Purification and characterization of two isoforms of glucose 6-phosphate dehydrogenase (G6PDH) from Chlorella vulgaris C-27

Article abstract of DOI:10.1271/bbb.67.1888

Two kinds of isoforms of glucose 6-phosphate dehydrogenase (G6PDH) were purified from cells of a freezing-tolerant strain, Chlorella vulgaris C-27, by sequential steps of chromatography on five kinds of columns, including a HiTrap Blue column which showed excellent separation of the isoforms from each other. The two isoforms (G6PDH₁ and G6PDH₂) were purified up to 109-fold and 197-fold with specific activity of 14.4 and 26.0 U/mg-protein, respectively. G6PDH₁ showed an apparent M_r of 200,000 with a subunit M_r of about 58,000, whereas G6PDH₂ showed an apparent M_r of 450,000 with a subunit M_r of about 52,000. The kinetic parameters were measured and several enzymatic features of the isoforms, such as effects of metal ions on the enzyme activity, were clarified, which showed that the two isoforms were different from each other in many respects. Among the effective ions, Cd²⁺ showed marked stimulating effects on both isoforms. G6PDH₁ and G6PDH₂ seem to be a cytosolic and a chloroplastic type, respectively, as judged by their sensitivity to DTT, and also from the results of sequence similarity searches using their N-terminal and internal amino acid sequences.

Full text of DOI:10.1271/bbb.67.1888

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Purification and Characterization of Two Isoforms of
Glucose 6-Phosphate Dehydrogenase (G6PDH) from
Chlorella vulgaris C-27
Ken-ichi HONJOHa, Ayano MIMURAb, Eiko KUROIWAb, Takahiro HAGISAKOb, Koushirou
SUGAbc, Hideyuki SHIMIZUb, Rama Shanker DUBEYd, Takahisa MIYAMOTOa, Shoji HATANOe
& Masayoshi IIOa
a Department of Bioscience and Biotechnology, Faculty of Agriculture, Graduate School,
Kyushu UniversityFukuoka 812-8581, Japan
b Department of Bioscience and Biotechnology, Graduate School of Bioresource and
Environmental Sciences, Kyushu UniversityFukuoka 812-8581, Japan
c Chlorella Industries Co., Ltd.1343 Hisatomi, Chikugo, Fukuoka 833-0056, Japan
d Department of Biochemistry, Faculty of Science, Banaras Hindu
UniversityVaranasi-221005, India
e Graduate School of Health and Social Welfare Science, Nishikyushu University4490-9
Ooazaozaki, Kanzaki-machi, Kanzaki-gun, Saga 842-8585, Japan
Published online: 22 May 2014.
To cite this article: Ken-ichi HONJOH, Ayano MIMURA, Eiko KUROIWA, Takahiro HAGISAKO, Koushirou SUGA,
Hideyuki SHIMIZU, Rama Shanker DUBEY, Takahisa MIYAMOTO, Shoji HATANO & Masayoshi IIO (2003) Purification and
Characterization of Two Isoforms of Glucose 6-Phosphate Dehydrogenase (G6PDH) from Chlorella vulgaris C-27, Bioscience,
Biotechnology, and Biochemistry, 67:9, 1888-1896, DOI: 10.1271/bbb.67.1888
To link to this article: http://dx.doi.org/10.1271/bbb.67.1888
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Biosci. Biotechnol. Biochem., 67 (9), 1888–1896, 2003
Puriˆcation and Characterization of Two Isoforms of Glucose 6-Phosphate
Dehydrogenase (G6PDH) from Chlorella vulgaris C-27
Ken-ichi HONJOH, ,
† Ayano MIMURA,2 Eiko KUROIWA,2 Takahiro HAGISAKO
1,
2
2,3
4
Koushirou SUGA
,
Hideyuki SHIMIZU,2 Rama Shanker DUBEY
,
1
Takahisa MIYAMOTO,1 Shoji HATANO,5 and Masayoshi IIO
1Department of Bioscience and Biotechnology, Faculty of Agriculture, Graduate School, Kyushu University,
Fukuoka 812-8581, Japan
2Department of Bioscience and Biotechnology, Graduate School of Bioresource and Environmental Sciences,
Kyushu University, Fukuoka 812-8581, Japan
3Chlorella Industries Co., Ltd., 1343 Hisatomi, Chikugo, Fukuoka 833-0056, Japan
4Department of Biochemistry, Faculty of Science, Banaras Hindu University, Varanasi-221005, India
5Graduate School of Health and Social Welfare Science, Nishikyushu University, 4490-9 Ooazaozaki,
Kanzaki-machi, Kanzaki-gun, Saga 842-8585, Japan
Received February 24, 2003; Accepted June 18, 2003
