Affinity purification of the key enzyme of nyctinasty controlling the rhythm of leaf movement using gluconamidine ligand

Article abstract of DOI:10.1016/j.tetlet.2007.01.085

Synthesis of affinity gel aimed for leaf-opening factor β-glucosidase (LOFG) and affinity purification of LOFG are presented. Gluconamidine-based β-glucosidase inhibitor was utilized for the ligand of the affinity gel. β-Glucosidase exhibiting activity sh

Full text of DOI:10.1016/j.tetlet.2007.01.085

Tetrahedron Letters 48 (2007) 2409–2413
Affinity purification of the key enzyme of nyctinasty controlling
the rhythm of leaf movement using gluconamidine ligand
Eisuke Kato, Takehiko Sasaki, Tadahiro Kumagai and Minoru Ueda*
Department of Chemistry, Tohoku University, Aoba-ku, Sendai 980-8578, Japan
Received 4 December 2006; revised 15 January 2007; accepted 18 January 2007
Available online 23 January 2007
Abstract—Synthesis of affinity gel aimed for leaf-opening factor b-glucosidase (LOFG) and affinity purification of LOFG are pre-
sented. Gluconamidine-based b-glucosidase inhibitor was utilized for the ligand of the affinity gel. b-Glucosidase exhibiting activity
shift throughout the day was selectively purified from Lespedeza cuneata Don by the affinity gel. The resulting LOFG exhibited a
high substrate specificity toward the leaf-opening factor.
ꢀ 2007 Published by Elsevier Ltd.
Leguminous plants are well known to close their leaves
at night and open them in the morning. This rhythmic
leaf movement is called nyctinasty whose rhythm is
known to be controlled by a biological clock. In the past
decade, a pair of leaf-movement factors, leaf-opening
factor (LOF), and leaf-closing factor (LCF), was identi-
fied as chemical factors controlling leaf movement.1,2 In
the case of Lespedeza cuneata, potassium lespedezate (1)
serves as LOF and potassium D-idarate (2) as LCF.3,4
The endogenous level of 1 in the plant body is controlled
by the metabolic enzyme, leaf-opening factor b-glucosi-
dase (LOFG), whereas that of 2 remains nearly constant
Figure 1. Leaf-movement factors (1 and 2) and circadian rhythmic
control of the hydrolysis on 1.
during the day (Fig. 1).5 This diurnal change in the level
of LOF in the plant body strongly affected the rhythm of
nyctinasty. Thus, the rhythm of nyctinastic leaf move-
attracted the attention of biochemists. Affinity chroma-
ment is generated by the change in the LOFG activity
tography8,9 has been widely used for this purpose. Some
during the day. Although this enzyme is considered as
glycosides and their analogs, such as thioglycoside, were
the key enzyme of nyctinasty, little is known about
used as affinity ligands in the purification of glycosi-
LOFG: Neither the circadian rhythmic activation mech-
dase.10,11 Glycosidase inhibitors, such as glycosyl-
anism nor the nature of LOFG was disclosed. Purifica-
amidine,12 deoxynojirimycin and its analogs13,14 are
tion of LOFG should provide detailed information on
also used as more effective affinity ligands. But in the
affinity purification of substrate-specific glycosidase,
these issues.
they are sometimes insufficient ligands due to the lack
of aglycon moieties. Recently we reported a concise
b-Glucosidase, which is a family of glycosidase, is
involved in a number of important biological processes
synthesis of N-substituted gluconamidine, a strong and
including storage and release of bioactive substances.6,7
selective b-glucosidase inhibitor.15,16 By this method,
Therefore, purification methods for glycosidases have
we synthesized gluconamidine (3) with the aglycon of
LOF, which showed a high and specific inhibitory
activity against b-glucosidase. Affinity chromatography
using 3 as a ligand would allow selective purification
of LOFG. In this Letter we report affinity purification
and some aspect of the nature of LOFG.
Keywords: Affinity chromatography; b-Glucosidase; Gluconamidine;
Nyctinast.
*
Corresponding author. Tel./fax: +81 22 795 6553; e-mail: ueda@
mail.tains.tohoku.ac.jp
0040-4039/$ - see front matter ꢀ 2007 Published by Elsevier Ltd.
doi:10.1016/j.tetlet.2007.01.085

