coupling agent in dimethyl sulfoxide, followed by the reaction with ChS in highly dilute aqueous solution at pH 6.8 via N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDAC) as a conjugation agent. The drug-ChS conjugates were confirmed by FTIR, 1H NMR and 13C NMR and the molar percent of drug substitution onto ChS was characterized by 1H NMR using the peak areas of the three protons of -PhiCHCH3 on the drugs to those of -NHCOCH3 on ChS. All drug-ChS conjugates are water-soluble. The release amounts of the free drugs from their corresponding drug-ChS conjugates were evaluated in the presence or absence of either esterase or chondroitinase, and the both enzymes in pH 7.4 Tris-buffer solutions at 37 degrees C by high performance liquid chromatography (HPLC). Keto-ChS conjugates released approximately 100% ketoprofen within 12h in the presence of esterase, but the combination with chondroitinase did not accelerate the release rate. The degradation of Keto-ChS conjugates by chondroitinase was confirmed by gel permeation chromatography (GPC). The Keto-ChS conjugates still retained the enzymatic recognition even at the substitution of ketoprofen as high as 56 mol%. The inhibition percent of carrageenan-induced edema of Keto-ChS-56 was comparable to that of a simple blend of ChS and ketoprofen, suggesting that biologically active ChS and ketoprofen could be liberated from the conjugate.
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PMID:Chondroitin sulfate-based anti-inflammatory macromolecular prodrugs. 1683 35