Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.2.1.31 (beta-glucuronidase)
7,680 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Plants possess two alternative biochemical pathways for sucrose (Suc) degradation. One involves hydrolysis by invertase followed by phosphorylation via hexokinase and fructokinase, and the other route-which is unique to plants-involves a UDP-dependent cleavage of Suc that is catalyzed by Suc synthase (SuSy). In the present work, we tested directly whether a bypass of the endogenous SuSy route by ectopic overexpression of invertase or Suc phosphorylase affects internal oxygen levels in growing tubers and whether this is responsible for their decreased starch content. (a) Oxygen tensions were lower within transgenic tubers than in wild-type tubers. Oxygen tensions decreased within the first 10 mm of tuber tissue, and this gradient was steeper in transgenic tubers. (b) Invertase-overexpressing tubers had higher activities of glyceraldehyde-3-phosphate dehydrogenase, lactate dehydrogenase, and alcohol dehydrogenase, and (c) higher levels of lactate. (d) Expression of a low-oxygen-sensitive Adh1-beta-glucuronidase reporter gene construct was more strongly induced in the invertase-overexpressing background compared with wild-type background. (e) Intact transgenic tubers had lower ATP to ADP ratios than the wild type. ATP to ADP ratio was restored to wild type, when discs of transgenic tubers were incubated at 21% (v/v) oxygen. (f) Starch decreased from the periphery to the center of the tuber. This decrease was much steeper in the transgenic lines, leading to lower starch content especially near the center of the tuber. (g) Metabolic fluxes (based on redistribution of (14)C-glucose) and ATP to ADP ratios were analyzed in more detail, comparing discs incubated at various external oxygen tensions (0%, 1%, 4%, 8%, 12%, and 21% [v/v]) with intact tubers. Discs of Suc phosphorylase-expressing lines had similar ATP to ADP ratios and made starch as fast as wild type in high oxygen but had lower ATP to ADP ratios and lower rates of starch synthesis than wild type at low-oxygen tensions typical to those found inside an intact tuber. (h) In discs of wild-type tubers, subambient oxygen concentrations led to a selective increase in the mRNA levels of specific SuSy genes, whereas the mRNA levels of genes encoding vacuolar and apoplastic invertases decreased. (i) These results imply that repression of invertase and mobilization of Suc via the energetically less costly route provided by SuSy is important in growing tubers because it conserves oxygen and allows higher internal oxygen tensions to be maintained than would otherwise be possible.
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PMID:A bypass of sucrose synthase leads to low internal oxygen and impaired metabolic performance in growing potato tubers. 1291 61

The anti-HIV drug 3'-azido-3'-deoxythymidine (AZT) is the drug of choice for preventing maternal-fetal HIV transmission during pregnancy. Our aim was to assess the cytotoxic effects of AZT on human placenta in vitro. The mechanisms of AZT-induced effects were investigated using JEG-3 choriocarcinoma cells and primary explant cultures from term and first-trimester human placentas. Cytotoxicity measures included trypan blue exclusion, MTT, and reactive oxygen species (ROS) assays. Apoptosis was measured with an antibody specific to cleaved caspase-3 and by rescue of cells by the general caspase inhibitor Boc-D-FMK. The effect of AZT on the activities of glutathione-S-transferase, beta-glucuronidase, UDP-glucuronosyl transferase, cytochrome P450 (CYP) 1A, and CYP reductase (CYPR) in the placenta was assessed using biochemical assays and immunoblotting. AZT increased ROS levels, decreased cellular proliferation rates, was toxic to mitochondria, and initiated cell death by a caspase-dependent mechanism in the human placenta in vitro. In the absence of serum, the effects of AZT were amplified in all the models used. AZT also increased the amounts of activity of GST, beta-glucuronidase, and CYP1A, whereas UGT and CYPR were decreased. We conclude that AZT causes apoptosis in the placenta and alters metabolizing enzymes in human placental cells. These findings have implications for the safe administration of AZT in pregnancy with respect to the maintenance of integrity of the maternal-fetal barrier.
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PMID:3'-azido-3'-deoxythymidine (AZT) induces apoptosis and alters metabolic enzyme activity in human placenta. 1455 Jul 50

