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A simple microassay for the determination of UDP-glucuronic acid was developed on the basis of the formation of benzo[a]pyrene 3-glucuronide catalysed by UDP-glucuronyltransferase of guinea-pig liver. As little as 1-5 pmol of UDP-glucuronic acid was detectable in extracts of heat-denatured probes of liver or cultured cells equivalent to 10-50 micrograms of cellular protein.
The optimization of the assay conditions to detect glucuronate-2-sulphatase (GS) activity present in cultured human skin fibroblast homogenates towards a heparin-derived disaccharide substrate O-(beta-D-glucuronic acid 2-sulphate)-(1----4)-D-O-2,5-anhydro[l-3H]mannitol 6-sulphate (GSMS) has shown that a complex relationship exists between pH, buffer composition, ionic strength and the influence of added BSA and salts (NaCl, Na2SO4, CuCl2 and ZnCl2) to achieve maximum sulphatase activity. Whereas albumin stimulated GS activity by more than 2-fold over the pH range 2.7-5.7, CuCl2 stimulated GS activity over the narrow pH range 3.0-4.2, and inhibited GS activity at higher pH. ZnCl2 stimulated GS activity more than 3-fold at pH 3.0 and by more than 10-fold at pH 4.8. NaCl inhibited GS activity at pH 3.0, while activity between pH 4.2 and 4.8 was stimulated by up to 10-fold, resulting in a shift in the observed pH optimum from 3.0 to 4.8 in the presence of 315 mM-NaCl. Skin fibroblast GS activity toward GSMS had apparent Km values of 0.5-1.2 microM at pH 3.0, and 27.0-33.2 microM at pH 4.8. Albumin stimulated GS activity at both low and high pH by an increase in the apparent Vmax. values without significant alteration in the respective Km values. At pH 4.8, NaCl stimulated GS activity as a result of an increase in Vmax. values. These observations raise the possibility that two forms of GS activity are present in skin fibroblast homogenates: a low-Km form that has a pH optimum of 3.0 and is stimulated by BSA and a high-Km form with a pH optimum of 4.8 which is stimulated by NaCl.
Urine extracts from children diagnosed with generalized peroxisomal disorders were screened by continuous flow-negative ion fast atom bombardment-mass spectrometry. In 45 of 60 children with generalized peroxisomal disorders, we observed one or more intense ions (m/z 489, 505, 461, and others) that are infrequently found in children with cholestatic liver disease or normal children. Compounds giving rise to these ions were isolated using reverse phase and anion exchange chromatography. After appropriate derivatization and/or methanolysis the compounds were analyzed using capillary gas chromatography-mass spectrometry. The major compounds were found to be 12,13-dihydroxy-9-octadecenoic acid and 9,10-dihydroxy-12-octadecenoic acid, with one of the hydroxyl groups in glycosidic linkage with glucuronic acid. Minor compounds were glucuronic acid conjugates of 9,10-dihydroxy-octadecanoic acid, and 12,13-dihydroxy-6,9-, 15,16-dihydroxy-9,12-, and 9, 10-dihydroxy-12,15-octadecadienoic acids. A series of hexadecanoic, hexadecenoic, and hexadecadienoic acid glucuronides which appear to be beta-oxidation products of the C18 fatty acids were also observed, with the major species being 10, 11-dihydroxy-7-hexadecenoic acid glucuronide. In all, 16 C16 and C18 dihydroxy fatty acids were identified by gas chromatography-mass spectrometry. A series of at least 11 trihydroxy fatty acids was also observed but not fully characterized. Measurement of these compounds may prove to be useful in the diagnosis of some peroxisomal disorders.
We found a novel glycosyltransferase gene having a hypothetical beta 1,4-galactosyltransferase motif (GenBank accession number ) by a BLAST search and cloned its full-length open reading frame using the 5'-rapid amplification of cDNA ends method. The truncated form was expressed in insect cells as a soluble enzyme. It transferred N-acetylgalactosamine, not galactose, to para-nitrophenyl-beta-glucuronic acid. The N-acetylgalactosamine-glucuronic acid linkage has been identified only in chondroitin sulfate; therefore, we examined its chondroitin elongation and initiation activities. N-Acetylgalactosaminyltransferase activity was observed toward chondroitin poly- and oligosaccharides, chondroitin sulfate oligosaccharides, and linkage tetrasaccharide (GlcA-Gal-Gal-Xyl-O-methoxyphenyl), and the chondroitin polysaccharide and linkage tetrasaccharide were better acceptor substrates than the others. Northern blot analysis and quantitative real-time PCR analysis revealed that its 4-kb transcripts were highly expressed in thyroid and placenta, although they were ubiquitously expressed in various tissues and cells. These results suggest that this enzyme has N-acetylgalactosaminyltransferase activity in both the elongation and initiation of chondroitin sulfate synthesis. Furthermore, we performed enzymatic synthesis of chondroitin pentasaccharide in vitro. In one tube reaction with four enzymes, beta 1,4-galactosyltransferase-VII, beta 1,3-galactosyltransferase-VI, glucuronyltransferase-I, and this enzyme, and a synthetic xylose-peptide acceptor, the structure GalNAc-GlcA-Gal-Gal-Xyl-peptide was constructed. This is the first report of a chondroitin pentasaccharide constructed with recombinant glycosyltransferases in vitro.
