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PubMed1968

Inositol Metabolism in Plants. V. Conversion of Myo-inositol to Uronic Acid and Pentose Units of Acidic Polysaccharides in Root-tips of Zea mays.

The metabolism of myo-inositol-2-(14)C, d-glucuronate-1-(14)C, d-glucuronate-6-(14)C, and l-methionine-methyl-(14)C to cell wall polysaccharides was investigated in excised root-tips of 3 day old Zea mays seedlings. From myo-inositol, about one-half of incorporated label was recovered in ethanol insoluble residues. Of this label, about 90% was solubilized by treatment, first with a preparation of pectinase-EDTA, then with dilute hydrochloric acid. The only labeled constituents in these hydrolyzates were d-galacturonic acid, d-glucuronic acid, 4-O-methyl-d-glucuronic acid, d-xylose, and l-arabinose, or larger oligosaccharide fragments containing these units. Medium external to excised root-tips grown under sterile conditions in myo-inositol-2-(14)C contained labeled polysaccharide.When label was supplied in the form of d-glucuronate, the pattern of labeled uronic acid and pentose units in cell wall polysaccharides resembled that obtained from labeled myo-inositol, indicating that both substances were metabolized along a common path during polysaccharide formation, and that methylation occurred at a step subsequent to uronic acid formation. When label was supplied in the form of l-methionine-methyl-(14)C, 4-O-methyl-d-glucuronic acid was the only labeled monosaccharide component that survived enzymatic or acid hydrolysis.Zea mays endosperm, a known source of phytin, developed maximal phytase activity after the third day of germination. Results obtained here suggest that myo-inositol released by hydrolysis of phytin represents the initial precursor of a normal, possibly predominant pathway for the formation of uronic acids in plants.

Journal Article↗
PubMed1978

Conversion of labeled substrates to sugars, cell wall polysaccharides, and tartaric Acid in grape berries.

[U-(14)C]Sucrose, myo-[U-(14)C]inositol, [6-(14)C]- and [U-(14)C]glucuronate, UDP-[U-(14)C]glucuronate, [U-(14)C]gluconate, and l-[1-(14)C]ascorbic acid were fed into grape berries, Vitis labrusca L. cv. Delaware, at intervals throughout the ripening process and incorporation of (14)C into several metabolites was studied.[U-(14)C]Sucrose was the most effective precursor of cellulose in young grape berries and of glucose and fructose in mature berries. On the other hand, UDP-[U-(14)C]glucuronate was the best precursor of pectic substance, followed by [(14)C]glucuronate and myo-[U-(14)C]inositol. l-[1-(14)C]Ascorbic acid was the most effective precursor of tartaric acid. In young berries, [U-(14)C]sucrose and [U-(14)C]gluconate also produced labeled tartaric acid, the latter a somewhat better precursor in the 3 weeks following flowering. The remaining test compounds were only poor sources of (14)C for tartaric acid although all three, glucuronate, UDP-glucuronate, and myo-inositol, were utilized by the grape berry for pectin biosynthesis.These results strongly indicate that tartaric acid is synthesized by a C-1 oxidation mechanism of hexose in young grape berries.

Journal Article↗
PubMed1996

Direct detection and identification of radicals generated during the hydroxyl radical-induced degradation of hyaluronic acid and related materials.

