THEJOURNALOF BIOLOGICAL CHEMISTRY Val. 267, No. 24, Issue of August 25, pp.

16943-16950,1992
0 1992 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

Hepatic MicrosomalBilirubin UDP-glucuronosyltransferase

T H E KINETICS OF BILIRUBIN MONO- AND DIGLUCURONIDE SYNTHESIS*

(Received for publication, March 18, 1992)

James M. Crawford$$, Bernard J. Ransilq, Julianne P. NarcisoII ,and JohnL. GollanII

From the Departmentsof $Pathology and I(Medicine, Brigham and Women’s Hospital, the’11CharlesA . Dana Research Institute
and the Harvard-Thorndike Laboratorv of Beth Israel Hospital, and the Harvard Digestive Diseases Center and Harvard
Medical School, Boston, Massachusetts-02115

Hepatic biotransformationof bilirubin to the hydro- ologic elimination of bilirubin,’ the end product of heme
philic species bilirubin mono- (BMG) and diglucuron- degradation. In the process, this potentially toxic, hydropho-
ide (BDG) by microsomal bilirubin UDP-glucuronosyl- bic molecule is rendered more water soluble, thereby facilitat-
transferase (GT) is a prerequisite for its physiologic ing its excretion into bile (1). Glucuronidation reactions are
excretion into bile. The reaction mechanism of biliru- catalyzed by the uridine diphosphate glucuronosyltransferases
bin-GT and theaccess of bilirubin andBMG (the inter- (GTs), a family of membrane-bound enzymes located primar-
mediate substrate) to the active site of bilirubin-GT ily in the rough and smooth endoplasmic reticulum (2). The
are undefined. Highly purified [14C]bilirubin and [3H] closely related isoforms of bilirubin-GT, which appear to be
BMG were coincubated with rat liver microsomes, and
the initial rates of radiolabeled bilirubin glucuronide multiple in humans (3, 4), but single in rats (5, 6), require
synthesis were measured. Although these substrates uridinediphosphate glucuronic acid (UDP-GlcA) or other
differ markedly in their hydrophilicity, no significant donor UDP-sugars ( 5 ) as essential cosubstrates. A glucuronyl
differences were observed in [‘“CI- and r3H]BDG rates moiety is conjugated to either of the two propionic acid side
of formation from equimolar [“C]bilirubin and [3H] chains of bilirubin, located on the C-8 and C-12 carbons of
BMG, in the absence or presence of soluble binding the two central pyrrole rings (7), thusresulting inthe synthe-
proteins (albumin and hepatic cytosol). In further ki- sis of two possible bilirubin monoglucuronide (BMG) isomers.
netic studies, [“Clbilirubin and [3H]BMG exhibited Both BMG isomers, generated in situ or from exogenous
mutually competitive inhibition of [3H]- and [“CIBDG sources, may be further glucuronidated by bilirubin-GT to the
synthesis,respectively, and [3H]BMG alsoinhibited diglucuronide (BDG, 8). In human and rat bile, BDG consti-
[14C]BMGformation. Finally, unlabeled BMG and BDG tutes the principal bile pigment (-80%), with smaller quan-
inhibited the glucuronidation of [“Clbilirubin, with all tities of the two BMG isomers (9, 10).
three pigments yielding virtual Michaelis-Menten dis- The hepatocellular synthesis of BMG and BDG by micro-
sociation constants in the10-20 p~ range. These find- somal bilirubin-GT is well documented (2, 11, 12), but the
ings indicate that: 1) bilirubin-GT follows Michaelis- reaction mechanism and mode of substrate access to theactive
Menten kinetics for both bilirubin and BMG glucuron-
site(s) are poorly defined, in part due to the difficulties in
idation over the range of substrate concentrationsem-
ployed; 2) the findings are consistent with a single preparing purified pigment substrates (13). Recent sequencing
active site for the enzymatic synthesis of both BMG data indicate thattheGT enzymes areanchoredin the
and BDG; 3) bilirubin, BMG, and BDG bind competi- membrane by a carboxyl-terminal hydrophobic domain, with
tively to this active site with comparable affinities;theand bulk of the polypeptide chain located within the cisternal
4) access of both bilirubin and BMG substrates to the lumen of the endoplasmic reticulum (1).Binding of the nu-
enzymatic active site is reduced by soluble binding cleotide-sugar cosubstrate to thecarboxyl-terminal half of GT
proteins. occurs either in the cisternal lumen (14, 15) or within the
membrane (16), and appears to be mediated by a specific
UDP-GlcA membrane transport system (16, 17). Binding of
aglycone substrates such as bilirubin occurs on the amino-
Hepatic glucuronidation is an obligatory step in thephysi- terminal half of the polypeptide chain (3, 18),but the mech-
anism whereby substrate gains access to the luminally ori-
* This study was supported in partby National Institutes of Health ented enzyme is unknown. In addition, while it is presumed
Grants DK-39512, DK-36887, AM-07502, a Milton Fund Award from that both the C-8 andC-12 isomers of BMG are formed from
Harvard Medical School, and by BRSG 807 RR-05950 awarded by bilirubin at a single active site (19), it remains to be shown
the Biomedical Research Support Grant Program, Division of Re- whether addition of the second glucuronyl moiety tothe
search Resources, National Institutes of Health.This work was
presented in part at the annualmeetings of the American Gastroen- intermediate substrate BMG occurs at this same catalytic
terological Association, New Orleans, May 1988, and the American site. Access of the more hydrophilic BMG to the active site
Association for the Studyof Liver Diseases, Chicago, November 1990. also remains to be characterized.
The costs of publication of this article were defrayed in part by the
payment of page charges. This articlemusttherefore be hereby The abbreviations used are: bilirubin, bilirubin-IXa; BMG, bili-
marked “advertisement” in accordance with 18 U.S.C. Section 1734 rubin-IXa C-8- and C-12-monoglucuronides; BDG, bilirubin-IXa dig-
solely to indicate this fact. lucuronide; GT, uridine diphosphate glucuronosyltransferase; UDP-
§ Recipient of an American Gastroenterological Association/Searle GlcA, Uridine-diphosphate glucuronic acid; UDP-GlcNAc, Uridine-
Industries Research Scholar Award. To whom correspondence should diphosphate N-acetylglucosamine; HPLC, high performance liquid
be addressed Dept. of Pathology, Brigham & Women’s Hospital, 75 chromatography; TLC, thin layer chromatography; CHAPSO, 3-[(3-
Francis St., Boston, MA 02115. Tel.: 617-732-6672; Fax: 617-732- cholamidopropyl)dimethylammonio]-2-hydroxy-l-propanesulfonic
6796. acid.

