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Separation, purification and characterization of three isoenzymes of UDP-glucuronyltransferase from rat liver microsomes
ARCHIVES OF BIOCHEMISTRY Vol. 227, No. 1, November,
AND BIOPHYSICS
pp. 248-258,
1983
Separation, Purification and Characterization of Three lsoenzymes of UDP-Glucuronyltransferase from Rat Liver Microsomes CHARLES The Toxicology
Center,
N. FALANY
Department
Received
May
AND
of Pharmacology,
THOMAS University
23, 1983, and in revised
form
R. TEPHLY’ of Iowa,
July
Imva
City,
Iowa
52242
19, 1983
Three isoenzymes of UDP-glucuronyltransferase (UDPGT) have been separated and purified from liver microsomes of untreated female rats or female rats pretreated with 3-methylcholanthrene. The UDPGT isoenzymes were purified utilizing Chromatofocusing, column isoelectric focusing, and UDP-hexanolamine Sepharose 4B affinity chromatography. UDPGT activities could also be separated during UDP-hexanolamine affinity chromatography by elution with different UDPGA (UDP-glucuronic acid) concentrations. One isoenzyme exhibits a subunit molecular weight of 56,000 and is capable of conjugating p-nitrophenol, 1-naphthol, and 4-methylumbelliferone. This isoenzyme is inducible by 3-methylcholanthrene treatment and requires high UDPGA concentrations for elution from the UDP-hexanolamine affinity column in contrast to the other UDPGT isoenzymes. A second isoenzyme was purified and displayed a subunit molecular weight of 50,000. This isoenzyme was not induced by 3-methylcholanthrene and was active towards testosterone, the 17-OH position of P-estradiol, p-nitrophenol, and lnaphthol. A third isoenzyme was also purified and exhibited a subunit molecular weight of 52,000. This isoenzyme conjugated androsterone and etiocholanolone and was not induced by 3-methylcholanthrene treatment. This study reports the purification of two separate and distinct rat liver UDPGT isoenzymes capable of conjugating p-nitrophenol, only one of which is inducible by 3-methylcholanthrene treatment. Also, this is the first report of the purification of a UDPGT isoenzyme active towards the 3-OH position of androgens.
UDP-Glucuronyltransferase (EC 2.4.1.17) represents a family of enzymes present in the endoplasmic reticulum of the liver which are responsible for the conjugation of glucuronic acid with a wide variety of xenobiotic and endogenous compounds. The heterogeneity of UDP-glucuronyltransferase (UDPGT)2 activities has been established by a number of studies which have focused on the differential rates of development of UDPGT activities (1,5),
differential inducibilities of UDPGT activities (2-4), the absence of certain forms of UDPGT activities in certain strains of animals (6, 7), and on physical separation of UDPGT activities. The separation of various UDPGT activities has been reported by de1 Villar et al. (8), Gorski and Kasper (9), Bock et al. (lo), Burchell and co-workers (11,12), and this laboratory (13). Recently, Matern et al. (14) have isolated a rat liver UDPGT activity which glucuronidates testosterone and chenodeoxycholate. Our laboratory has reported the separation, purification, and characterization of UDPGT activities obtained from rabbit liver, and has identified separate enzymes capable of catalyzing estrone and p-nitro-
’ To whom correspondence should be addressed. a Abbreviations used: UDPGT, UDP-glucuronyltransferase; UDPGA, UDP-glucuronic acid; 3-MC, 3methylcholanthrene; SDS, sodium dodecyl sulfate; PNP, pnitrophenol. 0003-9861/83 Copyright All rights
$3.00
c> 1983 by Academic Press, Inc. of reproduction in any form reserved.
248
THREE
ISOENZYMES
OF
RAT
LIVER
phenol glucuronidation (13). Both rabbit liver UDPGT isoenzymes displayed similar subunit molecular weights following SDSpolyacrylamide gel electrophoresis but were separable by ion-exchange chromatography and UDP-hexanolamine affinity chromatography. Limited proteolysis studies have confirmed that they are distinct proteins (13). A tentative classification of rat and rabbit liver UDPGT activities has been proposed by Burchell(15), based primarily on separation characteristics after different types of ion-exchange chromatography. In this classification, isoenzymes of UDPGT have been suggested to possess activity towards both endogenous compounds and xenobiotics. Also, a given compound may be conjugated by more than one isoenzyme. This classification scheme also suggests that the number of UDPGT isoenzymes in rat liver may be greater than in rabbit liver. Another classification of rat liver UDPGT activities has been suggested by Bock et al. (10). This classification relies on the inducibility and separation of rat liver UDPGT activities following 3-methylcholanthrene (3-MC) or phenobarbital treatment. These investigators have isolated 3-MC-inducible and phenobarbitalinducible isoenzymes of UDPGT, termed GT1 and GT2, respectively. In this classification, steroid glucuronidation may represent a third form of UDPGT activities since it is apparently not greatly affected by either 3-MC or phenobarbital treatment. Our laboratory has recently been studying the separation, purification, and inducibility of xenobiotic and steroid UDPGT activities present in female rat liver. The current study presents the purification and characterization of three isoenzymes of UDPGT from liver microsomes of untreated or 3-MC-treated female rats by Chromatofocusing and UDP-hexanolamine affinity chromatography. MATERIALS
AND
METHODS
Materials. [4-i4C]Estrone (57 mCi/mmol), [4-i4C]testosterone (50 mCi/mmol), [1,2-3H]etiocholanolone (40-60 Ci/mmol), [1,2-aH]androsterone (40-60 Ci/ mmol), [l-‘4C]naphtho1 (4.3 mCi/mmol), and [4-i4C]p-
UDP-GLUCURONYLTRANSFERASE
249
estradiol (50 mCi/mmol) were purchased from New England Nuclear, Boston, Massachusetts. p-Nitro[U‘*C]phenol (24 mCi/mmol) was obtained from California Bionuclear Corporation, Sun Valley, California. [N-methyl-“C]Morphine hydrochloride (40 mCi/mmol) was obtained from Amersham, Arlington Heights, Illinois. L-a-Phosphatidylcholine (egg yolk Type XI-E), UDP-glucuronic acid, estrone, fl-estradiol, pnitrophenol, testosterone, 1-naphthol, and I-methylumbelliferone were purchased from Sigma Chemical Company, St. Louis, Missouri. Morphine sulfate was obtained from Mallinckrodt Chemical Works, St. Louis, Missouri. Chromatofocusing materials were obtained from Pharmacia Fine Chemicals, Piscataway, New Jersey. Emulgen 911 was purchased from Kao Atlas Ltd., Tokyo, Japan. Bio-Rad dye reagent protein assay was obtained from Bio-Rad Laboratories, Richmond, California. All other chemicals were of analytical grade. UDP-hexanolamine Sepharose 4B affinity resin was prepared as outlined by Sadler et al. (16) with modifications as previously described (13). Assays. pNitropheno1 glucuronidation activity was assayed as previously described (17) except a final p nitrophenol concentration of 4 mM was utilized. Morphine glucuronidation was determined by the method of Sanchez and Tephly (18); 4-methylumbelliferone glucuronidation activity was assayed as described by Aitio (19); 1-naphthol UDPGT activity was analyzed by the procedure described by Bock et al (20). Estrone, estradiol, and testosterone glucuronidation assays were assayed by the method of Tukey et al (1’7), except a final concentration of 37.5 pM for each substrate was utilized. Etiocholanolone and androsterone glucuronidation was measured by the method of Tukey et al. (17) with a final substrate concentration of 55 PM. All reactions were performed at 37°C with 5 mM UDP-glucuronic acid (UDPGA) and in the presence of 100 fig of phosphatidylcholine. Buffer solutions containing phosphatidylcholine were clarified by sonication as previously described (13). However, when alternate substrate-inhibition studies were performed, 10 mM UDPGA was used. All reactions were linear with respect to time and protein concentration. Protein concentrations were determined using the Bio-Rad protein assay originally developed by Bradford (21). Preparation of microsomes. 3-MC, when used, was dissolved in corn oil and administered daily 40 mg/ kg; ip) to female Sprague-Dawley rats (150-200 g, Biolabs, Madison, Wise.) for 3 days. Following the last injection the rats were starved for 24 h and killed by cerebral concussion, and their livers were perfused in situ with 1.15% cold KCl. Livers were then removed and homogenized in 5 vol 1.15% KC1 with a motordriven Teflon pestle. Microsomes were isolated by centrifugation, washed with 1.15% KCl, and stored as pellets with a 1.15% KC1 overlay at -6O’C. The
250 microsomal no apparent
FALANY
AND
