Glucuronate, the precursor of vitamin C, is directly formed
from UDP-glucuronate in liver
Carole L. Linster and Emile Van Schaftingen
Laboratory of Physiological Chemistry, Universite
´
Catholique de Louvain and the Christian de Duve Institute of Cellular Pathology, Brussels,
Belgium
Formation of free glucuronate from UDP-glucuronate
can be considered as the first step in the synthesis of
vitamin C (Fig. 1), a pathway that occurs in most ver-
tebrates, although not in guinea pigs and primates,
including humans [1]. Free glucuronate can also be
converted to pentose phosphate intermediates via
the ‘pentose pathway’ [2]. The latter is inter-
rupted in subjects with pentosuria, who have a
deficiency in l-xylulose reductase and excrete abnormal
amounts of l-xylulose [3]. We recently reinvestigated
Keywords
glucuronate; glucuronate 1-phosphate;
UDP-glucuronosyltransferases; vitamin C;
xenobiotics
Correspondence
E. Van Schaftingen, Laboratory of
Physiological Chemistry, UCL-ICP, Avenue
Hippocrate 75, B-1200 Brussels, Belgium
Fax: +32 27 647 598
Tel: +32 27 647 564
E-mail:
(Received 12 January 2006, revised 2
February 2006, accepted 10 February 2006)
doi:10.1111/j.1742-4658.2006.05172.x

indirectly, by a combination of ATP-Mg and CoASH.
Abbreviations
ER, endoplasmic reticulum; 4-Np-UGT, 4-nitrophenylglucuronosyltransferase; UDPGlcNAc, UDP-N-acetylglucosamine.
1516 FEBS Journal 273 (2006) 1516–1527 ª 2006 The Authors Journal compilation ª 2006 FEBS
the mechanism by which some xenobiotics stimulate the
formation of vitamin C in animals and enhance the
excretion of l-xylulose in humans with pentosuria and
have shown that aminopyrine, metyrapone and other
xenobiotics cause an almost instantaneous increase in
the conversion of UDP-glucuronate to glucuronate in
isolated rat hepatocytes [4]. The precise mechanism by
which free glucuronate is formed remains unclear. It is
usually stated that glucuronate formation from UDP-
glucuronate is the result of two successive reactions
comprising the hydrolysis of UDP-glucuronate to glu-
curonate 1-phosphate and UMP by a pyrophospha-
tase, followed by dephosphorylation of glucuronate
1-phosphate [5,6]. However, neither the pyrophospha-
tase nor the phosphatase implicated in these reactions
has been identified. Furthermore, other mechanisms, in
which glucuronate is directly formed by hydrolysis of
UDP-glucuronate or indirectly through the transfer
of glucuronide to an endogenous (unknown) acceptor
by a UDP-glucuronosyltransferase, followed by the
hydrolysis of the glucuronidated acceptor, need to be
considered [4,7,8].
The purpose of this study was to check if the effect
of aminopyrine, metyrapone and chloretone to stimu-
late the formation of glucuronate from UDP-glucuro-
nate could be reproduced in cell-free systems and to

L-xylulose
ATP-Mg
+
CoASH
(-)
Aglycones
(-)
Metyrapone
Aminopyrine
Chloretone
(-)
ER
cytosol
(8)
(1)
Sorbinil
(-) (2)
(5)
(6)
(3)
(4)
xylitol
(7)
Pentosuria
Fig. 1. Pathways of vitamin C, L-xylulose and glucuronate 1-phosphate formation. 1, UDP-glucuronidase; 2, glucuronate reductase; 3, aldono-
lactonase; 4,
L-gulono-1,4-lactone oxidase; 5, L-gulonate 3-dehydrogenase; 6, 3-dehydro-L-gulonate decarboxylase; 7, L-xylulose reductase; 8,
nucleotide pyrophosphatase. As shown in this study (see Discussion), glucuronate appears to be formed directly from UDP-glucuronate by a
membrane-bound enzyme in the endoplasmic reticulum (ER). Metyrapone, aminopyrine and chloretone stimulate this formation by antagon-
izing the inhibitory effect exerted, presumably indirectly, by a combination of ATP-Mg and CoASH.