HO. attack on hyaluronic acid, related polymers and monomers has been studied by both direct, rapid-flow, EPR (ESR) and EPR spin trapping using a variety of traps. Evidence has been obtained, with the monomers, for essentially random hydrogen-atom abstraction at all the ring C -- H bonds with glucuronic acid, and at all sites except the N-acetyl side chain and C(2) with N-acetylglucosamine. The initial radicals do not undergo rapid rearrangement reactions at pH 4; however at both lower and higher pH values, acid- and base-catalysed rearrangement process, respectively, result in the loss of these species. The rate of loss of these species is dependent on the substrate, with those derived from N-acetylglucosamine undergoing slowed acid-catalysed rearrangement than the glucuronic acid-derived species. This is rationalised in terms of a rearrangement reaction of 1.2-dihydroxyalkyl(1.2-dio) radicals involving an electron-deficient radical-cation intermediate; the formation of this species would be disfavoured by the electron-withdrawing N-acetyl substituent. The base-catalysed process, which is believed to involve a radical-anion intermediate, occurs rapidly at pH 7.4, and appears to be less substrate dependent. In the case of glucuronic acid- (but not N-acetylglucosamine-) derived species this latter process results in the detection of ring-opened semidione species. With equimolar mixtures of the two monomers essentially random attack occurs on the two rings. However with chondroitin sulphate A, attack appears to be much more selective, with a radical generated at C(5) on the glucuronic acid ring present at highest concentration. The initial radicals obtained with this polysaccharide also undergo base- and acid-catalysed rearrangements; this leads to strand-breakage and the formation of low-molecular-weight material. Spin-trapping experiments carried out with hyaluronic acid, and a number of other polysaccharides, resulted in the detection of a number of novel spin adducts, the formation of which are consistent with attack on both the sugar rings in the polymer. The pH dependence of the observed spectra, and the detection of additional species at some pH values, suggest that at least some of the initial radicals undergo base-catalysed rearrangement reactions which result in strand-breakage and the formation of low-molecular-weight fragments. The extent of fragmentation at a particular pH, is also affected by the radical flux, with high radical yields giving more low-molecular-weight material. These observations suggest that pH-independent processes also contribute to strand-cleavage; this may be due to beta-cleavage of the radicals formed at C(1) on either ring, C(3) on N-acetylglucosamine or C(4) on the glucuronic acid ring.

Acetylglucosamine↗
PubMed1986

Glycoconjugates as noninvasive probes of intrahepatic metabolism: pathways of glucose entry into compartmentalized hepatic UDP-glucose pools during glycogen accumulation.

Recent studies have questioned the efficiency with which administered glucose generates hepatic glycogen through the direct nonrecycling route compared with the indirect route from glucose recycled through glycolysis followed by gluconeogenesis. Using fasted and refed rats, we examined the relative access of infused [1-3H]- and [U-14C]glucose by way of these two pathways to liver glycogen and to hepatic glucuronic acid, the latter recovered from the urine as the glucuronide conjugated with administered acetaminophen. In fasted animals and during early refeeding, extensive dilution of administered [3H]- and [14C]glucose recovered in glycogen showed that 60-70% of the labeled glucose had undergone recycling by the indirect route. As refeeding progressed with substantial glycogen deposition, the contribution of the recycling pathway to glycogen and glucuronic acid diminished considerably. Thus, there is a shift in pathways of hepatic glucose utilization as liver glycogen accumulates. Consequently, the ratio of 3H/14C in glucuronic acid was closely correlated with the glycogen content of the liver at sacrifice, indicating that this ratio may prove useful as a noninvasive indicator of liver glycogen concentration. Since glycogen and glucuronic acid are derived by single reactions from UDP-glucose, they should show a common labeling pattern with 3H and 14C under various nutritional conditions. However, detailed analysis of their labeling patterns showed a striking divergence, implying that there must be compartmentation of the UDP-glucose pools leading to each of these end products, either because they are made in separate compartments within the same cell or because each is made in different hepatocyte populations. We favor the former explanation because galactose secreted in glycoproteins shows 3H and 14C labeling patterns similar to those of glucuronic acid conjugated with acetaminophen, and both of these conjugations occur in the endoplasmic reticulum of the liver, whereas most glycogen is present in the cytosol.

Animals↗
PubMed2007

Vitamin C. Biosynthesis, recycling and degradation in mammals.