Two kinds of isoforms of glucose 6-phosphate
dehydrogenase (G6PDH) were puriˆed from cells of a
freezing-tolerant strain, Chlorella vulgaris C-27, by
sequential steps of chromatography on ˆve kinds of
columns, including a HiTrap Blue column which
showed excellent separation of the isoforms from each
other. The two isoforms (G6PDH1 and G6PDH2) were
puriˆed up to 109-fold and 197-fold with speciˆc
methods to prevent the injury are very important to
meet a worldwide food crisis. It is also quite interest-
ing from the point of view of plant biochemistry and
physiology to understand such a phenomenon as cold
acclimation of plants. Freezing injury leads to severe
dehydration within the cells, denaturation of pro-
teins, and precipitation of various molecules,2,3)
further leading to oxidative damage to the cells and
ˆnally killing them. Cold hardening, i.e. exposure of
plants to non-freezing low temperatures for a few
hours or days, results in alteration in gene expres-
sion, increased synthesis of certain proteins, changes
in activity behaviors of many enzymes, and accumu-
lation of soluble sugars.4–7) These events together
could potentially contribute to freezing tolerance.
Glucose 6-phosphate dehydrogenase (G6PDH) has
prime importance in carbon metabolism of plants
and it catalyzes the ˆrst reaction in the oxidative
pentose phosphate pathway.8) The main function of
the enzyme is to provide NADPH, which is a critical
modulator of the redox potential of the cells and is
the principal intracellular reductant for various
biosynthetic reactions.9,10) It has been observed by
various workers that physiological processes associ-
ated with low temperature treatment of plants and
animals lead to an increase in activity of G6PDH and
increased capacity of the pentose phosphate path-
way.4,11–15) Desiccation or oxidative stress, which are
often associated with chilling, also tend to increase
G6PDH activity in yeast, plants, and human
cells.16–18) Under a condition of cold hardening, to
activity of 14.4 and 26.0 U mg-protein, respectively.
W
G6PDH1 showed an apparent Mr of 200,000 with a
subunit Mr of about 58,000, whereas G6PDH2 showed
an apparent Mr of 450,000 with a subunit Mr of about
52,000. The kinetic parameters were measured and
several enzymatic features of the isoforms, such as
eŠects of metal ions on the enzyme activity, were clari-
ˆed, which showed that the two isoforms were diŠerent
from each other in many respects. Among the eŠective
＋
ions, Cd2 showed marked stimulating eŠects on both
isoforms. G6PDH1 and G6PDH2 seem to be a cytosolic
and a chloroplastic type, respectively, as judged by their
sensitivity to DTT, and also from the results of sequence
similarity searches using their N-terminal and internal
amino acid sequences.
Key words: Chlorella vulgaris C-27; freezing toler-
ance; glucose 6-phosphate dehydrogenase
(G6PDH); puriˆcation
Freezing injury is one of the most severe con-
straints limiting crop productivity.1) Investigating
mechanisms of freezing injury and developing
†
To whom correspondence should be addressed. TelWFax: 81-92-642-3025; E-mail: honjoh agr.kyushu-u.ac.jp
＋ ＠
Abbreviations: CBB, Coomassie brilliant blue; G6PDH, glucose 6-phosphate dehydrogenase; DTT, dithiothreitol; PMSF, phenylmethyl-
sulfonyl‰uoride

Puriˆcation and Characterization of Two G6PDHs from Chlorella
1889
which glycolysis is sensitive,12) increased activity of
and kept in the light (250
m
mol m2s). The hardened
W
G6PDH may provide sustainable levels of NADPH
to meet the biosynthetic events within the cells.15,19,20)
An increased NADPH level is also essential to main-
tain su‹cient reduced glutathione, which serves as
antioxidant to detoxify H2O2 within cells under
oxidative stress.21)
cells were used for puriˆcation of G6PDH.
Puriˆcation of G6PDH. The hardened cells of a
ˆve-liter culture were harvested by centrifugation
and suspended in 40 ml of 50 m
M
potassium phos-
PMSF.