16943

This is an Open Access article under the CC BY license.

16944 Analysis
Kinetic of Bilirubin UDP-glucuronosyltransferase
Wehave demonstrated previously that BDG is formed and the final supernatant (11 mg of protein/ml) stored at -70 "C
preferentially from purified [14C]bilirubin,compared to [3H] (20).
BMG, when these substrates are administered simultaneously Assay of Microsomal Bilirubin Glucuronide Synthesis-Radiola-
beled bilirubin was solubilized in 0.05 M NaOH (0.03 ml), followed
to intact rats (8),suggesting that BMG generated by the by the immediate addition of 0.167 mM TEA buffer (0.1 ml) to bring
enzyme insitu is the preferred substrate for the second the pH to 7.6. This was added to a second 0.1-ml volume of buffer
glucuronidation step. In this study we have examined the (pH 7.6) containing the desired concentrations of albumin or cytosol
generation of radiolabeled bilirubin glucuronides from puri- and/or radiolabeled or unlabeled BMG or BDG. The combined bili-
fied ['4C]bilirubin and [3H]BMG i n vitro in the absence or rubinbilirubin glucuronide/albumin or cytosol mixture was then
presence of soluble binding proteins(albumin or hepatic added to the enzyme assay mixture. The elapsed time for solubiliza-
tion of all pigmentsand delivery to theassay mixturewas consistently
cytosol), using a sensitive microsomal GT radioassay system 45-60 s. Bilirubin and bilirubin glucuronides remained intact during
(20). Initial rates of product formation from equimolar and this procedure, as verified by HPLC. When a single pigment species
from variable concentrations of ['*C]bilirubin and [3H]BMG was used, the identical solubilization procedure was followed, using
were examined in the absence or presence of unlabeled BMG buffer vehicle alone where appropriate.
or BDG, to characterize substrate access to microsomal bili- The initial rates of formation of ['4C]bilirubin monoglucuronides
rubin-GT and theenzymatic reaction mechanism. (C-8 and C-12 isomers) and ["C]- and [3H]bilirubin diglucuronides
were determined by radiochemical assay(20). The reaction was
initiated by addition of the solubilized radiolabeled or unlabeled
MATERIALS AND METHODS
bilirubinpigments (0.2 ml) to an assaymixture which had been
Chemical Reagents-Radiolabeled ["Clbilirubin (specific activity preincubated for 2 min at 37 "C, containing final concentrations of
8-13 pCi/pmol) and [3H]BMG (54-68 pCi/pmol) were prepared bio- 0.167 M TEA-HCl buffer (pH 7.6), 2 mM MgC12,8.3 mM D-saccharic
synthetically from the bile of rats injectedwith 6-amino-[5-14C] acid l,4-lactone, 1.7 mM P-NAD, 20 mM UDP-GlcA (cosubstrate), 1
levulinic acid (49 pCi/pmol) or d-amin0-[3,5-~H]levulinic acid (1.8 mM UDP-GlcNAc (the presumed in uiuo allostericeffector), and
mCi/pmol, Du Pont-New England Nuclear),respectively, as described microsomes (3 mg of protein/ml), in a total assay volume of 1.2 ml.
previously (8). Unlabeled bilirubin mono- and diglucuronides were Detergent activation of microsomes was not employed. Aliquots of
isolated from bile obtained from rats infused with 20 mM bilirubin the reaction mixture were obtained a t 30-5 intervals for up to2.5 min
intravenously in 3.2% bovine serum albumin, using a standard pro- and mixed promptly with ice-cold methanol. The content of radiola-
tocol (8). The purified pigments were consistently >99% pure, as beled bilirubin conjugates (C-8 plus C-12 BMG, BDG) were deter-
measured by HPLC (21). Although incompletely resolved by HPLC, mined by alkaline methanolysis, extractioninto chloroform and TLC
the C-8 andC-12 isomers of the BMG preparations were obtained in (2), using 12 p1 of bilinoid-enriched bile added to thesamples to serve
approximately equal quantities. as carrier (obtained from rats infused with 20 mM bilirubin intrave-
Aliquots of purified ["Clbilirubin (4-40 pg) were prepared by nously in 3.2% serum albumin). Radioactivity in separated TLC
evaporatingaconcentrated chloroform solutionunder an argon bands was determined as described previously (8), usinga liquid
stream at 37 "C and were stable for at least a year in Vacuo in the scintillation spectrometer (Beckman LS7500, Beckman Instruments,
dark at 25 "C. Purified bilirubin mono- and diglucuronides (6-50 pg) Fullerton, CA). Internal standards were calibrated with [3H]- and
were evaporated from a concentrated solution of acidified methanol [14C]tolueneas external standards, to enable appropriate correction
(1%glacial acetic acid/MeOH, v/v) under an argon stream at 37 "C, for the dualchannel (3H,14C)counting mode. The initial rates of
and remained intact for up to 3 months in the dark under argon a t formation of radiolabeled bilirubin monoglucuronides and bilirubin
-70 "C. Bovine albumin (essentially fatty acid free, Fraction V) was diglucuronide were obtained from the slopes of linear regression, and
dialyzed repeatedly against 0.167 M HC1-triethanolamine (TEA) expressed as picomoles/milligram of microsomal protein/minute
buffer (pH 7.6) prior to use. Albumin, UDP-GlcA (ammonium salt), (mean f S.D. for replicate assays).
UDP-GlcNAc (sodium salt) were obtained from Sigma. Silica Gel Experimental Protocol-In order to evaluate the kinetic properties
G60 TLC plates were purchased from MCB Manufacturing Chemists, of bilirubin-GT, it was necessary to vary the concentrations of the
Inc. (Cincinnati, OH). Dimiscint scintillation mixture was from Du competing substrates, bilirubin and bilirubin glucuronides, as well as
Pont-New England Nuclear. All other reagents were of the highest albumin, which is capable of binding substrate and therefore influ-
grade commercially available. encing availability for enzymatic glucuronidation. The rationale for
All aqueous solutions were prepared from argonized distilled water, the experimental approach is addressedin the main text; the protocol
which had been boiled for 30 min and cooled to room temperature is summarized herein for easy reference (Table I).
with argon bubbling. Reagents were stored in sealed vessels under an
argon atmosphere. With the exception of liver microsomal prepara- RESULTS
tions, all procedures were performed in subdued light under an argon
stream or in argonized stoppered tubes or chambers, to minimize Glucuronidation of P4C]Bilirubin andf'H]BMGandthe
oxidative and photodegradation of pigments. Effect of Albumin Binding-The primary experimental ap-
Isolation of Rat Liver Microsomes and Cytosol-Male Sprague- proach was to measure the generation of[14C]BMG and
Dawley rats weighing 200-230 g were fed standard laboratory chow [14C]- and [3H]BDG simultaneously in assay mixtures con-
and then fasted for 18 h before decapitation. After rapid resection of taining the two radiolabeled substrates, [14C]bilirubinand
the liver, microsomes from four pooled livers were prepared in ice- [3H]BMG. Based on in viuo studies (8),it is postulated that
cold 0.25 M sucrose, 1 mM EDTA (pH 7.4) according to a standard
procedure (22). The final suspension of microsomes (35-50mg of [14C]bilirubingains access to the active site of microsomal
protein/ml) was stored at -70 "C, and assays were performed within GT more readily than t3H]BMG, due to the greater hydro-
4 weeks. Protein concentrations were measured by the method of phobicity and affinity of [14C]bilirubinfor lipid bilayers (25).
Lowry et al. (23) with bovine serum albumin as standard. To assess Thus, [14C]BMGformed in situ should be converted to BDG
the integrity and intactness of microsomal membranes, mannose-6- more readily than exogenous [3H]BMG, so that the addition
phosphatase activity was determined in microsomes, and in micro- of equimolar [14C]bilirubinand [3H]BMG to the microsomal
somes pretreated for 60 min at 0 "C with 4 mM CHAPS0 before
diluting the microsomal mixture 1:20 with buffer to a final protein