protein). Even lower
activities were observed at concentrations of glucuro-
nate 1-phosphate < 0.5 mm, indicating that the glu-
curonate 1-phosphate phosphatase activity was not
underestimated because of substrate inhibition. These
results further argued against glucuronate 1-phosphate
being an intermediate in the formation of glucuronate
from UDP-glucuronate (see Discussion).
Localization of the enzyme forming glucuronate
in microsomes
Liver extract fractionation showed that the enzyme
responsible for glucuronate formation from UDP-glu-
curonate (henceforth called ‘UDP-glucuronidase’) was
mainly present in the microsomal fraction (Table 1), as
were UDP-glucuronosyltransferase and UDP-glucuro-
nate pyrophosphatase. Interestingly, metyrapone sti-
mulated UDP-glucuronidase activity in the microsomal
fraction by only 20%, despite the presence of ATP-
Mg. It is shown below that this is due to loss of the
inhibitory effect of ATP-Mg, consequent to the
removal of a heat-stable cofactor present in the high-
speed supernatant. Accordingly, the total recovery of
UDP-glucuronidase activity in the mitochondrial and
microsomal fractions was much higher than 100% if
metyrapone was omitted (first column of Table 1), but
was close to 100% if the assays were performed in the
presence of this xenobiotic. The microsomal fraction
contained only minimal glucuronate 1-phosphatase
activity (0.09 nmolÆmin
)1

M UDPGlcNAc, 0.5 mM sorbinil, without or with
10 m
M ATP-Mg and ⁄ or 1 mM of the indicated xenobiotic (open
diamonds, no xenobiotic added; filled triangles, aminopyrine; filled
circles, chloretone; filled squares, metyrapone). A control incubation
containing 0.5% dimethylsulfoxide (solvent for chloretone) was also
performed (open circles). When incubations were run without liver
extract, no glucuronate, but 6.7 ± 0.6 l
M (mean ± SEM, n ¼ 12)
glucuronate 1-phosphate, resulting from acid hydrolysis of UDP-
glucuronate, was measured. This value was subtracted from those
found in the presence of liver extract. Note that the scale of the
ordinate in Fig. 2B differs from the other panels by sixfold.
Glucuronate formation in liver cell-free systems C. L. Linster and E. Van Schaftingen
1518 FEBS Journal 273 (2006) 1516–1527 ª 2006 The Authors Journal compilation ª 2006 FEBS
and ATP-Mg inhibited UDP-glucuronate pyrophos-
phatase, 50% inhibition being reached at  4 and
0.5 mm, respectively (Fig. 3 and not shown). By con-
trast, ATP-Mg did not affect UDP-glucuronidase
activity in the microsomal fraction, although, as shown
above, it did inhibit this activity in crude extracts.
Implication of UDP-glucuronosyltransferases in
the formation of free glucuronate
Because they are located in the same subcellular com-
partment and use the same nucleotide substrate, it was
of interest to compare the properties of UDP-glucu-
ronidase and UDP-glucuronosyltransferases. The latter
are sensitive to several detergents [12,13], because they
are integral membrane proteins [14,15]. We therefore
compared the effect of various detergents on free glu-

)1
Æmg
)1
protein)
Heavy mitochondrial fraction 0.05 ± 0.01 0.08 ± 0.01 0.27, 0.25 2.76, 2.70
Light mitochondrial fraction 0.18 ± 0.01 0.23 ± 0.01 0.90, 0.78 4.93, 4.96
Microsomal fraction 0.87 ± 0.02 1.02 ± 0.01 3.07, 2.88 12.2, 11.8
Final supernatant 0.01 ± 0.00 0.01 ± 0.00 0.00, 0.04 0.71, 0.77
Total activity (nmolÆmin
)1
Æg
)1
liver)
Post-nuclear supernatant 14.3 ± 0.8 31.3 ± 0.3 74.3, 73.8 354.9, 364.2
Sum of fractions 25.4 ± 1.0 30.4 ± 0.8 89.2, 86.8 426.3, 420.9
Yield (%) 179 ± 4 97 ± 2 120, 118 120, 116
Fig. 3. Stimulation of glucuronate and b-glucuronide formation (A)
and inhibition of glucuronate 1-phosphate formation (B) by UDPGlc-
NAc in microsomes. Microsomes were incubated at 30 °C with
1m
M UDP-glucuronate, the indicated concentrations of UDPGlcNAc
and without (open symbols) or with (filled symbols) 10 m
M ATP-
Mg. For the assay of 4-nitrophenylglucuronoslytransferase
(4-Np-UGT), the medium additionally contained 0.2 m
M 4-nitrophe-
nol and 1 m
M saccharo-1,4-lactone. The reactions were initiated by
the addition of microsomes. Perchloric acid extracts were prepared
after 8 min to measure b-glucuronide (triangles) and after 20 min to