pellets could be stored for 3 months with loss of glucuronidation activity. Separation and purtfication of PNP-UDPGTactivity (S-MC inducible). Frozen microsomal pellets were thawed, the KC1 overlay was removed, and a 10% (w/ v) suspension was formed by gentle homogenization in 25 mM ethanolamine-HAc buffer, pH 9.4. All buffers utilized in the purification procedures contained 20% glycerol, 0.1 mM dithiothreitol, and 0.05% Emulgen 911. The microsomal suspension was solubilized by the further addition of 0.5 mg Emulgen 911/mg protein, stirred for 30 min, and then centrifuged at 100,OOOg for 30 min. Between 85 and 95% of the UDPGT activities and protein were routinely recoverable in the supernatant fluid following solubilization. Solubilized microsomal protein was applied directly to a Chromatofocusing PBE 94 column (0.9 X 40 cm) previously equilibrated in 25 mM ethanolamine-HAc, pH 9.4. The column was eluted with Polybuffer 96HAc, pH 6.0, as recommended by Pharmacia to generate a pH 9 - 6 gradient. A sharp peak of pnitrophenol-UDPGT activity eluted in the void volume prior to the start of the pH gradient. A second broad peak of pnitrophenol-UDPGT activity eluted at a pH maxima of approximately 8.1. Fractions from the void volume (pH 9.0) of the Chromatofocusing column that contained pnitrophenol-UDPGT activity were pooled, and MgClz was added to a final concentration of 5 mM. The pooled fractions were applied to a UDP-hexanolamine Sepharose 4B affinity column (1.6 X 10 cm) previously equilibrated with 20 mM Tris-HCl, pH 8.0, buffer. The affinity column was then washed with at least 5 column volumes of the same buffer containing 50 mM KC1 and 50 pg phosphatidycholine/ml. Next, the column was washed with two to three column volumes of the same buffer containing 0.1 mM UDPGA. This procedure removed small amounts of activities toward testosterone, androsterone, morphine, and estrone. Finally, the same buffer containing 4 mM UDPGA was applied to the UDP-hexanolamine affinity column. This higher UDPGA concentration allowed for the removal of a 3-MC-inducible p-nitrophenolUDPGT activity in an apparently homogeneous form. Separation and purification of IT-OH steroid UDPGT activity. Microsomal protein was solubilized as described above. The solubilized microsomal protein was applied directly to a Chromatofocusing PBE 94 column (0.9 X 40 cm) previously equilibrated in 25 mM ethanolamine-HCl, pH 9.4. The column was eluted with Polybuffer 96-HCl, pH 7.0, to form a pH 9 - 7 gradient. Two fractions of 1’7-OH steroid (testosterone) UDPGT activity were resolved during the pH 9 - 7 gradient, one eluted at pH 8.5 and the second eluted at approximately pH 8.1. Fractions which eluted from the Chromatofocusing column at or about pH 8.5 or at or about pH 8.1 were
TEPHLY
pooled, and MgClz was added to a final concentration of 5 mM. The pooled fractions were applied to separate UDP-hexanolamine affinity columns (1.6 X 10 cm) previously equilibrated in 20 mM Tris-HCl, pH 8.0, buffer. The columns were then washed with five to seven column volumes of the same buffer containing 50 mM KC1 and 50 fig phosphatidylcholine/ml. The columns were then rinsed with the same buffer containing a low concentration of UDPGA (0.1 mM) which removed the 17-OH steroid UDPGT activity. Higher concentrations of UPDGA (4 mM) did not elute any more 17-OH steroid UDPGT activity. Separation and purification of S-OH androgen UDPGT activity. Microsomal protein was solubilized with Emulgen 911 as previously described, and the solubilized microsomal protein was applied directly to a Chromatofocusing PBE 94 column (0.9 X 40 cm) previously equilibrated in 25 mM ethanolamine-HCl, pH 9.4. The column was eluted with Polybuffer 96HCl, pH 7.0, in order to generate a pH 9 - ‘7 gradient. Two peaks of androsterone UDPGT activity were obtained, the first peak eluted at about pH 8.1 and the second at approximately pH 7.8. Fractions eluting from the Chromatofocusing column at pH 8.1 or pH 7.8, which contained androsterone-UDPGT activity, were pooled and MgClz was added to a final concentration of 5 mM. The pooled fractions were applied to UDP-hexanolamine affinity columns (1.6 X 10 cm) previously equilibrated in 20 mM Tris-HCl, pH 8.0, buffer. The columns were then washed with five to seven column volumes of buffer containing 50 mM KC1 and 50 pg/ml phosphatidylcholine. The columns were then eluted with the same buffer containing 0.1 mM UDPGA. This UDPGA concentration eluted an isoenzyme of UDPGT which was capable of conjugating both androsterone and etiocholanolone. Rinsing the affinity columns with a higher concentration of UDPGA (4 mM) did not elute any further androsterone or etiocholanolone UDPGT activity. Occasionally, elution of UDPGT activity from a UDP-hexanolamine affinity column was performed utilizing a linear gradient of UDPGA in the presence of the 20 mM Tris-HCl, pH 8.0, buffer mixture; and this procedure yielded good recovery of enzyme. Gel electrophoresis. Polyacrylamide gel electrophoresis was performed in the presence of sodium dodecyl sulfate (SDS) in a Hoefer Scientific Model 600 apparatus as previously outlined by Laemmli (22). Samples were pretreated by the addition of P-mercaptoethanol and SDS to final concentrations of 5 and l%, respectively, and placed in a boiling-water bath for 10 min. Following electrophoresis, the slabs were stained for 2 h in a mixture of isopropanol:acetic acid:H,O (25:10:65, v/v) which contained 0.1% Coomassie G-250 and destained with isopropanol:acetic acid:water (10:10:80, v/v). Minimum subunit molecular weights were estimated by comparison to commercial standards (Pharmacia).
THREE
ISOENZYMES
OF
RAT
LIVER
phenol-UDPGT activity was observed in the first fraction while the fraction of pnitrophenol-UDPGT activity which eluted at pH 8.1 was apparently unaffected (Table II). The increase in p-nitrophenol-UDPGT activity induced by 3-MC treatment and which eluted in the first fraction was not due to an increased recovery of protein since similar amounts of protein were added to each column and equivalent amounts of protein were recovered in each fraction. UDP-hexanolamine Sepharose 4B affinity chromatography was used to completely purify the 3-MC-inducible p-nitrophenolUDPGT activity. Fractions that contained p-nitrophenol-UDPGT activity and eluted prior to the start of the pH gradient were pooled, MgClz was added to a final concentration of 5 mM, and the fraction was applied to the affinity column. The affinity column was then rinsed with five to seven column volumes of 20 mM Tris-HCl, pH 8.0, containing 50 mM KC1 and 50 wg/ml phosphatidylcholine to remove non-specifically bound proteins. Next, the column was washed with the same buffer mixture containing 0.1 mM UDPGA. This procedure provided for the complete separation of several contaminating UDPGT activities which were present. The addition of 4.0 mM UDPGA to the buffer mixture yielded high purification of a p-nitrophenolUDPGT activity.
RESULTS
Isolation, Purijcatim, and Characterization of 3-Methylcholanthrene-Inducible pNitropheno1 UDPGlucuronyltramferase
Following treatment of female rats with 3-MC, there was a marked induction of hepatic microsomal UDPGT activity toward p-nitrophenol, 4-methylumbelliferone, and 1-naphthol. This treatment did not lead to increases in UDPGT activities toward morphine, testosterone, estrone, androsterone, etiocholanolone, or estradiol (Table I). The rate of 4-methylumbelliferone glucuronidation was increased approximately sevenfold; p-nitrophenol glucuronidation was increased more than threefold, and 1-naphthol was increased about fourfold. Similar results following 3-MC treatment of rats have been obtained from other laboratories (2, 3, 23). Chromatofocusing procedures allowed for the separation of two UDPGT activities toward p-nitrophenol, one of which reacts with 4-methylumbelliferone and one which does not. One fraction of p-nitrophenolUDPGT activity eluted prior to the start of the pH gradient, and the second fraction eluted at a pH of approximately 8.1 (Fig. 1). When microsomes from 3-MC-treated rats were utilized, an increase in p-nitroTABLE The Effect
of 3-Methylcholanthrene Solubilized
I
Pretreatment Rat Liver
on Glucuronidation Microsomes
Untreated controls (units/mg)“.*
Substrate p-Nitrophenol 4-Methylumbelliferone 1-Naphthol Morphine Testosterone Estrone P-Estradiol Etiocholanolone Androsterone Note. Female rats were pretreated Materials and Methods. a One unit of activity represents *Means f SE of three to twelve from three to twelve rats.
Female
110.8 24.7 52.6 9.7 6.0 1.4 4.2 22.4 14.5 with
+ f f 2 f zk f It f
251
UDP-GLUCURONYLTRANSFERASE
in
3-Methylcholanthrene (units/mg)“,*
4.2 1.1 3.0 0.4 0.3 0.1 0.6 0.8 0.5
3-methylcholanthrene
Rates
358.9 168.7 225 9.1 6.0 1.7 4.2 22.9 14.3 and microsomes
1 nmol substrate conjugated/min. determinations of separate mierosomal
‘- 39.7 t- 13.3 f 16.4 f 0.3 k 0.2 + 0.2 f 0.6 f 0.9 * 0.7
prepared
preparations
treated
as described
consisting
under
of livers
252
FALANY
AND
TEPHLY
-9 o . 0 .