Vitamin C, a reducing agent and antioxidant, is a cofactor in reactions catalyzed by Cu(+)-dependent monooxygenases and Fe(2+)-dependent dioxygenases. It is synthesized, in vertebrates having this capacity, from d-glucuronate. The latter is formed through direct hydrolysis of uridine diphosphate (UDP)-glucuronate by enzyme(s) bound to the endoplasmic reticulum membrane, sharing many properties with, and most likely identical to, UDP-glucuronosyltransferases. Non-glucuronidable xenobiotics (aminopyrine, metyrapone, chloretone and others) stimulate the enzymatic hydrolysis of UDP-glucuronate, accounting for their effect to increase vitamin C formation in vivo. Glucuronate is converted to l-gulonate by aldehyde reductase, an enzyme of the aldo-keto reductase superfamily. l-Gulonate is converted to l-gulonolactone by a lactonase identified as SMP30 or regucalcin, whose absence in mice leads to vitamin C deficiency. The last step in the pathway of vitamin C synthesis is the oxidation of l-gulonolactone to l-ascorbic acid by l-gulonolactone oxidase, an enzyme associated with the endoplasmic reticulum membrane and deficient in man, guinea pig and other species due to mutations in its gene. Another fate of glucuronate is its conversion to d-xylulose in a five-step pathway, the pentose pathway, involving identified oxidoreductases and an unknown decarboxylase. Semidehydroascorbate, a major oxidation product of vitamin C, is reconverted to ascorbate in the cytosol by cytochrome b(5) reductase and thioredoxin reductase in reactions involving NADH and NADPH, respectively. Transmembrane electron transfer systems using ascorbate or NADH as electron donors serve to reduce semidehydroascorbate present in neuroendocrine secretory vesicles and in the extracellular medium. Dehydroascorbate, the fully oxidized form of vitamin C, is reduced spontaneously by glutathione, as well as enzymatically in reactions using glutathione or NADPH. The degradation of vitamin C in mammals is initiated by the hydrolysis of dehydroascorbate to 2,3-diketo-l-gulonate, which is spontaneously degraded to oxalate, CO(2) and l-erythrulose. This is at variance with bacteria such as Escherichia coli, which have enzymatic degradation pathways for ascorbate and probably also dehydroascorbate.

Animals↗
PubMed1971

Bilirubin glucuronyltransferase. Specific assay and kinetic studies.

1. Bilirubin glucuronide was synthesized in vitro in a system containing a rat liver microsomal fraction, UDP-glucuronic acid, Mg(2+) and bilirubin. The enzymic synthesis was accomplished without the addition of a bilirubin carrier. 2. Azobilirubin and azobilirubin glucuronide were separated by t.l.c. and paper chromatography and the measurement of the conjugate provided a specific assay for bilirubin UDP-glucuronyltransferase (EC 2.4.1.17). 3. This diazo compound was labelled when [U-(14)C]UDP-glucuronic acid was employed in the transglucuronidation reaction. 4. Identity of the glucuronide nature of the product was further confirmed by hydrolysis with beta-glucuronidase prepared from limpets and Helix pomatia. In each instance azobilirubin and glucuronic acid were liberated. 5. There was a close correlation between the bilirubin glucuronyl-transferase activity as measured by two procedures, colorimetric and radioisotopic. The specific activities so measured were 19nmol of bilirubin ;equivalents' conjugated/h per mg of protein and 16.9-18.4nmol of UDP-glucuronic acid incorporated/h per mg of protein, respectively. On this basis, it was concluded that the major product formed in vitro was bilirubin monoglucuronide; this represents about 77% of the total products formed. 6. The K(m) values for bilirubin and UDP-glucuronic acid at pH8.2 are 3.3x10(-4)m and 1.67x10(-3)m, respectively. 7. The addition of Mg(2+) at a final concentration of 5mm to the reaction mixture increased the rate of conjugation by 5.6-fold in the microsomal preparation that had been subjected to overnight dialysis against 10mm-EDTA (disodium salt). 8. Diethyl-nitrosamine at a final concentration of 1-20mm has no effect on the glucuronidation of bilirubin in vitro.

Animals↗
PubMed1986

Characterization of a high-Mr plasma-membrane-bound protein and assessment of its role as a constituent of hyaluronate synthase complex.

