Purification and characterization of an α-glucosidase from germinating millet seeds

Article abstract of DOI:10.1016/j.phytochem.2005.02.024

An α-glucosidase (α-d-glucoside glucohydrolase, EC 3.2.1.20) was isolated from germinating millet (Panicum miliaceum L.) seeds by a procedure that included ammonium sulfate fractionation, chromatography on CM-cellulofine/Fractogel EMD SO₃, Sephacryl S-200 HR and TSK gel Phenyl-5 PW, and preparative isoelectric focusing. The enzyme was homogenous by SDS-PAGE. The molecular weight of the enzyme was estimated to be 86,000 based on its mobility in SDS-PAGE and 80,000 based on gel filtration with TSKgel super SW 3000, which showed that it was composed of a single unit. The isoelectric point of the enzyme was 8.3. The enzyme readily hydrolyzed maltose, malto-oligosaccharides, and α-1,4-glucan, but hydrolyzed polysaccharides more rapidly than maltose. The K_m value decreased with an increase in the molecular weight of the substrate. The value for maltoheptaose was about 4-fold lower than that for maltose. The enzyme preferably hydrolyzed amylopectin in starch, but also readily hydrolyzed nigerose, which has an α-1,3-glucosidic linkage and exists as an abnormal linkage in the structure of starch. In particular, the enzyme readily hydrolyzed millet starch from germinating seeds that had been degraded to some extent.

Full text of DOI:10.1016/j.phytochem.2005.02.024

PHYTOCHEMISTRY
Phytochemistry 66 (2005) 851–857
www.elsevier.com/locate/phytochem
Purification and characterization of an a-glucosidase
from germinating millet seeds
*
Yoshiki Yamasaki , Mikio Fujimoto, Junji Kariya, Haruyoshi Konno
Research Institute for Bioresources, Okayama University, Kurashiki-shi, Okayama 710-0046, Japan
Received 24 February 2004; received in revised form 27 December 2004
Available online 28 March 2005
Abstract
An a-glucosidase (a-D-glucoside glucohydrolase, EC 3.2.1.20) was isolated from germinating millet (Panicum miliaceum L.) seeds
by a procedure that included ammonium sulfate fractionation, chromatography on CM-cellulofine/Fractogel EMD SO3, Sephacryl
S-200 HR and TSK gel Phenyl-5 PW, and preparative isoelectric focusing. The enzyme was homogenous by SDS–PAGE. The
molecular weight of the enzyme was estimated to be 86,000 based on its mobility in SDS–PAGE and 80,000 based on gel filtration
with TSKgel super SW 3000, which showed that it was composed of a single unit. The isoelectric point of the enzyme was 8.3. The
enzyme readily hydrolyzed maltose, malto-oligosaccharides, and a-1,4-glucan, but hydrolyzed polysaccharides more rapidly than
maltose. The Km value decreased with an increase in the molecular weight of the substrate. The value for maltoheptaose was about
4-fold lower than that for maltose. The enzyme preferably hydrolyzed amylopectin in starch, but also readily hydrolyzed nigerose,
which has an a-1,3-glucosidic linkage and exists as an abnormal linkage in the structure of starch. In particular, the enzyme readily
hydrolyzed millet starch from germinating seeds that had been degraded to some extent.
ꢀ 2005 Elsevier Ltd. All rights reserved.
Keywords: Panicum miliaceum L.; Gramineae; Millet; a-Glucosidase
1. Introduction
Dekker, 1966; Nomura et al., 1969) have suggested that
a-glucosidase is part of the non-phosphorolytic pathway
for the breakdown of starch, and plays a role in seed
germination by hydrolyzing the oligosaccharides pro-
duced by a- and b-amylases. However, Sun and Henson
(1990) reported that barley seed a-glucosidase can initi-
ate the attack of raw starch granules and that this attack
is independent of the presence of a-amylase. This has
been verified by Sissons and MacGregor (1994) and
Sun et al. (1995). Therefore, the in vivo significance of
a-glucosidase is not yet clear.
