Purification and characterization of UDP-glucuronate: Baicalein 7-O- glucuronosyltransferase from Scutellaria baicalensis Georgi. cell suspension cultures

Article abstract of DOI:10.1016/S0031-9422(99)00593-2

UDP-glucuronate: baicalein 7-O-glucuronosyltransferase (UBGAT) catalyzes the transfer of glucuronic acid from UDP-glucuronic acid to the 7-OH of baicalein. UBGAT was purified from cultured cells of Scutellaria baicalensis Georgi (Lamiaceae). It was purified 95-fold using various chromatography and chromatofocusing procedures to apparent homogeneity. The M(r) was estimated to be 110 kDa by gel filtration chromatography with a 52 kDa subunit by SDS- PAGE. The isoelectric point was pH 4.8. UBGAT was specific to UDP-glucuronic acid as a sugar donor and flavones with substitution ortho- to the 7-OH group such as baicalein (6-OH), scutellarein (6-OH) and wogonin (8-OMe). (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0031-9422(99)00593-2

Phytochemistry 53 (2000) 533±538
www.elsevier.com/locate/phytochem
Puri®cation and characterization of UDP-glucuronate: baicalein 7-
O-glucuronosyltransferase from Scutellaria baicalensis Georgi. cell
suspension cultures
Shigeyuki Nagashima, Masao Hirotani*, Takafumi Yoshikawa
School of Pharmaceutical Sciences, Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8641, Japan
Received 26 May 1999; received in revised form 22 September 1999
Abstract
UDP-glucuronate: baicalein 7-O-glucuronosyltransferase (UBGAT) catalyzes the transfer of glucuronic acid from UDP-
glucuronic acid to the 7-OH of baicalein. UBGAT was puri®ed from cultured cells of Scutellaria baicalensis Georgi (Lamiaceae).
It was puri®ed 95-fold using various chromatography and chromatofocusing procedures to apparent homogeneity. The Mr was
estimated to be 110 kDa by gel ®ltration chromatography with a 52 kDa subunit by SDS-PAGE. The isoelectric point was pH
4.8. UBGAT was speci®c to UDP-glucuronic acid as a sugar donor and ¯avones with substitution ortho- to the 7-OH group
such as baicalein (6-OH), scutellarein (6-OH) and wogonin (8-OMe). # 2000 Elsevier Science Ltd. All rights reserved.
Keywords: Scutellaria baicalensis; Lamiaceae; Baicalin; Baicalein; Cell culture; Glucuronosyltransferase
1. Introduction
glycosyl group from a nucleotide sugar to a small
hydrophobic molecule (aglycone). Mammalian UDP-
glucuronosyltransferases (UDPGTs: EC 2.4.1.17) have
been studied in detail because of their importance in
drug and xenobiotic metabolism (Mackenzie et al.,
1997). Flavonoid-speci®c UDP-sugar transferases from
plants have been widely investigated, and they are con-
sidered to be late or terminal steps in ¯avonoid biosyn-
thesis. Most are UDP-glucosyltransferases, for which
some cDNAs have already been cloned and character-
ized (Heller & Forkmann, 1994). However, there are
few reports of UBGATs from plants; in the case of ¯a-
vonoids, there is only a report of partial puri®cation
from rye primary leaves (Schulz & Weissenbock,
1988), for which a cDNA has not yet been cloned.
It is known that S. baicalensis possesses a b-glucuro-
nidase (EC 3.2.1.31) called baicalinase, which hydro-
lyzes baicalin to baicalein and glucuronic acid (Miwa,
1932, 1935, 1936). A baicalinase had already been pur-
i®ed and characterized from suspension cultured cells
of S. baicalensis (Morimoto, Harioka & Shoyama,
1995) and from dried Scutellariae radix (Ikegami, Mat-
The dried roots of Scutellaria baicalensis Georgi
(Lamiaceae) are frequently used as a Chinese herbal
medicine to treat bacterial infections. The dried roots
contain over 30 kinds of ¯avonoids (Tang & Eisen-
brand, 1992). It has been reported that the major ¯a-
vonoid baicalin has anti-allergic (Koda, Watanabe,
Yanagihara, Nagai & Sakamoto, 1977), anti-HIV
e€ect (Konoshima et al., 1992) and anti-tumor activi-
ties (Li, Fu, Yan, Baylor, Ruscetti & Kung, 1993). Bai-
calin has glucuronic acid as a sugar moiety and lacks a
hydroxyl group at the 4' position of the B-ring. Typi-
cal ¯avonoid glycosides have glucose as a sugar moiety
and a 4' hydroxyl group derived from 4-coumaroyl
CoA as a precursor.
