Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Heparin Lyase”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 19 recordsLinked to original sources
PubMed1990

Randomness in the heparin polymer: computer simulations of alternative action patterns of heparin lyase.

Heparin is a mixture of linear polysaccharides of undetermined sequence. Both biosynthetic data and computer simulation studies have established that each heparin polymer chain is comprised of oligosaccharides of defined sequence, representing ordered domains. One such ordered domian is a pentasaccharide corresponding to heparin's antithrombin III binding site. Previous computer simulation studies, performed under the assumption that heparin lyase (heparinase, EC 4.2.2.7), has a random endolytic action pattern, suggested that certain of these ordered oligosaccharide domains may themselves be nonrandomly arranged in the heparin polymer. The present work presents computer simulations of alternative action patterns for heparin lyase while assuming a random distribution of these oligosaccharide units within the heparin polymer. We consider action patterns that are determined solely by the primary structure of the substrate molecules. Results of the simulations are compared to (1) the experimental measurements of product chains formed throughout the reaction and (2) the change in weight average molecular weight Mw as a function of reaction completion as determined by absorbance at 232 nm. From the simulation of 60 action patterns for heparin lyase, we infer that one of the following statements concerning heparin and heparin lyase is true: (1) Heparin is a random arrangement of a small number of structurally defined oligosaccharide units. Heparin lyase changes its action pattern during the depolymerization of heparin (perhaps influenced by the secondary structure of substrate). (2) Heparin contain clusters of oligosaccharide sequences that are present in low concentrations (overall) in the polymer. Heparin lyase has a specificity for cleaving glycosidic linkages either exolytically at the nonreducing terminus of a chain or (endolytically) at the reducing side of these rare oligosaccharide sequence.

Carbohydrate Conformation↗
PubMed1982

Mode of action of heparin lyase on heparin.

Heparinase (heparin lyase, EC 4.2.2.7) prepared from Flavobacterium heparinum was used to digest heparin. The products of digestion were examined with a viscosometric assay at various stages of the reaction to measure their average molecular weight. By comparison with computer simulations of various models, heparinase was shown to act in a random endolytic mode. The relative abundance of intermediates in heparin degradation catalyzed by heparinase immobilized on Sepharose 4B was measured by high pressure liquid chromatography (HPLC) at various time points. The results obtained using HPLC were consistent with a random endolytic mechanism. The heparin digestion products were separated and identified using gel permeation chromatography. The final distributions of heparin degradation products for free and immobilized heparinase were identical. Contaminating sulfatases and glycuronidases which could have subsequently acted on heparin degradation products were not found in significant amounts in the heparinase preparation studied.

Chromatography, Gel↗
PubMed1993

Monoclonal antibodies prepared against heparin lyase I and their reactivity toward heparin lyase I, II and III.

1. Six different monoclonal IgG mouse antibodies to heparin lyase I from Flavobacterium heparinum were prepared. 2. The monoclonal antibodies were used to detect heparin lyases I, II and III by dot-blotting immunoassay and by Western blotting. 3. Individual antibodies showed different reactivity toward the three heparin lyases. 4. The reactivity of two of the monoclonal antibodies was destroyed by exposing heparin lyases to sodium dodecyl sulfate. 5. The antibodies can be used to rapidly distinguish between the three heparin lyases.

Animals↗
PubMed2004

Relative susceptibilities of the glucosamine-glucuronic acid and N-acetylglucosamine-glucuronic acid linkages to heparin lyase III.