In view of the role of a-glucosidase in germination,
we investigated a-glucosidase during the early stage of
germination of millet in the context of starch degrada-
tion, which exhibits little a-amylase activity but abun-
dant a-glucosidase activity. We describe here the
purification and properties of a-glucosidase from millet
seeds at an early stage of germination. Moreover, we
In a previous paper, we reported that two a-glucosi-
dases were isolated from millet seeds and that they each
showed a very small Km for maltose. In fact, the Km val-
ues for maltose are the smallest reported to date for any
plant a-glucosidase. Moreover, the enzymes hydrolyzed
millet starch to liberate glucose.; millet seeds contain
abundant a-glucosidase activity which approximately
doubles after 24 h when seeds are soaked in water for
germination. On the other hand, a-amylase activity
was detected only very weakly, but increased markedly
after 48 h. This shows that a-glucosidase in seeds may
play an important role during the early stage of germi-
nation. On the other hand, some authors (Swain and
*
Corresponding author. Fax: +86 434 1249.
E-mail address: yosikiy@rib.okayama-u.ac.jp (Y. Yamasaki).
0031-9422/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.phytochem.2005.02.024

852
Y. Yamasaki et al. / Phytochemistry 66 (2005) 851–857
attempted to examine its enzymatic activity toward a
starch purified from millet seeds.
2. Results and discussion
2.1. Purification of a-glucosidase
The a-glucosidase activity of seeds approximately
doubled after 24 h of germination, with the highest value
from days 1 to 5. Therefore, a crude enzyme solution
was prepared from seeds that had germinated for 24 h,
as described in Section 3 and this was dialyzed overnight
against 20 mM NaOAc buffer, pH 4.5.
The enzyme solution was applied to a CM-cellulofine
column (3.4 · 15 cm) that had been equilibrated with
20 mM NaOAc buffer, pH 4.5. The column was eluted
with a linear gradient of 0–0.5 M NaCl in 20 mM
NaOAc buffer, pH 4.5. The peak that exhibited a-gluco-
sidase activity was collected and applied to a Fractogel
EMD SO3 column (1.7 · 17 cm) that had been equili-
brated with 20 mM NaOAc buffer, pH 4.5. After the col-
umn was washed with 20 mM NaOAc buffer, pH 4.5, a
linear gradient of 0–1.2 M NaCl in the buffer was ap-
plied. The eluate with a-glucosidase activity was concen-
trated using an Amicon ultrafiltration device (PM-10
membrane; Amicon). The concentrate was subjected to
gel filtration on a Sephacryl S-200 HR (Pharmacia).
Fig. 1. SDS–PAGE of purified millet a-glucosidase. SDS–PAGE was
carried out as described in Section 3. E, a-glucosiclase; S, Precision
Plus Protein Standards (Dual Color, Bio-Rad).
The eluate was purified further on
a
column
(0.75 cm · 7.5 cm) of TSKgel Phenyl-5PW that had
been equilibrated with 0.1 M phosphate buffer, pH 7.0,
containing 1.5 M ammonium sulfate, by repeatedly
using an HPLC system (AKTA, Pharmacia). After the
column was washed with buffer containing 1.5 M
ammonium sulfate, a linear gradient of 1.5–0 M ammo-
nium sulfate was applied. The peak that exhibited a-glu-
cosidase activity was subjected to preparative isoelectric
focusing (IEF) (pH range, 6.5–9.0) using an HSI GT
Tube Gel Electrophoresis Unit (Hoefer Scientific Instru-
ments, CA). After insoluble materials were removed by
filtration, the enzyme solution was concentrated using
an Amicon ultrafiltration device and dialyzed overnight
against 20 mM NaOAc buffer, pH 5.3. The purification
procedure is summarized in Table 1. The purified en-
zyme was homogeneous by SDS–PAGE (Fig. 1). The
enzyme produced glucose as the sole product from amy-
lose EX-I, which shows that the purified enzyme did not
contain any a,b-amylases (Fig. 3).
2.2. Purification of starch
Two crude starch preparations were prepared from
intact seeds and seeds that had been allowed to germi-
nate for 24 h, as described in Section 3. The precipitates
were suspended in isoamylalcohol–H2O (1:5) to wash off
proteins, and centrifuged at ca. 1940g for 10 min. This
procedure was repeated three times. The precipitates
were washed with ethyl alcohol three times and lyophi-
lized. The starch preparations obtained from seeds and
Table 1
Purification of a-glucosidase from germinating millet seeds
Step
Total protein, mg
Total activity, units
Specific activity, U/mg prot.
