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)

Both UDP-glucuronyltransferase (GT) and beta-glucuronidase (betaG) were assayed in untreated liver microsomes. Optimum assay conditions were established with rat liver microsomes using p-nitrophenol (pNP) and its glucuronide (pNPGA) at the pH optima of GT (7.5) and betaG (4.5). The activities of the two enzymes were compared using microsomes from rats, mice, pigs, cattle and horses, with pNP, pNPGA, and phenolphthalein as substrate, in the presence of various cofactors and inhibitors at pH 7.5 and 4.5. These data disclose pronounced differences with respect to species, substrate and other experimental conditions, thereby precluding the establishment of general optimum conditions. The two enzymes were also assayed under strictly identical conditions using pNP and pNPGA and rat liver microsomes at pH 7.5 in the presence and absence of UDP-glucuronate disodium (UDPGA), activators (ATP;UDP-N-acetylglucosamine) and inhibitors. When provided with a functional level of UDPGA, both enzymes proved active under those conditions, and a conjugation-deconjugation interplay was indicated. The two processes could be selectively and totally inhibited by Zn2+ and saccharolactone. The results suggest that conjugation-deconjugation-reconjugation cycles may be operative in the metabolism of drugs in vivo, taking place already at the level of the liver endoplasmic reticulum.
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PMID:Liver microsomal beta-glucuronidase and UDP-glucuronyltransferase. 0 Feb 30

Differentiation of teratocarcinoma cells led to induction of hyaluronate synthesis. The synthase was recovered in the membrane fraction of cell lysates. Hyaluronate was synthesized at the membranes and was then released as a soluble product. The synthase could be stimulated by a variety of phosphate esters which prevented the degradation of the substrates UDP-GlcNAc and UDP-GlcA and the release of the growing hyaluronic acid chain from the membrane. Hyaluronidases or oligosaccharides derived from hyaluronate did not affect the synthesis. The chains grew at a rate of 60 repeating units/min. Continuous new chain initiation occurred during prolonged synthesis. Digestion of pulse-chase-labelled hyaluronate with beta-N-acetylglucosaminidase and beta-glucuronidase showed that the chains grew at the reducing end.
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PMID:Synthesis of hyaluronate in differentiated teratocarcinoma cells. Characterization of the synthase. 640 89

Bilirubin monoglucuronide is rapidly deconjugated when incubated with UDP and rat liver microsomal preparations at pH 5.1. The following evidence was found that this reaction is catalyzed by UDP-glucuronyltransferase: (i) unconjugated bilirubin and UDP-glucuronic acid were identified as the reaction products; (ii) Gunn rat microsomal preparations lack bilirubin UDP-glucuronyltransferase deficiency and do not catalyze the deconjugation reaction, and (iii) neither saccharo-1,4-lactone, a beta-glucuronidase inhibitor, nor butylated hydroxytoluene, an inhibitor of spontaneous isomerisation, affect the rate of the deconjugation reaction. Deconjugation appears to be the reverse of UDP-glucuronyltransferase-catalyzed glucuronidation. The conditions for the reverse reaction differ in the following aspects from those of the forward reaction: (i) nucleotide triphosphates stimulate the reverse reaction probably allosterically; (ii) UDP-N-acetylglucosamine stimulates the forward reaction but has no effect on the reverse reaction; (iii) the optimal pH for the reverse reaction is pH 5.1 and for the forward reaction is pH 7.8, and (iv) Mg++ ion is not required for the reverse reaction but stimulates the forward reaction. Detergents stimulate both reactions. Stimulation of the reverse reaction by nucleotide triphosphates and detergents is mutually independent and additive which suggests different mechanisms of action. Deconjugation reactions may become important during parenchymatous liver disease when, as a result of anaerobic glycolysis, intracellular pH decreases. Elevated levels of unconjugated bilirubin in the serum of patients with parenchymatous liver disease may be a sign of sick liver cells rather than decreased UDP-glucuronyltransferase activity.
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PMID:UDP-glucuronyltransferase-catalyzed deconjugation of bilirubin monoglucuronide. 643 92

