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
Trang 1from 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: vanschaftingen@bchm.ucl.ac.be
(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 reacforma-tion 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 concenconcen-trations 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 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.
Trang 2the 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 phosphapyrophospha-tase 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.
Trang 3were 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)1Æmg)1 protein irrespective of the
pres-ence or abspres-ence 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)1Æmg)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)1Æmg)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 m M sorbinil, without or with
10 m M ATP-Mg and ⁄ or 1 m M 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.
Trang 4and 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 m M 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 pyrophosphatase
No metyrapone 1 m M metyrapone Specific activity (nmolÆmin)1Æmg)1protein)
Total activity (nmolÆmin)1Æg)1liver)
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
1 m 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.
Trang 5glucuronate, 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 formaInhibi-tion 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
1 mm bis-p-nitrophenylphosphate, an esterase inhibitor [18], for 30 min at 37C 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 C as des-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 m M 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.
Trang 6Role 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 95C, 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 25C 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 m M 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 m M ; 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.
Trang 7rapidly 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 glucuroglucuro-nate 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
1 m 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
1 m 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
1 m 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.
Trang 81-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 glucuroUDP-glucuro-nate 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-nitrophenyl- 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 glucuro-nate glucuronyl hydrolase, which we desigglucuro-nate 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
Trang 9substrates (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
glucuronate to UDP and glucuronate A
UDP-glucuronosyltransferase purified from pig liver (GT2P)
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
‘GT2P’ [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
Trang 10purification 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-gluglu-curonate 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 K2CO3 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, MgCl2,
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
4C 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)1antipain and 5 lgÆmL)1leupeptin 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 37C through the conversion of UDP-glucuronate to glucuronate and glucuronate 1-phos-phate, respectively Unless otherwise stated, the assay med-ium contained 20 mm sodmed-ium phosphate, pH 7.1, 2 mm MgCl2, 10 mm 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 30C, through the formation of b-glucuronides in an incubation mixture containing 20 mm sodium phosphate, pH 7.1,
2 mm MgCl2, 10 mm ATP-Mg, 1 mm saccharo-1,4-lactone,