The Molecular Weights of UDP-glucuronyltransferase Determined with ...

Vol. 259, No. 19, Iwue of October 10, pp. 11701-11705,1984

THEJOURNAL OF BIOLOGICAL CHEMISTRY 0 1984 by The American Society of Biological Chemists. Inc.

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U.S.A.

The Molecular Weights of UDP-glucuronyltransferase Determined with Radiation-inactivationAnalysis A MOLECULARMODELOF

BILIRUBIN UDP-GLUCURONYLTRANSFERASE* (Received for publication, January 30,1984)

Wilbert H. M. Peters, Peter L. M. Jansen, and Henk NautaS From the Division of Gastrointestinal and Liver Diseases, Department of Medicine, University Hospital, Nijmegen and the $Laboratory of Biophysics, State Unioersity, Utrecht, The Netkrlands

The molecular masses of the UDP-glucuronyltransferases were determined by means of radiation-inactivation analysis of sodium dodecylsulfate-treated lyophilized rat liver microsomal preparations using a calibrated 6oCosource. Bilirubin glucuronidation with formation of bilirubin monoglucuronide is catalyzed by a 41,500-Da enzyme; conversion of bilirubin mono- to diglucuronide is catalyzed by a 175,000-Da enzyme. The glucuronidation of estrone and testosterone is mediated by enzymes with molecular masses of 73,500 and 142,000 Da, respectively, and the glucuronidation of p-nitrophenol and phenolphthalein is mediated by enzymes with molecular masses of 109,000 and 159,000 Da, respectively. Our results show that UDP-glucuronyltransferase consists of a heterogenous groupof enzymes with strikingly different molecular masses. Our data furthermore suggest that these enzymes may be oligomers composed of one to four subunits with similar molecular masses. Based on these findings, a molecular model of bilirubin UDP-glucuronyltransferaseis proposed, consisting of four subunits. For bilirubin diglucuronide formation, the complete tetrameric enzyme is required, whereas formation of monoglucuronide can be mediated by a single subunit. Themonomeric monoglucuronide-forming enzyme is resistant to sodium dodecyl sulfate, treatment whereas the tetrameric diglucuronide-forming enzyme is labile, but once inactivated, thediglucuronide-formingenzymecanbereconstituted by decreasing the sodium dodecyl sulfate concentration by means of dialysis.

52,000-57,000 Da for p-nitrophenol and estrone (6-9),and 50,000-54,000 Da for testosterone and chenodeoxycholic acid UDP-glucuronyltransferases(2,6). Membrane-bound enzymes usuallyare oligomers composed of multiple subunits (10). With gel-filtration techniques, the molecular masses of some UDP-glucuronyltransferase oligomers have been estimated at 230,000 Da for p-nitrophenol and estrone (7) and 318,000 Da for testosterone and chenodeoxycholic acid(2). Accurate determination of the molecular mass of the complete enzyme in its active form, however, is hampered by several sources of error. UDP-glucuronyltransferases after purification are delipidated and partially or completely inactivated (2, 3, 6). Delipidated membrane proteins readily undergo aggregation, and their molecular masses, determined with gel filtration, may be overestimated. Furthermore, interaction of hydrophobic proteins with gel matrix may also cause molecular-mass inaccuracies. Radiation-inactivation analysis is a method to determine molecular masses of membrane-bound enzymes in situ (11). When a membrane preparation is irradiated with ionizing radiation, the enzymes in the preparation are inactivated. The degree of inactivation is directly related to radiation dose and is dependent on target size or molecular mass of the enzyme (12). By irradiating membrane preparations for different periods of time, enzyme inactivation curves are obtained from which the molecular mass can accurately be determined as described by Kepner and Macey (13). The method has been validated by numerous studies correlating the molecular mass determined by the radiation-inactivation technique with other techniques (11,141. We have studied the molecular massesof bilirubin UDP-glucuronyltransferaseand several other UDPglucuronyltransferases using the radiation-inactivation method.