The UDP-glycosyltransferases (UGT) represent a
super family of enzymes that catalyze transfer of the
* Corresponding author. Fax: +81-3-3444-6192.
E-mail address: hirotanim@platinum.pharm.kitasato-u.ac.jp (M.
Hirotani).
0031-9422/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0031-9422(99)00593-2

534
S. Nagashima et al. / Phytochemistry 53 (2000) 533±538
Table 1
Puri®cation of UBGAT from S. baicalensis cell suspension cultures
1
Puri®cation step
Total activity (nkat)
Total protein (mg)
Speci®c activity (nkat mg
)
Recovery (%)
Puri®cation (-fold)
Crude
772.5
730.8
718.8
237.2
133.5
14.3
359.3
248.6
24.4
4.0
2.2
2.9
100
94.6
93.0
30.7
17.3
1.9
1
1.4
Ammonium sulfate
Sephadex A-50
Phenyl Sepharose CL-4B
Sephacryl S-200
Mono P
29.4
59.3
171.6
203.7
13.7
27.6
79.8
94.7
0.8
0.07
1
sunae, Hisamitsu, Kurihara, Yamamoto & Murakoshi,
1995). However, puri®cation of the corresponding
UDP-glucuronate: baicalein 7-O-glucuronosyltransfer-
ase (UBGAT) has not yet been reported. Previously,
we have reported that three strains of calli from S. bai-
calensis had variations in baicalinase and UBGAT ac-
tivities (Hirotani, Nagashima & Yoshikawa, 1998). We
here report on the puri®cation and characterization of
UBGAT from S. baicalensis cell suspension culture.
bu€er B, speci®c activities reached 200 nkat mg pro-
tein. Fig. 1 shows the results of the SDS-PAGE of
protein fractions from the chromatographic steps of
UBGAT puri®cation indicating that UBGAT was pur-
i®ed to apparent homogeneity.
The apparent Mr of native UBGAT was estimated
to be 110 kDa based on the gel ®ltration chromatog-
raphy on a calibrated Sephacryl S-200 column and the
Mr in the denatured condition was estimated to be 52
kDa from the results of SDS-PAGE, signifying that
UBGAT is composed of a homo-dimer in cells. It is
known that the dimeric form of a glycosyltransferase
is very rare even among mammalian UDPGTs (Kleene
& Berger, 1993). There is, however, a report of a
dimeric UGT in the plant kingdom; Kamsteeg, van
Brederode & van Nigtevecht (1978) reported that
UDP-glucose: cyanidin 3-O-glucosyltransferase from
petals of the red campion (Silene dioica ) eluted at
about 125 and 60 kDa from gel ®ltration chromatog-
raphy, showing that the enzyme could exist as a dimer
and an active monomer. In our experiments, UBGAT
was eluted as a single peak from gel ®ltration chroma-
tography. This result suggests that UBGAT does not
exist or function as a monomer whether UBGAT can
function as a monomer is a topic of interest requiring
further analysis, e.g., expression of UBGAT cDNA in
Escherichia coli.
2. Results and discussion
UBGAT was puri®ed from cultured cells of S. baica-
lensis. The puri®cation protocol is shown in Table 1.
UBGAT was puri®ed 95-fold with 1.9% recovery. In
the polybu€er 74 used for chromatofocusing on Mono
P, UBGAT showed lower activity than Sephacryl S-
200 fraction. However, after the bu€er exchange to
UBGAT was eluted at pH 4.8 from chromatofocus-
ing on Mono P, indicating a pI of 4.8. The UBGAT
activity was recovered in the supernatant after ultra-
centrifugation at 105,000 g indicating that UBGAT
exists in the soluble fraction of the cells.
UBGAT from the Sephacryl S-200 fraction was
stable at 48C for more than two weeks without loss of
activity. UBGAT activity remained at 50 and 25%
after incubation at room temperature for 3 and 24 h,
respectively. The pH optimum was 7.5 in 50 mM Tris±
HCl bu€er, and the optimum temperature was in the
range of 30±408C. Proteins of the Sephacryl S-200
fraction were used for further analysis, because of the
unstability of the UBGAT in the bu€er used for chro-
matofocusing.