C. The cells
hle (Vibrogen-
hler Co., Tubingen,
C. The homogenate was
phate buŠer (pH 7.5) that contained 1 m
All the subsequent steps were done at 4
were broken with a Vibrogen-Zellmu
Zellmuhle; Edmund Bu
Germany) for 12 min at 4
M
From bacteria, yeast, and animal tissues, G6PDHs
have been puriˆed to homogeneity, as a homodimer
or tetramer with a subunit molecular weight between
50,000 to 57,000.22–24) From plant cells, however,
G6PDH isoforms have been di‹cult to purify to
homogeneity,25,26) as they are very unstable during the
puriˆcation process. The enzymes appear to be
tetramers with molecular weights of from 210,000 to
240,000. Owing to the di‹culties in purifying
G6PDH from plant sources, there is little informa-
tion available on its characteristics and kinetic prop-
erties.8)
Our previous studies indicated possible involve-
ment of the pentose phosphate pathway (PPP) in the
process of hardening in Chlorella cells, marked by
increased activity of the PPP enzymes, i.e., glucose
6-phosphate dehydrogenase and 6-phosphogluconate
dehydrogenase.4) However, detailed studies regarding
involvement of G6PDH in the development of freez-
ing tolerance are still under way. Although G6PDH
seems to be involved in the development of freezing
tolerance of plants, the exact analysis of the enzymes
have not been tried. There are at least two isoforms
of G6PDH in higher plants; one is in the cytosol and
the other is in the chloroplast.9,27,28) However, there
are no reports on which isoform is involved in the
development of freezing tolerance. Thus, as a part of
the study to make clear the involvement of G6PDH
and further, that of the isoforms in the development
of freezing tolerance on a ˆrm molecular basis, we
tried ˆrst to purify two kinds of isoforms of G6PDH
from hardened cells of C. vulgaris and second, to
characterize their structural, kinetic, and other
enzymatic properties.
9
ä
ä
ä
ä
9
ˆltered using a sintered-glass funnel and then cen-
×
trifuged at 168,000
g for 30 min. The supernatant
was concentrated to about 15 ml by ultraˆltration
using a 20-kDa ultraˆlter membrane (Advantec Co.,
Tokyo, Japan).
The concentrated sample was put directly on a
×
column (2.5 66 cm) of Sepharose-4B (Amersham
Pharmacia Biotech, Uppsala, Sweden), which was
equilibrated with buŠer A (50 m
M
potassium phos-
phate buŠer (pH 7.5), 10
z
glycerol). Proteins were
eluted with the same buŠer. Active fractions were
mixed and used for a subsequent step.
The enzyme solution was put directly on a HiTrap
Blue column (5 ml; Amersham Pharmacia), which
had been equilibrated with buŠer A. The non-
adosorbed solution, which included one of the
isoforms of G6PDH, was used for another a‹nity
chromatography on a Blue Celluloˆne column. The
HiTrap Blue column was washed with 20 ml of buŠer
A and then adsorbed proteins were eluted by three
kinds of the following buŠers; ˆrst step: 30 ml of
buŠer A plus 1 m
buŠer A plus 1 m NADP, third step: 30 ml of buŠer
A plus 1 KCl. Active fractions eluted with 1 KCl
were mixed and dialyzed with buŠer A. We designat-
M
NAD, second step: 30 ml of
M
M
M
ed an isoform, which was eluted with 1
G6PDH2.
M
KCl, as
The non-adsorbed fraction, which contained
another isoform of G6PDH that did not bind to the
HiTrap Blue column, was put on a Blue-Celluloˆne
×
(Seikagaku Co., Tokyo, Japan) column (1.6 8 cm),
which had been equilibrated with buŠer A. The
column was washed with the same buŠer and then
Materials and Methods
Plant materials. Cells of Chlorella vulgaris
washed with buŠer A plus 1 m
adsorbed enzyme was eluted by an increasing
gradient of NADP in the buŠer (0–1 m , total
M
NAD. Then the
Beijerinck IAM C-27 were grown synchronously in
MC medium29) at 25
9
C, under a photosynthetic
M
photon ‰ux density of about 250
m
mol m2s, with
volume 60 ml). Active fractions were mixed. The
isoform separated on a Blue-Celluloˆne column was
designated G6PDH1.
