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

Burchell, Brian; Hallinan, T.

doi:10.1042/bj1710821

Cited by 20 publications

(12 citation statements)

References 22 publications

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“…In animals, the glucuronosyltransferases involved in the conjugation of drugs are located in the endoplasmic reticulum membrane, and several of these enzymes have been solubilized from the membrane using various detergents, e.g. Brij 58 (Bock et al 1979), Triton X-100 (Clarke et al 1992), Emulgen 911 (Ishii et al 1994), Lubrol PX (Gorski and Kasper 1977), Lubrol WX (Burchell and Hallinan 1978). According to these reports, the appropriate detergent for solubilization varies with the enzyme.…”

Section: Discussionmentioning

confidence: 96%

UDP-glucuronic acid:soyasapogenol glucuronosyltransferase involved in saponin biosynthesis in germinating soybean seeds

Kurosawa

Takahara

Shiraiwa

2002

Planta

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We detected UDP-glucuronic acid:soyasapogenol glucuronosyltransferase (UGASGT) activity in the microsomal fraction from germinating soybean (Glycine max [L.] Merr.) seed. A microsomal fraction was isolated from germinating soybean seed and treated with various detergents to solubilize the enzyme. UGASGT activity was monitored throughout purification using UDP-[U-(14)C]glucuronic acid and soyasapogenol B as substrates. Purification of UGASGT was achieved by HiTrap Q, Superdex 200, and HiTrap Blue chromatography procedures. This resulted in >205-fold enrichment relative to the starting homogenate. UGASGT was found to require divalent cations for activity. Studies on the substrate specificity of UGASGT demonstrated that the specificity for the sugar residue transferred was very high, as activity was scarcely found when UDP-glucuronic acid was replaced by other UDP sugars: UDP-glucose and UDP-galactose. Soyasapogenols, which are the aglycons of soybean saponin, are usable acceptors, but glycyrrhetinic acid, sophoradiol, beta-amyrin, and flavonoids are not. These findings suggest that this UGASGT was a specific enzyme for UDP-glucuronic acid as a donor and soyasapogenols as acceptors, and that it was related to the biosynthesis of the sugar chain in soybean saponin. This study provides a basis for the molecular characterization of a key enzyme in saponin biosynthesis in soybean. The isolation of the gene may enable its use in the elucidation of the biosynthesis and physiological role of saponins in soybean.

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Section: Discussionmentioning

confidence: 96%

UDP-glucuronic acid:soyasapogenol glucuronosyltransferase involved in saponin biosynthesis in germinating soybean seeds

Kurosawa

Takahara

Shiraiwa

2002

Planta

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“…Highly active, apparently homogeneous transferase proteins which contain almost no phospholipid have been isolated from rat liver microsomal membranes [32,34] and it has been suggested [33] that Lubrol (present throughout the purification) can substitute for phospholipid in supporting their transferase activity. Clearly, the data presented show that the detergent did not replace tnicrosomal phosphatidylcholine in maintaining the activity of the guinea-pig enzyme.…”

Section: Results a N D Discussionmentioning

confidence: 99%

The Phospholipid-Dependence of UDPGlucuronosyltransferase. Purification, Delipidation and Reconstitution of Microsomal Enzyme from Guinea-Pig Liver

1981

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In order to study the interaction of liver microsomal UDPglucuronosyltransferase and microsomal phospholipids under closely defined conditions, guinea-pig enzyme was purified to homogeneity (as judged by sodium dodecyl sulphate gel electrophoresis) by detergent-solubilisation, salt precipitation, chromatography on DEAE-cellulose and DEAE-Sephadex, and affinity chromatography on UDPglucuronosyl-diaminohexanyl -Sepharose 4B. The purified transferase, which catalysed the glucuronidation of p-nitrophenol with high specific activity, was associated with microsomal phospholipids, and phosphatidylcholine was the major species present ; the transferase protein had a subunit molecular weight of about 55000. The enzyme was almost completely inactivated by delipidation of the protein by hydroxyapatite chromatography and efficient reconstitution of high activity was observed only with fluid (microsomal and egg-yolk) phosphatidylcholines. These results confirm that microsomal UDPglucuronosyltransferase is phospholipid-dependent with a specific requirement for phosphatidylcholine.Liver microsomal U DPglucuronosyltransferase is regulated by the intact phospholipid structure of the microsomal membrane [l, 21 and a large body of data, obtained by studying impure enzyme fractions, indicates that for expression of full activity the enzyme requires phospholipids [3].When microsomal membranes containing non-latent enzyme were treated with phospholipases the transferase was inactivated and activity was restored with dispersions of phospholipid mixtures extracted from the membranes [4-71. Delipidated, unfractionated microsomal protein showed very low transferase activity and re-activation was observed with egg, bovine or soybean phosphatidylcholine, egg or bovine lysophosphatidylcholine, or mixtures of the two phospholipid species [8-131. It was also shown that reconstitution of UDPglucuronosyltransferase activity was associated with binding of phospholipid re-activators to these proteins [I I]. Solubilised, partially-purified rabbit-liver transferase preparations (catalysing the glucuronidation of p-nitrophenol and oestrone) were inactivated by removing phospholipids and their activities were reconstituted with egg, bovinc or synthetic phosphatidylcholine or with cgg lysophosphatidylcholine [14,15]. It has also been reported [I61 that phospholipids were removed from rat-liver transferase fractions during enzyme purification and that the activity of a crude detergentsolubulised enzyme fraction towards bilirubin, but not p-nitrophenol, was supported by egg phosphatidylcholine.In this paper we extend our work on the modulation of U DPglucuronosyltransferase by phospholipids by reporting results of a lipid-depletion/reconstitution study employing a closely defined syslem. We have uscd guinea-pig liver microsomal enzyme purified to apparent homogeneity and purified microsomal phospholipids. The enzyme is shown to be a phospholipid-protein complex whose high activity is specifically dependent upon fluid phosphatidylcholines.

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“…Results were obtained with preparations of solubilized microsomal fractions that were resolved into separate and distinct transferase activities as described previously (Burchell & Hallinan, 1978).…”

Section: Discussionmentioning

confidence: 99%

“…Gorski & Kasper (1977) and Burchell (1977) achieved considerable purification ofa UDP-glucuronyltransferase activity, but confined their studies to p-nitrophenol glucuronyltransferase activity. Gorski & Kasper (1977) reported a phospholipid content of 25.2,g of Pi/mg of protein in their purified preparation, but Burchell & Hallinan (1978) …”

mentioning

confidence: 99%

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

Tukey¹,

Billings²,

Autor³

et al. 1979

Biochemical Journal

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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 andp-nitrophenol glucuronyltransferase 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.

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

Cited by 20 publications

References 22 publications

UDP-glucuronic acid:soyasapogenol glucuronosyltransferase involved in saponin biosynthesis in germinating soybean seeds

UDP-glucuronic acid:soyasapogenol glucuronosyltransferase involved in saponin biosynthesis in germinating soybean seeds

The Phospholipid-Dependence of UDPGlucuronosyltransferase. Purification, Delipidation and Reconstitution of Microsomal Enzyme from Guinea-Pig Liver

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

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