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)

Cultured microvascular endothelial cells isolated from rat epididymal fat pads produce glycosaminoglycans that accelerate thrombin-antithrombin complex formation. The heparinlike nature of these macromolecules was established by complete destruction of their anticoagulant activity employing purified Flavobacterium heparinase. Only 15% of the biologic activity of these complex carbohydrates was expressed when the heparin binding domain on the protease inhibitor was chemically modified at the Trp 49 residue. The anticoagulantly active species contains disaccharides which constitute the unique antithrombin binding region of the mucopolysaccharide. Removal of the biologically active heparinlike components from endothelial cells with 0.05% trypsin suggests that these molecular species are present on the cell surface.
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PMID:Heparinlike molecules with anticoagulant activity are synthesized by cultured endothelial cells. 397 Jun 99

Calf microvasculature was isolated from retina and cerebral gray matter. These preparations contained 0.048-0.060 U of heparin-like anticoagulant activity per gram of wet tissue. The retinal microvascular material contained no detectable mast cells. The anticoagulant potency of this product was associated solely with endothelial cells. This property appears to be due to a heparinlike proteoglycan since molecular species with biologic activity are precipitated with 10% (wt/vol) trichloroacetic acid and are destroyed by incubation with Flavobacterium heparinase. Furthermore, the above component functions in a manner virtually identical to heparin since approximately 60% of these species with anticoagulant activity bind to antithrombin-concanavalin A-Sepharose 4B, and only 15% of their biologic potency is expressed in the presence of antithrombin modified near the mucopolysaccharide binding domain. The cerebral microvascular tissue contained a trace subpopulation of mast cells (approximately 0.3%). The anticoagulant activity of this preparation is most probably associated with both endothelial cells and mast cells. However, complete separation of these two cellular elements has proven difficult with current methodology.
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PMID:Microvascular heparin-like species with anticoagulant activity. 635 38

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

We have examined the role of heparinlike molecules in the regulation of coagulation by perfusing rat hindquarters with purified human thrombin and with its plasma inhibitor, antithrombin. Our data indicate that contact of the hemostatic components with the endothelium enhances the rate of thrombin-antithrombin complex formation by as much as 19-fold over the uncatalyzed rate of enzyme-inhibitor interaction. Heparinlike molecules are responsible for the antithrombin accelerating activity. The amount of thrombin-antithrombin complex generated within the hindlimb preparation after pretreatment of the vasculature with purified Flavobacterium heparinase or with addition of platelet Factor IV to the hemostatic components, was equal to the uncatalyzed levels. These heparinlike molecules appear to be tightly bound to the luminal surface of the endothelium, since they could not be detected within the physiologic buffer that was perfused through the animal. The above mucopolysaccharides function in a manner similar to commercial heparin, since modification of antithrombin at a site critical for heparin-dependent acceleration of the protease inhibitor resulted in a level of interaction product identical to the uncatalyzed amount. Finally, addition of diisofluorophosphate-thrombin to the enzyme perfusion stream reduced the amount of thrombin-antithrombin complex formed in the animal by 30-40%, which suggested that thrombin bound to the endothelium as well as enzyme free in solution are accessible to antithrombin that has interacted with heparinlike molecules present on the endothelium.
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PMID:Acceleration of thrombin-antithrombin complex formation in rat hindquarters via heparinlike molecules bound to the endothelium. 674 97

Whale heparin was separated by affinity chromatography on an antithrombin III-Sepharose column into two distinct fractions. The high-affinity fraction accounted for most the anticoagulant activity of the unfractionated material, while the low-affinity fraction was relatively inactive. The yields of the two fractions were substantially equivalent. No significant difference was observed between these fractions in terms of electrophoretic mobilities on cellulose acetate membrane and analytical data except for the contents of N-acetylglucosamine and N-sulfoglucosamine. The highly active form contained more N-acetylglucosamine and less N-sulfoglucosamine than the relatively inactive form. The two fractions were separately subjected to the sequential digestion with purified heparinase and heparitinase, and the oligosaccharide fractions were isolated from the digests by DEAE-cellulose column chromatography, followed by preparative paper chromatography. The purified compounds were then characterized by routine chemical and physical methods. Compound 1, delta 4,5hexosyuronic acid1 leads to 4N-acetylglucosamine, was exclusively obtained from the highly active form, whereas compound 3a, delta 4,5hexosyluronic acid1 leads to 4N-acetylglucosamine 6-sulfate, and compound 3b delta 4,5hexosyluronic acid1 leads to 4-sulfoglucosamine, were the only ones obtained from the relatively inactive form. The yields of other oligosaccharide fractions from both forms were comparable. The present data suggest that an N-acetylglucosamine-containing oligosaccharide structure in whale heparin is essential for binding to antithrombin II.
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PMID:Comparative studies on the structures of highly active and relatively inactive forms of whale heparin. 728 79