The two enzyme solutions (G6PDH1 and G6PDH2)
were, separately, applied to a HiTrap Q-Sepharose-
XL (Amersham Pharmacia Biotech) column which
had been equilibrated with buŠer A. The column was
washed initially with 20 ml of the same buŠer A and
then proteins were eluted by an increasing gradient of
W
×
1.3 CO2 in air, to a concentration of about 1.0
z
1010 cells l, under a 16-h light 8-h dark regime, as
W
W
described previously.30)
Hardening. Cold treatment was done using Chlo-
rella cells grown up to an intermediate stage in the
ripening phase of the cell cycle, the L2 stage.30) The
synchronized cells (1.0 1010 cells l) in the MC
×
W
medium were treated at 3
9
C for 24 h. During the
KCl in the buŠer (0.15–0.25
M
; the total volume was
; the total
treatment, cultures were aerated with 1
z
CO2-air
70 ml in the case of G6PDH1, 0.25–0.45
M

1890
K. HONJOH et al.
volume was 70 ml in the case of G6PDH2). Active
fractions were mixed and dialyzed against buŠer A.
The two enzyme solutions were respectively put on
Temperature-, pH-dependence, and kinetic
parameters. The eŠects of temperature on the activity
of G6PDH were measured by the enzyme activity at
a HiLoad 16 60 Superdex 200 pg (Amersham Phar-
diŠerent temperatures from 20 to 709C. For inves-
W
macia Biotech) column which had been equilibrated
with buŠer A. The column was eluted with the same
buŠer. Active fractions were mixed and used for
further experiments.
tigating the eŠects of temperature on the stability of
the enzymes, the puriˆed enzyme was incubated in
buŠer A at various temperatures for 30 min, and then
the remaining activity was assayed at 25
pH-dependence was investigated by doing assays in
55 m of diŠerent buŠers ranging from pH 3 to 11:
9
C. The
Molecular mass measurement. The molecular
M
masses of the puriˆed G6PDHs were measured on a
citric acid-di-sodium hydrogenphosphate (pH 3–7),
Tris-HCl (pH 7–9), and glycine-NaOH (pH 9–11).
For investigating the eŠects of pH on the stability of
the enzymes, the puriˆed enzyme was incubated in
HiLoad 16 60 Superdex 200 pg column equilibrated
W
with buŠer A containing 0.15
was calibrated with a mixture of proteins prepared in
the same buŠer: ferritin, Mr 450,000; bovine liver
M
KCl. The column
＝
M
9
the above buŠers (55 m ) at 4 C overnight, and then
＝
＝
catalase, Mr 240,000; rabbit muscle aldolase, Mr
a portion of the mixture was transferred into the
assay mixture at pH 7.8 to measure the remaining
activity.
＝
160,000; bovine serum albumin, Mr 67,000; and egg
＝
albumin, Mr 45,000. The puriˆed enzyme samples
were put on the column. The column was eluted with
The Km for G6P was measured in 0.2 m
The Km for NADP was measured in 3.3 m
The maximum velocity ( max) of G6PDH and
m) values were calculated by
M
NADP.
the above buŠer at a ‰ow rate of 1.0 ml min and the
M
G6P.
W
absorbance was monitored at 220 nm. The molecular
masses of the isoforms were then calculated from the
plot of the molecular masses of the known proteins
vs. the retention times.
V
Michaelis constant (
K
constructing Lineweaver-Burk plots.
The molecular masses of subunits of G6PDH
Amino acid sequencing of the N-terminus and
digested peptides, and homology search. For analysis
of the N-terminal amino acid sequences, the puriˆed
G6PDHs were separated by SDS-PAGE, blotted
electrophoretically onto a PVDF membrane (Bio-
Rad, Richmond, CA, U.S.A.) in an electroblot
apparatus (AE-6675P; ATTO Co., Tokyo, Japan),
and detected by staining with CBB R-250. The amino
acid sequences of the isoforms were analyzed with a
gas-phase protein sequence analyzer (model PPSQ-
21; Shimadzu, Kyoto, Japan).
isoforms were measured by SDS-PAGE on a 7.5
z
(w v) polyacrylamide gel, by the procedure of Laem-
W
mli.31) After separation, the gels were stained with
Coomassie Brilliant Blue (CBB) R-250.
