Tài liệu Báo cáo khoa học: The thioredoxin-independent isoform of chloroplastic glyceraldehyde-3-phosphate dehydrogenase is selectively regulated by glutathionylation docx

The thioredoxin-independent isoform of chloroplastic
glyceraldehyde-3-phosphate dehydrogenase is selectively
regulated by glutathionylation
Mirko Zaffagnini
1,2
, Laure Michelet
2
, Christophe Marchand
3
, Francesca Sparla
1
,
Paulette Decottignies
3
, Pierre Le Mare
´
chal
3
, Myroslawa Miginiac-Maslow
2
,
Graham Noctor
2
, Paolo Trost
1
and Ste
´
phane D. Lemaire
2
1 Laboratory of Molecular Plant Physiology, University of Bologna, Italy
2 Institut de Biotechnologie des Plantes, UMR 8618, CNRS ⁄ Universite

´
Paris-Sud, Orsay, France
3 Institut de Biochimie et Biophysique Mole
´
culaire et Cellulaire, CNRS ⁄ Universite
´
Paris-Sud, Orsay, France
Glutathione represents the major low-molecular-weight
thiol in most cells. In addition to its well-established
role in cellular defense against oxidative stress, gluta-
thione can also promote a reversible post-translational
modification, termed protein glutathionylation [1,2].
This modification consists of the formation of a mixed
disulfide between glutathione and cysteine residues of
proteins. In mammals, glutathionylation occurs under
oxidative stress conditions and may protect cysteines
from oxidation to cysteine sulfinic (-SO
2
H) or sulfonic
(-SO
3
H) acids. In fact, while oxidation into sulfinic or
sulfonic groups is irreversible, 2-electron reduction of
glutathionylated cysteines can regenerate protein thiols.
Glutathionylation has been shown to alter, either posi-
tively or negatively, the activity of several proteins
[3–8]. Recent proteomic approaches allowed the
Keywords
Arabidopsis thaliana; Calvin cycle; GAPDH;
glutathionylation; oxidative stress

Correspondence
S. D. Lemaire, Institut de Biotechnologie
des Plantes, Ba
ˆ
timent 630, Universite
´
Paris-Sud, F-91405 Orsay Cedex, France
Fax: +33 1 69153423
Tel. +33 1 69153338
E-mail:
(Received 3 October 2006, revised 25
October 2006, accepted 7 November 2006)
doi:10.1111/j.1742-4658.2006.05577.x
In animal cells, many proteins have been shown to undergo glutathionyla-
tion under conditions of oxidative stress. By contrast, very little is known
about this post-translational modification in plants. In the present work,
we showed, using mass spectrometry, that the recombinant chloroplast
A
4
-glyceraldehyde-3-phosphate dehydrogenase (A
4
-GAPDH) from Arabid-
opsis thaliana is glutathionylated with either oxidized glutathione or
reduced glutathione and H
2
O
2
. The formation of a mixed disulfide between
glutathione and A
4

-GAPDH resulted in the inhibition of enzyme activity.
A
4
-GAPDH was also inhibited by oxidants such as H
2
O
2
. However, the
effect of glutathionylation was reversed by reductants, whereas oxidation
resulted in irreversible enzyme inactivation. On the other hand, the major
isoform of photosynthetic GAPDH of higher plants (i.e. the A
n
B
n
-GAPDH
isozyme in either A
2
B
2
or A
8
B
8
conformation) was sensitive to oxidants
but did not seem to undergo glutathionylation significantly. GAPDH cata-
lysis is based on Cys149 forming a covalent intermediate with the substrate
1,3-bisphosphoglycerate. In the presence of 1,3-bisphosphoglycerate, A
4
-
GAPDH was fully protected from either oxidation or glutathionylation.

Site-directed mutagenesis of Cys153, the only cysteine located in close
proximity to the GAPDH active-site Cys149, did not affect enzyme inhibi-
tion by glutathionylation or oxidation. Catalytic Cys149 is thus suggested
to be the target of both glutathionylation and thiol oxidation. Glutathiony-
lation could be an important mechanism of regulation and protection of
chloroplast A
4
-GAPDH from irreversible oxidation under stress.
Abbreviations
BPGA, 1,3-bisphosphoglyceric acid; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GSH, reduced glutathione; GSSG, oxidized
glutathione; ROS, reactive oxygen species; TRX, thioredoxin.
212 FEBS Journal 274 (2007) 212–226 ª 2006 The Authors Journal compilation ª 2006 FEBS
identification of many proteins undergoing such post-
translational modifications in mammalian and yeast
cells [9–12]. One of the prominent glutathionylated pro-
teins in mammalian cells under stress is the glycolytic
enzyme, glyceraldehyde-3-phosphate dehydrogenase
(GAPDH), which is inactivated by glutathionylation, pre-
sumably of its active-site Cys149 [13–17].
In addition to cytosolic GAPDHs, plants also contain
chloroplast GAPDH isoforms that participate in the
Calvin cycle by catalyzing the reduction of 1,3-bisphos-
phoglyceric acid (BPGA) to glyceraldehyde-3-phosphate
using NAD(P)H as a reductant. All GAPDHs, including
chloroplastic isoforms, share a common reaction mech-
anism based on a highly reactive cysteine (Cys149),
which is made acidic by an interaction with His176 [18].
During the catalytic cycle, the highly reactive thiolated
group of Cys149 (Cys-S
–