Yield, %
1. Ammonium-sulfate fractionation
17,600
3760
800
290
150
48
22,100
11,250
9100
6720
4800
2330
230
1.3
3.0
100
2. 1st CM-cellulofine column chromatography
3. 2nd CM-cellulofine column chromatography
4. Fractogel EMD SO3 column chromatography
5. Sephacryl S-200 HR column chromatography
6. TSK gel Phenyl-5 PW column chromatography
7. Preparative isoelectric focusing
50.9
41.2
30.4
21.7
10.5
1.0
11.4
23.2
32.0
48.5
76.0
3

Y. Yamasaki et al. / Phytochemistry 66 (2005) 851–857
853
Fig. 2. SEM of starch granules. A, native starch of millet; B, starch of millet seeds germinated for 3 days; C, wheat starch; D, sweet potato starch.
germinating seeds were designated as starch I and starch
II, respectively. The scanning electron micrograph
clearly shows that starch II has been degraded to some
extent (Fig. 2).
These results show that the enzyme is identical to a-
glucosidase II protein from millet seeds.
The enzyme was again prepared from seeds after the
seeds had been soaked in water for 24 h, and the activity
had doubled. Therefore, it is assumed that the confor-
mation of the enzyme protein may have changed to
show higher enzyme activity than that in intact seeds.
The optimum pH of the enzyme was found to be 4.5–
5.0. After 20 h of preincubation at 30 ꢁC with 50 mM
McIlvaineÕs buffer, the enzyme was stable in a pH range
of 2.5–8.0. The optimum temperature for enzyme activ-
ity was 50–60 ꢁC after 30 min of incubation. After
15 min of preincubation with 50 mM NaOAc buffer,
pH 4.5, at various temperatures, the enzyme was found
to be stable at temperatures up to 45 ꢁC.
2.3. Chemical and physical properties of a-glucosidase
The molecular weight of purified a-glucosidase was
estimated to be 86,000 based on its mobility in SDS–
PAGE, and 80,000 based on gel filtration with TSKgel
Super SW 3000, which showed that it is composed of
a single unit. The molecular weight is similar to that
of a-glucosidase from millet seeds (Yamasaki et al.,
1996). The isoelectric point of the enzyme was deter-
mined to be 8.3 by analytical IEF.
The molecular weight and isoelectric point of the en-
zyme are similar to those of a-glucosidase from millet
seeds (Yamasaki et al., 1996). Moreover, the mobility
of the enzyme in native PAGE at pH 4.0 is similar to
that of a-glucosidase II from millet seeds.
The enzyme was preincubated in 50 mM NaOAc buf-
fer, pH 4.5, containing 5 mM metal ions at 37 ꢁC for
30 min, Na+, K+, Ca2+, Co2+, Mn2+, Ni2+, Mg2+, Sn2+
and Al3+ had no effect, but Hg2+ and Pb2+ reduced en-
zyme activity by 80% or more.

854
Y. Yamasaki et al. / Phytochemistry 66 (2005) 851–857
2.4. Substrate specificity
Various substrates (180 nmol) were incubated with
the enzyme under standard conditions (Table 2). The en-
zyme readily hydrolyzed maltose, nigerose, kojibiose,
malto-oligosaccharides, and solubilized polysaccharides,
such as amylose, soluble starch and amylopectin. How-
ever, the enzyme hydrolyzed polysaccharides more
rapidly than maltose, even at considerably low concen-
trations compared with maltose. The enzyme prepara-
tion did not contain any enzyme contaminant (Figs. 1
and 3). Moreover, inhibitors of a-amylase and b-amy-
lase were used in the experiment. PCMB completely
inhibited b-amylase of millet seeds at a low concentra-
tion (Yamasaki, 2003). a-Amylase of millet seeds was
strongly inhibited by Cu2+ (data not shown). The same
results were obtained when PCMB and Cu2+ were re-
acted with the substrates under the same conditions as
in Table 2. Consequently, PCMB and Cu2+ gave the
same results as in Table 2. Therefore, the results in Table
2 were obtained with single a-glucosidase.