Assay of G6PDH activity. The activity of G6PDH
was assayed by the method of Sagisaka.32) An assay
mixture in a total volume of 3 ml contained 55 m
M
Tris-HCl buŠer (pH 7.8), 3.3 m
NADP, 3.3 m
M
MgCl2, 0.2 m
M
M
glucose 6-phosphate (G6P), and
enzyme. The reaction was started by adding 0.1 ml
of an enzyme solution to the reaction mixture. The
reduction of NADP was followed by monitoring the
increase in absorbance at 340 nm in a double-beam
recording spectrophotometer (UV-160; Shimadzu,
Kyoto, Japan). One unit of the activity was deˆned
For analysis of internal amino acid sequences of
the G6PDHs, puriˆed G6PDH1 was cleaved by
cyanogen bromide,34) and puriˆed G6PDH2 was
digested by V8 protease in a gel according to the
35)
method of Kennedy et al
.
The digested peptide
fragments were separated by SDS-PAGE on a 15
z
as the amount of enzyme forming 1.0
NADPH per minute at 25 C.
m
mol of
polyacrylamide gel and blotted electrophoretically
onto a PVDF membrane. The peptides were stained
with CBB R-250 and sequenced as described above.
The sequences obtained were compared with those
of proteins in the databases, using the BLAST
9
Estimation of protein concentration. Protein
concentrations were measured by the method of
Bradford33) using bovine serum albumin as
standard.
a
program36) on the NCBI homepage
(http:
WW
www.ncbi.nlm.nih.gov BLAST ).
W
W
Treatment with DTT. In order to examine the
eŠects of DTT on G6PDH activity, puriˆed enzyme
solutions were diluted with an equal volume of buŠer
Results and Discussion
Puriˆcation of two isoforms of G6PDH from
Chlorella
Two diŠerent enzymes showing activity of G6PDH
have been puriˆed from Chlorella vulgaris C-27. A
typical puriˆcation protocol is described in Table 1.
Although the activity proˆle from the Sepharose 4B
column showed only one peak with G6PDH activity
A containing 200 m
M
DTT. After incubation of the
samples at 0 C for diŠerent times, portions were
9
withdrawn and used for measurement of G6PDH
activity. The corresponding controls were diluted
with buŠer alone.

Puriˆcation and Characterization of Two G6PDHs from Chlorella
Table 1. Puriˆcation of G6PDHs from Chlorella vulgaris C-27
1891
Total protein
(mg)
Total activity
(U)
Speciˆc activity
(U mg protein)
Puriˆcation
(fold)
Yield
Step
Crude extracts
Sepharose 4B
HiTrap Blue
G6PDH2
W
(z)
133
117
17.55
13.53
0.132
0.115
1.00
0.87
100
77.1
8.61
0.85
5.76
2.51
0.669
2.95
5.08
14.3
32.8
22.4
Blue Celluloˆne
G6PDH1
HiTrap Q-Sepharose XL
G6PDH1
0.10
0.52
1.04
3.24
10.2
6.21
77.1
47.1
5.50
18.5
G6PDH2
HiLoad 16
G6PDH1
G6PDH2
W
60 Superdex 200 pg
0.03
0.08
0.40
1.99
14.4
26.0
109
197
2.29
11.3
(data not shown), the second chromatography using
a HiTrap Blue column showed G6DPH activity both
in the adsorbed and non-adsorbed fractions. The
non-adsorbed isoform was designated as G6PDH1
and the adsorbed isoform as G6PDH2. The G6PDH1
solution was put on the third chromatography using
a Blue Celluloˆne column. After following ion
exchange and gel ˆltration chromatography, both
isoforms were respectively puriˆed to homogeneity.
Various workers have attempted to purify G6PDH
from plant sources. However, because of its high
instability and its unspeciˆc aggregation during
puriˆcation, little success has resulted so far.9,26) Only
two cytosolic G6PDHs from plant species have been
puriˆed in homogeneous states.9,26) Therefore, many
experiments related to characterization of plant
G6PDH have been done with partially puriˆed
preparations or crude extracts.9) In this study, as
Fig. 1B shows, two kinds of isoforms of G6PDH
have been puriˆed, especially owing to the use of a
HiTrap Blue column, which was very helpful and
eŠective for puriˆcation of G6PDH2. According to
the manufacturer's instructions, HiTrap Blue con-
tains Cibacron Blue F3G-A as a ligand, to which the
two isoforms showed diŠerent a‹nity, leading to
their eŠective separation from each other. Thus, we
succeeded in puriˆcation of the two isoforms without
the problem of the unspeciˆc aggregation of the en-
zymes. Another point for the successful puriˆcation
of the isoforms in this study lies in our use of
hardened Chlorella cells, because the total activity of
G6PDH2 of unhardened cells was low and thus it was
di‹cult to purify the isoform.