) forms a thioacylenzyme inter-
mediate by nucleophilic attack on the substrate [19,20].
As a side-effect, the acidic nature of Cys149 makes it
particularly prone to oxidation and to other redox modi-
fications of its thiol group [15,21,22]. In glycolytic
mammalian GAPDH, these modifications include S-glu-
tathionylation, S-nitrosylation [15,23–26] and formation
of an intrasubunit disulfide with the neighboring Cys153
[27]. However, although chloroplasts are a major site of
reactive oxygen species (ROS) production, particularly
under photoinhibitory conditions [28], it is not known
whether chloroplast GAPDH is also subject to thiol oxi-
dation or glutathionylation, or any other redox reaction
affecting catalytic Cys149.
The major isoform of chloroplast GAPDHs in
higher plants displays the A
n
B
n
structure and is regula-
ted by metabolites and thioredoxin f (TRX f), a small
disulfide oxidoreductase involved in the activation of
the Calvin cycle by light [29–33]. B subunits are almost
identical to A subunits, except for the presence of a
C-terminal extension containing a pair of cysteines,
which is the target of TRX regulation [34]. Oxidized
thioredoxin and low NADP(H) ⁄ NAD(H) ratios pro-
mote the aggregation of fully active A
2
B

2
-GAPDH
into A
8
B
8
-GAPDH hexadecamers with partially inhib-
ited NADPH-dependent activity, whereas the secon-
dary activity with NADH as a coenzyme is
constitutively low and not regulated [35]. A second iso-
form of chloroplast GAPDH is homotetrameric (A
4
-
GAPDH) and TRX-insensitive [36,37], but can be
regulated by the redox-sensitive peptide, CP12, which
promotes the formation of a supramolecular complex
with phosphoribulokinase [35,38–40]. Besides regula-
tion of chloroplast GAPDH by photosynthetically
reduced TRX and pyridine nucleotides, a type of regu-
lation that is primarily linked to light⁄ dark conditions
in the chloroplast, post-translational modifications of
catalytic Cys149, such as glutathionylation, might con-
stitute novel mechanisms of GAPDH regulation under
oxidative stress.
Compared with nonphotosynthetic organisms, very
little is known about glutathionylation in plants,
although recent studies have allowed the identification
of a number of plant proteins undergoing glutathiony-
lation [41–46]. We have recently shown that chloro-
plast f-type TRXs are modified by glutathionylation,

resulting in less efficient activation of TRX-sensitive
GAPDH and NADP-malate dehydrogenase in the
light [46]. Fructose-1,6-bisphosphate aldolase, a mem-
ber of the Calvin cycle, like GAPDH, has also been
reported to undergo glutathionylation [44].
In order to investigate whether chloroplast GAPDH
may undergo glutathionylation and other redox modi-
fications, we examined the effect of oxidized glutathi-
one (GSSG) and other oxidizing molecules, including
H
2
O
2
, on recombinant Arabidopsis thaliana A
4
-GAPDH
and on native spinach A
2
B
2
-GAPDH and A
8
B
8
-GAP-
DH. We examined the effect of these modifications on
the enzyme activities, their reversibility in the presence
of reductants, and the protective effect of the substrate
BPGA and cofactors. Glutathionylation was investi-
gated by MALDI-TOF mass spectrometry and site-

directed mutagenesis. The results indicate that
glutathionylation might constitute a previously unde-
scribed mechanism of redox regulation and ⁄ or
protection against oxidative damage of chloroplastic
A
4
-GAPDH.
Results
Inactivation of A
4
-GAPDH by GSSG and other
oxidants, and protection by substrate and
cofactors
Incubation of recombinant Arabidopsis A
4
-GAPDH
with 5 mm GSSG resulted in a rapid decrease in
enzyme activity. Indeed, the NADPH-dependent activ-
ity decreased to less than 20% after 10 min of incuba-
tion (Fig. 1A) and identical results were obtained
with NADH as the coenzyme (data not shown). The
decrease in GAPDH activity displayed a linear rela-
tionship with increasing GSSG concentrations in the
0–5 mm range (Fig. 1B). A slow, reproducible loss of
activity, observed when the enzyme was incubated in
buffer alone or with reduced glutathione (GSH) as a
control, suggested that A
4
-GAPDH underwent sponta-
neous inactivation upon dilution. The exposure of