UDP-glucuronyltransferases (EC 2.4.1.17) are microsomal membrane-bound drug-metabolizing enzymes with considerable heterogeneity with regard to substrate specificity, perinatal development, and effect of enzyme-inducing agents (1). Several UDP-glucuronyltransferases have been purified (27), and subunit molecular masses have been estimated by SDSI-polyacrylamide gel electrophoresis. The values are 57,000 Da for bilirubin UDP-glucuronyltransferase (3),

MATERIALSANDMETHODS

Chemic&-UDP-glucuronic acid (ammonium salt), sodium phenobarbital, DL-dithiothreitol, bilirubin (from bovine gallstones), Dsaccharic acid l,4-lactone, and digitonin were from Sigma. Sodium dodecyl sulfate was from BDH Chemicals Ltd.,Poole, United Kingdom. Zwittergent 3-12 was from Calbiochem-Behring. Ethanol, 1,2dichloroethane, dimethyl sulfoxide,p-nitrophenol, and phenolphthalein came from Merck, Darmstadt, Federal Republic of Germany. Testosterone and estrone were fromOrganon, Oss, The Netherlands. [3H]Testosterone (50.4 Ci/mmol) and [‘4C]estrone (52 mCi/mmol) * This study has been presented inpreliminary form at the annual came from New England Nuclear. A n i d - M a l e Wistar rats, weighing about 250 g, were obtained meeting of the American Association for Study of Liver Diseases, Chicago, 1983 (30). The costa of publication of this article were from the animal laboratory of the University of Nijmegen, The defrayed in part by the payment of page charges. This article must Netherlands. Phenobarbital treatment was performed by daily intratherefore be hereby marked “advertisement” in accordance with 18 peritoneal injections (80 mg/kg of body weight) for 5 days. Before U.S.C. Section 1734 solelyto indicate this fact. killing, the animals were fasted for 18 h with water ad libitum. ’ The abbreviations used are: SDS, sodium dodecyl sulfate; BMG, Preparation of Microsomes-Rat liver microsomes were prepared bilirubin monoglucuronide; BDG, bilirubin diglucuronide. essentially as described by Cuypers et al. (15). In all experiments,

11701

Molecular Weights of UDP-glucuronyltransferase

11702

microsomes from phenobarbital-treated rats were used. Detergent Treatment of Microsomes-Microsomes (3.5-10mgof protein/ml) were resuspended in 50 mM Tris/HCl buffer, pH 7.4, containing 0.25 M sucrose, 1 mM EDTA, 0.7 mM UDP-glucuronic acid, 1 mM dithiothreitol, and 1 mM D-saccharic acid 1,4-lactone. After preincubation for 5 min at room temperature (22 "C), diluted detergent solutions were added with shaking. For testingthe stability of microsomes treated with different detergents, the mixtures were incubated a t 4 "C for 0-68 h. Final detergent concentrations in this incubation mixture were 0.06% (w/v) for SDS, 0.25% (w/v) for digitonin, and 0.13% (w/v) for Zwittergent 3-12. The most commonly used SDS-activated microsomes were prepared by adding a 1% (w/v) SDS solution and incubating for 1 h a t 22 "C. Final SDS concentrations ranged from 0 to 0.12% as indicated in the legends of the figures. After SDS treatment, the microsomes were stored at -20 "C. Irradiation of Liver Microsomes-SDS-treated microsomes were frozen in liquid nitrogen and lyophilized in 1.5-1111 plastic tubes (Eppendorf, Hamburg, Federal Republic of Germany). After lyophilization, the tubes were kept at least 24 h in a Pz06-containingvacuum desiccator to achieve complete dryness. The tubes were closed and sealed with Parafilm (American Can Company, Greenwich, CT). Irradiation was performed with y-radiation (1.17 and 1.33 MeV) from a Gamma-cell 200 (Atomic Energy of Canada, Ottawa, Canada) containing a "Co source. A holder was used whichcould hold 12 tubes at equivalent positions so that all tubes received an equal radiation dose. The radiation dose a t sample location was determined by Fricke dosimetry (Fez++ Fe3+; 15.5 Fe3+ions being formed per 100 eVof absorbed radiation energy). The dose rate was corrected for cobalt decay by using a monthly factor of 0.98908. During our experiments, a dose rate of approximately 70 krad/h was achieved. Irradiation was performed at room temperature (22 "C) for time periods from 0 to 70 h. Control tubeswere treated inexactly the same way but were placed outside the "Co source for the duration of the irradiation. The dose rate of the "Co source was such that no increase of temperature could be detected in the sample during irradiation. In each series five non-irradiated control tubes were included. Before the enzyme assay, the lyophilized material was dissolved in distilled water (up to its original volume), left for 10 min a t 22 "C, and mixed well. This preparation can be stored at -20 "C for several months, without significant loss of activity. Calcuiation of Moiecular Weight-Enzyme activity was determined (seebelow), and the residual activity plotted semilogarithmically versus radiation dose gave reasonably straight lines. This indicates that the radiation inactivation is realized by a one-hit single-target mechanism. The residual enzymatic activity, A, decreases as a simple where AO exponential function of dose, D according to A = Ao. is the activity a t zero dose. The dose needed to reach 37% residual activity (D37)can thus be found. A simple relationship between D37 and molecular mass of membrane proteins, givenby Kepner and Macey (13), holds the following. Molecular mass =