intermediate. The latter would be hydrolysed by b-
glucuronidase or possibly by esterases, in which case it
would be an acylglucuronide. However, saccharo-1,4-
lactone (3 mm) did not affect glucuronate formation
from UDP-glucuronate in microsomes, whereas it
powerfully inhibited b-glucuronidase in this subcellular
fraction. Fifty per cent inhibition was observed at
pH 7.1 with 10–15 lm saccharo-1,4-lactone when
0.5 mm 4-nitrophenylglucuronide or 0.5 mm 4-methyl-
umbelliferylglucuronide were used as substrates (not
shown). Similarly, preincubation of microsomes with
1mm bis-p-nitrophenylphosphate, an esterase inhibitor
[18], for 30 min at 37 °C did not affect their UDP-
glucuronidase activity, whereas it suppressed their
capacity to hydrolyse 3 mm o-nitrophenylacetate (not
shown).
Fig. 4. Effect of various detergents on glucuronate (A, C) and b-glucuronide (B, D) formation. Microsomes were incubated at 30 °Casdes-
cribed in Experimental procedures, but without ATP-Mg. UDP-glucuronate and UDPGlcNAc, as well as the indicated concentrations of the
various detergents (squares, Triton X-100; circles, b-octylglucoside; diamonds, polyoxyethylene ether W-1; triangles, deoxycholate) were
included in the assays. UDP-glucuronidase (UDPGAse) was measured in the presence of 1 m
M metyrapone and 4-Np-UGT in the presence
of 0.2 m
M 4-nitrophenol and 1 mM saccharo-1,4-lactone. The reactions were initiated by addition of microsomes. Perchloric acid extracts
were prepared after 8 and 20 min to measure b-glucuronide and glucuronate, respectively. PE W-1, polyoxyethylene ether W-1.
Glucuronate formation in liver cell-free systems C. L. Linster and E. Van Schaftingen
1520 FEBS Journal 273 (2006) 1516–1527 ª 2006 The Authors Journal compilation ª 2006 FEBS
Role of a heat-stable cofactor in the sensitivity
of UDP-glucuronidase to metyrapone and other
xenobiotics
The data obtained with purified microsomes suggested

in glucuronate formation
Previous results obtained with isolated hepatocytes
have indicated that free glucuronate formation is
Fig. 5. Effect of 4-methylumbelliferone (4-MU) and valproate on the
formation of free glucuronate (A) and the rate of their glucuronid-
ation (B). Microsomes were incubated at 30 °C with 3 m
M UDP-glu-
curonate, 0.1% Triton X-100, 10 m
M ATP-Mg, 1 mM saccharo-1,
4-lactone, 1 m
M metyrapone and the indicated concentrations of
4-methylumbelliferone (squares) or valproate (triangles). The react-
ions were initiated by addition of UDP-glucuronate after 10 min pre-
incubation. Perchloric acid extracts were prepared 10 min later to
measure glucuronate and b-glucuronides.
Fig. 6. Transience of the inhibitory effect of 4-methylumbelliferone
(4-MU) but not of valproate on the formation of free glucuronate.
Microsomes were incubated in the same conditions as for Fig. 5
but without (open triangles) or with a fixed concentration of valpro-
ate (1 m
M; closed triangles) or 4-methylumbelliferone (0.5 mM;
closed squares). Perchloric acid extracts were prepared at various
times after the addition of UDP-glucuronate to determine glucuro-
nate (A) and b-glucuronide (B) concentrations. A control incubation
containing 1% dimethylsulfoxide (solvent for 4-methylumbelliferone)
was also performed (open squares). The dashed line represents an
extrapolation of the initial rate of glucuronate formation in the pres-
ence of 4-methylumbelliferone over the whole incubation period.
C. L. Linster and E. Van Schaftingen Glucuronate formation in liver cell-free systems
FEBS Journal 273 (2006) 1516–1527 ª 2006 The Authors Journal compilation ª 2006 FEBS 1521