A
4
-GAPDH to 1 mm H
2
O
2
, CuCl
2
, or diamide also
caused a rapid loss of enzyme activity (Fig. 2). Kinet-
ics of inactivation were comparable for the three
M. Zaffagnini et al. Glutathionylation of chloroplastic GAPDH
FEBS Journal 274 (2007) 212–226 ª 2006 The Authors Journal compilation ª 2006 FEBS 213
different oxidants and led to a complete loss of activity
within 2 min of incubation. In the presence of a 10-
fold lower concentration of H
2
O
2
(0.1 mm, Fig. 2), the
inactivation kinetics was comparable to that obtained
in the presence of 5 mm GSSG (Fig. 1). These results
are consistent with the extreme sensitivity to oxidants
described for glycolytic GAPDH [15,18].
In order to determine if substrate and cofactors
might protect A
4
-GAPDH from glutathionylation
and ⁄ or oxidation, the enzyme was incubated with
GSSG or diamide in the presence of BPGA or

NADPH. Both the substrate and the cofactor appeared
to protect efficiently the enzyme from the two inactivat-
ing treatments (Fig. 3A). In contrast, protection against
the small H
2
O
2
molecule was only observed in the pres-
ence of BPGA (Fig. 3B). Moreover, in the presence of
BPGA, but not in the presence of NADPH, the enzyme
remained totally active during the time-course of incu-
bation (Fig. 3), suggesting protection from the slow
spontaneous inactivation of A
4
-GAPDH observed in
the control samples (Fig. 1). Similar results were
obtained with NADH as a cofactor (data not shown).
Glutathionylation of chloroplastic A
4
-GAPDH
by GSSG is reversible
In order to test the reversibility of A
4
-GAPDH inacti-
vation, the enzyme was treated with 10 mm dithiothrei-
tol after pre-incubation with 5 mm GSSG, 1 mm
diamide or 1 mm H
2
O
2.

As shown in Fig. 4, enzyme
inactivation caused by 1 mm diamide or H
2
O
2
could
not be reversed by dithiothreitol, suggesting that these
oxidants induced irreversible oxidation, possibly affect-
ing the sulfhydryl group of the active site Cys149. Irre-
versibly oxidized cysteine thiols are typically converted
to sulfinic (-SO
2
H) or sulfonic (-SO
3
H) acids [22]. On
the other hand, inactivation caused by GSSG was par-
tially (60%) reversed by dithiothreitol, although the
remaining 40% of the initial activity was still irrevers-
ibly lost. These results clearly indicate that oxidants,
such as diamide or H
2
O
2
, led to A
4
-GAPDH inactiva-
tion by irreversible oxidation, whereas GSSG, at least
in part, inhibited A
4
-GAPDH by a different and

reversible mechanism, possibly involving glutathionyla-
tion.
A
B
GSSG Concentration (mM)
0246
A
4
nim01retfaytivitcaHDPAG-
)%(tnemtaertGSSGfo
0
20
40
60
80
100
Time (min)
0246810121416
A
4
)%()HPDAN(ytivitcAHPDAG-
0
20
40
60
80
100
Buffer or GSH
GSSG
Fig. 1. Inactivation of A

4
-GAPDH by GSSG. (A) Time-dependent
inactivation. A
4
-GAPDH was incubated with 5 mM GSSG (open cir-
cles), or with tricine buffer as a control (closed circles). Results
with 5 m
M GSH were identical to those of controls. (B) Concentra-
tion-dependent inactivation. A
4
-GAPDH was incubated with various
concentrations of GSSG for 10 min at 25 °C. Aliquots of the incuba-
tion mixtures were withdrawn at the indicated time points and the
remaining NADPH-dependent activity was determined. Activity is
given as a percentage of the initial activity.
Time (min)
0246810121416
A
4
)%()HPDAN(ytivitcAHPDAG-
0
20
40
60
80
100
Buffer
0.1 m
M H
2

O
2
1mM oxidant
Fig. 2. Inactivation of A
4
-GAPDH by oxidants. Time-dependent inac-
tivation by 1 m
M H
2
O
2
,1mM diamide or 1 mM CuCl
2
(open
squares), 0.1 m
M H
2
O
2
(closed squares) or tricine buffer as the
control (closed circles). Aliquots of the incubation mixtures were
withdrawn at the indicated time points and the remaining NADPH-
dependent activity was determined. Activity is given as a percent-
age of the initial activity.
Glutathionylation of chloroplastic GAPDH M. Zaffagnini et al.
214 FEBS Journal 274 (2007) 212–226 ª 2006 The Authors Journal compilation ª 2006 FEBS
In order to test this hypothesis, GSSG-treated A
4
-
GAPDH was analyzed by MALDI-TOF mass spectro-

metry (Fig. 5). A clear shift in molecular mass was
observed after GSSG treatment. The shift of the major
peak, corresponding to GAPDH subunit A (theoretical
molecular mass 36 346 Da), is consistent with the pres-
ence of one glutathione adduct per subunit (theoretical
additional mass ¼ 305 Da). The other peaks observed
correspond to matrix adducts on A
4
-GAPDH. More-
over, upon the addition of dithiothreitol, the molecular
mass of A
4
-GAPDH shifted back to the mass of the
untreated protein. These results clearly demonstrate
that Arabidopsis A
4
-GAPDH can undergo glutathiony-
lation, and that dithiothreitol can reverse this post-
translational modification.
Glutathionylation of chloroplastic A
4
-GAPDH by
GSH in the presence of low concentrations of
hydrogen peroxide is fully reversible
Besides thiol disulfide exchange mediated by GSSG, it
has been reported that protein glutathionylation can
also be achieved in the presence of GSH and oxidants,
conditions that are believed to promote the conversion
of protein thiols into sulfenic acids which then react
with GSH to give rise to mixed disulfides [47–49].