In contrast to the wealth of biochemical and genetic information on vertebrate glucuronosyltransferases (UGATs), only limited information is available on the role and phylogenetics of plant UGATs. Here we report on the purification, characterization, and cDNA cloning of a novel UGAT involved in the biosynthesis of flower pigments in the red daisy (Bellis perennis). The purified enzyme, BpUGAT, was a soluble monomeric enzyme with a molecular mass of 54 kDa and catalyzed the regiospecific transfer of a glucuronosyl unit from UDP-glucuronate to the 2''-hydroxyl group of the 3-glucosyl moiety of cyanidin 3-O-6''-O-malonylglucoside with a kcat value of 34 s(-1) at pH 7.0 and 30 degrees C. BpUGAT was highlyspecific for cyanidin 3-O-glucosides (e.g. Km for cyanidin 3-O-6''-O-malonylglucoside, 19 microM) and UDP-glucuronate (Km, 476 microM). The BpUGAT cDNA was isolated on the basis of the amino acid sequence of the purified enzyme. Quantitative PCR analysis showed that transcripts of BpUGAT could be specifically detected in red petals, consistent with the temporal and spatial distributions of enzyme activity in the plant and also consistent with the role of the enzyme in pigment biosynthesis. A sequence analysis revealed that BpUGAT is related to the glycosyltransferase 1 (GT1) family of the glycosyltransferase superfamily (according to the Carbohydrate-Active Enzymes (CAZy) data base). Among GT1 family members that encompass vertebrate UGATs and plant secondary product glycosyltransferases, the highest sequence similarity was found with flavonoid rhamnosyltransferases of plants (28-40% identity). Although the biological role (pigment biosynthesis) and enzymatic properties of BpUGAT are significantly different from those of vertebrate UGATs, both of these UGATs share a similarity in that the products produced by these enzymes are more water-soluble, thus facilitating their accumulation in vacuoles (in BpUGAT) or their excretion from cells (in vertebrate UGATs), corroborating the proposed general significance of GT1 family members in the metabolism of small lipophilic molecules.
The hypothesis tested was that feeding rats sucrose rather than invert sugar (50:50 mixture of glucose and fructose) or cornstarch would result in a more rapid excretion of glucuronides and tritium from intravenously injected [1,2-3H]aldosterone. Thirty 56-d-old male rats of the Sprague-Dawley strain were fed for 8 wk one of three diets containing 45% of dietary energy from sucrose, invert sugar or cornstarch; 15% of energy was from protein and 40% from fat. Body weights and systolic blood pressures were measured weekly. After 60 d of feeding the diets ad libitum, all rats were injected intravenously with [1,2-3H]aldosterone and the percent recovery of tritium in both urine and feces was determined over the next 4 d. Urinary and fecal excretion of both free and conjugated glucuronic acid was determined over those 4 d. Urinary excretion of sodium and potassium (mg/d) was also determined. There were no differences between groups in food or water intakes, body weights, systolic blood pressures, daily fecal weights and daily urine volumes. The cornstarch-fed group excreted less sodium and potassium than did the other groups (P less than 0.05). The sucrose-fed group had a greater 4-d excretion of tritium (urinary + fecal) than did the invert sugar- or cornstarch-fed groups (P less than 0.01). The sucrose-fed group had a greater percentage of excreted glucuronic acid that was conjugated (urinary + fecal) than did the invert sugar- or cornstarch-fed groups (P less than 0.05). These results tended to confirm the hypothesis.