Heparin lyases are valuable tools for generating oligosaccharide fragments and in sequence determination of heparan sulfate (HS). Heparin lyase III is known to cleave the linkages between N-acetylglucosamine (GlcNAc) or N-sulfated glucosamine (GlcNS) and glucuronic acid (GlcA) as the primary sites and the linkages between GlcNAc, GlcNAc(6S), or GlcNS and iduronic acid as secondary sites. N-Unsubstituted glucosamine (GlcN) occurs as a minor component in HS, and it has been associated with various bioactivities. Here we investigate the specificity of heparin lyase III toward the GlcN-GlcA linkage using a recombinant enzyme of high purity and as substrates the partially de-N-acetylated polysaccharide of Escherichia coli K5 strain and derived hexasaccharides. The specificity of lyase III toward the GlcN-GlcA linkage is deduced by sequencing of the oligosaccharide products using electrospray mass spectrometry with collision-induced dissociation and MS/MS scanning. The results demonstrate that under controlled conditions for partial digestion, lyase III does not act at the GlcN-GlcA linkage, whereas GlcNAc-GlcA is cleaved. Even under forced conditions for exhaustive digestion, the GlcN-GlcA linkage is only partly cleaved. It is this property of lyase III that has enabled the isolation of a unique, nonsulfated antigenic determinant DeltaUA-GlcN-UA-GlcNAc from HS and from partially de-N-acetylated K5 polysaccharide. It was unexpected that pentasaccharide fragments were also detected among the digestion products of the K5 polysaccharide used. It is possible that these are products of an additional glycosidase activity of lyase III, although other mechanisms cannot be completely ruled out.

Acetylation↗
PubMed1992

Purification and characterization of heparin lyases from Flavobacterium heparinum.

Heparin lyase I has been purified from Flavobacterium heparinum and has been partially characterized (Yang, V. C., Linhardt, R. J., Berstein, H., Cooney, C. L., and Langer, R. (1985) J. Biol. Chem. 260, 1849-1857). There has been no report of the purification of the other polysaccharide lyases from this organism. Although all three of these heparin/heparan sulfate lyases are widely used, with the exception of heparin lyase I, there is no information on their purity or their physical and kinetic characteristics. The absence of pure heparin lyases and a lack of understanding of the optimal catalytic conditions and substrate specificity has stood in the way of the use of these enzymes as reagents for the specific depolymerization of heparin and heparan sulfate into oligosaccharides for structure and activity studies. This paper describes a single, reproducible scheme to simultaneously purify all three of the heparin lyases from F. heparinum to apparent homogeneity. Heparin lyase I (heparinase, EC 4.2.2.7), heparin lyase II (no EC number), and heparin lyase III (heparitinase, EC 4.2.2.8) have molecular weights (by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and isoelectric points (by isoelectric focusing) of M(r) 42,800, pI 9.1-9.2, M(r) 84,100, pI 8.9-9.1, M(r) 70,800, pI 9.9-10.1, respectively. Their amino acid analyses and peptide maps demonstrate that while these proteins are different gene products they are closely related. The kinetic properties of the heparin lyases have been determined as well as the conditions to optimize their activity and stability. These data should improve the application of these important enzymes in the study of heparin and heparan sulfate.

Amino Acid Sequence↗
PubMed1993

Specificity studies on the heparin lyases from Flavobacterium heparinum.

An understanding of the substrate specificity study of the heparin lyases (heparinase and heparitinases) is crucial for elucidation of the sequence of heparin and heparan sulfate. Four chemically modified heparins have been used to study the substrate specificity of the three heparin lyases. These modified heparins include the N- and O-desulfated and then specifically N-sulfated or N-acetylated derivatives of heparin and a modified heparin containing L-galactopyranosyluronic acid residues. These chemically modified heparins were degraded to various extents by the three heparin lyases. Differences in degree of sulfation have profound impact on the ease of cleavage of glycosidic linkages. Heparin lyase I (EC 4.2.2.7) is selective in cleaving highly sulfated polysaccharide chains containing linkages to 2-O-sulfated alpha-L-idopyranosyluronic acid residues. Heparin lyase III (EC 4.2.2.8) cleaves linkages that have reduced density of sulfation and that contain beta-D-glucopyranosyluronic acid residues. The ability of heparin lyase III to act on linkages to unsulfated alpha-L-idopyranosyluronic acid residues is observed for the first time. Heparin lyase II (no assigned EC number) demonstrates an unparalleled, wide specificity for substrates comprised of linkages containing both alpha-L-idopyranosyluronic and beta-D- glucopyranosyluronic acid residues. Heparin lyase II can also act on substrates containing linkages to unnatural alpha-L-galactopyranosyluronic acid residues. The high level of specificity of heparin lyase I makes it particularly suitable for use in the sequencing of heparin and heparan sulfate, while caution must be exercised in using heparin lyases II and III to sequence heparin and heparan sulfate because of their relatively broad specificity.

