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The glucuronic acid adducts of 1-naphthol, 2-naphthol and 4-methylumbelliferone activate microsomal UDP-glucuronyltransferase (EC 2.4.1.17) when the enzyme is assayed with p-nitrophenol as aglycone. Phenyl glucuronide and oestriol 3beta-glucuronide also activate UDP-glucuronyltransferase. but to a lesser extent. Activation by glucuronides is not dependent on metal ions, but is blocked by prior treatment of microsomal fractions with p-chloromercuribenzoate. The kinetic mechanism of activation is concluded to be an increase in the affinity of the enzyme for UDP-glucuronic acid. Activation by 1-naphthyl glucuronide, at high concentrations of p-nitrophenol, is not affected by 1-naphthol. Apparently 1-naphthyl glucuronide activates the preparation by binding at a site that is separate from the site of glucuronidation of 1-naphthol. Further evidence for the existence of distinct effector sites for the glucuronides was provided by the finding that activation by glucuronides is inhibited competitively by aglycone glucosides. These glucosides do not inhibit the rate of glucuronidation of p-nitrophenol in the absence of glucuronide adducts, nor do they alter the rate of glucuronidation of 1-naphthol. When UDP-glucuronyltransferase is assayed with 1-naphthol as aglycone it is activated by p-nitrophenyl glucuronide, 4-methyl-umbelliferyl glucuronide and under appropriate conditions by its own glucuronide. These activations are similarly inhibited by aglycone glucosides. p-Nitrophenyl glucuronide also stimulates the rate of glucuronidation of o-aminophenol, o-aminobenzoate and bilirubin.
An endogenous compound (included in the fraction of uremic toxins often called the "uremic middle molecules") was separated from plasma of uremic patients and urine from normal persons. As elucidated by mass spectrometry, enzymatic hydrolysis, and 1H, 13C nuclear magnetic resonance, it appears to be a conjugate of o-hydroxybenzoic acid with glucuronic acid. Its presence in urine of healthy subjects indicates its physiological character.
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A new active site-directed photoaffinity analogue, [beta-32P]5-azido-UDP-glucuronic acid (UDP-GlcA), was enzymatically synthesized from [beta-32P]5-N3UDP-Glc using UDP-glucose dehydrogenase. The product was characterized by its mobility on ion exchange and two thin-layer chromatographic systems, by its UV absorbance at 288 nm, and the loss of this absorbance after UV irradiation of the compound. Photoincorporation of [beta-32P]5-N3UDP-GlcA into bovine liver UDP-Glc dehydrogenase (EC 1.1.1.22) was saturable with an apparent Kd of 12.5 microM, and was inhibited by the known active-site effectors UDP-GlcA, UDP-Glc, and UDP-xylose. When human liver microsomes with known UDP-glucuronosyltransferase (EC 2.4.1.17) activities were photolabeled with [beta-32P]5-N3UDP-GlcA, major photolabeled bands of 35-37 and 50-54 kDa were detected. When rat liver microsomes from phenobarbital-injected rats were photolabeled with [beta-32P]5-N3UDP-GlcA, there was a marked increase in photoincorporation of a 51-kDa protein as compared with control animals. Evidence is presented which suggests that the photolabeled 51-54-kDa proteins in the liver microsomes from both tissues are UDP-glucuronosyltransferase and that [beta-32P]5-N3UDP-GlcA represents a new alternative approach in the study of UDP-glucuronosyltransferase and other UDP-GlcA-utilizing enzymes.
A newly automated method for the detection of uronic acids, particularly glucuronic acid is described. Data showing the limitations of this method are also presented. The advantages of the method are: (1) greatly increased sensitivity compared to the carbazole methods and increased range; (2) lowered interference from other sugars, proteins and salts; (3) variability to allow small sample volumes and/or continuous flow; (4) a more stable color reagent solution.
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The mucopolysaccharide, hyaluronic acid, is an important component of both mammals and pathogenic streptococci. This high molecular weight polymer is synthesized by a membrane-associated, multisubunit hyaluronate synthase which utilizes UDP-glucuronic acid and UDP-N-acetylglucosamine as substrates. Using the photoaffinity probe, [beta-32P]5-azido-UDP-glucuronic acid, three streptococcal membrane proteins (42, 33, and 27 kDa) specifically photoincorporated this probe. Labeling of these proteins was enhanced in the presence of UDP-N-acetylglucosamine, whereas UDP-galactose or UDP-glucose had no effect on incorporation. UDP-glucuronic acid inhibited the labeling of the three proteins in a dose-dependent manner. Detergent-solubilized membrane proteins from transposon-inactivated hyaluronic acid capsule mutants no longer incorporated the probe. This was also the case when membranes from stationary phase organisms were tested. Finally, glucuronic acid no longer was incorporated into high molecular weight hyaluronic acid with either the mutant or stationary phase preparations. Further biochemical analysis will be required to demonstrate the exact role each of the proteins play in hyaluronic acid biosynthesis.
