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PubMed2004

UGD1, encoding the Cryptococcus neoformans UDP-glucose dehydrogenase, is essential for growth at 37 degrees C and for capsule biosynthesis.

We report the identification and disruption of the Cryptococcus neoformans var. grubii UGD1 gene encoding the UDP-glucose dehydrogenase, which catalyzes the conversion of UDP-glucose into UDP-glucuronic acid. Deletion of UGD1 led to modifications in the cell wall, as revealed by changes in the sensitivity of ugd1Delta cells to sodium dodecyl sulfate, NaCl, and sorbitol. Moreover, two of the yeast's major virulence factors-capsule biosynthesis and the ability to grow at 37 degrees C-were impaired in ugd1Delta strains. These results suggest that the UDP-dehydrogenase represents the major, and maybe only, biosynthetic pathway for UDP-glucuronic acid in C. neoformans. Consequently, deletion of UGD1 blocked not only the synthesis of UDP-glucuronic acid but also that of UDP-xylose. To differentiate the phenotype(s) associated with the UDP-glucuronic acid defect alone from those linked to the UDP-xylose defect, ugd1Delta mutants were phenotypically compared to strains from which the gene encoding UDP-xylose synthase (i.e., that required for synthesis of UDP-xylose) had been deleted. Finally, studies of strains from which one of the four CAP genes (CAP10, CAP59, CAP60, or CAP64) had been deleted revealed common cell wall phenotypes associated with the acapsular state.

Antibodies, Monoclonal↗

PubMed1976

Physiological and genetic regulation of the aldohexuronate transport system in Escherichia coli.

In Escherichia coli K-12, the specificity of the aldohexuronate transport system (THU) is restricted to glucuronate and galacturonate. There is a relatively high basal-level activity in uninduced wild-type or isomeraseless strains. Supplementary activity is obtained with the inducers mannonic amide (five-fold), galacturonate (fourfold), fructuronate (fivefold), and tagaturonate (sevenfold). Specific THU- mutants were selected as strains unable to grow on either aldohexuronate but able to grow on fructuronate or tagaturonate. The remaining transport activity in uninduced and induced THU- starins represents less than 20% of that found in the wild type. Conjugation and transduction experiments indicate that all of the THU- mutations are located in a unique locus, exuT, half-way between the tolC (59 min) and argG (61 min) markers. exuT is closely linked to the uxaC-uxaA operon (60 min) and to the regulatory gene exuR (60 min), which controls the above-mentioned operon and the uxaB operon (45 min). Growth on either aldohexuronate and transport activity are fully recovered when exuT mutants are allowed to revert to exuT+ on galacturonate or glucuronate. Reversion on glucuronate alone may lead to the mutational derepression of the 2-keto-3-deoxygluconate transport system, which is uninducible in the wild type, which also takes up glucuronate, and whose structural gene belongs to the kdg regulon. Such strains, which remain unable to grow on galacturonate, are exuT and kdgR (constitutive allele of the regulatory gene kdgR of the kdg regulon). THU activity is superrepressed in an exuR mutant in which the uxaC-uxaA operon and the uxaB operon are superrepressed; exuR+/exuR merodiploids are also superrepressed. In a thermosensitive exuR mutant in which the above-mentioned operons are constitutive at 42 degrees C, the THU activity is fully derepressed at this temperature. On the basis of these and other results, it is concluded that THU is coded for by the structural gene exuT, which is negatively controlled by the exuR gene product and which probably belongs to an operon distinct from the uxaA-uxaC operon.

Alcohol Oxidoreductases↗

PubMed1983

Effects of activation of UDP-glucuronyl transferase on metabolism of benzo[a]pyrene with rat liver microsomes.

The effects of Brij 58 on microsome-mediated benzo[a]pyrene (BP)-metabolism were investigated. At a concentration of 0.01%, which did not decompose the microsomal mixed-function oxidase system, Brij 58 increased UDP-glucuronyl transferase activity on 3-hydroxy-BP to 560% of the control level. Quantitative changes of BP-metabolites by glucuronidation were found by high performance liquid chromatography. On incubation in the presence of UDP-glucuronic acid, the amounts of phenols decreased but the amounts of dihydrodiols did not change. Brij 58 enhanced the decrease in phenol metabolites. Unexpectedly, the binding of BP-metabolites to DNA was found to be higher in the presence of UDP-glucuronic acid, and Brij 58 also enhanced the binding in the presence of UDP-glucuronic acid. The ratio of bound adducts with BP-7,8-dihydrodiol-9,10-oxide to those with 9-hydroxy-BP-4,5-oxide, which were separated by Sephadex LH-20 column chromatography, was increased by UDP-glucuronic acid and further enhanced by Brij 58. These results are discussed in relation to the role of glucuronidation in metabolism of BP.