Fig. 1. SDS-PAGE of protein from the UBGAT puri®cation steps,
silver stained. Lane 1: molecular weight marker proteins; Lane 2:
crude protein (6.7 mg); Lane 3: 30±50% (NH4)2SO4 fraction (20.5
mg); Lane 4: DEAE-Sehadex A-50 fraction (1.1 mg); Lane 5: Phenyl
Sepharose CL-4B fraction (0.7 mg); Lane 6: Sephacryl S-200 fraction
(0.2 mg); Lane 7: Mono P fraction (0.5 mg). An arrow shows the
UBGAT protein.
Table 2 shows the substrate speci®city and kinetic
data for UBGAT. UBGAT was highly speci®c for
UDP-glucuronic acid. Vmax and Km values were 2572

S. Nagashima et al. / Phytochemistry 53 (2000) 533±538
535
Table 2
Substrate speci®city of UBGAT from S. baicalensis cell suspension culturesa
1
)
Vmax (nkat mg
Km (mM)
52.422.1
Vmax=Km (ratio)
Baicalein
25724.7
290262
4.90
Scutellarein
109.0213.5
106.5224.7
2.67
1.71
Wogonin
18229.4
a Data are the means2SD of three replicate assays.
1
4.7 nkat mg and 72.9 2 9.4 mM, respectively, using
baicalein as the sugar acceptor. UDP-glucose and
UDP-galacturonic acid did not serve as substrates.
Regarding aglycones, UBGAT was speci®c for baica-
lein, scutellarein and wogonin, which are substituted at
the position ortho- to the 7-OH (6-OH, baicalein and
scutellarein; 8-OMe, wogonin). Vmax values of
UBGAT for baicalein and scutellarein were quite simi-
lar, and the Km value for baicalein was almost half of
that of scutellarein. The Vmax/Km ratios demonstrate
that baicalein is the best sugar acceptor among the ¯a-
vonoids tested. Other ¯avonoids lacking a substitution
at the position ortho- to the 7-OH (chrysin, apigenin,
luteolin, quercetin, formononetin and daidzein) did not
serve as substrates.
agent, and 4,4'-diisothiocyanostilbene-2,2'-disulfonic
acid (DIDS), a lysine modi®er (Go€ner et al., 1994),
inhibited UBGAT activity completely. However,
UBGAT activity was not a€ected by the other SH
group oxidation reagent, NEM. This phenomenon was
also reported by Latchinian-Sadek & Ibrahim (1991),
and may be due to the di€erent reactivity of NEM
and PCMBA. DTE (dithioerythritol) had no e€ect on
UBGAT activity, suggesting that a disul®de bond is
not necessary for the protein function.
There was no divalent cation requirement noted for
UBGAT activity. CaCl2 and MgCl2 had no e€ect on
enzyme activity, whereas CuSO4, ZnC12 and FeCl2
completely inhibited UBGAT (Table 4). The inhibition
of ¯avonoid glycosyltransferases by addition of Cu2+
ions is a well-known phenomenon (Latchinian-Sadek
& Ibrahim, 1991; Ishikura & Mato, 1993; Vogt, Zim-
mermann, Grimm, Meyer & Strack, 1997; Ford, Boss
& Hùj, 1998). It is thought that this phenomenon is
due to Cu2+-mediated substrate degradation rather
than by inhibition of the enzyme (Ford et al., 1998).
Further analysis is needed to clarify this phenomenon.
Various chemical treatments suggested that the free
SH group and lysine residues may play an important
role in UBGAT activity (Table 3). p-Chloromercuri-
benzoic acid (PCMBA), a free SH group oxidation re-
Table 3
The e€ects of various chemical treatments on UBGAT activity
Reagentsa
Speci®c activity Ratio Type of reagent
1 b
(nkat mg
)
(%)
Table 4
E€ects of divalent cations and EDTA ON UBGAT activity
None
PCMBA (100 mM) n.d.c
99.220.2
100
0
Free-SH group oxidizer
Free-SH group oxidizer
Lysine modi®er
NEM (100 mM)
DIDS (100 mM)
PG (100 mM)
PMSF (100 mM)
DEPC (100 mM)
DTE (10 mM)
91.925.9
n.d.
82.6
0
Reagent
Concentration
(mM)
Speci®c activity
1 a
(nkat mg )
Ratio
(%)
99.220.5
90.624.1
68.522.6
113.723.0
100
91.3
69.1
Arginine modi®er
Serine modi®er
Histidine modi®er
None
-
140.1211.9
n.d.b
n.d.
100
0
CuSO4
ZnCl2
FeCl2
CaCl2
MgCl2
EDTA
1
1
1
1
1
5
114.6 Disul®de bond reducer
0
n.d.
