Invited Review - American Chemical Society

Invited Review. Isolation and Purification of UDP-Glucuronosyltransferases. Thomas R. Tephly. Department of Pharmacology, University of Iowa, Iowa Cit...
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Chem. Res. Toxicol. 1990, 3,509-516

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Invited Review Isolation and Purification of UDP-Glucuronosyltransferases Thomas R. Tephly Department of Pharmacology, University of Iowa, Iowa City, Iowa 52242 Received May 30, 1990

Introduction The study of the metabolic fate of xenobiotics in mammals has been of interest for about 200 years, ever since benzoic acid conjugation to glycine was discovered (1). Glucuronide and sulfate conjugation was revealed in work dating back to the 1870s as was the oxidative transformation of benzene to phenol ( I ) . Over the last 40 years, the biological role of the hepatic endoplasmic reticulum in the metabolism of xenobiotics by oxidation, reduction, and conjugation has been studied extensively. Over the last 15 years, we have begun to appreciate the wide diversity of enzymes involved in glucuronide synthesis. This review will indicate progress made in the isolation and purification of UDP-glucuronosyltransferases (UDPGTs) since 1980; research prior to 1981 has been reviewed by Dutton (2),Kasper and Henton (31, and Burchell (4). Recent reviews (5,6) have also appeared which deal with numerous aspects of UDPGTs, including purification. Although this review will emphasize our knowledge concerning the functional and physical properties of highly purified UDPGTs, some molecular biological research will also be referred to. Isolation and expression of cDNAs for UDPGTs have been carried out in parallel with studies on enzyme purification, and each approach has enhanced our understanding of the nature and variety of UDPGTs which mediate the glucuronidation of xenobiotics and endobiotics. In this review the terminology used to denote the various UDPGTs employs the guidelines of Bock et al. (7), where a UDPGT is named after an endogenous substrate (bilirubin UDPGT) or a class of endogenous substrates (170or 3cu-hydroxysteroid UDPGTs) if a specific site on the molecule is linked to glucuronic acid. Where no endogenous substrate has been identified, the UDPGT is named for a highly reactive xenobiotic substrate (p-nitrophenol UDPGT, digitoxigenin monodigitoxoside UDPGT). Glucuronides are generally more water soluble than the parent aglycon and are readily excreted into the urine or bile. It should be noted, however, that in certain cases glucuronide metabolites may be more active pharmacologically than the parent drug or may possess toxic potential. Morphine 6-O-glucuronide has been shown to be a more potent analgesic than morphine (8). Whereas this metabolite is not formed to any great extent in rats, it is found in humans (9) after morphine administration and can be generated, in vitro, in human liver microsomes (10). Adverse reactions produced by glucuronideshave also been reported. Glucuronidation of the N-hydroxy group of N-hydroxyphenacetin resulted in a glucuronide conjugate that was covalently bound to protein (11).Estrogen D-ring glucuronides have been shown to produce an inhibition of canalicular bile flow and bile acid secretory rate in rats and to decrease hepatic excretory function in monkeys (12,13).

Acyl glucuronides formed from a number of nonsteroidal antiinflammatory drugs have been shown to bind covalently to proteins in vivo, and it has been suggested that the adduct acts to produce an immunological response which may result in anaphylactic reactions (14,25).Thus, although glucuronidation is a major route of drug elimination in animals, potential toxicities may result from elevated levels of certain glucuronides given the right xenobiotic or even with certain endobiotic substances. Assessment of rates of glucuronidation in tissue preparations or in vivo depends largely, but not exclusively, upon the type and amount of UDP-glucuronosyltransferase (UDPGT) present. Understanding the properties of UDPGTs, such as their substrate specificity and kinetics of reaction, has therefore been an important area of research. In addition, recent work has also revealed important information on their abundance in untreated and induced animals, qualitative and quantitative differences in lower animals and humans, and genetic regulation in a number of animal species (5,6). UDPGTs are membrane-bound enzymes of the endoplasmic reticulum and are found primarily in liver, a major source of tissue for work on purification. They catalyze the linkage of glucuronic acid from UDP-glucuronic acid to substances possessing OH, COOH, NH2, SH, and C moieties (2,3). These proteins are extremely labile, especially when purification procedures are employed to resolve them from other microsomal proteins and from each other. Overcoming the problems of lability in order to obtain preparations of UDPGTs retaining catalytic function has led many investigators to molecular biological work where expression of cDNAs has become a popular approach. On the other hand, our laboratory has been fortunate to ultimately purify some UDPGTs to homogeneity from rabbit, rat, and human liver microsomes. Each UDPGT, in general, has required a separate series of experimental procedures, and for each, basic questions have directed the work. Questions addressed have included the following: which detergent should be used, what concentration of detergent should be used for solubilization, what substances should be used for protection of activity, and what is the rate of decay in enzymic activity throughout a given procedure? In the early 1970s, Dr. Emilio Sanchez and Dr. Eugenia del Villar began work on morphine glucuronidation in this laboratory. Studies were performed to determine whether p-nitrophenol or p-nitrophenol glucuronide might competitively inhibit morphine glucuronidation in microsomal preparations. The reason for doing this experiment came from the work of Zakim et al. (16),who had shown that p-nitrophenol glucuronide inhibited competitively the glucuronidation of p-nitrophenol and that p-aminophenol glucuronide did not inhibit p-nitrophenol glucuronidation. 0 1990 American Chemical Society

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Morphine and morphine 3-glucuronide were tested as inhibitors of p-nitrophenol glucuronidation, and no inhibition of morphine glucuronidation by p-nitrophenol or its glucuronide was observed. p-Nitrophenol glucuronide competitively inhibited p-nitrophenol glucoronidation ( I 7) as had previously been reported by Zakim et al. (16). Morphine 3-glucuronide inhibited morphine glucuronidation, but neither morphine nor its glucuronide inhibited p-nitrophenol glucuronidation. Interestingly, bilirubin stimulated (18) the rate of glucuronidation of both p-nitrophenol and morphine and produced no inhibition at any concentration. On the basis of this work one might suppose that a t least three UDPGTs existed in rat liver microsomes. Next, experiments were performed to solubilize and separate p-nitrophenol and morphine UDPGTs from both rat and rabbit liver microsomes by using Emulgen 911 as detergent (19,20). This represented the first separation of UDPGTs from hepatic microsomes. We noted that the morphine UDPGT was extremely labile and allowed for only limited purification because the enzyme activity decreased rapidly once resolution was achieved. This problem retarded progress on study with morphine UDPGT, and it took a decade to finally find a partial solution to the problem, which resulted in the ultimate purification of the enzyme to homogeneity. By the early 1980s it was clear that a number of hepatic microsomal UDPGTs existed in rabbits and rats. Reports from our laboratory, Bock and his colleagues (21-23), and Burchell’s laboratory (24) led to this inescapable conclusion. However, evidence that rat hepatic microsomal UDPGTs could be isolated to homogeneity was still of major concern. From our point of view, three major breakthroughs occurred which ailowed for improvements in UDPGT purification. Gorski and Kasper (25) used a UDP-hexanolamine Sepharose 4B affinity procedure which facilitated the purification of a rat liver microsomal UDPGT reacting with p-nitrophenol. This procedure was useful but did not allow for separation of UDPGTs if several UDPGTs were present when applied to the resin. The procedure depended largely on binding of the UDPGT through the UDPGA binding site, and since this site was common to all UDPGTs, elution of the proteins by high concentrations of UDPGA generally yielded a mixture of UDPGTs. A second advance was the finding that UDPGTs could be separated on the basis of different isoelectric points (26). Lastly, we found that certain UDPGTs could be separated on the UDP-hexanolamine Sepharose 4B affinity columns by adjustment of salt and UDPGA concentrations (27).

