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)

In this report we describe a very sensitive high-performance liquid chromatographic method for the determination of 24 nonsulfated and variously sulfated disaccharides present in chondroitin sulfates, dermatan sulfates, and hyaluronic acid. The method is superior to others in that monosulfated disaccharides at either C-2 or C-3 of the uronic acid moieties and mono-, di-, and trisulfated disaccharides containing N-sulfated galactosamine as well as non-, mono-, and oversulfated disaccharides derived from iduronic acid can be determined. Following chondroitinase digestions of tissue extracts or purified hyaluronic acid, chondroitin sulfate, and dermatan sulfate, the non-, di-, and trisulfate delta-disaccharides, are separated by direct injections into HPLC, whereas the monosulfated delta-disaccharides are chromatographed after a simple reduction of the galactosamine carbonyl group with sodium borohydride. The various sulfated delta-disaccharides are separated on an amino column (Econosphere NH2) and recorded at 231 nm. The column is eluted isocratically with 5 mM sodium dihydrogen orthophosphate, pH 2.55, for nonsulfated delta-disaccharides; 50 mM sodium dihydrogen orthophosphate, pH 2.50, for reduced monosulfated; and 50 mM sodium sulfate-10 mM sodium acetate, pH 5.0, for the separation of di- and trisulfated delta-disaccharides. A linear detector response was obtained for injections up to 50 micrograms of delta-disaccharides. As little as 5-8 ng of nonsulfated, 8-11 ng of monosulfated, 12-15 ng of disulfated, and 25-30 ng of trisulfated delta-disaccharides can be reliably detected. Application of this HPLC method to the analysis of various glycosaminoglycans in conjunction with chondroitinase AC, ABC, or B digestions and sulfatase hydrolysis adds to the knowledge of the structural spectrum of the galactosaminoglycans. It was thus possible to identify 24 different disaccharides in chondroitinase-susceptible glycosaminoglycans, including all C-5 epimeric disaccharides and those sulfated at C-2 or C-3 of the uronic acids and at the amino group of the galactosamine.
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PMID:Determination of 24 variously sulfated galactosaminoglycan- and hyaluronan-derived disaccharides by high-performance liquid chromatography. 798 92

The effect of mouse interferon alpha/beta (MuIFN alpha/beta) on the production of glycosaminoglycans (GAGs) by mouse glioma G-26 in vitro was evaluated. Two GAG species secreted extracellularly by the mouse glioma G-26 were isolated using cellulose acetate electrophoresis. They were identified as hyaluronic acid (HA) and chondroitin sulfate (CS) following enzymatic digestion with enzymes: hyaluronidase and chondroitinase ABC. Further characterization of CS by enzymatic digestion with specific chondroitinases for chondroitin 4-sulfate (CSA) and chondroitin 6-sulfate (CSC), revealed that the isolated CS was neither CSA nor CSC. Therefore, it may be either chondroitin sulfate B (CSB) (dermatan sulfate) or one of the 'chondroitin sulfate isomers' (D-H). The three day incubation of glioma G-26 cells with 8 x 10-8 x 10(4) U/ml of MuIFN alpha/beta resulted in a dose dependent inhibition of cell proliferation measured by 3H-thymidine incorporation and the MTT assay. The significant decrease of the CS (p < 0.008) but not the HA level, (measured densitometrically), was observed following 72 hours (hrs) incubation of G-26 cells with 8 x 10(3) U/ml of MuIFN alpha/beta (IFN treated cells: 0.03 +/- 0.007 integrated optical density (IOD); control cells: 0.07 +/- 0.01 IOD). The decreased CS production may be the underlying cause of IFN mediated inhibition of glioma cell proliferation.
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PMID:Interferon effect on glycosaminoglycans in mouse glioma in vitro. 805 39

Previous studies have identified glycosaminoglycans in gingival crevicular fluid (GCF) associated with a variety of clinical conditions, notably those involving bone resorptive activity. GCF was here collected from around teeth undergoing active orthodontic movement. Proteoglycan metabolites were purified from GCF by anion-exchange chromatography using fast performance liquid chromatography. Sulphated glycosaminoglycan was associated with the most highly anionic protein fractions IV, V and VI, and biochemical analysis was restricted to these fractions. Analysis included glycosaminoglycan content by cellulose acetate electrophoresis, molecular size by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), Western blotting and amino acid analyses. Fraction IV contained hyaluronan (18.7%) and chondroitin sulphate (10.9%), fraction V heparan sulphate (29.5%) and chondroitin sulphate (19.6%) and fraction VI chondroitin sulphate only (21.3%). SDS-PAGE revealed two Coomassie blue bands in fraction V of 72 and 60 kDa and two further bands in fraction VI of 71 and 56 kDa. These proteoglycans appeared resistant to digestion by chondroitinase ABC or heparinase III, although the glycosaminoglycan chains underwent degradation after protein-core removal. The molecular mass and amino acid composition of the chondroitin sulphate proteoglycan fractions showed a close similarity to those of human alveolar bone proteoglycan. The presence of heparan sulphate proteoglycan in GCF in association with orthodontic movement is in accord with previous reports. The findings support the view that proteoglycans in GCF are 'biomarkers', notably those associated with active resorption of alveolar bone.
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PMID:Characterization of proteoglycan metabolites in human gingival crevicular fluid during orthodontic tooth movement. 806 Feb 58