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E. Kato et al. / Tetrahedron Letters 48 (2007) 2409–2413
Scheme 1. Synthesis of affinity gel with gluconamidine ligand based on 1.
Synthesis of affinity gel is shown in Scheme 1. From our
preliminary study, LOFG was predicted to have a high
substrate specificity and strictly recognizes the aglycon
moiety of 1 (data not shown). Hence, we synthesized 4
which has a linker moiety on the phenolic hydroxy
group connecting 3 with an affinity gel. The direct intro-
duction of the linker moiety to 3 resulted in failure due
to instability of the amidine moiety under coupling con-
dition. Thus, the linker must be introduced before the
formation of the amidine moiety. p-Hydroxycinnamic
acid (5) was fully protected and diol was introduced to
the olefin moiety by osmium tetroxide. After deprotec-
tion, the resulting phenol compound (6) was coupled
with linker (7) containing a protected thiol group to give
8. Then, the a and b-hydroxyl groups were substituted
by azide and fluoride, respectively. Following reduction
of the azide gave 9, which was coupled with thionolac-
tam (10)17,18 by NBS to give 11. Successive deprotection
and photoisomerization by UV light (312 nm) gave a 1:1
mixture of 4 and 12. Desired 419 was obtained by puri-
fication using HPLC and then coupled with gel 13 con-
taining a maleimide moiety, which was prepared from
the commercially available Toyopearl AF-Amino-
650M (Tosoh Co.) by the reported procedure,12 to give
affinity gel 14. Performance of 14 was tested by commer-
cially available b-glucosidase from Aspergillus niger
(Fluka Co.). b-Glucosidase (0.1 unit) dissolved in
50 mM acetate buffer (pH 5.0) was applied on a column
(B 5 · 100 mm) with 2 mL of affinity gel 14. After wash-
ing the column with 50 mM acetate buffer (pH 5.0), the
trapped b-glucosidase was eluted with the same buffer
containing 2.0 M D-glucose (10 mL). Eleven percent of
initial b-glucosidase activity (1.1 · 10ꢀ2 unit) was eluted
Scheme 2. Derivatization of a-keto acids to semicarbazone for
quantitative analysis of b-glucosidase activity.
by D-glucose, and the rest was recovered from the pass-
through fraction. Then the capacity of gel 14 was esti-
mated at 5.5 · 10ꢀ3 unit/mL gel. From this result, it
was revealed that gel 14 can be used in the affinity puri-
fication of b-glucosidase (Scheme 2).
Affinity purification of LOFG from L. cuneata by 14
was then performed. Our previous study showed that
LOFG activity in the plant body was monitored around
evening.5 Thus, we examined when the LOFG is acti-
vated. A series of acetone powder was prepared from
the leaves of L. cuneata collected every two hours from
10:00 to 20:00. Each acetone powder was extracted by
0.1 M Bis–Tris buffer (pH 7.0), and the b-glucosidase
activity of each extract was measured using 1 as a sub-
strate. However, the quantitative analysis of b-glucosi-
dase activity using 1 was very difficult because a-keto
acid 15, a hydrolytic product of 1, was unstable and eas-
ily decomposed in aqueous solution. We thus developed
a novel method for the quantitative analysis of unstable
a-keto acids. Coupling of an a-keto acid with semicar-
bazide hydrochloride (16) gave stable semicarbazone
(17).20 Thus, instead of 15, we analyzed 17 which is
formed by the in situ coupling of 15 and 16 in aqueous
solution.21 By using this novel method, a remarkable
increase in LOFG activity using 1 as substrate was
observed between 14:00 and 16:00 (Fig. 2). We then used
acetone powder prepared from the plants that were col-
lected between 14:00 and 16:00 for further purification.