Studies on Rabbit Liver UDP-Glucuronosyitransferases The first highly purified preparations of rabbit liver microsomal UDPGTs were described by Billings et al. (26) and Tukey et al. (28). In this work, highly purified pnitrophenol and estrone UDPGTs were isolated by using anion-exchange, isoelectric focusing, and affinity chromatographic procedures. This report was also one of the first to show the dependence of activity and reconstitution of several purified UDPGTs on phospholipids (29). Shortly after, Tukey and Tephly (30) reported on the purification to homogeneity of estrone and p-nitrophenol UDPGTs from rabbit liver microsomes. This work clearly established that the two proteins possessed different functional properties. Peptide mapping experiments clearly indicated different peptide patterns, supporting the notion that these UDPGTs were products of different

Tephly genes. Isoelectric points and amino acid composition were different, but surprisingly, these proteins had identical subunit molecular weights, 57 000. This observation was, in fact, an ominous sign and would in the future lead to more than the usual difficulty in proving homogeneity. Other instances of UDPGTs with identical monomeric molecular weights will be noted later. A procedure for the rapid separation and purification of estrone and p-nitrophenol UDPGTs from rabbit liver microsomes has also been published (31I. Substrate specificities of these proteins have been studied. Falany et al. (32)found that rabbit liver estrone UDPGT catalyzed the glucuronidation of estrogens almost exclusively at the 3-position of the steroid A ring, and more recently (33),we have shown the rank order of reactivity for estrogens to be estrune > 170-estradiol > estriol (approximately 11:51, respectively). When the A ring of the steroid is not aromatic, as with androsterone, no activity is observed, and testosterone with a 170-hydroxyl group also does not serve as a substrate. However, rabbit liver estrone UDPGT catalyzes the N-glucuronidation of cynaphthylamine, P-napthylamine, and 4-aminobiphenyl at respectable rates (33) but does not react with morphine or 4-hydroxybiphenyl. Further studies with highly purified rabbit liver microsomal p-nitropheno! UDPGT have also been performed (33). p-Nitrophenol and l-naphthol are excellent substrates as is 2-aminophenol. This is interesting since rat liver p-nitrophenol UDPGT does not react with 2aminophenol (unpublished results). Although slight activity was observed with estrone and 0-estradiol, no activity with estriol was observed. Furthermore, it does not react with androgens, naphthylamines, morphine, or 4hydroxybiphenyl (33). An antibody raised in sheep against rabbit liver pnitrophenol UDPGT has been very useful. This antibody preparation inhibits and immunoprecipitates p-nitrophenol UDPGT activity in solubilized rabbit liver microsomes. It also immunoprecipitates certain human liver UDPGT activities (34). Both estrone and p-nitrophenol UDPGTs have been shown to be glycosylated (35). Each reacts with endoglycosidases to yield peptides on SDS-PAGE with lower monomeric molecular weights than the untreated proteins. However, deglycosylation of the UDPGTs had no effect on the catalytic activity of these proteins. Thus, glycosylation appears not to play a role in the catalytic function of these enzymes.

Purification of Rat Liver UDP-Glucuronosyltransferases A practical extension of the principle of enzyme separation based on isoelectric focusing was the use of chromatofocusing chromatography, whereby UDPGTs could be separated on the basis of their pZ values. Subsequent application and resolution of certain UDPGTs with low and high concentrations of UDPGA on UDP-hexanolamine Sepharose 4B affinity resin allowed for three rat hepatic microsomal UDPGTs to be purified to homogeneity (27). These UDPGTs were identified by different substrate specificities, different subunit molecular weights on SDSPAGE, and unambiguous NH,-terminal amino acid sequence. A p-nitrophenol UDPGT with a monomeric molecular weight of 56000 and pZ value of about 9.0 was shown to employ p-nitrophenol, 4-methylumbelliferone, and l-naphthol as substrates but showed no reactivity with steroids or morphine (27). It was induced by pretreating rats with 3-methylcholanthrene (3-MC). Two other