a-Glucosidases in higher plants also readily hydro-
lyze polysaccharides (Chiba et al., 1978; Grinna and
Robbins, 1979; Yamasaki and Konno, 1989; Yamasaki
et al., 1996). The enzyme hydrolyzed amylose to liberate
a-glucose, which is an inherent property of a-glucosi-
dase (Yamasaki and Suzuki, 1978, 1980; Yamasaki
et al., 1995). The Km value for maltose is lower than
those of a-glucosidases from other plants, such as rice
seed (Eksittikul et al., 1993), buckwheat (Chiba et al.,
1979), sugar beet (Matsui et al., 1978; Yamasaki and
Konno, 1991), and barley (Henson and Sun, 1995).
The Km value for malto-oligosaccharide decreased with
an increase in the molecular weight of the substrate.
The value for maltoheptaose is about 4-fold lower than
that for maltose. Therefore, a-glucosidase from germi-
Fig. 3. HPLC of the product of the digestion of amylose by a-
glucosidase. A reaction mixture (1 ml) containing amylose EX-I
(10 mg) and the purified a-glucosidase was incubated at 37 ꢁC. After
60 min, the reaction was stopped by boiling for 5 min. A 20-ll sample
of the mixture (E) was assayed by eluting the column with CH3CN–
H2O (6:4) as described in the Section 3. A, reaction mixture (20 ll)
without enzyme; S, standard (each sugar, 4 lg). 1, glucose; 2, maltose;
3, maltotriose; 4, maltotetraose; 5, maltopentaose.
nating millet seeds has a stronger affinity for malto-oli-
gosaccharides liberated from starch by a-amylase than
for maltose produced by b-amylase. The a-glucosidase
may preferably hydrolyze oligo-saccharides liberated
from starch by a-amylase to glucose without the preced-
ing action of b-amylase during the germination of millet
seeds, although it has been suggested that a-glucosidase
needs the preceding action of a- and b-amylases to play
a role during the germination of plants (Swain and Dek-
ker, 1966; Nomura et al., 1969). Moreover, the Km value
for amylopectin (MW, 6,600,000) was about 1000-fold
lower than that for maltose. The enzyme hydrolyzed sol-
uble starch more rapidly than amylopectin. Therefore,
the enzyme is similar to glucoamylase, although gluco-
amylase has not yet been found in plants. Since gluco-
amylase readily hydrolyzes starch to glucose without
the preceding action of a-amylase and b-amylase, the
Table 2
Substrate specificity of a-glucosidase from germinating millet seeds
Relative rate of hydrolysis, %
Km value, mM
kcat, sÀ1
kcat/Km, mMÀ1 sÀ1
Maltosea
100
93
1.46
1.46
33.5
35.7
9.44
5.75
43.9
40.7
36.1
47.5
26.2
24.1
49.7
3.80
22.9
24.5
Nigerosea
Kojibiosea
Isomaltosea
79
3
0.34
9.76
27.4
0.589
38.8
47.4
Maltotriosea
Maltotetraosea
Maltohexaosea
Maltoheptaosea
Amylose EX-Ia
Amylopectinb
Soluble starchb
Phenyl a-glucosidea,c
138
154
236
247
233
129
175
10
1.13
0.86
0.45
0.39
80.4
123.4
92.3
191.3
0.28
1.3 · 10À3
3640d
3.90
0.974
A reaction mixture containing substrate, enzyme solution and 50 mM NaOAc buffer, pH 4.5, in a final volume of 0.5 ml was incubated at 37 ꢁC for
30 min. Liberated glucose was determined by the glucose oxidase–peroxidase method as described in Section 3.
a
Concentration = 180 nmol in 0.5 ml of reaction mixture.
Concentration = 3 mg (below 180 nmol) in 0.5 ml of reaction mixture.
Liberated glucose was measured according to the method of Somogyi (1952).
b
c
d
mg/1.