6.4 X 10" D37 (rad)

~

This relationship, which has been applied successfully (11, 12) for a number of enzymes, was also used in ourexperiments. Preparation of Bilirubin Monoglucuronide-BMG was prepared as described by Jansen et al. (16) and purified by thin-layer chromatography (17). Enzyme Assay-Bilirubin was dissolved in 0.05 M NaOH and immediately diluted with 0.1 M Tris/HCl, pH 7.8, to obtain a metastable solution which was used within a few minutes. Incubations using BMG as substrate were performed as follows. Undiluted microsomes (25-35 mg of protein/ml) which contained 2 ml of saccharic acid 1,4-lactone were added to a glass-stoppered tube containing a dry film of BMG. The microsomes were shaken a t 4 "C in the dark under an argon atmosphere for 0.5-2 h until all BMG was dissolved in the microsomes. Bilirubin glucuronidation was determined in a medium containing microsomes (1.3-4.0 mg of protein/ml), 50 Tris/HCl, pH 7.8, 10 mM MgClz, 3.5 mM UDP-glucuronic acid, 1mM saccharic acid l,4-lactone, and variable amounts of bilirubin or bilirubin monoglucuronide. Incubations were performed in glass-stoppered tubes at 37 "Cunder argon atmosphere for varying time periods. The reaction was stopped by adding 4.0 ml of methanol containing 80mg of Na ascorbate and

