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)

Heparinase was isolated from a transplantable mouse mastocytoma, by salt extraction of a particulate fraction sedimenting at 20,000 times g, followed by precipitation from saturated ammonium sulfate. By use of gel chromatography through Sepharose 4B, the enzyme was shown to degrade macromolecular. 35S-labeled, mastocytomal heparin (K-av about 0.25) to products similar in size to commercial heparin (K-av about 0.85), apparently by nonrandom cleavage of a limited number of glycosidic linkages per molecule. Prolonged incubation times (up to 5 days, with repeated addition of enzyme) did not result in further degradation of the product. No significant depolymerizing activity was observed with any other glycosaminoglycan tested, including chondroitin sulfate, dermatan sulfate, hyaluronic acid, heparan sulfate, and commercial heparin. The pH optimum for degradation of macromolecular heparin was around pH 5. The nature of the linkage cleaved by the heparinase was investigated by reduction of unlabeled polysaccharide degradation products with sodium [3H]borohydride. The degraded chains (but not the macromolecular substrate) incorporated significant amounts of tritium. An essentially monodisperse fraction of the labeled, degraded heparin was subjected to meniscus depletion sedimentation equilibrium ultracentrifugation, indicating a molecular weight of 14,500. By relating the molecular weight to the specific activity of the preparation, the amount of reducible groups was calculated to be approximately one per molecule. The 3H-labeled heparin was degraded to monosaccharides by a combination of acid hydrolysis and cleavage due to deamination with nitrous acid. Analysis of the degradation products, by paper electrophoresis and paper chromatography, showed a major radioactive component which behaved like L-gulonic acid. Since [3H]gulonic acid would be the expected reduction product of a polysaccharide molecule, containing a glucuronic acid residue in terminal position, these results tentatively suggest that the heparinase is an endoglucuronidase. By direct deaminative cleavage (no hydrolysis) of the 3H-labeled heparin, the glucosamine unit in penultimate position (i.e. adjacent to the [3H]gulonic acid residue) was shown to be 52% N-sulfated and 48% N-acetylated. As only 14% of the glucosamine was N-acetylated in the macromolecular heparin substrate, it is suggested that cleavage of this polysaccharide, by the heparinase, occurs in regions more abundant in N-acetylated glucosamine residues than other portions of the molecule. The possibility that formation and degradation of macromolecular heparin occurs also in mammalian species other than rodents in discussed.
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PMID:Cleavage of macromolecular heparin by an enzyme from mouse mastocytoma. 80 78

Heparinase production by Flavobacterium heparinum in complex protein digest medium, with heparin employed as the inducer, has been studied and improved. The maximum productivity of heparinase has been increased 156-fold over that achieved by previously published methods to 375 U/liter per h in the complex medium. Rapid deactivation of heparinase activity, both specific and total, was observed at the onset of the stationary phase. Nutritional studies on growth and heparinase production showed an obligate requirement for L-histidine and no vitamin requirement. L-Methionine partially relieved the L-histidine requirement. A defined medium containing glucose, ammonium sulfate, basal salts, L-methionine, and L-histidine was developed for growth and heparinase production. The growth rate in this medium was 0.21 h-1, which is 40%, higher than that in complex medium. The maximum volumetric productivity of heparinase in the defined medium was increased to 1,475 U/liter per h, providing a 640-fold increase over that achieved by previously published methods. No rapid deactivation was observed. An examination of alternate inducers for heparinase showed that heparin degradation products, hyaluronic acid, heparin monosulfate, N-acetyl-D-glucosamine, and maltose, induce heparinase in complex medium. An Azure A assay was modified and fully developed to measure the heparin concentration during fermentation and the heparinase specific activity of crude extracts of F. heparinum obtained from sonication, thus negating the need for further purification to measure activity."
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PMID:Heparinase production by Flavobacterium heparinum. 723 92

A simple and efficient method for the separation of hydrophobic derivatives of glycosaminoglycan-derived disaccharides is described. Hydroxyl-protected derivatives of a trisulfated disaccharide, prepared from heparin using heparin lyase, were separated by reversed-phase high-performance liquid chromatography. These disaccharide derivatives differed by the number, position, and stereochemistry of acetyl and pivaloyl groups. Separation was achieved on a C18 column using a reversed gradient of ammonium sulfate in water. This method has application in the purification of disaccharide derivatives being used as chiral synthons in the preparation of higher oligosaccharides.
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PMID:Separation of hydroxyl protected heparin derived disaccharides using reversed-phase high-performance liquid chromatography. 764 Jul 72

A sensitive high-performance liquid chromatographic method for the determination of unsaturated disaccharides produced from heparin and heparan sulfate is described. Heparan sulfate was depolymerized using a combination of heparin lyase I (EC 4.2.2.7), heparin lyase II and heparin lyase III (EC 4.2.2.8). Seven unsaturated disaccharides were separated under isocratic conditions within 25 min using acetonitrile-H2O-0.2 M sodium phosphate buffer (pH 7.0)-3.0 M ammonium chloride (32:10:1:1) and were monitored by fluorescence detection using 2-cyanoacetamide as a post-column derivatizing reagent. As little as 2 pmol of a disaccharide could be detected with excitation at 346 nm and emission at 410 nm. This method was applied to the analysis of normal human urine. It was revealed that the concentration of normal human urinary heparan sulfate is 1.53+/-0.36 mg/mg creatinine (n=4).
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PMID:Sensitive high-performance liquid chromatographic method with fluorometric detection for the determination of heparin and heparan sulfate in biological samples: application to human urinary heparan sulfate. 951 50

