Department of Medical Biochemistry and Microbiology, Uppsala University, The Biomedical Center, Box 582, S-751 23, Uppsala, Sweden, 2Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, Birmingham, AL 35294, USA, and 3Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, IL 60637, USA
Received on May 10, 1999; revised on July 5, 1999; accepted on July 22, 1999.
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: Escherichia coli K5/heparan sulfate/heparin/glucuronyl C5-epimerase/mastocytoma
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
During the epimerization, the C5 hydrogen of the hexuronic acid is abstracted and replaced by hydrogen from water (Jacobsson et al., 1979a; Prihar et al., 1980
). This feature of the reaction is the basis for the assay of epimerase activity currently used (Campbell et al., 1983
; Jensen et al., 1983
). The substrate for the enzyme is prepared by N-deacetylation and complete desulfation of heparin, followed by N-sulfation, and incubation with the epimerase in the presence of 3H2O then yields a product in which tritium has been incorporated at C5 of both GlcA and IdoA residues. Upon incubation of the labeled polysaccharide with epimerase preparations to be analyzed, the tritium is released as 3H2O, and the rate of this reaction provides a measure of the activity of the enzyme.
The glucuronyl C5-epimerase catalyzing formation of IdoA residues in the course of heparan sulfate biosynthesis has previously been purified to homogeneity from bovine liver (Campbell et al., 1994), and its primary structure has been determined by cDNA cloning (Li et al., 1997
). Some of its basic molecular and catalytic properties, however, have not yet been elucidated; e.g., we have only scant knowledge of the kinetics of the epimerase reaction.
In the present study, we have developed a new procedure for substrate preparation based on metabolic labeling of the capsular polysaccharide of Escherichia coli K5, which has the same structure, [GlcAß1,4-GlcNAc1,4]n, as the nonsulfated precursor polysaccharide in heparin/heparan sulfate biosynthesis (Vann et al., 1981
). The bacterial polysaccharide is labeled at C5 of the GlcA (and N-acetylglucosamine) residues by growing E.coli K5 bacteria in the presence of D-[5-3H]glucose, and the isolated polysaccharide is then converted into an epimerase substrate by chemical N-deacetylation and N-sulfation. Since glucuronic acid is its sole uronic acid constituent, the derivative of the bacterial polysaccharide is more akin to the physiological substrates of the epimerase than the O-desulfated [5-3H]uronyl-heparin used previously (Jensen et al., 1983
), which contains a large proportion of iduronic acid residues. Using the metabolically labeled K5 polysaccharides as substrate, we have examined the kinetics of the epimerase reaction in some detail and have also, with the use of partially N-deacetylated/N-sulfated derivatives, determined the effect of N-acetylated glucosamine residues located in the vicinity of the glucuronic acid residues targeted by the enzyme. Furthermore, we have compared the kinetic properties of epimerase preparations from mouse mastocytoma and bovine liver with a view to exploring further the problem why connective-tissue type mast cells make heparin (Enerbäck, 1989
; Nader and Dietrich, 1989
) while all other cell types, with an apparently similar synthetic apparatus, produce the closely related but less extensively modified heparan sulfates (Gallagher and Walker, 1985
; Gallagher et al., 1986
; Kjellén and Lindahl, 1991
). An additional incentive for undertaking this comparison was the finding that multiple forms of the N-deacetylase/N-sulfotransferase occur in various tissues (Hashimoto et al., 1992
; Eriksson et al., 1994
; Orellana et al., 1994
), pointing to the possibility that similar polymorphism may apply to the epimerase so as to modulate the biosynthetic process toward the formation of either heparan sulfate or heparin.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Characterization of tritium-labeled E.coli K5 polysaccharide and its N-deacetylated/ N-sulfated derivatives