Acetylation↗
PubMed2004

Purification and characterization of heparin lyase I from Bacteroides stercoris HJ-15.

Heparin lyase I was purified to homogeneity from Bacteroides stercoris HJ-15 isolated from human intestine, by a combination of DEAE-Sepharose, gel-filtration, hydroxyapatite, and CM-Sephadex C-50 column chromatography. This enzyme preferred heparin to heparan sulfate, but was inactive at cleaving acharan sulfate. The apparent molecular mass of heparin lyase I was estimated as 48,000 daltons by SDS-PAGE and its isoelectric point was determined as 9.0 by IEF. The purified enzyme required 500 mM NaCl in the reaction mixture for maximal activity and the optimal activity was obtained at pH 7.0 and 50 degrees C. It was rather stable within the range of 25 to 50 degrees C but lost activity rapidly above 50 degrees C. The enzyme was activated by Co(2+) or EDTA and stabilized by dithiothreitol. The kinetic constants, K(m) and V(max) for heparin were 1.3 10(-5) M and 8.8 micromol/min.mg. The purified heparin lyase I was an eliminase that acted best on porcine intestinal heparin, and to a lesser extent on porcine intestinal mucosa heparan sulfate. It was inactive in the cleavage of N-desulfated heparin and acharan sulfate. In conclusion, heparin lyase I from Bacteroides stercoris was specific to heparin rather than heparan sulfate and its biochemical properties showed a substrate specificity similar to that of Flavobacterial heparin lyase I.

Animals↗
PubMed1993

Substrate specificity of the heparin lyases from Flavobacterium heparinum.

A detailed knowledge about the substrate specificities of the heparin lyases is necessary when using these enzymes as tools for elucidating the sequence of heparin and heparan sulfate. The substrate specificity of heparin lyases I, II, and III have been profiled with structurally defined, heparin-derived oligosaccharides. The primary substrate specificities of heparin lyases I and III require the presence of 2-O-sulfated alpha-L-idopyranosyluronic acid and beta-D-glucopyranosyluronic acid residues, respectively, at the linkages being cleaved. Heparin lyase II demonstrates an intriguingly broad primary specificity for oligosaccharides, acting at linkages containing alpha-L-idopyranosyluronic and beta-D-glucopyranosyluronic acid as well as at linkages containing alpha-L-galactopyranosyluronic acid residues. In addition to their primary specificities, each lyase also demonstrates secondary specificities under forcing conditions. Differences in the sulfation pattern within uronic acid residues and sulfation of adjacent residues has profound impact on the ease of lyase cleavage of a glycosidic linkage. Specifically, heparin lyases I and III exhibit secondary specificity for oligosaccharides containing an unsulfated alpha-L-idopyranosyluronic acid residue. The lack of sulfation on residues adjacent to the linkage undergoing cleavage increases the action of heparin lyase III on a glycosidic linkage. In contrast, reduced sulfation on adjacent residues make glycosidic linkage resistant to heparin lyase I. The primary and secondary specificity can be rationalized on the basis of most favorable solution conformation of the uronic acid residues.

Carbohydrate Conformation↗
PubMed1988

Purification and properties of Bacteroides heparinolyticus heparinase (heparin lyase, EC 4.2.2.7).

Heparinase (heparin lyase, EC 4.2.2.7) was isolated from the cell extract of an oral bacterium, Bacteroides heparinolyticus. It was a basic protein with an isoelectric point of 9.5. Its molecular weight was 63,000. The enzyme was the most active against heparin among the tested mucopolysaccharides. Catalytic properties may be similar to those of heparinase of Flavobacterium heparinum, since the enzymatic degradation products obtained by using the two enzymes were the same on the basis of paper chromatography.

Bacteroides↗
PubMed2001

Modulations of glypican-1 heparan sulfate structure by inhibition of endogenous polyamine synthesis. Mapping of spermine-binding sites and heparanase, heparin lyase, and nitric oxide/nitrite cleavage sites.