The recognition marker for the targeting of lysosomal enzymes contains mannose 6-phosphate. The recent discovery of phosphate in diester linkage between N-acetylglucosamine (GlcNAc) and mannose in newly synthesized beta-glucuronidase led to the proposal that the phosphate might be acquired via N-acetylglucosamine-phosphate transfer from UDP-GlcNAc (Tabas, I., and Kornfeld, S. (1980) J. Biol. Chem. 255, 6633-6639). We describe the synthesis of [beta-32P]UDP-[3H]GlcNAc and the use of this compound to demonstrate a UDP-GlcNAc:glycoprotein N-acetylglucosamine-1-phosphotransferase. The basis of the enzyme assay is the incorporation of 32P and 3H into glycopeptides with a high affinity for Concanavalin A-Sepharose. This membrane-associated transferase is neither inhibited by tunicamycin nor stimulated by dolichol-phosphate, indicating that the reaction does not proceed via a dolichylpyrophosphoryl-N-acetylglucosamine intermediate. Characterization of the enzyme reaction products (derived from either endogenous or exogenous acceptors) demonstrated that alpha-linked N-acetylglucosamine 1-phosphate is transferred en bloc to the 6-hydroxyl of mannose in high mannose oligosaccharides of glycoproteins. We propose that the function of this enzyme is to donate N-acetylglucosamine 1-phosphate to mannose residues of newly synthesized lysosomal enzymes.
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PMID:UDP-N-acetylglucosamine:glycoprotein N-acetylglucosamine-1-phosphotransferase. Proposed enzyme for the phosphorylation of the high mannose oligosaccharide units of lysosomal enzymes. 645 59

The bisecting N-acetylglucosamine residue is formed by UDP-N-acetylglucosamine:beta-D-mannoside-beta-1, 4-N-acetylglucosaminyltransferase III (GnT-III), a key branching enzyme for N-glycans. We found that forskolin, an adenylyl cyclase activator, markedly enhanced GnT-III at the transcriptional level in various hepatoma cells and hepatocytes, resulting in an increase of bisecting GlcNAc residues in various glycoproteins, as judged from the lectin binding to erythroagglutinating phytohemagglutinin (E-PHA). In whole cell lysates, the E-PHA binding was increased, and leukoagglutinating phytohemagglutinin (L-PHA) binding was decreased at 12 h after forskolin treatment, by time, both GnT-III activity and mRNA had reached the maximum levels. In contrast, the binding capacity as to E-PHA, determined by fluorescence-activated cell sorting on the cell surface, was decreased, suggesting that bisecting GlcNAc structures in certain glycoproteins changed the expression levels of glycoproteins and decreased their sorting on the cell surface. Fractionated organelles of M31 cells showed that the binding capacity as to E-PHA was mainly localized in Golgi membranes and lysosomes. This was also supported by a fluorescence microscopy. In order to determine whether or not the bisecting GlcNAc residue acts as a sorting signal for glycoproteins, N-oligosaccharide structures of lysosomal-associated membrane glycoprotein 1 and beta-glucuronidase, gamma-glutamyltranspeptidase, and secretory glycoproteins such as ceruloplasmin and alpha-fetoprotein were measured by E-PHA and L-PHA blotting after immunoprecipitation. The expression levels of lysosomal membrane glycoprotein 1 and gamma-glutamyltranspeptidase on the cell surface were decreased at 12 h after forskolin treatment, indicating that the bisecting GlcNAc structure may act as a negative sorting signal for the cell surface glycoproteins and may alter the characteristics of hepatoma cells. This is the first report on glycoprotein sorting related to a specific structure of oligosaccharides, bisecting GlcNAc.
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PMID:Bisecting GlcNAc structures act as negative sorting signals for cell surface glycoproteins in forskolin-treated rat hepatoma cells. 900 30