Heparin-induced thrombocytopenia represents one of the most severe drug-induced disorders of platelets. This syndrome is believed to be mediated through antibodies generated against a heparin-platelet factor 4 complex. Complexation of a sulfated mucopolysaccharide chain of heparin with a platelet granular protein (platelet factor 4) produces an allosteric modification of platelet factor 4 resulting in neoepitope formation and the generation of antiheparin-platelet factor 4 antibodies. These antibodies are capable of activating platelets by binding to heparin, platelet factor 4 and the Fc receptor on platelets, resulting in a complex pathophysiology involving ischemic, thrombotic, and inflammatory processes. To characterize this antibody, IgG fractions were obtained from the serum of patients with heparin-induced thrombocytopenia using ammonium sulphate precipitation and heparin-platelet factor 4-sepharose 4B affinity chromatography methods. With the affinity purification, two major components, peaks I and II, with high antiheparin-platelet factor 4 antibody titers were eluted. The purity of all the fractionated immunoglobulins was established by sodium dodecylsulphate-polyacrylamide gel electrophoretic analyses. While peak I did not induce 14C-serotonin release from platelets in the heparin-dependent assay for heparin-induced thrombocytopenia antibodies (14C-serotonin release assay), peak II and the IgGs obtained with the ammonium sulphate precipitation method exhibited a strong and concentration-dependent activation in the presence and absence of heparin and low molecular weight heparin. These immunoglobulins were treated with heparinase, a cationic ion-exchange resin (Heparsorb), or dialyzed to remove traces of heparin, and when tested in the 14C-serotonin release assay, showed the same high degree of activity. These data are suggestive of the generation of heparin-induced thrombocytopenia antibodies capable of activating platelets directly in a nonheparin-dependent manner. These observations underscore the complex pathophysiology of heparin-induced thrombocytopenia syndrome and suggest that the severity of this syndrome in some patients may be due to the generation of "super-active" heparin-induced thrombocytopenia antibodies capable of activating platelets without the requirement of heparin. This could explain why the cessation of heparin in patients does not necessarily correct the symptoms of heparin-induced thrombocytopenia or associated thrombosis.
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PMID:Functional heterogeneity of antiheparin-platelet factor 4 antibodies: implications in the pathogenesis of the HIT syndrome. 1072 34

C(18) and C(8) bonded stationary phases dynamically coated with cetyltrimethylammonium (CTA) and strong anion exchange (SAX) were developed to obtain separations of oligosaccharide mixtures resulting from chemical or enzymatic depolymerization of heparin. With this method, the retention of sulfated oligosaccharides is directly adjustable depending on the amount of CTA adsorbed into the column. Oligosaccharides containing up to 20 sulfates were separated with a resolving power superior to that of conventional SAX analysis. The stability of the column coating enables hundreds of injections. Using ammonium methane sulfonate aqueous solutions as ultraviolet transparent mobile phases, it was possible to set up double detection, including selective detection of acetylated oligosaccharides. Analytical gel permeation chromatography was directly coupled to CTA-SAX, obtaining a two-dimensional profile of analyzed oligosaccharidic mixtures. A sequencing method of heparin oligosaccharides using partial depolymerization by heparinases according to their size and sulfation pattern and digest analysis by CTA-SAX was developed. A direct application of this method to the analysis of oligosaccharide mixtures obtained by complete digestion of heparins by heparinases I, II, and III was done. It allowed a reliable quantification of heparin building blocks. We also focused our attention on di- and tetrasaccharidic species containing the 3-O-sulfated glucosamines taken as markers of the active sites for antithrombin III. The method was also applied to more complex mixtures resulting from porcine heparin partially depolymerized with heparinase I. The specificity of the reaction was studied up to decasaccharidic fractions.
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PMID:Chromatographic analysis and sequencing approach of heparin oligosaccharides using cetyltrimethylammonium dynamically coated stationary phases. 1532 99

Glycosaminoglycans (GAGs) are known to play pivotal roles in physiological processes and pathological conditions. To study interactions of GAGs with proteins, immobilization of GAGs is often required. Current methodologies for immobilization involve modification of GAGs and/or surfaces, which can be time-consuming and may involve specialized equipment. Here, we use an efficient and low-cost method to immobilize GAGs without any (chemical) modification using highly concentrated salt solutions. A number of salts from the Hofmeister series were probed for their capacity to immobilize heparin and chondroitin-6-sulfate on microtiter plates applying single chain antibodies against GAGs for detection (ELISA). From all salts tested, the cosmotropic salt ammonium sulfate was most efficient, especially at high concentrations (80-100% (v/v) saturation). Immobilized GAGs were bioavailable as judged by their binding of FGF2 and VEGF, and by their susceptibility towards GAG lyases (heparinase I, II and III, chondroitinase ABC). Using 80% (v/v) saturated ammonium sulfate, block and continuous gradients of heparin were established and a gradient of FGF2 was created using a heparin block gradient as a template. In conclusion, high concentrations of ammonium sulfate are effective for immobilization of GAGs and for the establishment of gradients of both GAGs and GAG-binding molecules, which enables the study to the biological roles of GAGs.
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PMID:A versatile salt-based method to immobilize glycosaminoglycans and create growth factor gradients. 3105 97