Incubation of Arabidopsis A
4
-GAPDH with 0.1 mm
H
2
O
2
and 0.5 mm GSH (Fig. 6A) resulted in a rapid
decrease of enzyme activity with kinetics comparable
to that obtained in the presence of 0.1 mm H
2
O
2
alone
(Table 1). Moreover, as in the case of 0.1 mm H
2
O
2
alone, BPGA appeared to provide full protection from
inactivation, whereas almost no protection was
observed in the presence of NADPH.
The addition of dithiothreitol provided nearly 50%
recovery of the initial activity of samples inactivated
by 0.1 mm H
2
O
2
(Fig. 6B). This partial recovery, not
observed after treatment with 1 mm H
2

O
2
(Fig. 4),
indicated that part of the A
4
-GAPDH molecules were
reversibly oxidized, probably to sulfenic acid (-SOH)
or, by analogy to animal GAPDH [27], an intrasubunit
disulfide with Cys153 (conserved in chloroplast GAPDH
[50]) might have formed. Indeed, both sulfenic acids and
disulfides are generally reduced by dithiothreitol. On the
other hand, an almost complete recovery of the initial
A
B
Time (min)
0246810121416
A
4
)%()HPDAN(ytivitcAHPDAG-
0
20
40
60
80
100
BPGA then H
2
O
2
(0.1 mM)

NADPH then H
2
O
2
(0.1 mM)
H
2
O
2
(0.1 mM)
Time (min)
0246810121416
A
4
)%()HPDAN(ytivitcAHPDAG-
0
20
40
60
80
100
BPGA then (GSSG or Diamide)
NADPH then (GSSG or Diamide)
GSSG
Diamide
Fig. 3. Protection of A
4
-GAPDH by BPGA and NADPH. A
4
-GAPDH

was incubated in the presence of (A) 5 m
M GSSG or 1 mM diamide
in the presence of BPGA (open triangles) or 0.2 m
M NADPH
(closed triangles) at 25 °C, or (B) 0.1 m
M H
2
O
2
in the presence of
BPGA (open diamonds) or 0.2 m
M NADPH (closed diamonds). Inac-
tivation by 5 m
M GSSG (open circles), 1 mM diamide (closed
circles), or 0.1 m
M H
2
O
2
(open squares) are presented for compar-
ison. NADPH-dependent activity is given as a percentage of the
initial activity.
A
4
)%()HPDAN(ytivitcAHPDAG-
0
20
40
60
80

100
+dithiothreitol
+dithiothreitol
+dithiothreitol
GSSG
5 m
M
Diamide
1 m
M
H
2
O
2
1 mM
Fig. 4. Reversal, by dithiothreitol, of the inactivation of pretreat-
ed A
4
-GAPDH. A
4
-GAPDH was incubated with 5 mM GSSG, 1 mM
diamide or 1 mM H
2
O
2
for 10 min and subsequently treated with
10 m
M dithiothreitol for 10 min at 25 °C. The remaining NADPH-
dependent activity was determined before (black bars) and after
(white bars) treatment with dithiothreitol. Activities are given as a

percentage of the initial activity measured before the inactivation
treatment.
M. Zaffagnini et al. Glutathionylation of chloroplastic GAPDH
FEBS Journal 274 (2007) 212–226 ª 2006 The Authors Journal compilation ª 2006 FEBS 215
activity upon the addition of dithiothreitol was observed
for A
4
-GAPDH samples treated with 0.1 mm H
2
O
2
and
0.5 mm GSH, either in the presence or absence of
NADPH. This suggested that, under the latter condi-
tions, the mechanism of inactivation might have been
different a n d may involve glutathionylation. This h ypo-
thesis wa s confirmed by MALDI-TOF ma ss spectro-
metry, which demonstrated a 305 Da mass increase for
 50% of the A
4
-GAPDH subunits in the samples trea-
tedwith0.1mm H
2
O
2
and 0.5 mm GSH (Fig. 7).
Hence, these results clearly d emonstrate t hat A
4
-GAPDH
can undergo glutathionylation in the presence of low

concentrations of H
2
O
2
and GSH. Moreover, the com-
plete recovery of the initial activity after dithiothreitol
treatment indicates that glutathionylation is able to
protect A
4
-GAPDH from irreversible oxidation.
Identification of the cysteine residue involved
in the inactivation of A
4
-GAPDH
The observation that BPGA fully protects GAPDH
from oxidation and ⁄ or glutathionylation suggests
indeed that cysteines of the active site are the targets
of the redox modification. Besides Cys149, which
forms a covalent thioacylenzyme with BPGA during
the catalytic cycle, Cys153 is also in proximity to the
active site, whereas the remaining three cysteines of
Arabidopsis A
4
-GAPDH reside in different regions of
the protein [51]. In theory, inactivation of A
4
-GAPDH
by glutathione and ⁄ or oxidants could thus depend on
either Cys149 or Cys153 being redox modified. High
performance liquid chromatography and MALDI-

