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.     

Preparation and structure of heparin lyase-derived heparan sulfate oligosaccharides

Porcine intestinal mucosal heparan sulfate was exhaustivelydepolymerized on a large scale using beparin lyase II (heparinaseII) or heparin lyase III (heparitinase, EC 4.2.2.8 [EC] ). The oligosaccharidemixtures formed with each enzyme were fractionated by low pressuregel permeation chromatography. Size-uniform mixtures of disaccharides,tetrasaccharides, and hexasaccharides were obtained. Each size-fractionatedmixture was then purified on the basis of charge by repetitivesemipreparative strong-anion-exchange high-performance liquidchromatography. This approach has led to the isolation of 13homogenous oligosaccharides. The purity of each oligosaccharidewas demonstrated by the presence of a single peak on analyticalstrong-anion-exchange high-performance liquid chromatographyand reversed polarity capillary electrophoresis. The structuresof these oligosaccharides were established using 500 MHz one-and two-dimensional nuclear magnetic resonance spectroscopy.Three of the thirteen structures that were solved were novelwhile the remaining 10 have been previously described. All ofthe structures obtained using heparin lyase III contained a     

Analysis of glycosaminoglycan-derived oligosaccharides using reversed-phase ion-pairing and ion-exchange chromatography with suppressed conductivity detection

Oligosaccharides prepared from glycosaminoglycans (GAGs) including heparin, heparan sulfate, chondroitin sulfates, dermatan sulfate, and keratan sulfate were analyzed using reverse-phase ion-pairing HPLC and ion-exchange HPLC with suppressed conductivity detection. The results were compared with those obtained by strong anion-exchange HPLC using uv detection. These oligosaccharides were first prepared by enzymatically depolymerizing the GAGs with enzymes including heparin lyase (EC 4.2.2.7), heparan sulfate lyase (EC 4.2.2.8), chondroitin ABC lyase (EC 4.2.2.4), and keratan sulfate hydrolase (EC 3.2.1.103). Analysis was then performed without derivitization under isocratic conditions with a limit of sensitivity in the picomole range. Preliminary studies suggest that this approach may be particularly useful in examining oligosaccharides having no uv chromophore such as those prepared from keratan sulfate.     

Examination of the substrate specificity of heparin and heparan sulfate lyases

We have examined the activities of different preparations of heparin and heparan sulfate lyases from Flavobacterium heparinum. The enzymes were incubated with oligosaccharides of known size and sequence and with complex polysaccharide substrates, and the resulting degradation products were analyzed by strong-anion-exchange high-performance liquid chromatography and by oligosaccharide mapping using gradient polyacrylamide gel electrophoresis. Heparinase (EC 4.2.2.7) purified in our laboratory and a so-called Heparinase I (Hep I) from a commercial source yielded similar oligosaccharide maps with heparin substrates and displayed specificity for di- or trisulfated disaccharides of the structure----4)-alpha-D-GlcNp2S(6R)(1----4)-alpha-L-IdoAp2S( 1----(where R = O-sulfo or OH). Oligosaccharide mapping with two different commercial preparations of heparan sulfate lyase [heparitinase (EC 4.2.2.8)] indicated close similarities in their depolymerization of heparan sulfate. Furthermore, these enzymes only degraded defined oligosaccharides at hexosaminidic linkages with glucuronic acid:----4)-alpha-D-GlcNpR(1----4)-beta-D-GlcAp(1----(where R = N-acetamido or N-sulfo). The enzymes showed activity against solitary glucuronate-containing disaccharides in otherwise highly sulfated domains including the saccharide sequence that contains the antithrombin binding region in heparin. A different commercial enzyme, Heparinase II (Hep II), displayed a broad spectrum of activity against polysaccharide and oligosaccharide substrates, but mapping data indicated that it was a separate enzyme rather than a mixture of heparinase and heparitinase/Hep III. When used in conjunction with the described separation procedures, these enzymes are powerful reagents for the structural/sequence analysis of heparin and heparan sulfate.     

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.     

