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)

The glycosulphatase which hydrolyses the 2-O-sulphate of the disaccharide, 4-deoxy-2-O-sulphato-alpha-L-threohex-4-enopyranosyl uronic acid-(1----4)-2-deoxy-2-sulphamido-6-O-sulphato-D-glucose (delta UA-2S----GlcNS-6S), has been isolated from the soluble fraction of disrupted Flavobacterium heparinum. The activity was purified 3300-fold by chromatography on CM-Sepharose CL-6B, hydroxyapatite, taurine-Sepharose CL-4B and blue-Sepharose CL-6B. From sodium dodecylsulphate/polyacrylamide gel electrophoresis, the enzyme was homogeneous and of 62000 Mr. A novel assay was devised using the de-N-sulphonated [1-3H]alditol, 4-deoxy-2-O-sulphato-alpha-L-threo-hex-4-enopyranosyl uronic acid-(1----4)-2-amino-2-deoxy-6-O-sulphato-D-[1-3H]glucitol (delta UA-2S----[1-3H]GlcNH2-ol-6S). This alditol was shown by 13C-NMR to be desulphated in the analogous manner to the original reducing trisulphated disaccharide. The purified 2-O-sulphatase was completely free of heparinase I, heparinase II (heparitinase), chondroitinases AC, chondroitinase B, the delta 4,5-glycuronidase for heparin delta 4,5-disaccharides, the 6-O-sulphatase and the 2-sulphamidase. It was optimally active over the range pH 5.5-6.5 and was practically unaffected by Na, K, Ca or Mg ions. Inorganic phosphate inhibited the activity. The Km value for the alditol substrate was 1.22 mmol dm-3. Using 13C-NMR, the 2-O-sulphatase was found to hydrolyse the analogous esters of higher delta 4,5-oligosaccharides from heparin. This contrasts with the findings of other authors [Dietrich, C. P., Silva, M. E., and Michelacci, Y. M. (1973) J. Biol. Chem. 248, 6408-6415].
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PMID:Flavobacterium heparinum 2-O-sulphatase for 2-O-sulphato-delta 4,5-glycuronate-terminated oligosaccharides from heparin. 651 Apr 19

Three tetrasaccharides representing major structural sequences of heparin were isolated in good yield and characterized after degradation of heparin by purified flavobacterial heparinase. N-Desulfation was necessary to achieve good separation of these closely related compounds from each other. One of the tetrasaccharides was shown to be derived from the fully sulfated repeating segments; to contain L-iduronic acid and six sulfate groups, and have the structure delta 4,5- HexpA -(2-SO4)-(1----4)-alpha-D- GlcpN -(N-SO4)-(6-SO4)-(1- ---4)-alpha -L- IdopA -(2-SO4)-(1----4)-D- GlcN -(N-SO4)-(6-SO4). The second contained a D-glucuronic acid unit that was nonsulfated instead of the L-iduronic acid, and the third, obtained in a fairly low yield, contained five sulfate groups, three of which being located on the disaccharide at the nonreducing end, and having the structure delta 4,5- HexpA -(2-SO4)-(1----4)-alpha-D- GlcpN -(N-SO4)-(6-SO4)-(1- ---4)-alpha -L- IdopA -(2-SO4)-(1----4)-D- GlcN -(N-SO4). All tetrasaccharides had a sulfated, unsaturated uronic acid unit at the nonreducing end, confirming that the heparinase requires sulfated L-iduronic acid units for activity.
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PMID:Structural studies on heparin. Tetrasaccharides obtained by heparinase degradation. 671 43