The polysaccharide isolated from a 45-ml culture incubated with 3 mCi of D-[5-3H]glucose (see Materials and methods) contained a total of 1.5 mg of HexA and had a specific radioactivity of 70 x 106 cpm/mg HexA. Upon analytical anion-exchange chromatography on DEAE-Sephacel of a sample of the labeled polysaccharide in mixture with the isomeric glycosaminoglycan, hyaluronan, both polysaccharides were eluted in essentially the same position (Figure 1A). As expected, the ~50% and ~100% N-deacetylated/N-sulfated derivatives were eluted in more retarded positions. Gel chromatography of the native K5 polysaccharide and the two derivatives on Superose 6 showed that the hydrazinolysis procedure had resulted in substantial depolymerization (Figure 1B). The peak elution positions of the rather broad peaks corresponded to species of ~57 kDa for the native K5 polysaccharide (~150 disaccharide units), ~30 kDa for the ~50% N-sulfated derivative (~75 disaccharide units), and ~16 kDa for the fully N-sulfated derivative (~40 disaccharide units). This effect of hydrazinolysis is in accord with previous observations (Shaklee and Conrad, 1984). Deaminative cleavage of the ~100% N-sulfated derivative at pH 1.5 yielded predominantly nonsulfated disaccharides, as indicated by the gel chromatography profile shown in Figure 2A. Small amounts of tetrasaccharides and larger oligosaccharides were also formed, suggesting that the N-deacetylation had not been complete. However, part of the tetra- and larger oligosaccharides were due to "anomalous" ring contraction during the deaminative cleavage, as indicated by repeated gel chromatography following mild acid treatment (data not shown) (Shively and Conrad, 1976b
). When this was taken into account, the proportion of N-sulfated residues in the modified K5 polysaccharide was calculated to be ~92%. The ~50% N-sulfated species also yielded disaccharides but, in addition, appreciable amounts of tetrasaccharides and larger oligosaccharides (Figure 2B). Calculations based on the proportions of disaccharides and larger oligosaccharides indicated that approximately 50% of the GlcN units were N-sulfated. However, in view of the incomplete separation of the larger oligosaccharides, this ratio could not be established with certainty. Elution patterns similar to those in Figure 2 were observed when the partially and essentially completely N-deacetylated (1.5 and 5 h hydrazinolysis, respectively) but not yet N-sulfated K5 samples were reacted with HNO2 at pH 3.9 (data not shown) (see Shively and Conrad, 1976a
).
|
|
|
Product formation as a function of time
Three enzyme preparations were used in the experiments reported here, i.e. pure bovine liver epimerase and partially purified preparations from bovine liver and the Furth mouse mastocytoma. With the fully N-deacetylated/N-sulfated as well as the 50% N-deacetylated/N-sulfated E.coli K5 polysaccharide as substrates, the reaction with all three enzyme preparations proceeded linearly with time for at least 30 min at all substrate concentrations used in the kinetic experiments described below (data not shown).
Km and Vmax values
Determination of the Km and Vmax values for the fully and partially N-sulfated K5 polysaccharide at three different concentrations of the pure liver epimerase gave the results summarised in Table I. Surprisingly, the values increased substantially with increasing enzyme concentration, ranging from 4 to 19 µM HexA for the fully N-sulfated K5 derivative and from 11 to 33 µM HexA for the partially N-sulfated K5 derivative (Table I; see also Figure 5). This increase was in general agreement with the results of a number of single
measurements, carried out earlier at different times and at different enzyme concentrations, which had given values ranging from 3.4 to 78 µM HexA for the fully N-sulfated K5 derivative, an almost 25-fold variation. In contrast to the Km measurements, the calculations of Vmax values gave the expected results and showed good proportionality to the enzyme concentration (Table I).
|
|
|
Effect of N-acetyl groups on kinetic parameters
An absolute requirement for substrate recognition by the epimerase is the presence of an N-sulfate group on the glucosamine residue linked to C4 of the glucuronic acid unit targeted by the enzyme (Jacobsson et al., 1984). In contrast, the glucosamine residue on the other side of the uronic acid may be either N-sulfated or N-acetylated. It has not been previously established, however, whether there is a quantitative difference in reactivity between uronic acid residues flanked by two N-sulfated glucosamines and those with an N-acetylglucosamine residue on the reducing terminal side. In the present study, this problem was approached by experiments of two types, i.e., competition studies with nonradioactive E.coli K5 polysaccharide derivatives of appropriate structure and direct determination of Km and Vmax values with [5-3H]GlcA-labeled K5 polysaccharide derivatives as substrates.