Animals↗

PubMed1986

Identification of novel N-glucuronides in rat bile after administration of 450191-S, a 1H-1,2,4-triazolyl benzophenone derivative.

5-[2-Aminoacetamido)methyl]-1-[p-chloro-2-(o-chlorobenzoyl)phenyl] -N,N-dimethyl-1H-s-triazole-3-carboxamide hydrochloride dihydrate (450191-S) is a ring-opened derivative of 1,4-benzodiazepine, which is activated by desglycylation and subsequent cyclization. After 450191-S administration, rat bile contained three novel conjugates which released active metabolites possessing the 1,4-benzodiazepine structure through beta-glucuronidase hydrolysis. Since the released metabolites have no functional groups to conjugate with glucuronic acid, we speculated that the aglycone might be the ring-opened form of 1,4-benzodiazepine which spontaneously cyclizes after the release of glucuronic acid. This possibility was tested by chemically reducing the ketone group of the ring-opened 1,4-benzodiazepine glucuronate conjugates, which would prevent the spontaneous ring closure reaction after the release of the glucuronic acid moiety. The NaBH4 reduction of the ketone of the benzophenone moiety of the conjugates and subsequent treatment with beta-glucuronidase allowed identification of the reduced aglycones with authentic samples using gas chromatography-mass spectrometry.

Animals↗

PubMed1984

Kinetic properties of UDP-glucuronosyltransferase(S) in different membranes of rat liver cells.

Glucuronidation of 4-nitrophenol, borneol and morphine occurred in rough and smooth endoplasmic reticulum, Golgi apparatus and plasma membranes of rat liver cells. In all fractions, prior fixation of either substrate (UDP-glucuronic acid or the aglycone) enhanced the affinity for the second substrate. Whatever the membrane, glucuronidation of 4-nitrophenol was characterized by high Vmax and high affinity for UDP-glucuronic acid. On the other hand, glucuronidation of borneol exhibited a lower Vmax and a lower affinity for UDP-glucuronic acid. In the endoplasmic reticulum, conjugation of morphine had a low Vmax, but the enzyme had high affinities for both UDP-glucuronic acid and the aglycone.

Animals↗

PubMed1986

Mechanisms behind the inhibitory effect of ethanol on the conjugation of morphine in rat hepatocytes.

Liver microsomes were isolated by calcium aggregation, and isolated hepatocytes from male Wistar rats were prepared according to a two-step Ca++-free collagenase perfusion method. With the hepatocytes maximal inhibition of glucuronidation (about 40%) was reached at 10 mM ethanol after incubation at 37 degrees C for 60 min. UDP-glucuronic acid concentration and energy charge in the hepatocytes also did decrease maximally (about 90 and 50%, respectively) and the amount of UDP-glucose was tripled in the presence of 10 mM and higher concentrations of ethanol. The alcohol dehydrogenase inhibitor 4-methylpyrazole abolished ethanol-induced inhibition of morphine glucuronidation in the hepatocytes. Acetaldehyde (250-50 microM) and the pH decrease induced by ethanol did not reduce morphine-3-glucuronide formation by the cells. Cellular uptake of morphine and excretion of morphine metabolites were similar in the absence and presence of ethanol. Ethanol (60 mM) did not affect the glucuronidation of morphine (1.7 mM added) during a 30-min incubation at 37 degrees C with the microsomes (UDP-glucuronic acid, 5 mM). When the concentration of UDP-glucuronic acid in the microsomes was lowered from 1 to 0.1 mM, the decrease in morphine-3-glucuronide formation was similar to that observed in cells. The data indicate that the inhibition by ethanol of morphine glucuronidation was due to decreased levels of UDP-glucuronic acid. The mechanism is likely to be inhibition of UDP-glucose dehydrogenase activity by ethanol from increased intracellular NADH/NAD ratio accompanying ethanol oxidation.