Hydroxycinnamic acids are antioxidant phenolic compounds which are widespread in plant foods, contribute significantly to total polyphenol intakes, and are absorbed by humans. The extent of their putative health benefit in vivo depends largely on their bioavailability. However, the mechanisms of absorption and metabolism of these phenolic compounds have not been described. In this study, we used the in vitro Caco-2 model of human small intestinal epithelium to investigate the metabolism of the major dietary hydroxycinnamates (ferulate, sinapate, p-coumarate, and caffeate) and of diferulates. The appearance of metabolites in the medium versus time was monitored, and the various conjugates and derivatives produced were identified by HPLC-DAD, LC/MS, and enzyme treatment with beta-glucuronidase or sulfatase. Enterocyte-like differentiated Caco-2 cells have extra- and intracellular esterases able to de-esterify hydroxycinnamate and diferulate esters. In addition, intracellular UDP-glucuronosyltransferases and sulfotransferases existing in Caco-2 cells are able to form the sulfate and the glucuronide conjugates of methyl ferulate, methyl sinapate, methyl caffeate, and methyl p-coumarate. However, only the sulfate conjugates of the free acids, ferulic acid, sinapic acid, and p-coumaric acid, were detected after 24 h. The O-methylated derivatives, ferulic and isoferulic acid, were the only metabolites detected following incubation of Caco-2 cells with caffeic acid. These results show that the in vitro model system differentiated Caco-2 cells have the capacity to metabolize dietary hydroxycinnamates, including various phase I (de-esterification) and phase II (glucuronidation, sulfation, and O-methylation) reactions, and suggests that the human small intestinal epithelium plays a role in the metabolism and bioavailability of these phenolic compounds.
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PMID:Characterization of metabolites of hydroxycinnamates in the in vitro model of human small intestinal epithelium caco-2 cells. 1469 Mar 69

We investigated the chemoprotective effects of four common constituents of the human diet, i.e. a fermented milk, inulin, oligofructose and Brussels sprouts, towards 2-amino-3-methylimidazo[4,5-f]quinoline (IQ)-induced genotoxicity in male Fischer 344 rats harbouring a human intestinal microflora. We found that the four dietary components significantly reduced IQ-induced DNA damage in hepatocytes (reduction ranged from 74% with inulin to 39% with Brussels sprouts) and colonocytes (reduction ranged from 68% with inulin to 56% with Brussels sprouts). This chemoprotective effect correlated with the induction of hepatic UDP-glucuronosyl transferase following Brussels sprouts consumption, and with alterations of bacterial metabolism in the distal gut (acidification, increase of butyrate proportion, decrease of beta-glucuronidase activity) following inulin consumption.
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PMID:Protective effects of Brussels sprouts, oligosaccharides and fermented milk towards 2-amino-3-methylimidazo[4,5-f]quinoline (IQ)-induced genotoxicity in the human flora associated F344 rat: role of xenobiotic metabolising enzymes and intestinal microflora. 1503 16

Hyaluronan (HA), a functionally essential glycosaminoglycan in vertebrate tissues and a putative virulence factor in certain pathogenic bacteria, is an extended linear polymer composed of alternating units of glucuronic acid (GlcUA) and N-acetylglucosamine (GlcNAc). Uncertainty regarding the mechanism of HA biosynthesis has included the directionality of chain elongation, i.e. whether addition of monosaccharide units occurs at the reducing or non-reducing terminus of nascent chains. We have investigated this problem using yeast-derived recombinant HA synthases from Xenopus laevis (xlHAS1) and from Streptococcus pyogenes (spHAS). The enzymes were incubated with UDP-[3H]GlcUA and UDP-[14C]GlcNAc, under experimental conditions designed to yield HA chains with differentially labeled reducing-terminal and non-reducing terminal domains. Digestion of the products with a mixture of beta-glucuronidase and beta-N-acetylglucosaminidase exoenzymes resulted in truncation of the HA chain strictly from the non-reducing end and release of labeled monosaccharides. The change in 3H/14C ratio of the monosaccharide fraction, during the course of exoglycosidase digestion, was interpreted to indicate whether sugar units had been added at the reducing or non-reducing end. The results demonstrate that the vertebrate xlHAS1 and the bacterial spHAS extend HA in opposite directions. Chain elongation catalyzed by xlHAS1 occurs at the non-reducing end of the HA chain, whereas elongation catalyzed by spHAS occurs at the reducing end. The spHAS is the first glycosyltransferase that has been unanimously demonstrated to function at the reducing end of a growing glycosaminoglycan chain.
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PMID:Biosynthesis of hyaluronan: direction of chain elongation. 1562 18