The beta1,3-glucuronosyltransferases are responsible for the completion of the protein-glycosaminoglycan linkage region of proteoglycans and of the HNK1 epitope of glycoproteins and glycolipids by transferring glucuronic acid from UDP-alpha-D-glucuronic acid (UDP-GlcA) onto a terminal galactose residue. Here, we develop phylogenetic and mutational approaches to identify critical residues involved in UDP-GlcA binding and enzyme activity of the human beta1,3-glucuronosyltransferase I (GlcAT-I), which plays a key role in glycosaminoglycan biosynthesis. Phylogeny analysis identified 119 related beta1,3-glucuronosyltransferase sequences in vertebrates, invertebrates, and plants that contain eight conserved peptide motifs with 15 highly conserved amino acids. Sequence homology and structural information suggest that Y84, D113, R156, R161, and R310 residues belong to the UDP-GlcA binding site. The importance of these residues is assessed by site-directed mutagenesis, UDP affinity and kinetic analyses. Our data show that uridine binding is primarily governed by stacking interactions with the phenyl group of Y84 and also involves interactions with aspartate 113. Furthermore, we found that R156 is critical for enzyme activity but not for UDP binding, whereas R310 appears less important with regard to both activity and UDP interactions. These results clearly discriminate the function of these two active site residues that were predicted to interact with the pyrophosphate group of UDP-GlcA. Finally, mutation of R161 severely compromises GlcAT-I activity, emphasizing the major contribution of this invariant residue. Altogether, this phylogenetic approach sustained by biochemical analyses affords new insight into the organization of the beta1,3-glucuronosyltransferase family and distinguishes the respective importance of conserved residues in UDP-GlcA binding and activity of GlcAT-I.
TWO ROUTES HAVE BEEN PROPOSED FOR CONVERSION OF BILIRUBIN MONOGLUCURONIDE TO THE DIGLUCURONIDE: glucuronyl transfer (a) from UDP-glucuronic acid to bilirubin monoglucuronide, catalyzed by a microsomal UDP-glucuronyltransferase, and (b) from one molecule of bilirubin monoglucuronide to another (transglucuronidation), catalyzed by an enzyme present in liver plasma membranes. The evidence regarding the role of the latter enzyme for in vivo formation of bilirubin diglucuronide is conflicting. We therefore decided to reexamine the transglucuronidation reaction in plasma membranes and to study the conversion of bilirubin monoglucuronide to diglucuronide in vivo. Purified bilirubin monoglucuronide was incubated with homogenates and plasma membrane-enriched fractions from liver of Wistar and Gunn rats. Stoichiometric formation of bilirubin and bilirubin diglucuronide out of 2 mol of bilirubin monoglucuronide was paralleled by an increase of the IIIalpha- and XIIIalpha-isomers of the bilirubin aglycone, thus showing that dipyrrole exchange, not transglucuronidation, is the underlying mechanism. Complete inhibition by ascorbic acid probably reflects intermediate formation of free radicals of dipyrrolic moieties. The reaction was nonenzymic because it proceeded independently of the protein concentration and heat denaturation of the plasma membranes did not result in decreased conversion rates. Collectively, these findings show spontaneous, nonenzymic dipyrrole exchange when bilirubin monoglucuronide is incubated in the presence of rat liver plasma membranes. Because bilirubin glucuronides present in biological fluids contain exclusively the bilirubin-IXalpha aglycone, formation of the diglucuronide from the monoglucuronide by dipyrrole exchange does not occur in vivo. Rapid excretion of unchanged bilirubin monoglucuronide in Gunn rat bile after injection of the pigment provides confirmatory evidence for the absence of a UDP-glucuronic acid-independent process.
For the purpose of obtaining liposomes with long circulation time in blood, we synthesized 1-O-palmityl-D-glucuronic acid (PGlcUA) and incorporated it into the liposomal membranes. The clearance of the PGlcUA-liposomes composed of dipalmitoylphosphatidylcholine (DPPC), cholesterol, and PGlcUA (40:40:20 as a molar ratio) from blood and their tissue distribution were compared with those of dipalmitoylphosphatidylglycerol (DPPG)-liposomes (DPPC/cholesterol/DPPG = 40:40:20). When [3H]inulin-loaded PGlcUA-liposomes and DPPG-liposomes were intravenously injected into rats, the half-life of the PGlcUA-liposomes in the blood appeared to be 1.7-fold longer than that of DPPG-liposomes. Radioactivities present in plasma and various tissues were measured 22 h after administration of these liposomes, and radioactivity remaining in the plasma was 2.5-fold greater when PGlcUA-liposomes were injected. The distribution pattern of [3H]inulin in PGlcUA-liposomes was similar to that in DPPG-liposomes. The radioactivity recovered in urine was 25% lower in rats treated with PGlcUA-liposomes than in those treated with DPPG-liposomes. Since both PGlcUA- and DPPG-liposomes exhibited similar size distribution and zeta-potential, glucuronic acid, rather than negative charge, on the liposomal surface appears to endow liposomes with a longer circulation time in the bloodstream.