a trace of EDTA and subsequent immersion of the tube in an ice bath. All bilirubin handling was done under dim red light. Other enzyme assays were performed in 0.4 ml of 50 mM Tris/HCl buffer, pH 7.8, containing 10 mM MgClz, 3.5 mM UDP-glucuronic acid, 0.25-0.5 mg of microsomal protein/ml, 1 mM saccharic acid 1,4lactone, and the following substrates: 60 p~ estrone, 0.5 mM testosterone, 1 mM p-nitrophenol, or 2.0 mM phenolphthalein. Inthe testosterone or estrone assay, each tube contained 100,000 cpm of [3H]testosteroneor ["Clestrone, respectively.Testosterone andphenolphthalein weredissolved in ethanol, estrone wasdissolved in ethanol/dimethyl sulfoxide (3:l (v/v)), and p-nitrophenol was dissolved in 0.01 M NaOH. Labeled and nonlabeled steroids were added to the assay medium as described by Rao et 01. (18). After incubation for 10-30 min at 37 "C, the reaction was stopped by adding 0.4 ml of 6% (v/v) trichloroacetic acid (for p-nitrophenol or phenolphthalein assays) or by heating for 10 min at 100 "C (for testosterone or estrone assays). The assays were terminated as follows. For p-nitrophenol, 4.0 ml of 0.3 M NaOH were added, followed by centrifugation for 10 min at 10,000 X g and measurement a t A m m. For phenolphthalein, 3.0 ml of a glycine/NaOH buffer, pH 10.4, were added, followed by centrifFor ugation for 10 min at 10,000 X g and measurement at Am estrone and testosterone, 4.0 and 2.0 ml of 1,2-dichloroethane were added, respectively, and after vigorous shaking, the organic layer was separated by centrifugation for 5 min at 3,000 x g.100 p1 of the aqueous phase were counted in duplicate in 5.0 ml of a liquid scintillation mixture. 0-Glucuronidase activity was determined at 37 "C in 0.4 ml of a medium containing microsomes (0.5-0.6 mg of protein/ml), 50 mM Na acetate/acetic acid, pH 5.0, 2 mM p-nitrophenol-&glucuronide, and 5 mM EDTA. The reaction was stopped by adding 0.4 ml of 6% (v/v) trichloroacetic acid. After adding 1.0 ml of 0.3 M NaOH, the mixture was centifuged for 10 min at 10,000 X g, and Am was measured in the clear supernatant. Determination of Bilirubin and ItsConjugates-Bilirubin glucuronides were converted to their methyl esters by alkaline methanolysis according to the method of Blanckaert (19). Determination of bilirubin and itsmethyl esters was performed by high-performance liquid chromatography as described previously (15). RESULTS

In order to study the molecular properties of UDP-glucuronyltransferases, microsomal membranes have to be treated with detergents. This activates the enzyme (20). Certain activation procedures, however (for example, activation with trypsin under aerobic conditions), causes accumulation of oxidants, probably peroxides, which rapidly oxidize bilirubin and glucuronides upon subsequent incubation(15). Therefore, the properties of untreated anddetergent-treated microsomal preparations were studied with regard to bilirubin diglucuronide formation and recovery of bilirubin and metabolites (Table I). Digitonin, SDS, and Zwittergent 3-12 caused 20-30 times activation of bilirubin &glucuronide formation. Most active in this respect was Zwittergent 3-12. When stored detergenttreated preparations were used as enzyme source, appreciable differences were found with regard to recovery of bilirubin and glucuronides. Total bilirubin recovery decreased dramatically when stored digitonin-treated microsomes were used. Incomplete recoveries were also seen with stored SDS- or Zwittergent-treated microsomes. These recovery problems can be avoided when storage is done inan non-aqueous environment. Table I1 shows that lyophilized, SDS-treated preparations are extremely stable and can be stored at 22 "C for up to 15 days. The recovery of bilirubin and glucuronides was excellent after incubation with stored lyophilized SDStreated microsomal preparations. In addition, these preparations were stable and optimally active enzyme sources for radiation-inactivation studies ofp-nitrophenol, phenolphthalein, estrone, and testosterone glucuronidation. To test the validity of the radiation-inactivation method,

Molecular Weights of UDP-glucuronyltransferme TABLEI Bilirubin diglucuronide synthesis and recovery of bilirubin and its conjugates after detergent treatment of rat liver microsomes Enzyme assays were performed at 37 ”C for 20 min with 4.0 mg/ ml protein and 30 PM bilirubin. Storage a t 4 “C

Detergent

BGD synthesis rate

Bilirubin recovery‘‘

SDS (0.06% (w/v)) 239

94

212

76

Digitonin 203 (0.25% (w/v)) 247 92

%Residual activity

100

90 80 70

60 50

..