UDPGT ACT 3 -MC IND ___-------UDPGT ACT CONTROL EH---3 -MC -_ IND _ PH CONTROL
-8 I
[I
FIG. 1. Chromatofocusing of liver microsomes from untreated and 3-methylcholanthrene-treated rats. One hundred twenty milligrams of microsomal protein from treated or untreated rats was applied to a 0.9 X 40-cm Chromatofocusing column. The columns were then eluted with Polybuffer 96-HAc, pH 6.0, to generate a pH 9 - 6 gradient. (A) indicates those fractions containing pnitrophenol UDPGT activity eluting prior to the start of the pH gradient. (B) indicates those fractions containing p-nitrophenol UDPGT activity eluting at or about pH 8.1.
p-Nitrophenol-UDPGT activity which eluted from the UDP-hexanolamine affinity column following high UDPGA displayed a single band with a subunit molecular weight of about 56,000 after SDSgel electrophoresis (Fig. 2). This UDPGT isoenzyme has been termed PNP-UDPGT, and studies on the substrate specificity of
TABLE ~NITROPHENOL
this enzyme are presented in Table III. This isoenzyme conjugated p-nitrophenol, lnaphthol, and 4-methylumbelliferone, but there was no glucuronidation of the steroids tested or of morphine. The presence of 1-naphthol (0.5 mM) or 4-methylumbelliferone (0.1 mM) inhibited PNP glucuronidation activity, whereas PNP glucuroni-
II
UDPGT
ACTIVITYFOLLOWINGCHROMATOFOCUSINGOFSOLUBILIZEDLIVERMICROSOMES FROM~NTREATEDANDS-METHyLCHOLANTHRENE-TREATED
p-Nitrophenol Untreated
Solubilized
microsomes
Chromatofocusing Fraction A (pH Fraction B (pH
9.0) 8.1)
microsomes (units)”
UDPGT
RATS activity
3-Methylcholanthrene-treated microsomes (units)’
13,430
34,020
2,500 2,760
5,589 2,724
Note. The chromatofocusing procedure was performed as described in Fig. 1. The fractions nitrophenol UDPGT activity were pooled, and the levels of p-nitrophenol glucuronidation described under Materials and Methods. a One unit of activity represents 1 nmol substrate conjugated/min.
containing determined
p as
THREE
A
ISOENZYMES
OF
RAT
LIVER
253
UDP-GLUCURONYLTRANSFERASE
B
TABLE SUBSTRATE
Substrate
SPECIFICITY
III OF PNP-UDPGT
Specific (units/mg
pNitropheno1 4-Methylumbelliferone 1-Naphthol Morphine Testosterone P-Estradiol Androsterone Etiocholanolone
activity protein)” 5733 1129 4331 ND* ND ND ND ND
a One unit of activity equals 1 nmol substrate conjugated/min. *ND represents no detectable activity. The limit of detection for steroid substrates is 0.5-1.0 units/mg; p-nitrophenol, 15 units/mg; 4-methylumbelliferone, 2 units/mg; morphine, 1 units/mg.
PM), estrone (75 PM), or morphine (0.5 mM). The apparent Km’s for p-nitrophenol and UDPGA were 0.4 and 0.4 mM, respectively. The purified protein did not display any visible or reduced carbon monoxide spectra.
Isolation, Purification, and Characterization of U-OH (PNP) UDPGT Activity
FIG. 2. SDS-polyacrylamide gel electrophoresis of PNP-UDPGT (3-MC inducible) and commercial standards. SDS-gel electrophoresis was performed by the method of Laemmli (22). (A) contains purified PNPUDPGT (2 pg), and (B) contains commercial standards (Pharmacia) with minimum molecular weights of 94,000, 67,000, 43,000, 30,000 and 20,100. Migration is from top to bottom.
dation was unaffected by the addition of testosterone (150 PM), estradiol (75 PM), androsterone (150 PM), etiocholanolone (150
Steroid
The 17-OH steroid (testosterone) UDPGT activity in solubilized microsomes could be resolved into two fractions by Chromatofocusing utilizing a pH 9 - 7 gradient, the first at pH 8.5 and the second at pH 8.1. When the fractions eluting at or about pH 8.5 were pooled and applied to the UDPhexanolamine affinity column, it was possible to separate and purify an isoenzyme of UDPGT that yielded a single band with a subunit molecular weight of 50,000 on SDS-gel electrophoresis (Fig. 3). When fractions eluting from the Chromatofocusing column at or about pH 8.1 were pooled and purified by UDP-hexanolamine affinity chromatography, two bands were observed following SDS-gel electrophoresis. One band displayed a subunit mo-
254
FALANY
A
B
C
AND
TEPHLY
pH 8.1 fraction was apparently associated with another isoenzyme which will be discussed later. We have termed the UDPGT isoenzyme present in the pH 8.5 fraction 17-OH steroid (PNP) UDPGT because it has the capacity to specifically glucuronidate the 17-OH position of steroids, such as testosterone and fl-estradiol, as well as the xenobiotics p-nitrophenol and 1-naphthol. This is a suggested nomenclature for UDPGT isoenzymes which favors naming them after endogenous substrates (24). The UDPGT activity toward P-estradiol was studied with respect to the site of glucuronide addition, whether at the 3-OH or 17-OH position of the steroid nucleus, and it was observed that greater than 97% of the glucuronide formed was due to glucuronidation of the 17-OH position (25). The substrate specificity of the 17-OH steroid (PNP) UDPGT is demonstrated in results presented in Table IV. This enzyme was capable of reacting with p-nitrophenol, lnaphthol, testosterone, and P-estradiol, but it had no reactivity with 4-methylumbelliferone, estrone, morphine, androsterone, or etiocholanolone. It was observed that pnitrophenol, 1-naphthol, testosterone, and ,&estradiol were capable of mutual inhibition of the respective glucuronidation of the substrates employed. The apparent Michaelis constants for testosterone, p-niTABLE SUBSTRATE
SPECIFICITY
IV
OF 17-OH
Substrate
FIG. 3. SDS-polyacrylamide gel electrophoresis of 17-OH steroid UDPGT, Chromatofocusing pH 8.1 fraction after UDP-hexanolamine chromatography, and commercial standards. (A) contains the pH 8.1 fraction (4 rg) following purification by affinity chromatography, (B) the purified 17-OH steroid UDPGT (2 pg), and (C) the commercial standards as listed in Fig. 2.
lecular weight of 50,000, and the second had a subunit molecular weight of 52,000 (Fig. 3). The 52,000-Da band present in the
STEROID
Specific (units/mg
activity protein)” 1650 ND* 1120 ND 190 64 ND ND
p-Nitrophenol 4-Methylumbelliferone l-Naphthol Morphine Testosterone P-Estradiol Androsterone Etiocholanolone a One unit of activity represents conjugatedimin. bND, represents no detectable Table 3.
UDPGT
1 nmol activity.
substrate Refer
to
THREE
ISOENZYMES
OF
RAT
LIVER
trophenol, and UDPGA were 10 PM, 2.0 mM, and 0.5 mM, respectively. The 17-OH steroid UDPGT isoenzyme was not inducible by 3MC treatment.
255
UDP-GLUCURONYLTRANSFERASE
AB
c
Isolation, PurQicaticm, and Characterization of &OH Androgenic UDPGT Activity
Following Chromatofocusing of solubilized microsomes (pH 9 - 7), it was possible to isolate a UDPGT activity for the 3-OH androgenic steroids androsterone and etiocholanolone. During the gradient, two peaks of glucuronidation activity toward androsterone were separated, one eluting at pH 8.1 and one at pH 7.8. When applied to the UDP-hexanolamine affinity column, both the pH 8.1 and pH ‘7.8 fractions could be eluted with low (0.1 mM) UDPGA concentrations. Following UDPhexanolamine affinity chromatography and SDS-gel electrophoresis, a single band with a subunit molecular weight of 52,000 was observed in the pH 7.8 fraction (Fig. 4). The UDPGT isoenzyme eluting at pH 7.8 has been termed a 3-OH androgen UDPGT since it appears to react with the 3-hydroxy position of the androgenic steroids androsterone and etiocholanolone. No activity was observed toward estrone, pestradiol, testosterone, pnitrophenol, lnaphthol, 4-methylumbelliferone, or morphine (Table V). The apparent Michaelis constant (Km) for androsterone was 10 WM, and the Km for etiocholanolone was 3 PM. Etiocholanolone and androsterone display mutual competitive inhibition, whereas testosterone (150 PM), p-nitrophenol (0.5 mM), 1-naphthol (0.5 mM), morphine (0.5 mM), and estrone (75 PM) do not exert any inhibitory influence on androsterone or etiocholanolone UDPGT activity. Preliminary evidence indicates that this enzyme is also capable of reacting with bile acids such as lithocholate and chenodeoxycholate (26). Following UDP-hexanolamine affinity chromatography and SDS-gel electrophoresis the pH 8.1 fraction contained two bands with subunit molecular weights of 50,000 and 52,000 (Figs. 3 and 4). The pH
FIG. 4. SDS-polyacrylamide gel electrophoresis of 3-OH androgen UDPGT, Chromatofocusing pH 8.1 fraction after UDP-hexanolamine affinity chromatography, and commercial standards. (A) contains the pH 8.1 fraction (4 pg) following purification by affinity chromatography, (B) the purified 3-OH androgen UDPGT (2 fig), and (C) the commercial standards as listed in Fig. 2.
256
FALANY TABLE
SUBSTRATE SPECIFICITY
AND
V
OF 3-OH
ANDROGEN Specific (units/mg
Substrate Androsterone Etiocholanolone pNitropheno1 4-Methylumbelliferone l-Naphthol Morphine Testosterone B-Estradiol
UDPGT activity protein)”
560 851 ND* ND ND ND ND ND
’ One unit of activity represents conjugated/min. *ND represents no detectable Table 3.