Animals↗

PubMed1981

A novel, highly modified, bacteriophage DNA in which thymine is partly replaced by a phosphoglucuronate moiety covalently bound to 5-(4',5'-dihydroxypentyl)uracil.

Bacteriophage SP-15, which infects Bacillus subtilis, contains a highly modified DNA in which 62% of its thymine residues are replaced by 5-(4',5'-dihydroxypentyl)uracil to which is attached a phosphoglucuronate via a phosphodiester linkage to one of the hydroxyl groups of the pentyl side chain. Glucose is also bound to this residue probably by glycosidic linkage to the other hydroxyl group of the pentyl side chain. In 0.3 M KOH at 37 degrees C, glucuronic acid 1-phosphate is slowly released from this DNA. After enzymatic or acid-induced dephosphorylation, this sugar was identified by chromatography in two thin layer chromatography systems, conversion to glucuronolactone under conditions known to lactonize glucuronic acid, and reaction in four colorimetric assays for hexuronic acids. Phage SP-15 DNA is the first DNA found to have a uronic acid moiety or a phosphate which is not part of the phosphodiester backbone. The glucuronic acid phosphate might be derived from uridine pyrophosphoglucuronic acid, whose glucuronic acid moiety is normally destined for synthesis of teichuronic acid in the host cell wall.

Bacillus subtilis↗

PubMed1975

Biosynthesis of heparin. Studies on the microsomal sulfation process.

Nonsulfated, partially N-deacetylated, 14C-labeled polysaccharide was formed by incubation of a mouse mastocytoma microsomal fraction with UDP-[14C]glucuronic acid and UDP-N-acetylglucosamine. After 60 min at 37 degrees the incorporation of radioactivity was interrupted, and sulfation was initiated, by the addition of unlabeled UDP-glucuronic acid and 3-phosphoadenylylsulfate, respectively. After varying periods of incubation with the nucleotide sulfate, polysaccharide was isolated and analyzed by chromatography on DEAE-cellulose. During incubation with 3-phosphoadenylylsulfate, sulfated 14C-polysaccharides were formed, while the nonsulfated 14C-polysaccharide was eliminated, each type of component retaining the same elution position throughout the entire sulfation period (60 min). No accumulation of low sulfated material was observed. Fully sulfated heparin was detected after only 0.5 min of sulfation. These results suggest that the sulfation of heparin is a rapid process, involving a limited number of polysaccharide molecules at a time. Besides heparin-like products, having both N- and O-sulfate groups, and [14C]iduronic acid as predominant uronic acid constituent, an additional sulfated component was detected, having N-sulfate but no O-sulfate groups. This novel polysaccharide contained [14C]glucuronic acid but little or no [14C]iduronic acid. Previous studies have shown that L-iduronic acid residues in heparin are formed by C-5 inversion of D-glucuronic acid units, previously incorporated into the polymer; the inversion reaction requires concomitant sulfation of the polymer. The present results demonstrate that the formation of iduronic acid residues is related to the sulfation of hydroxyl groups. Pulse-chase experiments with 3-phosphoadenylyl[35S]sulfate indicated that the N-sulfated polymer, lacking O-sulfate groups, could be converted into heparin-like products by further sulfation of hydroxyl groups only. The biosynthesis of heparin may thus involve a pathway, in which the formation of N-sulfated intermediates precedes the introduction of O-sulfate groups.

Animals↗

PubMed1982

Polyuronide biosynthesis by cell-free extracts of Mucor rouxii.

Cell-free extracts from Mucor rouxii contain enzymes that catalyse the synthesis of uridine diphosphate glucuronic acid (UDPGlcA) from UDPglucose and the incorporation of glucuronic acid from UDPGlcA into polymer(s). Two different polyuronide fraction isolated from the cell walls of this fungus were used as primers. Mucoran, a heteropolymer, was much more efficient than mucoric acid, which is largely a homopolymer of D-glucuronic acid. The primer ability of native cell walls was comparable to that of mucoric acid. Most of the glucuronosyltransferase activity in the cell-free extract was found in a 20000 g particulate fraction. Optimum pH for polyuronide synthesis was 7.0. Mn2+ or Mg2+ stimulated incorporation of GlcA. The products synthesized from mucoric acid and mucoran primers were different and yielded different disaccharides upon hydrolysis.