A high-Mr phosphoprotein (Mr 442,000) was purified from Nonidet-P-40-solubilized plasma membranes of cultured human skin fibroblasts. The protein comprised one 200,000-Mr subunit consisting of 116,000- and 84,000-Mr polypeptides and two identical 121,000-Mr subunits each consisting of 66,000- and 55,000-Mr polypeptides. The 200,000-Mr subunit and its polypeptides contained phosphotyrosine residues and were also [32P]phosphorylated at these residues from [gamma-32P]ATP in vitro by an intrinsic tyrosine kinase activity of the protein molecule in response to the presence of hyaluronate precursors, UDP-glucuronic acid and UDP-N-acetylglucosamine. The 121,000-Mr subunits and their polypeptides contained phosphoserine residues that could not be [32P]phosphorylated during autophosphorylation of the protein in vitro. The protein molecules separated from exponential- and stationary-growth-phase cells were identical in their quaternary structure, but appeared to exist in different proportions with respect to the state of phosphorylation of their 121,000-Mr subunits during different growth phases of the cell. Phosphorylation of polypeptides appeared to predispose in favour of their UDP-glucuronic acid- and UDP-N-acetylglucosamine-binding activities. The phosphorylated 116,000- and 84,000-Mr polypeptides of 200,000-Mr subunits possessed a single binding site for UDP-glucuronic acid and UDP-N-acetylglucosamine respectively. The phosphorylated 200,000-Mr subunit could also cleave the UDP moiety from UDP-glucuronic acid and UDP-N-acetylglucosamine precursors. The phosphorylated 121,000-Mr subunit possessed two binding sites with equal affinity towards UDP-glucuronic acid and UDP-N-acetylglucosamine but did not possess UDP-moiety-cleavage activity. The phosphorylation of 200,000-Mr subunit by an intrinsic kinase activity of the protein molecule appeared to elicit its oligosaccharide-synthesizing activity, whereas phosphorylation of 121,000-Mr subunits, presumably carried out in vivo, abolished this activity of the protein molecule. The oligosaccharides synthesized by the protein were about Mr 5000 and about 12 disaccharide units in length. Neither nucleotide sugars nor glycosyl residues nor newly synthesized oligosaccharides were bound covalently to the protein molecule. The UDP moiety of nucleotide sugar precursors did not constitute a link between protein molecule and oligosaccharide during its synthesis. Although isolated 442,000-Mr protein did not synthesize high-Mr hyaluronate in vitro, this protein molecule can be considered as a constituent of membrane-bound hyaluronate synthase complex because of its observed properties.

Amino Acids↗
PubMed1977

Escherichia coli K-12 structural kdgT mutants exhibiting thermosensitive 2-keto-3-deoxy-D-gluconate uptake.

A specific method is described for selecting thermosensitive mutants of Escherichia coli K-12 able to grow on 2-keto-3-deoxy-D-gluconate (KDG) and D-glucuronate at 2, but not at 42 degrees C. The extensive analysis of one such mutant is consistent with the conclusion that the carrier molecule responsible for KDG and glucuronate uptake becomes thermolabile. (i) Growth on a variety of carbon sources is perfectly normal at 28 and 42 degrees C, whereas in the same temperature range it gradually diminishes on KDG and glucuronate. (ii) The apparent Km value for KDG is about twofold in the range 25 to 40 degrees C. In the same temperature range, the Vmax values for KDG influx are higher for the mutant compared with those of the wild-type strain, but the optimum temperature is 34 degrees C instead of 38 degrees C. On the contrary, the Vmax values for glucuronate influx are lower for the mutant than for the parental strain, and the optimum temperature for both strains is shifted beyond 40 degrees C. (iii) The activation energies for KDG and glucuronate uptake are about twofold higher in the mutant than in the wild-type strain. (iv) Kinetics of counterflow under deenergized conditions (overshoot) at different temperatures indicate that the defect is located in the translocation step rather than in the processes involved in energy coupling. (v) The first-order rate constants for thermal denaturation are, respectively, 2.5- and 5-fold higher at 40 and 30 degrees C in the mutant than in the wild-type strain, and the activation energy for thermal denaturation is lower. (vi) The carrier molecule in the mutant is also much more sensitive to denaturation by N-ethylmaleimide. (vii) Four independent thermosensitive mutations and one revertatn were located by transduction in or near the kdgT locus, defined previously as the site of nonconditional KDG transport-negative mutations. These results support the conclusion that kdgT represents the structural gene coding for the KDG transport system.

Biological Transport↗
PubMed2004

GntP is the Escherichia coli Fructuronic acid transporter and belongs to the UxuR regulon.

Escherichia coli has four gluconate transporters, GntP, GntU, GntT, and IdnT, which are members of the major facilitator superfamily. The physiological function of GntP was previously unknown and is the subject of this study. GntP is not induced by gluconate, and despite being located adjacent to genes involved in glucuronate catabolism, gntP does not encode a glucuronate transporter. Here we identify gntP as the gene which encodes the fructuronate transporter. We show that gntP is induced by fructuronate and is a new member of the UxuR regulon: gntP is derepressed in an uxuR strain, UxuR binds in vitro specifically to an operator site that overlaps the gntP promoter, and UxuR binding is eliminated by fructuronate. Transcription of gntP requires activation by cyclic AMP (cAMP)-cAMP receptor protein. A gntP mutant cannot grow on fructuronate but grows normally on glucuronate and gluconate. Thus, the UxuR regulon is a module of sugar acid catabolism whose physiological role is for growth on fructuronate. Glucuronate, because it proceeds through a fructuronate intermediate, must induce the UxuR regulon and must also induce the ExuR regulon, which encodes the glucuronate transporter, ExuT, and the first step in its catabolism, UxaC. Thus, hexuronate catabolism in E. coli requires both the ExuR and UxuR regulons, while fructuronate catabolism requires only the UxuR regulon.