Invited Review

UDPGTs, not induced by 3-MC treatment, were shown

to react with steroid substrates and named for their specific reactivity with either 170- or 3a-hydroxyl substituents of testosterone and androsterone, respectively (27). The l'l&hydroxysteroid UDPGT reacted with testosterone and the 170-hydroxyl position of estradiol. Interestingly, this UDPGT, M , 50OO0, reacted well with p-nitrophenol as well as with 170-hydroxylatedsteroids. In livers from untreated rats, this enzyme is probably the major form reacting with p-nitrophenol since the p-nitrophenol UDPGT in untreated rats is in very low abundance. These studies alerted us to the unsuitability of comparing reaction rates of p-nitrophenol in hepatic microsomal preparations from untreated and 3-MC-pretreated rats. The 176-hydroxysteroid UDPGT does not react with 4-methylumbelliferone, morphine, or 3a-hydroxylated steroids. A 3n-hydroxysteroid UDPGT, M, 52 000, was shown to react with steroids possessing a 3a-hydroxyl moiety (27). This protein reacts well with androsterone and etiocholanolone but does not react with p-nitrophenol, 4methylumbelliferone, l-naphthol, morphine, or 6-estradiol. Later, it was shown that this protein was capable of reacting with bile acids if they had a 3a-hydroxyl substituent. Kirkpatrick et al. (36)showed that lithocholic acid and the 24-methyl ester of lithocholate were excellent substrates but that chenodeoxycholate was a marginal substrate. More recently, studies of the reactivity of this protein with bile acids of different chain lengths have been published (37, 38). No glucuronidation of the terminal carboxyl moiety occurs in the reaction of bile acids with this protein (38). Another interesting and important observation relevant to 3a-hydroxysteroid UDPGT was made by Matsui and Hakozaki (39),who found that 50% of Wistar rats possess a deficiency of this enzyme in liver microsomes. Wistar rat liver microsomal preparations (LA) devoid of 3ahydroxysteroid UDPGT have been extremely useful. Hepatic microsomes prepared from these LA Wistar rats have been shown to possess low glucuronidation activity toward substrates reacting with this enzyme (40),i.e., androsterone and bile acids. We have shown that 4-aminobiphenyl glucuronidation is mediated by 3a-hydroxysteroid UDPGT by using hepatic microsomes from LA Wistar rat livers and with the purified enzyme. Furthermore, purification of other UDPGTs from LA Wistar rat liver microsomes has had the advantage of lacking a major interfering UDPGT. The use of these livers was a major factor in allowing us to purify morphine and 4-hydroxybiphenyl UDPGTs. In 1979, Bock et al. (23) reported on the separation and purification of two UDPGTs from rat liver microsomes, a GT-1 which catalyzed p-nitrophenol glucuronidation and a GT-2 which catalyzed morphine, 4-hydroxybiphenyl,and chloramphenicol glucuronidation. The GT-1 was induced by 3-MC and the GT-2 was induced by phenobarbital. The GT-1, p-nitrophenol UDPGT, has been discussed above (p-nitrophenol UDPGT). The GT-2 has turned out to be at least two and, possibly, four other UDPGTs. Ten years after the separation of morphine UDPGT, Puig and Tephly were able to purify this enzyme to appear homogeneity from rat liver (41). We took advantage of three strategies. Livers from LA Wistar rats were employed. Phenobarbital was used to pretreat these rats providing a marked induction of morphine UDPGT activity. Lastly, a relatively high concentration of exogenous phosphatidylcholine was employed throughout most of the purification procedures, a feature that provided for a substantial degree of stabilization of the morphine

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UDPGT. Although many detergents were tried over the years, Emulgen 911 still provided the best means of solubilization. The morphine UDPGT had a monomeric molecular weight of 56 000, a value identical with that of the p-nitrophenol UDPGT, but by use of successive chromatofocusing procedures, a separation of morphine UDPGT from p-nitrophenol UDPGT and 176-hydroxysteroid UDPGT was possible. The enzyme catalyzed the glucuronidation of morphine and was competitively inhibited by codeine, but no reactivity toward steroids, bilirubin, p-nitrophenol, 4-aminobiphenyl, or a-naphthylamine was observed. Of major importance was the observation that this enzyme did not react with 4-hydroxybiphenyl. Another UDPGT (41) was resolved by chromatofocusing which mediated 4-hydroxybiphenyl but not morphine glucuronidation. More recently, we have been able to photoaffinity label the morphine UDPGT of rat liver microsomes. It had been suspected in studies performed in the 1970s that N-alkyl moieties on certain morphine agonists and antagonists determined their ability to react with morphine UDPGT (42). del Villar et al. (43) showed that cyproheptadine, a tertiary amine that is not glucuronidated in rat hepatic microsomes, was a potent and competitive inhibitor of morphine UDPGT activity (Ki = 80 pM). When desmethylcycloheptadine, the secondary amine, was studied, inhibitory potency was greatly decreased (Ki = 400 pM). del Villar and Sanchez and their colleagues later showed that benzodiazepines which are not glucuronidated but which have tertiary amine structure were potent competitive inhibitors of morphine glucuronidation (44-46). Furthermore, they demonstrated that these benzodiazepines did not inhibit p-nitrophenol, testosterone, or estrone glucuronidation. More recently, competitive inhibition by benzodiazepines of human liver morphine glucuronidation has been demonstrated by Rane et al. (47). Of the benzodiazepines, flunitrazepam (FNZ) was shown to be one of the most potent (45) in inhibiting morphine UDPGT. We have confirmed the findings of Vega et al. (45)that FNZ produces a competitive inhibition of morphine glucuronidation (Ki = 130 pM) in solubilized rat hepatic microsomes (48). As expected, FNZ had no effect on 4hydroxybiphenyl glucuronidation. Since flunitrazepam (FNZ) has been used as a photoaffinity probe for peripheral benzodiazepine receptors (49) and since it competitively inhibits morphine glucuronidttion, we have studied its potential in photolabeling morphine binding sites on morphine UDPGT. Under UV irradiation, the concentration of FNZ that produced 50% inhibition of morphine glucuronidation (using 10 mM morphine) was reduced from 2.5 mM to 100 pM. A time-dependent increase in [3H]FNZbinding to microsomal protein and a coincident decrease in morphine UDPGT activity was observed. Morphine antagonized both binding of [3H]FNZ and the inhibition of morphine UDPGT activity (48). No inhibition of testosterone, androsterone, or 4-hydroxybiphenyl glucuronidation occurred when FNZ was used in either ambient or UV light. Most importantly, UDPGA did not protect against light-enhanced FNZ inhibition of morphine glucuronidation or [3H]FNZ binding to solubilized microsomal protein. p-Nitrophenol glucuronidation is also unaffected by FNZ after light irradiation. When purification procedures were carried out as described by Puig and Tephly (41),only fractions of protein containing morphine UDPGT were labeled by [3H]FNZ. Protein fractions with other UDPGTs present did not bind [3H]FNZ. A comparison of morphine UDPGT specific