Biopolymers↗

PubMedDate not supplied

Bilirubin mono- and diglucuronide formation by human liver in vitro: assay by high-pressure liquid chromatography.

Bilirubin diglucuronide, the major pigment in human bile is formed in two steps. Bilirubin is converted to bilirubin monoglucuronide by transfer of the glucuronosyl moiety of uridine diphosphoglucuronic acid catalyzed by the microsomal enzyme, uridine diphosphoglucuronate glucuraonosyl transferase (UDP glucuronyl transferase, EC 2.4.1.17). Bilirubin monoglucuaronide is converted to bilirubin diglucuronide in vitro by two enzymatic mechanisms: (a) UDP glucuronyl transferase-mediated transfer of a second mole of glucuronic acid form UDP-glucuronic acid to bilirubin monoglucuronide; (b) dismutation of 2 moles of bilirubin monoglucuronide to 1 mole of bilirubin diglucuronide and 1 mole of unconjugated bilirubin, catalyzed by bilirubin monoglucuronide dismutase (bilirubin glucuronoside glucuronosyl transferase EC 2.4.1.95). Assay methods for the three enzymatic mechanisms in human liver homogenate by high-pressure liquid chromatography analysis of underivatized bilirubin tetrapyrroles have been developed. UDP glucuronyl transferase was activated in five human liver homogenates with digitonin, Triton X-100, or UDP-N-acetylglucosamine. Greatest activation was observed with Triton X-100. The pH optimum for conversion of bilirubin to bilirubin monoglucuronide was 7.4, and UDP glucuronyl transferase activity was 625 +/- 51 nmoles per 20 min per gm liver. At high initial bilirubin concentrations (342 microM), the product of UDP glucuronyl transferase assay with bilirubin as substrate was predominantly bilirubin monoglucuronide. At lower initial bilirubin concentrations (6.5 to 34 microM), up to 15% bilirubin diglucuronide was formed. Glucuronyl transferase-mediated UDP glucuronic acid-dependent conversion of bilirubin monoglucuronide to diglucuronide was assayed using UDP-14-C-glucuronic acid. The pH optimum was 7.4, and the rate was 21 +/- 7 nmoles per gm liver per 20 min. The rate of bilirubin diglucuronide formation by enzymatic dismutation of bilirubin monoglucuronide was 470 +/- 112 nmoles per gm liver per min. The pH optimum was 6.6. The products of enzymatic dismutation were of the IX alpha configuration.

Bilirubin↗

PubMedDate not supplied

Substrates and products of purified rat liver bilirubin UDP-glucuronosyltransferase.

To determine whether the isoform of UDP-glucuronosyltransferase which catalyzes the formation of bilirubin monoglucuronide also mediates the formation of bilirubin diglucuronide and other specific sugar conjugates of bilirubin, Wistar rats were treated with clofibrate (300 mg per kg i.p. X 7 days); this resulted in a 200% increase in hepatic transferase specific activity for bilirubin. Proteins from hepatic microsomal fractions were solubilized, and the transferase isoform with activity toward bilirubin was purified by a combination of chromatofocusing, affinity chromatography and hydrophobic chromatography, to apparent homogeneity as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified isoform catalyzed the formation of monoglucuronide and diglucuronide (with UDP-glucuronic acid as a cosubstrate), and glucoside and xyloside (with UDP-glucose and UDP-xylose as respective cosubstrates) of bilirubin and glucuronidation of the carcinogen metabolite 4'-hydroxydimethylaminoazobenzene. It also catalyzed the conversion of bilirubin monoglucuronide to diglucuronide (with UDP-glucuronic acid as cosubstrate, pH optimum 7.8), to mixed glucuronide-glucoside conjugate (with UDP-glucose as a cosubstrate) and to unconjugated bilirubin (with UDP as a cosubstrate, pH optimum 5.5). Each transferase activity was copurified at each purification step. Results of enzyme kinetic studies suggest that UDP-glucuronic acid, UDP-glucose and UDP-xylose recognize a common site. Transferase activities toward bilirubin were not detectable in homozygous Gunn rats liver microsomal fractions; in heterozygous Gunn rats, these activities were reduced by 40 to 60%. The results suggest that conjugation of bilirubin with glucuronic acid, glucose or xylose is catalyzed by a single transferase isoform.