E. Kato et al. / Tetrahedron Letters 48 (2007) 2409–2413
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strong LOFG activity of 1.6 unit/mg protein: 80-fold
enhancement of the activity was achieved compared
with the first extract (Table 2). Although 1 used for elu-
tion of LOFG was contained, the fraction was further
purified by Superdex 200 10/300 GL (GE Healthcare
Co.) to give LOFG which exhibited a 240-fold activity
(4.8 unit/mg protein) compared with the first extract.
The coincident elution of the protein (monitored at
280 nm) and glucosidase activity toward 1 was con-
firmed in this last step of purification (Fig. 3). The elu-
tion volumes of peaks in gel filtration corresponding
to LOFG activity, b-glucosidase activity measured by
using p-nitrophenylglucoside, and UV were all coin-
cided. SDS-PAGE analysis of the resulting LOFG gave
a smear band between 100 and 150 kDa (Fig. 4),
whereas this LOFG gave a sharp single peak in the
gel-filtration
HPLC
analysis
using
TSK-Gel
Figure 2. Diurnal change in the b-glucosidase activity in the leaves of
Lespedeza cuneata L.
SuperSW3000 (Tosoh Co.) (Fig. 4). The molecular
weight of LOFG estimated from the elution volume in
HPLC analysis corresponded to that from SDS-PAGE.
This led us to presume that LOFG is subjected to some
kinds of post translational modification because many
of glycoproteins are known to exhibit a smear band on
SDS-PAGE.22–24 We thus examined the presence of
the sugar chain. The modified method of O’Shannessy25
was employed for detection of the sugar chain. The
LOFG was subjected to SDS-PAGE and western blot-
ting was performed on a PVDF membrane. The mem-
brane was treated with 10 mM NaIO4 and then by
0.1 mM biotinhydrazide. Chemiluminescence detection
(ECL AdvanceTM Western Blotting Detection Kit, GE
Healthcare Co.) with streptavidin-HRP conjugate
clearly demonstrated the presence of the sugar chain
on LOFG (Fig. 5). Dissociation of the sugar chain from
LOFG and MS analysis of the sequence of resulting
protein are now under way.
Table 1. Relative activity of LOFG against various substrates: activity
was evaluated by analyzing corresponding aglycon by HPLC or
colorimetry with p-nitrophenol
Entry Substrate
Relative activity
1
2
3
4
5
6
7
8
9
Potassium lespedezate (1)
Potassium isolespedezate (18)
Dihydro lespedezate (19)
Potassium galactolespedezate (20)
Potassium galactoisolespedezate (21)
p-Nitrophenyl-b-D-glucopyranoside
p-Nitrophenyl-a-D-glucopyranoside
p-Nitrophenyl-b-D-galactopyranoside
p-Nitrophenyl-a-D-galactopyranoside
X-Glucose (22)
100
0
26
0
0
179
0
0
0
0a
49
10
11
Benzyl-b-D-glucopyranoside (23)
a Blue precipitate not detected.
We also examined substrate specificity of LOFG. Activ-
ity of LOFG against some glycosides is seen Table 1.
LOFG strictly differentiates the olefin moiety as shown
in the comparison among potassium lespedezate (1),
potassium isolespedezate (18) and 19 (entries 1–3).
Potassium galactolespedezate (20) and potassium galac-
toisolespedezate (21), which have galactose instead of
glucose, cannot be hydrolyzed (entries 4 and 5). Selectiv-
ity on the glycon moiety and stereochemistry of the gly-
cosidic linkage were also investigated using a variety of
p-nitrophenyl glycosides. LOFG specifically hydrolyzed
b-glucoside (entries 6–9). Additionally, X-glucose (22)
was not hydrolyzed at all (entry 10). However, a simple
glycoside such as benzyl-b-D-glucopyranoside (23) was
moderately hydrolyzed by LOFG (entry 11). These
results showed that LOFG is a b-glucosidase, which
The acetone powder was extracted with 0.1 M Bis–Tris
buffer (pH 7.0) containing 1.0 M sodium chloride,
2 mM DTT, 5 mM EDTA, and 8% w/v Polyclar-VT
(Wako Chemical Co.). After centrifugation, acetone
was added to the supernatant to the extent of 60%
v/v. The resulting precipitate was dissolved in 25 mM
Bis–Tris buffer (pH 7.0) and then partially purified by
HiPrep 16/10 Q XL (GE Healthcare Co.) anion
exchange chromatography. All fractions with LOFG
activity were then applied to an affinity column with
gel 14. After washing the gel with 25 mM acetate buffer
(pH 5.0), LOFG was eluted by the same buffer contain-
ing 2.0 M D-glucose. However, no LOFG activity was
observed in the fractions eluted by D-glucose. Then we
used 10 mM aqueous solution of potassium lespedezate
(1) as an eluent. The fraction eluted by 1 showed a
Table 2. Summary of purification of LOFG
Step
Total protein (mg)
Specific activity (unit/mg protein)
Purification (-fold)
Recovery (%)
Extract
16224.0
4824.0
197.0
1.6
0.02
0.05
0.15
1.6
1.0
2.1
6.5
80.0
240.0
100
72.7
7.9
0.4
0.1
Acetone precipitation
Anion exchange
Affinity
Gel filtration
0.1
4.8

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E. Kato et al. / Tetrahedron Letters 48 (2007) 2409–2413
Figure 3. Final purification of LOFG: Column:Superdex 200 10/300 GL, Eluent: 25 mM Bis–Tris (pH 7.0), 100 mM sodium chloride, flow rate:
0.5 mL/min, detection: 280 nm. LOFG activity of each fraction was shown as black dot.
Figure 4. Purification of LOFG [right: SDS-PAGE analysis of LOFG
(lane 1, Marker proteins; lane 2, LOFG); left: HPLC analysis of
LOFG (column: TSK-Gel SuperSW3000; eluent: 50 mM phosphate
buffer (pH 7.0), 150 mM sodium chloride; flow rate: 0.2 mL/min;
detection: 215 nm)].
In conclusion, we have successfully purified LOFG, a
key enzyme of nyctinasty, using affinity chromatogra-
phy. Gluconamidine ligand 4, which is designed on the
structure of 1, showed a strong inhibitory activity
toward b-glucosidase, and was revealed to be quite effec-
tive for selective purification of LOFG. It was also
shown that LOFG strongly recognizes an aglycon part
of the substrate and exhibited a high substrate specificity
toward 1. These results strongly suggested that this
Figure 5. Detection of a sugar chain in LOFG. Right: membrane
enzyme is the LOFG which is involved in the diurnal
stained by colloidal gold total protein stain (Bio-Rad Lab.). Left:
control of the level of 1 in the plant body.
biotinylated sugar chain detected by chemiluminescence; lane 1,
Marker proteins; lane 2, LOFG; lane 3, negative control (BSA); lane
4, positive control (Ovalbumin).
Acknowledgements
recognizes an aglycon moiety together with a glycon
moiety, and showed a high selectivity toward 1.
This work was supported by a Grant-in-Aid for Scien-
tific Research on Priority Area 16073216 from the Min-

E. Kato et al. / Tetrahedron Letters 48 (2007) 2409–2413
2413
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