dards (Fig. 1B). The isoform thus seems to be a
tetramer. On the other hand, the retention time of
G6PDH2 showed an apparent Mr of 450,000 for the
native isoform. SDS-PAGE showed a subunit Mr of
52,000. These results suggested that G6PDH2 formed
an octamer.
These oligomeric properties and the subunit
Mr are
very similar to characteristics of G6PDHs from other
species (bacteria, yeast, animal tissues, and plants),
which have been puriˆed as a homodimer or tetramer
with
a
subunit Mr of 50,000 to 57,000.9,16–18)
Cyanobacterial G6PDHs seems to form oligomers
(from dimer to tetramer, ˆnally to hexamer), leading
to higher activity.37) Likewise, in Chlorella the forma-
tion of the octamer might occur during hardening,
leading to the activation of G6PDH2, although the
reason for the activation of G6PDH2 during harden-
ing remains unclear. Therefore, in order to make
clear the involvement of G6PDH2 in the development
of freezing tolerance, the expression of its corre-
sponding gene and the formation of the octamer of
G6PDH2 will be studied in the future.
Properties of the puriˆed G6PDH isoforms
The eŠects of pH on activity and stability of the
puriˆed isoforms were measured. As Fig. 2A shows,
both isoforms were most active at pH 8.0. The activi-
ties of both isoforms, G6PDH1 and G6PDH2, were
reduced to 50
z
above pH 9.0 and below pH 6.0. The
below pH
stability of G6PDH1 was reduced to 50
z
5.0 and above pH 10.0 (Fig. 2B). On the other hand,
G6PDH2 was stable between pH 4.0 and 11.0.
The eŠects of temperature on activity and stability
of both isoforms were also examined. G6PDH1 was
The molecular mass of each puriˆed G6PDH
isoform was measured by gel-ˆltration chro-
most active at 50
most active at 40 C. The two isoforms were stable up
to 20 C, but the stability of G6PDH1 began to
9
C (Fig. 3A), while G6PDH2 was
matography using a HiLoad 16 60 Superdex 200 pg
9
W
column. The retention time of G6PDH1 in relation to
the protein molecular mass standards indicated an
apparent Mr of 200,000 for the native isoform
(Fig. 1A). When the isoform was put through SDS-
PAGE, a single band was observed, corresponding to
9
decrease at 30
(Fig. 3B).
9C and that of G6PDH2 at 509C
The results of kinetic studies are shown in Table 2;
the puriˆed G6PDH1 of Chlorella has structural and
kinetic properties comparable with those reported
a subunit
Mr of 58,000 compared to the protein stan-

1892
K. HONJOH et al.
Fig. 1. Molecular Mass Determination of G6PDHs from C. vulgaris
.
(A) Determination of molecular masses of G6PDH isoforms, G6PDH1 and G6PDH2 from C. vulgaris by chromatography on HiLoad
16 60 Superdex 200 pg. Marker proteins: ferritin from horse spleen (Mr 450,000) (1), catalase from bovine liver (Mr 240,000) (2),
W
＝ ＝
＝
＝
＝
aldolase from rabbit muscles (Mr 160,000) (3), bovine serum albumin (Mr 67,000) (4), and ovalbumin (Mr 45,000) (5). (B) SDS-
PAGE of the puriˆed G6PDHs. The gel was stained with CBB R-250.
Fig. 2. EŠects of pH on Activity and Stability of G6PDHs.
(A) EŠects of pH on activity. The activity of the enzyme was assayed as described in Materials and Methods, except for the buŠer.