Chondroitin ABC and AC lyases split hexosaminidic linkages in galactosaminoglycans and hyaluronic acid. Even-numbered oligosaccharides from hyaluronic acid with either D-glucuronic acid or N-acetylglucosamine in non-reducing position were used, prior to and after reduction with sodium borohydride, as substrates for chondroitin ABC and AC lyases. These substrates allowed elucidation of the effects of the nearest neighborhood of the bond to be split on the action of the enzymes. The results indicate that chondroitin ABC lyase acts strictly as an endolyase towards hyaluronate and requires the presence of a disaccharide in both reducing and non-reducing positions of the endohexosaminidic bond to be split. None of the hexosaminidic bonds of the tetrasaccharide GlcNAc-GlcUA-GlcNAc-GlcUA is split by chondroitin ABC lyase. In contrast chondroitin AC lyase acts also as an exoglycosidase towards hyaluronate and recognizes only the amino sugar and the uronic acid residue that are linked via the hexosaminidic bond which is split. Thus, the N-acetylglucosamine and glucuronic acid residues at both ends of a tetrasaccharide with the structure GlcNAc-GlcUA-GlcNAc-GlcUA are liberated.
1. Urinary levels of xylitol and glucuronic, glucaric, gulonic and ascorbic acids were measured in the rat, rabbit, guinea-pig and marmoset by an improved g.l.c. technique. 2. Administration of a compound (2-methylbenzanilide) known to be conjugated and excreted as a beta-glucuronide had some effect on the output of these compounds of the glucuronic acid pathway in all four species, and caused a significant decrease in gulonic acid in the rat.
1. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) was administered to rats to study its effects on the enzyme activities of the D-glucuronic acid pathway in the liver, small intestine and kidney. 2. The UDP-glucuronosyl transferase activity of male albino rats given TCDD (80 mug/kg, one dose, i.p.) 6 days before killing was significantly increased in all tissues examined, and UDP-glucuronic acid pyrophosphatase activity was markedly decreased in the liver. D-Glucuronolactone and L-gulonate dehydrogenase activities in the liver and small intestine were slightly decreased after TCDD treatment. 3. The activities of UDP-glucose dehydrogenase and beta-glucuronidase were unchanged. 4. The 24 h urinary excretion of L-ascorbic acid was enhanced 8-fold, although no difference was detected in the excretion of D-glucaric acid between the control and experimental animals. 5. These results suggest an increased capacity for glucuronide conjugation after treatment with TCDD. 6. The lack of increase in the urinary excretion of D-glucaric acid further challenges its use as a reliable indicator of enhanced drug metabolism.
Nutritionally harmful substances are formed when edible vegetable oils are heated (cyclization, polymerization of unsaturated fatty acids, ...). How are these substances metabolized in animals? This work is a first attempt to experiment if these altered fatty acids or their metabolites are excreted as conjugated compounds in the urine. Weaning rats (weighing 45 g) were divided into 3 groups of 9 animals each. They received balanced diets containing 10 p. 100 by weight of lipids. In the first group, the dietary of lipids was linseed oil which had been heated for 12 hours at 275 degrees C under nitrogen (group HC275); in the second group, the linseed oil had been heated 100 hours at 200 degrees C in presence of air (group HC200). These two groups of animals were fed ad libitum. The control group (group HF) was fed a diet containing fresh linseed oil and its food consumption was restricted to the quantity eated by the HC275 group (pair feeding). Urines were collected daily from the 8th to the 15th day of experiment and pooled for each rat. Hippuric acid and glucuronic acid contents were determined in these urines. During this period the hippuric acid excretion in HC275 and HC200 groups is twice as high as in HF group (32, 35 and 17 mumoles respectively). On the other hand urinary excretion of glucoronic acid is 8 times higher in HC275 and 3 times in HC200 than in HF (2214, 739 and 269 mumoles respectively). Hippuric acid excretion is very small as compared to that of glucuronic acid. Therefore it seems that most of the compounds formed during heating of the oil or their metabolites are excreted as glucoronic acid conjugates.