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activity and [SH]FNZbinding throughout a series of purification steps gave an excellent correlation. Fluorography of an SDS-PAGE gel of solubilized rat liver microsomes which had been photolabeled with [3H]FNZ showed on band appearing between 54 and 58 kDa which corresponded to the reported subunit molecular weight of homogeneous morphine UDPGT, 56 000. It appears that the photoaffinity labeling of morphine UDPGT may be relatively specific and may lead to many new avenues of research with this enzyme. An important observation is that FNZ only binds to active morphine UDPGT and, as activity is lost, FNZ binding is diminished. Thus, photolabeling has recently been carried out early in the purification process, and radioactivity is followed through the procedure rather than the more labor-intensive enzymic activity measurement. Also, we have performed preliminary studies with human liver microsomal preparation where morphine UDPGT activity is relatively low (50). So far, results have been similar to those of rat liver microsomal preparations. Recently, Coughtrie et al. (10) have reported on the use of (+)-morphine and (-)-morphine with rat liver microsomal preparations. Although natural (-)-morphine formed only the 3-O-glucuronide, (+)-morphine formed glucuronides at both the 3-OH and 6-OH positions. Using a series of induction, ontogenic, selective inhibition (1naphthylacetic acid), and genetic deficiency parameters, they determined that there may be two isoenzymes responsible for morphine glucuronidation in rat liver. Indeed, they suggest that bilirubin UDPGT may be responsible for (+)-morphine glucuronidation. Further studies will be needed to resolve this issue. Digitoxin (DT-3) is a cardiac glycoside containing three digitoxose sugar residues and is known to be metabolized in animals, including humans, through a series of stepwise cleavages to the bis- (DT-2) and monodigitoxoside (DT-1). The first two steps appear to be mediated by hepatic microsomal P-450 species (51). Previous studies by Schmoldt and colleagues (52,53) and Castle (54) demonstrated that, once DT-1 is formed, a UDPGT-dependent reaction leading to DT-1 glucuronide functions for ultimate elimination of this substance. Schmoldt and Promies (52) proposed that a specific UDPGT might be responsible for DT-1 glucuronidation. Watkins and Klaassen (55) suggested that there may be a separation UDPGT which catalyzed DT-1 glucuronidation on the basis of development of DT-1 glucuronidation in rats. Watkins et al. (56) had previously suggested that DT-1 and bilirubin glucuronidation might be mediated by the same UDPGT. The separation and purification of a DT-1 UDPGT from rat liver microsomes was accomplished by von Meyerink et al. (57). Spironolactone and pregenolone-16a-carbonitrile have been shown to induce DT-1 glucuronidation in rats (51-53). Rats pretreated with spironolactone were employed since Schmoldt and Promies (52)had previously shown that steroid, bile acid, bilirubin, p-nitrophenol, and 4-hydroxybiphenyl glucuronidation was not increased by spironolactone treatment. After many preliminary studies exploring various detergents and conditions for stabilization it was found that Emulgen 911 was the detergent of choice for solubilization and that high thiol concentrations were required for stabilization of DT-1 UDPGT throughout purification. DT-1 UDPGT eluted at an extremely high pH on chromatofocusing columns (pl approximately 10) which allowed for a separation from p-nitrophenol UDPGT which eluted a t about pH 9.0. After affinity column procedures were employed, a protein displaying DT-1 glucuronidation was obtained and studied for its

Tephly

substrate specificity. DT-1 UDPGT displayed a very narrow substrate specificity; it reacted only with DT-1 and DT-2. No reactivity with DT-3 was observed. Also, no reaction with bilirubin, morphine, p-nitrophenol, 4methylumbelliferone, 4-hydroxybiphenyl, carcinogenic amines, steroids, or bile acids was observed. Furthermore, none of these xenobiotics or endobiotics inhibited DT-1 UDPGT catalyzed glucuronidation. Recently, we have purified a 4-hydroxybiphenyl UDPGT from rat liver microsomes from animals treated with phenobarbital (58,59). Procedures used were similar to those described by Puig and Tephy (41) except that an affinity column was used for purification of this enzyme to apparent homogeneity. This protein has a monomeric molecular weight of about 52 500. It does not react with morphine or chloramphenicol (preliminary data). It does not react with steroids but possesses a high reactivity with 4-methylumbelliferone and p-nitrophenol. Mackenzie (60) has reported on the expression of a cDNA (UDPGT,-2) which demonstrated activity toward testosterone, chloramphenicol, 4-hydroxybiphenyl, and 4-methylumbelliferone. Since the substrate specificity of the protein expressed in his work is different from that of the protein we isolated, one can suggest that there are two UDPGTs capable of catalyzing 17&hydroxysteroids, one of which would have broader substrate specificity to include chloramphenicol and 4-hydroxybiphenyl. Recent work in our laboratory with chloramphenicol glucuronidation suggests that a protein corresponding to Mackenzie’s cDNA may be resolvable from rat liver microsomes obtained from phenobarbital-treated animals. Thus, there appears to be a separate UDPGT that reacts with testosterone, 4-hydroxybiphenyl, and chloramphenicol. The subject of bilirubin glucuronidation has been of major importance due to the possible toxicity of bilirubin in states where relative or absolute deficiency of bilirubin UDPGT occur. The isolation of apparently homogeneous bilirubin UDPGT from rat liver microsomes of phenobarbital-treated rats was reported by Burchell and Blackaert in 1984 (61). They found a Coomassie Blue staining band on SDS-PAGE with a subunit molecular weight of 53 000 although a recent review indicates the M, to be 54 000 (5). Roy Chowdhury et ai. (62) have reported purification of a rat hepatic bilirubin UDPGT and a Coomassie Blue stained protein band with a subunit molecular weight of 53000. Although both groups found UDP-glucose and UDP-xylose to serve as substrates, no extensive aglycon substrate specificity has yet been reported. Roy Chowdhury et al. (62) have shown reactivity of bilirubin UDPGT with 4-hydroxydimethylaminoazobenzene. This protein must receive more study considering its importance. Recently, Sat0 et al. (63) have reported on the isolation of a cDNA for rat liver bilirubin UDPGT. The cDNA was transfected into COS7 cells, where glucuronidation of bilirubin was observed in cell homogenates. Interestingly, this cDNA shares an identical 913-bp sequence with 3MC-inducible p-nitrophenol UDPGT corresponding to 247 amino acid residues from the carboxy-terminal portion of the protein. A UDPGT has recently been purified from hepatic microsomes of 3-methylcholanthrenez-treated rats which reacts with serotonin, p-nitrophenol, l-naphthol, and eugenol (64). It has a subunit molecular weight of 54000 on SDS-PAGE gels and differs from other UDPGTs in that it has a threonine residue at the NH2 terminus. All other UDPGTs analyzed to date have had either a glycine or aspartate residue at the NH2 terminus.

Invited Review

Chem. Res. Toxicol., Vol. 3, No. 6, 1990 513 Table I. Rat Hepatic UDP-Glucuronosyltransferases" substrate specificity

UDPGT

Mr

17@-OHsteroid 3a-OH steroid p-nitrophenol bilirubin morphine

DT-1 4-OH-biphenyl-1 4-OH-biphenyl-2 serotonin a

50 000 52000 56000 54 000 56 000 ? 52 500 52 000 54 000

endogenous testosterone, @-estradiol androsterone, 3-OH bile acids ? bilirubin, bilirubin mononlucuronide morphine ? ? testosterone, @-estradiol serotonin

exogenous p-nitrophenol, l-naphthol, a- and @-naphthylamine 4-aminobiphenyl, a- and @-naphthylamine p-nitrophenol, 1-naphthol, 4-methylumbelliferone 4-hydroxydimethylaminoazobenzene n a 1oxon e digitoxigenin monodigitoxoside, digitoxigenin bisdigitoxoside 4-0H-biphenyl, 4-methylumbelliferone, p-nitrophenol 4-OH-biphenyl, chloramphenicol p-nitrophenol, l-naphthol, eugenol

For details see refs 27, 60-62, 41, 57-59, and 64.