1 nmol activity.
substrate Refer
to
8.1 fraction also contained testosteroneUDPGT activity, and it is believed that the 50,000-Da band represents the 17-OH steroid-UDPGT activity. When a UDPGA gradient was utilized to elute the testosterone- and androsterone-UDPGT activities present in the pH 8.1 fraction from the UDP-hexanolamine affinity column, both activities elute at an UDPGA concentration of 30-40 PM. DISCUSSION
Clear differences exist with respect to the properties of microsomal UDPGT enzymes as isolated from female rabbit and rat liver. Tukey and Tephly (13) separated estrone and p-nitrophenol UDPGTs from rabbit liver microsomes and showed that these proteins differed considerably on the basis of substrate specificity, peptide maps, and amino acid composition. Recently (25), we have shown that the rabbit liver estrone UDPGT is highly specific for glucuronidation of the 3-OH position of estrone and /3-estradiol, and displayed no activity toward the 17-OH position of testosterone, p-nitrophenol, morphine, or 4-methylumbelliferone. This data corresponded well with results obtained when rabbit liver microsomes were studied. P-Estradiol is glucuronidated essentially completely at
TEPHLY
the 3-OH position in rabbit liver microsomal preparations. On the other hand, /3-estradiol is glucuronidated by rat liver microsomes at both the 3-OH and 17-OH positions. It was possible to separate isoenzymes from rat liver microsomal preparations that were capable of catalyzing either the 3-OH or 17-OH position of pestradiol (25). Thus, whereas rabbit liver possesses essentially a single UDPGT capable of conjugating P-estradiol at the 3OH position, rat liver has at least two separate enzymes, one that reacts with the 3OH and one that reacts with the 17-OH position of estrogenic steroids. During our preliminary studies with detergent-solubilized microsomes from rat liver, the use of ion-exchange and UDP-hexanolamine affinity chromatography procedures was not adequate to allow for complete separation of these rat liver isoenzymes of UDPGT. As a result, new methodology was developed which utilized Chromatofocusing procedures and UDP-hexanolamine Sepharose 4B chromatography with differential elution concentrations of UDPGA. These procedures allowed us to separate and purify at least three isoenzymes of UDPGT from rat liver microsomes. The isolation of a rat liver microsomal p-nitrophenol UDPGT (3-MC inducible) by this method appears to be a faster and more efficient way to purify this enzyme than has been reported previously. The enzyme, PNP-UDPGT, as isolated to apparent homogeneity by this technique, has activity toward p-nitrophenol, 1-naphthol, and 4-methylumbelliferone but has no reactivity toward morphine, testosterone, estrone, /3-estradiol, androsterone, or etiocholanolone. In a previous report, Bock et al. (10) used a number of purification steps to isolate a UDPGT activity that reacted with p-nitrophenol and 1-naphthol. However, they also observed activity toward morphine, testosterone, and estrone. The PNP-UDPGT enzyme, as we have purified it, is similar to the isoenzyme (GT-1) reported by this group since it is inducible by 3-methylcholanthrene; and on SDS-gel electrophoresis, a similar subunit molec-
THREE
ISOENZYMES
OF
RAT
LIVER
ular weight is noted. However, it would appear that their preparation may be somewhat contaminated with other UDPGT isoenzymes. Burchell (11, 12, 27) has reported on the separation of several UDPGT activities using ion-exchange and affinity chromatography, one of which reacts with p-nitrophenol, 1-naphthol, and morphine. However, he indicates that this form also reacts with testosterone in the presence of added phospholipids (12). After application of UDPGT activity to the UDPhexanolamine affinity column, all our buffer solutions contained added phospholipids; and we were able to completely separate residual testosterone UDPGT activity from the PNP-UDPGT activity by using low concentrations of UDPGA during affinity chromatography. Burchell (11) reported a similar subunit molecular weight (57,000) to that which we obtained in the current work for the 3-MC-inducible PNPUDPGT. However, the PNP-UDPGT does not glucuronidate testosterone or morphine. A second isoenzyme capable of catalyzing the glucuronidation of the 17-OH position of testosterone and P-estradiol has been isolated to apparent homogeneity. This enzyme also conjugated p-nitrophenol and 1-naphthol but not 4-methylumbelliferone and is not inducible by 3-methylcholanthrene treatment. The 17-OH steroid UDPGT has a subunit molecular weight of 50,000 after SDS-gel electrophoresis. The enzyme also displays no reactivity toward estrone, morphine, androsterone, or etiocholanolone. It is difficult to say whether this enzyme is similar to that reported by Weatheril and Burchell(12), who have isolated and identified an activity toward testosterone, since all their purified UDPGT isoenzymes display the same subunit molecular weight (57,000) (28). Recently, Matern et al. (14) have reported the isolation of an enzyme which reacts with testosterone and chenodeoxycholate. Preliminary studies from our laboratory have shown that it is possible to separate bile acid glueuronyltransferase activity from the 17-OH steroid UDPGT reported here (24), and that glucuronidation activity to-
UDP-GLUCURONYLTRANSFERASE
257
wards the bile acids lithocholate and chenodeoxycholate eluted with fractions containing 3-OH androgen UDPGT activity (26). Further work is needed to confirm and extend this observation. The activity of the 17-OH steroid UDPGT towards p-nitrophenol and lnaphthol appears to be inherent with this enzyme and not due to contamination with PNP-UDPGT. Evidence supporting this is that no 4-methylumbelliferone glucuronidation is observed, no 56,000 it!, species is observed on SDS-gel electrophoresis, the Km for p-nitrophenol is much higher with this enzyme (2.0 mM) than with the PNPUDPGA (0.4 mM), and no induction (increased specific activity) of p-nitrophenol glucuronidation occurs in this enzyme fraction after 3-MC induction. Also, there was mutual inhibition of p-nitrophenol and 17-OH steroid glucuronidation when this UDPGT isoenzyme was studied, results which were not obtained when the PNPUDPGT was examined. We report here the first isolation and purification to apparent homogeneity of a UDPGT capable of catalyzing 3-OH androgen glucuronidation. The enzyme reacts with etiocholanolone and androsterone but displays no activity towards p-nitrophenol, 1-naphthol, 4-methylumbelliferone, estrone, /3-estradiol, testosterone, or morphine. It is not inducible with 3-methylcholanthrene, and preliminary evidence suggests that it is also responsible for conjugation of some bile acids, such as lithocholate and chenodeoxycholate (26). Its subunit molecular weight on SDS-gel electrophoresis is 52,000; and based on the glucuronidation rate of androgens in liver microsomes obtained from female rats, it is present at levels which appear to be higher than that noted for the 17-OH steroid UDPGT. Matsui and colleagues have reported that rat liver androsterone UDPGT activity in rat liver microsomes is variable within (29) and between (30) strains of rats, suggesting that there may be genetic regulation of this isoenzyme. Such a situation could lead to different results between laboratories investigating UDPGT activities in different rat strains. It could also be
258
FALANY
possible that other strains and/or sex differences may be present and that such differences may account for some apparent discrepancies that exist between results reported from various laboratories. ACKNOWLEDGMENTS The authors would like to thank Clark E. Tedford and Catherine A. 0. Hawtrey for their expert technical assistance. This research was supported by NIH Grant GM26221.
REFERENCES 1. DUTTON, G. J. (1978) Annu. Rev. Pharmacol ToxicoL 18, 17-35. 2. BOCK, K. W., FR~HLING, W., REMMER, H., AND REXER, B. (1973) Biochem. Biophys. Acta 327, 46-56. 3. WISHART, G. J. (1978) B&hem. J. 174, 671-672. 4. BOCK, K. W., VON CLAUSBRUCH, U. C., JOSTING, D., AND OTTENWKLDER, H. (197’7) Biochem. Pharmacol. 26,1097-1100. 5. LUCIER, G. W. (1978) in Conjugation Reactions in Drug Metabolism (A. Aitio, ed.), pp. 167-178, Elsevier/North Holland, New York/Amsterdam. 6. CORNELIUS, C. E., AND ARIAS, I. M. (1972) Amer. .J. Path01 69, 369-371. 7. NAKATA, D., ZAKIM, D., AND VESSEY, D. A. (1976) Proc. NatL Acad Sci. USA 73, 289-292. 8. DEL VILLAR, E., SANCHEZ, E., AUTOR, A. P., AND TEPHLY, T. R. (1975) Mol. Pharmacol 11, 226240. 9. GORSKI, J. P., AND KASPER, C. B. (1977) J. BioL Chem. 252, 1336-1343. 10. BOCK, K. W., JOSTING, D., LILLIENBLUM, W., AND PFEIL, H. (1979) Eur. J. B&hem. 98, 19-26. 11. BURCHELL, B. (1978) Biochem. J. 173. 749-757.
AND
TEPHLY
12. WEATHERILL, P. J., AND BURCHELL, B. (1980) Biochem. J. 189,377-380. 13. TUKEY, R. H., AND TEPHLY, T. R. (1981) Arch B&hew Biophys. 209, 565-578. 14. MATERN, H., MATERN, S., AND GEROK, W. (1982) J. BioL Chem. 257, 7422-7429. 15. BURCHELL, B. (1981) in Reviews in Biochemical Toxicology (Hodgson, E., Bend, J. R., and Philpot, R. M., eds.), pp. l-32, Elsevier/North-Holland, New York/Amsterdam. 16. SADLER, E. J., REARICK, J. I., PAULSON, J. C., AND HILL, R. L. (1979) J. BioL Chem 254,4434-4443. 17. TUKEY, R. H., BILLINGS, R. E., AND TEPHLY, T. R. (1978) B&hem. .I 171, 659-663. 18. SANCHEZ, E., AND TEPHLY, T. R. (1974) L%ug Metub. Dispos. 2, 247-253. 19. AITIO, A. (1974) Int. J. B&hem. 5, 325-330. 20. BOCK, K. W., VAN ACKEREN, G., LORCH, F., AND BIRKE, F. W. (1976) Biochem Pharmacol 25, 2351-2356. 21. BRADFORD, M. (1976) Anal. B&hem. 72,248-253. 22. LAEMMLI, U. K. (1970) Nature (London) 227,680685. 23. WATKINS, J. B., GREGUS, Z., THOMPSON, T. N., AND KLAASSEN, C. D. (1982) ToxicoL AppL PharmacoL 64, 439-446. 24. BOCK, K. W., BURCHELL, B., DUTTON, G. J., HKNNINEN, O., MULDER, G. J., OWENS, I. S., SIEST, G., AND TEPHLY, T. R. (1983) B&hem. PharmacoL 32, 953-955. 25. FALANY, C. N., ROYCHOWDHURY, J., ROYCHOWDHURY, N., AND TEPHLY, T. R. (1983) Lh-ug Metub. Dispos., in press. 26. KIRKPATRICK, R. B., FALANY, C. N., AND TEPHLY, T. R. (1983) The Pharmucologist 25, 171. 27. BURCHELL, B. (1980) FEBS Lett. 111,131-135. 28. WEATHERILL, P. J., KENNEDY, S. M. E., AND BURCHELL, B. (1980) Biochem. J. 191, 155-163. 29. MATSUI, M., AND HAKOZAKI, M. (1979) B&hem. PharrnacoL 28, 411-415. 30. MATSUI, M., NAGAI, F., AND AOYAGI, S. (1979) Biochem. J. 179, 483-487.
