Biochem. J. (1986) 233, 827-837 (Printed in Great Britain) 827 Isolation and characterization of multiple forms of rat liver UDP-glucuronate glucuronosyltransferase Jayanta ROY CHOWDHURY,*t Namita ROY CHOWDHURY,* Charles N. FALANY,t Thomas R. TEPHLYt and Irwin M. ARIAS*§ *Liver Research Center, Albert Einstein College of Medicine, Bronx, NY 10461, U.S.A., and tToxicology Center, Department of Pharmacology, University of Iowa, Iowa City, IA 52242, U.S.A. UDP-glucuronosyltransferase (EC 2.4.1.17) activity was solubilized from male Wistar rat liver microsomal fraction in Emulgen 911, and six fractions with the transferase activity were separated by chromatofocusing on PBE 94 (pH 9.4 to 6.0). Fraction I was further separated into Isoforms Ia, Ib and Ic by affinity chromatography on UDP-hexanolamine-Sepharose 4B. UDP-glucuronosyltransferase in Fraction III was further purified by rechromatofocusing (pH 8.7 to 7.5). UDP-glucuronosyltransferases in Fractions IV and V were purified by UDP-hexanolamine-Sepharose chromatography. The transferase isoforms in Fractions II, III, IV and V were finally purified by h.p.l.c. on a TSK G 3000 SW column. Purified UDP- glucuronosyltransferase Isoforms Ia (Mr 51000), lb (Mr 52000), Ic (Mr 56000), II (Mr 52000), IV (Mr 53000) and V (Mr 53 000) revealed single Coomassie Blue-stained bands on sodium dodecyl sulphate/polyacrylamide- gel electrophoresis. Isoform III enzyme showed two bands ofMr 52000 and 53000. Comparison ofthe amino acid compositions by the method ofCornish-Bowden [(1980) Anal. Biochem. 105,233-238] suggested that all UDP-glucuronosyltransferase isoforms are structurally related. Reverse-phase h.p.l.c. of tryptic peptides of individual isoforms revealed distinct 'maps', indicating differences in primary protein structure. The two bands of Isoform III revealed distinct electrophoretic peptide maps after limited enzymic proteolysis. After reconstitution with phosphatidylcholine liposomes, the purified isoforms exhibited distinct but overlapping substrate specificities. Isoform V was specific for bilirubin glucuronidation, which was not inhibited by other aglycone substrates. Each isoform, except Ia, was identified as a glycoprotein by periodic acid/Schiff staining. INTRODUCTION or detergent content. Therefore, determination of the UDP-glucuronosyltransferase (EC 2.4.1.17) is concen- number of completely distinct forms requires structural trated in hepatic microsomal fraction (referred to below analysis of purified isoforms. simply-as 'microsomes'), and it catalyses the transfer of In the present study, by combining chromatofocusing glucuronic acid from UDP-glucuronic acid to many and affinity chromatography and using additional metabolites and xenobiotics, forming 0-, N-, S- and substrates, we have separated seven isoforms of C-glucuronides (Dutton, 1980; Dutton & Burchell, 1977). UDP-glucuronosyltransferase from rat liver microsomes; In rat liver, UDP-glucuronosyltransferase activities for five of these were purified after an additional affinity- various aglycone substrates develop at different perinatal chromatographic or gel-permeation h.p.l.c. step. Analysis periods (Wishart, 1978a) and are differentially affected by of peptides produced by enzymic proteolysis indicates various enzyme-inducing agents (Lucier & McDaniel, that at least four isoforms differ in primary protein 1977; Wishart, 1978b; Lillienblum et al., 1982), suggesting structure; differences in substrate specificities suggest the heterogeneity of the enzyme. Supportive evidence for presence of three additional UDP-glucuronosyltransfer- multiplicity ofUDP-glucuronosyltransferases came from ase isoforms. chromatographic separation of two (Burchell, 1978; Bock et al., 1979; Weatherill & Burchell, 1980; Roy EXPERIMENTAL Chowdhury et al., 1983) or three (Falany & Tephly, 1983; Falany et al., 1983) forms of the enzyme with distinct Materials substrate specificities from rat livermicrosomes. However, Testosterone, oestrone, oestradiol-17/1, androsterone, antisera raised against one form of purified rat liver bilirubin,4-nitrophenol, I -naphthol, aniline,4-methylum- UDP-glucuronosyltransferase react with all forms of rat belliferone, UDP-glucuronic acid and egg phosphatidyl- liver UDP-glucuronosyltransferases (Weatherill et al., choline were obtained from Sigma Chemical Co. (St. 1980; Roy Chowdhury et al., 1983, 1985), and thus the Louis, MO, U.S.A.). Aniline was recrystallized from structural multiplicity of rat liver UDP-glucuronosyl- ethanol before use. 3H-labelled testosterone, oestrone, transferases has not been established. Identical proteins oestradiol-17,/, androsterone and 1-naphthol were could possibly be chromatographically separated and obtained from New England Nuclear (Boston, MA, exhibit functional difference because of different lipid U.S.A.). 