Acidic glycoconjugates (glycosaminoglycans, sulfated glycopeptide, and sialoglycopeptide) were isolated by precipitation with cetylpyridinium chloride from human pancreatic juice after digestion with pronase. The acidic glycoconjugates were found exclusively in the proteinaceous precipitate that occurred during dialysis against a buffer of low ionic strength. The concentration of the acidic glycoconjugates in normal pancreatic juice was about 2.4 mg/L. The acidic glycoconjugates were characterized by electrophoresis on cellulose acetate membrane and chemical analysis before and after digestion with Streptomyces hyaluronidase, chondroitinase AC, chondroitinase ABC, and heparitinase. It was found that the major acidic glycoconjugates were heparan sulfate (39.3%), sulfated glycopeptide (34.4%), chondroitin sulfate (14.2%), and the minor ones hyaluronic acid (6.4%) and sialoglycopeptide (5.7%).
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PMID:Enzymic determination of acidic glycoconjugates in human pancreatic juice. 811 24

1. Urinary glycosaminoglycans were recovered from the papain digest of polyanions precipitated sequentially by cetylpyridinium chloride and sodium acetate-saturated ethanol. Those from the early morning urine of 48 stone formers and 43 normal control subjects measured 11 and 16 micrograms of uronic acid/ml of urine, respectively. 2. Preparative agarose gel electrophoresis of the recovered glycosaminoglycans in barium acetate buffer (pH 5.8) yielded fractions containing purely chondroitin sulphate, co-polymeric chondroitin/dermatan sulphates and heparan sulphate. Identification was based on the susceptibility of the fractions to chondroitinase or nitrous acid treatment. Similar compositions of glycosaminoglycan classes were observed in samples from stone formers and normal control subjects. 3. The fractionated glycosaminoglycans were dissolved in urine ultrafiltrate to assay for nucleation-promoting and growth-inhibiting activities towards crystallization of urinary calcium oxalate. When compared at the same uronic acid concentration, both the urinary chondroitin sulphate isomers and heparan sulphates of stone formers demonstrated the capacity to enhance crystal nucleation from calcium oxalate endogenous in urine ultrafiltrates, whereas only urinary heparan sulphates of normal control subjects demonstrated this capacity. 4. Tissue-derived reference chondroitin sulphate, dermatan sulphate and heparin, when similarly tested, showed negligible crystal nucleation-promoting activity. The tissue-derived heparan sulphate was similar to the urinary heparan sulphates in showing marked crystal nucleation-promoting activity. 5. Crystal-growth inhibitory activity was evident in all urinary glycosaminoglycan fractions studied. In particular, urinary heparan sulphate of normal control subjects showed higher activity than that of stone formers or the chondroitin sulphate isomers of both stone formers and normal control subjects (P < 0.005).
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PMID:Separate effects of urinary chondroitin sulphate and heparan sulphate on the crystallization of urinary calcium oxalate: differences between stone formers and normal control subjects. 814 91

Age-related changes in proteoglycans of human ligamentum flavum were studied using specimens obtained from patients divided into four age groups. The proteoglycans were purified by ion-exchange and gel-filtration chromatography. A low-molecular-weight proteoglycan with the same molecular size was present in all each age groups. Conversely, high-molecular-weight proteoglycan increased with advancing age. Properties of the sugar chains, glycosaminoglycans, in these proteoglycans were studied using cellulose acetate membrane electrophoresis and chondroitinase digestion. It was found that the high-molecular-weight proteoglycan consisted mainly of chondroitin 6-sulfate, whereas the low-molecular-weight proteoglycan consisted mainly of dermatan sulfate, although the ratio of chondroitin 6-sulfate increased with age. These results indicate that proteoglycans of human ligamentum flavum show changes in amount and composition with age.
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PMID:Age-related changes in proteoglycans of human ligamentum flavum. 827 43

Dermatan sulfate-proteoglycans (DS-PGs) were extracted from rabbit, rat and bovine defatted livers by magnesium chloride extraction and DEAE-cellulose chromatography, and then submitted successively to Asahipak GS-520 gel filtration chromatography, Asahipak ES-502N anion exchange chromatography, and cellulose acetate membrane electrophoresis. The disaccharide composition of the glycosaminoglycan chains was determined by differential digestion by chondroitinase ABC, AC, ACII and/or B followed by HPLC for analysis of the resulting unsaturated disaccharides. The hepatic dermatan sulfate chains contained disulfated disaccharide units; Di-diSB and Di-diSE. The hepatic DS-PGs were divided into two groups; Di-diSE-poor DS-PGs and Di-diSE-rich DS-PGs. The iduronic acid content of Di-diSE-poor dermatan sulfate chains was higher than that of Di-diSE-rich ones.
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PMID:Structural diversity of mammalian hepatic dermatan sulfates. 835 80