Table I summarizes information concerning substrate specificitiesfor purified rat hepatic UDPGTs or for cDNAs which have been isolated and expressed. It can be seen that certain substrates such as p-nitrophenol or l-naphthol are capable of reacting with more than one isoenzyme in rat hepatic microsomes. On the other hand, several UDPGTs have relatively narrow substrate specificities (morphine and DT-1 UDPGTs).

Rat Kidney UDPGTs Coughtrie et al. (65) have recently purified two transferases from rat kidney, a phenol and a bilirubin UDPGT. The subunit molecular weights on SDS-PAGE were 54 0oO (phenol) and 55 000 (bilirubin). However, when Western blot analysis was performed using specific antibodies, subunit molecular weights of 53 000 (phenol) and 54000 (bilirubin) were observed. Yokota et al. (66) have isolated a phenol-reactive UDPGT from kidney microsomes of rats treated with P-naphthoflavone. This protein has a subunit molecular weight of 54 000 on SDS-PAGE gels and reacts with pnitrophenol, l-naphthol, 4-methylumbelliferone, and serotonin but not with 4-hydroxybiphenylor chloramphenicol. Activity toward bilirubin was minimal. The NHz-terminal amino acid sequence was similar to that of rat hepatic microsomal p-nitrophenol UDPGT (67) but different from that reported by Yokota et al. (64) for the serotonin UDPGT from rat liver microsomes.

Purification of Human Liver UDPGTs Two human liver microsomal UDPGTs have been purified to apparent homogeneity (68). One protein present in relatively high abundance was eluted from chroinatofocusing chromatographic columns at pH 7.4 and one with lesser abundance that eluted at pH 6.2. Both were purified on UDP-hexanolamine Sepharose 4B affinity columns. The pZ7.4 UDPGT was found to react with 4-methyumbelliferone, p-nitrophenol, a-naphthylamine, and estriol. No activity with 4-aminobiphenyl, estrone, testosterone, androsterone, 17B-estradio1,or 5a-androstane-3a,l7P-diol was observed. The pZ6.2 UDPGT possessed the same substrate specificity as the pZ7.4 UDPGT except that it dit not react with estriol. It was distinguished functionally from the pZ7.4 UDPGT on the basis of its reactivity with 4-aminobiphenyl, which was not a substrate for the PI7.4 UDPGT. We have called the pZ7.4 UDPGT by the name estriol UDPGT. The pZ6.2 UPDGT could be termed a phenol UDPGT. The estriol UDPGT has a monomeric molecular weight on SDS-PAGE of 53000 and required a relatively high concentration of phosphatidylcholine for reconstitution of maximal activity. The product of estriol glucuronidation was estriol l6a-glucuronide and not the 3- or 17p-glucuronide. Thus, this protein is characteristic of the steroid

type of UDPGT but possesses a distinctive reaction with the 16whydroxyl position of the steroid structure. This type of reactivity has not been noted for UDPGTs from hepatic microsomes of other species. Recent studies with purified human liver estriol UDPGT have shown that it does not react with morphine, 4hydroxybiphenyl, or tertiary amines such as tripelennamine (69). It demonstrates immunoreactivity with antibodies raised against rat hepatic microsomal 3a- and 17/j-hydroxysteroid UDPGTs but not with antibodies raised against rat hepatic microsomal p-nitrophenol UDPGT. NH,-terminal analysis yielded an amino acid sequence which aligned to the deduced amino acid sequence of a cDNA cloned from a human liver library in Xgt ll(HLUG4). Sequence analysis showed that HLUG4 is 2094 base pairs in length, encoding a protein of 523 amino acids which includes a 16 amino acid leader sequence. Recently, Ritter et al. (70) isolated a human liver cDNA clone encoding a UDPGT which expressed glucuronidation in COS cells toward estriol as well as toward 3,4-catechol estrogens such as 4-hydroxyestrone, 2-hydroxyestradiol, and 4-hydroxyestradiol. The deduced amino acid sequence shows that it is different from the estriol UDPGT isolated by Irshaid and Tephly (68) and the HLUG4 which corresponds to the estriol UDPGT. Thus, there are two estriol UDPGTs in human liver. The pZ6.2 UDPGT has a subunit molecular weight of 54 OOO and reacts with 4-aminobiphenyl. This UDPGT has been shown to immunoprecipitate with sheep anti-rabbit p-nitrophenol UDPGT (34) and to be immunoreactive on Western blots with anti-rabbit and anti-rat p-nitrophenol UDPGTs. It does not demonstrate immunoreactivity with anti-rat 3a- or 176-hydroxysteroid UDPGTs. One might be tempted to say that the pZ6.2 UDPGT protein corresponds to a human hepatic UDPGT cDNA (termed HLUGP1) which has been expressed by Harding et al. (71)in COS cells and which expressed activity toward small phenolic compounds. However, NHz-terminalamino acid sequence analysis of this protein shows that it does not possess the same sequence as the deduced amino acid sequence of HLUGPl (preliminary results). Thus, it appears that there are a number of UDPGTs in human liver capable of reacting with small phenolic compounds. Another human liver UDPGT cDNA (HLUG25) has been expressed in COS cells (72). This cDNA expressed activity toward hyodeoxycholic acid, producing a 6-0-0glucuronide, a reaction previously shown by Radominska-Pyrek et al. (73) to occur in human liver microsomal fractions. It is interesting to note that the estriol UDPGT (HLUG4) has about 82% identity with HLUG25, suggesting that these proteins are members of the same gene subfamily. The protein corresponding to HLUG25 has not yet been purified from human liver microsomes. There are at least three other UDPGTs that are known

514

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Table 11. Human Liver UDP-Glucuronosyltransferasesn M. endogenous exogenous estriol (pl 7.4) 53000 estriol p-nitrophenol, (16a-OH) 4-methylumbelliferone, or-naphthylamine estriol 52 000 estriol, catechol ? (UDPGTh-2) estrogens phenol (4-amino- 54000 ? phenols, 4-aminobiphenyl biphenyl) phenols HLUGPl 53000 ? HLUG25 52000 6-OH bile acids ?

For details see refs 68-72.

to be present in human liver microsomes which catalyze the glucuronidation of bilirubin, morphine, and tertiary amines other than morphine (74). These proteins have not yet been purified from human liver, but their presence is indicated by a number of studies. They are very labile proteins, and their abundance appears to be low. Table I1 summarizes information on UDPGTs of human liver which have either been purified to homogeneity or whose cDNAs have been expressed.