Binding Sites↗
PubMed2003

Synthesis of the metabolites of a free radical scavenger edaravone (MCI-186, Radicut).

Two metabolites of a free radical scavenger, edaravone, were synthesized. Edaravone glucuronate was synthesized by glycosylation of a glucuronic acid precursor using silver (I) trifluoromethane-sulfonate with edaravone. Edaravone sulfate was synthesized by sulfonylation of edaravone using a sulfur trioxide-pyridine complex. The two synthesized metabolites were identical to isolated metabolites. X-ray analysis identified edaravone glucuronate as beta-O-glucuronate, although there were three possible edaravone glucuronate tautomers.

Antipyrine↗
PubMed1979

Cyclical changes in the carbohydrate composition of dog endometrium.

In the first part of prooestrus glucuronic acid could not be detected, but small amounts of glucose, fructose, inositol and sorbitol were found. When progesterone was formed a small amount of glucuronic acid could be observed and sorbitol had disappeared. During the first weeks of metoestrus the amounts of fructose and glucose increased gradually, but those of glucuronic acid remained low. When more progesterone and pregnanedione were found in the endometrium the amounts of glucuronic acid began to increase and the highest values were reached in anoestrus. During the last days of anoestrus a sudden change in the carbohydrate composition of the endometrium occurred: glucuronic acid and fructose could no longer be detected, the amounts of glucose and inositol decreased and sorbitol was found. These findings show that next to the cyclical ovarian events, there is a cyclical change in te carbohydrate composition of dog endometrium.

Animals↗
PubMed1984

Mechanism and subcellular site of bilirubin diglucuronide formation in rat liver.

Two different subcellular sites and mechanisms have been proposed for the hepatic conversion of bilirubin monoglucuronide to bilirubin diglucuronide: a microsomal system requiring UDP-glucuronate and a UDP-glucuronate-independent transglucuronidation or dismutase reaction localized at the bile canalicular plasma membrane. To further define these, canalicular plasma membranes were highly purified from rat liver, and the capacity of these to form bilirubin diglucuronide was compared with that of simultaneously isolated hepatic microsomes. The canalicular liver plasma membranes were 48-116-fold enriched over homogenate in various canalicular marker enzyme activities; microsomal contamination was less than 10% based on the NADPH-cytochrome c reductase activity. No evidence of any conversion of highly purified bilirubin IX alpha monoglucuronide to bilirubin diglucuronide was found with canalicular liver plasma membranes either in the absence or presence of UDP-glucuronate. In contrast, digitonin-treated microsomes isolated under similar conditions converted 31% of added bilirubin monoglucuronide (9.4-17.1 microM) into bilirubin diglucuronide in 30 min, the reaction being dependent on UDP-glucuronate. When bilirubin (12.5 microM) was added to the microsomes, 42.3% was converted to bilirubin monoglucuronide and 40.9% to bilirubin diglucuronide in 30 min. These data establish that the endoplasmic reticulum and not the canalicular liver plasma membranes forms bilirubin diglucuronide from bilirubin monoglucuronide and that the reaction requires UDP-glucuronate.

Animals↗
PubMed1993

Topography of glycosylation and UDP-xylose production.

