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PubMed1976

UDP-glucuronyltransferase in perfused rat liver and in microsomes. Glucuronidation of bilirubin.

In perfused rat liver and in fistula rats the formation of bilirubin conjugates was studied after labeling with [14C]bilirubin,5-amino [14C]levulinic acid and [14C]hemin. The latter two compounds were used to study heme degradation to bilirubin from intrahepatic and extrahepatic sources, respectively. Bilirubin glucuronides were the major conjugates in fistula bile. In liver perfusion bile the proportion of non-glucuronide conjugates was increased. After a high dose of hemin (2.5 mumol) bilirubin glucuronides were decreased compared with other bilirubin conjugates both in fistula bile and in liver perfusion bile. In addition green pigments were formed. These alterations were reversed in chronically hemin-treated rats in which heme oxygenase had been induced. The interference of UDP-glucose and UDP-glucuronic acid with bilirubin glucuronidation and glucosidation was studied in liver microsomes. UDP-glucose did not affect bilirubin glucuronidation in native microsomes in which UDP-glucuronyltransferase activity is constrained. When this constraint was released by various treatments altering membrane structure UDP-glucose markedly inhibited bilirubin glucuronidation. However, under these conditions bilirubin glucosidation was unaffected by UDP-glucuronic acid. The results suggest that the release of the constraint of UDP-glucuronyltransferase in vivo may lead to a decrease of the proportion of bilirubin glucuronides to other bilirubin conjugates in bile.

Aminolevulinic Acid↗
PubMed1990

Unique role of oxygen in regulation of hepatic monooxygenation and glucuronidation.

The purpose of this study was to evaluate the hypothesis that NADPH supply in intact cells is regulated by oxygen tension. This was accomplished by studying monooxygenation in perfused livers from Ah locus-responsive C57BL/6J mice, where rates of monooxygenation are high. Elevation of flow rate decreases the hepatic O2 gradient and increases O2 delivery to the organ. Under these conditions, rates of p-nitroanisole O-demethylation were 2-3 times higher in perfused livers from fed or fasted mice at high (10 ml/min) compared with normal (5 ml/min) flow rates. Rates of monooxygenation were directly proportional to oxygen tension (half-maximal rates occurred with approximately 400 microM O2). On the other hand, rates were independent of oxygen concentration in isolated microsomes where NADPH was supplied in excess. The decrease in rate due to diminished O2 concentration in the intact organ could not be attributed to hypoxia, because O2 tension in the effluent perfusate exceeded 50 microM even when influent perfusate was saturated with 25% O2 and ATP/ADP ratios were in the normal range. Thus, monooxygenation of p-nitroanisole in perfused mouse liver is dependent on oxygen tension. Similarly, glucuronidation of p-nitrophenol was oxygen dependent in the intact organ but not in isolated microsomes supplemented with UDP-glucuronic acid. Taken together, these data support the hypothesis that, at high oxygen tensions (e.g., in periportal regions of the liver lobule), mitochondrial activity is increased, which in turn enhances NADPH and UDP-glucuronic acid turnover, leading to accelerated rates of monooxygenation and glucuronidation in intact cells. In support of this idea, NH4Cl, which utilizes NADPH for urea synthesis, inhibited monooxygenation in the perfused mouse liver at high but not low flow rates. Thus, important phase I and II detoxification reactions are regulated indirectly by the hepatic oxygen gradient, via mechanisms involving cofactor supply, when cytochrome P-450 is not limiting.

Animals↗
PubMed1984

The structure of the polysaccharide moiety of Rhodopseudomonas sphaeroides ATCC 17023 lipopolysaccharide.

The chemical structure of the polysaccharide moiety of the lipopolysaccharide Rhodopseudomonas sphaeroides ATCC 17023 was established. Mild acetic acid hydrolysis of isolated lipopolysaccharide, followed by preparative high-voltage paper electrophoresis afforded three oligosaccharides. They were characterized by chemical and physicochemical studies to be: GlcA(alpha 1----4)dOclA8P, Thr(6') GlcA(alpha 1----4)GlcA and GlcA(alpha 1----4)dOclA, where GlcA is D-glucuronic acid and dOc1A is 3-deoxy-D-manno-octulosonic acid. Carboxyl-reduction of the lipopolysaccharide followed by acid hydrolysis gave a trisaccharide: GlcA(alpha 1----4)Glc(alpha 1----4)Glc, showing the presence of three residues of glucuronic acids in the O-specific chain and indicating that only two of them are reducible by NaBH4. The linkage between the polysaccharide and lipid A was shown to be through a single 1,4-linked residue of dOc1A attached by a 2,6'-linkage to the lipid A moiety.