TOF mass spectrometry analyses of tryptic peptides of
A
4
-GAPDH could not clarify this ambiguity because
both Cys149 and Cys153 belong to a single long pep-
tide which was very weakly desorbed ⁄ ionized from the
matrix. This point was thus addressed by site-directed
mutagenesis of Cys153. Mutation of Cys149 was not
attempted, because this residue is known to be abso-
lutely essential for catalysis [20]. On the other hand,
the activity of the purified recombinant C153S mutant
was similar to that of the wild-type protein (data not
shown), and the kinetics of inactivation in the presence
of 1 mm H
2
O
2
, 0.1 mm H
2
O
2
, or 0.1 mm H
2
O
2
+
0.5 mm GSH were identical for both proteins
(Fig. 8A,B and Table 1). Similarly to wild-type GAPDH,
BPGA (but not NADPH) protected the mutant from
irreversible oxidation by H

2
O
2
and glutathionylation
by H
2
O
2
+ GSH. MALDI-TOF mass spectrometry
confirmed that C153S A
4
-GAPDH underwent gluta-
thionylation after treatment with 0.1 mm H
2
O
2
+
0.5 mm GSH (Fig. 9). The spectra are comparable to
those obtained for the wild-type enzyme, showing a
similar reversion of the 305 Da shift after dithiothreitol
treatment. Overall, these results rule out the possibility
that Cys153 might be the target of glutathionylation or
% Intensity
B
+GSSG dithiothreitol
36400
36353
36663
36800 37200 3800037600
36000

36400 36800 37200 3800037600
36000
% Intensity
A
0
20
40
60
80
100
0
20
40
60
80
100
Mass (m/z)
Mass (m/z)
Fig. 5. MALDI-TOF mass spectrometry indi-
cates that A
4
-GAPDH undergoes glutath-
ionylation. Mass spectra of GSSG treated
A
4
-GAPDH were performed before and after
treatment with 10 m
M dithiothreitol
(20 min). After dithiothreitol treatment (A),
A

4
-GAPDH was at its expected mass
(36 346 Da) and shows a decrease of
310 Da in comparison with the spectrum
before dithiothreitol treatment (B). Accuracy
of the measurement is ± 7 Da (0.02%).
Glutathionylation of chloroplastic GAPDH M. Zaffagnini et al.
216 FEBS Journal 274 (2007) 212–226 ª 2006 The Authors Journal compilation ª 2006 FEBS
irreversible oxidation. Although a conformational
change after glutathionylation of a cysteine far distant
from the active site cannot be completely excluded, the
results strongly suggest that Cys149 is the target of
chloroplast GAPDH glutathionylation or irreversible
oxidation.
Nonetheless, Cys153 seemed to play some role in
GAPDH protection against irreversible oxidation. In
fact, the activity of mutant C153S treated with 0.1 mm
H
2
O
2
+ 0.5 m m GSH could not be completely recov-
ered by adding dithiothreitol, even when glutathionyla-
tion was performed in the presence of NADPH
(compare Figs 8C and 6B). This indicates that under
mild oxidizing conditions (0.1 mm H
2
O
2
), mutant

C153S was not completely protected by GSH and par-
tially underwent irreversible oxidation. Moreover, the
recovery by dithiothreitol after treatment with 0.1 mm
H
2
O
2
alone was also much lower in mutant C153S
than in wild-type A
4
-GAPDH (Figs 8C and 6B,
respectively). It is possible that the role played by
Cys153 in preventing the irreversible oxidation of
Cys149 might depend on the formation of a (transient)
disulfide between Cys149 and Cys153, as observed in
human GAPDH [27].
A
n
B
n
-GAPDH is sensitive to oxidants, but is not
significantly prone to glutathionylation
Besides A
4
-GAPDH, the major chloroplast GAPDH
isoform of higher plants comprises A and B subunits in
a stoichiometric ratio and is regulated by thioredoxins
and metabolites [35]. As attempts to produce recombin-
ant Arabidopsis A
n

B
n
GAPDH were unsuccessful, we
purified native A
n
B
n
GAPDH from spinach chloroplasts
in order to test its sensitivity to oxidants and its ability
to undergo glutathionylation. A
n
B
n
-GAPDH exists in
different conformations, and experiments were conduc-
ted on either fully active A
2
B
2
-GAPDH (conformation
prevailing in chloroplasts in the light) or A
8
B
8
-GAPDH
(‘dark’ conformation with partially inhibited NADPH-
dependent activity). Although both forms were inacti-
vated by H
2
O