None

100 100 100 100 100

0 4 20 44 68

8 16 10 11 8

0 4 20 44 68

214

134

70 83 55

0 4 20 44 68

184

97

15

80 30 8

0 4 20 44 68

267 258 262 293 195

88 197

LO

301 10

a\;

x

96

100 90 87 70

Total recovery of substrate plus products, bilirubin and bilirubin mono- and diglucuronide, after incubation. TABLE I1 Stability of SDS-activated microsomes and bilirubin recovery Values are mean f S.E. for six different preparations. Preparations were treated with SDS as described under “Materials and Methods.” SDS concentrations giving optimal bilirubin mono- and diglucuronide synthesis were used. This was achieved by making SDS dose-response curves (see Fig. 2) for each microsomal preparation. Lyophilized preparations were storedin Eppendorf plastic tubes sealed with Parafilm. Activity of the microsomes stored at -80 “C is taken as 100%. Enzyme assays were performed a t 37 “C for 20 min with 1.02.0 mg/ml protein and 30 p M bilirubin. Rest activity Treatment recove* BDG BMG synthesis synthesis

Bilirubin

%

Stored at -80 “C 100 100 90 f 3 Lyophilized and assayed 98.3 f 1.1 93.4 f 3.4 91 f 2.5 immediately 93.4 f 3.378.5 f 3.4 91 f 2 Lyophilized and stored a t 22 “Cfor 15 days Total recovery as explained in the legend of Table I.

the molecular mass of endogenous rat liver microsomal 8glucuronidase was determined. A value of74,800 Da was found, and thisis in close agreement withthe results of other authors using a similar method (14, 21) as well as with the value of 75,000 Da found for purified rat liver 8-glucuronidase (22). Fig. 1 shows the radiation-inactivation curves of bilirubin UDP-glucuronyltransferases. The inactivation curves for conversion of bilirubin to BMG and for BMG to BDG were significantly different. Both curves are linear when plotted semilogarithmically, indicating an exponential dependence of enzyme activity on radiationdose. This proves the validity of the one-hitsingle-target theoryfor this enzyme, and therefore molecular mass determination with the formula outlined un-

BMG*BDG: Mr=175.000 O \

I 0.7 1.4 2.1

Zwittergent 3-12 (0.13% (w/v))

11703

2.8 3.5 4.2 4.9 5.6 Radiation dose (Mrad)

FIG. 1. Radiation inactivation of bilirubin UDP-glucuronyltransferasein SDS-treated microsomes. Decrease of BMG (0) and BDG (0)activity (ordinate) is plotted semilogarithmically versw radiation dose (abscissa). Preparation and radiation of microsomes are described under “Material and Methods.” Four different microsomal preparations were used. Bilirubin ( B ) UDP-glucuronyltransferase assay was performed with 1.4-2.0 mg/ml protein and 25-40 PM bilirubin.

der “Material and Methods” is allowed (11). For the first step (conversion of unconjugated bilirubin to monoglucuronides), the molecular mass is 41,500 k 1,000 Da, and for the second step (conversion of mono- to diglucuronide), the molecular mass is 175,000 k 9,000 Da. The latter value was obtained with unconjugated bilirubin as substrate. In this reaction, formation of BMG is rapid when compared to conversion of BMG to BDG. Therefore, enough BMG is available to function as nonrate-limiting substrate for the second step (15). With biosynthetized purified BMG as substrate, similar molecular mass values for conversion of BMG to BDGwere found. The influence of SDS concentration of the activities of the 41,500-Da first-step enzyme and the 175,000-Da second-step enzyme was studied. Fig. 2 shows that the low-molecularmass enzyme is less latent and more stable than the highmolecular-mass enzyme. Fig. 3 shows that inactivation of the high-molecular-mass enzyme at higher SDS concentration is reversible. Decreasing the SDS concentration in the microsomal preparation by dialysis caused reactivation of bilirubin diglucuronide formation. UDP-glucuronyltransferasemolecular mass was also studied with testosterone, phenolphthalein, estrone, andp-nitrophenolas substrates. The molecular masses found with the radiation-inactivation procedure are shown in Table 111. On the basis of the results, we propose that UDP-glucuronyltransferaseconsists of different oligomeric enzymes composed of a different number of subunits, as will be discussed below. DISCUSSION

Radiation-inactivation analysis is a powerful technique to study the molecular properties of enzymes and is particularly suited to study membrane-bound enzymes (11). These en-

Molecular Weights of UDP-glucuronyltramferase

11704

.