assay mixture would be expected to produce greater initial
concentration of 0.1 mg/ml. Latency of mannose-6-phosphatase ac- rates of [14C]BDG than [3H]BDGsynthesis.
tivity was consistently >96% ( n = 4),using a standard procedure Albumin is frequently included in bilirubin glucuronidation
(24). Four separate pooled microsomal preparations were utilized, one assays in order to stabilize and maintain bilirubin substrate
for each of the four experimental studies shown in Figs. 1-4. in solution (20, 26-28), although it has been omitted in some
To prepare cytosol, animals were fasted, the abdomen opened under studies (12, 19, 29-31). To exclude the possibility that pref-
light ether anesthesia, and the portalvein was perfused briefly with erential binding of bilirubin to albumin might artifactually
ice-cold 154 mM KC1 prior to excision of the liver. A 50% (w/v)
homogenate was prepared in sucrose-EDTA and centrifuged as for reduce the availability of bilirubin for enzymatic glucuroni-
the preparation of microsomes (41,000 X g for 7 min). The decanted dation, these dual-labeled experiments were performed in the
supernatant was then centrifuged at 100,000 X g for 120 min at 4 "C, absence of albumin and with albumin at concentrations of
 Analysis
Kinetic of Bilirubin UDP-glucuronosyltransferase 16945
TABLEI albumin concentration increased. The initial rates of [“C]
Summary of emerimental RrOtOCOk. BMG formation, measured simultaneously, also were linear,
Figure Assay mixture Conc. Products and decreased upon addition of albumin (not shown). Slopes
Fixed obtained from linear regression of these data, representing
1 [14C]Bilirubin
[3H]BMG Fixed initial reaction rates, are presented in Table 11. The computed
Albumin Variable rates of [I4C]BDGand [3H]BDG formation at each albumin
concentration were not significantly different by unpaired t
2 [“CIBilirubin Fixedb [14C]BMGand [14C]BDG‘ test, indicating that [14C]bilirubin and [3H]BMG are con-
Albumin Fixed
verted to BDG in comparable molar quantities under these
3 PanelA experimental conditions.
[14C]Bilirubin Fixed [14C]BMGand [l4C]BDGC Since glutathione-S-transferase B (ligandin)is the primary
[3H]BMG Variable [3H]BDG bilirubin-binding protein in the cytosol of hepatocytes (32),
Panel B theseexperiments were repeated in the presence of liver
[’4C]Bilirubin Variable [“CIBMG and [I4C]BDG cytosol, prepared as ahigh speed supernatant of liver homog-
[3H]BMG Fixed [3H]BDG
enate. Ligandin represents -5% of cytosolic protein and has
4 PanelA a molecular mass of 47 kDa (33), and thus cytosolic protein
[14C]Bilirubin Variable [“CIBMG‘ (7.05 mg/ml final concentration) was added to approximate a
BMG or BDG Variable ligandin concentration of7.5 PM (20). Although the rate of
(unlabeled) formation of [14C]BDG was higher relative to [I4C]BMG in
Albumin Fixed
the presence of cytosol than for a similar concentration of
Panel B albumin, there was again no statistically significant difference
[14C]Bilirubin Variable [“CIBDG in the rates of [I4C]BDGand [3H]BDG formation (Table 11).
BMG or BDG Variable Power calculations for the four conditions reported in Table
(unlabeled) I established that a one-way difference of 30% or greater
Albumin Fixed between therates of [I4C]BDG and [3H]BDG formation
a Results are given as concentration of radiolabeled product versus should have been detected, if one existed. Since 41% more
incubation time. [14C]BDG isformed relative to [3H]BDG i n uiuo, when [“C]
Time for solubilization of [“Clbilirubin-albumin at 25 “C varied
from 60 s to 4 days. bilirubin and [3H]BMG are administered intravenously ( 8 ) ,
Results are given as rateof radiolabeled product formation versus these findings indicate that a difference exists between the in
substrate concentration. vivo and in vitro glucuronidation of [I4C]bilirubin and [3H]
BMG.
PhysicalState of r4C]Bilirubin-Although bilirubin has
600r 1.8 r T exceedingly low aqueous solubility under equilibrium condi-
tions (34), we considered the possibility that [’4C]bilirubin
was being introduced to theassay system in a non-physiolog-
ical conformation. Specifically, the physical state of [“C]
. 1.2 bilirubin after rapid neutralization from an alkali solution is
ill-defined (35), and evidence has been presented to suggest
that bilirubin does not achieve a stablephysical conformation
with internal hydrogen bonding in neutral aqueous solution
for 1-4 days (34,36,37). In theradioassay employed in these
. 0.6
studies, substrate solutions were prepared by solubilization of
[14C]bilirubinin dilute alkali solution, followed by the imme-
diate addition of pH 7.6 buffer with or without albumin and
bilirubin conjugates. This substrate solution (0.20 ml) was
added within 60 s to the microsomal assay mixture (1.2-ml
0 1 .o 2.0 final volume) to initiate the enzyme reaction. Because internal
Time (min) hydrogen bonding may have been incomplete, aqueous solu-
bility and affinity for hydrophobic environments may not
FIG. 1. Microsomal synthesis of bilirubin diglucuronide
resemble that of bilirubin in its physiologic conformation,
(BDG) from [“Clbilirubin and [‘HIBMG and the effect of
albumin. The simultaneous generation of radiolabeled BDG from thereby altering the relative rates of [I4C]bilirubin and [3H]
7.5 pM [“Clbilirubin (0,W, A) and 7.5 pM f‘H]BMG (0, 0, A) was BMG glucuronidation.
measured as described under “Materials and Methods” and is ex- To exclude this possibility, the rates of [14C]bilirubinglu-
pressed in p~ and in pmol/mg protein (mean & S.D., see Table I1 for curonidation were measured following extended solubilization
n values) as a functionof time after addition of radiolabeled substrates
t o the microsomal assay mixture. Albumin was included in theinitial
intervals at pH 7.6. Initially, the spectrophotometric absorb-
substrate solution to achieve a final concentration of 0 p~ (0,0), ance of bilirubin/albumin solution (90 pM, molar ratio 1:l in
3.75 pM (W, O), or 7.5 p~ (A,A). The lines were obtained by linear TEA buffer, pH 7.6) was shown to remain constant over a 4-
regression analysis of the mean values for each time point. day period (data not shown), indicating nosignificant oxida-
tive degradation (38). The rates of [14C]BMG and [I4C]BDG
3.75 or 7.5 p ~ The . results are presented inFig. 1. formation from [14C]bilirubin bound to albumin (7.5 p ~ ,
The generation of BDG from [I4C]bilirubin (7.5 p ~ and ) molar ratio l:l), which had remained in solution at pH 7.6
[3H]BMG (7.5 p ~ was ) linear over the measurement period for intervals of up to 4 days in sealed, argonized flasks, are
of 0.5-2.5 min, permitting definition of the data as initial shown in Fig. 2. There was no significant change in the
rates of formation.Contrary to expectations, radiolabeled reaction rates over this period, so that all further experiments
BDG appeared to be formed at comparable rates from [“C] were performed using a 1-min solubilization period.
bilirubin and [3H]BMG. These rates decreased in parallel as Competitionbetweenr4ClBilirubinand PHJBMG Sub-
16946 Kinetic Analysis of Bilirubin UDP-glucuronosyltransferase
TABLEI1
initial rates of formation of bilirubin glucuronides from ['4Clbilirubin and thePH]BMG: effects of albumin and liver cytosol
Rates of formation of radiolabeled bilirubin glucuronides, computed by linear regression analysisof individual assays, are presented for the
experiments shown in Fig. 1 and for experiments conducted with cytosol prepared from liver homogenate (see "Materials and Methods");
data are expressedas pmol/mg protein.min (mean & S.D.). The ratio of 13HlBDG uersus I"C1BDG formation was comDuted from the mean
initial rates; a value of 1.0 reflects equal rates of formation.n indicates the number of replicate determinations of foriation rates for ["C]
BMG, ["CIBDG, and 13HlBDG,
" ~- . resDectivelv.
-

