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:4.2.2.7 (heparinase)
1,270 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Glomeruli isolated from rat kidney were incubated with [14C]glucosamine and [35S]sulfate. Linear incorporation of [14C]glucosamine into total glycosaminoglycans was observed during incubation up to 24 h. More than 95% of the 35S-labeled sulfated glycoconjugates were extracted from the tissue with 4 M guanidine HCl, 50 mM sodium acetate, pH 6.0, and 0.5% Triton X-100, and separated clearly on DEAE-Sephacel into three major fractions, i.e. sulfated glycoprotein (11% of the total radioactivity), proteoheparan sulfate (33%), and proteochondroitin sulfate (38%) fractions. The molecular weight of the 35S-labeled proteoheparan sulfate thus isolated was estimated to be about 185,000, whereas that released into the medium was estimated to be about 87,000. When the 35S-labeled heparan sulfate isolated on Sephadex G-75 after mild alkaline borohydride treatment was digested with a combination of heparitinase and heparinase, approximately 70% of the radioactivity was converted to 2-acetamido-2-deoxy-4-O-(alpha-D-gluco-4-enepyranosyluronic acid)-6-O-sulfo-D- glucose.
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PMID:Isolation and characterization of proteoheparan sulfate synthesized in vitro by rat glomeruli. 622 84

We have isolated from nitrous acid cleavage products of heparin two major octasaccharide fragments which bind with high affinity to human antithrombin. Octasaccharide S, with the predominant structure iduronic acid----N-acetylglucosamine 6-O-sulfate----glucuronic acid-----N-sulfated glucosamine 3,6-di-O-sulfate----iduronic acid 2-O-sulfate----N-sulfated glucosamine 6-O-sulfate----iduronic acid 2-O-sulfate----anhydromannitol 6-O-sulfate, is sensitive to cleavage by Flavobacterium heparinase as well as platelet heparitinase and binds to antithrombin with a dissociation constant of (5-15) X 10(-8) M. Octasaccharide R, with the predominant structure iduronic acid 2-O-sulfate----N-sulfated glucosamine 6-O-sulfate----iduronic acid----N-acetylglucosamine 6-O-sulfate----glucuronic acid----N-sulfated glucosamine 3,6-di-O-sulfate----iduronic acid 2-O-sulfate----anhydromannitol 6-O-sulfate, is resistant to degradation by both enzymes and binds antithrombin with a dissociation constant of (4-18) X 10(-7) M. The occurrence of a 15-17% replacement of N-sulfated glucosamine 3,6-di-O-sulfate with N-sulfated glucosamine 3-O-sulfate and a 10-12% replacement of iduronic acid with glucuronic acid in both octasaccharides indicates that these substitutions have little or no effect on the binding of the oligosaccharides to the protease inhibitor. When bound to antithrombin, both octasaccharides produce a 40% enhancement in the intrinsic fluorescence of the protease inhibitor and a rate of human factor Xa inhibition of 5 X 10(5) M-1 s-1 as monitored by stopped-flow fluorometry. This suggests that the conformation of antithrombin in the region of the factor Xa binding site is similar when the protease inhibitor is complexed with either octasaccharide.
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PMID:Sequence variation in heparin octasaccharides with high affinity for antithrombin III. 652 37

Polysaccharides and other complex carbohydrates were released by proteolysis of the chloroform-methanol insoluble residue of 10 day-old worms and eggs of Hymenolepis diminuta. Gas-liquid chromatographic analysis of alditol acetate derivatives of monosaccharides released from the polysaccharides by hydrolysis revealed that in the 10 day-old worm, glucose was the most abundant sugar, followed by galactose, glucosamine, galactosamine, fucose and possibly rhamnose. Mannose was least abundant and xylose was absent. In the egg, glucose and galactose were equally abundant, followed by the same sugars found in 10 day-old worms, and xylose was present. Uronic acid was detected in both fractions by specific chemical tests. None of the saccharide material from eggs and worms was susceptible to degradation by Streptomyces hyaluronidase, chondroitinase AC, and slightly susceptible to chondroitinase ABC, as shown by electrophoretic analysis on composite 2.2% acrylamide-agarose slab gels and 4.5/12.5% polyacrylamide gels before and after enzymatic treatment. One of the gel-separable bands, however, was degradable by both nitrous acid and Flavobacterium heparinase. Both bands from eggs were degradable by nitrous acid. These results suggest that eggs contain heparin and/or heparan sulfate and perhaps dermatan sulfate and that 10 day-old worms also have these polyglycans but possibly not chondroitin sulfate or hyaluronic acid.
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PMID:Characterization of polysaccharides of the eggs and adults of Hymenolepis diminuta. 653 86

