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SOME DIFFERENCES IN THE CONJUGATION OF O-AMINOPHENOL AND P-NITROPHENOL BY THE URIDINE DIPHOSPHATE TRANSGLUCURONYLASE OF MOUSE-LIVER HOMOGENATES.

ABSTRACT: 1. Glucuronide synthesis from uridine diphosphate glucuronate and o-aminophenol or p-nitrophenol in the presence of uridine diphosphate transglucuronylase of mouse-liver homogenates has been studied with respect to inhibition by compounds known to be conjugated under the experimental conditions, and also by thiophenol. 2. Raising the o-aminophenol concentration decreased the inhibition of o-aminophenyl glucuronide synthesis by the alternative glucuronyl acceptors phenol, menthol and benzoic acid, but was without effect on that caused by p-nitrophenol and thiophenol. 3. Raising the p-nitrophenol concentration decreased or abolished the inhibition of p-nitrophenyl glucuronide synthesis due to phenol, menthol, benzoic acid, anthranilic acid, o-aminophenol and thiophenol. 4. The o-aminophenol system was much more readily inhibited by all compounds than the p-nitrophenol system. 5. In tris buffer, pH7.4, over 30% activation of the o-aminophenol system was achieved by 2mm-Mg(2+), but 10mm-Mg(2+) was inhibitory. The p-nitrophenol system showed only inhibition from 2mm-Mg(2+) upwards. 6. The results are discussed as suggesting that there are at least two uridine diphosphate transglucuronylases.

SUBMITTER: STOREY ID

PROVIDER: S-EPMC1215195 | biostudies-other | 1965 Apr

REPOSITORIES: biostudies-other

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Project description:BACKGROUND: Uridine diphosphate (UDP) is an extracellular nucleotide signaling molecule implicated in diverse biological processes via specific activation of pyrimidinergic receptor P2Y, G Protein-Coupled, 6 (P2Y6). There is very little knowledge about the function and mechanism of UDP in rheumatoid arthritis (RA). METHODS: This study used a quasi-targeted liquid chromatography-mass spectrometry (LC-MS) approach to investigate the unique expression of metabolites in RA synovial fluids (SF) (n = 10) with samples from osteoarthritis (OA) as controls (n = 10). RA fibroblast-like synoviocytes (FLSs) were collected from synovial tissues (n = 5) and cultured with UDP or MRS2578, a P2Y6 antagonist, and FLSs from OA were used as controls (n = 5). Rats with collagen-induced arthritis (CIA) were injected with UDP, MRS2578 or both (n = 9 for each group). P2Y6 expression was examined using real-time PCR, Western blotting and immunohistochemistry. Cell proliferation, apoptosis and migration of RA FLSs were measured using CCK-8 assay, real-time cell analysis, flow cytometry, wound healing assay and Transwell assay, respectively. The UDP levels in the culture medium, synovial fluid (n = 36) and peripheral blood (n = 36) of RA and CIA rats were measured using a Transcreener UDP Assay. Levels of proinflammatory cytokines were measured using a flow assay. Interleukin-6 (IL-6) levels were measured using ELISA and flow. RESULTS: LC-MS analysis detected significantly increased UDP levels in RA SF compared with OA SF, and the level was positively correlated with anticyclic citrullinated peptide (anti-CCP) and rheumatoid factor (RF)levels in RA. The increased UDP concentration was verified in the blood and synovial fluids of RA patients compared with samples from OA patients and healthy volunteers, respectively. UDP stimulated cell proliferation, migration and IL-6 secretion in RA FLSs and inhibited their apoptosis in culture, and MRS2578 inhibited these effects of UDP. UDP injection accelerated CIA and stimulated IL-6 production rather than other proinflammatory cytokines in the rat model, but simultaneous injection of MRS2578 suppressed these effects and alleviated CIA. P2Y6 expression was increased in RA and CIA synovial tissues. CONCLUSION: These results suggest that UDP is highly expressed in RA and stimulates RA pathogenesis by promoting P2Y6 activities to increase IL-6 production.

Project description:1. In incubation mixtures containing digitonin-activated or untreated preparations from rat liver, albumin-solubilized bilirubin as the acceptor substrate and (a) UDP-glucuronic acid, (b) UDP-glucose or (c) UDP-xylose as the sugar donor, formation of the following ester glycosides was demonstrated: with (a), bilirubin beta-d-monoglucuronoside, with (b), bilirubin beta-d-monoglucoside and with (c), bilirubin monoxyloside or mixtures of the mono-and di-xyloside. 2. With UDP-glucuronic acid prolonged incubation and variation of the composition of the incubation mixtures yielded equimolar amounts of azodipyrrole (I) and azodipyrrole beta-d-monoglucuronoside (II) after treatment of the incubation mixtures with the diazonium salt of ethyl anthranilate. The azo-derivatives were identified by t.l.c. by reference to known compounds and by the following chemical tests. After ammonolysis the conjugated azo-derivative (II) yielded d-glucuronic acid and the carboxylic acid amide of azodipyrrole, indicating transfer of a glucuronic acid residue to the carboxylic acid groups of bilirubin. The beta-d-configuration of the sugar moiety and binding at C-1 were demonstrated by enzymic hydrolysis tests. 3. Analogous evidence established the structure of the reaction product obtained with UDP-glucose as the sugar donor, as bilirubin beta-d-monoglucoside. 4. With UDP-xylose as the sugar donor xylosyl transfer to the carboxylic acid groups of bilirubin with attachment at C-1 was demonstrated in an analogous way. A beta-d-configuration is considered very likely, but requires confirmation. 5. Monoxyloside formation was predominant at pH7.4, whereas at decreasing pH values increasing fractions of the substrate were converted into the dixyloside. Prolonged incubation, low concentrations of bilirubin and high concentrations of UDP-xylose favoured diconjugate formation. The available evidence supports the synthesis sequence: bilirubin --> bilirubin monoxyloside --> bilirubin dixyloside.

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SOME DIFFERENCES IN THE CONJUGATION OF O-AMINOPHENOL AND P-NITROPHENOL BY THE URIDINE DIPHOSPHATE TRANSGLUCURONYLASE OF MOUSE-LIVER HOMOGENATES.

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