In the first type of experiments, reaction mixtures contained partially purified liver epimerase and O-desulfated [5-3H]HexA-labeled heparin as the substrate, and two nonradioactive K5 polysaccharide derivatives (one essentially completely N-deacetylated and N-sulfated, and the other containing 25% N-acetylated and 75% N-sulfated GlcN residues) were tested for their ability to compete with the labeled substrate. As shown in Figure 6, the presence of these polysaccharides in the reaction mixtures led to decreased formation of 3H2O from the 3H-labeled substrate. The fully N-sulfated derivative caused 50% inhibition at a concentration of ~0.1 µg per assay mixture (Figure 6A), while the partially N-acetylated polysaccharide was ~10-fold less efficient and yielded the same degree of inhibition only at a concentration of ~1.3 µg per assay mixture (Figure 6B). Thus, the results obtained are those expected if the presence of N-acetyl groups in the substrate polysaccharide weakens its interaction with the enzyme.
|
Comparison of O-desulfated [3H]heparin and metabolically labeled E.coli K5 polysaccharide derivatives as epimerase substrates
Determination of the kinetic parameters for the heparin-derived epimerase substrate showed that comparable values obtained at different enzyme concentrations were substantially higher than those of the K5 polysaccharide derivatives (Table I). This was true for all three enzyme concentrations used in these experiments, and, again, a significant increase in
values was observed with increasing enzyme concentration. Somewhat surprisingly, the Vmax values for the heparin-derived substrate were 2- to 4-fold higher (see Tables I, II) than those determined for the fully N-sulfated K5 polysaccharide derivative, although the latter may be considered to be more akin to the physiological epimerase substrates.
|
|
Kinetic studies on partially purified Furth mastocytoma and liver epimerase
One of the objectives of the present investigation was to compare the kinetic properties of epimerase preparations from the Furth mouse mastocytoma, which produces heparin, and bovine liver, which synthesizes heparan sulfate but not heparin. It was not practically feasible to prepare sufficient quantities of pure epimerase from the mastocytoma, and the comparison therefore had to be limited to partially purified materials. The results are shown in Figure 8 and Table II, from which it is apparent that the kinetic behavior of the mastocytoma enzyme vis-à-vis the three substrates used was similar to that of the liver enzyme. Thus, the fully N-sulfated K5 polysaccharide derivative exhibited the lowest value, while that of the 50% N-acetylated/N-sulfated derivative was more than twice as high and the heparin-derived substrate gave an intermediate value. Essentially similar Vmax values were observed for the two K5 polysaccharide substrates, while that of the O-desulfated [3H]heparin was about 2 to 3 times as high. The increase in
values with increasing enzyme concentration reported for the pure liver enzyme was observed also for the mastocytoma enzyme (data not shown) and the partially purified liver enzyme (see Table II). In view of this variation, it was not possible to make a valid comparison between the partially purified enzymes with respect to their true Km values, but it may be noted that the values for the mastocytoma enzyme fell within the same range as that observed for the liver enzyme (see Tables I and II).
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
A major but seemingly simple task in the present investigation was to determine the Km values for the N-deacetylated/N-sulfated derivatives of the E.coli K5 polysaccharide in the GlcA C5-epimerase reaction. This proved more difficult than we had anticipated, mainly because the experimentally determined values varied with the concentration of epimerase in the reaction mixtures. Since, however, the increase in apparent Km values with increasing enzyme concentration shown in Table I was close to linear (and perfectly linear in another experiment with a 1 h rather than a 30 min incubation time), it seemed reasonable to assume that a true Km value could be determined by extrapolation to zero enzyme concentration (see also legend to Figure 9). From the data in Table I, a Km of 23 µM was thus obtained for the fully N-sulfated derivative. If, instead of the uronic acid concentration, the concentration of polysaccharide molecules was used as the basis for the calculation of the Km values, the numbers were obviously substantially lower. For example, if the extrapolated value for the polysaccharide fraction with ~100 disaccharides per chain is taken to be ~2.5 mM HexA (see Figure 5A), then the value based on polysaccharide concentration is ~25 nM. This is an unusually low Km value and suggests (but does not prove) a high affinity of the enzyme for its substrate.