Microscale preparation of even- and odd-numbered N-acetylheparosan oligosaccharides

In order to prepare a series of N-acetylheparosan (NAH)-related oligosaccharides, bacterial NAH produced in Escherichia coli strain K5 was partially depolymerized with heparitinase I into a mixture of even-numbered NAH oligosaccharides, having an unsaturated uronic acid (DeltaUA) at the non-reducing end. A mixture of odd-numbered oligosaccharides was derived by removing this DeltaUA in the aforementioned mixture by a 'trimming' reaction using mercury(II) acetate. Each oligosaccharide mixture was subjected to gel-filtration chromatography to generate a series of size-uniform NAH oligosaccharides of satisfactory purity (assessed by analytical anion-exchange HPLC), and their structures were identified by MALDITOF-MS, ESIMS, and 1H NMR analysis. As a result, a microscale preparation of a series of both even- and odd-numbered NAH oligosaccharides was achieved for the first time. The developed procedure is simple and systematic, and thus, should be valuable for providing not only research tools for heparin/heparan sulfate-specific enzymes and their binding proteins, but also precursor substrates with medical applications.     

Enzymatic preparation of heparin disaccharides as building blocks in glycosaminoglycan synthesis.

Pharmaceutical heparin and heparan sulfate, isolated from a side-stream of a commercial heparin manufacturing process, have been enzymatically depolymerzed with heparin lyases obtained from Flavobacterium heparinun. Heparin afforded a trisulfated disaccharide product that was recovered from the reaction mixture using gel permeation chromatography. Heparan sulfate afforded unsulfated disaccharide that was conveniently recovered from the product mixture by ion exchange chromatography. Both disaccharides were obtained in gram amounts at 90% or higher purity. Both enzymatically prepared disaccharides were chemically protected to prepare building blocks required for the future chemical synthesis of therapeutically valuable heparin oligosaccharides.     

Separation and properties of five glycosaminoglycan sulfatases from rat skin.

Chondroitin sulfates, dermatan sulfate, heparan sulfate, heparin, keratan sulfate, and oligosaccharides derived from these sulfated glycosaminoglycans have been used for the measurement of sulfatase activity of rat skin extracts. Chromatographic fractionation of the extracts followed by specificity studies demonstrated the existence of five different sulfatases, specific for 1) the nonreducing N-acetylglucosamine 6-sulfate end groups of heparin sulfate and keratan sulfate, 2) the nonreducing N-acetylgalactosamine (or galactose) 6-sulfate end groups of chondroitin sulfate (or keratan sulfate), 3) the nonreducing N-acetylgalactosamine 4-sulfate end groups of chondroitin sulfate and dermatan sulfate, 4) certain suitably located glucosamine N-sulfate groups of heparin and heparan sulfate, or 5) certain suitably located iduronate sulfate groups of heparan sulfate and dermatan sulfate. Two arylsulfatases, one of which was identical in its chromatographic behaviors with the third enzyme described above, were also demonstrated in the extracts. These results taken together with those previously obtained from studies on human fibroblast cultures suggest that normal skin fibroblasts contain at least five specific sulfatases and diminished activity of any one may result in a specific storage disease.     

Heterogeneity of depolymerized heparin SEC fractions: to pool or not to pool?

In the structural analysis of heparin and heparan sulfate, it is customary to combine or pool like-sized fractions obtained by size-exclusion chromatography (SEC) of enzymatically derived heparin oligosaccharides. In this study, we examine the heterogeneity of preparative-scale SEC fractions obtained from enzymatic digests of porcine intestinal mucosa heparin. Each fraction was profiled by capillary electrophoresis with UV detection (CE−UV) using a 60 mM formic acid running buffer at pH 3.43. Differences in the composition and relative concentration of components of the SEC fractions were observed for disaccharides and larger oligosaccharides. The heterogeneity of the fractions becomes more pronounced when heparin is digested using a heparin lyase cocktail. The heterogeneity of preparative SEC fractions was further investigated by reversed-phase ion-pairing ultraperformance liquid chromatography coupled with mass spectrometry (RPIP−UPLC−MS) using the ion-pairing reagent, tributylamine (Bu₃N). Our results suggest that preliminary profiling of preparative SEC fractions prior to pooling may simplify efforts to identify and/or isolate rare structures.     