In order to define the location and organization of the numerous reactions involved in polysaccharide assembly during synthesis of proteoglycans and glycoproteins, the topography of some of the glycosylation reactions in chondroitin sulfate synthesis was examined using a relatively new technique for generating permeable cells. Permeable chondrocytes were shown to directly take up nucleotide sugar precursors and incorporate them into chondroitin sulfate proteoglycan (CSPG), allowing specific labeling at each step in chondroitin sulfate synthesis. Subcellular fractionation following labeling with UDP-[14C]xylose, UDP-[14C]galactose, UDP-[14C]glucuronic acid, or [35S]PAPS localized the labeled CSPG to the compartment where each glycosylation reaction occurred. From these experiments it appears that xylose addition begins in the endoplasmic reticulum and continues in the Golgi apparatus where galactose, glucuronic acid, and sulfate are added. This conclusion was confirmed by direct visualization of xylose incorporation using electron microscopic autoradiography (Vertel, B. M., Walters, L. M., Flay, N., Kearns, A. E., and Schwartz, N. B. (1993) J. Biol. Chem. 268, 11105-11112). Further examination of xylose addition showed that permeable chondrocytes can utilize both exogenous UDP-xylose transported into the lumen and UDP-xylose generated from UDP-glucuronic acid within the lumen. The enzyme responsible for this reaction, UDP-glucuronate carboxy-lyase, co-localized with xylosyltransferase activity in subcellular fractions. Orientation toward the lumen in subcellular compartments was determined by trypsin sensitivity in the permeable chondrocytes. Therefore, we conclude that UDP-xylose can be produced in the lumen of the compartment where it is utilized in CSPG synthesis, obviating the need for a direct transport mechanism for this nucleotide sugar and providing close regulation of UDP-xylose and UDP-glucuronic acid levels.

Animals↗
PubMed1983

Evidence for an active site arginine in UDP-glucuronyltransferase.

2,3-Butanedione inactivates the pure form of UDP-glucuronyltransferase used in these experiments (GT2P) (EC 2.4.1.17) purified from pig liver microsomes. The kinetics of the reaction indicates that 2,3-butanedione reacts with two amino acids that affect activity. A rapid, partial inactivation is followed by a slower rate of inactivation that leads eventually to completely inactive enzyme. UDP-glucuronic acid and glucuronic acid, as compared with UDP, are effective as protectors against the slow, secondary phase of inactivation; no ligand tested protected against the rapid phase of inactivation. The lipid environment of GT2P was a determinant of the pseudo-first order rate constant for the slow phase of inactivation, but did not affect the rate of the rapid phase of inactivation. The data suggest that GT2P contains an active site arginine that interacts with the -COO- at C-6 of the glucuronic acid moiety of UDP-glucuronic acid.

Amino Acids↗
PubMed1986

Regulation of bilirubin glucuronide synthesis in primate (Macaca fascicularis) liver. Kinetic analysis of microsomal bilirubin uridine diphosphate glucuronyltransferase.

Hepatic bilirubin uridine diphosphate glucuronyl-transferase (UDP-glucuronyltransferase) catalyzes the formation of bilirubin monoglucuronides (BMG, C-8 and C-12 isomers) and bilirubin diglucuronide (BDG) from bilirubin and the cosubstrate, UDP-glucuronic acid. Distinctive patterns of bile pigment excretion occur in different species and in pathologic disorders (e.g., Gilbert's syndrome). In normal human and monkey (Macaca fascicularis) bile, the proportion of BDG exceeds that of BMG and the C-8/C-12 BMG isomer ratio approaches unity. To investigate the mechanisms responsible for the patterns of BDG and BMG isomers in bile, we used a radiochemical assay to analyze the kinetics and regulation of bilirubin UDP-glucuronyltransferase in microsomes prepared from monkey liver. The synthesis of BMG from bilirubin was a higher capacity, lower affinity step (Vmax = 295 pmol/mg protein X min, Km = 24 microM) than BDG synthesis from endogenously formed BMG (Vmax = 170 pmol/mg protein X min, Km = 14 microM). This observation was confirmed when biosynthetically prepared BMG was used as substrate. The rate of formation of BDG relative to BMG was modulated by both bilirubin and UDP-glucuronic acid concentration, whereas the C-8/C-12 BMG isomer ratio was influenced exclusively by UDP-glucuronic acid concentration. The data obtained with increasing UDP-glucuronic acid concentration did not obey conventional single-site kinetics, suggesting the presence of more than one binding site on the enzyme or a membrane transporter for this nucleotide sugar. These findings demonstrate that microsomal bilirubin UDP-glucuronyltransferase has a greater capacity for BMG than BDG synthesis, and thus support the concept that the decreased BDG/BMG ratio in the bile of patients with reduced hepatic enzyme activity (i.e., Gilbert's syndrome and type II Crigler-Najjar disease) reflects the diminished capacity of the enzyme to synthesize BDG from BMG. Bilirubin and UDP-glucuronic acid concentrations both appear to be pivotal in regulating the synthesis of individual bilirubin glucuronides and hence the patterns of bilirubin conjugates excreted in bile.