Heparinase and heparitinase were separated from an extract of Flavobacterium heparinum, induced with heparin by using column chromatography on hydroxylapatite. As the heparinase preparation contained chondroitinases B and C, chondroitinase B was removed by rechromatography on a hydroxylapatite column. Chondroitinase C was then eliminated by column chromatography on O-phosphono("phospho")-cellulose. The heparinase preparation thus obtained was free from sulfoamidase for 2-deoxy-2-sulfoamino-D-glucose (GlcN-2S), sulfatase for 2-amino-2-deoxy-6-O-sulfo-D-glucose (GlcN-6S), as well as delta 4,5glycosiduronase for the unsaturated disaccharides obtained from heparin. The remaining sulfatase for 4-deoxy-alpha-L-threo-hex-4-enopyranosyluronic acid 2-sulfate (delta UA-2S) in the heparinase preparation was removed by affinity chromatography with dermatan sulfate-bound AH-Sepharose 4B coated with dermatan sulfate. The heparitinase preparation separated by column chromatography on hydroxylapatite was purified by affinity chromatography with heparin-bound AH-Sepharose 4B coated with heparin. Sulfatase for 2-amino-2-deoxy-6-O-sulfo-D-glucose (GlcN-6S) and delta 4,5glycosiduronase for the unsaturated disaccharides obtained from heparin were removed by this chromatography. Sulfatase for 4-deoxy-alpha-L-threo-hex-4-enopyranosyluronic acid 2-sulfate (delta UA-2S) remaining in the heparitinase preparation was finally removed by column chromatography on hydroxylapatite. The recoveries of the purified preparations of heparinase and heparitinase were estimated to be 39 and 50%, respectively, from the crude enzyme fractions obtained by the first column chromatography on hydroxylapatite. The purified heparinase and heparitinase were free from all enzymes that could degrade the sulfated unsaturated disaccharides produced from heparin with heparinase.
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PMID:Purification of heparinase and heparitinase by affinity chromatography on glycosaminoglycan-bound AH-Sepharose 4B. 721 77

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

Three discrete tetrasaccharide structures which are resistant to Flavobacterium heparinase and heparitinases I and II were isolated from porcine intestinal heparin after exhaustive digestion with a mixture of all the above enzymes, and the tri-, tetra-, and penta-sulfated structures were determined by negative ion mode fast atom bombardment mass spectrometry and 500-MHz 1H NMR analysis as delta 4,5GlcA beta 1-4GlcNAc (6-sulfate)alpha 1-4GlcA beta 1-4GlcN(N,3-disulfate), delta 4,5 GlcA beta 1-4GlcNAc(6-sulfate)alpha 1-4GlcA beta 1-4GlcN (N,3,6-trisulfate), and delta 4,5GlcA beta 1-4GlcN (N,6-disulfate)alpha 1-4GlcA beta 1-4GlcN(N,3,6-trisulfate). The three components share the 3-O-sulfated reducing GlcN and the 6-O-sulfated internal GlcN, indicating that they are structural variants derived from the nonreducing portion of the minimal pentasaccharide sequence required for binding to antithrombin III. Isolation of the pentasulfated component has never been reported. Their unexpected resistance to heparitinases I and II indicates that 3-O-sulfation of the reducing GlcN contributes to the resistant nature of these tetrasaccharides to the enzymes. The present study demonstrates that the nonreducing trisaccharide portion of the structural variants of the antithrombin III-binding pentasaccharide sequence can be isolated in tetrasaccharides resistant to heparinase/heparitinases I and II, while the rest of the repeating region is degraded into disaccharide units. The lyase treatment is applicable to evaluation of heparin/heparan sulfate preparations in terms of the presence or absence of the specific structure containing the 3-O-sulfated GlcN representing biosynthetic precursors, intermediates or final products of the binding site.
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PMID:Structural studies on the bacterial lyase-resistant tetrasaccharides derived from the antithrombin III-binding site of porcine intestinal heparin. 844 55

The soil bacterium Flavobacterium heparinum produces several enzymes that degrade heparan sulfate glycosaminoglycans (HSGAGs) in a sequence-specific manner. Among others, these enzymes include the heparinases and an unusual glycuronidase that hydrolyzes the unsaturated Delta4,5 uronic acid at the nonreducing end of oligosaccharides resulting from prior heparinase eliminative cleavage. We report here the molecular cloning of the Delta4,5 glycuronidase gene from the flavobacterial genome and its recombinant expression in Escherichia coli as a highly active enzyme. We also report the biochemical and kinetic characterization of this enzyme, including an analysis of its substrate specificity. We find that the Delta4,5 glycuronidase discriminates on the basis of both the glycosidic linkage and the sulfation pattern within its saccharide substrate. In particular, we find that the glycuronidase displays a strong preference for 1-->4 linkages, making this enzyme specific to heparin/heparan sulfate rather than 1-->3 linked glycosaminoglycans such as chondroitin/dermatan sulfate or hyaluronan. Finally, we demonstrate the utility of this enzyme in the sequencing of heparinase-derived HSGAG oligosaccharides.
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PMID:Molecular cloning of the heparin/heparan sulfate delta 4,5 unsaturated glycuronidase from Flavobacterium heparinum, its recombinant expression in Escherichia coli, and biochemical determination of its unique substrate specificity. 1204 76