Purification and properties of soluble and bound γ- glutamyltransferases from radish cotyledon

Article abstract of DOI:10.1271/bbb.70.369

Soluble and cell wall bound γ-glutamyltransferases (GGTs) were purified from radish (Raphanus sativus L.) cotyledons. Soluble GGTs (GGT I and II) had the same M_r of 63,000, and were composed of a heavy subunit (M_r, 42,000) and a light one (M_r, 21,000). The properties of GGT I and II were similar. Bound GGTs (GGT A and B) were purified to homogeneity from the pellet after the extraction of soluble GGTs. GGT A and B were monomeric proteins with an M_r of 61,000. The properties of GGT A and B were similar. Thus, bound GGTs were distinguished from soluble GGTs. The optimal pHs of soluble and bound GGTs were about 7.5. Both soluble and bound GGTs utilized glutathione, γ-L-glutamyl-p-nitroanilide, oxidized glutathione and the conjugate of glutathione with monobromobimane as substrates, and were inhibited by acivicin, but soluble GGTs were also distinguished from bound GGTs with regard to these properties.

Full text of DOI:10.1271/bbb.70.369

Biosci. Biotechnol. Biochem., 70 (2), 369–376, 2006
Purification and Properties of Soluble and Bound ꢀ-Glutamyltransferases
from Radish Cotyledon
y
Yoshihiro NAKANO, Satoshi OKAWA, Takayoshi YAMAUCHI, Yukio KOIZUMI, and Jiro SEKIYA
Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan
Received June 8, 2005; Accepted October 24, 2005
Soluble and cell wall bound ꢀ-glutamyltransferases
(GGTs) were purified from radish (Raphanus sativus L.)
cotyledons. Soluble GGTs (GGT I and II) had the same
Mr of 63,000, and were composed of a heavy subunit
(Mr, 42,000) and a light one (Mr, 21,000). The properties
of GGT I and II were similar. Bound GGTs (GGT A
and B) were purified to homogeneity from the pellet
after the extraction of soluble GGTs. GGT A and B
were monomeric proteins with an Mr of 61,000. The
properties of GGT A and B were similar. Thus, bound
GGTs were distinguished from soluble GGTs. The
optimal pHs of soluble and bound GGTs were about
7.5. Both soluble and bound GGTs utilized glutathione,
ꢀ-L-glutamyl-p-nitroanilide, oxidized glutathione and
the conjugate of glutathione with monobromobimane as
substrates, and were inhibited by acivicin, but soluble
GGTs were also distinguished from bound GGTs with
regard to these properties.
space in E. coli.4) Complete purification of plant GGT
has been achieved only from onion bulb scales18,19) and
tomato fruits.15) Onion and tomato GGTs that were
extracted with 50–100 mM Tris–HCl buffer containing
0.5–1.0 M NaCl were composed of a single polypeptide,
unlike the heterodimeric GGTs in mammals,2,20) S. cer-
evisiae,3) and E. coli.21) On the other hand, Arabidopsis
thaliana GGT expressed in the tobacco plant is a
heterodimeric enzyme.22) These results suggest the
occurrence of at least two forms of GGTs, a monomer
type and a heterodimer type, in higher plants, although
no report has indicated the occurrence of two forms in a
single plant source. Recently we found that both forms
of GGTs exist in radish (Raphanus sativus L.): soluble
GGT extracted with low ionic strength buffer and bound
GGT solubilized by high ionic strength buffer contain-
ing NaCl.23) In the present study, we attempt to purify
and characterize these two forms of GGTs, the soluble
GGT and the bound one. We describe herein the
purification and characterization of two soluble GGTs
and two bound GGTs from radish cotyledons. Our
results reveal that soluble GGTs are different from
bound GGTs in molecular structures and properties.