A key step in the targeting of soluble lysosomal enzymes is their recognition and phosphorylation by a 540 kDa multisubunit enzyme, UDP-N-acetylglucosamine-phosphotransferase (phosphotransferase). The molecular mechanism of recognition is still unknown, but previous experiments suggested that the phosphotransferase-binding sites on lysosomal proteins are represented by structurally conserved surface patches of amino acids. We identified four such regions on nonhomologous lysosomal enzymes, cathepsins A, B, and D, which were superimposed by rotating their structures around the Calpha atom of the glycosylated Asn residue. We proposed that these regions represent putative phosphotransferase-binding sites and tested synthetic peptides, derived from these regions on the basis of surface accessibility, for their ability to inhibit in vitro phosphorylation of purified cathepsins A, B, and D. Our results indicate that cathepsin A and cathepsin D have one closely related phosphotransferase recognition site represented by a structurally and topologically conserved beta-hairpin loop, similar to that previously identified in lysosomal beta-glucuronidase. The most potent inhibition of phosphorylation was demonstrated by homologous peptides derived from the regions located on cathepsin molecules opposite the oligosaccharide chains which are phosphorylated by the phosphotransferase. We propose that recognition and catalytic sites of the phosphotransferase are located on different subunits, therefore, providing an effective mechanism for binding and phosphorylation of lysosomal proteins of different molecular size.
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PMID:Identification of UDP-N-acetylglucosamine-phosphotransferase-binding sites on the lysosomal proteases, cathepsins A, B, and D. 989 Aug 84

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

UDP-GlcNAc: lysosomal enzyme N-acetylglucosamine-1-phosphotransferase (GlcNAc-1-phosphotransferase) is an alpha(2)beta(2)gamma(2) hexameric enzyme that catalyzes the first step in the synthesis of the mannose 6-phosphate targeting signal on lysosomal hydrolases. In humans, mutations in the gene encoding the alpha/beta subunit precursor give rise to mucolipidosis II (MLII), whereas mutations in the gene encoding the gamma subunit cause the less severe mucolipidosis IIIC (MLIIIC). In this study we describe the phenotypic, histologic, and serum lysosomal enzyme abnormalities in knockout mice lacking the gamma subunit and compare these findings to those of mice lacking the alpha/beta subunits and humans with MLII and MLIIIC. We found that both lines of mutant mice had elevated levels of serum lysosomal enzymes and cytoplasmic alterations in secretory cells of several exocrine glands; however, lesions in gamma-subunit deficient (Gnptg(-/-)) mice were milder and more restricted in distribution than in alpha/beta-subunit deficient (Gnptab(-/-)) mice. We found that onset, extent, and severity of lesions that developed in these two different knockouts correlated with measured lysosomal enzyme activity; with a more rapid, widespread, and severe storage disease phenotype developing in Gnptab(-/-) mice. In contrast to mice deficient in the alpha/beta subunits, the mice lacking the gamma subunits were of normal size, lacked cartilage defects, and did not develop retinal degeneration. The milder disease in the gamma-subunit deficient mice correlated with residual synthesis of the mannose 6-phosphate recognition marker. Of significance, neither strain of mutant mice developed cytoplasmic vacuolar inclusions in fibrocytes or mesenchymal cells (I-cells), the characteristic lesion associated with the prominent skeletal and connective tissue abnormalities in humans with MLII and MLIII. Instead, the predominant lesions in both lines of mice were found in the secretory epithelial cells of several exocrine glands, including the pancreas, and the parotid, submandibular salivary, nasal, lacrimal, bulbourethral, and gastric glands. The absence of retinal and chondrocyte lesions in Gnptg(-/-) mice might be attributed to residual beta-glucuronidase activity. We conclude that mice lacking either alpha/beta or gamma subunits displayed clinical and pathologic features that differed substantially from those reported in humans having mutations in orthologous genes.
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PMID:Comparative pathology of murine mucolipidosis types II and IIIC. 1926 45