)1
) were incubated in
the same conditions as the crude liver extracts in Fig. 2. The effect
of a high-speed supernatant (untreated, heated for 5 min at 95 °C
or heated and subsequently treated with 2% charcoal) on micro-
somal glucuronate formation was tested in the presence of 10 m
M
ATP-Mg and in the absence (black bars) or presence (grey bars) of
1m
M metyrapone (A). The effect of the heated high-speed super-
natant was further analysed in the absence (B) or presence (C) of
10 m
M ATP-Mg and in the absence (black bars) or presence of
1m
M metyrapone (light grey bars), aminopyrine (AP, white bars) or
chloretone (CL, dark grey bars). Perchloric acid extracts were pre-
pared 0 and 20 min after initiation of the reaction by addition of
UDP-glucuronate and UDPGlcNAc to measure glucuronate. The dif-
ference between the concentrations determined at 0 and 20 min of
incubation is shown.
A
B
Fig. 8. ATP-dependent inhibition of free glucuronate formation by
CoASH. Microsomes were incubated in the same conditions as the
crude liver extracts in Fig. 2 except that sorbinil was omitted from
the incubation medium. Glucuronate formation was measured with-
out (light grey bars) or with (dark grey bars) 100 l
M CoASH and in
the absence (A) or presence (B) of 10 m
M ATP-Mg. The effect of

substrate. Similarly, the low glucuronate 1-phosphate
phosphatase activity detected in liver extracts and
microsomes most likely corresponds to a nonspecific
phosphatase.
Lack of involvement of a glucuronidated
intermediate
The enzyme forming free glucuronate from UDP-glu-
curonate shares several properties with UDP-glucuron-
osyltransferases (see below). Because liver microsomes
contain b-glucuronidase [24–26], the formation of free
glucuronate from UDP-glucuronate observed in this
preparation could be the result of a glucuronidation–
deglucuronidation cycle, with a hypothetical acceptor
present in the microsomal fraction. Against this is the
finding that saccharo-1,4-lactone did not affect the for-
mation of glucuronate despite completely blocking
hydrolysis of 4-nitroph enyl- and 4-methylumbelliferyl-
glucuronide. As esterases are also present in micro-
somes [27], and some UDP-glucuronosyltransferases
use carboxylic acids as acceptors [28], we had to consi-
der the possibility that an acylglucuronide could form
as an intermediate. The finding that bis-p-nitrophenyl-
phosphate, although blocking esterase activity, did not
affect the formation of glucuronate from UDP-glu-
curonate allowed us to discard this second possibility.
Although we may not formally exclude that glucuro-
nate formation involves the hydrolysis of a hypothet-
ical glucuronidated intermediate by an unknown
enzyme that would not be affected by these inhibitors,
our observations indicate that UDP-glucuronate is