Action pattern of polysaccharide lyases on glycosaminoglycans

The action pattern of polysaccharide lyases on glycosaminoglycansubstrates was examined using viscosimetric measurements andgradient polyacrylamide gel electrophoresis (PAGE). Heparinlyase I (heparinase, EC 4.2.2.7 [EC] ) and heparin lyase II (no ECnumber) both acted on heparin in a random endolytic fashion.Heparin lyase II showed an ideal endolytic action pattern onheparan sulphate, while heparin lyase I decreased the molecularweight of heparan sulphate more slowly. Heparin lyase III (heparitinase,EC 4.2.2.8 [EC] ) acted endolytically only on heparan sulphate anddid not cleave heparin. Chondroitin ABC lyase (chondroitinaseABC, EC 4.2.2.4 [EC] ) from Proteus vulgaris acted endolytically onchondroitin-6-sulphate (chondroitin sulphate C) and dermatansulphate at nearly identical initial rates, but acted on chondroitin-4-sulphate(chondroitin sulphate A) at a reduced rate, decreasing its molecularweight much more slowly. Two chondroitin AC lyases (chondroitinaseAC, both EC 4.2.2.5 [EC] ) were examined towards chondroitin-4- and-6-sulphates. The exolytic action of chondroitin AC lyase Afrom Arthrobacter aurescens on both chondroitin-4- and -6-sulphateswas demonstrated viscosimetrically and confirmed using bothgradient PAGE and gel permeation chromatography. ChondroitinAC 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 endolyticallyon dermatan sulphate giving a nearly identical action patternas observed for chondroitin ABC lyase acting on dermatan sulphate. action pattern chondroitin lyase glycosaminoglycan heparin lyase.     

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.     

New oligosaccharides from heparin and heparan sulfate and their use as substrates for heparin-degrading enzymes

Oligosaccharides were isolated from heparin and heparan sulfate by a procedure consisting of three major steps: (a) acid hydrolysis; (b) gel chromatography; and (c) cation exchange chromatography on an amino acid analyzer. To date, six new oligosaccharides have been isolated by this procedure and have been sequenced by a combination of NaB3H4-labeling and deaminative cleavage with nitrous acid. The structures of these oligosaccharides were as follows: 1. GlcN-GlcUA-GlcN 2. GlcN-IdUA-GlcN 3. GlcN-GlcUA-GlcN-GlcUA-GlcN 4. GlcN-IdUA-GlcN-GlcUA-GlcN 5. GlcN-GlcUA-GlcN-IdUA-GlcN 6. GlcN-IdUA-GlcN-IdUA-GlcN The linkage positions and anomeric configurations were assumed to be the same as in the polysaccharides from which the oligosaccharides originated. The usefulness of some of these oligosaccharides as enzyme substrates was tested after appropriate modifications and radioactive labeling. Oligosaccharides 2 and 3 were N-[35S]sulfated and were found to serve as substrates for heparan N-sulfate sulfatase (heparin sulfamidase), with a homogenate of cultured skin fibroblasts as enzyme source. Similarly, reduction of oligosaccharide 2 with NaB3H4 yielded a substrate for acetyl-CoA:alpha-D-glucosaminide N-acetyltransferase. Finally, the previously known disaccharide, 4-O-alpha-D-glucosaminyl-L-iduronic acid, which was isolated in the course of this work, was N-acetylated with [3H] acetic anhydride and was shown to be a substrate for N-acetyl-alpha-D-glucosaminidase.     

Computational analyses of the catalytic and heparin-binding sites and their interactions with glycosaminoglycans in glycoside hydrolase family 79 endo-β-D-glucuronidase (heparanase)