Chemical Phenomena↗
PubMed1992

Isolation, characterization and properties of the oversulphated chondroitin sulphate proteoglycan from squid skin with peculiar glycosaminoglycan sulphation pattern.

Oversulphated chondroitin sulphate proteoglycan from squid skin was isolated from 4 M guanidine hydrochloride extract by ion-exchange chromatography, gel chromatography and density gradient centrifugation. The proteoglycan had Mr 3.5 x 10(5), contained on average six oversulphated chondroitin sulphate chains (Mr 4 x 10(4)) bound on a polypeptide of Mr 2.8 x 10(4), and oligosaccharides consisting of both hexosamines, glucuronic acid, sulphates and fucose as the only neutral monosaccharide. The major amino acids of the proteoglycan protein core are glycine (corresponding to about one third of the total amino acids), aspartic acid/asparagine and serine, together amounting to 50% of the total. The proteoglycan was resistant to the proteolytic enzymes V8 protease, trypsin (treated with diphenylcarbamoyl chloride), alpha-chymotrypsin and pronase, while it was completely degraded by papain and to a large extent by collagenase. Pretreated proteoglycan with chondroitinase AC was degraded by pronase to a large extent and slightly by V8 protease and trypsin. The proteoglycan did not interact with hyaluronic acid and did not form self-aggregates. Oversulphated chondroitin sulphate chains were composed of unusual sulphated disaccharide units which were isolated and characterized by HPLC. In particular, it contained 2-acetamido-2-deoxy-3-O-(alpha-L-threo-4-enopyranosyluronic acid)-D-galactose 4-sulphate (delta di-4S) and disulphated disaccharides (delta di-diS) [90% 2-acetamido-2-deoxy-3-O-(alpha-L-threo-4-enopyranosyluronic acid 2/3-sulphate)-D-galactose 6-sulphate (delta di-diSD) and 10% 2-acetamido-2-deoxy-3-O-(alpha-L-threo-4-enopyranosyluronic acid 2/3-sulphate)-D-galactose 4-sulphate (delta di-diSK)] as the major disaccharides, significant amounts of trisulphated disaccharides (delta di-triS) and small amounts of 2-acetamido-2-deoxy-3-O-(alpha-L-threo-4-enopyranosyluronic acid)-D-galactose 6-sulphate (delta di-6S) and 2-acetamido-2-deoxy-3-O-(alpha-L-threo-4-enopyranosyluronic acid)-D-galactose (delta di-OS). Trisulphated disaccharides contained sulphate groups at C-4 and C-6 of the galactosamine and at C-2 or C-3 of the glucuronic acid. By HPLC analysis of a pure preparation of oversulphated chondroitin sulphate, it was found that it contains glucose, galactose, mannose and fucose most likely as branches.

Amino Acids↗
PubMed1987

Reaction of unsaturated uronic acid residues with mercuric salts. Cleavage of the hyaluronic acid disaccharide 2-acetamido-2-deoxy-3-O-(beta-D-gluco-4-enepyranosyluronic acid)-D-glucose.

Degradation of connective-tissue polysaccharides with bacterial or fungal eliminases and subsequent characterization of the reaction products are now part of standard methodology for the analysis of these compounds. However, the scope of preparative and analytical work based on the use of eliminases has been limited by the lack of procedures for specific removal of the unsaturated uronic acid residues generated in the eliminase reactions. In the present investigation, we have shown that these residues are cleaved by mercuric salts under mild conditions that are not likely to affect other structures in an oligo- or poly-saccharide molecule. Thus the disaccharide generated from hyaluronic acid by digestion with chondroitinase AC or ABC was cleaved into a keto acid and free N-acetylglucosamine within 10 min at room temperature upon exposure to 14 mM-mercuric acetate at pH 5. The reaction of the disaccharide with mercuric salts was used for ready determination of the distribution of radioactivity between the glucuronic acid and N-acetylglucosamine moieties in radioactive hyaluronic acid that had been synthesized by IMR-90 fibroblasts from 3H-labelled monosaccharides. When the precursor was [3H]galactose, over 95% of the incorporated radioactivity was found in the glucuronic acid moiety. In contrast, cells grown in the presence of [3H]glucosamine synthesized a polysaccharide in which almost all of the label was located in the N-acetylglucosamine units. It is apparent from these experiments that the reaction of unsaturated uronic acid residues with mercuric salts provides a new tool with potential for many applications in the study of the structure and metabolism of connective-tissue polysaccharides.