Initial rates of formation of bilirubin glucuronides Ratio for rates of

Albumin
from ['4C]bilirubin (7.5 PM)
[3H]BMG
and (7.5 p ~ ) [3H]BDG/['4C]BDG n
["CIBMG [I4C]BDG
synthesis [3H]BDG
PM pmljmg protein. min
0 584 & 99 164 & 17 186 & 36 1.1 494, 4
3.75 349 & 92 79 & 24 78 & 8 1.0 8, 13, 8
7.50 145 k 58 40 & 17 44 & 16 1.1 7. 7. 3
Cytosol (7.5)" 163 & 39 66 & 9 84 & 20 1.3 4; 4; 4
Denotes liver cytosol (7.05 mg/ml final concentration),which was selected to provide a ligandin concentration of approximately 7.5 p~
(20).

C
.-0 400 r
A: [14C]Bilirubin = 7.5 p M E: [3H]BMG = 7.5 p M
v, I 800 r r

- u y 600.
0 *-
c n I I '

2
.-
-
1-
0 ._
m 10' lo2 lo5
10 lo4 lo6
Bilirubin solubilization time (sec)
0
v
7.5 15.0 22.5 0 7.5 15.0 22.5
FIG. 2. Effect of varying solubilization time at pH 7.6 on
subsequent glucuronidation of ['"Clbilirubin by microsomal
GT. ["CIBilirubin (12.6 pg)wassolubilizedin 0.05 M NaOHand
immediately neutralized with0.167 M TEA buffer, pH7.6, containing FIG. 3. Mutual substratecompetition between [14C]bilirubin
albumin (90 pM, 1:l molar ratio). This solution was stored inthe dark and [3H]BMG for bilirubin glucuronide synthesis. The simul-
in sealed argonized flasks for up to 4 days prior to addition to the taneous formation of ["CIBMG (m),[14C]BDG(O), and [3H]BDG
assaymixture (7.5 p~ final['4C]bilirubin/albuminconcentration, (0)was determined while maintaining ["Clbilirubin constant at 7.5
molar ratio 1:l).Synthesis of ["CIBMG (V),[14C]BDG(A),and total P M with increasing [3H]BMG concentration(panel A ) or [3H]BMG
glucuronidation rate (the sum of ["CIBMG formation and twice ["C] constant at 7.5 p~ with increasing [14C]bilirubin concentration (panel
BDG formation rate, 0 ) over the subsequent 2.5 min are shown, in B). Data are expressed as pmol/mg protein.min as a function of
pmol/mg protein Smin, as a function of solubilization time. The values substrate concentration. Each point represents a single assay deter-
for n, representing replicate assays, from left to right in the figure, mination. No albumin was present in these assays.
are 6,4,6, 2,4,2 and 2; mean k S.D. for n 2 4, or mean k difference
for n = 2. The solid lines are based on linear regression analysis of and B where the substrate concentrations are the same as in
the mean values for eachset of data. Table I1 (7.5 ~ L each).
M The initial ratesfor formation of ["C]
BDG and [3H]BDG in panel A are 134 and 155 pmol/mg
strates-The concentrations of [14C]bilirubin and [3H]BMG protein. min,respectively, and in panelB 198 and 220 pmol/
used in these experiments (Fig. l ) , 7.5 p~ each, were selected
mg protein. min,respectively. Thus, infive out of six instances
so that the total pigment concentration approximated the (four in Table I1 and two in Fig. 3) where substrate concen-
presumed physiologic plasma concentration of -15 p~ (22,
trations are equal, the initial rate of BDG formation from
39). Because the equivalent rates of BDG formation from
[3H]BMGwas greater than thatfrom ['4C]bilirubin, with the
both substrates may fortuitously reflect these equimolar con-
ratios of rates for [3H]BDG/['4C]BDG formation in the range
centrations, the concentration of one substrate was varied
while holding the other constant in the absence of albumin of 1.0-1.3. While five out of six trials are not statistically
(Fig. 3). Formation of [I4C]BMG and [I4C]BDG from 7.5 p~ significant, this trend is in the oppositedirection to that
[14C]bilirubin (panel A ) decreased progressively as the [3H] observed in vivo (8). Indeed, this finding suggests that [3H]
BMG concentration increased from 0 to 22.5 p ~ indicating , BMG maybeaslightlyfavored substrate in uitro, which
that [3H]BMG interfered with both ['4C]bilirubin glucuroni- might be expected on the basis of the stoichiometry of [3H]
dation steps. Formation of [3H]BDG increased concomitantly. BMG uersus [14C]bilirubin conversion to BDG.
The converse behavior is demonstrated in panelB,in which Fig. 3 demonstrates that there is reciprocal inhibition of
['HJBMG was maintained at 7.5 p ~ In. results complemen- ['4C]bilirubin and [3H]BMG glucuronidation by the converse
tary to those shown inpanel A , [3H]BDG synthesis was substrate. This behavior and the Michaelis-Menten kinetics
progressively inhibited, and [14C]BMGand [14C]BDGforma- observed in bothFigs. 1 and 3 are consistentwith the hypoth-
tion increased, as theconcentration of [14C]bilirubin in- esis of binding of both substrates to a single activesite, which
creased. is capable of converting both bilirubin toBMG, and nascent
It is instructive to look at the two situations in panels A and exogenous BMG toBDG. A final setof experiments was
 Analysis
Kinetic of Bilirubin UDP-glucuronosyltransferase 16947
therefore performed to examine the binding of bilirubin, TABLE111
BMG, and BDG to bilirubin-GT. Apparent Michaelis-Mentenparameters for inhibitionof ["C]
Glucuronidation of r4C]Bilirubin in the Presence of Unla- bilirubin glucuronidation by unlabeled BMG and BDG
beled BMG and BDG-Purified unlabeled BMG or BDG was Apparentmaximum velocity (VmJ forformationof["CIBMG
added to assay mixtures containing concentrations of["C] (panel A) and [I4C]BDG(panel B ) , and enzyme-substrate dissocia-
tion constants for bilirubin ( K , ) were derived from the data shown
bilirubin ranging from 5-40 p ~ Albumin
. (5 p ~ was
) present
in Fig. 4 (40). Constants for competitive inhibition of ["CIBMG and
in all assay tubes to stabilize the higher concentrations of [14C]BDG formation by unlabeled BMG and BDG ( K I ) are also
pigment. Fig. 4 shows the rates of formation of[14C]BMG shown.
(panel A ) and [14C]BDG(panel B). The data obey Michaelis- Product K I
Menten kinetics over the range of measurement, with a sys- Fig' data formed from Vmax
K,
source 1"Clbilirubin
(bilirubin) BMG BDG
tematic and equivalent percent reduction in both [14C]BMG
and [14C]BDGformation rates of -20% (mean reduction) in pmollmg. rnin FM
the presence of 5 p~ BMG, and -50% in the presence of both PanelA [14C]BMG 992 & 30 13.7 11.8 9.4
20 p~ BMG or 20 p~ BDG. Panel B [I4C]BDG 453 & 18 19.3 10.2 15.3
The systematic decrease in these velocity curves, as unla-
beled pigment is added, is consistent with competitive inhi- dissociation constants for [14C]bilirubin(&), and inhibition
bition, i.e. the competitive binding of unlabeled pigment to constants for unlabeled BMG and BDG binding ( K r )are all
the active site of GT. The competitive nature of unlabeled in the range of 10-20 p M ,suggesting that each pigment species
pigment inhibitionof [14C]bilirubinglucuronidation is readily interacts with the active site with similar affinity. Comparable
apparent when the results are expressed in Lineweaver-Burk competition experiments using [3H]BMG and unlabeled bili-
form, as shown in Fig. 4, panels C and D for the datain panels rubin were not performed, due tothe prohibitively large
A and B, respectively. For clarity, only the inhibition by quantities of radiolabeled BMG required for such a study.
unlabeled BMG is shown; the curve for inhibition by BDG
(20 p M ) closely approximates that for BMG (20 p ~ ) .The DISCUSSION
maximal limiting rate (V,,,; the inverse of the y intercept)
does not change, as expected. Apparent Michaelis-Menten Conjugation of bilirubin with one or two sugar moieties by
parameters were derived for inhibition of [14C]bilirubinglu- microsomal GT differs from the conjugation of other sub-
curonidation by unlabeled BMG and BDG (40),and areshown strates by this enzyme family, in that two glucuronyl groups
in Table 111. The virtual Michaelis-Menten enzyme-substrate may be linked to bilirubin, with the intermediates inthis two-
step process, C-8 or C-12 BMG, also serving as substrates (5,
11, 12, 30). While kinetic studies have provided insight into
1
C