Mammalian heparanase is an endo-β-glucuronidase associated with cell invasion in cancer metastasis, angiogenesis and inflammation. Heparanase cleaves heparan sulfate proteoglycans in the extracellular matrix and basement membrane, releasing heparin/heparan sulfate oligosaccharides of appreciable size. This in turn causes the release of growth factors, which accelerate tumor growth and metastasis. Heparanase has two glycosaminoglycan-binding domains; however, no three-dimensional structure information is available for human heparanase that can provide insights into how the two domains interact to degrade heparin fragments. We have constructed a new homology model of heparanase that takes into account the most recent structural and bioinformatics data available. Heparin analogs and glycosaminoglycan mimetics were computationally docked into the active site with energetically stable ring conformations and their interaction energies were compared. The resulting docked structures were used to propose a model for substrates and conformer selectivity based on the dimensions of the active site. The docking of substrates and inhibitors indicates the existence of a large binding site extending at least two saccharide units beyond the cleavage site (toward the nonreducing end) and at least three saccharides toward the reducing end (toward heparin-binding site 2). The docking of substrates suggests that heparanase recognizes the N-sulfated and O-sulfated glucosamines at subsite +1 and glucuronic acid at the cleavage site, whereas in the absence of 6-O-sulfation in glucosamine, glucuronic acid is docked at subsite +2. These findings will help us to focus on the rational design of heparanase-inhibiting molecules for anticancer drug development by targeting the two heparin/heparan sulfate recognition domains.     

Structural characterization of human liver heparan sulfate

The isolation, purification and structural characterization of human liver heparan sulfate are described. 1H-NMR spectroscopy demonstrates the purity of this glycosaminoglycan (GAG) and two-dimensional 1H-NMR confirmed that it was heparan sulfate. Enzymatic depolymerization of the isolated heparan sulfate, followed by gradient polyacrylamide gel, confirmed its heparin lyase sensitivity. The concentration of resulting unsaturated disaccharides was determined using reverse phase ion-pairing (RPIP) HPLC with post column derivatization and fluorescence detection. The results of this analysis clearly demonstrate that the isolated GAG was heparan sulfate, not heparin. Human liver heparan sulfate was similar to heparin in that it has a reduced content of unsulfated disaccharide and an elevated average sulfation level. The antithrombin-mediated anti-factor Xa activity of human liver heparan sulfate, however, was much lower than porcine intestinal (pharmaceutical) heparin but was comparable to standard porcine intestinal heparan sulfate. Moreover, human liver heparan sulfate shows higher degree of sulfation than heparan sulfate isolated from porcine liver or from the human hepatoma Hep 2G cell line.     

Structural determinants of heparan sulfate interactions with Slit proteins

We have previously demonstrated that the Slit proteins, which are involved in axonal guidance and related processes, are high-affinity ligands of the heparan sulfate proteoglycan glypican-1. Glypican-Slit protein interactions have now been characterized in greater detail using two approaches. The ability of heparin oligosaccharides of defined structure (ranging in size from disaccharide to tetradeccasaccharide) to inhibit binding of a glypican-Fc fusion protein to recombinant human Slit-2 was determined using an ELISA. Surface plasmon resonance (SPR) spectroscopy, which measures the interactions in real time, was applied for quantitative modeling of heparin-Slit binding on heparin biochips. Heparin was covalently immobilized on these chips through a pre-formed albumin-heparin conjugate, and the inhibition of Slit binding by heparin, LMW heparin, and heparin-derived oligosaccharides (di-, tetra-, hexa-, and octa-) was examined utilizing solution competition SPR. These competition studies demonstrate that the smallest heparin oligosaccharide competing with heparin binding to Slit was a tetrasaccharide, and that in the ELISA maximum inhibition (approximately 60% at 2 microM concentration) was attained with a dodecasaccharide.     

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.     

Microanalysis of glycosaminoglycan-derived oligosaccharides labeled with a fluorophore 2-aminobenzamide by high-performance liquid chromatography: application to disaccharide composition analysis and exosequencing of oligosaccharides

A series of disaccharides derived from chondroitin sulfate and heparin/heparan sulfate were derivatized at their reducing ends with a fluorophore 2-aminobenzamide to develop a sensitive microanalytical method for glycosaminoglycans. The resulting labeled compounds derived from chondroitin sulfate or heparin/heparan sulfate were well-separated and quantified by HPLC equipped with a fluorescence detector. The detection limit was a low picomole level. This method was applied to the analysis of the disaccharide composition of tetra- and hexasaccharides derived from chondroitin sulfate and heparin/heparan sulfate as well as these glycosaminoglycan polysaccharides. The method was also successfully applied to the exosequencing of chondrohexasaccharides, where the fluorophore-labeled oligosaccharides were degraded exolytically from the nonreducing ends using bacterial eliminases. The resultant labeled fragments were identified by HPLC.     