indicates that this enzyme is present in the same type
of vesicles as UDP-glucuronosyltransferases.
Further analogy between the two types of enzymes
is found in the similarity of the effect of detergents.
All tested detergents stimulated both enzymatic activit-
ies at low concentrations, consistent with the idea that
both types of enzymes have their catalytic site oriented
towards the lumen of the vesicles and that disruption
of the vesicular membrane increases accessibility to
UDP-glucuronate. Some of the detergents exerted inhi-
bition of the enzymatic activity at higher concentra-
tions and it is striking that the same order of potency
(deoxycholate > b-octylglucoside > Triton X-100) was
observed for UDP-glucuronosyltransferases and for
UDP-glucuronidase. This indicates that their activity
has the same type of requirement in terms of phos-
pholipidic environment.
That the UDP-glucuronidase activity may actually
be a side activity of UDP-glucuronosyltransferases
themselves is suggested by the fact that glucuronidable
C. L. Linster and E. Van Schaftingen Glucuronate formation in liver cell-free systems
FEBS Journal 273 (2006) 1516–1527 ª 2006 The Authors Journal compilation ª 2006 FEBS 1523
substrates (4-methylumbelliferone, valproate) inhibited
formation of free glucuronate. 4-Methylumbelliferone
was more potent than valproate as an inhibitor of glu-
curonate formation consistent with the former being a
substrate for many UDP-glucuronosyltransferase iso-
forms [31], which is not the case for valproate [32].
Taken together, these findings indicate that UDP-
glucuronosyltransferase (or at least some UDP-glucu-

‘a-glucuronidase’ activity was enhanced by the pres-
ence of phenylethers and lysophosphatidylcholines up
to  0.03% of its transferase activity. This value is
much lower than that observed in this study for a non-
purified enzyme, indicating that if indeed free glucuro-
nate production is due to an a-glucuronidase activity
of UDP-glucuronosyltransferases, the hydrolytic activ-
ity must be stimulated by phospholipids or other com-
pounds present in the microsomal membrane. Another
possibility is that the a-glucuronidase activity may be
more substantial in the case of some UDP-glucurono-
syltransferases than others, or that one or several
members of the UDP-glucuronosyltransferase family
only act as hydrolases.
Our conclusions on the involvement of UDP-glucu-
ronosyltransferases in the formation of glucuronate
are only tentative at this stage. Purification attempts
involving solubilization of UDP-glucuronidase with
detergents followed by chromatography (CL Linster &
E Van Schaftingen, unpublished results) failed because
the UDP-glucuronidase activity was inhibited by the
detergents or because the detergents were unable to
solubilize the enzyme properly. Ongoing experiments
with overexpressed UGT1A6 in HEK cells (CL Lin-
ster, CP Strassburg & E Van Schaftingen, unpublished
results) indicate that this enzyme has modest UDP-glu-
curonidase activity that is stimulated by menadione (a
stimulator of glucuronate formation in isolated hepato-
cytes) [4] and lysophosphatidylcholine (reported to be
a stimulator of the UDP-glucuronidase activity of

heated liver extract and CoASH also restored (in the
presence of ATP-Mg) the sensitivity to metyrapone
and other xenobiotics and it is likely that the effect
of the heated liver extract can be entirely ascribed to
CoASH or CoA derivatives. This identification may,
for instance, account for the loss of inhibitor upon
anion-exchange chromatography of the heated high-
speed supernatant and its partial recovery upon
treatment of the fractions with dithiothreitol, as
CoASH was found to be largely oxidized during this
Glucuronate formation in liver cell-free systems C. L. Linster and E. Van Schaftingen
1524 FEBS Journal 273 (2006) 1516–1527 ª 2006 The Authors Journal compilation ª 2006 FEBS
purification procedure. The finding that the effect of
CoASH depends on the presence of ATP (although
not of other NTPs such as GTP and UTP; not
shown) suggests that it is indirectly mediated via the
formation of acyl-CoAs from fatty acids present in
the microsomal preparation by microsomal acyl-CoA
synthetase. Interestingly, acyl-CoAs are known to
inhibit UDP-glucuronosyltransferases [36].
Our conclusions on glucuronate formation and its
regulation are summarized in Fig. 1. We have provided
evidence for the fact that glucuronate formation in
liver appears to proceed through direct hydrolysis of
UDP-glucuronate rather than via an intermediate, and
that UDP-glucuronosyltransferase or a closely related
enzyme seems to be involved in this conversion. How-
ever, the enzyme responsible for the synthesis of glu-
curonate 1-phosphate from UDP-glucuronate remains
a pending problem that needs further research. The