The potential cancer therapeutic agent, 6,7-(dimethoxy-2, 4-dihydroindeno[1,2-c]pyrazol-3-yl)-(3-fluoro-phenyl)-amine (JNJ-10198409), formed three N-glucuronides that were positively identified by liquid chromatography-tandem mass spectrometry and NMR as N-amine-glucuronide (Glu-A), 1-N-pyrazole-glucuronide (Glu-B), and 2-N-pyrazole-glucuronide (Glu-C). All three N-glucuronides were detected in rat liver microsomes, whereas only Glu-A and -B were found in monkey and human liver microsomes. In contrast to common glucuronides, Glu-B was completely resistant to beta-glucuronidase. Kinetic analyses revealed that glucuronidation of JNJ-10198409 in human liver microsomes exhibited atypical kinetics that may be described by a two-site binding model. For the high affinity binding, K(m) values were 1.2 and 5.0 microM, and V(max) values were 2002 and 2,403 nmol min(-1) mg(-1) for Glu-A and Glu-B, respectively. Kinetic constants of low affinity binding were not determined due to low solubility of the drug. Among the human UDP-glucuronosyltransferases (UGTs) tested, UGT1A9, 1A8, 1A7, and 1A4 were the most active isozymes to produce Glu-A; for the formation of Glu-B, UGT1A9 was the most active enzyme, followed by UGT1A3, 1A7, and 1A4. Glucuronidation of JNJ-10198409 by those UGT1A enzymes followed classic Michaelis-Menten kinetics. In contrast, no glucuronides were formed by all UGT2B isozymes tested, including UGT2B4, 2B7, 2B15, and 2B17. Collectively, these results suggested that glucuronidation of JNJ-10198409 in human liver microsomes is catalyzed by multiple UGT1A enzymes. Since UGT1A enzymes are widely expressed in various tissues, it is anticipated that both hepatic and extrahepatic glucuronidation will likely contribute to the elimination of the drug in humans. Additionally, conjugation at the nitrogens of the pyrazole ring represents a new structural moiety for UGT1A-mediated reactions.
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PMID:N-glucuronidation of the platelet-derived growth factor receptor tyrosine kinase inhibitor 6,7-(dimethoxy-2,4-dihydroindeno[1,2-C]pyrazol-3-yl)-(3-fluoro-phenyl)-amine by human UDP-glucuronosyltransferases. 1645 2

The conversion of UDP-glucuronate to glucuronate, usually thought to proceed by way of glucuronate 1-phosphate, is a site for short-term regulation of vitamin C synthesis by metyrapone and other xenobiotics in isolated rat hepatocytes. Our purpose was to explore the mechanism of this effect in cell-free systems. Metyrapone and other xenobiotics stimulated, by approximately threefold, the formation of glucuronate from UDP-glucuronate in liver extracts enriched with ATP-Mg, but did not affect the formation of glucuronate 1-phosphate from UDP-glucuronate or the conversion of glucuronate 1-phosphate to glucuronate. This and other data indicated that glucuronate 1-phosphate is not an intermediate in glucuronate formation from UDP-glucuronate, suggesting that this reaction is catalysed by a 'UDP-glucuronidase'. UDP-glucuronidase was present mainly in the microsomal fraction, where its activity was stimulated by UDP-N-acetylglucosamine, known to stimulate UDP-glucuronosyltransferases by enhancing the transport of UDP-glucuronate across the endoplasmic reticulum membrane. UDP-glucuronidase and UDP-glucuronosyltransferases displayed similar sensitivities to various detergents, which stimulated at low concentrations and generally inhibited at higher concentrations. Substrates of glucuronidation inhibited UDP-glucuronidase activity, suggesting that the latter is contributed by UDP-glucuronosyltransferase(s). Inhibitors of beta-glucuronidase and esterases did not affect the formation of glucuronate, arguing against the involvement of a glucuronidation-deglucuronidation cycle. The sensitivity of UDP-glucuronidase to metyrapone and other stimulatory xenobiotics was lost in washed microsomes, even in the presence of ATP-Mg, but it could be restored by adding a heated liver high-speed supernatant or CoASH. In conclusion, glucuronate formation in liver is catalysed by a UDP-glucuronidase which is closely related to UDP-glucuronosyltransferases. Metyrapone and other xenobiotics stimulate UDP-glucuronidase by antagonizing the inhibition exerted, presumably indirectly, by a combination of ATP-Mg and CoASH.
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PMID:Glucuronate, the precursor of vitamin C, is directly formed from UDP-glucuronate in liver. 1668 37