Cell Line↗
PubMed1982

Biosynthesis of polysaccharides in Acetobacter xylinum. Sequential synthesis of a heptasaccharide diphosphate prenol.

The sequential synthesis in vitro of a heptasaccharide diphosphate prenol, containing glucose, mannose, glucuronic acid and rhamnose in the ratio 4:1:1:1 is described. The enzyme preparation consisted of EDTA-treated Acetobacter xylinum cells and UDP-glucose, GDP-mannose, UDP-glucuronic acid and TDP-rhamnose were employed as sugar donors. The compounds soluble in chloroform/methanol/water (1:2:0.3) formed from incubations carried out under different conditions in the presence of a variety of combinations of the donors labeled with 14C, 3H or 32P were analysed by DEAE-cellulose column chromatography, gel filtration, partial acid hydrolysis, acetolysis, periodate oxidation, etc. The following structure is proposed for the most complex compound characterized: rhamnosyl-(1 leads to 6)-beta-glucosyl-(1 leads to 6)-alpha-glucosyl-(1 leads to 4)-beta-glucuronyl-(1 leads to 6)-beta-mannosyl-(1 leads to 3)-beta-glucosyl-(1 leads to 4)-alpha-glucosyl diphosphate prenol. The smaller oligosaccharide diphosphate prenols formed as intermediate steps are also characterized in this or in previous work [Garcia, R. C., Recondo, E. and Dankert, M. A. (1974) Eur. J. Biochem. 43, 93-105; Couso, R. O., Ielpi, L., and Dankert, M. A. (1980) Arch. Biochem. Biophys. 204, 434-443]. The role of these compounds in the biosynthesis of a complex exopolysaccharide that this microorganism forms in addition to cellulose is discussed.

Chemical Phenomena↗
PubMed1990

Purification and substrate specificity of heparitinase I and heparitinase II from Flavobacterium heparinum. Analyses of the heparin and heparan sulfate degradation products by 13C NMR spectroscopy.

The purification of two heparitinases and a heparinase, in high yields from Flavobacterium heparinum was achieved by a combination of molecular sieving and cation-exchange chromatography. Heparinase acts upon N-sulfated glucosaminido-L-iduronic acid linkages of heparin. Substitution of N-sulfate by N-acetyl groups renders the heparin molecule resistant to degradation by the enzyme. Heparitinase I acts on N-acetylated or N-sulfated glucosaminido-glucuronic acid linkages of the heparan sulfate. Sulfate groups at the 6-position of the glucosamine moiety of the heparan sulfate chains seem to be impeditive for heparitinase I action. Heparitinase II acts upon heparan sulfate producing disulfated, N-sulfated and N-acetylated-6-sulfated disaccharides, and small amounts of N-acetylated disaccharide. These and other results suggest that heparitinase II acts preferentially upon N,6-sulfated glucosaminido-glucuronic acid linkages. The total degradation of heparan sulfate is only achieved by the combined action of both heparitinases. The 13C NMR spectra of the disaccharides formed from heparan sulfate and a heparin oligosaccharide formed by the action of the heparitinases are in accordance to the proposed mode of action of the enzymes. Comparative studies of the enzymes with the commercially available heparinase and heparitinase are described.

Carbohydrate Sequence↗
PubMed2006

Deconjugation of N-glucuronide conjugated metabolites with hydrazine hydrate--biomarkers for exposure to the food-borne carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP).