|
The use of the fully and partially N-deacetylated and subsequently N-sulfated derivatives of the E.coli K5 polysaccharide made it possible to examine the effect of N-acetylated glucosamine residues on the epimerase reaction. Experiments in which nonradioactive K5 polysaccharide derivatives were found to inhibit tritium release from O-desulfated [3H]heparin suggested that a fully N-sulfated polysaccharide is a better substrate than one in which GlcNAc residues are present in the vicinity of the target GlcA residues. This finding was confirmed by direct measurements of Km values using the [5-3H]GlcA-labeled substrates. Epimerase from either bovine liver or mouse mastocytoma thus consistently showed values for the fully N-sulfated substrate that were substantially lower than those for the 50% N-acetylated derivative (Tables I, II). It should be noted, however, that the
values were expressed as total concentration of GlcA in the substrate polysaccharides and that, in the 50% N-acetylated/N-sulfated derivative, not all GlcA residues serve as potential substrates for the enzyme. Previous studies demonstrated that a GlcA residue is susceptible to attack by the enzyme only if it has an N-sulfated GlcN residue on its nonreducing terminal side (Jacobsson et al., 1984
). It may therefore be argued that the calculation of Km values for the GlcNAc-containing derivative should be based only on the concentration of those GlcA residues that are actually substrates for the epimerase, which is 50% of the total GlcA concentration. Even when calculated on this basis, however, the
values for the 50% N-deacetylated/N-sulfated derivative determined in the experiments with the partially purified liver enzyme were close to three times as high as those observed for the fully N-sulfated derivative (Table II). In the experiments with the mastocytoma enzyme (Table II) and the pure liver enzyme (Table I), the difference in
values was less pronounced, but in all cases was the fully N-sulfated K5 polysaccharide a better substrate than the GlcNAc-containing derivative in terms of their
values. In all but one case (see Table I) this was also true even when the
values were calculated on the basis of the concentration of reactive uronic acid residues.
Comparison of the kinetic properties of the epimerase preparations from bovine liver and mouse mastocytoma tissue turned out to be more complicated than we had anticipated because of the unexpected finding that the values varied with the enzyme concentration. Thus, in seven experiments with each of the two enzyme preparations, with the fully N-sulfated K5 polysaccharide as the substrate, the
values ranged from 5.8 to 34.2 µM HexA (mastocytoma enzyme) and from 3.4 to 77.9 µM (partially purified liver enzyme) (data not shown). It is therefore apparent that a valid comparison would have to involve the use of pure enzyme from both sources, examined at the same concentrations, but it was not feasible in terms of cost and labor to produce sufficient quantities of mastocytoma tissue for such experiments. If we assume, however, that the kcat values for the liver and mastocytoma enzymes are the same, we may then compare the
values obtained in experiments where the Vmax values were the same for both enzymes. One such experiment, with the 50% N-sulfated K5 polysaccharide as the substrate, is seen in Table II, which shows that the
values for the two enzymes were almost identical (30 and 29 µM for the mastocytoma and the liver enzyme, respectively). Therefore, if the basic premise is correct, i.e., that the two enzymes have identical kcat values, we may then conclude that there is no significant difference between them with regard to their Km values.