The mechanism of hyaluronan biosynthesis in vertebrates had been proposed to occur at the reducing end of growing chains. This mechanism was questioned because a recombinant synthase appeared to add new monosaccharides to the non-reducing end. I reinvestigated this problem with membranes from the eukaryotic B6 cell line. The membranes were incubated with UDP-[3H]GlcNAc and UDP-[14C]GlcA to yield differentially labelled reducing terminal and non-reducing terminal domains. Digestion of the product with a mixture of the exoglycosidases beta-glucuronidase and beta-N-acetylglucosaminidase truncated the hyaluronan chain strictly from the non-reducing end. The change in 3H/14C ratio of the remaining hyaluronan fraction, during the course of exoglycosidase digestion, confirmed the original results that the native eukaryotic synthase extended hyaluronan at the reducing end. This mechanism demands that the UDP-hyaluronan terminus is bound to the active site within the synthase and should compete with the substrates for binding. Accordingly, increasing substrate concentrations enhanced hyaluronan release from the synthase. A model is proposed that explains the direction of chain elongation at the reducing end by the native synthase and at the non-reducing end by the recombinant synthase based on a loss of binding affinity of the synthase towards the growing UDP-hyaluronan chain.
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PMID:Biosynthesis of hyaluronan: direction of chain elongation. 1671 38

The uricosuric agent sulfinpyrazone (SFZ) is metabolized via C-glucuronidation, an uncommon metabolic pathway, in humans. The present study aimed to characterize SFZ glucuronidation by human liver microsomes (HLMs) and identify the hepatic forms of UDP-glucuronosyltransferase responsible for this pathway. Incubations of SFZ with HLMs formed a single glucuronide that was resistant to beta-glucuronidase and acid hydrolysis, consistent with formation of a C-glucuronide. Mass spectral analysis confirmed the identity of the metabolite as SFZ glucuronide (sulfinpyrazone beta-D-glucuronide; SFZG). SFZ C-glucuronidation by HLMs exhibited Michaelis-Menten kinetics, with mean (+/- S.D.) Km and Vmax values of 51 +/- 21 microM and 2.6 +/- 0.6 pmol/min . mg, respectively. Fifteen recombinant human UDP-glucuronosyltransferases (UGTs), expressed in HEK293 cells, were screened for their capacity to catalyze SFZ C-glucuronidation. Of the hepatically expressed enzymes, only UGT1A9 formed SFZG. UGTs 1A7 and 1A10, which are expressed in the gastrointestinal tract, also metabolized SFZ, but rates of metabolism were low compared with UGT1A9. SFZ glucuronidation by UGT1A9 exhibited "weak" negative cooperative kinetics, which was modeled by the Hill equation (S50 16 microM). The data indicate that UGT1A9 is the enzyme responsible for hepatic SFZ C-glucuronidation and that SFZ may be used as a substrate "probe" for UGT1A9 activity in HLMs.
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PMID:Sulfinpyrazone C-glucuronidation is catalyzed selectively by human UDP-glucuronosyltransferase 1A9. 1698 98

2-amino-3-methylimidazo[4,5-f]quinoline (IQ) is a genotoxic/carcinogenic compound formed in meat and fish during cooking. Following absorption in the upper part of the gastrointestinal tract, IQ is mainly metabolized in the liver by xenobiotic-metabolizing enzymes. Among them, UDP-glucuronosyl transferases lead to harmless glucuronidated derivatives that are partly excreted via the bile into the digestive lumen, where they come into contact with the resident microbiota. The purpose of this study is to investigate if microbial beta-glucuronidase could contribute to IQ genotoxicity by releasing reactive intermediates from IQ glucuronides. We constructed a beta-glucuronidase-deficient isogenic mutant from a wild-type Escherichia coli strain carrying the gene uidA encoding this enzyme and compared the genotoxicity of IQ in gnotobiotic rats monoassociated with the wild-type or the mutant strain. The Comet assay performed on colonocytes and hepatocytes showed that the presence of beta-glucuronidase in the digestive lumen dramatically increased (3-fold) the genotoxicity of IQ in the colon. This deleterious effect was paralleled by slight modifications of the pharmacokinetics of IQ. The urinary and faecal excretion of the parent compound and its conjugated derivatives reached a maximum 24-48 h after gavage in rats harbouring the beta-glucuronidase-deficient strain. In rats associated with the wild-type strain, the kinetics of urinary excretion showed a biphasic curve with a second, smaller peak after 144 h. This is the first in vivo demonstration that bacterial beta-glucuronidase plays a pivotal role in the genotoxicity of a common food-borne carcinogen.
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PMID:Beta-glucuronidase in human intestinal microbiota is necessary for the colonic genotoxicity of the food-borne carcinogen 2-amino-3-methylimidazo[4,5-f]quinoline in rats. 1766 May 8


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