Tài liệu Báo cáo khoa học: Glucuronate, the precursor of vitamin C, is directly formed from UDP-glucuronate in liver pptx

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

The conversion of UDP-glucuronate to glucuronate, usually thought to
proceed by way of glucuronate 1-phosphate, is a site for short-term regula-
tion of vitamin C synthesis by metyrapone and other xenobiotics in isola-
ted rat hepatocytes [Linster CL & Van Schaftingen E (2003) J Biol Chem
278, 36328–36333]. Our purpose was to explore the mechanism of this
effect in cell-free systems. Metyrapone and other xenobiotics stimulated, by
approximately threefold, the formation of glucuronate from UDP-glucuro-
nate in liver extracts enriched with ATP-Mg, but did not affect the forma-
tion of glucuronate 1-phosphate from UDP-glucuronate or the conversion
of glucuronate 1-phosphate to glucuronate. This and other data indicated
that glucuronate 1-phosphate is not an intermediate in glucuronate forma-
tion from UDP-glucuronate, suggesting that this reaction is catalysed by a
‘UDP-glucuronidase’. UDP-glucuronidase was present mainly in the micro-
somal fraction, where its activity was stimulated by UDP-N-acetylglucosa-
mine, known to stimulate UDP-glucuronosyltransferases by enhancing the
transport of UDP-glucuronate across the endoplasmic reticulum mem-
brane. UDP-glucuronidase and UDP-glucuronosyltransferases displayed
similar sensitivities to various detergents, which stimulated at low concen-
trations and generally inhibited at higher concentrations. Substrates of
glucuronidation inhibited UDP-glucuronidase activity, suggesting that
the latter is contributed by UDP-glucuronosyltransferase(s). Inhibitors of
b-glucuronidase and esterases did not affect the formation of glucuronate,
arguing against the involvement of a glucuronidation–deglucuronidation
cycle. The sensitivity of UDP-glucuronidase to metyrapone and other stim-
ulatory xenobiotics was lost in washed microsomes, even in the presence of
ATP-Mg, but it could be restored by adding a heated liver high-speed
supernatant or CoASH. In conclusion, glucuronate formation in liver
is catalysed by a UDP-glucuronidase which is closely related to UDP-
glucuronosyltransferases. Metyrapone and other xenobiotics stimulate
UDP-glucuronidase by antagonizing the inhibition exerted, presumably

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

progress in the identification of the enzyme(s) implica-
ted in this conversion.
Results
Glucuronate and glucuronate 1-phosphate
formation in crude liver extracts
Our first attempts were aimed at identifying conditions
under which aminopyrine, metyrapone and chloretone
stimulated the formation of glucuronate from UDP-
glucuronate in crude liver extracts. These experiments
UDP-D-glucuronate
D-glucuronate-1-P
+
UMP
(-)
ATP
UDP-
D-GlcNAc
UDP-
D-glucose
Plasma
membrane
UDP-D-glucuronate
D-glucuronate + UDP
UDP-
D-GlcNAc
(+)
D-glucuronate
L-gulonate L-gulono-1,4-lactone
L-gulono-1,4-lactone L-ascorbate
3-dehydro-L-gulonate

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.

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 1517
were performed in the presence of sorbinil, an inhibitor
of aldose reductase and aldehyde reductase [9], to
block the conversion of glucuronate to l-gulonate,
and in the presence of UDP-N-acetylglucosamine
(UDPGlcNAc), which stimulates glucuronate forma-
tion (see below). As shown in Fig. 2, xenobiotics had
no effect on the formations of glucuronate and glucur-
onate 1-phosphate in extracts that were not supple-
mented with ATP-Mg. ATP-Mg inhibited the
formation of free glucuronate and, more powerfully,
that of glucuronate 1-phosphate, but the first effect
was counteracted by xenobiotics, whereas the second
was not, suggesting that glucuronate formation was
independent of glucuronate 1-phosphate formation.
In the presence of ATP-Mg, the rate of hydrolysis
of 0.5 mm glucuronate 1-phosphate amounted to
0.04 nmolÆmin
)1
Æmg
)1
protein irrespective of the pres-
ence or absence of xenobiotics (not shown), therefore
being much lower than the rate of glucuronate
formation from UDP-glucuronate in the presence of
xenobiotics (0.2 nmolÆmin
)1
Æmg
)1