The glycosaminoglycans produced by human fetal uveal melanocytes and by human melanoma cells were examined. The cells were grown in the presence of [3H]glucosamine and [35S]sulfate, and the labeled glycosaminoglycans were isolated from the cells, spend medium, and intracellular material. The distribution of the glycosaminoglycans was similar in both cells and spent media, which together accounted for 95% of the total. Of the total 3H]labeled glycosaminoglycans produced by the melanocyte culture, 42% was in chondroitin 4-sulfate, 25% in heparan sulfate, 16% in chondroitin 6-sulfate, and 17% in hyaluronic acid. In contrast, HM7 human melanoma cultures produced no chondroitin 6-sulfate, increased quantities of heparan sulfate, and less hyaluronic acid. A heparan sulfate fraction obtained from melanocytes required both heparitinase and heparinase for complete degradation, indicating the presence of heparin-like molecules in this fraction. The corresponding fraction from melanoma cells was totally degraded by heparitinase alone.
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PMID:Glycosaminoglycans of cultured human fetal uveal melanocytes and comparison with those produced by cultured human melanoma cells. 729 96

The endometrial scrapings obtained from the uteri of estrogen-progesterone-treated and estrogen-treated rabbits were incubated with N-acetyl-D-[1-3H]glucosamine and [35S]sulfate, and then the incubation medium (M-Fr) was separated from the tissue. The tissue was subsequently homogenized exhaustively in 0.25 M sucrose, and the insoluble residue (R-Fr) was separated. The supernatant at 8,5000 X g for 10 min of the homogenate was subjected to subcellular fractionation by discontinuous sucrose gradient ultracentrifugation, and a Golgi-rich fraction (G-Fr) was obtained. Crude glycoconjugates (glycosaminoglycans and glycoproteins) were then separated from M-Fr, R-Fr and G-Fr after pronase digestion. The amounts of the radioactivities incorporated into these glycoconjugates suggested that progesterone markedly suppressed the estrogen effect within 6 hr in R-Fr and G-Fr, whereas the suppression slightly delayed in M-Fr. The amounts of the radioactivities incorporated into acidic GC, which obtained by CPC-precipitation of the crude GC after digestion with crude heparinase, indicated that the biosynthesis of the CPC-precipitable sulfated glycoproteins was almost completely ceased by progesterone, although other glycoconjugates were still actively synthesized under the progesteronic condition.
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PMID:Progesterone effect on the biosynthesis of glycoconjugates, specifically of sulfated glycoprotein, in the endometrium of rabbit uterus. 744 38

We earlier reported calcium-dependent, heparin-like L-selectin ligands in cultured bovine endothelial cells (Norgard-Sumnicht, K. E., Varki, N. M., and Varki, A. (1993) Science 261,480-483). Here we show that these are heparan sulfate proteoglycans (HSPGs) associated either with the cultured cells or secreted into the medium and extracellular matrix. Activation of the endothelial cells with bacterial lipopolysaccharide (LPS) does not markedly alter the amount or distribution of this material. A major portion of the glycosaminoglycan (GAG) chains released from these HSPGs by alkaline beta-elimination rebinds to L-selectin in the presence of calcium, indicating that these saccharides alone can mediate the high affinity recognition. Heparin lyase digestions indicate that these GAG chains are enriched in heparan sulfate, not heparin sequences. Current understanding of the biosynthesis of heparan sulfate chains indicates that all glucosamine amino groups must be either N-acetylated or N-sulfated. However, nitrous acid deamination at pH 4.0 suggests the presence of some unsubstituted amino groups in these L-selectin-binding GAG chains from endothelial cell HSPGs. This is confirmed by chemical N-reacetylation and by reactivity with sulfo-N-hydroxysuccinimide-biotin. These unsubstituted amino groups are also found on HSPGs from human umbilical vein endothelial cells, but are not detected in those from Chinese hamster ovary cells. In both bovine and human endothelial cells, these novel groups are enriched for in the HS-GAG chains which bind to L-selectin. Despite this, studies with N-reacetylation and nitrous acid deamination do not show conclusive evidence for the direct involvement of the unsubstituted amino groups in L-selectin binding. This may be because the chemical reactions used to modify the amino groups do not go to completion. Alternatively, the unsubstituted amino groups may only be indirectly involved in generating binding, by dictating the biosynthesis of another critical group. Regardless, these studies shown that HSPGs from cultured endothelial cells which can bind to L-selectin are enriched with unsubstituted amino groups on their GAG chains. The possible biochemical mechanisms for generation of these novel groups are discussed.
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PMID:Endothelial heparan sulfate proteoglycans that bind to L-selectin have glucosamine residues with unsubstituted amino groups. 753 30

