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The phospholipid-dependence of uridine diphosphate glucuronyltransferase. Reactivation of phospholipase A-inactivated enzyme by phospholipids and detergents.

ABSTRACT: Specific degradation of the phospholipid membrane of guinea-pig liver microsomal fraction with phospholipase A inactivated glucuronyltransferase. The inactivation was reversed by phosphatidylcholine and mixed microsomal phospholipid micelles at concentrations similar to those present in intact microsomal preparations. The other commonly occurring phospholipids did not reactivate phospholipase A-treated enzyme. Since the mixed microsomal phospholipids consisted mainly of phosphatidylcholine, it is concluded that the reactivation by phospholipids is phosphatidylcholine-specific. Reactivation was also achieved by low concentrations of the cationic detergents cetylpyridinium chloride and cetyltrimethylammonium bromide. Higher concentrations of these detergents inactivated the glucuronyltransferase activity of intact and phospholipase A-treated microsomal fractions. Anionic detergents were potent inactivators of the glucuronyltransferase activity of untreated and phospholipase A-treated microsomal fractions, whereas non-ionic detergents had little effect on the activity of either preparation. Measurements of the zeta-potentials of the micellar species used in this study showed that no obvious relationship existed between the zeta-potentials and the ability to reactivate glucuronyltransferase. However, high positive or negative zeta-potentials were correlated with the ability of the amphipathic compound to inactivate glucuronyltransferase.

SUBMITTER: Attwood D

PROVIDER: S-EPMC1177088 | biostudies-other | 1971 Aug

REPOSITORIES: biostudies-other

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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

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

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

Phospholipid content and activity of pure uridine diphosphate-glucuronyltransferase from rat liver.

Project description:Rat liver phospholipids were radioactively labeled in vivo before purification of UDP-glucuronyltransferase to homogeneity. The pure enzyme contained very little phospholipid (approx. 0.7 mol of phospholipid/mol of protein). The solubilization detergent Lubrol 12A9 appeared to act as a phospholipid substitute, capable of supporting UDP-glucuronyltransferase activity. Phospholipase C did not inhibit the pure enzyme activity and pure UDP-glucuronyltransferase was stimulated by 40--100% by the addition of phospholipid dispersions.

| S-EPMC1184033 | biostudies-other

The phospholipid-dependence of uridine diphosphate glucuronyltransferase. Effect of protein deficiency on the phospholipid composition and enzyme activity of rat liver microsomal fraction.

Project description:After force-feeding a protein-free diet to male rats for 5-7 days a substantial (2.4-fold) increase in the specific activity of the liver microsomal enzyme UDP-glucuronyltransferase (EC 2.4.1.17) was observed. A similar activation of the enzyme occurred when rats were fed on a low-protein (5%, w/w, casein) diet for 60 days. Although both the short- and long-term protein-deficient diets decreased the contents of microsomal protein and phospholipid in liver tissue they did not significantly alter the ratio of these major membrane components. Protein deficiency profoundly altered the phospholipid composition of microsomal membranes. The most striking difference in microsomal phospholipid composition between control and protein-deficient rats was their content of lysophosphatides. Whereas microsomal membranes from protein-deficient rats contained significant proportions of lysophosphatidylcholine and lysophosphatidylethanolamine very little or no lysophosphatides were detected in control preparations. Pretreatment of microsomal fractions from normal rats with phospholipase A markedly increased their UDP-glucuronyltransferase activity as did their pretreatment with lysophosphatidylcholine. It is concluded that the quantities of lysophosphatides present in microsomal membranes from protein-deficient rats were sufficient to have caused the increased UDP-glucuronyltransferase activities of these preparations. Evidence is presented suggesting that these changes in microsomal phospholipid composition and UDP-glucuronyltransferase activity caused by protein deficiency reflect changes that occur in vivo. The possible physiological significance of these findings is discussed.

| S-EPMC1166158 | 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

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

Phospholipid-dependence of oestrone UDP-glucuronyltransferase and p-nitrophenol UDP-glucuronyltransferase.

Project description:Hepatic UDP-glucuronyltransferase activity was resolved into two fractions, one exhibiting oestrone glucuronyltransferase activity and the other exhibiting p-nitrophenol glucuronyltransferase activity. Hydroxyapatite-column chromatography removed greater than 95% of the phospholipids from both preparations. The partially purified delipidated enzymes were essentially devoid of catalytic activity, but activities were restored by the addition of phospholipids or phosphatidylcholine mixtures containing various saturated and unsaturated fatty acids. Both oestrone and p-nitrophenol glucuronyl-transferase activities were reconstituted to similar degrees with the phosphatidylcholine mixtures. When purified phospholipids were tested, phosphatidylcholine and lysophosphatidylcholine were most effective in restoring activity, whereas phosphatidylethanolamine was the least effective. These results further suggest that oestrone and p-nitrophenol UDP-glucuronyltransferases are dependent on phospholipids for their activity.

| S-EPMC1186595 | 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

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