;:L
.-
c
0
8oo[ A: [ 4C]BMG B: [14C]13DG the binding of the UDP-sugar cosubstrates to bilirubin-(;?'
n
(5, 16,29, 41), only the conjugation of the first sugar group to
bilirubin has been amenable to kinetic studies (14). Events
leading to the second glucuronidation step remain poorly
understood. The biosynthesis and preparation of highly pu-
rified unlabeled and radiolabeled bilirubin and bilirubin glu-
curonides (8) provided an opportunity for directly examining
the reaction kinetics of GT i n vitro and, in particular, the
simultaneous conjugation ofboth ['4C]bilirubin and [3H]BMG
substrates. The radioassay system selected provides an accu-
rate assessment of initial rates of radiolabeled bilirubin glu-
curonide formation, with multiple time points over the initial
." [' 4C]Bilirubin (pM) 2.5 min of the reaction (Fig. 1, see Refs. 2, 20). Initial rate
studies avoid the confounding effects of product formation
b D-j/ (BMG and BDG), as may occur with conventional GT assays
of longer incubation periods (5, 14, 27-31, 41).
In a prior study of [14C]bilirubinand [3H]BMGprocessing
L
0,
by the intact liver i n uiuo, [14C]bilirubinwas converted more
a!,
L 0 .c efficiently to BDG, suggesting that [14C]bilirubinis the pre-
o c E
,+ e3 ae ferred substrate of bilirubin-GT (8). The preferential glucu-
:-; a ronidation of bilirubin was attributedtothe fact thatit
exhibits low aqueous solubility and readily associates with