The glucuronic acid conjugate of N-hydroxy-4-aminobiphenyl (NOH-4-ABP) has been isolated in relatively pure form from the urine of dogs given 4-aminobiphenyl, utilizing molecular size, ion exchange, adsorption, and partition chromatography. This conjugate is an active mutagen in Salmonella typhimurium strains TA1538 and TA98 but not in TA1535 or TA1537. NOH-4-ABP and 4-nitrosobiphenyl are also highly active in TA1538 and TA98 and inactive in TA1535 and TA1537. These observations support the concept that this conjugate is the water-soluble carrier that delivers the active metabolite to the bladder. A substance of identical chromatographic and spectral properties to the conjugate isolated from dog urine has been synthesized in low yield by the direct condensation of NOH-4-ABP with glucuronic acid. This substance yields NOH-4-ABP on dilute acid hydrolysis. Sodium (N-4-biphenylhydroxylamino-beta-D-glucopyranosid) uronate, the N-O-C isomer, was also synthesized. It was found to have differing chromatographic and chemical properties to the natural conjugate. This evidence suggests that the urinary conjugate is the compound in which conjugation has occurred with the nitrogen atom of the hydroxylamine group rather than the oxygen atom.
Previously, we had described the structures of the haptenic oligosaccharides of the surface glycopeptidolipid antigens from serotypes 9 and 25 of the Mycobacterium avium complex and had synthesized these units as putative antigenic probes. The lack of chemical concordance between the synthetic products and the haptens has prompted a re-examination of these structures utilizing the instrumental techniques not previously available of fast atom bombardment-mass spectrometry, Fourier transform infra-red, and high resolution NMR spectroscopy. With the additional information thus available, more extensive chemical fragmentations by base degradation, followed by alkylation, have furnished supportive evidence to allow formulation of revised and novel structures, all of which contain glucuronic acid: serotype 9, 2,3-di-O-Me-L-Fucp(alpha 1----4)-D-GlcAp(beta 1----4)-2,3-di-O-Me-L-Fucp(alpha 1----3)-L-Rhap(alpha 1----2)-6dTal; and serotype 25, 4-acetamido-4,6-dideoxy-2-O-Me-hexosyl(alpha 1----4)-D- GlcAp(beta 1----4)2-O-Me-L-Fucp(alpha 1----3)-L-Rhap(alpha 1----2)6dTal. Glucuronic acid, acetamido sugars, and other novel sugars appear to be widespread in the glycopeptidolipid antigens of Mycobacterium spp. The revised structures will allow renewed synthesis of artificial antigen probes and rational approaches to preparing monoclonal antibodies, both necessary for the new diagnostics required to trace the sources of widespread infections due to M. avium and Mycobacterium intracellulare.
The glucuronic acid conjugate of methylazoxymethanol was synthesized by oxidizing the primary alcohol of the glucose moiety of cycasin (methylazoxymethanol-beta-D-glycopyranoside) to a carboxylic acid. The oxidation was carried out by bubbling oxygen gas through a cycasin solution in the presence of a platinum-on-carbon catalyst. A band at 1715 cm-1, not present in the cycasin infrared spectrum, appeared in the spectrum of the oxidized cycasin product, establishing the presence of a carboxylic acid group. The oxidation product is methylazoxymethanol-beta-D-glucosiduronic acid because, when hydrolyzed with Escherichia coli beta-glucuronidase, it produced methylazoxymethanol and glucuronic acid and also indicated retention of the beta-linkage of cycasin. Varying quantities of the synthesized methylazoxymethanol-glucosiduronic acid, injected into Wistar rats of both sexes and of varying weights, were not acutely toxic. The compound was mutagenic to Salmonella typhimurium when preincubated with E. coli beta-glucuronidase, but not when preincubated with bovine liver glucuronidase.
Both in normal and experimentally jaundiced rats cis-2-hydroxy-2-phenyl-cyclohexanecarboxilic acid (cicloxilic acid) induces a greater and longer lasting increase in bile flow and a greater increase in bilirubin and glucuronic acid secretion than do the choleretics sodium 2-(2-hydroxy-3n hexoxy-propoxy)-benzoate and dehydrocholic acid. The intense increase in bilirubin secretion is interpreted as the outcome of stimulation of the biliary secretion mechanisms whilst the effect on bile flow and on the biliary secretion of glucuronic acid are attributed to the kinetics of biliary secretion of cicloxilic acid.
According to the ultrastructure of cell walls, glucuronate-positive species of the genus Candida include both ascomycetous organisms (C. ciferrii, C. incommunis, C. steatolytica) and basidiomycetous organisms (C. bogoriensis, C. curiosa, C. diffluens, C. javanica, C. marina). The character of budding and the structure of septa suggest that the perfect forms of glucuronate-positive ascomycetous Candida species should be looked for within the family Ascoideaceae.