AND BIOPHYSICS
pp. 248-258,
1983
Separation, Purification and Characterization of Three lsoenzymes of UDP-Glucuronyltransferase from Rat Liver Microsomes CHARLES The Toxicology
Center,
N. FALANY
Department
Received
May
AND
of Pharmacology,
THOMAS University
23, 1983, and in revised
form
R. TEPHLY’ of Iowa,
July
Imva
City,
Iowa
52242
19, 1983
Three isoenzymes of UDP-glucuronyltransferase (UDPGT) have been separated and purified from liver microsomes of untreated female rats or female rats pretreated with 3-methylcholanthrene. The UDPGT isoenzymes were purified utilizing Chromatofocusing, column isoelectric focusing, and UDP-hexanolamine Sepharose 4B affinity chromatography. UDPGT activities could also be separated during UDP-hexanolamine affinity chromatography by elution with different UDPGA (UDP-glucuronic acid) concentrations. One isoenzyme exhibits a subunit molecular weight of 56,000 and is capable of conjugating p-nitrophenol, 1-naphthol, and 4-methylumbelliferone. This isoenzyme is inducible by 3-methylcholanthrene treatment and requires high UDPGA concentrations for elution from the UDP-hexanolamine affinity column in contrast to the other UDPGT isoenzymes. A second isoenzyme was purified and displayed a subunit molecular weight of 50,000. This isoenzyme was not induced by 3-methylcholanthrene and was active towards testosterone, the 17-OH position of P-estradiol, p-nitrophenol, and lnaphthol. A third isoenzyme was also purified and exhibited a subunit molecular weight of 52,000. This isoenzyme conjugated androsterone and etiocholanolone and was not induced by 3-methylcholanthrene treatment. This study reports the purification of two separate and distinct rat liver UDPGT isoenzymes capable of conjugating p-nitrophenol, only one of which is inducible by 3-methylcholanthrene treatment. Also, this is the first report of the purification of a UDPGT isoenzyme active towards the 3-OH position of androgens.
UDP-Glucuronyltransferase (EC 2.4.1.17) represents a family of enzymes present in the endoplasmic reticulum of the liver which are responsible for the conjugation of glucuronic acid with a wide variety of xenobiotic and endogenous compounds. The heterogeneity of UDP-glucuronyltransferase (UDPGT)2 activities has been established by a number of studies which have focused on the differential rates of development of UDPGT activities (1,5),
differential inducibilities of UDPGT activities (2-4), the absence of certain forms of UDPGT activities in certain strains of animals (6, 7), and on physical separation of UDPGT activities. The separation of various UDPGT activities has been reported by de1 Villar et al. (8), Gorski and Kasper (9), Bock et al. (lo), Burchell and co-workers (11,12), and this laboratory (13). Recently, Matern et al. (14) have isolated a rat liver UDPGT activity which glucuronidates testosterone and chenodeoxycholate. Our laboratory has reported the separation, purification, and characterization of UDPGT activities obtained from rabbit liver, and has identified separate enzymes capable of catalyzing estrone and p-nitro-
’ To whom correspondence should be addressed. a Abbreviations used: UDPGT, UDP-glucuronyltransferase; UDPGA, UDP-glucuronic acid; 3-MC, 3methylcholanthrene; SDS, sodium dodecyl sulfate; PNP, pnitrophenol. 0003-9861/83 Copyright All rights
$3.00
c> 1983 by Academic Press, Inc. of reproduction in any form reserved.
248
THREE
ISOENZYMES
OF
RAT
LIVER
phenol glucuronidation (13). Both rabbit liver UDPGT isoenzymes displayed similar subunit molecular weights following SDSpolyacrylamide gel electrophoresis but were separable by ion-exchange chromatography and UDP-hexanolamine affinity chromatography. Limited proteolysis studies have confirmed that they are distinct proteins (13). A tentative classification of rat and rabbit liver UDPGT activities has been proposed by Burchell(15), based primarily on separation characteristics after different types of ion-exchange chromatography. In this classification, isoenzymes of UDPGT have been suggested to possess activity towards both endogenous compounds and xenobiotics. Also, a given compound may be conjugated by more than one isoenzyme. This classification scheme also suggests that the number of UDPGT isoenzymes in rat liver may be greater than in rabbit liver. Another classification of rat liver UDPGT activities has been suggested by Bock et al. (10). This classification relies on the inducibility and separation of rat liver UDPGT activities following 3-methylcholanthrene (3-MC) or phenobarbital treatment. These investigators have isolated 3-MC-inducible and phenobarbitalinducible isoenzymes of UDPGT, termed GT1 and GT2, respectively. In this classification, steroid glucuronidation may represent a third form of UDPGT activities since it is apparently not greatly affected by either 3-MC or phenobarbital treatment. Our laboratory has recently been studying the separation, purification, and inducibility of xenobiotic and steroid UDPGT activities present in female rat liver. The current study presents the purification and characterization of three isoenzymes of UDPGT from liver microsomes of untreated or 3-MC-treated female rats by Chromatofocusing and UDP-hexanolamine affinity chromatography. MATERIALS
AND
METHODS
Materials. [4-i4C]Estrone (57 mCi/mmol), [4-i4C]testosterone (50 mCi/mmol), [1,2-3H]etiocholanolone (40-60 Ci/mmol), [1,2-aH]androsterone (40-60 Ci/ mmol), [l-‘4C]naphtho1 (4.3 mCi/mmol), and [4-i4C]p-
UDP-GLUCURONYLTRANSFERASE
249
estradiol (50 mCi/mmol) were purchased from New England Nuclear, Boston, Massachusetts. p-Nitro[U‘*C]phenol (24 mCi/mmol) was obtained from California Bionuclear Corporation, Sun Valley, California. [N-methyl-“C]Morphine hydrochloride (40 mCi/mmol) was obtained from Amersham, Arlington Heights, Illinois. L-a-Phosphatidylcholine (egg yolk Type XI-E), UDP-glucuronic acid, estrone, fl-estradiol, pnitrophenol, testosterone, 1-naphthol, and I-methylumbelliferone were purchased from Sigma Chemical Company, St. Louis, Missouri. Morphine sulfate was obtained from Mallinckrodt Chemical Works, St. Louis, Missouri. Chromatofocusing materials were obtained from Pharmacia Fine Chemicals, Piscataway, New Jersey. Emulgen 911 was purchased from Kao Atlas Ltd., Tokyo, Japan. Bio-Rad dye reagent protein assay was obtained from Bio-Rad Laboratories, Richmond, California. All other chemicals were of analytical grade. UDP-hexanolamine Sepharose 4B affinity resin was prepared as outlined by Sadler et al. (16) with modifications as previously described (13). Assays. pNitropheno1 glucuronidation activity was assayed as previously described (17) except a final p nitrophenol concentration of 4 mM was utilized. Morphine glucuronidation was determined by the method of Sanchez and Tephly (18); 4-methylumbelliferone glucuronidation activity was assayed as described by Aitio (19); 1-naphthol UDPGT activity was analyzed by the procedure described by Bock et al (20). Estrone, estradiol, and testosterone glucuronidation assays were assayed by the method of Tukey et al (1’7), except a final concentration of 37.5 pM for each substrate was utilized. Etiocholanolone and androsterone glucuronidation was measured by the method of Tukey et al. (17) with a final substrate concentration of 55 PM. All reactions were performed at 37°C with 5 mM UDP-glucuronic acid (UDPGA) and in the presence of 100 fig of phosphatidylcholine. Buffer solutions containing phosphatidylcholine were clarified by sonication as previously described (13). However, when alternate substrate-inhibition studies were performed, 10 mM UDPGA was used. All reactions were linear with respect to time and protein concentration. Protein concentrations were determined using the Bio-Rad protein assay originally developed by Bradford (21). Preparation of microsomes. 3-MC, when used, was dissolved in corn oil and administered daily 40 mg/ kg; ip) to female Sprague-Dawley rats (150-200 g, Biolabs, Madison, Wise.) for 3 days. Following the last injection the rats were starved for 24 h and killed by cerebral concussion, and their livers were perfused in situ with 1.15% cold KCl. Livers were then removed and homogenized in 5 vol 1.15% KC1 with a motordriven Teflon pestle. Microsomes were isolated by centrifugation, washed with 1.15% KCl, and stored as pellets with a 1.15% KC1 overlay at -6O’C. The
250 microsomal no apparent
FALANY
AND