--
m E
.c
n o
1
'. lipid membranes (25, 35, 42), whereas bilirubin glucuronides
.-.-- are considerably more hydrophilic (43). On the basis of these
m observations, we anticipated that i n vitro studies of microso-
-0.1 0 0.1 0.2 -0.1 0 0.1 0.2 mal bilirubin-GT would result in preferential conversion of
([14C]Bilirubin)-1 (pM)" [14C]bilirubin to BDG. However, there was no significant
FIG. 4. Inhibition of [14C]bilirubin glucuronidation by un- difference in initial rates of BDG synthesis from equimolar
labeled bilirubin mono- and diglucuronide. The simultaneous [14C]bilirubinand [3H]BMG inthis assay system (Fig. 1).
formation of ["CIBMG (panel A) and [14C]BDG (panel B ) is shown Two concerns emerged regarding the interpretation of this
as a function of ["Clbilirubinconcentration and is expressed in pmol/ finding. First, thepremise that bilirubin preferentially parti-
mg protein. min. The concentration of unlabeled pigments was as tions intomicrosomal membranes to undergo glucuronidation
follows: 0 , none; B, 5 p~ BMG; A, 20 PM BMG; and A, 20 p~ BDG.
Albumin was present in all assays at 5 p ~ The . data for [14C]BMG (20, 42, 44, 45) assumes that the physical conformation of
and ["CIBDG synthesis, in the presence of 0,5, and 20 p~ BMG, are bilirubin assayed in vitro resembles that of bilirubin i n uiuo.
plotted in Lineweaver-Burk format in panels C and D, respectively. However, the rapid solubilization of bilirubin in alkali and
Each point represents a single assay determination. subsequent neutralization may generate an unphysiological
16948 Kinetic Analysis of Bilirubin UDP-glucuronosyltransferase
conformation of bilirubin (37), and thereby lead to erroneous Second, kinetic studies performed with detergent-activated
kinetic data.Specifically, physicochemical studies of bilirubin microsomes indicate that the interaction of bilirubin with the
typically employ equilibration periods of 20 h to 4 days (34, active site of GT does not depend on thebilirubin substrate-
36), whereas the period of solubilization for G T assays in vitro albumin complex (19). Indeed, in our study, substitution of
is typically <5 min (2, 14, 15, 19,20, 22, 28, 44,46). As shown hepatic cytosol at a protein concentration approximating a
in Fig. 2, solubilization of bilirubin with albumin (molar ratio bilirubin binding capacity of 7.5 pM (see “Results”) yielded
1:l) for 1 minto 4 days a t 25 “Chadno effect onthe comparable rates of BDG formation from [14C]bilirubin and
conversion of bilirubin to either BMG or BDG. Thus, it is [3H]BMG (Table 11). This experiment, which simulates the
probable thatbilirubin achieves its final conformationat pH intracellular milieu of the hepatocyte, leads to a similar con-
7.6 within 1 min of neutralization from alkaline solution, or clusion regarding substrate binding to protein and access to
that theaqueous, hydrogen-bonded conformation of bilirubin microsomal GT.
is not a primary determinantof its access to theactive site of Although the initial ratesof BDG formation from equimolar
G T (also inferred in Ref. 19). Our findings clearly document [14C]bilirubin (7.5 p ~ and ) [3H]BMG (7.5 p ~ were ) compa-
that aqueous bilirubin may be used within 1 min of adjusting rable (Fig. I), variation in relative substrate concentrations
a n alkaline solution to pH7.6, without alteration in bilirubin was required to evaluate thepossibility of substrate competi-
glucuronidation rates. Rapid utilization of substrate is essen- tion. As shown in Fig. 3, a progressive decrease in bilirubin
tial toavoid degradation of pigments if the less stable bilirubin glucuronide formation was observed when one substrate was
glucuronides are included in the substrate mixture. held constant ([14C]bilirubin in panel A and [3H]BMG in
The second concern was the necessity of albumin when panel B ) , and the alternate substrate concentration was in-
bilirubin was being solubilized. At alkaline pH (>9), equilib- creased, demonstrating mutual competition for glucuronida-
rium bilirubin solubility is in the mM range, but decreases to tion between[14C]bilirubin and [3H]BMG. Of noteisthe
the nM range upon neutralization to pH 7-8 (34, 37). In the inhibition of both [14C]BMG and [l4C]BDG synthesis by
absence of a binding protein such as albumin, the medium exogenous [3H]BMG (panel A ) ; theoretically, thismay result
may contain dissolved bilirubin sodium salt in a supersatu- from end product inhibition at a monoglucuronidation site,
rated solution, several orders of magnitude above the equilib- and substrate competition at a distinct diglucuronidation site.
rium solubility. Over time, self-aggregation of bilirubin mol- However, it is far more plausible that a single active site is
ecules can form either a colloid suspension ormicrocrystalline involved in theprocess of both mono- and diglucuronidation.
precipitates (35, 37). Alternatively, a metastable supersatu- T o provide further insight into the kinetic behavior ob-
rated solution may persist, transforming to the crystalline served in Fig. 3, more detailed studies were performed, using
state over a prolonged period of time (47,48).Binding proteins unlabeled BMG or BDG as inhibitors of [‘4C]bilirubin glu-
providelong term stabilization of bilirubin insolution a t curonidation (Fig. 4). Both BMG and BDG inhibited [“C]
physiological pH (49). The efficient glucuronidation of bili- BMG and [14C]BDGformation in a competitive manner and
rubin observed in the absence of albumin (Fig. 1) indicates followed Michaelis-Menten kinetics. Assuming a single active
that a binding protein is not essential for the initial solubi- site, estimates were obtained of apparent maximal velocities
lization of bilirubin in aqueous solution. Moreover,the highest Vmaxfor [14C]bilirubin conversion to both [14C]BMG (992 &
rates of glucuronidation were observed intheabsence of 30 pmol/mg protein. min) and[14C]BDG (453 k 18 pmol/mg
albumin, indicating that bilirubin has readyaccess to the protein. min), andof the apparentK values for [ 14C]bilirubin