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Large, rapid decreases in hepatic UDP-glucuronic acid concentrations occur in rats following exposure to myriad chemicals. In fact, 80% reductions in UDP-glucuronic acid occur within minutes after exposure to inhalation anesthetics. The present study was designed to determine whether this decrease in hepatic UDP-glucuronic acid may be due to (a) a decrease in the precursor UDP-glucose; (b) decreased activity of UDP-glucose dehydrogenase, which oxidizes UDP-glucose to UDP-glucuronic acid; (c) increased activity of UDP-glucuronosyltransferases; or (d) increased activity of nucleotide pryophosphatase, which degrades UDP-glucuronic acid to glucuronic acid-1-phosphate. Exposure to halothane, isoflurane and sevoflurane decreased UDP-glucuronic acid concentrations by 40-52% as compared to that in unanesthetized control mice. No sex-dependent or anesthetic-induced effects of UDP-glucose levels and the activities of UDP-glucose dehydrogenase and UDP-glucuronosyltransferase were observed. Nucleotide pyrophosphatase activity was increased by 47-65% in female mice after inhalation of halothane, isoflurane and sevoflurane. The apparent Vmax for hydrolysis of 4-nitrophenol thymidine 5'-monophosphate ester by nucleotide pyrophosphatase was increased by 56-80% in female mice, whereas the apparent Km was unchanged. These alterations in nucleotide pyrophosphate kinetics may be responsible, in part, for the marked decrease of hepatic UDP-glucuronic acid concentrations by the volatile anesthetics.
In some patients with neuropathy and plasma cell dyscrasia, the serum IgM M-proteins are known to bind to the myelin associated glycoprotein and to peripheral nerve glycolipids. We have isolated two acidic glycolipids which bind to the M-protein from human cauda equina by DEAE-Sephadex, Iatrobeads, and high performance liquid column chromatographies. The major acidic glycolipid migrated between GM1 and GD1a and the minor acidic glycolipid migrated between GD1a and GD1b. Their structures were elucidated by sugar analysis, enzymatic digestion, mild acid hydrolysis, permethylation, fast atom bombardment mass spectrometry, and NMR studies. Their core structure was confirmed to be paragloboside by high performance thin-layer chromatography-immunostaining using anti-paragloboside monoclonal antibody. Both acidic glycolipids lacked sialic acid but contained sulfated glucuronic acid as their acidic moiety. The sulfate group in the glucuronic acid was established by periodate oxidation and permethylation studies to be attached to the 3 position. The structures of the two acidic glycolipids are therefore consistent with the following: IV3GlcUA(3-sulfate)nLcOse4Cer and VI3GlcUA(3-sulfate)nLcOse6Cer. Additionally, the free carboxyl group on the glucuronic acid residue was shown to be necessary to bind the IgM M-proteins from neuropathy patients.
Because hepatic UDP-glucuronic acid levels decrease upon exposure to volatile anesthetics, the present study was designed to determine the mechanism by which enflurane decreases UDP-glucuronic acid in mice by measuring the concentrations of intermediates and the activities of enzymes in the UDP-glucuronic acid pathway. UDP-glucuronic acid concentrations were decreased by 40% in both male and female mice after 10 min of enflurane-induced narcosis. Concentration of UDP-glucose and the activities of diethylstilbestrol UDP-glucuronosyltransferase and UDP-glucose dehydrogenase were not affected by enflurane treatment. In contrast, nucleotide pyrophophatase activity was increased approximately 50% in both sexes. Thus, the decrease in hepatic UDP-glucuronic acid upon exposure of mice to enflurane is probably due to increased degradation by nucleotide pyrophosphatase.
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.
1. Glucuronic acid was excreted in the urine of the brushtail possum, Trichosurus vulpecula, in response to dietary terpenes (essential oils found in Eucalyptus foliage). 2. The relationship between urinary glucuronic acid and the estimated terpene content of the diet was not equimolar, but varied from 1-17%, with levels of 5-39% found in animals maintained on terpenes or eucalypt leaves for several weeks. 3. Blood levels of ascorbate and the turnover rate of ascorbate were increased in the brushtail possum in response to dietary terpenes. 4. The presence of an active glucuronic acid pathway and associated glucose-ascorbate-glucose cycle was postulated for the brushtail possum and the other arboreal marsupials, Pseudocheirus peregrinus (common ringtail possum) and Petauroides volans (greater glider). 5. By means of these pathways these animals may utilise the high ascorbate content of Eucalyptus leaves to conserve glucose required for synthesis of glucuronic acid used for conjugation of dietary terpenes.
Salicylamide, clofibric acid, valproic acid and chloramphenicol are all known to be glucuronidated. The effects of these compounds on the hepatic concentration of UDP-glucuronic acid, the cosubstrate for glucuronidation, were studied in mice and found to lower hepatic UDP-glucuronic acid in a dose- and time-dependent fashion. Valproic acid, chloramphenicol, salicylamide and clofibric acid depleted hepatic UDP-glucuronic acid significantly at dosages as low as 0.5, 0.5, 0.75 and 4.0 mmol/kg, respectively. Hepatic UDP-glucuronic acid was decreased by 90% by valproic acid, 91% by chloramphenicol, 98% by salicylamide and 41% by clofibric acid (after dosages of 4, 2, 1 and 5 mmol/kg, respectively). UDP-glucuronic acid was depleted maximally by 15 after drug administration. Salicylamide also was used as a model compound to study the effect of drug loading on the hepatic concentrations of UDP-glucose and glycogen, precursors of UDP-glucuronic acid. It was found that, in addition to UDP-glucuronic acid, salicylamide (4 mmol/kg) also depleted UDP-glucose and glycogen by about 50%. These data suggest that large drug loads cause an increase flux through the glucuronic acid pathway and that hepatic UDP-glucuronic acid is consumed more rapidly than it is produced.