pellets could be stored for 3 months with loss of glucuronidation activity. Separation and purtfication of PNP-UDPGTactivity (S-MC inducible). Frozen microsomal pellets were thawed, the KC1 overlay was removed, and a 10% (w/ v) suspension was formed by gentle homogenization in 25 mM ethanolamine-HAc buffer, pH 9.4. All buffers utilized in the purification procedures contained 20% glycerol, 0.1 mM dithiothreitol, and 0.05% Emulgen 911. The microsomal suspension was solubilized by the further addition of 0.5 mg Emulgen 911/mg protein, stirred for 30 min, and then centrifuged at 100,OOOg for 30 min. Between 85 and 95% of the UDPGT activities and protein were routinely recoverable in the supernatant fluid following solubilization. Solubilized microsomal protein was applied directly to a Chromatofocusing PBE 94 column (0.9 X 40 cm) previously equilibrated in 25 mM ethanolamine-HAc, pH 9.4. The column was eluted with Polybuffer 96HAc, pH 6.0, as recommended by Pharmacia to generate a pH 9 - 6 gradient. A sharp peak of pnitrophenol-UDPGT activity eluted in the void volume prior to the start of the pH gradient. A second broad peak of pnitrophenol-UDPGT activity eluted at a pH maxima of approximately 8.1. Fractions from the void volume (pH 9.0) of the Chromatofocusing column that contained pnitrophenol-UDPGT activity were pooled, and MgClz was added to a final concentration of 5 mM. The pooled fractions were applied to a UDP-hexanolamine Sepharose 4B affinity column (1.6 X 10 cm) previously equilibrated with 20 mM Tris-HCl, pH 8.0, buffer. The affinity column was then washed with at least 5 column volumes of the same buffer containing 50 mM KC1 and 50 pg phosphatidycholine/ml. Next, the column was washed with two to three column volumes of the same buffer containing 0.1 mM UDPGA. This procedure removed small amounts of activities toward testosterone, androsterone, morphine, and estrone. Finally, the same buffer containing 4 mM UDPGA was applied to the UDP-hexanolamine affinity column. This higher UDPGA concentration allowed for the removal of a 3-MC-inducible p-nitrophenolUDPGT activity in an apparently homogeneous form. Separation and purification of IT-OH steroid UDPGT activity. Microsomal protein was solubilized as described above. The solubilized microsomal protein was applied directly to a Chromatofocusing PBE 94 column (0.9 X 40 cm) previously equilibrated in 25 mM ethanolamine-HCl, pH 9.4. The column was eluted with Polybuffer 96-HCl, pH 7.0, to form a pH 9 - 7 gradient. Two fractions of 1’7-OH steroid (testosterone) UDPGT activity were resolved during the pH 9 - 7 gradient, one eluted at pH 8.5 and the second eluted at approximately pH 8.1. Fractions which eluted from the Chromatofocusing column at or about pH 8.5 or at or about pH 8.1 were
TEPHLY
pooled, and MgClz was added to a final concentration of 5 mM. The pooled fractions were applied to separate UDP-hexanolamine affinity columns (1.6 X 10 cm) previously equilibrated in 20 mM Tris-HCl, pH 8.0, buffer. The columns were then washed with five to seven column volumes of the same buffer containing 50 mM KC1 and 50 fig phosphatidylcholine/ml. The columns were then rinsed with the same buffer containing a low concentration of UDPGA (0.1 mM) which removed the 17-OH steroid UDPGT activity. Higher concentrations of UPDGA (4 mM) did not elute any more 17-OH steroid UDPGT activity. Separation and purification of S-OH androgen UDPGT activity. Microsomal protein was solubilized with Emulgen 911 as previously described, and the solubilized microsomal protein was applied directly to a Chromatofocusing PBE 94 column (0.9 X 40 cm) previously equilibrated in 25 mM ethanolamine-HCl, pH 9.4. The column was eluted with Polybuffer 96HCl, pH 7.0, in order to generate a pH 9 - ‘7 gradient. Two peaks of androsterone UDPGT activity were obtained, the first peak eluted at about pH 8.1 and the second at approximately pH 7.8. Fractions eluting from the Chromatofocusing column at pH 8.1 or pH 7.8, which contained androsterone-UDPGT activity, were pooled and MgClz was added to a final concentration of 5 mM. The pooled fractions were applied to UDP-hexanolamine affinity columns (1.6 X 10 cm) previously equilibrated in 20 mM Tris-HCl, pH 8.0, buffer. The columns were then washed with five to seven column volumes of buffer containing 50 mM KC1 and 50 pg/ml phosphatidylcholine. The columns were then eluted with the same buffer containing 0.1 mM UDPGA. This UDPGA concentration eluted an isoenzyme of UDPGT which was capable of conjugating both androsterone and etiocholanolone. Rinsing the affinity columns with a higher concentration of UDPGA (4 mM) did not elute any further androsterone or etiocholanolone UDPGT activity. Occasionally, elution of UDPGT activity from a UDP-hexanolamine affinity column was performed utilizing a linear gradient of UDPGA in the presence of the 20 mM Tris-HCl, pH 8.0, buffer mixture; and this procedure yielded good recovery of enzyme. Gel electrophoresis. Polyacrylamide gel electrophoresis was performed in the presence of sodium dodecyl sulfate (SDS) in a Hoefer Scientific Model 600 apparatus as previously outlined by Laemmli (22). Samples were pretreated by the addition of P-mercaptoethanol and SDS to final concentrations of 5 and l%, respectively, and placed in a boiling-water bath for 10 min. Following electrophoresis, the slabs were stained for 2 h in a mixture of isopropanol:acetic acid:H,O (25:10:65, v/v) which contained 0.1% Coomassie G-250 and destained with isopropanol:acetic acid:water (10:10:80, v/v). Minimum subunit molecular weights were estimated by comparison to commercial standards (Pharmacia).
THREE
ISOENZYMES
OF
RAT
LIVER
phenol-UDPGT activity was observed in the first fraction while the fraction of pnitrophenol-UDPGT activity which eluted at pH 8.1 was apparently unaffected (Table II). The increase in p-nitrophenol-UDPGT activity induced by 3-MC treatment and which eluted in the first fraction was not due to an increased recovery of protein since similar amounts of protein were added to each column and equivalent amounts of protein were recovered in each fraction. UDP-hexanolamine Sepharose 4B affinity chromatography was used to completely purify the 3-MC-inducible p-nitrophenolUDPGT activity. Fractions that contained p-nitrophenol-UDPGT activity and eluted prior to the start of the pH gradient were pooled, MgClz was added to a final concentration of 5 mM, and the fraction was applied to the affinity column. The affinity column was then rinsed with five to seven column volumes of 20 mM Tris-HCl, pH 8.0, containing 50 mM KC1 and 50 wg/ml phosphatidylcholine to remove non-specifically bound proteins. Next, the column was washed with the same buffer mixture containing 0.1 mM UDPGA. This procedure provided for the complete separation of several contaminating UDPGT activities which were present. The addition of 4.0 mM UDPGA to the buffer mixture yielded high purification of a p-nitrophenolUDPGT activity.
RESULTS
Isolation, Purijcatim, and Characterization of 3-Methylcholanthrene-Inducible pNitropheno1 UDPGlucuronyltramferase
Following treatment of female rats with 3-MC, there was a marked induction of hepatic microsomal UDPGT activity toward p-nitrophenol, 4-methylumbelliferone, and 1-naphthol. This treatment did not lead to increases in UDPGT activities toward morphine, testosterone, estrone, androsterone, etiocholanolone, or estradiol (Table I). The rate of 4-methylumbelliferone glucuronidation was increased approximately sevenfold; p-nitrophenol glucuronidation was increased more than threefold, and 1-naphthol was increased about fourfold. Similar results following 3-MC treatment of rats have been obtained from other laboratories (2, 3, 23). Chromatofocusing procedures allowed for the separation of two UDPGT activities toward p-nitrophenol, one of which reacts with 4-methylumbelliferone and one which does not. One fraction of p-nitrophenolUDPGT activity eluted prior to the start of the pH gradient, and the second fraction eluted at a pH of approximately 8.1 (Fig. 1). When microsomes from 3-MC-treated rats were utilized, an increase in p-nitroTABLE The Effect
of 3-Methylcholanthrene Solubilized
I
Pretreatment Rat Liver
on Glucuronidation Microsomes
Untreated controls (units/mg)“.*
Substrate p-Nitrophenol 4-Methylumbelliferone 1-Naphthol Morphine Testosterone Estrone P-Estradiol Etiocholanolone Androsterone Note. Female rats were pretreated Materials and Methods. a One unit of activity represents *Means f SE of three to twelve from three to twelve rats.
Female
110.8 24.7 52.6 9.7 6.0 1.4 4.2 22.4 14.5 with
+ f f 2 f zk f It f
251
UDP-GLUCURONYLTRANSFERASE
in
3-Methylcholanthrene (units/mg)“,*
4.2 1.1 3.0 0.4 0.3 0.1 0.6 0.8 0.5
3-methylcholanthrene
Rates
358.9 168.7 225 9.1 6.0 1.7 4.2 22.9 14.3 and microsomes
1 nmol substrate conjugated/min. determinations of separate mierosomal
‘- 39.7 t- 13.3 f 16.4 f 0.3 k 0.2 + 0.2 f 0.6 f 0.9 * 0.7
prepared
preparations
treated
as described
consisting
under
of livers
252
FALANY
AND
TEPHLY
-9 o . 0 .