Dermatan sulfate excreted in normal human urine was isolated and characterized by TLC and cellulose acetate strip electrophoresis after cetylpyridinium chloride precipitation and pronase digestion. In these separation methods, dermatan sulfate and chondroitin sulfate were extracted and then monitored by sensitive HPLC methods with post column fluorometric derivatization coupled with chondroitinase ABC, ACII and B digestion. From the results, we demonstrated that human urinary dermatan sulfate contains iduronic acid as its major uronic acid (80-90% of total uronic acid), and is composed mainly of repeated mono-sulfated disaccharide units [Di-4S (structure shown in Fig. 1), 89%] and small numbers of di-sulfated disaccharide units (Di-diSB, 7% and Di-diSE, 1%).
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PMID:Separation and characterization of dermatan sulfate in normal human urine. 835 81

Using an in vitro rat incisor odontoblast system, the effect of fluoride on proteoglycans was investigated at both the metabolic and structural level. Incisors were removed from 4-week-old rats, split longitudinally, and the pulps removed. Teeth were incubated at 37 degrees C, 5% CO2 in Eagle's Minimum Essential Medium containing 35S-sulfate for 7 hours in the presence of 0 mM, 3 mM, or 6 mM sodium fluoride. Teeth were demineralized in EDTA, proteoglycan was extracted from the residue with 4 M guanidinium chloride, and further purified by anion exchange chromatography. Uptake of radiolabel was monitored by liquid scintillation counting. The resultant products were examined by cellulose acetate electrophoresis, SDS-PAGE, chondroitinase digestion, and amino acid analysis. Differential effects of fluoride were observed in both metabolism and biochemical characterization of proteoglycans following incubation at the two concentrations. Fluoride decreased uptake of the radiolabel but led to an accumulation of glycosaminoglycan within the proteoglycan of the matrix. Chondroitin sulfate was the predominant glycosaminoglycan identified, with the additional presence of dermatan sulfate and heparan sulfate identified. Dermatan sulfate levels increased in 3 mM-treated teeth. Fluoride-treated proteoglycans had a reduced molecular weight (200-90K to 180-79K); this reduction is primarily a result of smaller glycosaminoglycan chains, with limited reduction in the size of the core protein of 6 mM-treated teeth occurring. Such alterations in the biochemical metabolism and hence structure and function of proteoglycan may be implicated in the hypomineralization seen in fluorosis.
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PMID:The influence of fluoride on proteoglycan structure using a rat odontoblast in vitro system. 850 77

Basic fibroblast growth factor (bFGF) has been identified as an important cytokine for blood cells. To determine whether hematopoietic cells have receptors that recognize bFGF, the ability of human leukemia cell lines to bind 125I-bFGF was investigated. Specific bFGF-binding sites were identified on K562 and HL60 cells, but not on U937 cells. DAMI cells bound low amounts of 125I-bFGF specifically. Binding of 125I-bFGF to K562 cell surfaces was reduced in a dose-dependent manner by unlabeled bFGF or by heparin. Scatchard analysis of binding to K562 cells revealed two classes of binding sites: 1,650 high affinity binding sites per cell with a dissociation constant (kd) of 192 pmol/L, and 36,600 low affinity sites per cell with a kd of 9.3 nmol/L. Chemical crosslinking experiments with K562, HL60, and DAMI cells revealed receptor-growth factor complexes with molecular masses of 140 to 160 kD, similar in size to complexes formed by known receptor species. Binding of 125I-bFGF to K562 cells was sensitive to heparinase treatment but not to chondroitinase treatment, suggesting that heparan sulfate proteoglycans (HSPGs) may be responsible for the low affinity binding sites. To further investigate whether K562 cells make HSPG, the incorporation of 35SO4 into proteoglycans was assessed. Metabolically labeled cell-surface proteoglycans with molecular masses of 180 to 300 kD were identified in K562 cells. These proteoglycans were sensitive to heparinase, demonstrating that K562 cells synthesize bFGF-binding HSPG. Treatment of K562 cells with phorbol-12-myristate-13-acetate (PMA) caused a loss of bFGF-binding capacity. This decreased binding capacity reflected a rapid loss of high affinity receptors. The ability to form bFGF-receptor complexes decreased by 65% to 70% within 1 hour and declined continuously thereafter. The decrease in binding of bFGF was not due to an autocrine downregulation of bFGF receptors, because there was no increase in bFGF after PMA treatment as detected by Western blotting, and suramin, which blocks bFGF binding to receptors, did not prevent the loss of receptors after exposure to PMA. In addition, inhibitors of either protein synthesis or protease activity did not prevent the loss of bFGF receptors in PMA-treated cells. In summary, this work demonstrates that leukemia cell lines have receptors that specifically bind bFGF and supports the hypothesis that bFGF acts directly on certain blood cells to stimulate their proliferation.
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PMID:Human leukemia cell lines bind basic fibroblast growth factor (FGF) on FGF receptors and heparan sulfates: downmodulation of FGF receptors by phorbol ester. 854 48


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