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

Æmg
)1
protein, i.e.  10% of
the UDP-glucuronidase activity in the same fraction).
This activity was not modified in the presence of 0.1%
Triton X-100.
UDP-glucuronate is used in the lumen of the endo-
plasmic reticulum (ER) by UDP-glucuronosyltrans-
ferase [10] and its transport into this organelle appears
to be stimulated by UDPGlcNAc, explaining the sti-
mulation that this nucleotide exerts on glucuronidation
[11]. To test whether the enzymes catalysing the forma-
tion of glucuronate and glucuronate 1-phosphate were
present in the lumen of the ER (or had their catalytic
site oriented towards the lumen of this organelle), we
checked the effect of UDPGlcNAc on their activity.
As shown in Fig. 3, UDPGlcNAc exerted a marked
stimulatory effect on UDP-glucuronidase, similar to
that observed for UDP-glucuronosyltransferase, but
did not stimulate the formation of glucuronate 1-phos-
phate. This indicated that UDP-glucuronate must
cross the ER membrane to reach the catalytic site of
UDP-glucuronidase, but not of UDP-glucuronate
pyrophosphatase. As a matter of fact, UDPGlcNAc
Fig. 2. Effect of metyrapone, aminopyrine and chloretone on the
formation of free glucuronate and glucuronate 1-phosphate in crude
liver extracts incubated in the absence (A, B) or presence (C, D) of
ATP-Mg. Crude liver extracts were incubated with 1 m
M UDP-glu-
curonate, 1 m

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-

curonate formation and glucuronidation of 4-nitrophe-
nol. All incubations were performed in the presence of
UDPGlcNAc to stimulate the entry of UDP-glucuro-
nate into undisrupted microsomes. The four tested
detergents had similar effects on both activities: stimu-
lation was observed with low concentrations and,
except for polyoxyethylene ether W-1, inhibition was
observed at higher concentrations with the following
order of potency: deoxycholate > b-octylglucoside >
Triton X-100 (Fig. 4). By contrast, 0.5% deoxycholate
and 1.8% b-octylglucoside, which both completely
inhibited UDP-glucuronidase and UDP-glucuronosyl-
transferase activities, only slightly affected the activity
of glucose-6-phosphatase (10 and 20% inhibition,
respectively), another integral membrane protein of the
ER [16].
To determine whether UDP-glucuronosyltransferas-
es are directly implicated in the formation of free
Table 1. Subcellular distributions of UDP-glucuronidase, 4-Np-UGT and UDP-glucuronate pyrophosphatase. UDP-glucuronidase was assayed
at 37 °C in the presence of 0.5 m
M sorbinil without or with 1 mM metyrapone. ATP-Mg was omitted from the UDP-glucuronate pyrophos-
phatase assay, also performed at 37 °C. 4-Np-UGT was assayed with 0.2 m
M 4-nitrophenol and the assay was started by addition of the
enzyme preparation. Results are means ± SEM for three experiments or individual values obtained in two independent experiments.
UDP-glucuronidase
4-Np-UGT
UDP-glucuronate
pyrophosphataseNo metyrapone 1 m
M metyrapone
Specific activity (nmolÆmin

)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

measure glucuronate (squares) and glucuronate 1-phosphate (dia-
monds). Glucuronate 1-phosphate formation from UDP-glucuronate
was corrected for acid hydrolysis as in Fig. 2. UDPGAse, UDP-
glucuronidase.
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 1519
glucuronate, we tested the effect of glucuronidation
substrates on UDP-glucuronidase activity. These
experiments were performed in the presence of ATP-
Mg, to inhibit UDP-glucuronate breakdown by the
pyrophosphatase, saccharo-1,4-lactone, an inhibitor of
b-glucuronidase [17], to block hydrolysis of the b-glu-
curonides and 0.1% Triton X-100, to prevent any limi-
tation in UDP-glucuronate supply due to saturation of
a transport mechanism. As shown in Fig. 5, 4-methyl-
umbelliferone and valproate both dose-dependently
inhibited the formation of free glucuronate. Remark-
ably, the effect of 4-methylumbelliferone disappeared
after it had been completely glucuronidated (Fig. 6),
indicating that inhibition was truly due to the presence
of this substrate of glucuronidation. No such decrease
in the inhibition was observed with time in the case of
valproate, which was more slowly metabolized. Inhibi-
tion of glucuronate formation was also observed with
other substrates of glucuronidation including resorci-
nol, 4-nitrophenol and chloramphenicol (not shown).
A potential explanation for the involvement of
UDP-glucuronosyltransferases in the formation of free
glucuronate could be a glucuronidation–deglucuroni-
dation cycle involving an unknown glucuronidated