The metabolism of the carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) has been investigated in rabbit liver S9. Two phase I metabolites, N(2)-OH-PhIP and 4'-OH-PhIP were identified based on UV and mass spectra and co-elution with reference standards. Fortification of the incubation with UDGPA resulted in a complete glucuronidation of PhIP and N(2)-OH-PhIP, while 4'-OH-PhIP was only partly glucuronidated. Also, the PhIP metabolite 5-OH-PhIP was completely glucuronidated by rabbit liver S9, while 5-OH-PhIP was a poor substrate for CYP mediated hydroxylation. The glucuronic acid conjugates of PhIP metabolites were unsusceptible to treatment with beta-glucuronidase indicating that these are N-glucuronides. Treatment of the conjugates with hydrazine hydrate, however, resulted in complete hydrolysis of the glucuronic acid conjugates as well as in reduction to the parent amine of metabolites hydroxylated in the exocyclic amino group. Urine was collected from a male volunteer following consumption of fried chicken. Treatment of the urine with beta-glucuronidase/sulfatase resulted in release of 4'-OH-PhIP, while treatment with hydrazine hydrate in addition resulted in release of substantial amounts of PhIP and 5-OH-PhIP. The data show that hydrazine hydrate can hydrolyse N-glucuronides of metabolites of PhIP, glucuronides that are unsusceptible to enzymatic hydrolysis. In addition the data indicate that humans metabolise a large fraction of ingested PhIP to genotoxic metabolites. The chemical hydrolysis of glucuronide conjugates of PhIP metabolites with hydrazine hydrate observed in this study may also be a useful approach in the development of biomarkers for exposure and effect of other xenobiotics.

Animals↗
PubMed1983

2,3-diamino-2,3-dideoxy-D-glucofuranurono-6,3-lactam from the hydrolysate of Pseudomonas aeruginosa P14 lipopolysaccharide.

An unknown amino sugar, U-7, which had been detected in the hydrolysate of the polysaccharide fraction (F-A) of Pseudomonas aeruginosa P14 lipopolysaccharide, was isolated from the hydrolysate of whole cells of this micro-organism and converted into the N-acetyl derivative (U-7NAc). On the basis of i.r.-absorption spectrometry, 13C-n.m.r. and 1H-n.m.r. spectroscopy and mass spectrometry, the structure of compound U-7NAc was identified as 2-acetamido-3-amino-2,3-dideoxyhexofuranurono-6,3-lactam. The configuration of compound U-7NAc was then unequivocally identified as 2-acetamido-3-amino-2,3-dideoxy-D-glucofuranurono-6,3-lactam by comparing the synthetic and natural compounds. Compound U-7 and synthetic 2,3-diamino-2,3-dideoxy-D-glucofuranurono-6,3-lactam showed the same behaviour on chromatography. G.l.c.--mass-spectral analyses of fraction F-A and synthetic 2,3-diacetamido-2,3-dideoxy-D-glucuronic acid after methanolyses and trimethylsilylations showed the presence of the same derivative. It was concluded that the amino sugar U-7 was produced from the 2,3-diacetamido-2,3-dideoxy-D-glucuronic acid residue present in fraction F-A.

Chemical Phenomena↗
PubMed1999

Variability of human hepatic UDP-glucuronosyltransferase activity.

The availability of a unique series of liver samples from human subjects, both control patients (9) and those with liver disease (6; biliary atresia (2), retransplant, chronic tyrosinemia type I, tyrosinemia, Wilson's disease) allowed us to characterize human hepatic UDP-glucuronosyltransferases using photoaffinity labeling, immunoblotting and enzymatic assays. There was wide inter-individual variation in photoincorporation of the photoaffinity analogs, [32P]5-azido-UDP-glucuronic acid and [32P]5-azido-UDP-glucose and enzymatic glucuronidation of substrates specific to the two subfamilies of UDP-glucuronosyltransferases. However, the largest differences were between subjects with liver disease. Glucuronidation activities toward one substrate from each of the UDP-glucuronosyltransferases subfamilies, 1A and 2B, for control and liver disease, respectively, were 1.7-4.5 vs 0.4-4.7 nmol/mg x min for hyodeoxycholic acid (2B substrate) and 9.2-27.9 vs 8.1-75 nmol/mg x min for pchloro-m-xylenol (1A substrate). Microsomes from a patient with chronic tyrosinemia (HL32) photoincorporated [32P]5-azido-UDP-glucuronic acid at a level 1.5 times higher than the other samples, was intensely photolabeled by [32P]5-azido-UDP-glucose and had significantly higher enzymatic activity toward p-chloro-m-xylenol. Immunoblot analysis using anti-UDP-glucuronosyltransferase antibodies demonstrated wide inter-individual variations in UDP-glucuronosyltransferase protein with increased UDP-glucuronosyltransferase protein in HL32 microsomes, corresponding to one of the bands photolabeled by both probes. Detailed investigation of substrate specificity, using substrates representative of both the 1A (bilirubin, 4-nitrophenol) and 2B (androsterone, testosterone) families was carried out with HL32, HL38 (age and sex matched control) and HL18 (older control). Strikingly increased (5-8-fold) glucuronidation activity was seen in comparison to HL18 only with the phenolic substrates. The results indicate that one or more phenol-specific UDP-glucuronosyltransferase 1A isoforms are expressed at above normal levels in this tyrosinemic subject.