2
treatment, with or without GSH
(Fig. 10A,B), hexadecameric A
8
B
8
-GAPDH was clearly
less sensitive to H
2
O
2
than A
2
B
2
-GAPDH (Table 1).
Similar results were obtained with NADH as a cofactor
(data not shown), indicating that treatment with H
2
O
2
did not affect the redox regulation (mediated by C-ter-
minal extensions) of the enzyme, a process which is
thioredoxin-dependent and specific for the NADPH-
linked activity of the enzyme [34]. Furthermore, BPGA
protected A
2
B
2
-GAPDH from inactivation, as in the

case of A
4
-GAPDH, suggesting that catalytic Cys149
was the target of the redox modification (Fig. 10A). Pro-
tection by BPGA of the A
8
B
8
isoform could not be
tested because BPGA incubation is known to convert
the hexadecamer into active tetramers [32].
In order to test the reversibility of A
n
B
n
inactiva-
tion, the enzymes were treated with 10 mm dithiothrei-
tol for 10 min after the oxidative treatment. Similarly
to A
4
-GAPDH, dithiothreitol treatment of A
2
B
2
GAPDH, after incubation with 0.1 mm H
2
O
2
, allowed
A

4
)%()HPDAN(ytivitcAHPDAG-
0
20
40
60
80
100
A
B
H
2
O
2
(0.1 mM)
H
2
O
2
(0.1 mM)
+GSH
H
2
O
2
(0.1 mM)
+ GSH
+ NADPH
+dithiothreitol
+dithiothreitol

+dithiothreitol
Time (min)
0246810121416
A
4
)%()HPDAN(ytivitcAHPDAG-
0
20
40
60
80
100
BPGA then H
2
O
2
(0.1 mM)+GSH
H
2
O
2
(0.1 mM)+GSH
NADPH then H
2
O
2
(0.1 mM)+GSH
Fig. 6. Inactivation of A
4
-GAPDH in the presence of H

2
O
2
and
GSH. (A) Protective effect of NADPH and BPGA. A
4
-GAPDH was
incubated with 0.1 m
M H
2
O
2
and 0.5 mM GSH alone (open trian-
gles), or in the presence of 0.2 m
M NADPH (closed circles) or
BPGA (closed triangles). NADPH-dependent activity is given as a
percentage of the initial activity. (B) Reversal of A
4
-GAPDH inactiva-
tion by dithiothreitol. A
4
-GAPDH was inactivated by incubation with
0.1 m
M H
2
O
2
, alone or in the presence of 0.5 mM GSH, or 0.2 mM
NADPH and 0.5 mM GSH for 10 min at 25 °C and subsequently
treated with 10 m

M dithiothreitol for 10 min at 25 °C. The NADPH-
dependent activity was determined before (black bars) and after
(white bars) treatment with dithiothreitol. Activities are given as a
percentage of the initial activity measured before the inactivation
treatment.
M. Zaffagnini et al. Glutathionylation of chloroplastic GAPDH
FEBS Journal 274 (2007) 212–226 ª 2006 The Authors Journal compilation ª 2006 FEBS 217
a partial recovery of the initial enzyme activity
(Fig. 10C). However, when dithiothreitol treatment
was performed after inactivation with 0.1 mm H
2
O
2
+
0.5 mm GSH, the recovery was only slightly improved.
This result contrasts with the almost complete recovery
observed for A
4
-GAPDH in the same conditions
(Fig. 6B) and suggests that A
2
B
2
GAPDH might not
be significantly glutathionylated. In the case of A
8
B
8
GAPDH, the lower sensitivity of the enzyme to oxi-
dants led to a more complete recovery of the initial

activity after dithiothreitol treatment of samples trea-
ted either with 0.1 mm H
2
O
2
alone or in the presence
of 0.5 mm GSH (Fig. 10C). Therefore, A
n
B
n
-GAPDH
samples incubated with 0.1 mm H
2
O
2
+ 0.5 mm GSH,
with or without subsequent dithiothreitol treatment,
were analyzed by MALDI-TOF mass spectrometry
(Fig. 11). No significant shift of the peak correspond-
ing to B subunits (theoretical mass ¼ 39 357 Da) was
Table 1. Half-time inactivation (in min) of wild-type A
4
-glyceraldehyde-3-phosphate dehydrogenase (A
4
-GAPDH), C153S A
4
-GAPDH,
A
2
B

2
-GAPDH and A
8
B
8
-GAPDH under different oxidative treatments. Results are presented as mean ± standard deviation (SD), representa-
tive of at least three independent experiments. Because A
2
B
2
-GAPDH and A
8
B
8
-GAPDH were prepared by the addition of 0.2 mM NADP(H)
or NAD(H), respectively, the kinetics in the absence of cofactors could not be determined (ND).
Oxidative Treatment
0.1 m
M H
2
O
2
0.1 mM H
2
O
2
+ 0.2 mM NAD(P)H
H
2
O

2
0.1 mM
+ 0.5 mM GSH
0.1 m
M H
2
O
2
+ 0.5 mM GSH
+ 0.2 m
M NAD(P)H
Wild-type A
4
-GAPDH
half time of inactivation (min)
2.28 ± 0.37 3.59 ± 0.29 1.64 ± 0.16 3.94 ± 0.23
C153S A
4
-GAPDH
half time of inactivation (min)
2.04 ± 0.21 3.70 ± 0.10 1.87 ± 0.11 3.76 ± 0.81
A
2
B
2
-GAPDH
half time of inactivation (min)
ND 4.75 ± 0.66 ND 3.53 ± 0.11
A
8