Animals↗
PubMed1979

Hybrid glycosaminoglycans synthesized by monolayers of chick embryo arterial fibroblasts.

Monolayer cultures of arterial fibroblasts from 13-day chick embryonic aorta incorporated 35SO42- into glycosaminoglycans containing both glucuronic and iduronic acids. Bacterial chondroitinase ABC converted more than 98% of the 35SO4-labeled polymer to mono- or disaccharides, including (1) N-acetyl-D-galactosamine 4-sulfate, (2) delta 4,5-glucuronic acid 2- or 3-sulfate leads to N-acetylgalactosamine 6-sulfate, and (3) the unsaturated disaccharides normally obtained from chondroitin 4-sulfate and chondroitin 6-sulfate sequences. Chondroitinase AC converted only 77% of the 35SO4-labeled polymer to the same mono- and disaccharides and yielded, in addition, the following oligosaccharide products: (1) delta 4,5-glucuronic acid leads to N-acetylgalactosamine 4- or 6-sulfate leads to iduronic acid leads to N-acetylgalactosamine 6- or 4-sulfate; (2) N-acetylgalactosamine 4-sulfate leads to iduronic acid 2- or 3-sulfate leads to N-acetylgalactosamine 6-sulfate; (3) delta 4,5-glucuronic acid leads to N-acetylgalactosamine 4-sulfate leads to (iduronic acid leads to N-acetylgalactosamine 4-sulfate)2; (4) delta 4,5-glucuronic acid leads to N-acetylgalactosamine 4- or 6-sulfate leads to (iduronic acid leads to N-acetylgalactosamine 6- or 4-sulfate)2; (5) higher oligosaccharides containing iduronic acid and N-acetylgalactosamine 4-sulfate.

Animals↗
PubMed1984

Conjugation of 1-naphthol by human colon and tumour tissue using different experimental systems.

The metabolism of 1-naphthol, a model phenolic substrate, to its glucuronic acid and sulphate ester conjugates has been studied in short-term organ cultures of normal human colon and tumour tissue, subcellular fractions of these tissues, human colonic tumour cell lines and human colonic tumour xenografts. Normal colonic tissue, in short-term organ culture, formed more 1-naphthyl sulphate than glucuronic acid conjugates. In contrast the colonic tumours, under the same conditions, produced more 1-naphthyl beta-D-glucuronide than 1-naphthyl sulphate. A marked interindividual variation in sulphate ester and glucuronic acid conjugation was noted in both normal and tumorous colon. The conjugation of 1-naphthol was also investigated, using subcellular fractions, where the metabolism found with normal colon reflected that observed utilizing short-term organ culture, but that from colonic tumour samples did not. Cell lines derived from human colonic adenocarcinomas metabolised 1-naphthol almost exclusively to its glucuronic acid conjugate. Xenografts derived from human colonic tumours formed similar conjugates to surgical samples in culture. Thus somewhat different results were obtained dependent on the experimental model chosen. However, in all colonic tumour systems studied, when the cells remained intact and where tissue architecture was maintained, 1-naphthol was metabolised predominantly to its glucuronic acid conjugate.

Animals↗
PubMed1978

Inhibition of glucuronokinase by substrate analogs.

Glucuronokinase from Lilium longiflorum pollen was purified 30- to 40- fold on a blue dextran-Sepharose column. Substrate analogs were tested for inhibitory effects, and nucleotide substrate specificity of the enzyme was determined. Nine nucleotides were tested, and all were inhibitory when the substrate was ATP. ADP was competitive with ATP and had a K(i) value of 0.23 mm. None of the other nucleotide triphosphates could effectively substitute for ATP as a nucleotide substrate. Ten mm dATP and ITP reacted only 3% as rapidly as 10 mm ATP, while the rates for 10 mm GTP, CTP, UTP, and TTP were less than 1%. The glucuronic acid analogs, methyl alpha-glucuronoside, methyl beta-glucuronoside, beta-glucuronic acid-1-phosphate, and 4-O-methylglucuronic acid were tested as possible enzyme inhibitors. The three methyl derivatives showed little or no inhibition. The beta-glucuronic acid-1-phosphate was inhibitory, with 50% inhibition obtained at 1 to 3 mm depending on the concentration of the glucuronic acid. It is concluded that the glucuronic acid-binding site on the enzyme is highly selective.

Journal Article↗