active site of microsomal GT. (substrate) and unlabeled BMG and BDG (inhibitors, Table
Initial rates of both [14C]bilirubin and [3H]BMG glucuron- 111).
idation decreased hyperbolicallywith the additionof 3.75 and The presence of 5 p~ albumin would be anticipated to
7.5 p~ albumin to the assay mixture (Fig. 1, Table 11). A decrease reaction rates by -60-65%, due to decreased sub-
reduction in bilirubin glucuronidation with increasing molar strate availability to theenzyme (seeFig. 1).Computed values
ratios of bilirubin/albumin has been documented previously for Vmexwill not be affected by albumin, but the K values
(20, 29) and is consistent with the binding of bilirubin to (Table 111) may be modestlyelevated.Nevertheless, V,,,
albumin anddecreased availabilityof substrate to the enzyme. values obtained in this study slightly
are lower, and substrate/
Although the non-covalent binding of BMG (or BDG) to inhibitor K values an order of magnitude higher than those
albumin is poorly characterized, the observed reduction of reported for digitonin (29) or CHAPS0 (19) pretreated rat
[“HIBDG synthesis by albumin in a manner comparable to microsomes, raising theissue of whether detergent treatment
that of [l4C]BDGwas unexpected. Albumin-bound conjugated of microsomal membranes is appropriate for studies of bili-
bilirubin in serum may be covalently linked (“biliprotein” or rubin-GT. Detergent activation is traditionallyperformed to
“delta fraction,” 50, 51) as a consequence of an acyl shift of obtain measurable and reliable rates of microsomal BDG
the glucuronyl moiety (52, 53). However, conversion of non- synthesis (14,22,27,29). However, this study and our previous
covalently bound bilirubin glucuronides to the covalent albu- applications of this radioassay (44, 60) clearly demonstrate
min conjugate form occurs over several hours a t 37 “C (52), that formation of BDG can bemeasured accurately using
and hence is unlikely to occur during the brief exposure of intact microsomes. Although intact microsomes are reportedly
BMG to albumin in the course of this assay. Rather, it is heterogeneous and exist in both latent and partially disrupted
probable that [14C]bilirubin and [3H]BMG interact non-co- forms (19, 61), the high latency toward mannose-6-phospha-
valently with albumin in a similar manner, as reflected by tase (>96%, “MaterialsandMethods”)andthe higher K
two lines of evidence. values (Table 111) observed in this study suggest that the
First, while unconjugated bilirubin may bind to albumin potential contribution of “disrupted” (and thereby activated)
via reversible ionic interactions (37, 54-56), hydrogen bonds microsomal vesicles to the kinetic results is minimal or absent.
(55,57) and van der Waalforces (55,58), removal, ionization, Thus, the findings reported herein reflect the glucuronidation
or methyl esterification of the propionic acid carboxyl groups of bilirubin and itscongeners under conditionswhich approx-
appears to have little effect on its binding (13, 55). Variable imate those extanti n uiuo, and indicate that the intact micro-
side chains also play only a minor role in the hydrophobic somal membrane is likely to play a significant role in modu-
interactions between albumin and porphyrin monomers(59). lating the access of substrates to GT.
 Kinetic
Analysis of Bilirubin UDP-glucuronosyltransferase 16949
With these considerations in mind, it is appropriate to is comparable for exogenous BMG and that generated in situ
examine the structure of microsomal bilirubin-GT and the from bilirubin.
interaction of bilirubin and bilirubin glucuronides with micro- Insight into the structural requirements for access of bili-
somal membranes. Based on sequencing studies, the bulk of rubin congeners to the active site of GT has beengained
bilirubin-GT is located within the cisternal lumen, with short recently from experiments with synthetic analogs. Systematic
cytosolic andtrans-membrane carboxyl-terminal segments substitution of the side chains of bilirubin demonstrates that
(62-64). Comparison of cDNA clones for different GTs sug- internal hydrogen bonding of both carboxyl groups facilitates
gests that binding specificity for the various aglycone sub- glucuronidation but impairs biliary excretion; aberrant sol-
strates resides in the amino-terminal half of the enzyme, with vent exposure of one carboxyl group enables direct biliary
the more conserved carboxyl-terminal portion of the polypep- excretion but does not prevent further glucuronidation (78).
tide chain containing the binding site for the cosubstrate, Studies with arylcarboxylic acid competitive inhibitors also
UDP-GlcA (65, 66). Access of the cosubstrate UDP-sugars to underscore the necessity of a single carboxyl group on a
G T requires carrier-mediated delivery across the microsomal lipophilic molecule for binding to bilirubin-GT. The best
membrane (14,16). In theevolving view, therefore, the cosub- inhibitors (e.g. 7,7,7-triphenylheptanoicacid) are half the size
strate and substrate-binding sites are in the lumen of the of the bilirubin molecule (79). Thus, these reports suggest
endoplasmic reticulum (18). that the physicochemical properties residing in half of an
Localization of the aglycone-binding site to the amino- unconjugated bilirubin congener, or a similar-sized analog,
terminal half of polypeptide chain, however, does not con- are sufficient for access to the active site of microsomal
strain bilirubin binding to the cisternal lumen. Sequencing bilirubin-(=?', thereby corroborating the current findings of
data reveals regions of significant hydrophobicity inthis comparable rates of BDG biosynthesis from bilirubin and
portion of the molecule which, although not sufficiently long BMG. Resolution of issues pertaining to access of bilirubin
and hydrophobic to traverse the membrane, may permit mem- pigments to GT and the role of the microsomal membrane
may be possible byexamining the movement of bilirubin and