B
8
-GAPDH
half time of inactivation (min)
ND 7.88 ± 0.31 ND 9.56 ± 1.32
% Intensity
0.54363
0.44663
2.14
3
63
3560035000 36200 36800
38000
37400
Mass (m/z)
35600 36200 36800 380003740035000
Mass (m/z)
0
20
40
60
80
100
% Intensity
0
20
40
60
80
100

H
2
O
2
+ GSH
+dithiothreitol
Fig. 7. MALDI-TOF mass spectrometry indi-
cates that A
4
-GAPDH also undergoes glu-
tathionylation in the presence of H
2
O
2
and
GSH. Mass spectra of A
4
-GAPDH treated
with 0.1 m
M H
2
O
2
and 0.5 mM GSH for 1 h
at 25 °C were performed before and after a
10 m
M dithiothreitol treatment (30 min at
25 °C). Accuracy of the measurement is
± 7 Da (0.02%).
Glutathionylation of chloroplastic GAPDH M. Zaffagnini et al.

218 FEBS Journal 274 (2007) 212–226 ª 2006 The Authors Journal compilation ª 2006 FEBS
observed after the glutathionylation treatments. For A
subunits (theoretical mass ¼ 36 141 Da), besides the
matrix adduct peak (sinapinic acid, theoretical mass
increase 205 Da), a very discrete peak, corresponding
to a 305 Da mass increase, was observed after treat-
ment with H
2
O
2
and GSH, but disappeared after sub-
sequent treatment with dithiothreitol. These results are
consistent with the recovery of activity observed after
dithiothreitol treatment (Fig. 10C). Therefore, it can
be concluded that the B subunits of A
n
B
n
-GAPDH do
not undergo glutathionylation and that glutathionyla-
tion of the A subunits is very limited. Overall, these
in vitro results indicate that glutathionylation does not
seem to play a significant role in the protection of
A
n
B
n
GAPDH isoforms against oxidative stress.
Discussion
The aims of the present study were to establish whe-

ther chloroplastic GAPDH isoforms undergo gluta-
thionylation and thiol oxidation, and to examine the
effect of these modifications on enzyme activity. The
results demonstrate that Arabidopsis A
4
-GAPDH can
undergo glutathionylation and that this post-transla-
tional modification results in inhibition of the enzyme
activity. MALDI-TOF mass spectrometry revealed the
presence of one glutathione adduct per A
4
-GAPDH
subunit, which could be removed by dithiothreitol with
a concomitant recovery of enzyme activity. Glutath-
ionylation of the protein can occur either in the pres-
ence of GSSG or in the presence of H
2
O
2
and GSH.
A
4
-GAPDH can also be irreversibly inactivated by oxi-
dants, including H
2
O
2
. The substrate BPGA, forming
a covalent intermediate with catalytic Cys149, fully
protects the enzyme from either oxidation or glutath-

ionylation. Mutant C153S, having no other cysteines
than Cys149 in the catalytic site, was oxidized and
H
2
O
2
(0.1 mM)
H
2
O
2
(0.1 mM)
+GSH
H
2
O
2
(0.1 mM)
+ GSH
+ NADPH
H
2
O
2
(1 mM)
A
4
)%(
)HPDAN(yti
v

itcAS35
1C
HPDAG-
0
20
40
60
80
100
Time (min)
0246810121416
0
20
40
60
80
A
4
)%()HPDAN(ytivitcAS351CHPDAG-
100
A
B
C
BPGA then H
2
O
2
(0.1 mM)+GSH
H
2

O
2
(0.1 mM)+GSH
NADPH then H
2
O
2
(0.1 mM)+GSH
Time (min)
0246810121416
A
4
)%()HPDAN(ytivitcAS351CHPDAG-
0
20
40
60
80
100
Buffer
NADPH then H
2
O
2
(0.1 mM)
H
2
O
2
(0.1 mM)

H
2
O
2
(1 mM)
+dithiothreitol
+dithiothreitol
+dithiothreitol
+dithiothreitol
Fig. 8. Inactivation of the C153S A
4
-GAPDH mutant and reversal by
dithiothreitol. (A) Time-dependent inactivation of C153S A
4
-GAPDH
in the presence of H
2
O
2
. C153S A
4
-GAPDH was incubated with
1m
M H
2
O
2
(open circles), 0.1 mM H
2
O

2
alone (open squares) or in
the presence of 0.2 m
M NADPH (closed squares), or in tricine buf-
fer as a control (closed circles). Aliquots of the incubation mixtures
were withdrawn at the indicated time points and the remaining
NADPH-dependent activity was determined. Activity is given as a
percentage of the initial activity. (B) Time-dependent inactivation of
C153S A
4
-GAPDH in the presence of H
2
O
2
and reduced glutathione
(GSH). C153S A
4
-GAPDH was incubated either with 0.1 mM H
2
O
2
and 0.5 mM GSH alone (open triangles) or in the presence of
0.2 m
M NADPH (closed diamonds) or BPGA (open diamonds).
Aliquots of the incubation mixtures were withdrawn at the indica-
ted time points and the remaining NADPH-dependent activity was
determined. Activity is given as a percentage of the initial activity.
(C) Reversal of C153S A
4
-GAPDH inactivation by dithiothreitol.