,
4-nitrophenol, sodium deoxycholate and sodium phosphate
were from Merck (Darmstadt, Germany). Aminopyrine,
metyrapone, charcoal, saccharo-1,4-lactone, polyoxyethyl-
ene ether W-1, b-octylglucoside, Triton X-100 and the
sodium salts of CoASH (from yeast), UDP-glucuronic acid
and UDPGlcNAc were from Sigma-Aldrich (St Louis,
MO). Chloretone and Dowex 1 · 8 were from Acros
Organics (Geel, Belgium). 4-Methylumbelliferone was from
Koch-Light (Colnbrook, UK) and sodium valproate from
Labaz-Sanofi (Brussels, Belgium). Sorbinil was a kind gift
of Pfizer. All other reagents, whenever possible, were of
analytical grade.
Preparation of crude liver extracts, microsomes
and other subcellular fractions
All steps of the described procedures were carried out at
4 °C. Liver extracts were prepared from overnight-fasted
male Wistar rats. Livers were homogenized in a Potter-Elv-
ehjem apparatus with 3 vol. (v ⁄ w) of a buffer containing
25 mm Hepes, pH 7.1, 25 mm KCl, 0.25 m sucrose,
5 lgÆmL
)1
antipain and 5 lgÆmL
)1
leupeptin. The homogen-
ate was centrifuged for 20 min at 18 000 g. The resulting
supernatant (crude liver extract) was centrifuged for another
45 min at 100 000 g to obtain a high-speed supernatant and
a microsomal pellet. The latter was washed twice in the
homogenization buffer and resuspended in the same buffer

nents except UDP-glucuronate and UDPGlcNAc, and the
assay was initiated by addition of these two nucleotides.
Where indicated, the assay was initiated by the addition of
the enzyme preparation to an otherwise complete assay
mixture. The reaction was stopped after 0–30 min by mix-
ing a portion of the incubation medium with 0.5 vol. of
cold 10% (w ⁄ v) perchloric acid. Glucuronate-1-phosphatase
was measured through the formation of glucuronate under
similar conditions, except that UDP-glucuronate was
replaced by 0.5 mm glucuronate 1-phosphate. UDP-glucu-
ronosyltransferase was also similarly assayed, at 30 °C,
through the formation of b-glucuronides in an incubation
mixture containing 20 mm sodium phosphate, pH 7.1,
2mm MgCl
2
,10mm ATP-Mg, 1 mm saccharo-1,4-lactone,
C. L. Linster and E. Van Schaftingen Glucuronate formation in liver cell-free systems
FEBS Journal 273 (2006) 1516–1527 ª 2006 The Authors Journal compilation ª 2006 FEBS 1525
1mm UDPGlcNAc, 1 mm UDP-glucuronate and 0.2 mm
4-nitrophenol, 0.5 mm 4-methylumbelliferone or 1 mm val-
proate. In all cases, perchloric acid extracts were centri-
fuged at 4 °C and the supernatants neutralized by the
addition of K
2
CO
3
. These perchloric acid extracts were
treated with 2% charcoal in experiments in which 4-methyl-
umbelliferone was used, because the latter absorbs light at
340 nm and thus interferes with the spectrophotometric glu-

Molecular Bases of Inherited Disease (Scriver CR, Beau-
det AL, Sly WS & Valle D, eds), 8th edn, Vol. I, pp.
1589–1599. McGraw-Hill, New York.
3 Wang YM & Van Eys J (1970) The enzymatic defect in
essential pentosuria. N Engl J Med 282, 892–896.
4 Linster CL & Van Schaftingen E (2003) Rapid stimula-
tion of free glucuronate formation by non-glucuronid-
able xenobiotics in isolated rat hepatocytes. J Biol Chem
278, 36328–36333.
5 Ginsburg V, Weissbach A & Maxwell ES (1958) Forma-
tion of glucuronic acid from uridinediphosphate glu-
curonic acid. Biochim Biophys Acta 28, 649–650.
6 Puhakainen E & Ha
¨
nninen O (1976) Pyrophosphatase
and glucuronosyltransferase in microsomal UDPglu-
curonic-acid metabolism in the rat liver. Eur J Biochem
61, 165–169.
7 Pogell BM & Leloir LF (1961) Nucleotide activation of
liver microsomal glucuronidation. J Biol Chem 236,
293–298.
8 Horio F, Shibata T, Makino S, Machino S, Hayashi Y,
Hattori T & Yoshida A (1993) UDP glucuronosyltrans-
ferase gene expression is involved in the stimulation of
ascorbic acid biosynthesis by xenobiotics in rats. J Nutr
123, 2075–2084.
9 Bhatnagar A, Liu S, Das B, Ansari NH & Srivastava
SK (1990) Inhibition kinetics of human kidney aldose
and aldehyde reductases by aldose reductase inhibitors.
Biochem Pharmacol 39, 1115–1124.