Proteoglycans (PGs), biosynthetically labelled with [35S]sulphate, were isolated from mouse mastocytoma tissue. Chromatography on antithrombin (AT)-Sepharose resulted in the separation of the 35S-labelled PGs into three fractions: PGs with no affinity for the gel (NA-PGs), PGs with low affinity (LA-PGs), and PGs with high affinity (HA-PGs) for antithrombin. Whereas NA-PGs contained almost exclusively chondroitin sulphate (CS), the AT-binding PGs contained 80-85% heparin and 15-20% CS. [35S]CS-containing macromolecules obtained from the HA-PG fraction after removal of the heparin polysaccharide chains were rechromatographed on AT-Sepharose. A majority of these 35S-labelled macromolecules no longer showed affinity for AT. These experiments indicate that the [35S]CS recovered in the AT-binding PGs is present in hybrid PGs. Polysaccharide chain-length determination demonstrated that the heparin chains were somewhat larger (M(r) approximately 30,000) than the CS chains in the NA-PGs (M(r) approximately 25,000). CS chains in the hybrid PGs were slightly smaller (M(r) approximately 20,000). Characterization of the sulphated CS disaccharides from NA- and HA-PGs showed that they contained similar amounts (20%) of disulphated disaccharides of [GlcA-GalNAc(4,6-di-OSO3)] type. The monosulphated CS-disaccharides were O-sulphated at C-4 of the galactosamine units. Analysis by gel chromatography of the [35S]CS components isolated from HA-PGs after heparinase treatment showed that a major portion of these contained one CS chain only. Calculations of the number of CS and heparin chains in AT-binding PGs, based on polysaccharide composition and polysaccharide chain length, indicate that all heparin-containing PGs are hybrids.
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PMID:Heparin proteoglycans synthesized by mouse mastocytoma contain chondroitin sulphate. 757 59

Human umbilical vein endothelial cells cultured in growth media that did not contain exogenous heparin were found to grow less well while binding significantly more antithrombin (AT) than comparable cells cultured in growth media that did contain exogenous heparin (90 micrograms/ml). The binding of AT to plasma membranes of cultured endothelial cells was measured immunologically by flow cytometry. This binding was eliminated completely by reacting the cells with heparinase III before incubating them with AT, indicating that the most likely heparinase-sensitive process responsible for AT binding to plasma membranes was heparan sulfate proteoglycan. Increased AT binding also was promoted by addition of heparin-binding molecules (protamine, AT, or ECGF) to growth media, and the effects of other glycosaminoglycans and dextran on AT binding were found to be dependent on their sulfation. Thus, one response of endothelial cells to heparin deficiency is up-regulation of the ability to bind AT to plasma membranes.
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PMID:Antithrombin binding by human umbilical vein endothelial cells: effects of exogenous heparin. 767 4