A long-term goal of our investigations of the biosynthesis of heparin and heparan sulfate is to arrive at an understanding of the physiological biosynthetic process. The present study is a step in this direction, but it is important to remember that the polymer-modifying enzymes in the living cell reside in the Golgi apparatus and are membrane-bound. The kinetic parameters established for the soluble experimental systems used in this study therefore do not necessarily reflect the behavior of the biosynthetic apparatus in vivo. Furthermore, it has been proposed that the various enzymes involved in biosynthesis of heparin/heparan sulfate, including the epimerase, act in a processive fashion along the nascent polysaccharide chain that is still growing by addition of GlcA and GlcNAc units at the nonreducing terminus (Lidholt and Lindahl, 1992; Salmivirta et al., 1996
). Evidence for such processivity in the soluble experimental system is lacking, and we have assumed that the interaction between the enzymes and the polysaccharide substrates occurred in a more random fashion (see also Hannesson et al. (1996)
regarding epimerase action in dermatan sulfate biosynthesis). If, however, the enzyme does indeed act in a processive mode in free solution, processivity might be hampered by "excess" enzyme molecules in the reaction mixture, thus causing a rise in the
. Despite these uncertainties, some valuable information has emerged from the present work. In particular, a marked dependence of the
values on the concentration of enzyme protein in the reaction mixtures has been observed, which must be taken into account in future studies of the kinetic properties of the enzyme.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
GlcA C5-epimerase was purified from bovine liver as described (Campbell et al., 1994) and gave one major band on SDS-PAGE. The specific activity of the enzyme preparation was 4.4 x108 cpm 3H released/mg protein/30 min using [5-3H]HexA-labeled O-desulfated heparin as substrate. The partially purified C5-epimerase from bovine liver and mouse mastocytoma were prepared by affinity chromatography on O-desulfated heparin-Sepharose, Procion Red 120-Sepharose, and phenyl-Sepharose as described previously (Campbell et al., 1994
). The specific activities of the liver and mastocytoma enzyme preparations were 8 x 107 and 6 x 106 cpm 3H released/mg protein/30 min, respectively, as determined by assay with the heparin-derived substrate. Prior to use, the enzyme preparations were passed through PD-10 columns equilibrated with assay buffer.
Preparation of metabolically radiolabeled E.coli K5 polysaccharide
Escherichia coli K5 bacteria (20 µl of a stock suspension) were precultured at 37°C overnight in 2 ml of LB-medium [10 g/l tryptone (Difco), 5 g/l yeast extract (Difco), 10 g/l NaCl]. For preparative labeling, a 1 ml aliquot of the preculture was added to 45 ml of LB medium lacking glucose. After incubation for 70 min (OD600 nm ~0.25), 2 mCi of D-[5-3H]glucose was added along with unlabeled glucose to a final concentration of 0.01%, followed by an additional 1 mCi D-[5-3H]glucose after 2 h of further incubation. The culture was then maintained for a 24 h period and was then centrifuged at 3000 x g for 30 min. The supernatant was applied to a 2 ml column of DEAE-Sephacel in 50 mM LiCl, 50 mM sodium acetate, pH 4.0. The column was washed with the same buffer, and the polysaccharide was eluted with a 50 ml gradient of 0.051.5 M LiCl. Fractions containing labeled polysaccharide (near the middle of the gradient) were pooled and dialyzed against water. The 53H-labeled polysaccharide obtained had a specific activity of 70 x 106 cpm/mg HexA. A 14C-labeled analog (specific activity 18.5 x 106 cpm/mg HexA) was prepared in a similar fashion, but with D-[1-14C]glucose substituted for D-[5-3H]glucose.
Chemical modification of tritium-labeled K5 polysaccharide
For N-deacetylation of the 53H-labeled K5 polysaccharide, samples of 1015 x 106 cpm were transferred to screw-cap tubes, dried, and treated with 2 mg of hydrazine sulfate and 200 µl of hydrazine hydrate (~36% water) at 96°C. Based on pilot experiments with N-[3H]acetyl-labeled K5 polysaccharide, reaction times of 1.5 and 5 h were chosen to achieve ~50% and essentially complete N-deacetylation. The samples were then cooled, evaporated to dryness in a rotary evaporator, dissolved in water, desalted on PD-10 columns in 10% aqueous ethanol, and finally lyophilized. After lyophilization, the polysaccharides were treated with 0.25 M HIO3 to eliminate hydrazides (Shaklee and Conrad, 1984), again desalted on PD-10 columns, and lyophilized. N-Sulfation was carried out by treatment with 26 mg of trimethylamine·SO3 at pH 9.5 in 500 µl of water containing 22 mg NaHCO3 (Levy and Petracek, 1962
). The reaction mixtures were kept at 55°C under vigorous shaking for 24 h. The products were desalted by gel chromatography and lyophilized.