Summary UDPGTs are members of a class of enzymes located in the endoplasmic reticulum and are encoded by a multigene family. These proteins are responsible for the glucuronidation of hundreds of xenobiotics of many chemical classes and many endogenous substances such as steroid hormones, bile acids, and bilirubin. There are a number of UDPGTs which have been identified by purification and characterization studies and a significant number which have been characterized by expression of cDNAs. On the basis of the primary structures elucidated they appear to have marked similarities ( 5 ) and are highly conserved. However, key differences in their functional properties appear to depend primarily on differences in amino acid sequences at or about the NH,-terminal area of the protein (5). Many of the UDPGTs have an extraordinarily broad substrate specificity; a few, however, are relatively specific for a given class of substrate (morphine, DT-1 UDPGTs). This places a burden on investigators to clearly identify which substrate and how many UDPGTs will be involved in any analysis of rates of glucuronidation in microsomal preparations. Caution should also be advised for extrapolation of data from hepatic microsomes of experimental animals to human hepatic microsomal preparations because human liver microsomes possess UDPGTs which are qualitatively different and, in certain cases, UDPGTs are present in human liver which are not present in lower animals. Acknowledgment. I gratefully acknowledge the participation of many colleagues who have made much of the work reported here possible. These include Drs. Sanchez, del Villar, Billings, Tukey, Falany, Puig, Ishaid, and Thomassin. The research contributions of Mitchell Green and Birgit Coffman have been invaluable. The research was supported by NIH Grant GM 26221. Registry No. UDPGT, 9030-08-4.

References (1) Williams, R. T . (1959) Introductory and Historical. In Detoxication Mechanisms, pp 13-22, Chapman & Hall, London.

(2) Dutton, G. J. (1980) Glucuronidation of Drugs and Other Compounds, CRC Press, Boca Raton, FL. (3) Kasper, C. B., and Henton, D. (1980) Glucuronidation. In Enzymatic Basis of Detoxication W. B., (Jacoby, Ed.) Vol. 2, pp 3-26, Academic Press, New York.

Tephly (4) Burchell, B. (1981) Identification and purification of multiple forms of UDP-glucuronosyltransferase. Reu. Biochem. Toxicol. 3, 1-39. (5) Burchell, B., and Coughtrie, M. W. H. (1989) UDP-glucuronosyltransferases. Pharmacol. Ther. 43, 261-289. (6) Mackenzie, P. I., Roy Chowdhury, N., and Roy Chowdhury, J. (1989) Characterization and regulation of rat liver UDP-glucuronosyltransferases. Clin. Exp. Pharmacol. 16, 501-504. (7) Bock, K. W., Burchell, B., Dutton, G. J., Hanninen, O., Mulder, G . J., Owens, I. S., Siest, G., and Tephly, T. R. (1983) UDPGlucuronosyltransferase activities. Guidelines for consistent interim terminology and assay conditions. Biochem. Pharmacol. 32, 953-955. (8) Abbott, F. V., and Palmour, R. M. (1988) Morphine-6-glucuronide: Analgesic effects and receptor binding profile in rats. Life Sci. 43, 1685-1695. (9) Yeh, S. V. H. (1975) Urinary excretion of morphine and its metabolites in morphine-dependent subjects. J. Pharmacol. Exp. Ther. 192, 201-210. (IO) Coughtrie, M. W. H., Ask,B., b e , A., Burchell, B., and Hume, R. (1989) The enantioselective glucuronidation of morphine in rats and humans: Evidence for the involvement of more than one UDP-glucuronosyltransferase isoenzyme. Biochem. Pharmacol. 38, 3272-3280. (11) Mulder, G. J., Hinson, J. A., and Gillette, J. R. (1977) Generation of reactive metabolites of N-hydroxy-phenacetin by glucuronidation and sulfation. Biochem. Pharmacol. 26, 189-196. (12) Meyers, M., Slikker, W., and Vore, M. (1981) Steroid D-ring glucuronides: Characterization of a new class of cholestatic agents in the rat. J. Pharnacol. Exp. Ther. 218,63-73. (13) Slikker, W., Vore, M., Bailey, J. R., Meyers, M., and Montgomery, C. (1983) Hepatotoxic effects of estradiol-17/3-D-glucuronide in the rat and monkey. J . Pharmacol. Exp. Ther. 225, 138-143. (14) Smith, P. C., McDonagh, A. F., and Benet, L. Z. (1986) Irreversible binding of zomepirac to plasma protein in vitro and in uiuo. J . Clin. Inuest. 77, 934-939. (15) Hyneck, M. L., Smith, P. C., Munafo, A., McDonagh, A. F., and Benet, L. Z. (1988) Disposition and irreversible plasma protein binding of tolmetin in humans. Clin. Pharmacol. Ther. 44, 107-114. (16) Zakim, D., Goldenberg, J., and Vessey, D. A. (1973) Differentation of homologous forms of UDP-glucuronyltransferase. I. Evidence for the glucoronidation of o-aminophenol and p-nitrophenol by separate enzymes. Biochem. Pharmacol. 309,67-74. (17) Sanchez, E., and Tephly, T. R. (1974) Morphine metabolism. I. Evidence for separate enzymes in the glucuronidation of morphine and p-nitrophenol by rat hepatic microsomes. Drug Metab. Dispos. 2, 247-253. (18) Sanchez, E., and Tephly, T. R. (1973) Activation of hepatic microsomal glucuronyltransferase by bilirubin. Life. Sci. 13, 1488-1490. (19) del Villar, E., Sanchez, E., Autor, A. P., and Tephly, T. R. (1975) Morphine metabolism. 111. Solubilization and separation of morphine and p-nitrophenol uridine diphosphoglucuronyltransferases. Mol. Pharmacol. 11, 236-240. (20) del Villar, E., Sanchez, E., and Tephly, T. R. (1977) Morphine metabolism. V. Isolation of separate glucuronyltransferase activities for morphine and p-nitrophenol from rabbit liver microsomes. Drug Metab. Dispos. 5, 273-278. (21) Bock, K. W., Clausbruch, U. C., Kaufmann, R., Lilienblum, W., Oesch, F., Pfeil, H., and Platt, K. L. (1980) Functional heterogeneity of UDP-glucuronyltransferase in rat tissues. Biochem. Pharmacol. 29, 495-500. (22) Lilienblum, W., Walli, A. K., and Bock, K. W. (1982) Differential induction of rat liver microsomal UDP-glucuronosyltransferase activities by various inducing agents. Biochem. Pharmacol. 31, 907-913. (23) Bock, K. W., Josting, D., Lilienblum, W., and Pfeil, H. (1979) Purification of rat-liver microsomal UDP-glucuronyltransferase. Separation of two enzyme forms inducible by %methylcholanthrene or phenobarbital. Eur. J. Biochem. 98, 19-26. (24) Weatherill, P. J., and Burchell, B. (1980) The separation and purification of rat liver UDP-glucuronyltransferase activities towards testosterone and oestrone. Biochem. J. 189, 377-390. (25) Gorski, J. P., and Kasper, C. B. (1977) Purification and properties of microsomal UDP-glucuronyltransferase from rat liver. J. Biol. Chem. 252, 1336-1343.