0
a PCMBA: p-chloromercuribenzoic acid; NEM: N-ethylmaleimide;
DIDS: 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid; PG: phenyl-
glyoxal; PMSF: phenylmethylsulfonyl¯uoride; DEPC: diethylpyro-
carbonate; DTE: dithioerythritol.
b Data are the means2SD of three replicate assays.
c n.d.: Enzyme activity was not detected.
129.226.2
109.927.7
116.921.0
92.2
78.4
83.4
a Data are the means2SD of three replicate assays.
b n.d.: Enzyme activity was not detected.

536
S. Nagashima et al. / Phytochemistry 53 (2000) 533±538
3. Experimental
3.1. Plant material
by the addition of 75 ml of MeOH. Identi®cation and
quantitation of the enzyme reaction products were car-
ried out by reversed phase-HPLC as described above.
The callus tissue was derived from the petiole of a
sterile young plant of S. baicalensis Georgi in April
1991. It was subcultured at 3-week intervals on MS
medium (Murashige & Skoog, 1962) containing 3 mg
3.6. Puri®cation of UBGAT
All the puri®cation procedures were carried out at
48C. Protein concentration was determined by the
Bradford method (Bradford, 1976) with BSA as a stan-
dard.
1
1
l
naphthaleneacetic acid, 0.1 mg l kinetin and 3%
sucrose. For the suspension culture, approximately 4 g
of 2-week-old callus tissues were transferred to 125 ml
of liquid medium in a 500 ml ¯ask and cultured at
258C in the dark on a reciprocal shaker at 80 spm.
Ten-day-old suspension cultured cells of S. baicalen-
sis were ®ltered and washed with distilled water. Two
ml of bu€er A per g fr. wt. was added. Homogeniz-
ation was performed using a glass homogenizer. The
homogenate was ®ltered through four-layers of cheese
cloth and centrifuged at 12,000 g for 10 min to remove
the cell debris. Proteins were precipitated by 30±50%
saturated ammonium sulfate, centrifuged at 12,000 g
for 10 min, resuspended in a minimum volume of buf-
fer B, centrifuged at 12,000 g for 10 min and desalted
on a Sephadex G-25 column (30 cm length, 4.7 cm
i.d.) with bu€er B.
Desalted protein solution was applied to a DEAE
Sephadex A-50 (8.0 cm length, 3.2 cm i.d.) anion-
exchange column, pre-equilibrated in bu€er B. Protein
fractions were eluted with a linear salt gradient of
NaCl in bu€er B (from 0 to 0.4 M) within 1000 ml.
Ten ml samples of the fractions were collected and
assayed for enzyme activity and protein concentration.
The pooled fractions containing enzyme activity
from the anion-exchange chromatography were preci-
pitated by 0±50% saturated ammonium sulfate, centri-
fuged at 12,000 g for 15 min, and resuspended in 1 ml
of bu€er B containing 1 M of ammonium sulfate.
Samples were applied to a Phenyl Sepharose CL-4B
column (4.5 cm length, 1.4 cm i.d.) pre-equilibrated
with 1 M ammonium sulfate in bu€er B. Protein frac-
tions were eluted with a linear salt gradient of am-
monium sulfate (from 1 to 0 M) within 200 ml. Two
ml fractions were collected and assayed as above.
The pooled fractions containing enzyme activity
from hydrophobic chromatography were concentrated
by ultra®ltration (Amicon, MA, USA) to 1 ml. Con-
centrated samples were applied to a Sephacryl S-200
column pre-equilibrated with bu€er B. Protein frac-
tions were eluted with bu€er B and 1.5 ml samples of
the fractions were collected.
3.2. Column materials and substrates
Sephadex G-25, DEAE Sephadex A-50, Phenyl
Sepharose CL-4B, Sephacryl S-200HR and Mono P
HR 5/20 were obtained from Pharmacia (Uppsala,
Sweden). Baicalin and baicalein were from Kanto
(Tokyo, Japan). UDP-glucuronic acid, UDP-glucose
and UDP-galacturonic acid were obtained from Naca-
lai Tesque (Kyoto, Japan). Wogonin and wogonin 7-
O-glucuronoside were from our laboratory collection.
Scutellarin and scutellarein were kindly provided by
Professor Tomimori (Hokuriku University, Ishikawa,
Japan). All the amino acid modifying reagents were
obtained from Sigma (MO, USA).