Key words: Raphanus sativus L.; radish; purification;
glutathione; ꢀ-glutamyltransferase
ꢀ-Glutamyltransferase (EC 2.3.2.2, GGT), which cat-
alyzes the transfer of the ꢀ-glutamyl residue of ꢀ-
glutamyl peptides to appropriate acceptors, is distributed
in a wide range of living cells. It is well-known that
GGT catalyzes the first step of GSH catabolism in
mammals and composes the ꢀ-glutamyl cycle.1,2) Sim-
ilar results were obtained with Saccharomyces cerevi-
siae3) and Escherichia coli.4) In addition, GGT plays an
important role in supplying L-cysteine from GSH,
particularly in mammal cells.5) Reduction in growth
and L-cysteine content has been observed in GGT-
deficient mice.6) In E. coli, GGT is essential for normal
growth.7) In higher plants, it is assumed that GGT
catalyzes the synthesis and degradation of numerous
ꢀ-glutamyl peptides.8–12) It has also been reported that
GGT is involved in GSH catabolism in higher
plants.13–15)
Materials and Methods
Chemicals. GSH and ꢀ-L-glutamyl-L-cysteine (ꢀ-
GluCys) were purchased from Nacalai Tesque (Kyoto,
Japan); acivicin, L-cysteinylglycine (CysGly), glycyl-
glycine (GlyGly), and 6-diazo-5-oxo-L-norleucine
(DON) from Sigma-Aldrich (St. Louis, MO); 1-amino-
cyclopropane-1-carboxylic acid (ACC) from Wako Pure
Chemical (Osaka, Japan); monobromobimane from
Molecular Probes (Eugene, OR); and Mimetic Green
1A6XL from Prometic Life Sciences (Cambridge, UK).
Plant material. Radish seedlings with developed
green cotyledons (Kaiware-daikon in Japanese) were
purchased from local producers. They were germinated
and cultured for 7 to 10 d in a greenhouse with nutrient
solution containing 16.4 mM N (1.4 mM NH4-N and
GGT is a membrane-bound enzyme in mammals1) and
S. cerevisiae,16,17) whereas it occurs in the periplasmic
y
To whom correspondence should be addressed. Fax: +81-75-753-6128; E-mail: jiro@kais.kyoto-u.ac.jp
Abbreviations: ACC, 1-aminocyclopropane-1-carboxylic acid; CysGly, L-cysteinylglycine; DON, 6-diazo-5-oxo-L-norleucine; DTT, dithiothreitol;
GGT, ꢀ-glutamyltransferase; ꢀ-GluCys, ꢀ-L-glutamyl-L-cysteine; ꢀ-GluNphA, ꢀ-L-glutamyl-ꢁ-naphthylamide; ꢀ-GlupNA, ꢀ-L-glutamyl-p-nitro-
anilide; GlyGly, glycylglycine; GS-bimane, the conjugate of GSH with monobromobimane

370
Y. NAKANO et al.
15 mM NO3-N), 0.65 mM P, 4 mM K, 3.9 mM Ca, 2.0 mM
Mg, 2.1 mM S, 21 mM Mn, 0.6 mM Zn, 48 mM Fe, 0.3 mM
Cu, 14 mM B, and 0.2 mM Mo in tap water. Cl was
supplied from the tap water.
212 ml of 200 mM Tris–HCl buffer, pH 9.2, and then
518 ml of 5% acetic acid was added. The mixture was
injected into an HPLC to quantify the monobromobi-
mane derivative of CysGly formed, according to the
method described previously.25)
Enzyme assay.
Assay method 1. GGT activity was routinely measured
according to the method of Nakano et al.23) The standard
reaction mixture (final volume, 150 ml) contained 3 mM
ꢀ-L-glutamyl-p-nitroanilide (ꢀ-GlupNA), 50 mM Gly-
Gly, 100 mM Tris–HCl buffer, pH 8.0, and 5 to 20 ml of
the enzyme solution. The enzyme reaction was termi-
nated by adding 500 ml of 10% acetic acid after
incubation for 20 min at 37 ꢀC. Then 250 ml of 0.1%
NaNO2 was added and the mixture was incubated for
3 min at room temperature. To this, 250 ml of 1%
ammonium amidosulfate was added. After 1 min incu-
bation at room temperature, 250 ml of 0.05% N-naph-
thylethylenediamine was added and the absorbance at
540 nm was measured. The amount of p-nitroaniline
released was calculated from the standard curve ob-
tained using p-nitroaniline.