Cell surface heparan sulfate proteoglycans facilitate uptake of growth-promoting polyamines (Belting, M., Persson, S., and Fransson, L.-A. (1999) Biochem. J. 338, 317-323; Belting, M., Borsig, L., Fuster, M. M., Brown, J. R., Persson, L., Fransson, L.-A., and Esko, J. D. (2001) Proc. Natl. Acad. Sci. U. S. A., in press). Here, we have analyzed the effect of polyamine deprivation on the structure and polyamine affinity of the heparan sulfate chains in various glypican-1 glycoforms synthesized by a transformed cell line (ECV 304). Heparan sulfate chains of glypican-1 were either cleaved with heparanase at sites embracing the highly modified regions or with nitrite at N-unsubstituted glucosamine residues. The products were separated and further degraded by heparin lyase to identify sulfated iduronic acid. Polyamine affinity was assessed by chromatography on agarose substituted with the polyamine spermine. In heparan sulfate made by cells with undisturbed endogenous polyamine synthesis, free amino groups were restricted to the unmodified, unsulfated segments, especially near the core protein. Spermine high affinity binding sites were located to the modified and highly sulfated segments that were released by heparanase. In cells with up-regulated polyamine uptake, heparan sulfate contained an increased number of clustered N-unsubstituted glucosamines and sulfated iduronic acid residues. This resulted in a greater number of NO/nitrite-sensitive cleavage sites near the potential spermine-binding sites. Endogenous degradation by heparanase and NO-derived nitrite in polyamine-deprived cells generated a separate pool of heparan sulfate oligosaccharides with an exceptionally high affinity for spermine. Spermine uptake in polyamine-deprived cells was reduced when NO/nitrite-generated degradation of heparan sulfate was inhibited. The results suggest a functional interplay between glypican recycling, NO/nitrite-generated heparan sulfate degradation, and polyamine uptake.

Animals↗
PubMed1998

Characterization of a Bacteroides species from human intestine that degrades glycosaminoglycans.

Polysaccharide lyases that can degrade glycosaminoglycans (GAGs) were identified in an anaerobic strain living in the human intestine. The strain was isolated from the stool of a healthy male and identified as Bacteroides sp. strain HJ-15. A detailed taxonomical study indicated the species is a strain of Bacteroides stercoris. The isolate was cultured and the polysaccharide lyase activity was partially purified. This enzyme preparation could act on GAGs containing either glucosamine or galactosamine suggesting the presence of both heparinases and chondroitinases. Various GAGs were incubated with the partially purified enzyme and the products formed were analyzed by strong anion-exchange high performance liquid chromatography and proton nuclear magnetic resonance spectroscopy. These studies demonstrated the presence of at least two types of polysaccharide lyases: heparin lyase and chondroitin sulfate lyase. The eliminative mechanism of these lyase enzymes was confirmed through the isolation of unsaturated disaccharide products. The heparin lyase acted on both heparin and acharan sulfate, a GAG recently isolated from Achatina fulica. The Bacteroides chondroitin lyase, acted on chondroitin sulfates A, B (dermatan sulfate), and C, resembling chondroitin lyase ABC. The presence of a GAG-degrading organism in human intestine may pose problems for the effective oral administration of GAG drugs.

Anaerobiosis↗
PubMed1997

Preparation and structure of heparin lyase-derived heparan sulfate oligosaccharides.

Porcine intestinal mucosal heparan sulfate was exhaustively depolymerized on a large scale using heparin lyase II (heparinase II) or heparin lyase III (heparitinase, EC 4.2.2.8). The oligosaccharide mixtures formed with each enzyme were fractionated by low pressure gel permeation chromatography. Size-uniform mixtures of disaccharides, tetrasaccharides, and hexasaccharides were obtained. Each size-fractionated mixture was then purified on the basis of charge by repetitive semipreparative strong-anion-exchange high-performance liquid chromatography. This approach has led to the isolation of 13 homogenous oligosaccharides. The purity of each oligosaccharide was demonstrated by the presence of a single peak on analytical strong-anion-exchange high-performance liquid chromatography and reversed polarity capillary electrophoresis. The structures of these oligosaccharides were established using 500 MHz one- and two-dimensional nuclear magnetic resonance spectroscopy. Three of the thirteen structures that were solved were novel while the remaining 10 have been previously described. All of the structures obtained using heparin lyase III contained a delta UAp residue (where delta UAp is 4-deoxy-alpha-L-threo-hex-4-eno-pyranosyluronic acid) at their nonreducing termini. Structures obtained using heparin lyase II contained both delta UAp and delta UAp2S (where S is sulfate) at their nonreducing termini. These results are consistent with the reported specificity of both enzymes.