Animals↗

PubMed1986

Attachment and long term survival of adult rat hepatocytes in primary monolayer cultures: comparison of different substrata and tissue culture media formulations.

Long-term monolayer cultures of adult rat hepatocytes were tested for their ability to glucuronize phenol red and to maintain initial levels of cell proteins, glucose consumption, and lactic acid production. Lactate dehydrogenase leakage served as an index of culture status because a high value indicates cell death. Three tissue culture (TC) media formulations were the main variables introduced to determine ideal conditions for cell survival in vitro. Investigations of long-term cultures were preceded by studies of hepatocyte attachment to polystyrene surfaces. This attachment was influenced by the amount of substrate deposited and the number of cells seeded, but not by the uniformity of the substrate coating. A statistical analysis of our data revealed that in the absence of fetal bovine serum (FBS), air dried collagen (ADC) and Biomatrix (BMX) were superior to saline precipitated collagen and fibronectin as attachment substrates. In the presence of 10% FBS, all of the substrates performed equally. Chee's Medium (CEM) proved to be the best for preserving cell proteins over a time course of 28 d and Williams' E medium also performed adequately up to 14 d. The glucuronization of phenol red was at 50% of initial values at Day 7 in CEM-ADC hepatocytes in contrast to 30% for cells in Williams' E medium and 5% for cells grown in Waymouth's. At 14 d glucuronization was still present at 40% of original values in CEM-ADC cells but had ceased in the other two media. When BMX was used, none of the TC media supported glucuronization levels comparable to ADC cells.

Animals↗

PubMed2003

Compositional difference of the exopolysaccharides produced by the virulent and virulence-deficient strains of Xanthomonas oryzae pv. oryzae.

Xanthomonas oryzae pv. oryzae, the bacterial blight pathogen of rice, is known to produce phytotoxic polysaccharides. The extracellular polysaccharide (EPS) was isolated from virulent (BXO1) and virulence-deficient gum G mutant (BXO1002) strains of X. oryzae pv. oryzae and characterized using fourier transform infrared (FT-IR) and nuclear magnetic resonance (NMR). Data from the FT-IR suggested that the aldehyde (R-CHO) group and C=O of acid anhydride are present in BXO1 but absent in BXO1002. The (1)H-NMR spectra showed the presence of an acetyl amine of hexose or pentose, free amines of glucose, an beta-anomeric carbon of hexose and pentose, hydrogen next to hydroxyl group, an acetyl amine of hexose and pentose in the polysaccharides of both BXO1 and BXO1002, and the absence of alpha-anomeric carbon of hexose or pentose and the glucuronic acid in the polysaccharides produced by BXO1002. The test for glucuronic acid also confirmed the absence of glucuronic acid in the polysaccharides of BXO1002 and the presence glucuronic acid (32 microg/mg) in the polysaccharides produced by BXO1.

Bacterial Proteins↗

PubMed1984

Kinetic studies of latent microsomal UDP-glucuronyltransferases. Kinetics of glucuronidation in intact and perturbant-treated membranes.

Double-reciprocal plots (with UDP-glucuronate as varied substrate) of the rate of glucuronidation of p-nitrophenol by the latent UDP-glucuronyltransferases of intact guinea pig and rat liver microsomal membranes (prepared with 154 mM KCl and 0.25 M sucrose) were continuously curved concave-downwards. Good fits to the kinetic data were obtained by using two different calculation methods which assume that two forms (high K and low K) of the transferase catalyse the reaction simultaneously. No evidence of cooperativity in binding of UDP-glucuronate to the enzyme was found. When latency of the enzymes of these preparations was destroyed by disrupting the membranes with Triton X-100 or lysophosphatidylcholine, double-reciprocal plots were linear. With guinea pig membranes, lysophosphatidylcholine generated an activated single-enzyme form obeying the simple Michaelis-Menten rate law; K for the activated species was close to that (K1) for the native low K form and its value of V was greater than the combined maximum velocities (V1 + V2) of the two forms in intact membranes. With rat membranes, both perturbants produced a single activated form also with V greater than (V1 + V2) and with K2 greater than K greater than K1. These results are discussed and are consistent with the view of transferase latency which envisages that there are two populations (buried and exposed) of enzyme molecules in intact microsomal membranes. The effects of membrane perturbants on the kinetic parameters of the two native transferase forms were assessed by accounting for the possibility that the reactivity of the buried transferase is controlled by the rate of transport of UDP-glucuronate across the membrane matrix. The data are compatible with a model which supposes that UDP-glucuronate gains access to the buried population by a process with the kinetic characteristics of a facilitated transport system.