Activity (pmol/mgprotein min)

300

-

250

-

TABLE111 Molecular weights and subunit composition of several UDPglwuronyltransferases obtained by radiation-inactivation anulysis Molecular masses are given in daltons f S.E., and the values are the result of determinations in three different preparations. B, bili-

rubin; T, testosterone;Ph,phenolphthalein;E,estrone;PNP, nitrophenol; G indicates glucuronide.

200 Substrate

150

Bilirubin

100 4'

d

50-

Reaction catalyzed

Testosterone

B -t BMG BMG + BDG T -t TG

Phenolphthal-

Ph -+ PhG

p-

mass

Prowsed sudunit composition of active enzyme

Da f S.E. 41,500 f 1,000 175,000 f 9,000 142,000 k 5,000

Monomer

Tetramer Trimer or tetramer 159,000 f 14,000 Tetramer

ein

0.b

012

014

1

OI6

018 1.0 SDS (mg /ml)

FIG. 2. Titration of microsomes with SDS:effect on bilirubin UDP-glucuronyltransferaseactivity. Formation of BMG (0)and BDG (0)(ordinate)is plotted versus the SDS concentration present during preincubation(abscissu).Microsomal proteinconcentrationduringSDSpreincubation was 8 mg/ml protein.Enzyme assay was performed for 15 min at 37 "C with 3.2 mg/ml protein and

Estrone E-tEG 73,500 f 9 , m p-NitrophenolPNP * PNPG 109,OOO k 7,000

+

Dimer Dimer or trimer

(Na+ K+)-ATPaseis responsible for the K+-occluding mechanism of the enzyme (28). For radiation-inactivation analysis, lyophilized membrane preparations are required, and onlyenzymes resistant to lyophilization procedures can be studied with this technique. 15 p~ bilirubin. In anaqueous environment, radiation hits in the water phase cause free-radical chain reactions which secondarily may inActivity (pmol/mg protein. min) activate enzymes so that the one-hit single-target theory is not valid anymore. UDP-glucuronyltransferaseproved to be extremely stable in lyophilized microsomes. Lyophilization may introduce artifacts in membrane proteins such as aggre300 gation. We have included dithiothreitol in our preparations to avoid disulfide bridge formation which would cause overestimation of target size. Radiation-inactivation analysis of UDP-glucuronyltransferase revealed significant molecular-weight differences among the enzymes mediating the glucuronidation of different substrates. The values, however, appear to be mathematical multiples of each other. This suggests that these enzymes are composed of one to four subunits with similar molecular weights (Table 111). UDP-glucuronyltransferasesfor estrone and testosterone are enzymes with molecular massesof 73,500 and 142,000 Da, respectively. The enzymes catalyzing the glucuronidation of testosterone and phenolphthalein have similar molecular I I I I I masses of 142,000 and 159,000 Da, respectively.The enzyme 0 10 20 30 40 50 for glucuronidation of p-nitrophenol falls with 109,000 Da in Dialysis time (hrs) FIG. 3. Effect of lowering the microsomal SDS concentra- between the values for low- and high-molecular-weight UDPtion on bilirubin UDP-glucuronyltransferaseactivity. Micro- glucuronyltransferases. somes (10mg/ml protein) were preincubated with SDS (1.0 mg/ml) Our finding that estrone and testosterone glucuronidation followed by dialysis against a 50 mM Tris/HCl, pH 7.4, buffer con- is catalyzed by enzymes with different molecular masses is taining 0.25 M sucrose and1mM dithiothreitolfor the indicated times supported by the data of Falany et al. (6)and Matern et al. (abscissa).BMG (0)and BDG (0)activity (ordinate) was determined by incubating dialyzed microsomes(4 mgfml protein) for 20 min at (2). They found that on purification both activities are separated in different protein fractions. With gel-filtration tech37 "Cwith 25 p~ bilirubin. niques, the molecular mass of the p-nitrophenol and estrone glucuronidation enzyme has been estimated at 230,000Da zymes can be studied in situ avoiding the introduction of and for testosterone was estimated at 318,000 Da (Refs. 7 and artifacts inherent to purification procedures. In addition to 2, respectively). These values are considerably higher than molecular mass,interactions between enzymesubunits can be the values we found and might represent molecular masses of studied (11, 23-27). For example, with the radiation-inacti- enzyme aggregatesrather than single enzymes. vation method, a smaller functionally distinct part,possessing Interestingly, Hochman et al. (9) reported two nucleotidep-nitrophenylphosphatase activity, couldbe distinguished binding sites/217,000-Da p-nitrophenol glucuronidation enwithin the larger plasma membrane-bound enzymes (Na+ + zyme; this isone binding site/109,000-Da enzyme.This value K+)-ATPase and (K' H+)-ATPase (27). By using this closely approximates the value we found for the p-nitrophenol method, it was in addition found that only a small part of enzyme. According to Hochman et al. (9), the p-nitrophenol ~