Enzyme assay
The assay of epimerase activity is based on the release of 3H (recovered as 3H2O) from a substrate polysaccharide of the appropriate structure, 3H-labeled at C5 of HexA units (Jacobsson et al., 1979a). Enzyme and substrate, in the amounts stated in the descriptions of the individual experiments, were incubated for 30 min at 37°C in 50 µl of 0.05 M Hepes, 0.015 M EDTA, 0.1 M KCl, 0.015% Triton X-100, pH 7.4. The reaction was stopped by addition of 50 µl of cold 0.05 M sodium acetate, 0.05 M LiCl, pH 4.0, and the reaction mixtures were transferred, along with a 100 µl rinse, to ~400 µl columns of DEAE-cellulose, equilibrated in the same buffer and placed over 6 ml polypropylene scintillation vials. The 3H2O was recovered with an additional 900 µl buffer rinse and quantified by liquid scintillation counting. The background derived from substrate with this method was <1%. Kinetic coefficients were evaluated from steady-state data by nonlinear least squares iterative fitting to a rectangular hyperbolic function by the Enzfitter program (Biosoft). Conversion of Vmax values from cpm to molar terms was done taking into account that only ~60% of the 3H was located at C5 of GlcA units in the K5 polysaccharide.
Analytical methods
The N-substituent patterns of the K5 polysaccharide derivatives were investigated by gel chromatography of degradation products generated by deaminative cleavage. Samples were treated with nitrous acid at pH 1.5, resulting in cleavage of the polysaccharide chains at N-sulfated GlcN units (GlcNAc units being resistant) and conversion of the susceptible units to 2,5-anhydro-D-mannose residues (Shively and Conrad, 1976a). The products were reduced with NaBH4, essentially as previously described (Pejler et al., 1987
), yielding terminal aManR units. They were then analyzed by gel filtration on a prototype of a Superdex 30 column (1.6 x 60 cm), designed for fast performance liquid chromatography (Pharmacia), eluted with 0.5 M NH4HCO3.
The HexA composition of the O-desulfated heparin was assessed by treatment with HNO2 at pH 1.5, and the resultant hexuronyl-anhydromannose disaccharides (~95% disaccharides, data not shown) were reduced with 0.5 mCi NaB3H4 (Maccarana et al., 1996) or with 50 µmol NaBH4 (Pejler et al., 1987
). The labeled HexA-[1-3H]aManR or HexA-[5-3H]aManR disaccharide products were isolated by gel chromatography on a column (1 x 200 cm) of Sephadex G-15 in 0.2 M NH4HCO3, and desalted by repeated lyophilization. The samples were analyzed by paper chromatography on Whatman no.1 paper in ethyl acetate/acetic acid/water (3:1:1 by volume), along with GlcA-[1-3H]aManR and IdoA-[1-3H]aManR standards (Jacobsson et al., 1979b
). After the separation, the paper strips were dried and cut into 1 cm pieces that were transferred to 6 ml scintillation vials. The samples were extracted in the vials with 1 ml of water for at least 15 min at room temperature and were then analyzed for radioactivity by scintillation counting following the addition of 4 ml of scintillation fluid. The relative proportions of GlcA and IdoA were calculated from the corresponding disaccharide peak areas.
Polysaccharides were quantified by the carbazole reaction for HexA (Bitter and Muir, 1962). Protein was determined by the method of Bradford (Bradford, 1976
). Radioactivity was measured in a Beckman model LS 6000 liquid scintillation counter using Optiphase HiSafe scintillation fluid.
![]() |
Acknowledgments |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Abbreviations |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Footnotes |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Bradford,M.M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem., 72, 248254.[ISI][Medline]
Campbell,P., Feingold,D.S., Jensen,J.W., Malmström,A. and Rodén,L. (1983) New assay for uronosyl 5-epimerases. Anal. Biochem., 131, 146152.[ISI][Medline]
Campbell,P., Hannesson,H.H., Sandbäck,D., Rodén,L., Lindahl,U. and Li,J.-P. (1994) Biosynthesis of heparin/heparan sulfate. Purification of the D-glucuronyl C-5 epimerase from bovine liver. J. Biol. Chem., 269, 2695326958.