Adolescent↗
PubMed1995

Gluconeogenesis and glucuronidation in liver in vivo and the heterogeneity of hepatocyte function.

In order to examine metabolic zonation in human liver, [2-14C]glycerol, which labels carbons 2 and 5 of glucose-6-P, and [1-14C]lactate, which labels carbons 3 and 4 of glucose-6-P, in the process of gluconeogenesis, were infused intravenously into healthy subjects who ingested acetaminophen and had fasted 36 h. Distributions of 14C were determined in glucose in blood and in the glucuronic acid moiety of acetaminophen glucuronide excreted in urine. Ratios of 14C in carbons 2 and 5 to 14C in carbons 3 and 4 were significantly higher in blood glucose than in glucuronide. Since glucose and glucuronic acid are formed from glucose-6-P in liver without randomization of carbon, the differences in the ratios indicate that the pool of glucose-6-P in liver is not homogeneous. The glucuronide sampled glucose-6-P with more label from lactate than glycerol compared to the glucose-6-P sampled by the glucose. The apparent explanation is the greater decrease in glycerol compared with lactate concentration as blood streams from the periportal to the perivenous zones of the liver lobule. Glucuronidation is then expressed in humans relatively more in the perivenous than periportal zones and gluconeogenesis from glycerol more in the periportal than perivenous zones.

Acetaminophen↗
PubMed1990

Pulsed amperometric detection of carbohydrates in lysosomal storage disease fibroblasts: a new screening technique for carbohydrate storage diseases.

A first step in determining the metabolic defect in patients with an unknown storage disease is to identify the stored material. In the case of fibroblasts storing carbohydrates, this can be accomplished by trifluoroacetic acid (TFA) hydrolysis producing monosaccharides which are separated by anion-exchange chromatography and quantitated by pulsed amperometric detection. This technique separates neutral, amino, and acidic monosaccharides in a single run with a detection limit of 50 pmol. The method, applied to hydrolyzed 100,000 g supernatants of ten normal fibroblast sonicates, revealed a mean +/- S.D. content of the following monosaccharides (in nmol/mg of protein): fucose, 7 +/- 3; galactosamine, 4 +/- 2; glucosamine, 20 +/- 3; galactose, 11 +/- 3; mannose, 27 +/- 6; glucuronic acid, 56 +/- 28; iduronic acid, 17 +/- 11. Six mucopolysaccharidosis fibroblast strains (types I, II, IIIB, IVA, VI and VII) contained 2 to 8 times the normal glucuronic acid levels, and types I and II exhibited 10- to 30-fold normal levels of iduronic acid and 40-fold increases in galactosamine. All the mucopolysaccharidoses could be distinguished from normal based upon an increased concentration of some monosaccharide. Fibroblasts from patients with mannosidosis and fucosidosis contained 7-fold normal amounts of mannose and 11-fold normal amounts of fucose, respectively. The quantitation of monosaccharides in fibroblasts after TFA hydrolysis can identify cells that store excess amounts of a glycosaminoglycan, glycoprotein, oligosaccharide or, presumably, a glycolipid. This may comprise the first step toward identifying novel lysosomal storage disorders and point the way toward new glycoconjugate degradative pathways.

Carbohydrate Metabolism↗
PubMed1980

The synthesis of bile acid glucuronides by agarose-bound UDP-glucuronyltransferase.