Animals↗

PubMed1992

Insect glycolipids.

Systematic elucidations of the chemical structures of glycosphingolipids from members of the phyla Arthropoda (class:insecta) of the Protostomia have shown several characteristic differences to those of the Deuterostomia, e.g., the Vertebrata. The ceramide constituents of the arthropod sphingolipids are C14:1- and C16:1-sphingoid and a majority of saturated C18-, C20-, C22- and C24-fatty acids. The GSL of the dipteran insects, L. caesar and C. vicina, consist of a series of homologous compounds of increasing sugar chain length. The carbohydrate moieties of these arthropod GSL are all derived from one unique neutral sugar core sequence, the arthro-series. The longest member known, an arthrononaosylceramide has the structure Gal beta,3GalNAc beta,3GlcNAc beta,3Gal beta,3GalNAc alpha,4GalNAc beta,4GlcNAc beta,3Man beta,4Glc beta Cer. Three pentahexosylceramides have been characterized that differ in their nonreducing terminus, i.e., IV4GalNAc alpha-Ap4Cer, IV3Gal alpha-Ap4Cer and IV3Gal beta-Ap4Cer. A large repertoire of structural variations of the arthro-series GSL is created by two types of derivatisations of the neutral carbohydrate core: addition of a zwitterionic 2-aminoethylphosphate group in phosphodiester linkage to the 6-position of the III-N-acetylglucosamine of the arthrotriaosylceramide core and/or substitution of a terminal galactose in 3-position by a glucuronic acid residue. The latter acidic arthro-series GSL were given the name arthrosides. Preliminary results have indicated that the blowfly GSL component distribution is strongly organ specific. GSL of various larval organs are distinguished by the length of their neutral core carbohydrate chain, as well as, the degree of PEtn- and GlcA-substitutions. Normal human sera contain antibodies that recognize epitopes on arthro-series GSL. Thereby, sites of the neutral carbohydrate chain, as well as, the zwitterionic and acidic epitopes are involved in the immune recognition. In a pathological condition, a M-protein of patients with autoallergic demyelinating peripheral neuropathy was found to react strongly with a glucuronic acid-containing molecular structure on arthrosides. Some indication was obtained for a possible functional role of glucuronic acid in dipteran-cell adhesion by using a mouse monoclonal antibody, mAb CAF-I. This antibody, directed against an arthroside epitope involving glucuronic acid, could induce an in vitro detachment of insect cells from one another or from their substratum. Obviously, present research on the GSL of insects is only at an early stage. Nevertheless, from the results available so far it was become increasingly apparent that a new chapter in our knowledge of GSL structures has been opened.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1987

Glycoconjugates as noninvasive probes of intrahepatic metabolism: I. Kinetics of label incorporation with evidence of a common precursor UDP-glucose pool for secreted glycoconjugates.

Conditions are described under which hepatic metabolism of administered labeled sugars can be monitored in the intact rat by measuring output of label from the liver in two secreted glycoconjugates derived from intracellular UDP-glucose, namely glucuronic acid linked to an administered drug (acetaminophen), and galactose secreted in glycoproteins. Rats with indwelling intrajugular catheters were given constant infusions of acetaminophen at nontoxic doses, and acetaminophen-glucuronide was isolated from rat urine by HPLC. When an unprimed continuous infusion of [U-14C]glucose or [1-3H]galactose is begun during acetaminophen infusion, labeling of urinary acetaminophen-glucuronide attains a plateau with an apparent half-life of 2.1 h from labeled glucose as the 14C-donor, and 1.2 h from labeled galactose. The times required to attain steady-state are attributable either to formation or to hepatic secretion of the glucuronyl conjugate rather than to mixing in extracellular pools or to delays in renal excretion since the half-time of urinary excretion of intravenously injected acetaminophen-glucuronide is only 21 minutes. Consequently, the excreted glucuronide provides noninvasive evidence of the recent specific activity in the precursor pool of hepatic UDP-glucose feeding glucuronic acid. The second glycoconjugate used for comparison was galactose, isolated from the acute-phase plasma protein alpha 1-acid glycoprotein. This represents hepatic UDP-galactose labeling. Comparison of the labeling patterns of secreted galactose and glucuronic acid demonstrates that they are derived from the same intracellular pool of UDP-glucose. This is in contrast to the patterns of labeling of hepatic glycogen from (1-3H) and [U-14C]glucose which have been shown to diverge from those of secreted glucuronic acid.