Y. Yamasaki et al. / Phytochemistry 66 (2005) 851–857
855
a-glucosidase from germinating millet seeds may also
hydrolyze starch to glucose independently during the
early stage of millet seed germination, when only weak
a-amylase activity was detected. On the other hand,
the a-glucosidase from germinating millet seeds readily
hydrolyzed nigerose, which contains an a-1,3-glucosidic
linkage. Since this linkage is a universal abnormal link-
age in the structure of starch, nigerose-hydrolyzing
activity may be vital for the digestion of starch.
On the other hand, polysaccharide powder (100 mg)
was incubated with the enzyme in 0.3 ml of 50 mM
acetate buffer, pH 4.5, at 28 ꢁC for 48 h (Table 3). The
enzyme hydrolyzed native starch granules, similar to
a-glucosidases of barley seed (Sun and Henson, 1990),
malt (Sissons and MacGregor, 1994) and pea (Sun
et al., 1995). The enzyme hydrolyzed starch I (millet
starch) and wheat starch at half the rate of soluble
starch to liberate only glucose, but hydrolyzed sweet po-
tato starch very weakly. The a-glucosidase may by itself
initiate the attack of native starch granules during an
early stage of germination. It has been reported that en-
zyme activity can be affected by the origin of the starch
substrate. An a-amylase from poplar leaves hydrolyzes
corn starch at about a 4-fold higher rate than potato
starch (Witt and Sauter, 1996). The authors speculated
that this difference is due to the large size and the result-
ing relatively small surface area of potato starch. The
size of millet starch was similar to that of wheat starch,
but much smaller than that of sweet potato (Fig. 2).
Therefore, a-glucosidase may also exhibit stronger
hydrolytic activity toward the small starches of millet
and wheat than the large starch of sweet potato. The en-
zyme hydrolyzed starch II that had been degraded to
some extent at more than twice the rate of soluble
starch. This fact supports the notion that the enzyme
may carry out vigorous digestion toward native starch
by itself in the middle stage of germination.
Fig. 4. Determination of amylose content in wheat starch degraded by
a-glucosidase. AL, Amylose content in the mixture; AP, amylopectin
content in the mixture; 1, wheat starch; 2, wheat starch degraded 25%
by the purified a-glucosidase.
2.5. Mode of starch digestion by a-glucosidase
The enzyme was incubated with wheat starch under
standard conditions and the remaining starch was deter-
mined by measuring the iodine color reaction. At 25%
hydrolysis of wheat starch, the amylose content in the
starch increased from 20% to 28% (Fig. 4). Moreover,
the enzyme hydrolyzed amylopectin (mw. 6,600,000)
10-times faster than amylose EX-III (Dpn., ca. 100),
when the enzyme was incubated with insoluble polysac-
charides (Table 3). Therefore, the a-glucosidase from
germinating millet seeds may play an important role
via the preferential hydrolysis of amylopectin and hydro-
lysis of an a-1,3-glucosidic linkage in the structure of
starch during the early stage of germination.
3. Experimental
3.1. Preparation of crude a-glucosidase
Table 3
Activity of a-glucosidase for insoluble polysaccharides
Millet (Panicum miliaceum L.) seeds were soaked in
distilled water and grown on moist absorbent cotton
at 28 ꢁC for 24 h.
Glucose liberated, lg
Ratea, %
Soluble starch
Starch I
Starch II
1116
563
2715
584
65
100
The seeds were homogenized in a homogenizer (Nis-
sei Excel Auto-Homogenizer; Nihonseiki Co., Tokyo)
with 25 mM NaOAc buffer, pH 5.3, containing 0.5 M
NaCl and 5 mM mercaptoethanol. The homogenate
was left overnight at 4 ꢁC and the debris was removed
by filtration. The supernatant was brought to 90% satn
with (NH4)2SO4. The precipitate was collected by centri-
fugation and dissolved in 25 mM NaOAc buffer, pH 4.5.