O-Sulfation at C-3 of N-sulfated GlcN units concludes polymer modification and the formation of antithrombin binding regions in the biosynthesis of heparin/heparan sulfate. The resulting GlcNSO3(3-OSO3) units are largely restricted to heparin chains with high affinity for antithrombin (HA heparin). Low affinity (LA) heparin fails to serve as a substrate in the 3-O-sulfotransferase reaction yet contains potential 3-O-sulfate acceptor sites (Kusche, M., Torri, G., Casu, B., and Lindahl, U. (1990) J. Biol. Chem. 265, 7292-7300), as verified in the present study using a novel sequencing procedure. O-Desulfated, re-N-sulfated LA heparin, as well as an octasaccharide fraction isolated after heparinase I digestion of LA heparin, both yielded labeled HA components following incubation with solubilized mouse mastocytoma microsomal enzymes and [35S]adenosine 3'-phosphate 5'phosphosulfate (PAPS), suggesting that the 3-O-sulfo-transferase may be inhibited by sulfated saccharide sequences outside the 3-O-sulfate acceptor region. Indeed, the addition of LA heparin precluded enzymatic 3-O-sulfation of a synthetic pentasaccharide substrate. The Km for the pentasaccharide was determined to approximately be 6 microM. Incubations of mixed pentasaccharide substrate and saccharide inhibitors revealed Ki values for intact LA heparin and for a heparin octasaccharide fraction of approximately 1.3 and approximately 0.7 microM, respectively. Inhibition experiments with selectively desulfated heparin indicated that both IdoA 2-O-sulfate and GlcN 6-O-sulfate groups contributed to the inhibition of the 3-O-sulfotransferase. By contrast, chondroitin sulfate or dermatan sulfate showed no significant inhibitory activity. It is proposed that the regulation of GlcN 3-O-sulfation during biosynthesis of heparin/heparan sulfate depends on the topological organization of the membrane-bound enzyme machinery in the intact cell.
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PMID:Biosynthesis of heparin/heparan sulfate. The D-glucosaminyl 3-O-sulfotransferase reaction: target and inhibitor saccharides. 774 62

Heparin, NAcHep, DS, and CS were labeled with deuterium by N-reacetylating, with the deuterated acetic anhydride (CD3CO)2O, GAGs previously N-deacetylated (by hydrazinolysis) to the desired extent. Degrees of deuteration of the present preparations, as determined by 2H- and 1H-NMR were 15%, 51%, 49%, and 79% for heparin, NAcHep, DS, and CS, respectively. The NMR analysis (including the 13C spectra) of the labeled products indicated that deuterium labeling did not involve any substantial modification of the GAG structures. Also NMR signals associated with specific sequences of heparin for antithrombin and of DS for heparin cofactor II were essentially the same in the unlabeled and in the deuterated GAGs. The substantial retention of the original structure was confirmed by data on the degree of sulfation (by conductimetry) and on the electrophoretic mobility in acid buffer. On the other hand, HPLC/SEC data indicated some depolymerization of heparin and DS in the N-deacetylation step of the labeling reactions. HPLC/MS spectrometry permitted a clear identification of disaccharide and tetrasaccharide fragments obtained from deuterated GAGs by enzymic (heparinase, chondroitinase ABC) or chemical depolymerization (deaminative cleavage, Smith degradation), opening new prospects for studies of human pharmacokinetics, with differentiation of exogenous from endogenous GAGs.
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PMID:Preparation and characterization of deuterium-labeled glycosaminoglycans. 799 88

Immobilized enzyme reactors can form the basis of useful blood detoxification systems. One such reactor was developed for heparin neutralization by immobilized heparinase. In this article, reactor kinetics were studied under clinically relevant conditions. Heparin neutralization was assessed in vitro in whole human blood using (a) a well-mixed batch reactor, and (b) an oscillating, continuous-flow reactor. The kinetics of heparin neutralization in human blood were first order over the entire range of heparin and enzyme concentrations and particle fractions tested. The kinetic rate was not sensitive to physiological variations in the concentration of antithrombin, a heparin binding protein in blood. Enzyme activity did not decrease significantly over the 2 hour test period. Kinetic control of the system with minimal intraparticle diffusional limitations was suggested by the Thiele moduli (0.11-0.67) and effectiveness factors (0.98 +/- 0.01). The ratio kcat/Km obtained in batch studies was 0.0028 +/- 0.0008 cm3/microgram-min. A continuous-flow oscillating reactor within a closed recirculation loop performed as a single well mixed batch reactor; there was a short mixing time of recirculating blood when compared to reaction time. A model based on this mixing pattern and the kinetics obtained in independent batch studies accurately predicted heparin neutralization profiles observed in the continuous-flow system.
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PMID:Kinetics of immobilized heparinase in human blood. 843 22


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