Assay method 2. When GSH or ꢀ-GluCys was used as
the substrate, assay method 2 was employed. The
reaction mixture (final volume, 100 ml) contained 3 mM
GSH or ꢀ-GluCys, 50 mM GlyGly, 1 mM dithiothreitol
(DTT), 20 ml of the enzyme solution, and 40 mM Tris–
HCl buffer, pH 8.0. After reacting at 37 ꢀC for 20 min,
20 ml of 25% trichloroacetic acid was added to terminate
the reaction. Then 20 ml of the reaction mixture was
pipetted into a derivatization solution containing 200 ml
of 200 mM Tris–HCl buffer, pH 9.2, and 12 ml of 15 mM
monobromobimane.24) After 15 min incubation at
37 ꢀC, 518 ml of 5% acetic acid was added. The mixture
was injected into an HPLC to quantify L-cysteine and
CysGly formed, according to the method described
previously.25) When GSSG was used as the substrate,
DTT was omitted from the enzyme reaction mixture.
After terminating the enzyme reaction by adding 20 ml
of 25% trichloroacetic acid, oxidized sulfhydryl com-
pounds in the mixture were reduced at 37 ꢀC for 15 min
by adding 200 ml of Tris–HCl buffer, pH 9.2, containing
1 mM DTT. Then 12 ml of 15 mM monobromobimane
was added to the mixture for derivatization of sulfhydryl
compounds. The monobromobimane derivatives were
analyzed according to methods described previously.25)
Assay method 3. The conjugate of GSH with mono-
bromobimane (GS-bimane) was prepared by incubating
10 mM GSH and 10 mM monobromobimane in 20 mM
Tris–HCl buffer, pH 8.0, at 30 ꢀC for 1 h in the dark. The
GS-bimane thus obtained was used without further
purification because we confirmed that no GSH was left.
The reaction mixture (final volume, 100 ml) contained
3 mM GS-bimane, 50 mM GlyGly, 1 mM DTT, 20 ml of
the enzyme solution, and 40 mM Tris–HCl buffer,
pH 8.0. After incubation for 15 min at 37 ꢀC, 20 ml of
25% trichloroacetic acid was added to terminate the
reaction. The reaction mixture (20 ml) was pipetted into
Purification of soluble GGT. All procedures were
performed at 4 ꢀC unless otherwise specified. Radish
cotyledons (1 kg) were homogenized in three volumes of
grinding buffer (50 mM Tris–HCl buffer, pH 8.0, con-
taining 10 mM 2-mercaptoethanol, 1 mM EDTA, and
1 mM phenylmethylsulfonyl fluoride) and 5% (w/w)
insoluble polyvinylpyrrolidone. The homogenate was
centrifuged at 10;000 g for 10 min. The supernatant
containing soluble GGT,23) was subjected to 50–80%
(NH4)2SO4 fractionation. The precipitate collected by
centrifugation, was dissolved in buffer A (20 mM Tris–
HCl buffer, pH 8.0, containing 1 mM 2-mercaptoetha-
nol) containing 1.5 M (NH4)2SO4. The enzyme solution
was applied to a Toyopearl Butyl-650M column (2:5 ꢁ
20 cm, Tosoh, Tokyo). The column was washed with
buffer A, containing 1.5 M (NH4)2SO4, and then eluted
with a linear gradient of 1.5 to 0 M (NH4)2SO4 in 600 ml
of buffer A. The proteins were precipitated by 80%
saturation with (NH4)2SO4, followed by centrifugation.
The precipitated proteins were dissolved in a small
volume of buffer A and desalted with Sephadex G-25.