The purpose of this study was to determine the biochemical and molecular characteristics of mucin synthesized by cystic fibrosis cells (CFPAC-1), a pancreatic cancer cell line derived from a patient with cystic fibrosis, and pancreatic cancer (SW-1990) cell lines. High molecular weight glycoproteins (HMG) were quantified by [3H]-glucosamine labeling and chromatography on sepharose CL-4B. Mucin gene expression was determined by using cDNA probes for 2 distinct intestinal mucins (MUC2 and MUC3) and one stomach mucin (MUC1). The specific mucin core epitopes were confirmed by immunoblots using antibodies that recognize T, Tn, sialosyl Tn, MUC1, MUC2, and MUC3. The results of these experiments demonstrate that CFPAC-1 cells contained 1.25 fold and 1.4 fold more HMG in the membrane and cytosolic fractions, however, secreted 4-fold more HMG into the medium compared to SW-1990 cells. The HMG of SW-1990 was found to be mucinous in nature and not proteoglycans, as it was not susceptible to hyalurinidase, heparinase and chondroitinase ABC. The HMG of CFPAC-1 was also predominantly (80%) mucinous but with small amounts of proteoglycans. mRNA and immunoblot analysis suggest that these CFPAC-1 and SW-1990 cells predominantly express MUC1 apomucin, small amounts of MUC2 apomucin, and no MUC3. Pulse chase labeling and immunoprecipitation of MUC1 type mucin using the 139H2 monoclonal antibody demonstrated that different sizes of mucin gene product were present in both cell lines, corresponding to the known length polymorphism of this mucin. Both T and Tn antigens were significantly higher in CFPAC-1 and SW-1990 cells as compared to sialosyl Tn antigen. These findings were associated with the increased activities of polypeptidyl N-acetylgalactosaminyl transferase and b1,3-galactosyltransferase. These investigations demonstrate for the first time that cystic fibrosis cells (CFPAC-1) secrete and synthesize high amounts of mucin which is associated with high levels of MUC1 mRNA, low levels of MUC2 mRNA and non detectable MUC3 mRNA.
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PMID:Cystic fibrosis and pancreatic cancer cells synthesize and secrete MUC1 type mucin gene product. 754 50

Four hexasaccharides representing major structural sequences of heparin were isolated and characterized after degradation of heparin by heparinase. The structures were determined from two-dimensional 1H NMR spectroscopy including TOCSY (total correlated spectroscopy), COSY (correlated spectroscopy), and ROESY (rotating frame nuclear Overhauser enhancement spectroscopy) methods, providing new data on hexasaccharides. One of the hexasaccharides, the last eluting component from anion exchange chromatography, was derived from the tri-sulfated repeating disaccharide, alpha-L-idopyranosyluronic acid 2-sulfate-(1-->4)-2-amino-2-deoxy-D-glucopyranose 6,N-disulfate, and having the structure delta UAp2S-(1)-->4)-alpha-D-GlcNp2S6S-(1-->4)-alpha-L- IdoAp2S-(1-->4)-alpha-D-GlcNp2S6S-(1-->4)-alpha-L- IdoAp2S-(1-->4)-alpha-D-GlcNp2S6S. The second hexasaccharide contained a nonsulfated D-glucuronic acid unit instead of the L-iduronic acid adjacent to the reducing end, and having the structure delta UAp2S-(1-->4)-alpha-D-GlcNp2S6S-(1-->4)-alpha-L- IdoAp2S-(1-->4)-alpha-D-GlcNp2S6S-(1-->4)-beta-D- GlcAp-(1-->4)-alpha-D-GlcNp2S6S. The last two hexasaccharides were obtained in lower yield and they have not been isolated and characterized before. The structure of the third saccharide corresponded to a trimer of the repeating disaccharide except for the lack of a 6-O-sulfate group at the reducing end glucosamine residue; deltaUAp2S-(1-->4)-alpha-D-Glcnp2S6S-(1-->4)-alpha-L- IdoAp2S-(1-->4)-alpha-D-GlcNp2S6S-(1-->4)-alpha-L-IdoAp2S -(1-->4)-alpha- D-GlcNp2S. The fourth and last hexasaccharide were less sulfated and the following structure was established delta UAp2S-(1-->4)-alpha-D-GlcNp2S6S-(1-->4)-alpha-L- Idop2S-(1-->4)-alpha-D-GlcNp2S6S-(1-->4)-alpha-L- IdoAp-(1-->4)-alpha-D-GlcNpAc6S. Analysis of the ROESY spectra revealed conformational difference of the glucosidic linkage alpha-L-IdoAp-(1-->4)-alpha-D-GlcNp between the hexasaccharides and longer heparin chains.
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PMID:Isolation and characterization of hexasaccharides derived from heparin. Analysis by HPLC and elucidation of structure by 1H NMR. 769 49