The activity at pH 8.5 in Tris-HCl buŠer was taken as 100
z
. (B) EŠects of pH on stability. The remaining activity at pH 7.0 in Tris-HCl
buŠer was taken as 100
activity of G6PDH2.
z
. The open square symbols represent the activity of G6PDH1 and the closed circular symbols represent the
from other sources38,39) including a potato cytosolic
enzyme.9) For example, Chlorella G6PDH1 has simi-
lar parameters to cytosolic G6PDH from potato (Km
EŠects of DTT on G6PDH isoforms
Puriˆcation of chloroplast G6PDH had not been
successful, partly because this isoform is inactivated
by light because of redox modulation by thioredoxin,
values of 260
m
M
for G6P and 6
m
M
for NADP).9)
This shows the low Km value of Chlorella G6PDH1
for its substrate G6P compared to the potato en-
and partly because it easily aggregates unspeciˆcally
42)
during puriˆcation.41) Anderson et al
.
reported an
zyme. The
K
m values of G6PDH2 for both substrates
isoform of G6PDH as the cytosolic one because of its
insensitivity to DTT. Incubation with DTT in vitro
has been widely used to mimic the redox modulation
of plastidic G6PDH to distinguish between the two
(G6P and NADP) were higher than those of
40)
G6PDH1. As Esposito et al
.
shows, the Km values
of a plastid G6PDH isoform for G6P and NADP are
higher than those of the cytosol isoform. These
results also supported the idea that G6PDH2 of Chlo-
rella is a chloroplast type, as will be discussed later.
plant isoforms, cytosolic and chloroplast types.41)
A
disulˆde bond between cysteine residues of the pota-
to chloroplast G6PDH seems to be reduced by DTT,

Puriˆcation and Characterization of Two G6PDHs from Chlorella
1893
Fig. 3. EŠects of Temperature on Activity and Stability of G6PDHs.
(A) EŠects of temperature on activity. The activity was assayed as described in Materials and Methods at various temperatures as indi-
cated in the ˆgure. The maximal activity of each isoform was taken as 100 . (B) EŠects of temperature on stability. The residual activi-
ty at 5 C was taken as 100 . The open square symbols represent the activity of G6PDH1 and the closed circular symbols represent the
activity of G6PDH2.
z
9
z
Table 2. Kinetic Parameters for Puriˆed G6PDHs from
C. vulgaris
Km
G6P
( mM)
Vmax
for G6P
WKm
Vmax
(U
W
mg protein)
NADP
－
2
2
×
3.07 10
－
G6PDH1
G6PDH2
154
335
12.4
15.6
4.73
13.9
×
4.15 10
leading to inactivation of the enzyme.41) As shown in
Fig. 4, the G6PDH1 preparation probably represents
the cytosolic isoform; but G6PDH2, which was inac-
tivated by DTT, seems to have the characteristics of
the chloroplast isoform.9,26) The insensitivity of
G6PDH1 towards DTT suggests that this isoform is
not under the control of redox modulation, similarly
to potato cytosolic G6PDH9) and contrarily to
cyanobacterial G6PDH (chloroplast G6PDH).8)
Although it is necessary to investigate the localization
of G6PDH2, this isoform is likely to be a chloroplast
type as judged by its sensitivity to DTT.
Fig. 4. EŠects of DTT on the Activity of G6PDHs.
The open square symbols represent the activity of G6PDH1
and the closed circular symbols represent the activity of
G6PDH2.
＋
Among the eŠective ions, Cd2 showed marked
stimulating eŠects on both isoforms. Similar stimula-
＋
tion (up to 10-fold) by Cd2 was also reported with
Escherichia coli threonine dehydrogenase,43) which is
EŠect of metal ions on G6PDH activity
supposed to have a zinc-binding site in which a
＋
In order to compare other characteristics of the
puriˆed isoforms, the eŠects of metal ions on the
activity were examined (Table 3). Several ions
showed quite diŠerent eŠects on the two isoforms;
cysteine residue is located.44) In addition, Cd2 is
known to bind to the sulfhydryl groups of several
proteins.45) An analysis of cDNA clones corre-
sponding to genes encoding cytosolic and chloroplast
G6PDHs from potato showed that six cysteine
residues exist on the deduced amino acid sequence.41)
Out of them, two residues are suggested to be in-
volved in the reductive inactivation of the chloroplast
isoform by DTT.41) At present, we suppose that the
Cd2＋ ion activated both isoforms through binding to
free sulfhydryl groups, although we do not know the
exact site(s) of the Cd2＋ binding. The fact that EDTA
did not dramatically aŠect the activities of both
＋
for example, Mn2 ion had a slight eŠect on
G6PDH1, but it stimulated the activity of G6PDH2.