The proteins were applied to a Toyopearl DEAE-650M
column (1:5 ꢁ 20 cm), and the column was washed with
buffer A. The proteins were eluted with a linear gradient
of 0 to 300 mM KCl in 400 ml of buffer A. The active
fractions were pooled, and NaCl was added to make
350 mM. The mixture was applied to a concanavalin
A-agarose column (1:5 ꢁ 7 cm). The column was wash-
ed with buffer A, containing 500 mM NaCl and eluted
with 50 mM ꢁ-methyl-D-glucoside in buffer A contain-
ing 500 mM NaCl. The active fractions were dialyzed
overnight against 25 mM Bis–Tris buffer, pH 6.1. The
proteins were applied to a Mono P HR5/20 column
(Amersham Biosciences, St. Louis, MO) and eluted with
a pH gradient of 6.0 to 4.0, according to the manufac-
turer’s instructions. The active fractions eluted at pH 4.9
and 4.7 were referred to as GGT I and GGT II, respec-
tively. These fractions were concentrated to 100 ml
with a centrifugal vacuum evaporator, and applied to a
Sephacryl S-200 HR column (1 ꢁ 31 cm). The proteins
were eluted with buffer A. The active fractions were
combined, concentrated, and used as purified enzymes.
Purification of bound GGT. All procedures were
performed at 4 ꢀC unless otherwise specified. The pellet
after the centrifugation of the crude homogenate at
10;000 g for 10 min as described above was washed
twice with grinding buffer. Then, it was homogenized
in grinding buffer containing 1 M NaCl, and the homo-
genate was centrifuged at 10;000 g for 10 min. The
supernatant was subjected to 40–80% (NH4)2SO4 frac-
tionation. The precipitate collected by centrifugation

Radish Soluble and Bound ꢀ-Glutamyltransferases
371
was dissolved in buffer A, containing 1.5 M (NH4)2SO4,
and applied to a Toyopearl Butyl-650M column (1:5 ꢁ
40 cm). The column was washed with buffer A, con-
taining 1.5 M (NH4)2SO4, and eluted with a linear
gradient of 1.5 to 0 M (NH4)2SO4 in 700 ml of buffer A.
The proteins in the active fractions were precipitated by
80% saturation with (NH4)2SO4, followed by centrifu-
gation. The protein precipitate was dissolved in a small
amount of buffer A and desalted with Sephadex G-25.
The desalted proteins were applied to a Toyopearl
DEAE-650M column (1 ꢁ 4 cm), and the column was
washed with buffer A. The proteins were eluted with a
linear gradient of 0 to 300 mM KCl in 60 ml of buffer A.
The active fractions were diluted ten times with 20 mM
K-phosphate buffer, pH 7.0, and applied to a Toyopearl
CM-650M column (1 ꢁ 4 cm). The column was washed
with 20 mM K-phosphate buffer, pH 7.0, and eluted
with a linear gradient of 0 to 500 mM KCl in 60 ml of
20 mM K-phosphate buffer, pH 7.0. The active fractions
were diluted two times with buffer A and applied to
a Mimetic Green 1A6XL column (1 ꢁ 1:3 cm). The
column was washed with buffer A and eluted with a
linear gradient of 0.1 to 1.5 M NaCl in 30 ml of buffer A.
The active fractions were poured into concanavalin A-
agarose (wet gel volume, 3.6 ml) suspended in buffer A,
containing 500 mM NaCl, and incubated for 12 h. The
suspension was packed in an empty column (1 ꢁ 2:5
cm). The column was washed with buffer A containing
500 mM NaCl and then eluted stepwise with 17 ml of
15 mM D-glucose and 19 ml of 100 mM ꢁ-methyl-D-
glucoside in buffer A containing 500 mM NaCl. The
enzymes that were eluted with D-glucose and ꢁ-methyl-
D-glucoside were referred to as GGT A and GGT B
respectively. The active fractions, A and B, were
combined and used as purified bound GGT A and B
after desalting and concentrating respectively.
ing with a protein sequencer (Hewlett Packard Model
HP241 N&C).
Results
Purification of soluble GGTs
We reported previously that two forms of GGTs,
soluble and bound, were detected in radish cotyle-
dons.23) These two forms of GGTs were readily
separated by differential extraction varying the ionic
strength of the buffer. Soluble GGTs were extracted
from radish cotyledons with grinding buffer (50 mM
Tris–HCl buffer, pH 8.0, containing 10 mM 2-mercap-
toethanol, 1 mM EDTA, and 1 mM phenylmethylsulfonyl
fluoride), and then purified by (NH4)2SO4 fractionation
and various column chromatographies. On Mono P
chromatofocusing, GGT activity was detected in two
peaks that were eluted at pH 4.9 and 4.7, and these were
referred to as GGT I and II respectively (Fig. 1). Further
purification of GGT I and II by Sephacryl S-200 column
chromatography resulted in 10,000-fold purification
with a specific activity of about 200 nkat/mg protein.
