Purification of Rat‐Liver Microsomal UDP‐glucuronyltransferase

Abstract

Glucuronidation reactions catalysed by rat liver microsomal UDP-glucuronyltransferase are differentially inducible by 3-methylcholanthrene and phenobarbital. To elucidate the molecular basis of this functional heterogeneity the enzyme was purified from livers of rats pretreated with the inducing agents.Using cholate solubilization, chromatography on Bio-Gel A-1.5m and on DEAE-cellulose in the presence of the nonionic detergent Brij 58, two enzyme forms could be separated. Both forms were subsequently purified to apparent homogeneity by affinity chromatography on UDP-hexanolamine Sepharose 4B. 3-Methylcholanthrene-inducible enzyme activity towards 1-naphthol, 4-nitrophenol, 3-hydroxybenzo(a)pyrene and N-hydroxy-2-naphthylamine copurified with one enzyme form (enzyme 1). In contrast phenobarbital-inducible enzyme activity towards morphine, chloramphenicol and 4-hydroxybiphenyl was associated with the other enzyme fraction (enzyme 2). Sodium dodecylsulfate/polyacrylamide gels showed similar molecular weights of 54000 for enzyme 1 and 56000 for enzyme 2.The results suggest the presence of at least two forms of UDP-glucuronyltransferase in rat liver. Factors affecting enzyme activity in purified and membrane-bound states are discussed.

Isolation and characterization of multiple forms of rat liver UDP-glucuronate glucuronosyltransferase

“…Thus the differential effects of ontogenic development, enzymeinducing agents, detergents and physical agents on enzyme activity towards various substrates may result from either multiplicity of the enzyme or changes in membrane lipid or cofactors. UDP-glucuronosyltransferase activities towards various substrates have been chromatographically separated, and corresponding isoforms have been purified in several laboratories (Bock et al, 1979;Burchell, 1978Burchell, , 1981Tukey & Tephly, 1981;Roy Chowdhury et al, 1983). However, since different lipid or detergent contents may possibly result in differences in chromatographic mobility and function of an identical protein, true multiplicity of the transferases can be proved only by structural analysis of purified isoforms.…”

“…In rat liver, UDP-glucuronosyltransferase activities for various aglycone substrates develop at different perinatal periods (Wishart, 1978a) and are differentially affected by various enzyme-inducing agents (Lucier & McDaniel, 1977;Wishart, 1978b;Lillienblum et al, 1982), suggesting heterogeneity of the enzyme. Supportive evidence for multiplicity of UDP-glucuronosyltransferases came from chromatographic separation of two (Burchell, 1978;Bock et al, 1979;Roy Chowdhury et al, 1983) or three forms of the enzyme with distinct substrate specificities from rat livermicrosomes. However, antisera raised against one form of purified rat liver UDP-glucuronosyltransferase react with all forms of rat liver UDP-glucuronosyltransferases Roy Chowdhury et al, 1983, 1985, and thus the structural multiplicity of rat liver UDP-glucuronosyltransferases has not been established.…”

Isolation and purification of bilirubin UDP‐glucuronyl‐transferase from rat liver

“…If 4-nitrophenol UDP-glucuronyltransferase activity can be representative of 'enLyme 1' [6], which glucuronidates Wishart's planar molecules [33] then bilirubm UDP-glucuronyltransferase may be 'enzyme 2' proposed [6] which glucuronidates non-planar substrates. In this case morphine with planar and nonplanar portions in its molecular structure may be glucuronidated by both enzymes.…”

“…Hepatic UDP-glucuronyltransferase with activity towards phenolic substrates has been purified to apparent homogeneity in [3-71. In these transferase preparations, when looked for, activity towards bilirubin was not found [4,6]. These transferase activities depend on the presence of phospholipids [8-121. Transferase activity towards bilirubin can be restored to delipidated hepatic microsomal preparations by addition of lecithin [8].…”

Simultaneous Expression of Guinea Pig UDP-Glucuronosyltransferase 2B21 and 2B22 in COS-7 Cells enhances UDP-Glucuronosyltransferase 2B21-Catalyzed Morphine-6-Glucuronide Formation

“…Recently, Nagano et al (2000) demonstrated that the formation of M-3-G and M-6-G is comparable when brain homogenate of rat was incubated with an endogenous level of [ 3 H]morphine. UGT isoforms involved in M-3-G formation have been purified from experimental animals (Bock et al, 1979;Mackenzie et al, 1984;Puig and Tephly, 1986;Ishii et al, 1993;Oguri et al, 1996), although the UGT isoform involved in M-6-G formation remains to be purified. On the basis of the substrate specificity of expressed cloned UGT isoforms, human UGT2B7 and monkey UGT2B9 were shown to catalyze the glucuronidation of morphine at the 6-hydroxyl and 3-hydroxyl groups (Coffman et al, 1997;Green et al, 1997).…”