4-Nitro[14C]phenol was purchased from Cali- Abbreviations used: NaDodSO4, sodium dodecyl sulphate. t To whom correspondence should be addressed. § Present address: Department of Physiology, Tufts University Medical School, Boston, MA 021 11, U.S.A. Vol. 233 828 J. Roy Chowdhury and others fornia Bionuclear (Sun Valley, CA, U.S.A.) and was Fraction III (eludon pH 8.0). Since Fraction III yielded purified by diethyl ether extraction before use. Emulgen two protein bands on NaDodSO4/polyacrylamide-gel 911 was obtained from Kao-Atlas, Tokyo, Japan. Poly- electrophoresis (see the Results section), we attempted to buffer Exchanger (PBE 94) and Polybuffer 96 were pur- separate these bands by rechromatofocusing. The pooled chased from Pharmacia (Uppsala, Sweden). H.p.l.c. was fractions were adjusted to pH 8.7 by addition of performed on a Waters (Milford, MA, U.S.A.) chroma- 25 mM-ethanolamine containing 0.05% Emulgen 911; tograph, with a 60 cm TSK G 3000 SW gel-permeation chromatofocusing was performed as described in a pH chromatographic column. UDP-hexanolamine was syn- range from 8.7 to 7.5. Fractions with transferase activity thesized and linked to Sepharose 4B as described by for testosterone were purified by h.p.l.c. Sadler et al. (1979) and modified by Tukey & Tephly (1981). Male Wistar rats (200-250 g) were purchased from Charles River Laboratories (Cambridge, MA, Fraction IV (elution pH 7.8) and Fraction V (elution U.S.A.) pH 7.5). UDP-glucuronosyltransferase from these frac- tions were purified by UDP-hexanolamine-Sepharose 4B Solubilization of rat liver microsomes chromatography as described above, except that equilib- Hepatic microsomes were prepared from male Wistar ration and wash buffers did not contain KCl and the rats and solubilized in 0.5% Emulgen 911 as previously transferases were eluted with 1 mM-UDP-glucuronic described (Falany et al., 1983). For most experiments, acid. UDP-glucuronosyltransferase isoforms were puri- livers from two weight- and age-matched rats were fied from enzymically active fractions by h.p.l.c. as de- pooled. To evaluate individual variation, single livers scribed above. were used, with no difference in results. Fraction VI (elution pH 6.5). This fraction had activity Chromatofocusing for oestrone and oestradiol-17,f, contained many bands Chromatofocusing was performed with Polybuffer on NaDodSO4/polyacrylamide-gel electrophoresis and Exchanger 94 (20 ml bed volume) as previously described was not further purified. (Falany et al., 1983), with the following modifications: glycerol and dithiothreitol were omitted from the Enzyme assays equilibration and elution buffers, the pH range was UDP-glucuronosyltransferase activities were reconsti- extended (9.4 to 6.0), the protein load was decreased to tuted as follows. Phosphatidylcholine liposomes were 80 mg (160 mg/m column length) of solubilized micro- prepared as described by Burchell (1980). For enzyme somal protein, and the elution rate was decreased to assay in all chromatographic fractions (but not for 0.2 ml/min. UDP-glucuronosyltransferase activities for solubilized microsomes), phosphatidylcholine liposomes various substrates were determined as described below; were added to the incubation mixture (1 mg/ml for six enzymically active chromatofocusing peaks were bilirubin and 0.1 mg/ml for other substrates). Since all eluted (Fig. 1); the corresponding transferase isoforms samples contained Emulgen 91 1, no additional detergent were purified as follows. was added. UDP-glucuronosyltransferase activities towards bili- Fraction I (elution pH 8.9). Fractions were applied at rubin (Jansen et al., 1977) and aniline (Axelrod et al., 0.6 ml/h to a 15 ml UDP-hexanolamine-Sepharose 4B 1958) were assayed colorimetrically; the activity towards column that had been equilibrated with 25 mM-ethanol- 4-methylumbelliferone was determined fluorimetrically amine/acetate buffer, pH 8.0, containing 5 mM-MgCl2 (Arias, 1961). The transferase activities towards 4- and 0.5 mg ofEmulgen 91 1/ml. UDP-glucuronosyltrans- nitrophenol, 1-naphthol, oestrone, testosterone, oestra- ferases were eluted sequentially with 2 bed volumes each diol-17fl and androsterone were quantified by radioassay of30 mM-KCl (Fraction Ia), 50 ,uM-UDP-glucuronic acid (Dutton et al., 1981). Glucuronidation of bilirubin was (Fraction Ib) and S mM-UDP-glucuronic acid (Fraction assayed at pH 7.8, of 4-nitrophenol at pH 7.4, and of all Ic) in equilibration buffer. Eluate was collected in 2.5 ml other substrates at pH 8.0. For all assays, product fractions. Enzymically active fractions were pooled and formation was linear with enzyme concentrations and concentrated by negative pressure dialysis against incubation time. 25 mM-ethanolamine/acetate