The purification of soluble GGTs (GGT I and II) is
summarized in Table 1. Purified GGT I and II were
found to be homogeneous by native PAGE, and the
protein bands of GGT I and II corresponded to the bands
obtained by activity staining with ꢀ-L-glutamyl-ꢁ-
naphthylamide (ꢀ-GluNphA) (Fig. 2). Purified soluble
GGTs were stored at ꢂ80 ꢀC until use without any
significant loss of activity.
Purification of bound GGTs
Purification of bound GGTs from radish cotyledons
was performed. Bound GGTs were solubilized with a
grinding buffer containing 1.0 M NaCl from the pellet
after extraction of soluble GGTs,15,18,19,23) and purified
by (NH4)2SO4 fractionation and various column chro-
matographies. Upon concanavalin A-agarose column
chromatography, GGT activity was found in three
Measurements of kinetic parameters. Assay method 1
was employed for substrate ꢀ-GlupNA, assay method 2
for substrates ꢀ-GluCys, GSH, and GSSG, and assay
method 3 for substrate GS-bimane. Concentrations of
the substrates ranged from 0.01 to 3.0 mM for Km and
Vmax determination, and the amount of the enzyme used
was 10–15 ng/assay. Km and Vmax were calculated from
Hanes–Woolf plots. Ki values were determined by
Lineweaver–Burk plots with 0.01–3.0 mM ꢀ-GlupNA
at inhibitor concentrations of 0.01, 0.05, and 0.1 mM.
15
12
0.4
0.3
0.2
0.1
0
I
II
9
6
3
0
6
5
4
General experimental procedures. SDS–PAGE and
native PAGE were performed according to Laemmli26)
and Davis27) respectively. After electrophoresis, the gels
were visualized by silver staining or activity staining.21)
Mr was measured by gel filtration on a Sephacryl S-200
HR column (1 ꢁ 31 cm) connected to an FPLC. Protein
was assayed according to the method of Bradford28)
with bovine serum albumin as the standard. A purified
enzyme protein was blotted onto the PVDF membrane
after SDS–PAGE and subjected to amino acid sequenc-
0
20
40
60
80
Fraction no. (0.5 ml/tube)
Fig. 1. Mono P Column Chromatography of Soluble GGTs.
The active fraction of soluble GGT obtained after concanavalin
A-agarose column chromatography was applied to a Mono P
column. Peaks I and II were collected and further purified with
Sephacryl S-200. , GGT activity; ꢃ ꢃ ꢃ, absorbance at 280 nm;
ꢂ, pH.

372
Y. NAKANO et al.
Table 1. Typical Purification of Soluble GGT I and II from Radish Cotyledons
Protein
(mg)
Total activity
(nkat)
Specific activity
(nkat/mg protein)
Yield
(%)
Procedure
Crude extract
6644
640
119
121
125
78.8
55.3
18.9
16.4
9.9
0.0179
0.189
1.62
100
102
105
66
46
16
14
8
(NH4)2SO4 fractionation
Toyopearl Butyl-650M
Toyopearl DEAE-650M
Concanavalin A-agarose
77.0
26.7
2.95
0.506
0.240
0.220
0.048
0.052
109
Mono P
I
78.8
74.5
II
Sephacryl S-200
I
206
190
II
9.9
8
3
a
b
c
0.12
a
b
B
A
66,000
0.09
0.06
0.03
0
2
1
0
46,000
29,000
0
20
40
60
Fraction no. (1 ml/tube)
1 2
1 2
1
2
Fig. 3. Concanavalin A-Agarose Column Chromatography of Bound
GGT.
Fig. 2. Electrophoreses of Purified Soluble GGT I and II.
Proteins were subjected to native PAGE (a and b) and SDS–
PAGE (c). Protein bands were visualized by silver staining (a and c)
and activity staining (b). Lane 1, GGT I; lane 2, GGT II.
