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)

Human lung fibroblasts produce heparan sulphate proteoglycans (HSPG) that are associated with the plasma membrane. A monoclonal-antibody (Mab)-secreting hybridoma, S1, was produced by fusion of SP 2/0-AG 14 mouse myeloma cells with spleen cells from mice immunized with partially purified cellular HSPG fractions. The HSPG character of the material carrying the epitope recognized by Mab S1 was demonstrated by: (i) the co-purification of the S1 epitope with the membrane HSPG of human lung fibroblasts; (ii) the decrease in size of the material carrying the S1 epitope upon treatment with heparinase or heparitinase, and the resistance of this material to heparinase treatment after N-desulphation. The S1 epitope appears to be part of the core protein, since it was destroyed by proteinase treatment and by disulphide-bond reduction, but not by treatments that depolymerize the glycosaminoglycan chains and N-linked oligosaccharide chains. Polyacrylamide-gel electrophoresis of non-reduced heparitinase-digested membrane HSPG followed by Western blotting and immunostaining with Mab S1 revealed a single band with apparent molecular mass of 64 kDa. Membrane proteoglycans isolated from detergent extracts or from 4 M-guanidinium chloride extracts of the cells yielded similar results. Additional digestion with N-glycanase lowered the apparent molecular mass of the immunoreactive material to 56 kDa, suggesting that the core protein also carries N-linked oligosaccharides. Fractionation of 125I-labelled membrane HSPG by immuno-affinity chromatography on immobilized Mab S1, followed by heparitinase digestion and polyacrylamide-gel electrophoresis of the bound material, yielded a single labelled band with apparent molecular mass 64 kDa. Treatment with dithiothreitol caused a slight increase in apparent molecular mass, suggesting that the core protein of this membrane proteoglycan of a single subunit containing (an) intrachain disulphide bond(s).
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PMID:Identification of a 64 kDa heparan sulphate proteoglycan core protein from human lung fibroblast plasma membranes with a monoclonal antibody. 244 76

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 application of capillary electrophoresis to total compositional analysis of heparin and low-molecular-weight heparin samples has been studied. Optimum resolution of 17 defined oligosaccharides was obtained with the buffer system composed of 10 mM sodium borate and 50 mM sodium dodecyl sulfate at pH 8.81 and at a constant voltage of 20 kV. The ratio of oligosaccharide charge to the number of saccharide residues correlated with the migration time. For oligosaccharides having the same charge to saccharide ratio, the larger of the oligosaccharides eluted earlier. A hexasaccharide having a 3-O-sulfated glucosamine residue at the reducing end and arising from heparin's antithrombin III binding site, migrated in an unusual fashion. The limit of oligosaccharide detection was from 600 fmol to 1 pmol. Quantitative analysis could conveniently be performed on 10 pmol of an oligosaccharide sample. Oligosaccharide composition using capillary electrophoresis was obtained by nearly complete depolymerization of heparins with a mixture of heparin lyase I, II, and III. The analysis resulted in 95% mass balance for both heparin and low-molecular-weight heparin. Capillary electropherograms of heparin and different low-molecular-weight heparins depolymerized with heparin lyase I alone showed a high level of structural heterogeneity in the products formed. The oligosaccharide maps thus obtained might find use in fingerprinting the heparin and low-molecular-weight samples.
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PMID:Oligosaccharide composition of heparin and low-molecular-weight heparins by capillary electrophoresis. 823 64

Heparinase was purified to homogeneity from the cell extract of an oral bacterium, Prevotella heparinolytica, by a combination of anion exchange chromatography, gel filtration chromatography, and hydroxyapatite chromatography. Properties of the purified P. heparinolytica heparinase (P. heparinase) were investigated. The enzyme exhibited a maximum activity in 50 mM Tris-HCl buffer, pH 7.5-8.0, containing 75 mM sodium acetate, 0.1 M NaCl, and 1 mM CaCl2. Optimum conditions for the maximum activity of P. heparinase were similar to those of the heparinase from Flavobacterium heparinum (F. heparinase). The two enzymes also yielded similar digestion profiles of various glycosaminoglycans and heparin tetrasaccharides, suggesting that they have a similar substrate specificity. Kinetic study of the P. heparinase reaction using porcine intestinal heparin as substrate gave a Km value of 3.8 x 10(-5) M and a Vmax value of 11.4 micromol/min x mg protein. The Michaelis constant of P. heparinase was slightly larger than but not significantly different from that of F. heparinase. The amino acid composition of P. heparinase was also similar to that of F. heparinase, but its N-terminal sequence of 20 amino acid residues was different and hitherto unreported. These results together indicate that these heparinases are different proteins with closely similar enzymatic properties. Since F. heparinum produces not only heparinase but also heparitinase II, which has a broad substrate specificity, F. heparinase may be contaminated with this enzyme. In contrast, P. heparinolytica does not produce heparitinase II, and P. heparinase should prove a useful tool for degrading heparin without the risk of contamination with heparitinase II.
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PMID:Characterization of heparinase from an oral bacterium Prevotella heparinolytica. 953 4