This paper describes low-density mucus glycoconjugates released from feline trachea by dirhamnolipid (DRL), a toxin from Pseudomonas aeruginosa. Mucus glycoconjugates in feline tracheas were radiolabeled in vivo with 3H-proline and 14C-glucose. Control mucus and that released by 200 micrograms/ml DRL were dissolved in guanidine hydrochloride buffer (GuHCl) and chromatographed on Sepharose CL-2B. Molecules eluting in the void volume (V0) of the column were isolated by isopycnic density gradient centrifugation in CsCl/GuHCl. All samples gave peaks of radiolabeled and periodic acid/Schiff (PAS)-reactive material at rho = approximately 1.50 and approximately 1.60 g/ml, but DRL-stimulated samples contained low-density material (rho < 1.32 g/ml), also PAS-reactive and radiolabeled. Control secretions incubated with DRL in vitro did not form low-density material. In Triton X-100 (1% vol/vol), a nonionic detergent, low-density material behaved as smaller molecules, running in the partially included volume (Vi) of the column of Sepharose CL-2B, but still in the V0 of Sephacryl S-300. Incubation with chondroitinase ABC, heparinase II and III, and keratanase failed to change its elution profile on S-300, evidence against glycosaminoglycans; but proteolysis with trypsin or proteinase K gave two peaks, peptide fragments near the totally included volume of the column and glycopeptides in V0. The V0 glycopeptides banded between 1.50 and 1.55 g/ml in a CsCl gradient and eluted as a single peak in the Vi of Sephacryl S-400, suggesting a distinct homogeneous glycopeptide, smaller than those from normal mucins. The main 14C-labeled sugars in this glycopeptide were fucose, glucosamine, galactosamine, and galactose, consistent with a mucin. Thus, DRL releases stable but noncovalent complexes containing one or more distinct mucinlike glycoconjugates, probably combined with lipids and peptides. We discuss their possible relevance to airway diseases, including cystic fibrosis.
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PMID:Mucus glycoconjugate complexes released from feline trachea by a bacterial toxin. 787 96

Human skin fibroblasts in different growth states were incubated with [3H]glucosamine and/or Na(2)35SO4 and extracted with Triton X-100 for various periods of time. Free heparan-sulphate oligosaccharides and protein-bound heparan-sulphate chains were separated by chromatography on octyl-Sepharose and analyzed. A pool of endogenously produced oligosaccharides, present in the cultured cells and isolated after brief extraction, contained fragments of uniform size (approximately 7-10 kDa corresponding to approximately 14-20 disaccharides). Analysis by heparinase I and heparinase III degradations followed by electrophoretic separation (oligosaccharide mapping) showed that the oligosaccharides were rich in glucuronic acid but had a few sulphated iduronic acid residues at the periphery of each molecule. These results indicated that endoheparanase cleavage points were located close to linkages between N-sulphated glucosamine and sulphated iduronic acid, generating fragments that comprise a major portion of the unmodified segments and a minor portion of the highly modified segments. Prolonged extraction (24-48 h) of cells with Triton X-100 at 4 degrees C in the presence of proteinase inhibitors resulted in further degradation. There was an increase in the amount of heparan-sulphate oligosaccharides and a concomitant decrease in the amount of protein-bound heparan-sulphate chains present in the same extract. The heparan-sulphate oligosaccharides obtained after prolonged extraction were more heterogeneous in size comprising, in addition to the major species of approximately 7-10 kDa, intermediate and larger fragments of approximately 17 kDa and 30-40 kDa. This observation suggests that endoheparanase acted at periodically appearing, specific regions in the intact heparan-sulphate chain. Furthermore, the enzyme and substrate should remain closely associated during cold Triton X-100 extraction. To determine if the endogenously produced heparan-sulphate oligosaccharides were derived from a particular heparan-sulphate species degraded during the growth phase, proteoglycan-derived heparan-sulphate chains obtained from proliferating or quiescent fibroblasts were also examined. These chains showed similar oligosaccharide maps, except for a small increase in the amount of glucuronic acid as cell growth was arrested. Hence, an endoheparanase with restricted specificity may generate slightly different oligosaccharides in the various growth states.
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PMID:Analysis of heparan-sulphate chains and oligosaccharides from proliferating and quiescent fibroblasts. A proposed model for endoheparanase activity. 803 94


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