The active fraction of bound GGT that was obtained after
Mimetic Green column chromatography was applied to a concana-
valin A-agarose column and eluted with the elution buffers
described in the text. D-Glucose (15 mM) was added to the elution
buffer at point (a) and 100 mM ꢁ-methyl-D-glucoside at point (b).
Peaks A and B were collected, desalted, concentrated, and used as
purified bound GGT A and B, respectively. , GGT activity; ꢃ ꢃ ꢃ,
absorbance at 280 nm.
fractions: the non-adsorbed fraction and fractions eluted
with D-glucose (A) and ꢁ-methyl-D-glucoside (B)
(Fig. 3). These three fractions were concentrated and
desalted with Sephadex G-25. GGT in the non-adsorbed
fraction was not adsorbed to concanavalin A-agarose
when it was again applied to the same column.
Furthermore, the GGT in this fraction was not homoge-
neous and labile. Hence, no further purification was
carried out. GGTs eluted with D-glucose and ꢁ-methyl-
D-glucoside were electrophoretically homogeneous
(Fig. 4). These were referred to as GGT A and B
respectively, and were used in the subsequent experi-
ments. GGT A and B were purified 100- to 150-fold
with a final yield of 1%. The specific activities of GGT
A and B were 257 and 342 nkat/mg protein respectively,
comparable to those of onion18,19) and tomato GGTs.15)
A typical purification is summarized in Table 2. GGT A
and B were also detected by activity staining with ꢀ-
GluNphA, although to a much lesser extent than GGT I
and II (data not shown). This result suggests a difference
in substrate specificity for ꢀ-GluNphA between soluble
and bound GGTs. Purified GGT A and B were not stable
since half of the activities were lost within one week at
either 4 or ꢂ20 ꢀC. This might have been due to loss of
the stable enzyme structure due to dissociation from
cell walls.
a
b
66,000
46,000
29,000
1
2
1
2
Fig. 4. Electrophoreses of Purified Bound GGT A and B.
Proteins were subjected to native PAGE (a) and SDS–PAGE (b).
Protein bands were visualized by silver staining. Lane 1, GGT A;
lane 2, GGT B.

Radish Soluble and Bound ꢀ-Glutamyltransferases
373
Table 2. Typical Purification of Bound GGT A and B from Radish Cotyledons
Protein
(mg)
Total activity
(nkat)
Specific activity
(nkat/mg protein)
Yield
(%)
Procedure
NaCl extract
1073
265
2478
1040
500
2.31
3.92
34.0
62.4
264
100
42
20
24
8
(NH4)2SO4 fractionation
Toyopearl Butyl-650M
Toyopearl DEAE-650M
Toyopearl CM-650M
Mimetic Green
14.7
9.40
587
202
0.765
0.288
0.096
0.084
132
458
257
5
Concanavalin A-agarose
A
B
24.7
28.7
1
342
1
Table 3. Kinetic Parameters of Soluble and Bound GGTs
Soluble GGT
Bound GGT
Parameter
Substrate
I
II
A
B
Km (mM)
ꢀ-GlupNA
ꢀ-GluCys
GSH
0.046
0.046
0.042
0.038
0.21
0.024
0.038
0.032
0.042
0.22
4.4
2.4
4.3
2.3
0.60
0.44
0.47
0.66
0.39
0.41
GSSG
GS-bimane
ꢀ-GlupNA
ꢀ-GluCys
GSH
kcat (1/s)
15
16
39
38
30
31
31
32
13
11
23
21
13
12
30
20
GSSG
27
33
32
40
GS-bimane
ꢀ-GlupNA
ꢀ-GluCys
GSH
kcat=Km (1/mM/s)
326
652
738
711
157
666
815
1,000
762
182
8.8
5.4
18
8.8
5.6
18
GSSG
52
45
77
49
GS-bimane
ꢀ-GlupNA
ꢀ-GlupNA
Ki(acivicin) (mM)
Ki(DON) (mM)
0.017
0.081
0.016
0.085
0.096
0.57
0.103
0.47
Values are means of duplicate measurements.
Structural properties of GGTs
GGT I and II adsorbed well to concanavalin A-agarose,
suggesting that they are glycosylated proteins composed
of the same polypeptides modified with different
carbohydrate side chains.
