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A rapid NAD+-linked assay for microsomal uridine diphosphate glucuronyltransferase of rat liver and some observations on substrate specificity of the enzyme.

ABSTRACT: 1. A new and rapid continuous assay of rat liver microsomal UDP-glucuronyltransferase (EC 2.4.1.17) has been developed. It is based on measurement of UDP production from UDP-glucuronate during the glucuronidation reaction; UDP production was continuously measured by coupling it to the conversion of NADH into NAD+ through pyruvate kinase and lactate dehydrogenase. This assay is independent of the acceptor substrate used; several findings confirm its applicability. 2. The glucuronidation rate of a series of phenol derivatives was determined with this assay, by using a Triton X-100-activated microsomal preparation as enzyme source. Conjugation of a series of nitrophenol derivatives was also investigated by the 'classical' assay (measurement of disappearance of the yellow colour of the nitrophenol during glucuronidation). The substrate with the highest conversion rate was 3-methyl-2-nitrophenol. 3. Both electron releasing and electron withdrawing ring substituents increased the glucuronidation rate of the phenol derivatives, as compared with phenol. 4. Lipid solubility seems important for determining the conversion rate: poorly lipid-soluble substrates were glucuronidated only at a low rate and high lipid solubility seems to be a prerequisite for high conversion rate. Glucuronidation of poorly lipid-soluble compounds may be limited by diffusion. 5. The consequences of these findings for the interpretation of studies on heterogeneity of the enzyme are discussed.

SUBMITTER: Mulder GJ

PROVIDER: S-EPMC1172334 | biostudies-other | 1975 Oct

REPOSITORIES: biostudies-other

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Stimulation of microsomal uridine diphosphate glucuronyltransferase by glucuronic acid derivatives.

Project description: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.

| S-EPMC1166273 | biostudies-other

Substrate specificity and properties of uridine diphosphate glucuronyltransferase purified to apparent homogeneity from phenobarbital-treated rat liver.

Project description:1. The purification to homogeneity of stable highly active preparations of UDP-glucuronyltransferase from liver of phenobarbital-treated rats is briefly described. 2. A single polypeptide was visible after sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, of mol.wt.57000. 3. Antiserum raised against the pure enzyme produces a single sharp precipitin line after Ouchterlony double-diffusion analysis. 4. The pure UDP-glucuronyltransferase isolated from livers of untreated and phenobarbital-pretreated rats appears to be the same enzyme. 5. The Km (UDP-glucuronic acid) of the pure enzyme is 5.4 mM. 6. The activity of the pure enzyme towards 2-aminophenol can still be activated 2-3-fold by diethylnitrosamine. 7. UDP-glucose and UDP-galacturonic acid are not substrates for the purified enzyme. 8. The final preparation catalysed the glucuronidation of 4-nitrophenol, 1-naphthol, 2-aminophenol, morphine and 2-aminobenzoate. 9. Activities towards 4-nitrophenol, 1-naphthol and 2-aminophenol were all copurified. The proposed heterogeneity of UDP-glucuronyltransferase is discussed.

| S-EPMC1185840 | biostudies-other

Assay and properties of dititonin-activated bilirubin uridine diphosphate glucuronyltransferase from rat liver.

Project description:1. The bilirubin UDP-glucuronyltransferase assay described by Van Roy & Heirwegh (1968) has been improved. 2. Extraction of final azo-derivatives is rendered more simple and efficient by thorough emulsification and by cooling. 3. Pretreatment of homogenates and cell fractions with digitonin increases the sensitivity of the assays and gives less variable results than those with untreated preparations. The activation procedure is flexible. 4. Blank values (obtained from incubation mixtures from which activating bivalent metal ion and UDP-glucuronic acid were omitted) are low. No endogenous conjugate formation could be detected except with untreated, fresh liver homogenates. Control incubation mixtures containing the latter preparations are preferably kept at 0 degrees C. 5. With activated microsomal preparations, rates of breakdown of UDP-glucuronic acid (as monitored by release of P(i)) were low. Little if any increase in enzyme activity was found when UDP-N-acetylglucosamine was included in the incubation mixtures. 6. Slight deviation from Michaelis-Menten kinetics with respect to bilirubin observed at low substrate concentrations is probably related to the use of binding protein in the assay mixtures. Michaelis-Menten kinetics were followed with respect to UDP-glucuronic acid. Part of the enzyme in microsomal preparations from rat liver functioned independently of added bivalent metal ions. Mn(2+) was slightly more, and Ca(2+) somewhat less, stimulatory than Mg(2+). The Mg(2+)-dependent fraction showed Michaelis-Menten kinetics with respect to the added Mg(2+). 7. The enzyme activities found were higher than values reported in the literature for untreated or purified preparations from rat liver. They were above reported values of the maximal biliary excretion rate of bilirubin.

| S-EPMC1174163 | biostudies-other

Properties of lubrol-extracted uridine diphosphate glucuronyltransferase.

Project description:1. A partially purified UDP-glucuronyltransferase was obtained by extracting rat liver microsomal preparations with Lubrol, a non-ionic detergent. 2. The soluble enzyme catalysed conjugation of both o-aminophenol and p-nitrophenol and was extremely stable when compared with untreated microsomal preparations. 3. The characteristics of the conjugation of the two phenols were found to differ with respect to pH optimum, bivalent cation requirement and Michaelis constants, suggesting that more than one enzyme is involved in the conjugation reaction.

| S-EPMC1178261 | biostudies-other

The phospholipid-dependence of uridine diphosphate glucuronyltransferase. Temperature-dependence of microsomal enzyme activity (reverse reaction).