1

50i

I

+

Molecular Weights of UDP-glucuronyltramferase enzyme is composed of maximally three subunits, a possibility which fits well with our data (Table 111). Formation of bilirubin monoglucuronide and conversion of mono- to diglucuronide are catalyzed by UDP-glucuronyltransferase (15, 29). Formation of monoglucuronide is a fast reaction with a low KJ;I"";" (where UDPGA represents UDPglucuronic acid) of 43 PM. Conversion of mono- to diglucuronide is much slower with a high KpPGAof 1 mM (15). Our present results show that the first glucuronidation step is catalyzed by a small enzyme of 41,500 Da, whereas for the second step an enzyme with a molecular mass of 175,000 Da is required. The question arises whether these are different enzymes or different forms of the same enzyme. Wepostulate that the first and second glucuronidation steps are catalyzed by mono- and tetrameric forms,respectively, of the same enzyme rather than by completely different enzymes. In the mutant homozygous Gunn rat, both glucuronidationsteps are absent (30). If both stepswere catalyzedby different enzymes, a mutation in the first-step enzyme would leave the second step intact. However, this is not the case, and this supports our hypothesis that first- and second-step enzymes have a common subunit. A mutation in this subunit renders both the first and second glucuronidation steps inactive. More structural information about the bilirubin glucuronidation enzymes was obtained in experiments in which microsomal membranes were titrated with SDS.Conversion of bilirubin to monoglucuronidewas more resistant toSDS than conversion of mono- to diglucuronide(Fig. 2). This is in agreement with the idea that the first step is catalyzed by a monomer which is relatively resistant to SDS, whereas the second step is catalyzed by a morelabile tetramer which dissociates in SDS butreassembles when the SDS concentration is lowered by dialysis (Fig. 3). The present data and the literaturecan be recombined in the following hypothetical model of bilirubin glucuronyltransferase. The bilirubin &glucuronide-forming enzymeis a tetramer composedof two pairs of identical subunits. One pair has a low for conversion of bilirubin of monoglucuronide (subunits a and b). These subunits are defective in the Gunn rat. The other subunit pair (c and d) has a high ED"" and catalyzes conversion of mono- to diglucuronide. For the latter activity, the complete tetrameric enzyme is required. Subunits c and d by themselves are inactive. For the first-step activity, the presence of subunits c and d is not necessarysince subunits a and b as monomers can catalyze this reaction. In conclusion, we have collected further evidence for the existence of different UDP-glucuronyltransferasesby demonstrating significant differences in the molecular structures of the enzymes. UDP-glucuronyltransferaseappear to consist of a family of oligomeric enzymes composedof, at present, an unknown number of different subunits with similar molecular masses. The oligomeric structure maybeof advantage in