Lithocholic acid, chenodeoxycholic acid and deoxycholic acid but not cholic acid or the glycine or taurine conjugates of these bile acids are glucuronidated by agarose-bound UDPglucuronyltransferase from rt liver in the presence of UDPglucuronic acid. The identification of bile acid glucuronides is based on the specific enzymatic hydrolysis, incorporation of 14C-labeled glucuronic acid into the molecule and by mass spectrometry. Mass spectrometry indicates that bile acid glucuronides formed by agarose-bound UDPglucuronyltransferase are 3-glucuronides. The glucuronidation of lithocholic acid, chenodeoxycholic acid and deoxycholic acid is linear up to 45 min and at a protein concentration between 0.5 and 2.0 mg per assay. The pH optimum of the enzyme reaction is 7.0. The Km for lithocholic acid, chenodeoxycholic acid and deoxycholic acid, which are 0.040, 0.031 and 0.037 mM, respectively, are similar to those found in microsomal preparations. The use of agarose-bound UDP-glucuronyltransferase offers the possibility to synthesize bile acid glucuronides which are needed for kinetic studies in man and animal.

Animals↗
PubMed1968

Glucuronidation of 3-O-methylnoradrenaline, harmalol and some related compounds.

1. The following compounds were glucuronidated in the presence of UDP-glucuronic acid and a microsomal preparation made from guinea-pig liver: (14)C-labelled 3-O-methyladrenaline, 3-O-methylnoradrenaline, 3-methoxytyramine and 4-hydroxy-3-methoxyphenethanol, as well as unlabelled harmalol and harmol. 2. [(14)C]Homovanillic (4-hydroxy-3-methoxyphenylacetic) acid was not a substrate for the microsomal glucuronyltransferase. 3. The K(m) values for harmalol and harmol were 0.69x10(-4)m and 0.50x10(-4)m respectively. 4. The K(m) values for UDP-glucuronic acid, in the presence of (14)C-labelled 3-O-methylnoradrenaline, harmalol and harmol as aglycones, were 0.57x10(-4)m, 0.44x10(-4)m and 2.20x10(-4)m respectively. 5. Mg(2+) added at 2.5-10mm activated glucuronyltransferase, with harmalol as substrate. Concentrations above 10mm inhibited the enzymic activity. 6. The overall, or net, transglucuronidating activity of microsomal preparations of the liver, with harmalol as substrate, was greatest for guinea pig, and very much lower for rabbit, mouse and rat.

Animals↗
PubMedDate not supplied

Glycoside conjugation in microsomes from hepatic and renal carcinoma of man.

Human hepatoma which had been xenografted into nude mice have been estimated for their ability to catalyze glucuronic acid and glucose conjugation of endogenous compounds and p-nitrophenol. The xenobiotic p-nitrophenol was glucuronidated with a comparable rate in microsomes from human hepatoma, human liver and host liver. With regard to glucuronic acid or glucose conjugation of the endogenous compounds of bile acids, bilirubin and steroid hormones, glucosidation of bile acids was the only conjugation mechanism that was not decreased or deficient in microsomes from hepatoma, but showed about a 2-fold increase in reaction rate compared to normal human liver. Human hepatoma and host liver were shown to respond to phenobarbital treatment which led to about a 2-fold increase in UDP-glucuronosyltransferase activity toward bilirubin in hepatoma and in host liver. Compared to normal tissues, alterations in the pattern of glycoside conjugating enzymes were not only observed in microsomes from human hepatoma, but also in microsomes from human adenocarcinoma of the kidney, exhibiting negligible UDP-glucuronosyltransferase activities toward bile acids and steroid hormones. Bile acid glucoside formation was measurable in kidney adenocarcinoma with an activity which was similar to the activity observed in hepatoma. In comparison to normal renal tissue, glucose-conjugating activity toward bile acids decreased about 2-fold in kidney adenocarcinoma.

Adenocarcinoma↗
PubMed1983

Structure-activity relationship in heparin: a synthetic pentasaccharide with high affinity for antithrombin III and eliciting high anti-factor Xa activity.