UDPGT ACT 3 -MC IND ___-------UDPGT ACT CONTROL EH---3 -MC -_ IND _ PH CONTROL
-8 I
[I
FIG. 1. Chromatofocusing of liver microsomes from untreated and 3-methylcholanthrene-treated rats. One hundred twenty milligrams of microsomal protein from treated or untreated rats was applied to a 0.9 X 40-cm Chromatofocusing column. The columns were then eluted with Polybuffer 96-HAc, pH 6.0, to generate a pH 9 - 6 gradient. (A) indicates those fractions containing pnitrophenol UDPGT activity eluting prior to the start of the pH gradient. (B) indicates those fractions containing p-nitrophenol UDPGT activity eluting at or about pH 8.1.
p-Nitrophenol-UDPGT activity which eluted from the UDP-hexanolamine affinity column following high UDPGA displayed a single band with a subunit molecular weight of about 56,000 after SDSgel electrophoresis (Fig. 2). This UDPGT isoenzyme has been termed PNP-UDPGT, and studies on the substrate specificity of
TABLE ~NITROPHENOL
this enzyme are presented in Table III. This isoenzyme conjugated p-nitrophenol, lnaphthol, and 4-methylumbelliferone, but there was no glucuronidation of the steroids tested or of morphine. The presence of 1-naphthol (0.5 mM) or 4-methylumbelliferone (0.1 mM) inhibited PNP glucuronidation activity, whereas PNP glucuroni-
II
UDPGT
ACTIVITYFOLLOWINGCHROMATOFOCUSINGOFSOLUBILIZEDLIVERMICROSOMES FROM~NTREATEDANDS-METHyLCHOLANTHRENE-TREATED
p-Nitrophenol Untreated
Solubilized
microsomes
Chromatofocusing Fraction A (pH Fraction B (pH
9.0) 8.1)
microsomes (units)”
UDPGT
RATS activity
3-Methylcholanthrene-treated microsomes (units)’
13,430
34,020
2,500 2,760
5,589 2,724
Note. The chromatofocusing procedure was performed as described in Fig. 1. The fractions nitrophenol UDPGT activity were pooled, and the levels of p-nitrophenol glucuronidation described under Materials and Methods. a One unit of activity represents 1 nmol substrate conjugated/min.
containing determined
p as
THREE
A
ISOENZYMES
OF
RAT
LIVER
253
UDP-GLUCURONYLTRANSFERASE
B
TABLE SUBSTRATE
Substrate
SPECIFICITY
III OF PNP-UDPGT
Specific (units/mg
pNitropheno1 4-Methylumbelliferone 1-Naphthol Morphine Testosterone P-Estradiol Androsterone Etiocholanolone
activity protein)” 5733 1129 4331 ND* ND ND ND ND
a One unit of activity equals 1 nmol substrate conjugated/min. *ND represents no detectable activity. The limit of detection for steroid substrates is 0.5-1.0 units/mg; p-nitrophenol, 15 units/mg; 4-methylumbelliferone, 2 units/mg; morphine, 1 units/mg.
PM), estrone (75 PM), or morphine (0.5 mM). The apparent Km’s for p-nitrophenol and UDPGA were 0.4 and 0.4 mM, respectively. The purified protein did not display any visible or reduced carbon monoxide spectra.
Isolation, Purification, and Characterization of U-OH (PNP) UDPGT Activity
FIG. 2. SDS-polyacrylamide gel electrophoresis of PNP-UDPGT (3-MC inducible) and commercial standards. SDS-gel electrophoresis was performed by the method of Laemmli (22). (A) contains purified PNPUDPGT (2 pg), and (B) contains commercial standards (Pharmacia) with minimum molecular weights of 94,000, 67,000, 43,000, 30,000 and 20,100. Migration is from top to bottom.
dation was unaffected by the addition of testosterone (150 PM), estradiol (75 PM), androsterone (150 PM), etiocholanolone (150
Steroid
The 17-OH steroid (testosterone) UDPGT activity in solubilized microsomes could be resolved into two fractions by Chromatofocusing utilizing a pH 9 - 7 gradient, the first at pH 8.5 and the second at pH 8.1. When the fractions eluting at or about pH 8.5 were pooled and applied to the UDPhexanolamine affinity column, it was possible to separate and purify an isoenzyme of UDPGT that yielded a single band with a subunit molecular weight of 50,000 on SDS-gel electrophoresis (Fig. 3). When fractions eluting from the Chromatofocusing column at or about pH 8.1 were pooled and purified by UDP-hexanolamine affinity chromatography, two bands were observed following SDS-gel electrophoresis. One band displayed a subunit mo-
254
FALANY
A
B
C
AND
TEPHLY
pH 8.1 fraction was apparently associated with another isoenzyme which will be discussed later. We have termed the UDPGT isoenzyme present in the pH 8.5 fraction 17-OH steroid (PNP) UDPGT because it has the capacity to specifically glucuronidate the 17-OH position of steroids, such as testosterone and fl-estradiol, as well as the xenobiotics p-nitrophenol and 1-naphthol. This is a suggested nomenclature for UDPGT isoenzymes which favors naming them after endogenous substrates (24). The UDPGT activity toward P-estradiol was studied with respect to the site of glucuronide addition, whether at the 3-OH or 17-OH position of the steroid nucleus, and it was observed that greater than 97% of the glucuronide formed was due to glucuronidation of the 17-OH position (25). The substrate specificity of the 17-OH steroid (PNP) UDPGT is demonstrated in results presented in Table IV. This enzyme was capable of reacting with p-nitrophenol, lnaphthol, testosterone, and P-estradiol, but it had no reactivity with 4-methylumbelliferone, estrone, morphine, androsterone, or etiocholanolone. It was observed that pnitrophenol, 1-naphthol, testosterone, and ,&estradiol were capable of mutual inhibition of the respective glucuronidation of the substrates employed. The apparent Michaelis constants for testosterone, p-niTABLE SUBSTRATE
SPECIFICITY
IV
OF 17-OH
Substrate
FIG. 3. SDS-polyacrylamide gel electrophoresis of 17-OH steroid UDPGT, Chromatofocusing pH 8.1 fraction after UDP-hexanolamine chromatography, and commercial standards. (A) contains the pH 8.1 fraction (4 rg) following purification by affinity chromatography, (B) the purified 17-OH steroid UDPGT (2 pg), and (C) the commercial standards as listed in Fig. 2.
lecular weight of 50,000, and the second had a subunit molecular weight of 52,000 (Fig. 3). The 52,000-Da band present in the
STEROID
Specific (units/mg
activity protein)” 1650 ND* 1120 ND 190 64 ND ND
p-Nitrophenol 4-Methylumbelliferone l-Naphthol Morphine Testosterone P-Estradiol Androsterone Etiocholanolone a One unit of activity represents conjugatedimin. bND, represents no detectable Table 3.
UDPGT
1 nmol activity.
substrate Refer
to
THREE
ISOENZYMES
OF
RAT
LIVER
trophenol, and UDPGA were 10 PM, 2.0 mM, and 0.5 mM, respectively. The 17-OH steroid UDPGT isoenzyme was not inducible by 3MC treatment.
255
UDP-GLUCURONYLTRANSFERASE
AB
c
Isolation, PurQicaticm, and Characterization of &OH Androgenic UDPGT Activity
Following Chromatofocusing of solubilized microsomes (pH 9 - 7), it was possible to isolate a UDPGT activity for the 3-OH androgenic steroids androsterone and etiocholanolone. During the gradient, two peaks of glucuronidation activity toward androsterone were separated, one eluting at pH 8.1 and one at pH 7.8. When applied to the UDP-hexanolamine affinity column, both the pH 8.1 and pH ‘7.8 fractions could be eluted with low (0.1 mM) UDPGA concentrations. Following UDPhexanolamine affinity chromatography and SDS-gel electrophoresis, a single band with a subunit molecular weight of 52,000 was observed in the pH 7.8 fraction (Fig. 4). The UDPGT isoenzyme eluting at pH 7.8 has been termed a 3-OH androgen UDPGT since it appears to react with the 3-hydroxy position of the androgenic steroids androsterone and etiocholanolone. No activity was observed toward estrone, pestradiol, testosterone, pnitrophenol, lnaphthol, 4-methylumbelliferone, or morphine (Table V). The apparent Michaelis constant (Km) for androsterone was 10 WM, and the Km for etiocholanolone was 3 PM. Etiocholanolone and androsterone display mutual competitive inhibition, whereas testosterone (150 PM), p-nitrophenol (0.5 mM), 1-naphthol (0.5 mM), morphine (0.5 mM), and estrone (75 PM) do not exert any inhibitory influence on androsterone or etiocholanolone UDPGT activity. Preliminary evidence indicates that this enzyme is also capable of reacting with bile acids such as lithocholate and chenodeoxycholate (26). Following UDP-hexanolamine affinity chromatography and SDS-gel electrophoresis the pH 8.1 fraction contained two bands with subunit molecular weights of 50,000 and 52,000 (Figs. 3 and 4). The pH
FIG. 4. SDS-polyacrylamide gel electrophoresis of 3-OH androgen UDPGT, Chromatofocusing pH 8.1 fraction after UDP-hexanolamine affinity chromatography, and commercial standards. (A) contains the pH 8.1 fraction (4 pg) following purification by affinity chromatography, (B) the purified 3-OH androgen UDPGT (2 fig), and (C) the commercial standards as listed in Fig. 2.
256
FALANY TABLE
SUBSTRATE SPECIFICITY
AND
V
OF 3-OH
ANDROGEN Specific (units/mg
Substrate Androsterone Etiocholanolone pNitropheno1 4-Methylumbelliferone l-Naphthol Morphine Testosterone B-Estradiol
UDPGT activity protein)”
560 851 ND* ND ND ND ND ND
’ One unit of activity represents conjugated/min. *ND represents no detectable Table 3.