Casu,B., Grazioli,G., Razi,N., Guerrini,M., Naggi,A., Torri,G., Oreste,P., Tursi,F., Zopetti,G. and Lindahl,U. (1994). Heparin-like compounds prepared by chemical modification of capsular polysaccharide from E.coli K5. Carbohydr. Res., 263, 271278.[ISI][Medline]
Cleland,R.L. (1983) Viscometric study of the proteoglycan-hyaluronate (2:1) "dimer": minimum hyaluronate chain length. Biopolymers, 22, 25012506.[ISI][Medline]
Enerbäck,L. (1989) The mast cell system. In Lane,D.A. and Lindahl,U. (eds.), Heparin: Chemical and Biological Properties, Clinical Applications. Edward Arnold, London, pp. 97113.
Eriksson,I., Sandbäck,D., Ek,B., Lindahl,U. and Kjellén,L. (1994) cDNA cloning and sequencing of mouse mastocytoma glucosaminyl N-deacetylase/N-sulfotransferase, an enzyme involved in the biosynthesis of heparin. J. Biol. Chem., 269, 1043810443.
Furth,J., Hagen,P. and Hirsch,E.I. (1957) Transplantable mastocytoma in the mouse containing histamine, heparin, 5-hydroxytryptamine. Proc. Soc. Exp. Biol. Med., 95, 824828.
Gallagher,J.T., Lyon,M. and Steward,W.P. (1986) Structure and function of heparan sulphate proteoglycans. Biochem. J., 236, 313325.[ISI][Medline]
Gallagher,J.T. and Walker,A. (1985) Molecular distinctions between heparan sulphate and heparin. Analysis of sulphation patterns indicates that heparan sulphate and heparin are separate families of N-sulphated polysaccharides. Biochem. J., 230, 665674.[ISI][Medline]
Hannesson,H.H., Hagner-McWhirter,Å., Tiedemann,K., Lindahl,U. and Malmström,A. (1996) Biosynthesis of dermatan sulphate. Defructosylated Escherichia coli K4 capsular polysaccharide as a substrate for the D-glucuronyl C-5 epimerase and an indication of a two-base reaction mechanism. Biochem. J., 313, 589596.[ISI][Medline]
Hashimoto,Y., Orellana,A., Gil,G. and Hirschberg,C.B. (1992) Molecular cloning and expression of rat liver N-heparan sulfate sulfotransferase. J. Biol. Chem., 267, 1574415750.
Jacobsson,I., Bäckström,G., Höök,M., Lindahl,U., Feingold,D.S., Malmström,A. and Rodén,L. (1979a) Biosynthesis of heparin. Assay and properties of the microsomal uronosyl C-5 epimerase. J. Biol. Chem., 254, 29752982.[ISI][Medline]
Jacobsson,I., Höök,M., Pettersson,I., Lindahl,L., Larm,O., Wirén,E. and von Figura,K. (1979b) Identification of N-sulfated disaccharide units in heparin-like polysaccharides. Biochem. J., 179, 7787.[ISI][Medline]
Jacobsson,I., Lindahl,U., Jensen,J.W., Rodén,L., Prihar,H. and Feingold,D.S. (1984) Biosynthesis of heparin. Substrate specificity of heparosan N-sulfate D-glucuronosyl 5-epimerase. J. Biol. Chem., 259, 10561063.
Jensen,J.W., Rodén,L., Jacobsson,I., Lindahl,U., Prihar,H. and Feingold,D.S. (1983) Biosynthesis of heparin. A new substrate for heparosan-N-sulfate-D-glucopyranosyl-uronate 5-epimerase. Carbohydr. Res., 117, 241253.[ISI]
Kjellén,L. and Lindahl,U. (1991) Proteoglycans: Structures and interactions. Annu. Rev. Biochem., 60, 44375.[ISI][Medline]
Levy,L. and Petracek,F.J. (1962) Chemical and pharmacological studies on N-resulfated heparin. Proc. Soc. Exp. Biol. Med., 190, 901905.