Invited Review (26) Billings, R. E., Tephly, T. R., and Tukey, R. H. (1978) The separation and purification of estrone and p-nitrophenol UDPglucuronyltransferase activities. In Conjugation Reactions in Drug Biotransformation (Aitio, A., Ed.) pp 365-376, Elsevier/ North-Holland Press, Amsterdam. (27) Falany, C. N., and Tephly, T. R. (1983) Separation, purification and characterization of three isoenzymes of UDP-glucuronyltransferase from rat liver microsomes.~Arch. Biochem. Biophys. 227, 248-258. (28) Tukey, R. H., Billings, R. E., and Tephly, T. R. (1978) Separation of oestrone UDP-glucuronyltransferase and p-nitrophenol UDP-glucuronyltransferase activities. Biochem. J. 171,659-663. (29) Tukey, R. H., Billings, R. E., Autor, A. P., and Tephly, T. R. (1979) Phospholipid-dependence of oestrone UDP-glucuronyltransferase and p-nitrophenol UDP-glucuronyltransferase. Biochem. J . 179, 59-65. (30) Tukey, R. H., and Tephly, T. R. (1981) Purification and properties of rabbit liver estrone and p-nitrophenol UDP-glucuronyltransferases. Arch. Biochem. Biophys. 209, 565-578. (31) Tukey, R. H., Robinson, R., Holm, B., Falany, C. N., and Tephly, T. R. (1982) A procedure for the rapid separation and purification of UDP-glucuronosyltransferases from rabbit liver microsomes. Drug Metab. Dispos. 10,97-101. (32) Falany, C. N., Chowdhury, J. R., Chowdhury, N. R., and Tephly, T. R. (1983) Steroid 3- and 17-OH UDP-glucuronyltransferase activities in rat and rabbit liver microsomes. Drug Metab. Dispos. 11,426-432. (33) Tephly, T. R., Green, M., Puig, J., and Irshaid, Y. (1988) Endogenous substrates for UDP-glucuronosyltransferases. Xenobiotica 18, 1201-1210. (34) Green, M. D., Coffman, B. L., Irshaid, Y. M., and Tephly, T. R. (1988) Characterization of antibodies to a rabbit hepatic UDP-glucuronosyltransferase and the identification of an immunologically similar enzyme in human liver. Arch. Biochem. Biophys. 262, 367-374. (35) Green, M. D., and Tephly, T. R. (1989) N-glycosylation of purified rat and rabbit hepatic UDP-glucuronosyltransferases. Arch. Biochem. Riophys. 273, 72-78. (36) Kirkpatrick, R. B., Falany, C. N., and Tephly, T. R. (1984) Glucuronidation of bile acids by rat liver 3-OH androgen UDPglucuronyltransferase. J . Biol. Chem. 259, 6176-6180. (37) Kirkpatrick, R. B., Green, M. D., Hagey, L. R., Hofmann, A. F., and Tephly, T. R. (1988) Effect of side chain length on bile acid conjugation: Glucuronidation, sulfation and coenzyme A formation of nor-bile acids and their natural CZ4homologs by human and rat liver fractions. Hepatology 8, 353-357. (38) Radominska, A., Green, M. D., Zimniak, P., Lester, R., and Tephly, T. R. (1988) Biosynthesis of hydroxy-linked glucuronides of short-chain bile acids by rat liver 3-hydroxysteroid UDP-glucuronosyltransferase. J . Lipid Res. 29, 501-508. (39) Matsui, M., and Hakozaki, M. (1979) Discontinuous variation in hepatic uridine diphosphate glucuronyltransferase toward androsterone in Wistar rats. A regulatory factor for in vivo metabolism of androsterone. Biochem. Pharmacol. 28, 411-415. (40) Green, M. D., Falany, C. N., Kirkpatrick, R. B., and Tephly, T. R. (1985) Strain differences in purified rat hepatic 3cuhydroxysteroid UDP-glucuronosyltransferase. Biochem. J . 230, 403-409. (41) Puig, J. F., and Tephly, T. R. (1986) Isolation and purification of rat liver morphine UDP-glucuronosyltransferase. Mol. Pharmacol. 30, 558-565. (42) Sanchez, E., del Villar, E., & Tephly, T. R. (1978) Structural requirements in the reaction of morphine uridine diphosphate glucuronyltransferase with opioid substances. Biochem. J . 169, 173-177. (43) del Villar, E., Sanchez, E., and Tephly, T. R. (1977) The inhibition of morphine: UDP-glucuronyltransferase in rabbit liver microsomes by cyproheptadine. Life Sci. 21, 1801-1806. (44) del Villar, E., Sanchez, E., Letelier, M. E., and Vega, P. (1984) Differential inhibition by diazepam and nitrazepam of UDPglucuronosyltransferase activity in rats. Res. Commun. Chem. Pathol. Pharmacol. 33, 433-447. (45) Vega, P., Carrasco, M., Sanchez, E., and del Villar, E. (1984) Structure activity relationship in the effect of 1,4-benzodiazepines on morphine, aminopyrine and oestrone metabolism. Res. Commun. Chem. Pathol. Pharmacol. 44, 179-198. (46) Vega, P., Gaule, G., Sanchez, E., and del Villar, E. (1986) Inhibition and activation of UDP-glucuronosyltransferase in alloxan diabetic rats. Cen. Pharmacol. 17, 641-645. (47) Rane, A., Sawe, J., Pacifici, G. M., Svenson, J. O., and Kager, L. (1986) Regioselective glucuronidation of morphine and inter-