After this was dialyzed overnight against 20 mM
NaOAc buffer, pH 4.5, CM-cellulofine resin was added
to the dialyzate, the resulting mixture was gently stirred
for 30 min and the resin was removed by filtration. After
the resin was washed with 20 mM NaOAc buffer, pH
50.4
243.3
52.3
5.8
Wheat starch
Sweet potato starch
Amylose EX-III
Amylopectin
628
5588
56.3
500.0
A reaction mixture containing polysaccharide powder 100 mg, enzyme
solution 0.669 units and 50 mM NaOAc buffer, pH 4.5, in a final
volume of 0.3 ml was incubated at 28 ꢁC. After 48 h, deionized water
5 ml was added to the reaction mixture, which was then thoroughly
stirred. After insoluble materials were removed by centrifugation,
glucose in the supernatant was measured by the glucose oxidase–per-
oxidase method (Papadopoulos and Hess, 1960; Dahlqvist, 1961).
a
Relative rate of hydrolysis.

856
Y. Yamasaki et al. / Phytochemistry 66 (2005) 851–857
4.5, the a-glucosidase was released by treatment with a
solution, 20 mM NaOAc buffer, pH 4.5, containing
0.5 M NaCl. a-Glucosidase in the eluate was precipi-
tated with (NH4)2SO4 and the resulting precipitate was
collected and dissolved in 25 mM NaOAc buffer, pH
4.5 as mentioned above.
3.8. High-performance liquid chromatography
The product formed from amylose EX-I with a-glu-
cosidase was determined by HPLC. Chromatography
was performed on a column (0.46 · 15 cm) of COSMO-
SIL 5NH2-MS, using an HPLC System (Tosoh Co., To-
kyo). The column was eluted with CH3CN:H2O (6:4).
3.2. Preparation of native starch
3.9. Scanning electron microscopy
Millet seeds and germinating millet seeds were soaked
in deionized water and homogenized in a homogenizer.
The homogenates were filtered through gauze and the
filtrates were centrifuged.
The digestion of starch by the enzyme was examined
using a scanning electron microscope (SEM). After the
reaction, the starch was washed twice with deionized
water and dried. The surface was coated with gold and
viewed under a Hitachi S-570 Scanning Electron Micro-
scope operating at 5 kV.
3.3. Assay of enzyme activity
a-Glucosidase activity was determined as follows.
The reaction mixture containing 1 mg of maltose and
enzyme solution in 0.5 ml of 50 mM acetate buffer, pH
4.5, was incubated at 37 ꢁC for 30 min. After incubation,
the reaction was stopped by boiling the mixture for
5 min. The amount of glucose formed was measured
using glucose oxidase–peroxidase (Papadopoulos and
Hess, 1960; Dahlqvist, 1961). One unit of a-glucosidase
activity was defined as the amount of enzyme that liber-
ated 1 lmol/min of glucose from maltose under the con-
ditions described above.
References
Awdeh, Z.L., Williamson, A.R., Askonas, B.A., 1968. Isoelectric
focusing in polyacrylamide gel and its application to immunoglob-
ulines. Nature (London) 219, 66–67.
Chiba, S., Inomata, S., Matsui, H., Shimomura, T., 1978. Purification
and properties of an a-glucosidase (glucoamylase) in sugar beet
seed. Agric. Biol. Chem. 42, 241–245.
Chiba, S., Kanaya, K., Hiromi, K., Shimomura, T., 1979. Substrate
specificity and subsite affinities of buckwheat a-glucosidase. Agric.
Biol. Chem. 43, 237–242.
The ratio of amylose to amylopectin was determined
by measuring the iodine color of the reaction mixture
and comparing it with a standard curve of the two poly-
saccharides mixed in various ratios.
Dahlqvist, A., 1961. Determination of maltase and isomaltase activ-
ities with a glucose-oxidase reagent. Biochem. J. 80, 547–551.
Eksittikul, T., Svendsby, O., Yazmaguchi, H., lizuka, M., Minamim-
ura, N., 1993. Thai rice seed a-glucosidase and its specificity.
Biosci. Biotechnol. Biochem. 57, 319–321.
Fawcett, J.S., 1968. Isoelectric fractionation of proteins on polyacryl-
amide gels. FEBS Lett. 1, 81–82.
3.4. Determination of protein
Grinna, L.S., Robbins, P.W., 1979. Glycoprotein biosynthesis. Rat
liver microsomal glucosidases which process oligosaccharides. J.
Biol. Chem. 254, 8814–8818.
Protein was determined by the method of Warburg
and Christian (1942). The protein profiles in column
chromatography were determined by measuring the
absorbance of the eluates at 280 nm.