Animals↗
PubMed1997

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.

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).

Adult↗
PubMed1991

Ultrastructural localisation of anionic sites at the dermo-epidermal junction in normal human skin.

Glycosaminoglycans have been demonstrated throughout the cutaneous BMZ at the ultrastructural level. Colloidal iron and cationised ferritin proved of limited value, whilst staining with Alcian blue and application of the critical electrolyte concentration principle has provided evidence for the presence of sulphated GAGs at the lamina lucida and lamina reticularis. Digestions with chondroitin ABC lyase and heparin lyase have confirmed the existence of chondroitin and/or dermatan sulphates and heparan sulphates, although the results obtained with hyaluronate lyase have indicated that hyaluronates are also present.

Adult↗
PubMed2000

The fusion glycoprotein of human respiratory syncytial virus facilitates virus attachment and infectivity via an interaction with cellular heparan sulfate.

Human respiratory syncytial virus (RSV) F glycoprotein (RSV-F) can independently interact with immobilized heparin and facilitate both attachment to and infection of cells via an interaction with cellular heparan sulfate. RSV-glycosaminoglycan (GAG) interactions were evaluated using heparin-agarose affinity chromatography. RSV-F from A2- and B1/cp-52 (cp-52)-infected cell lysates, RSV-F derived from a recombinant vaccinia virus, and affinity-purified F protein all bound to and were specifically eluted from heparin columns. In infectivity inhibition studies, soluble GAGs decreased the infectivity of RSV A2 and cp-52, with bovine lung heparin exhibiting the highest specific activity against both A2 (50% effective dose [ED(50)] = 0.28 +/- 0.11 microg/ml) and cp-52 (ED(50) = 0.55 +/- 0. 14 microg/ml). Furthermore, enzymatic digestion of cell surface GAGs by heparin lyase I and heparin lyase III but not chondroitinase ABC resulted in a significant reduction in cp-52 infectivity. Moreover, bovine lung heparin inhibited radiolabeled A2 and cp-52 virus binding up to 90%. Taken together, these data suggest that RSV-F independently interacts with heparin/heparan sulfate and this type of interaction facilitates virus attachment and infectivity.

Animals↗
PubMed1994

Action pattern of polysaccharide lyases on glycosaminoglycans.

The action pattern of polysaccharide lyases on glycosaminoglycan substrates was examined using viscosimetric measurements and gradient polyacrylamide gel electrophoresis (PAGE). Heparin lyase I (heparinase, EC 4.2.2.7) and heparin lyase II (no EC number) both acted on heparin in a random endolytic fashion. Heparin lyase II showed an ideal endolytic action pattern on heparan sulphate, while heparin lyase I decreased the molecular weight of heparan sulphate more slowly. Heparin lyase III (heparitinase, EC 4.2.2.8) acted endolytically only on heparan sulphate and did not cleave heparin. Chondroitin ABC lyase (chondroitinase ABC, EC 4.2.2.4) from Proteus vulgaris acted endolytically on chondroitin-6-sulphate (chondroitin sulphate C) and dermatan sulphate at nearly identical initial rates, but acted on chondroitin-4-sulphate (chondroitin sulphate A) at a reduced rate, decreasing its molecular weight much more slowly. Two chondroitin AC lyases (chondroitinase AC, both EC 4.2.2.5) were examined towards chondroitin-4- and -6-sulphates. The exolytic action of chondroitin AC lyase A from Arthrobacter aurescens on both chondroitin-4- and -6-sulphates was demonstrated viscosimetrically and confirmed using both gradient PAGE and gel permeation chromatography. Chondroitin AC lyase F from Flavobacterium heparinum (Cytophagia heparinia) acted endolytically on the same substrates. Chondroitin B lyase (chondroitinase B, no EC number) from F.heparinum acted endolytically on dermatan sulphate giving a nearly identical action pattern as observed for chondroitin ABC lyase acting on dermatan sulphate.

Arthrobacter↗