brane interaction (63, 64, 67). Bilirubin-GT activity is mark-
its congeners across model and reconstituted membranes,
edly dependent on the phospholipid membrane environment
using rapid fluorescence-quenching techniques (75,80,81).
(68, 69), and GT-catalyzed conjugation reactions generally
It has been proposed that a tetrameric enzymemaybe
occur faster for the more lipophilic aglycones (70). Thus, itis
required to catalyze the formation of BDG (but not BMG),
possible that the hydrophobic binding site for bilirubin and
based on radiation inactivation analysis (82, 83) andthe
its glucuronides (71) resides at a membrane interface (29),
enhanced sensitivity of BDG synthesis to membrane disrup-
thereby facilitating ready access of membrane-bound bilirubin tion (29, 30, 84). A single active site would still be involved,
to theenzyme. Furthermore, the cosubstrate UDP-GlcA may but higher order interactions between enzyme subunits may
be delivered to a binding site within the membrane, rather be necessary. Preliminary kinetic analysis of bilirubin glucu-
than entering the cisternal compartment (16). BMG, when ronidation by hepatic microsomes from the Mucacu fascicu-
formed, interchanges rapidly with exogenous BMG present in lurk monkey suggests that simultaneous bimolecular glucu-
the environment (with no apparent preference for the C-8 or ronidation of ['4C]bilirubin directly to [I4C]BDG does occur
C-12 isomers, 19), yielding high initial rates of BMG forma- (85), presumably involving oligomeric interactions between
tion in uitro (Table 11), and permitting rapid excretion of GT subunits. Further experimentation is required to clarify
newly formed BMG into bile in vivo (8). these observations and to define the structural-functional
A carrier transport system for movement of bilirubin pig- behavior of bilirubin-GT. Finally, kinetic studies of bilirubin-
ments across the microsomal membrane has not been identi- GT as a crude microsomal fraction, uersus purified bilirubin-
fied. Conversely, it remains to be established whether physi- GT reconstituted in model membranes, will beneeded to
cochemically based membrane interactions are sufficient to determine whether multiple isozymes contribute to the ob-
explain the observed kinetics of bilirubin-GT. Bilirubin ex- served kinetic behavior.
hibits exceedingly low aqueous solubility and readily parti-
tions into lipid phases (42). However, bilirubin binding ap- CONCLUSION
pears to be at the phospholipid-water interface, rather than This study constitutes the most extensive characterization
in the lipid membrane itself (35). Reported data suggest that of bilirubin glucuronidation by microsomal bilirubin-GT re-
bilirubin may transiently reside deeper within the membrane ported to date. Our findings indicate that: 1) bilirubin-(;?'
bilayer (72, 73) and may have considerable free motion, since obeys Michaelis-Menten kinetics for [14C]bilirubinand [3H]
it is not constrained to a particular orientation (42, 43), and BMG glucuronidation over the range of substrate concentra-
potentially may traverse lipid membranes easily (74)and tions employed; 2) ['4C]bilirubin and [3H]BMGappear to be
rapidly (75). Thus, our observation that microsomal bilirubin equivalent substrates for the formation of BDG, although
conjugation is optimal utilizing liposomal bilirubin substrate, [3H]BMG may be slightly favored in uitro; 3) the inhibition
prepared with purified endogenous microsomal lipids (20, 44, of [I4C]bilirubin glucuronidation by unlabeled BMG (Fig. 4)
76), suggests that membrane interactions alone account for or [3H]BMG (Fig. 3), and of [3H]BMG glucuronidation by
the efficient access of bilirubin to theactive site of bilirubin- [I4C]bilirubin(Fig. 3), is consistent with competitive binding
GT. of both substrates to a single active site on the enzyme; 4)
Considerably less is known about the interaction of BMG bilirubin, BMG, and BDG appear to bind to this active site
and BDG with lipid membranes, since bilirubin is rendered with comparable affinities in intact microsomal preparations;
water soluble by the addition of one or two glucuronyl groups and 5) the availability of [14C]bilirubinand [3H]BMG for
(43). Bilirubin conjugates may adsorb to the interface of lipid enzymatic glucuronidation are reduced to a similar extent by
assemblies, such as mixed lecithin-bile salt micelles (35), and the presence of albumin and hepatic cytosol, suggesting com-
interact with intracellular membranes in uiuo en route to parable affinities for binding to albumin and cytosolic pro-
biliary excretion (77). Although access of BMG to the active teins. A plausible explanation for the preferential i n vivo
site may be modulated by the intact microsomal membrane, glucuronidation of bilirubin, relative to exogenous BMG, is
the present study indicates that such modulation, if operative, the existence of efficient mechanisms for bilirubin substrate
16950 Kinetic Analysisof Bilirubin UDP-glucuronosyltransferase
delivery to the microsomal enzyme which are operative only
in the intact hepatocyte. Finally, these studies illustrate the
critical importance of correlating in vitro findings of mem-
brane-associated enzyme function with behavior in vivo (8,
77) since data obtainedin vitro may not reflect the integrated
contribution of intracellular components.
Acknowledgments-We gratefully acknowledge Stephen C. Hauser,
M. D., for helpful discussions during the course of this study and the
expert technical assistance of Julie Ziurys, Ellen Wrchota, and Bar-
bara Frishkopf. Data organization and analysis were performed on
the PROPHET system, a national computer resource sponsored by
the National Center for Research Resources, National Institutes of
Health. The experiments reported herein were conducted according
to the principles set forth in the Guide for the Care and Use of
Laboratory Animals, Institute ofAnimal Resources, National Re-
search Council.
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