＋
Ca2 ion had almost no eŠect on G6PDH1, while it
inhibited the activity of the other isoform. These
results, in addition to the diŠerent behavior in a‹nity
chromatography and in electrophoresis, indicated
that the two isoforms are diŠerent kinds of polypep-
tides, which was deˆnitely clariˆed from amino acid
sequence analysis as described later.

1894
K. HONJOH et al.
isoforms suggests a possibility that they are not
metalloenzymes, although whether the isoforms are
metalloenzymes remains to be elucidated.
sequences derived from G6PDH1 were found in the
deduced amino acid sequence of an isolated cDNA
clone, which showed similarity to cytosolic types of
G6PDH from several plants, in our laboratory (un-
published data). Thus, the puriˆed G6PDH1 was
thought to be localized in the cytosol.
Amino acid sequences of peptide fragments of the
two isoforms
For identiˆcation of the puriˆed proteins as
G6PDHs, analysis of the N-terminal amino acid
sequence was done and that of G6PDH2 was identi-
ˆed as NH2–GLQEENWEKAALSIV–, while that
of G6PDH1 was found to be blocked. In order to
analyze the internal amino acid sequences of
G6PDH1, the isoform was cleaved with cyanogen
bromide and put through amino acid sequencing.
Two peptide fragments derived from G6PDH1
showed the sequences –NDDKLREKLK– (fragment
1) and –IVKKPGLEFD– (fragment 2). Homology
search of the sequence of fragment 2 showed similari-
ties to those of cytosolic G6PDHs from several
higher plants (Fig. 5A). The two internal amino acid
For identiˆcation of the internal amino acid
sequences of G6PDH2, the isoform was digested by
V8 protease. Three fragments obtained were se-
quenced. Fragment 1 was found to be as –LVIRIQ-
PNEGIYLKVNNKVP-, but fragments 2 and 3 could
not be identiˆed so far. Homology search of the
sequence showed similarity to the sequences of
plastid G6PDHs from several higher plants (Fig. 5B).
This result also suggested that G6PDH2 was localized
in the chloroplast. Based on the sequences of
G6PDH2 obtained, the cDNA corresponding to
G6PDH2 will be isolated and characterized.
This study shows the puriˆcation and characteris-
tics of two G6PDHs from hardened Chlorella vul-
garis C-27. The obtained results suggested that
G6PDH1 was a cytosolic type as a tetramer and
G6PDH2 was a chloroplast type as an octamer in har-
dened Chlorella cells. A cDNA clone corresponding
to G6PDH1 has been isolated and characterization of
the clone is in progress. Furthermore, based on the
obtained amino acid sequences, isolation of a cDNA
clone corresponding to a gene encoding G6PDH2, a
chloroplast isoform, is also in progress. By inves-
tigating the expression patterns of the corresponding
genes and producing transgenic plants, the relation-
ship of G6PDHs with the development of freezing
tolerance will become clearer.
Table 3. EŠects of Metal Ions and EDTA on Activity of
G6PDHs from C. vulgaris
Relative activity (
z)
Reagent
Conc. (mM)
G6PDH1
G6PDH2
Control
ZnCl2
MnCl2
FeCl3
CuCl2
BaCl2
CdCl2
CaCl2
MgCl2
EDTA
—
1
1
1
1
1
1
1
1
1
100
202
102
209
87.2
99.1
529
102
99.1
90.0
100
109
574
470
80.9
93.0
435
37.4
99.1
100
Acknowledgments
The puriˆed enzymes were dialyzed against 50 m
M
potassium phosphate
buŠer (pH 7.5) and used to measure the eŠects of metal ions on the enzyme
activity. The enzyme activity was assayed in the presence of each metal ion
tested under the conditions described in Materials and Methods without
MgCl2.
We are grateful to Dr. M. Nakao, Kyushu Univer-
sity for amino acid sequencing. R. S. Dubey was
supported by the JSPS fellowship.
Fig. 5. Alignment of the Amino Acid Sequences of Fragment 2 of G6PDH1 (A) and of Fragment 1 of G6PDH2 (B) with Those of the
Proteins Identiˆed by a Computer Search.
Accession numbers of databases (DDBJ
WGenBankWEMBL) are in parentheses. Identical amino acids are shown in capital letters and
shaded, and similar amino acids are in lower case and shaded.

Puriˆcation and Characterization of Two G6PDHs from Chlorella
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