3.3. Bu€ers
Bu€er A: 100 mM phosphate bu€er, 10 mM 2-ME,
pH 7.0. Bu€er B:10 mM phosphate bu€er, 1 mM 2-
ME, pH 7.0.
3.4. Reversed phase-HPLC
The HPLC system consisted of a model 510 pump
(Waters, MA, USA) and an SPD-2A spectrophoto-
metric detector equipped with a Mightysil RP-18 col-
umn (150 mm length, 4.6 mm i.d.; Kanto). The elution
was performed with CH3CN±60 mM phosphoric acid
(29:71) at a ¯ow rate of 2.2 ml min 1; the eluent was
monitored by absorbance at 274 nm. The peak inten-
sity was determined with a C-R3A Chromatopac (Shi-
madzu, Kyoto, Japan). The amounts of enzymatic
reaction products were estimated from standard curves
obtained with authentic samples.
The pooled fractions containing enzyme activity
from gel ®ltration chromatography were concentrated
by ultra®ltration to 3.0 ml, followed by the bu€er
exchange on a PD-10 column (Pharmacia) pre-equili-
brated with start bu€er. Proteins were applied to a
Mono P HR 5/20 column (20 cm length, 5 mm i.d.)
pre-equilibrated with 25 mM 2-methylpiperazine, pH
5.7 (the pH was adjusted with saturated iminodiacetic
acid (IDA)). The pH gradient was created by elution
3.5. Assay of UBGAT activity
The standard assays consisted of 50 mM Tris±HCl
bu€er (pH 7.5), 0.16 mM substrate, 0.5 mM UDP-
sugar and 5 ml of enzyme solution in a ®nal volume of
125 ml. The sample mixtures were incubated for 10
min at 378C, and the enzyme reaction was terminated

S. Nagashima et al. / Phytochemistry 53 (2000) 533±538
537
Bronze-1 locus that may primarily serve to glucosylate anthocya-
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1
rated IDA) at a ¯ow rate of 1.5 ml min and 1.5 ml
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3.7. Determination of the molecular mass
To determine the Mr of the puri®ed proteins, SDS-
PAGE and gel ®ltration chromatography were per-
formed. SDS-PAGE was done according to Laemmli
(1970) with standard protein markers (New England
Biolabs, MA, USA). Proteins were stained with Silver
Stain Kanto II (Kanto, Tokyo, Japan). Gel ®ltration
chromatography was done as above, and the Mr was
determined with the standard protein markers (Phar-
macia), chymotrypsinogen A (25 kDa), ovalbumin (45
kDa), BSA (67 kDa), aldolase (158 kDa) and ferritin
(440 kDa).
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3.8. Kinetic properties
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3.9. E€ects of various chemicals on UBGAT activity
Various chemicals were added to the standard assay
mixture. The ®nal concentrations in the assay mixture
of PCMBA, N-ethylmaleimide, DIDS, phenylglyoxal,
phenylmethylsulfonyl¯uoride and diethylpyrocarbonate
were 100 mM and that of DTE was 10 mM.
Copper sulfate, zinc chloride, iron (II) chloride, cal-
cium chloride and magnesium chloride were added to
the standard assay mixture at a concentration of 1
mM.
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Latchinian-Sadek, L., & Ibrahim, R. K. (1991). Partial puri®cation
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bulbs. Phytochemistry, 30, 1767±1771.
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Kung, H. F. (1993). Inhibition of HIV infection by baicalin Ð a
¯avonoid compound puri®ed from Chinese herbal medicine. Cell.
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Iyanagi, T., Lancet, D., Louisot, P., Magdalou, J., Chowdhury,
J. R., Ritter, J. K., Schachter, H., Tephly, T. R., Tipton, K. F.,
& Nebert, D. W. (1997). The UDP glycosyltransferase gene
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Acknowledgements
We thank Professor Tomimori (Hokuriku Univer-
sity) for providing us with the ¯avonoid substrates.
This research was ®nancially supported by a Sasakawa
Scienti®c Research Grant from the Japan Science So-
ciety.
Miwa, T. (1932). Baicalinase, a ¯avone glucuronide-splitting enzyme:
Part I. Acta Phytochim. (Japan), 6, 155±171.
Miwa, T. (1935). Baicalinase, a ¯avone glucuronide-splitting enzyme:
Part II. Acta Phytochim. (Japan), 8, 231±243.
Miwa, T. (1936). Baicalinase, a ¯avone glucuronide-splitting enzyme:
Part III. Acta Phytochim. (Japan), 9, 89±102.
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characterization of ¯avone-speci®c b-glucuronidase from callus
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