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

that a cofactor required for inhibition of UDP-glucu-
ronidase by ATP-Mg had been lost during the prepar-
ation of this subcellular fraction. Accordingly, addition
of a liver high-speed supernatant inhibited microsomal
UDP-glucuronidase in the presence of ATP-Mg
(Fig. 7A). This inhibition was much less important in
the presence of metyrapone. Similar results were
obtained with a high-speed supernatant that had been
heated for 5 min at 95 °C, indicating that the cofactor
was heat stable. This heat-stable cofactor was dependent
on ATP-Mg for its action and the inhibition that it exer-
ted together with ATP-Mg was antagonized by metyra-
pone, aminopyrine and chloretone (Fig. 7B,C). Further
characterization of the cofactor indicated that it was
retained on charcoal (Fig. 7A) and on the anion-exchan-
ger Q-Sepharose (not shown). No inhibitor was appar-
ently eluted from the column by applying a salt
gradient. However, incubation of the eluted fractions
for 90 min with 5 mm dithiothreitol at 25 °C allowed us
to recover  15% of the initial inhibitory activity in the
fraction eluted with 500 mm NaCl. As this inhibitory
fraction contained CoASH, we tested the effect of this
nucleotide on glucuronate formation. Like the heat-sta-
ble cofactor, CoASH inhibited free glucuronate forma-
tion in an ATP-dependent manner and its inhibitory
effect was antagonized by metyrapone, aminopyrine
and chloretone (Fig. 8). The effect of CoASH was half-
maximal at 30 lm.
Discussion
Lack of involvement of glucuronate 1-phosphate

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

rapidly stimulated by aminopyrine, metyrapone and
other xenobiotics, and that this formation takes place
at the expense of UDP-glucuronate [4]. We were able
to reproduce this effect in liver extracts enriched with
ATP-Mg (see below). However, although metyrapone
and other xenobiotics stimulated the formation of glu-
curonate in these preparations, they did not affect the
formation of glucuronate 1-phosphate. This indicates
that glucuronate 1-phosphate is not an intermediate in
the formation of glucuronate. If it were, its concentra-
tion would either increase or decrease, depending on
whether the stimulation by xenobiotics was exerted on
its formation or its hydrolysis.
Other observations further argue against glucuro-
nate 1-phosphate being an intermediate in glucuronate
formation. First, is the finding that the rate of glucuro-
nate 1-phosphate hydrolysis is several-fold slower than
the rate of glucuronate formation from UDP-glucuro-
nate under various conditions (e.g. in liver extracts
incubated in the presence of ATP-Mg and xenobiotics;
in microsomes). Second, is the finding that glucuronate
A
B
C
Fig. 7. Requirement of a heat-stable cofactor for the effect of
metyrapone (MP) and other xenobiotics on microsomal UDP-glucu-
ronidase. Microsomes (ms, 2.5 mg proteinÆmL
)1
)and⁄ or a high-
speed supernatant (HSS, 12.2 mg proteinÆmL

)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

1m
M metyrapone (MP), aminopyrine (AP) or chloretone (CL) on
glucuronate formation in the presence of CoASH was also tested.
Perchloric acid extracts were prepared 20 min after initiation of the
reaction by addition of UDP-glucuronate and UDPGlcNAc to meas-
ure glucuronate.
Glucuronate formation in liver cell-free systems C. L. Linster and E. Van Schaftingen
1522 FEBS Journal 273 (2006) 1516–1527 ª 2006 The Authors Journal compilation ª 2006 FEBS
1-phosphate formation in microsomes is profoundly
inhibited by ATP-Mg, whereas glucuronate formation,
under the same conditions, is unaffected by this nuc-
leotide (Fig. 3). Furthermore, low concentrations of
UDPGlcNAc stimulate the formation of glucuronate
although not that of glucuronate 1-phosphate in
microsomes.
These data indicate, therefore, that UDP-glucuro-
nate hydrolysis to glucuronate 1-phosphate is unre-
lated to free glucuronate formation. The enzyme that
forms glucuronate 1-phosphate from UDP-glucuronate
most likely corresponds to nucleotide pyrophosphatase
(Fig. 1). This enzyme, which is mainly present on the
outer face of the plasma membrane, hydrolyses a series
of nucleotide diphosphate sugars, as well as triphos-
phonucleotides [19–23]. The finding that ATP-Mg and
UDPGlcNAc (Fig. 3), as well as UDP-glucose (not
shown), inhibit the formation of glucuronate 1-phos-
phate supports this interpretation. It is therefore likely
that nucleotide pyrophosphatase does not serve physi-
ologically to hydrolyse UDP-glucuronate, because it is
not present in the same compartment as this potential