Project description:Arrhenius plots of the non-latent UDP-glucuronlytransferase reverse reaction (p-nitrophenyl glucuronide donor) activity of guinea-pig microsomal membranes prepared with 15 mM-KCl were linear from 5 to 40 degrees C. These plots for other preparations from guinea-pig and rat liver (i.e. preparations that show transferase latency) exhibited two linear regions intersecting at a transition point near 19--21 degrees C. This discontinuity was abolished when latency was removed by treating the membranes with perturbants of phospholipid-bilayer structure. Thus the temperature-depdendnces of the reverse reaction catalysed by the enzymes of these various preparations are similar to those of the corresponding forward reactions [Pechey, Graham & Wood (1978) Biochem. J. 175, 115--1124]. Perturbants activated the enzyme of KCl-prepared guinea-pig microsomal membranes only slightly and caused no significant alteration to Arrhenius plots of its forward or reverse reaction activities. These results support the 'compartmentation' theory of UDP-glucuronyltransferase lactency.

| S-EPMC1161381 | biostudies-other

Effect on uridine diphosphate glucuronyltransferase activity of depleting and restoring phospholipids to guinea-pig liver microsomal preparations.

Project description:Phospholipid depletion substantially inhibited the maximum demonstrable activities of the forward (glucuronidation) and reverse reactions of UDP-glucuronyltransferase towards p-nitrophenol in guinea-pig liver microsomal preparations. Dispersions of liver phospholipids restored activity, whereas non-phospholipid amphipaths failed to do so effectively. These results suggest that the system is probably phospholipid-dependent rather than conformationally constrained by phospholipids.

| S-EPMC1172782 | biostudies-other

The heterogeneity of uridine diphosphate glucuronyltransferase from rat liver.

Project description:1. The glucuronide conjugation of p-nitrophenol, phenolphthalein, o-aminophenol and 4-methylumbelliferone by rat liver microsomes has been studied. The detergent Triton X-100 activated UDP-glucuronyltransferase activity towards all these substrates, therefore the optimum activating concentration was added in all experiments. 2. Mg(2+) enhanced the conjugation of the substrates. 3. With phenolphthalein substrate inhibition occurred but this could be relieved by adding albumin, which binds excess of phenolphthalein. 4. Kinetic constants of the substrates and UDP-glucuronate have been determined. Mutual inhibition was found with the substrates p-nitrophenol, 4-methylumbelliferone and phenolphthalein. p-Nitrophenol conjugation was inhibited competitively by phenolphthalein and 4-methylumbelliferone. 5. o-Aminophenol did not inhibit the conjugation of the other three substrates because these are conjugated preferentially to o-aminophenol. 6. It is concluded that the four substrates are conjugated by one enzyme at the same active site.

| S-EPMC1178020 | biostudies-other

The phospholipid-dependence of uridine diphosphate glucuronyltransferase. Phospholipid depletion and re-activation of guinea-pig liver microsomal enzyme.

Project description:More than 80% of the phospholipid component of guinea-pig liver microsomal membranes (prepared with 154mM-KCl) was removed by treatment with phospholipase A followed by extraction of the lysophosphatides and fatty acids produced with albumin. Delipidation strongly inactivated the highly active UDP-glucuronyltransferase of these preparations and activity was restored by mixtures of phosphatidylcholine and lysophosphatidylchlone. However, small quantities of lysophosphatides were still associated with the delipidated fractions after extraction with albumin and might have influenced the inactivation and re-activation observed. To eliminate these uncertainties, microsomal proteins and phospholipids were separated by gel filtration on Sephadex G-150 in the presence of cholate. This technique also strongly inactivated the enzyme but did not generate membrane-active phospholipid degradation products. High transferase activity was again restored to the delipidated protein by choline glycerophosphatides. These results confirm the view that the fully active form of microsomal UDP-glucuronyltransferase is phospholipid-dependent.

| S-EPMC1164666 | biostudies-other

Studies on the purification of rat liver uridine diphosphate glucuronyltransferase.

Project description:1. A stable, more highly purified, preparation of UDP-glucuronyltransferase was obtained than previously reported. 2. Enzyme activity towards o-aminophenyl and p-nitrophenyl was increased 43- and 46-fold respectively. 3. The final preparation contains only three staining polypeptide bands visible after sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. 4. The only known major accompanying protein appears to be epoxide hydratase. 5. The purified enzyme activity towards o-aminophenol can still be activated 3 fold by diethylnitrosamine. 6. On evidence from purification, o-aminophenol and p-nitrophenol appear to be glucuronidated by the same enzyme protein. The possible recognition of the UDP-glucuronyltransferase enzyme is discussed.

| S-EPMC1164539 | biostudies-other

The phospholipid-dependence of uridine diphosphate glucuronyltransferase. Temperature-dependence of microsomal enzyme activity and thermotropic changes in membrane structure.

Project description:Arrhenius plots of the non-latent UDP-glucuronyltransferase (p-nitrophenol acceptor) activity of guinea-pig microsomal membranes prepared with 154 mM-KCl were linear from 5 to 40 degrees C. Arrhenius plots for other microsomal preparations from guinea pig and rat liver that show various degrees of transferase latency, exhibited two linear regions intersecting at a sharp transition point near 20-25 degrees C. This discontinuity was abolished or greatly decreased when transferase latency was removed by treating the membranes with perturbants of phospholipid bilayer strucutre. The fluorescent probe N-phenyl-1-naphthyl-amine detected a thermotropic change in the fluidity of the phospholipid acyl chains of all the microsomal membrane preparations studied, at temperatures close to those of the Arrhenius-plot transitions. It is concluded that the thermotropic change in the structure of the membrane bilayer probably is a 'phase separation' or clustering of phospholipids, which affects a permeability barrier that restricts access of substrate to the transferase molecules.

| S-EPMC1186046 | biostudies-other

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