1 nmol activity.
substrate Refer
to
8.1 fraction also contained testosteroneUDPGT activity, and it is believed that the 50,000-Da band represents the 17-OH steroid-UDPGT activity. When a UDPGA gradient was utilized to elute the testosterone- and androsterone-UDPGT activities present in the pH 8.1 fraction from the UDP-hexanolamine affinity column, both activities elute at an UDPGA concentration of 30-40 PM. DISCUSSION
Clear differences exist with respect to the properties of microsomal UDPGT enzymes as isolated from female rabbit and rat liver. Tukey and Tephly (13) separated estrone and p-nitrophenol UDPGTs from rabbit liver microsomes and showed that these proteins differed considerably on the basis of substrate specificity, peptide maps, and amino acid composition. Recently (25), we have shown that the rabbit liver estrone UDPGT is highly specific for glucuronidation of the 3-OH position of estrone and /3-estradiol, and displayed no activity toward the 17-OH position of testosterone, p-nitrophenol, morphine, or 4-methylumbelliferone. This data corresponded well with results obtained when rabbit liver microsomes were studied. P-Estradiol is glucuronidated essentially completely at
TEPHLY
the 3-OH position in rabbit liver microsomal preparations. On the other hand, /3-estradiol is glucuronidated by rat liver microsomes at both the 3-OH and 17-OH positions. It was possible to separate isoenzymes from rat liver microsomal preparations that were capable of catalyzing either the 3-OH or 17-OH position of pestradiol (25). Thus, whereas rabbit liver possesses essentially a single UDPGT capable of conjugating P-estradiol at the 3OH position, rat liver has at least two separate enzymes, one that reacts with the 3OH and one that reacts with the 17-OH position of estrogenic steroids. During our preliminary studies with detergent-solubilized microsomes from rat liver, the use of ion-exchange and UDP-hexanolamine affinity chromatography procedures was not adequate to allow for complete separation of these rat liver isoenzymes of UDPGT. As a result, new methodology was developed which utilized Chromatofocusing procedures and UDP-hexanolamine Sepharose 4B chromatography with differential elution concentrations of UDPGA. These procedures allowed us to separate and purify at least three isoenzymes of UDPGT from rat liver microsomes. The isolation of a rat liver microsomal p-nitrophenol UDPGT (3-MC inducible) by this method appears to be a faster and more efficient way to purify this enzyme than has been reported previously. The enzyme, PNP-UDPGT, as isolated to apparent homogeneity by this technique, has activity toward p-nitrophenol, 1-naphthol, and 4-methylumbelliferone but has no reactivity toward morphine, testosterone, estrone, /3-estradiol, androsterone, or etiocholanolone. In a previous report, Bock et al. (10) used a number of purification steps to isolate a UDPGT activity that reacted with p-nitrophenol and 1-naphthol. However, they also observed activity toward morphine, testosterone, and estrone. The PNP-UDPGT enzyme, as we have purified it, is similar to the isoenzyme (GT-1) reported by this group since it is inducible by 3-methylcholanthrene; and on SDS-gel electrophoresis, a similar subunit molec-
THREE
ISOENZYMES
OF
RAT
LIVER
ular weight is noted. However, it would appear that their preparation may be somewhat contaminated with other UDPGT isoenzymes. Burchell (11, 12, 27) has reported on the separation of several UDPGT activities using ion-exchange and affinity chromatography, one of which reacts with p-nitrophenol, 1-naphthol, and morphine. However, he indicates that this form also reacts with testosterone in the presence of added phospholipids (12). After application of UDPGT activity to the UDPhexanolamine affinity column, all our buffer solutions contained added phospholipids; and we were able to completely separate residual testosterone UDPGT activity from the PNP-UDPGT activity by using low concentrations of UDPGA during affinity chromatography. Burchell (11) reported a similar subunit molecular weight (57,000) to that which we obtained in the current work for the 3-MC-inducible PNPUDPGT. However, the PNP-UDPGT does not glucuronidate testosterone or morphine. A second isoenzyme capable of catalyzing the glucuronidation of the 17-OH position of testosterone and P-estradiol has been isolated to apparent homogeneity. This enzyme also conjugated p-nitrophenol and 1-naphthol but not 4-methylumbelliferone and is not inducible by 3-methylcholanthrene treatment. The 17-OH steroid UDPGT has a subunit molecular weight of 50,000 after SDS-gel electrophoresis. The enzyme also displays no reactivity toward estrone, morphine, androsterone, or etiocholanolone. It is difficult to say whether this enzyme is similar to that reported by Weatheril and Burchell(12), who have isolated and identified an activity toward testosterone, since all their purified UDPGT isoenzymes display the same subunit molecular weight (57,000) (28). Recently, Matern et al. (14) have reported the isolation of an enzyme which reacts with testosterone and chenodeoxycholate. Preliminary studies from our laboratory have shown that it is possible to separate bile acid glueuronyltransferase activity from the 17-OH steroid UDPGT reported here (24), and that glucuronidation activity to-
UDP-GLUCURONYLTRANSFERASE
257
wards the bile acids lithocholate and chenodeoxycholate eluted with fractions containing 3-OH androgen UDPGT activity (26). Further work is needed to confirm and extend this observation. The activity of the 17-OH steroid UDPGT towards p-nitrophenol and lnaphthol appears to be inherent with this enzyme and not due to contamination with PNP-UDPGT. Evidence supporting this is that no 4-methylumbelliferone glucuronidation is observed, no 56,000 it!, species is observed on SDS-gel electrophoresis, the Km for p-nitrophenol is much higher with this enzyme (2.0 mM) than with the PNPUDPGA (0.4 mM), and no induction (increased specific activity) of p-nitrophenol glucuronidation occurs in this enzyme fraction after 3-MC induction. Also, there was mutual inhibition of p-nitrophenol and 17-OH steroid glucuronidation when this UDPGT isoenzyme was studied, results which were not obtained when the PNPUDPGT was examined. We report here the first isolation and purification to apparent homogeneity of a UDPGT capable of catalyzing 3-OH androgen glucuronidation. The enzyme reacts with etiocholanolone and androsterone but displays no activity towards p-nitrophenol, 1-naphthol, 4-methylumbelliferone, estrone, /3-estradiol, testosterone, or morphine. It is not inducible with 3-methylcholanthrene, and preliminary evidence suggests that it is also responsible for conjugation of some bile acids, such as lithocholate and chenodeoxycholate (26). Its subunit molecular weight on SDS-gel electrophoresis is 52,000; and based on the glucuronidation rate of androgens in liver microsomes obtained from female rats, it is present at levels which appear to be higher than that noted for the 17-OH steroid UDPGT. Matsui and colleagues have reported that rat liver androsterone UDPGT activity in rat liver microsomes is variable within (29) and between (30) strains of rats, suggesting that there may be genetic regulation of this isoenzyme. Such a situation could lead to different results between laboratories investigating UDPGT activities in different rat strains. It could also be
258
FALANY
possible that other strains and/or sex differences may be present and that such differences may account for some apparent discrepancies that exist between results reported from various laboratories. ACKNOWLEDGMENTS The authors would like to thank Clark E. Tedford and Catherine A. 0. Hawtrey for their expert technical assistance. This research was supported by NIH Grant GM26221.
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AND
TEPHLY
12. WEATHERILL, P. J., AND BURCHELL, B. (1980) Biochem. J. 189,377-380. 13. TUKEY, R. H., AND TEPHLY, T. R. (1981) Arch B&hew Biophys. 209, 565-578. 14. MATERN, H., MATERN, S., AND GEROK, W. (1982) J. BioL Chem. 257, 7422-7429. 15. BURCHELL, B. (1981) in Reviews in Biochemical Toxicology (Hodgson, E., Bend, J. R., and Philpot, R. M., eds.), pp. l-32, Elsevier/North-Holland, New York/Amsterdam. 16. SADLER, E. J., REARICK, J. I., PAULSON, J. C., AND HILL, R. L. (1979) J. BioL Chem 254,4434-4443. 17. TUKEY, R. H., BILLINGS, R. E., AND TEPHLY, T. R. (1978) B&hem. .I 171, 659-663. 18. SANCHEZ, E., AND TEPHLY, T. R. (1974) L%ug Metub. Dispos. 2, 247-253. 19. AITIO, A. (1974) Int. J. B&hem. 5, 325-330. 20. BOCK, K. W., VAN ACKEREN, G., LORCH, F., AND BIRKE, F. W. (1976) Biochem Pharmacol 25, 2351-2356. 21. BRADFORD, M. (1976) Anal. B&hem. 72,248-253. 22. LAEMMLI, U. K. (1970) Nature (London) 227,680685. 23. WATKINS, J. B., GREGUS, Z., THOMPSON, T. N., AND KLAASSEN, C. D. (1982) ToxicoL AppL PharmacoL 64, 439-446. 24. BOCK, K. W., BURCHELL, B., DUTTON, G. J., HKNNINEN, O., MULDER, G. J., OWENS, I. S., SIEST, G., AND TEPHLY, T. R. (1983) B&hem. PharmacoL 32, 953-955. 25. FALANY, C. N., ROYCHOWDHURY, J., ROYCHOWDHURY, N., AND TEPHLY, T. R. (1983) Lh-ug Metub. Dispos., in press. 26. KIRKPATRICK, R. B., FALANY, C. N., AND TEPHLY, T. R. (1983) The Pharmucologist 25, 171. 27. BURCHELL, B. (1980) FEBS Lett. 111,131-135. 28. WEATHERILL, P. J., KENNEDY, S. M. E., AND BURCHELL, B. (1980) Biochem. J. 191, 155-163. 29. MATSUI, M., AND HAKOZAKI, M. (1979) B&hem. PharrnacoL 28, 411-415. 30. MATSUI, M., NAGAI, F., AND AOYAGI, S. (1979) Biochem. J. 179, 483-487.