Preparation and structural determination of large oligosaccharides derived from acharan sulfate

The structures of a series of large oligosaccharides derived from acharan sulfate were characterized. Acharan sulfate is an unusual glycosaminoglycan isolated from the giant African snail, Achatina fulica. Oligosaccharides from decasaccharide to hexadecasaccharide were enzymatically prepared using heparin lyase II and purified. Capillary electrophoresis and gel electrophoresis confirmed the purity of these oligosaccharides. Their structures, determined by ESI-MS and NMR, were consistent with the major repeating sequence in acharan sulfate, -->4)-alpha-d-GlcN(p)Ac-(1-->4)-alpha-l-IdoA(p)2S-(1-->, terminated by 4-linked alpha-d-GlcN(p)Ac residue at the reducing end and by 4,5-unsaturated pyranosyluronic acid 2-sulfate at the non-reducing end.     

Isolation of a plasma-membrane fraction from gastric smooth muscle. Comparison of the calcium uptake with that in endoplasmic reticulum.

Heparan sulphate by-products from the commercial manufacture of pig mucosal heparin were freed of chondroitin sulphate and fractionated according to anionic density. The fractions were treated with HNO2 at pH 1.5, and the resulting mixtures of oligosaccharides were reduced with NaB3H4 and analysed for their disaccharide composition by paper chromatography and by high-pressure liquid chromatography. The results show that the molar ratio of 2-O-sulpho-alpha-L-iduronosylanhydromannose to 6-O-sulpho-(2-O-sulpho-alpha-L-iduronosyl)anhydromannose decreased from 2.5 to 0.04 as the degree of sulphation of the fractions increased. In contrast, the molar ratio of 6-O-sulpho-(beta-D-glucuronosyl)anhydromannose to 6-O-sulpho-(alpha-L-iduronosyl)anhydromannose was approx. 2.4 in all heparan sulphate fractions and decreased to only half of this value in the most highly sulphated heparin fractions. These results are consistent with biosynthetic studies, which have shown that the N-sulpho-(2-O-sulpho-alpha-L-iduronosyl)D-glucosamine disaccharide is the metabolic precursor of the NO-disulpho-(2-O-sulpho-alpha-L-iduronosyl)-D-glucosamine disaccharide in heparin biosynthesis. The high-pressure liquid chromatography of the heparan sulphate oligosaccharides also revealed a number of unidentified oligosaccharides in the deamination mixtures.     

Biosynthesis of heparin/heparan sulfate: kinetic studies of the glucuronyl C5-epimerase with N-sulfated derivatives of the Escherichia coli K5 capsular polysaccharide as substrates

The D-glucuronyl C5-epimerase involved in the biosynthesis of heparin and heparan sulfate was investigated with focus on its substrate specificity, its kinetic properties, and a comparison of epimerase preparations from the Furth mastocytoma and bovine liver, which synthesize heparin and heparan sulfate, respectively. New substrates for the epimerase were prepared from the capsular polysaccharide of Escherichia coli K5, which had been labeled at C5 of its D-glucuronic and N-acetyl-D-glucosamine moieties by growing the bacteria in the presence of D-[5-(3)H]glucose. Following complete or partial ( approximately 50%) N-deacetylation of the polysaccharide by hydrazinolysis, the free amino groups were sulfated by treatment with trimethylamine.SO(3)complex, which yielded products that were recognized as substrates by the epimerase and released tritium from C5 of the D-glucuronyl residues upon incubation with the enzyme. Comparison of the kinetic properties of the two substrates showed that the fully N-sulfated derivative was the best substrate in terms of its K(m)value, which was significantly lower than that of its partially N-acetylated counterpart. The V(max)values for the E.coli polysaccharide derivatives were essentially the same but were both lower than that of the O-desulfated [(3)H]heparin used in our previous studies. Surprisingly, the apparent K(m)values for all three substrates increased with increasing enzyme concentration. The reason for this phenomenon is not entirely clear at present. Partially purified C5-epimerase preparations from the Furth mastocytoma and bovine liver, respectively, behaved similarly in terms of their reactivity towards the various substrates, but the variation in apparent K(m)values with enzyme concentration precluded a detailed comparison of their kinetic properties.