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

directly hydrolysed to glucuronate and UDP, i.e. that
glucuronate formation is catalysed by a UDP-glucuro-
nate glucuronyl hydrolase, which we designate UDP-
glucuronidase for the sake of simplicity.
Probable identity of UDP-glucuronidase and
UDP-glucuronosyltransferases
To date, very few enzymes have been described that
hydrolyse a nucleotide diphosphate sugar to a free sugar
and a nucleotide diphosphate. A well-characterized
example of this type of reaction is the one catalysed by
GDP-mannose hydrolase, an enzyme that was initially
characterized in Escherichia coli [29], but whose physio-
logical function is not known. Like other members of
the Nudix family, GDP-mannose hydrolase is a soluble
protein, and therefore very different from liver UDP-
glucuronidase, a membrane-bound enzyme.
UDP-glucuronidase shares several properties with
UDP-glucuronosyltransferases. Both enzymes are pre-
sent in liver microsomes and are stimulated by UD-
PGlcNAc. The stimulatory effect of UDPGlcNAc on
UDP-glucuronosyltransferases depends on the integrity
of the microsomal membrane [30] and has been attrib-
uted to the ability of this nucleotide to stimulate UDP-
glucuronate influx into microsomes [11]. This involves
conversion of UDP-glucuronate to UMP in the lumen
of the vesicles and exchange of the latter with cytosolic
UDPGlcNAc. Once inside microsomes, the latter can,
in turn, be exchanged with cytosolic UDP-glucuronate
thanks to a UDP-glucuronate–UDPGlcNAc antiport.
Stimulation of UDP-glucuronidase by UDPGlcNAc

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-

ronosyltransferase isoforms) may actually catalyse not
only the transfer of a glucuronosyl group to an accep-
tor, but also the hydrolysis of the glycosidic linkage in
UDP-glucuronate. From the data shown in Fig. 5 this
reaction would be substantial, amounting to  7% of
the rate of glucuronidation of 4-methylumbelliferone,
one of the best substrates for glucuronidation. The
involvement of UDP-glucuronosyltransferases in glu-
curonate formation is consistent with the finding that
3-methylcholanthrene (an inducer of UDP-glucurono-
syltransferases of the UGT1A family) stimulates vita-
min C formation in normal rats, although not in
Gunn rats [8], in which all UGT1A isoforms are defici-
ent [33,34]. However, Gunn rats produce vitamin C,
which, if our hypothesis is correct, would mean that
UGT2 family isozymes may also be involved in the
formation of glucuronate. Interestingly, vitamin C for-
mation is induced in Gunn rats by phenobarbital [8],
an inducer of UGT2s, which is indirect evidence for
the involvement of members of the UGT2 family.
To the best of our knowledge, very few studies on
purified UDP-glucuronosyltransferases have investi-
gated the capacity of these enzymes to hydrolyse
UDP-glucuronate to UDP and glucuronate. A UDP-
glucuronosyltransferase purified from pig liver (GT
2P
)
was shown to hydrolyse UDP-glucuronate to free glu-
curonate and UDP at a rate corresponding to
 0.001% of its activity as a transferase [35]. This

‘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

‘GT
2P
’ [35]). Under the ‘best’ conditions, the UDP-
glucuronidase activity amounted to  0.4% of the
UDP-glucuronosyltransferase activity, which is  10-
fold higher than the highest ratios observed by
Hochman & Zakim, but which is still far from the
 7% UDP-glucuronidase ⁄ UDP-glucuronosyltrans-
ferase activity described for intact liver microsomes
(this study). Further work is needed to identify the
UGT isozymes and potential cofactor(s) involved in
free glucuronate formation.
Conditions required to observe the effect
of xenobiotics in cell-free systems
The stimulation exerted by several xenobiotics on
vitamin C formation has recently been attributed to a
rapid effect of these agents to stimulate glucuronate
formation in intact liver cells [4]. We were able to
reproduce the stimulation of glucuronate formation in
liver extracts and microsomes. With the first type of
preparation we noticed that ATP-Mg behaved as an
inhibitor of the UDP-glucuronidase activity, and that
metyrapone, aminopyrine and chloretone could then
show a ‘stimulatory effect’ (a deinhibitory effect in
fact) of about the same order of magnitude as in
intact hepatocytes. This inhibitory effect of ATP-Mg
was no longer present in washed liver microsomes,
but could be restored in this last preparation by add-
ing a heated liver high-speed supernatant or low
(physiological) concentrations of CoASH. Both the