Henson, C.A., Sun, Z., 1995. Barley seed a-glucosidases: their
characteristics and roles in starch degradation. In: Saddler, J.N.,
Penner, M.H. (Eds.), Developed from a Symposium Sponsored by
the Division of Agricultural and Food Chemistry at the 207th
National Meeting of the American Chemical Society, San Diego,
California, March 13–17, 1994, Enzymatic Degradation of Insol-
uble Carbohydrates. American Chemical Society, Washington,
DC.
3.5. Electrophoresis
SDS–PAGE was performed on 7.5% gels according
to the method of Laemmli (1970). After the run, the gels
were stained for proteins with Phast Blue R (R-350).
Laemmli, U.K., 1970. Cleavage of structural proteins during
the assembly of the head of bacterio phage. Nature 227,
680–685.
Matsui, H., Chiba, S., Shimomura, T., 1978. Substrate speci-
ficity of an a-glucosidase in sugar beet seed. Agric. Biol.
Chem. 42, 1855–1860.
3.6. Estimation of Mr
The Mr of the purified enzyme was estimated by
SDS–PAGE (Laemmli, 1970) and gel filtration with
TSKgel SuperSW 3000.
Nomura, T., Kono, Y., Akazawa, T., 1969. Enzymic mechanism of
starch breakdown in germinating rice seeds. II. Scutellum as the
site of sucrose synthesis. Plant Physiol. 44, 765–769.
Papadopoulos, N.M., Hess, W.C., 1960. Determination of neuraminic
(sialic) acid, glucose, and fructose in spinal fluid. Arch. Biochem.
Biophys. 88, 167–171.
3.7. Estimation of the isoelectric point
Sissons, M.J., MacGregor, A.W., 1994. Hydrolysis of barley starch
granules by a-glucosidases from malt. J. Cereal Sci. 19, 161–169.
Somogyi, M., 1952. Notes on sugar determination. J. Biol. Chem. 195,
19–23.
The isoelectric point of the purified enzyme was esti-
mated as described in the literature (Awdeh et al., 1968;
Fawcett, 1968).

Y. Yamasaki et al. / Phytochemistry 66 (2005) 851–857
857
Sun, Z., Henson, C.A., 1990. Degradation of native starch granules by
barley a-glucosidases. Plant Physiol. 94, 320–327.
Yamasaki, Y., Suzuki, Y., 1980. Two forms of a-glucosidase from
sugar-beet seeds. Planta 148, 354–361.
Sun, Z., Duke, S.H., Henson, C.A., 1995. The role of Pea chloroplast
a-glucosidase in transitory starch degradation. Plant Physiol. 108,
211–217.
Yamasaki, Y., Konno, H., 1989. a-Glucosidase of suspension-cultured
sugar-beet cells. Phytochemistry 28, 2583–2585.
Yamasaki, Y., Konno, H., 1991. Purification and properties of a-
glucosidase from suspension-cultured sugar-beet cells. Phytochem-
istry 30, 2861–2863.
Swain, R.R., Dekker, E.E., 1966. Seed germination studies II.
Pathways for degradation in germinating pea seedlings. Biochim.
Biophys. Acta 122, 87–100.
Yamasaki, Y., Elbein, A.D., Konno, H., 1995. Purification and
properties of a-glucosidase from Trichoderma viride. Biosci. Bio-
technol. Bichem. 59, 2181–2182.
Warburg, O., Christian, W., 1942. Isolation and crystallization of
enolase. Biochem. Z. 310, 384–421.
Witt, W., Sauter, J.J., 1996. Purification and characterization of a-
amylase from poplar leaves. Phytochemistry 41, 365–372.
Yamasaki, Y., Suzuki, Y., 1978. Purification and properties of a-
glucosidase and glucoamylase from Lentinus edodes (Berk.) Sing.
Agric. Biol. Chem. 42, 971–980.
Yamasaki, Y., Konno, H., Masima, H., 1996. Purification and
properties of a-glucosidase from millet seeds. Phytochemistry 41,
703–705.
Yamasaki, Y., 2003. b-Amylase in germinating millet seeds. Phyto-
chemistry 64, 935–939.