Chem. Res. Toricol., Vol. 3, No. 6, 1990 515 action with benzodiazepines in human liver. Adv. Pain Res. Ther. 8, 57-64. (48) Thomassin, J., and Tephly, T. R. (1990) Photoaffinity labeling of rat liver microsomal morpine UDP-glucuronosyltransferase by [3H]flunitrazepam. Mol. Pharmacol. (in press). (49) Snyder, S. H., Verma, A., and Trifiletti, R. R. (1987) The peripheral-type benzodiazepine receptor: A protein of mitochondrial outer membranes utilizing_ porphyrins _ - - as endogenous ligands. FASEB J . 1, 282-288. (50) Thomassin, J., Styczyski, P., and Tephly, T. R. (1989) UVlight-activated inhibition of human liver morahine UDP-glue uFonosyltransferase (UDPGT) activity by flunftrazepam (FhZ). Pharmacologist 31, 174. (51) Schmoldt, A., and Roholoff, C. (1978) Dehydro-digitoxosides of digitoxigenin: Formation and importance for the digitoxin metabolism in the rat. Naunyn Schmiedeberg's Arch. Pharmacol. 305, 167-172. (52) Schmoldt, A., and Promies, J. (1982) On the substrate specificity of the digitoxigenin monodigitoxoside conjugating UDPglucuronosyltransferase in rat liver. Biochem. Pharmacol. 31, 2285-2289. (53) Schmoldt, A. (1978) Increased digitoxin cleavage by liver microsomes of spironolactone-pretreated rats. Naunyn-Schmiedeberg's Arch. Pharmacol. 305, 261-263. (54) Castle, M. C. (1980) Glucuronidation of digitalis glycosides by rat liver microsomes: Stimulation by spironolactone and pregnenolone-16a-carbonitrile.Biochem. Pharmacol. 29,1497-1502. (55) Watkins, J. B., and Klaassen, C. D. (1985) Development of UDP-glucuronosyltransferase activity toward digitoxigenin-monodigitoxoside in neonatal rats. Drug. Metab. Dispos. 13,186-191. (56) Watkins, J. B., Gregus, Z., Thompson, T. N., and Klaassen, C. D. (1982) Induction studies on the functional heterogeneity of rat liver UDP-glucuronosyltransferases. Toxicol. Appl. Pharmacol. 64,439-446. (57) von Meyerinck, L., Coffman, B. L., Green, M. D., Kirkpatrick, R. B., Schmoldt, A., and Tephly, T. R. (1985) Separation, purification and characterization of digitoxigenin-monodigitoxoside UDP-glucuronosyltransferase activity. Drug Metab. Dispos. 13, 700-704. (58) Tephly, T. R., Townsend, M., Coffman, B., Puig, J., and Green, M. (1988) Characterization of UDP-glucuronosyltransferases from animal and human liver. In Cellular and Molecular Aspects of Glucuronidation (Siest, G., Magdalou, J., and Burchell, B., Eds.) Vol. 173, pp 37-42, Colloques INSERM/John Libbey Eurotext, London. (59) Tephly, T. R., Townsend, M., and Green, M. D. (1989) UDPglucuronosyltransferases in the metabolic disposition of xenobiotics. In Drug Metabolism Reviews (Di Carlo, F. J., Ed.) Vol. 20, pp 689-695, Marcel Dekker, New York. (60) Mackenzie, P. I. (1984) Rat liver UDP-glucuronosyltransferase: identification of cDNAs encoding two enzymes which glucuronidate testosterone, dihydrotestosterone and &estradiol. J. Biol. Chem. 262,9744-9749. (61) Burchell, B., and Blanckaert, N. (1984) Bilirubin mono- and diglucuronide formation by purified rat liver microsomal bilirubin UDP-glucuronotransferase. Biochem. J. 223, 461-465. (62) Chowdhury, N. R., Arias, I. M., Lederstein, M., and Chowdhury, J. R. (1986) Substrates and products of purified rat liver bilirubin UDP-glucuronosyltransferase. Hepatology 6, 123-128. (63) Sato, H., Koiwai, O., Tanabe, K., and Kashiwamata, S. (1990) Isolation and sequencing of rat liver bilirubin UDP-glucuronosyltransferase cDNA Possible alternate splicing of a common primary transcript. Biochem. Biophys. Res. Common. 169, 260-264. (64) Yokota, H., Yuasa, A., and Sato, R. (1988) Purification and properties of a form of UDP-glucuronosyltransferase from liver microsomes of 3-methylcholanthrene-treated rats. J . Biochem. 104, 531-536. (65) Coughtrie, M. W. H., Burchell, B., and Bend, J. R. (1987) Purification and properties of rat kidney UDP-glucuronosyltransferase. Biochem. Pharmacol. 36, 245-251. (66) Yokota, H., Ohgiua, N., Ishihara, G., Ohta, K., and Yuasa, A. (1989) Purification and properties of UDP-glucuronosyltransferse from kidney microsomes of b-naphtho-flavone-treatedrat. J . Biochem. 106, 248-252. (67) Iyanagi, T., Haniv, M., Sagawa, K., Fujii-Kuriyama, Y., Watanabe, s.,Shively, J. E., and Anan, K. F. (1987) Cloning and characterization of cDNA encoding of 3-methylcholanthrene-inducible rat mRNA for UDP-glucuronosyltransferase. J. Biol. Chem. 261, 15607-15614. (68) Irshaid, Y. M., and Tephly, T. R. (1987) Isolation and purifi-

516 Chem. Res. Toxicol., Vol. 3, No. 6, 1990 cation of two human liver UDP-glucuronosyltransferases. Mol. Pharmacol. 31, 27-34. (69) Coffman, B. L., Tephly, T. R., Irshaid, Y. M., Green, M. D., Smith, C., Jackson, M. R., Wooster, R., and Burchell, B. (1990) Characterization and primary sequence of human hepatic microsomal estriol UDP-glucuronosyltransferase. Arch. Biochem. Biophys. 281, 170-175. (70) Ritter, J. K., Sheen, Y. Y., and Owens, I. S. (1990) Cloning and expression of human liver UDP-glucuronosyltransferase in COS-1 cells: 3,4-catechol estrogens and estriol as primary substrates. J . Biol. Chem. 265, 7900-7906. (71) Harding, D., Fournel-Gigleux, S., Jackson, M. R., and Burchell, B. (1988) Cloning and substrate specificity of a human phenol

Tephly UDP-glucuronosyltransferase expressed in COS-7 cells. Proc. Natl. Acad. Sci. U.S.A. 85, 8381-8385. (72) Fournel-Gigleux, S., Jackson, M. R., Wooster, R., and Burchell, B. (1989) Expression of a human liver cDNA encoding UDPglucuronosyltransferase catalyzing the glucuronidation of hyodeoxycholic acid in cell culture. FEBS Lett. 243,119-112. (73) Radominska-Pyrek, A., Zimniak, P., Irshaid, Y. M., Lester, R., Tephly, T. R., and Pyrek, J., St. (1987) Glucuronidation of 6ahydroxy bile acids by human liver microsomes. J . Clin. Inoest. 80, 234-241. (74) Stycynski, P. B., Coffman, B. L., Green, M. D., and Tephly, T. R. (1989) Studies on quaternary ammonium-linked glucuronidation in human liver microsomes. Pharmacologist 31, 131.