(1998) Transmembrane topology of glucose-6-phospha-
tase. J Biol Chem 273, 6144–6148.
17 Levvy GA (1952) The preparation and properties of
b-glucuronidase. 4. Inhibition by sugar acids and their
lactones. Biochem J 52, 464–472.
18 Heymann E, Mentlein R, Schmalz R, Schwabe C &
Wagenmann F (1979) A method for the estimation of
esterase synthesis and degradation and its application to
evaluate the influence of insulin and glucagon. Eur J
Biochem 102, 509–519.
19 Touster O, Aronson NN Jr, Dulaney JT & Hendrickson
H (1970) Isolation of rat liver plasma membranes. Use
of nucleotide pyrophosphatase and phosphodiesterase I
as marker enzymes. J Cell Biol 47, 604–618.
Glucuronate formation in liver cell-free systems C. L. Linster and E. Van Schaftingen
1526 FEBS Journal 273 (2006) 1516–1527 ª 2006 The Authors Journal compilation ª 2006 FEBS
20 Evans WH, Hood DO & Gurd JW (1973) Purification
and properties of a mouse liver plasma-membrane gly-
coprotein hydrolysing nucleotide pyrophosphate and
phosphodiester bonds. Biochem J 135, 819–826.
21 Evans WH (1974) Nucleotide pyrophosphatase, a sialo-
glycoprotein located on the hepatocyte surface. Nature
250, 391–394.
22 Bachorik PS & Dietrich LS (1972) The purification and
properties of detergent-solubilized rat liver nucleotide
pyrophosphatase. J Biol Chem 247, 5071–5078.
23 Bischoff E, Tran-Thi TA & Decker KFA (1975)
Nucleotide pyrophosphatase of rat liver. A comparative
study on the enzymes solubilized and purified from
plasma membrane and endoplasmic reticulum. Eur J

of UDP-glucuronyltransferase. Eur J Biochem 38 , 59–
63.
31 Burchell B, Brierley CH & Rance D (1995) Specificity
of human UDP-glucuronosyltransferases and xenobiotic
glucuronidation. Life Sci 57, 1819–1831.
32 Ethell BT, Anderson GD & Burchell B (2003) The effect
of valproic acid on drug and steroid glucuronidation by
expressed human UDP-glucuronosyltransferases. Bio-
chem Pharmacol 65, 1441–1449.
33 Iyanagi T, Watanabe T & Uchiyama Y (1989) The 3-
methylcholanthrene-inducible UDP-glucuronosyltrans-
ferase deficiency in the hyperbilirubinemic rat (Gunn
rat) is caused by a -1 frameshift mutation. J Biol Chem
264, 21302–21307.
34 Iyanagi T (1991) Molecular basis of multiple UDP-
glucuronosyltransferase isoenzyme deficiencies in the
hyperbilirubinemic rat (Gunn rat). J Biol Chem 266,
24048–24052.
35 Hochman Y & Zakim D (1984) Studies of the catalytic
mechanism of microsomal UDP-glucuronyltransferase.
a-Glucuronidase activity and its stimulation by phosp-
holipids. J Biol Chem 259, 5521–5525.
36 Yamashita A, Nagatsuka T, Watanabe M, Kondo H,
Sugiura T & Waku K (1997) Inhibition of UDP-glucur-
onosyltransferase activity by fatty acyl-CoA. Kinetic
studies and structure-activity relationship. Biochem
Pharmacol 53, 561–570.
37 Lowry OH, Rosebrough NJ, Farr AL & Randall RJ
(1951) Protein measurement with the Folin phenol
reagent. J Biol Chem 193, 265–275.