The Mr value of soluble GGT I and II was estimated
to be 63,000 by Sephacryl S-200 gel filtration. Two
bands, with Mr values of 42,000 and 21,000, were
detected when purified GGT I and II were subjected to
SDS–PAGE (Fig. 2), indicating that GGT I and II were
heterodimeric proteins composed of a heavy (Mr,
42,000) and a light (Mr, 21,000) subunit. N-Terminal
amino acid sequence analyses of the light subunits of
GGT I and II gave the same sequence, TSHFSIVDSD,
homologous to those of E. coli and mammalian GGTs.
There are three putative GGT genes (At4g29210,
At4g39640, and At4g39650) in A. thaliana genome,
all of which have post-translational processing sites and
are therefore predicted to encode a heterodimeric type of
GGT. The N-terminal amino acid sequences of the light
subunits of GGT I and II were also highly homologous
to those of the light subunits deduced from A. thaliana
putative GGT genes, but the N-terminus of the heavy
subunit was blocked. When each subunit of soluble
GGT I and II was excised from the gel after SDS–
PAGE, digested with V8 protease, and subjected to
SDS–PAGE,29) the same protein band patterns for GGT
I and II were observed (data not shown). In addition,
The Mr value of bound GGT A and B was estimated
to be 61,000 by Sephacryl S-200 gel filtration. SDS–
PAGE of GGT A and B revealed that the Mr value of the
polypeptides was 61,000 (Fig. 4). Thus GGT A and B
are monomeric proteins, similar to onion and tomato
GGTs. The N-terminal amino acid sequences of GGT A
and B were the same, KFSGRPGINYGQLGN, but this
amino acid sequence was not found in the A. thaliana
putative GGT genes. GGT A and B were also glyco-
sylated since they adsorbed well to concanavalin A-
agarose.
Substrate specificity
ꢀ-GlupNA, GSH, ꢀ-GluCys, GSSG, and GS-bimane
were utilized by GGT I, II, A, and B as ꢀ-glutamyl
donors (Table 3). There was no appreciable difference in
substrate specificity between soluble GGT I and II, or
between GGT A and B. GGT I and II showed similar
apparent Km (0.024–0.046 mM), kcat (15–32 1/s), and
kcat=Km (326–1,000 1/mM/s) values for all of the

374
Y. NAKANO et al.
substrates, except for GS-bimane. GS-bimane was a less
reactive substrate for GGT I and II. These results
indicate that GGT I and II utilized all of the substrates
except for GS-bimane at almost the same efficiency. In
contrast, GGT A and B showed preference for GSH,
GSSG, and GS-bimane (Km, 0.39–0.66 mM and kcat=Km,
18–77 1/mM/s) but not for ꢀ-GlupNA and ꢀ-GluCys
(Km, 2.3–4.4 mM and kcat=Km, 5.4–8.8 1/mM/s). In
addition, GGT I and II catalyzed the ꢀ-glutamyl transfer
from these substrates to GlyGly more efficiently than
GGT A or B.
also inhibited by DON, an inhibitor of mammalian
GGT.2) The Ki values for DON were about 0.08 mM for
GGT I and II and about 0.5 mM for GGT A and B. The
optimal pHs were 7.5 for soluble GGTs and 7.5–8.0 for
bound GGTs. At pH 9.0, about 80% of maximum
activity was detected for all four GGTs, and the
activities fell to 50% for GGT I and II at pH 7.0 and
55% for GGT A and B at pH 6.0.
Discussion
The occurrence of two forms of GGTs, soluble and
bound, has been reported in several plants, including
radish.23) Our objective was to characterize GGTs that
occur in multiple forms in a single plant source. In this
study, we purified soluble heterodimeric GGTs (GGT I
and II) from radish cotyledons to homogeneity for the
first time. We also purified radish-bound GGTs (GGT A
and B) that were structurally similar to the enzymes of
onion18) and tomato.15) This supports our earlier finding
that soluble GGTs and bound GGTs occur in a single
plant source.23) Shaw et al., however, have reported that
onion-bound GGT is a heterodimeric enyme,19) although
onion GGT is a monomeric enzyme.18) This discrepancy
remains to be resolved.