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

identification of conditions that allow one to observe
the stimulation of glucuronate formation by xenobiot-
ics in cell-free systems is an important step towards the
identification of the detailed mechanisms by which
these compounds act and of the enzyme implicated in
glucuronate formation.
Experimental procedures
Materials
Glucuronate 1-phosphate was prepared by incubating
80 mm UDP-glucuronate in the presence of 3.3% perchloric
acid (w ⁄ v) at 50 °C for 1 h in a total volume of 0.6 mL.
After neutralization with K
2
CO
3
and elimination of the
resulting salt precipitate by centrifugation, the preparation
was treated twice with 5% charcoal in the presence of
25 mm Hepes, pH 7.1 to eliminate nucleosides and nucleo-
tides, and centrifuged to remove charcoal. The resulting
supernatant was chromatographed on a Dowex 1 · 8 resin
(1 mL), from which glucuronate 1-phosphate was eluted
with a NaCl gradient. Four fractions of 0.5 mL, eluted with
250–350 mm NaCl and containing between 4 and 6 mm glu-
curonate 1-phosphate, were obtained in this way. These
fractions did not contain any free glucuronate.
E. coli b-glucuronidase, alkaline phosphatase and ATP
(disodium salt) were purchased from Roche Applied Sci-
ence (Mannheim, Germany). Dimethylsulfoxide, MgCl
2

,
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

to get a microsomal preparation containing  40 mg pro-
teinÆmL
)1
. For subcellular fractionation (Table 1), livers
from two overnight fasted male Wistar rats were homo-
genized as described but in a buffer containing 10 mm
Hepes, pH 7.1, 0.25 m sucrose, 2.5 lgÆmL
)1
antipain and
2.5 lgÆmL
)1
leupeptin. The homogenate was submitted to
differential centrifugation [24]. The extracts and subcellular
fractions were stored at )80 °C. Protein was measured
according to Lowry et al. [37], with bovine serum albumin
as a standard.
Assay of enzymatic activities
UDP-glucuronidase and UDP-glucuronate pyrophosphatase
were assayed at 30 or 37 °C through the conversion of
UDP-glucuronate to glucuronate and glucuronate 1-phos-
phate, respectively. Unless otherwise stated, the assay med-
ium contained 20 mm sodium phosphate, pH 7.1, 2 mm
MgCl
2
,10mm ATP-Mg, 1 mm UDPGlcNAc, 1 mm UDP-
glucuronate and  3 (microsomes) or 15 (crude extracts)
mg proteinÆmL
)1
. In most experiments, the enzyme prepar-
ation was preincubated for 10 min with all assay compo-

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-

curonate assay. Glucuronate was assayed enzymatically
with E. coli uronate isomerase and mannonate dehydroge-
nase [38]. This method was also used to assay glucuro-
nate 1-phosphate and b-glucuronides, by measuring
glucuronate before and after hydrolysis of these compounds
by alkaline phosphatase and E. coli b-glucuronidase,
respectively. The incubation conditions for hydrolysis by
these two enzymes were the same as those described previ-
ously [38] except that, because of the presence of saccharo-
1,4-lactone, an inhibitor of b-glucuronidase, in the samples
where b-glucuronides had to be determined, the amount of
b-glucuronidase and the incubation time with the latter
enzyme were doubled. Unless otherwise indicated, the
results shown in this study are means ± SEM for three
experiments using different enzymatic preparations.
Acknowledgements
This work was supported by the Concerted Research
Action Program of the Communaute
´
Franc¸ aise de Bel-
gique; the Interuniversity Attraction Poles Program,
Belgian Science Policy; and the Fonds de la Recherche
Scientifique Me
´
dicale. CLL is a fellow of the Fonds
National de la Recherche Scientifique (FNRS).
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