The structures of the tetrasaccharide (beta-D-glucuronic acid)1 leads to 4 (N-sulfate-3,6-di-0-sulfate-alpha-D-glucosamine)1 leads to 4(2-0-sulfate-alpha-L-iduronic acid)1 leads to 4(N-sulfate-6-0-sulfate-D-glucosamine) and of the pentasaccharide (N-sulfate-6-0-sulfate-alpha-D-glucosamine)1 leads to 4(beta-D-glucuronic acid)1 leads to 4(N-sulfate-3,6-di-0-sulfate-alpha-D-glucosamine)1 leads to 4(2-0-sulfate-alpha-L-iduronic acid)1 leads to 4(N-sulfate-6-0-sulfate-D-glucosamine), both prepared for the first time, by chemical synthesis from D-glucose and D-glucosamine, have been confirmed by nuclear magnetic resonance. The synthetic tetrasaccharide neither binds to AT-III nor induces anti-factor Xa activity enhancement of this inhibitor. In contrast, the synthetic pentasaccharide strongly binds to AT-III (Ka: 7.10(6)M-1) forming an equimolar complex and also enhances the AT-III inhibitory activity towards factor Xa. These results confirm that the synthetic pentasaccharide with the above structure corresponds to the actual minimal sequence required in heparin for binding to AT-III.

Antithrombin III↗
PubMed1975

Biosynthesis of glycosaminoglycans: uridine diphosphate glucose 4'-epimerase from cornea and epiphysial-plate cartilage.

UDP-glucose 4'-epimerase (EC 5.1.3.2.) was extracted from newborn-pig epiphysial-plate cartilage and whole bovine cornea. The formation of radioactive UDP-galactose from UDP[U-14C]glucose was demonstrated by radioautography after separation of the sugar nucleotides by paper chromatography or t.l.c. The pH optimum and the Km values for UDP-glucose, UDP-galactose and NAD+ were determined in both tissues. UDP-galactose and UDP-glucuronic acid formation after incubation with different UDP-glucose concentrations was followed; the same experiment was carried out using different UDP-galactose concentrations and following the formation of UDP-glucose and UDP-glucuronic acid. At equilibrium, the ratio UDP-glucose/UDP-galactose reaches a value of about 3.5. The results obtained seem to indicate that UDP-glucose 4'-epimerase activity is strongly dependent on that of UDP-glucose dehydrogenase. The physiological meaning of UDP-glucose 4'-epimerase in glycosaminoglycan biosynthesis in the two tissues under study is discussed on the basis of the Km values of UDP-glucose 4'-epimerase and UDP-glucose dehydrogenase and on the basis of the rate of UDP-glucose and UDP-galactose utilization.

Animals↗
PubMed1986

Identification of pyruvated monosaccharides in polysaccharides by gas-liquid chromatography mass spectrometry.

Twelve bacterial polysaccharides of known structure containing a representative range of pyruvated monosaccharides, were methanolysed, trimethylsilylated, and analysed by g.l.c. and g.l.c.-m.s. Except for 3,4-O-(1-carboxyethylidene)-L-rhamnose, which was unusually labile, the pyruvic acid substituents were largely retained during methanolysis and the Me3Si derivatives of the resulting pyruvated methyl glycosides gave distinctive g.l.c. peaks with characteristic mass spectra. The pyranose rings of 4,6-O-(1-carboxyethylidene)-D-glucose, 4,6-O-(1-carboxyethylidene)-D-mannose, 4,6-O-(1-carboxyethylidene)-D-galactose, and 3,4-O-(1-carboxyethylidene)-D-galactose survived the methanolysis, but that of 2,3-O-(1-carboxyethylidene)-D-glucuronic acid was cleaved to give the methyl ester of 2,3-O-(1-carboxyethylidene)-aldehydo-D-glucuronic acid dimethyl acetal. In the case of 2,3-O-(1-carboxyethylidene)-D-galactose, cleavage of the pyranose ring was less complete; under the conditions used in these experiments two-thirds of the pyranose rings were intact while one-third were cleaved to give the methyl ester of 2,3-O-(1-carboxyethylidene)-aldehydo-D-galactose dimethyl acetal. A very small amount of 3,4-O-(1-carboxyethylidene)-L-rhamnose from one polysaccharide retained its pyruvic acid substituent after gentle methanolysis to give the methyl ester of 3,4-O-(1-carboxyethylidene)-aldehydo-L-rhamnose dimethyl acetal. Susceptibility to cleavage of the pyranose ring during methanolysis appears to be a property of pyruvated monosaccharides with trans-fused 1,3-dioxolane rings.

Chromatography, Gas↗