11705

giving the enzyme system evolutionaland adaptational flexibility. Further studies are warranted to fully establish the proposed enzymatic structures. REFERENCES 1. Dutton, G. J., and Burchell, B. (1977)Prog. Drug. Metab. 2, 1-

70 2. Matern, H., Matern, S., and Gerok, W. (1982)J. Biol. Chem. 267,7422-7429 3. Burchell, B. (1981)Methods Enzymol. 77, 188-192 4. Hochman, Y., Zakim, D., and Vessey, D. A. (1981)J. Biol. Chem. 266,4783-4788 5. Bock, K. W., Josting, D., Lilienblum, W., and Pfeil, H. (1979) Eur. J. Biochem. 98,19-26 6. Falany, C. N., Chowdhury, J. R., Chowdhury, N. R., and Tephly, T. R. (1983)Drug. Metab. Dispos. 11,426-432 7. Tukey, R. H., and Tephly, T. R. (1981)Arch. Biochem. Biophys. 209,565-578 8. Burchell, B., and Weatherill, P. (1981)Methods Enzymol. 77, 169-177 9. Hochman, Y.,Kelley, M., and Zakim, D. (1983)J. Biol. Chem. 268,6509-6516 10. Klingenberg, M. (1981)Nature ( L o n d . ) 290,449-454 11. Kempner, E. S., and Schlegel, W. (1979)Anal. Biochem. 92, 210 12. Steer, C. J., Kempner, E. S., and Ashwell, G. (1981)J. BWL Chem. 266,5851-5856 13. Kepner, G. R., and Macey, R. I. (1968)Biochim. Biophys. Acta 163,188-203 14. Beauregard, G., and Potier, M. (1982)Anal. Biochem. 122,379384 15. Cuypers, H. T. M., Ter Haar, E. M., and Jansen,P. L. M. (1983) Biochim. Biophys. Acta 768, 135-143 16. Jansen, P. L. M., Chowdhury, J. R., Fischberg, E. B., and Arias, I. M. (1977)J. Bwl. Chem. 262, 2710-2716 17. Chowdhury, J. R., Chowdhury, N. R., Giirtner, U., Wolkoff, A. W., and Arias, I. M. (1982)J. Clin. Znuest. 69,595-603 18. b o , G. S., Haueter, G., b o , M. L., and Breuer, H. (1976)Anal. Biochem. 74,35-40 19. Blanckaert, N. (1980)Biochern. J. 186,115-128 20. Winsnes, A. (1969)Biochim. Biophys. Acta 191,279-291 21. Shikita, M., and Hatano-Sata, F. (1973)FEBS Lett. 36,187-189 22. Stahl, P. D., and Touster, 0.(1971)Fed. Proc. 30,1121 23. McIntyre, J. O., Churchill, P., Maurer, A., Berenski, C. J., Yung, C. Y., and Fleischer, S. (1983)J. Biol. Chem. 268,953-959 24. Kempner, E. S., Cole, K. W., and Gaertner, F. H. (1982)J. BWL Chem. 267,8919-8921 25. Barhanin, J., Schmid, A., Lombet, A., Wheeler, K. P., Lazdunski, M., and Ellory, J. C. (1983)J. Biol. Chem. 268, 700-702 26. Maret A., Potier, M., Salvayre, R., and Douste-Blazy, L. (1983) FEBS Lett. 160.93-97 27. Schrijen, J. J., van Groningen-Luyben, W. A. H. M., Nauta, H., de Pont, J. J. H. H. M., and Bonting, S. L. (1983)Biochirn. Biophy~.Acta 731,329-337 28. Richards, D. E.,Ellory; J. C., and Glynn, I. M. (1981)Biochirn. Bwphys. Acta 148,284-286 29. Blanckaert, N., Gollan, J., and Schmid, R. (1979) Proc. NatL Acad. Sci. U. S. A. 76,2037-2041 30. Peters, W. H. M., Ter Haar, E. M., Nauta, H., and Jansen, P. L. M. (1983)Hepatology (Baltimore) 3,811