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

Abstract

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.

Purification and Properties of a Form of UDP-Glucuronyltransferase from Liver Microsomes of 3-Methylcholanthrene-Treated Rats

“…This is especially so in view of the finding that microheterogeneity exists among the major phenobarbital-inducible forms of rabbit liver microsomal cytochrome P-450 (34,35). In agreement with the reports by Erickson et al (32) and Singh et al (36), our transferase preparation is also activated most efficiently by phosphatidylcholine and lysophosphatidylcholine among the phospholipids examined.…”

First molecular characterization of a uridine diphosphate galacturonate 4‐epimerase: an enzyme required for capsular biosynthesis in Streptococcus pneumoniae type 1

“…Many different attempts were made to purify the Cap1J protein, e.g. size exclusion or ion exchange chromatography, affinity chromatography on blue Sepharose 6 or on UDP‐GlcA covalently linked to agarose (Sigma) (Singh et al ., 1981), etc. Only DEAE‐cellulose could be used to avoid a complete loss of the enzymatic activity of Cap1J.…”

Purification of limonoid glucosyltransferase from navel orange albedo tissues

“…The enzyme activity occurred in the Mr range of 4(~60 k was determined by SDS-PAGE. Nucleotide sugar affinity chromatography has been used successfully in the purification scheme for a ftavanone-specific 7-O-glucosyltransferase from grapefruit [5] and the purification of a UDP-glucosyltransferase from liver microsomes [6]. The enzyme fraction obtained from the UDP-glucuronic acid affinity column was applied to a DEAE ion exchange HPLC and run at pH 7.…”