Acetaminophen↗

PubMed1988

Glycoconjugates as noninvasive probes of intrahepatic metabolism: III. Application to galactose assimilation by the intact rat.

A tracer methodology has been developed for noninvasive assessment of intrahepatic metabolism of administered labeled sugars. In this procedure, we measure the output of the label from the liver in two glycoconjugates derived from hepatic UDP-glucose, namely, glucuronic acid formed through UDP-glucuronic acid and excreted in the urine following acetaminophen administration, and galactose formed through UDP-galactose and then secreted in the carbohydrate portion of glycoproteins in the plasma. Comparison of the distribution of label from various sugar precursors in these end-products can indicate exchanges between hepatic UDP-glucose, UDP-galactose, and UDP-glucuronic acid. In this study we apply the technique to explore whether the enzyme UDP-galactose-4-epimerase catalyzing the step UDP-galactose to UDP-glucose is nonequilibrium and therefore potentially has a regulatory role for utilization of free galactose. The specific activity in the two glycoconjugates was compared when either [1-3H]galactose or [U-14C]glucose was the infused precursor sugar. In rats under a variety of conditions (fasting, oral refeeding, intravenous administration of galactose), label from [1-3H]galactose accumulated in glycoprotein-bound galactose much more than in acetaminophen-bound glucuronic acid, in comparison to label from [U-14C]glucose, demonstrating limitation of the rate of transfer from UDP-galactose to UDP-glucose at the epimerase step. Accordingly, epimerase is suggested to have a regulatory role in the galactose assimilation pathway.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1994

The relationship between proteoglycan synthesis in Swarm chondrocytes and pathways of cellular energy and UDP-sugar metabolism.

The effect of anaerobic culture conditions and various metabolic inhibitors on 35S-proteoglycan synthesis, UDP-sugar pools, and the ATP pool were investigated in confluent, primary, day 1 cultures of Swarm chondrosarcoma chondrocytes. (i) Incubation under a nitrogen atmosphere for 6 h did not affect 35S-proteoglycan synthesis or the pool size for UDP-glucuronate, other UDP-sugars, or ATP. Incubation with 5 mM sodium azide brought about a 40% reduction of proteoglycan synthesis in the first 30 min but no further change over the subsequent 90 min. UDP-Glucuronate, other UDP-sugar pools, and the ATP level were not affected by azide treatment. The results indicate that proteoglycan synthesis and its energy requirements can be supported entirely by anaerobic metabolism in these cells. (ii) 35S-Proteoglycan synthesis, UDP-sugar production, and nucleotide triphosphate pools were inhibited in a concentration-dependent fashion with sodium iodoacetate. A > 70% reduction of the ATP pool after 30 min treatment suggests that glycolysis is a major target for iodoacetate. Lactate production was inhibited by 40% after 3 h treatment with 10(-4) M iodoacetate. (iii) Glutamine deprivation resulted in a 60% contraction in the UDP-N-acetylhexosamine pool and markedly inhibited 35S-proteoglycan and 3H-protein synthesis. At the same time the UDP-glucose pool expanded to 200%, but the UDP-glucuronate pool was unchanged. The sum of the UDP-N-acetylhexosamine and UDP-hexose pools remained constant. Restoration of glutamine to previously depleted cultures resulted in excessive expansion of the UDP-N-acetylhexosamine pool and excessive contraction of the UDP-hexose pool before both adjusted to normal levels. The UDP-xylose pool was very small. No increases were observed during inhibition of proteoglycan synthesis induced by glutamine deprivation. (iv) 6-Diazo-5-oxo-L-norleucine (DON), a glutamine analogue and amino transferase inhibitor, induced a further contraction of the UDP-N-acetylhexosamine pool and a further decrease in proteoglycan synthesis in glutamine-deprived cultures. Thus cultures use endogenous glutamine during exogenous glutamine deprivation. DON unaccountably stimulated expansion of the UDP-glucuronate pool by 180%, irrespective of whether glutamine was present or not.

Aggrecans↗