The properties of GGT I and II, including Mr, subunit
composition, substrate specificity, ꢀ-glutamyl acceptor
specificity, and inhibitor sensitivity, were similar. The
properties of GGT A and B were also similar, but there
were critical differences between soluble GGTs and
bound GGTs. First, their subunit compositions were
different; soluble GGTs, I and II, were composed of a
heavy and a light subunit, whereas bound GGTs, A and
B, were monomeric enzymes composed of a single
polypeptide. Tomato GGTs have been reported to be a
monomeric enzyme, and were solubilized by high ionic
strength buffer containing 0.5 or 1.0 M NaCl.15) The
monomeric GGT was different from the GGTs reported
to date in mammalian cells, S. cerevisiae and E. coli,
in which the GGTs were all heterodimeric enzymes. It
has been reported that a heterodimeric type of GGT
occurred in a tobacco plant expressing A. thaliana cDNA
(D22), one homologous to At4g39640.22) A. thaliana
has three other putative GGT genes, At1g69820,
At4g39650, and At4g29210, in addition to At4g39640,
but At1g69820 might be a fragment of one of the GGT
genes, since the length of polypeptide encoded by this
gene is shorter than that encoded by the others. Purified
soluble GGTs conserved the N-terminal amino acid
sequences of the light subunit, T(STA)HX(ST)(VIA)-
X(DSA)X as S. cerevisiae.32) Thus these genes,
At4g39640, At4g39650, and At4g29210, are assumed
to encode heterodimeric GGTs, since the threonine
residue required for post-translational processing was
found.31) Deglycosylation of renal GGTs with subunits
with different Mr values resulted in subunits having
identical Mr values.33) The structural differences between
radish GGT I and II and between GGT A and B can be
Acceptor specificity
When ꢀ-glutamyl acceptor specificities in the trans-
peptidation reaction were measured with ꢀ-GlupNA as
the ꢀ-glutamyl donor, GlyGly was found to be a good ꢀ-
glutamyl acceptor. Enzyme activities without an accep-
tor were reduced to 35–38% of those with GlyGly as
the acceptor for soluble GGTs, whereas for bound GGTs
the reactions did not proceed significantly without
an acceptor (Table 4). For soluble GGT I and II, L-
methionine, L-arginine, L-aspartic acid, and ACC were
better acceptors than GlyGly; no marked difference was
observed between GGT I and II. In contrast, bound GGT
A and B did not utilize these amino acids well. None of
the compounds tested showed acceptor activity higher
than GlyGly. Therefore, there was a significant differ-
ence in ꢀ-glutamyl acceptor specificity between soluble
and bound GGTs.
Other properties
GGT I, II, A, and B were strongly inhibited by
acivicin, an inhibitor of mammalian and E. coli
GGTs.30,31) Both soluble and bound GGT activities were
inhibited by acivicin in a mixed manner. Soluble GGTs
were more sensitive to acivicin than bound GGTs; Ki
values were 0.017 mM for GGT I and II and 0.10 mM for
GGT A and B (Table 3). Soluble and bound GGTs were
Table 4. ꢀ-Glutamyl Acceptor Specificities of Soluble and Bound
GGTs
Acceptor
(50 mM)
Relative activity (%)
Soluble GGT Bound GGT
I
II
A
B
GlyGly
None
CysGly
100
35
100
38
100
1
100
1
16
48
15
43
36
14
7
39
16
12
15
69
56
17
7
L-Glycine
L-Cysteine
L-Glutamic acid
L-Methionine
L-Arginine
L-Aspartic acid
ACC
7
13
7
11
13
43
44
13
8
141
124
144
161
137
169
140
176
ꢀ-GlupNA was used as the substrate for the ꢀ-glutamyl donor. Relative
activity of 100% represents about 200 nkat/mg protein for GGT I and II and
about 300 nkat/mg protein for GGT A and B. Values are means of duplicate
measurements.

Radish Soluble and Bound ꢀ-Glutamyltransferases
375
attributed to differences in the carbohydrate side chains.
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Acknowledgments
This work was partially supported by a JSPS-
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