Li,J.-p., Hagner-McWhirter,Å., Kjéllen,L., Palgi,J., Jalkanen,M. and Lindahl,U. (1997) Biosynthesis of Heparin/Heparan Sulfate. cDNA cloning and expressing of D-glucuronosyl C5-epimerase from bovine lung. J. Biol. Chem., 272, 2815828163.
Lidholt,K. and Lindahl,U. (1992) Biosynthesis of heparin. The D-glucuronosyl- and N-acetyl-D-glucosaminyltransferase reactions and their relation to polymer modification. Biochem. J., 287, 2129.[ISI][Medline]
Lindahl,U. (1989) Biosynthesis of heparin and related polysaccharides. In Lane,D.A. and Lindahl,U. (eds.), Heparin: Chemical and Biological Properties, Clinical Applications. Edward Arnold, London, pp. 159189.
Lindahl,U., Jacobsson,I., Höök,M., Bäckström,G. and Feingold,D.S. (1976) Biosynthesis of heparin. Loss of C-5 hydrogen during conversion of D-glucuronic to IdoA residues. Biochem. Biophys. Res. Commun., 70, 492499.[ISI][Medline]
Linhardt,R.J., Turnbull,J.E., Wang,H.M., Loganathan,D. and Gallagher,J.T. (1990) Examination of the substrate specificity of heparin and heparan sulfate lyases. Biochemistry, 29, 26112617.[ISI][Medline]
Maccarana,M., Sakura,Y., Tawada,A., Yoshida,K. and Lindahl,U. (1996) Domain structure of heparan sulfates from bovine organs. J. Biol. Chem., 271, 1780417810.
Nader,H.B. and Dietrich,C.P. (1989) Natural occurrence and possible biological role of heparin. In Lane,D.A. and Lindahl,U. (eds.), Heparin: Chemical and Biological Properties, Clinical Applications. Edward Arnold, London, pp. 8196.
Orellana,A., Hirschberg,C.B., Wei,Z., Swiedler,S.J. and Ishihara,M. (1994) Molecular cloning and expression of a glycosaminoglycan N-acetylglucosaminyl N-deacetylase/N-sulfotransferase from a heparin-producing cell line. J. Biol. Chem., 269, 22702276.
Pejler,G., Bäckström,G., Lindahl,U., Paulsson,M., Dziadek,M., Fujiwara,S. and Timpl,R. (1987) Structure and affinity for antithrombin of heparan sulfate chains derived from basement membrane proteoglycans. J. Biol. Chem., 262, 50365043.
Prihar,H.S., Campbell,P., Feingold,D.S., Jacobsson,I., Jensen,J.W., Lindahl,U. and Rodén,L. (1980) Biosynthesis of heparin. Hydrogen exchange at carbon 5 of the glycuronosyl residues. Biochemistry, 19, 495500.[ISI][Medline]
Salmivirta,M., Lidholt,K. and Lindahl,U. (1996) Heparan sulfatea piece of information. FASEB J., 10, 12701279.
Shaklee,P.N. and Conrad,H.E. (1984) Hydrazinolysis of heparin and other glycosaminoglycans. Biochem. J., 217, 187197.[ISI][Medline]
Shively,J.E. and Conrad,H.E. (1976a) Formation of anhydrosugars in the chemical depolymerization of heparin. Biochemistry, 15, 39323942.[ISI][Medline]
Shively,J.E. and Conrad,H.E. (1976b) Nearest neighbor analysis of heparin: identification and quantitation of the products formed by selective depolymerization procedures. Biochemistry, 15, 39433950.[ISI][Medline]
Vann,W.F., Schmidt,M.A., Jann,B. and Jann,K. (1981) The structure of the capsular polysaccharide (K5 antigen) of urinary-tract-infective Escherichia coli 010:K5:H4. A polymer similar to desulfo-heparin. Eur. J. Biochem., 116, 359364.[Abstract]