C153S A
4
-GAPDH was inactivated by incubation with 1 mM H
2
O
2
,
0.1 m
M H
2
O
2
, alone or in the presence of 0.5 mM GSH, or 0.2 mM
NADPH and 0.5 mM GSH for 10 min at 25 °C, and subsequently
treated with 10 m
M dithiothreitol for 10 min at 25 °C. The NADPH-
dependent activity was determined before (black bars) and after
(white bars) treatment with dithiothreitol. Activities are given as a
percentage of the initial activity measured before the inactivation
treatment.
M. Zaffagnini et al. Glutathionylation of chloroplastic GAPDH
FEBS Journal 274 (2007) 212–226 ª 2006 The Authors Journal compilation ª 2006 FEBS 219
glutathionylated similarly to wild-type A
4
-GAPDH.
Taken together, these results strongly suggest that
Arabidopsis A
4
-GAPDH can be reversibly glutathionyl-
ated and irreversibly oxidized on its catalytic Cys149.

Moreover, analysis of the C153S mutant also suggests
that the formation of a disulfide between catalytically
essential Cys149 and the neighboring Cys153 could
contribute to the protection of A
4
-GAPDH from irre-
versible oxidation.
By contrast, the results of the present study show
that A
n
B
n
GAPDH isoforms, representing the major
isoforms of photosynthetic GAPDH in chloroplasts of
higher plants, though being sensitive to oxidants, do
not undergo significant modification by glutathionyla-
tion. As A and B subunits are almost identical, except
for the C-terminal extension of subunits B, it is very
likely that in A
n
B
n
-GAPDH isoforms, the C-terminal
extension of subunits B could partially protect catalytic
Cys149 from oxidation by H
2
O
2
and effectively pre-
vent the attack of glutathione. The C-terminal exten-

sion bears a pair of TRX-sensitive cysteines and allows
A
2
B
2
-GAPDH to associate into hexadecamers in the
presence of NAD(H) [35]. The low sensitivity of A
8
B
8
-
GAPDH to H
2
O
2
alone, or in the presence of GSH, is
thus likely to depend on partial steric protection of
active sites within the hexadecamer.
Because the results presented here were obtained
in vitro, the question arises as to how important such
modifications are in vivo. In all the initial experiments
described above, we performed A
4
-GAPDH glutath-
ionylation assays in the presence of 5 mm GSSG. This
corresponds to classical conditions generally used to
test protein glutathionylation in vitro. However, this
concentration of GSSG is significantly higher than the
estimated concentration in chloroplasts. Indeed, the
concentration of the glutathione pool has been estima-

ted to be between 1 and 4.5 mm in the stroma [52] and
GSSG only represents  10% of this pool. All these
considerations suggest that glutathionylation of A
4
-
GAPDH, through nonenzymatic thiol disulfide exchange
mediated by GSSG, is probably of limited physio-
logical significance. The low efficiency of GSSG, as a
mediator of protein glutathionylation, has been repor-
ted previously [8,48]. Other possible mechanisms lead-
ing to glutathionylation might involve more reactive
oxidized forms of glutathione, such as S-nitrosoglu-
tathione and glutathione disulfide S-oxide [8], or the
initial oxidation of a protein thiol that would subse-
quently react with GSH [47–49,53]. Which of these
mechanisms determines glutathionylation of GAPDH
in vivo remains to be determined. However, the results
presented here clearly show that A
4
-GAPDH is
35000 35600 36200 36800 37400
38000
+
dithiothreitol
Mass (m/z)
% Intensity
0
20
40
60

80
100
35000 35600 36200
36324.5
36633.4
36326.0
36800 37400
38000
Mass (m/z)
% Intensity
0
20
40
60
80
100
H
2
O
2
+ GSH
Fig. 9. MALDI-TOF mass spectrometry
indicates that C153S A
4
-GAPDH undergoes
glutathionylation in the presence of H
2
O
2
and

GSH. Mass spectra of C153S A
4
-GAPDH
treated with 0.1 m
M H
2
O
2
and 0.5 mM GSH
for 1 h at 25 °C were performed before and
after treatment with 10 m
M dithiothreitol
(30 min at 25 °C). The accuracy of the
measurement is ± 7 Da (0.02%).
Glutathionylation of chloroplastic GAPDH M. Zaffagnini et al.
220 FEBS Journal 274 (2007) 212–226 ª 2006 The Authors Journal compilation ª 2006 FEBS
glutathionylated in the presence of physiologically rele-
vant concentrations of H
2
O
2
and GSH [52,54], suggest-
ing a mechanism of glutathionylation based on the
primary oxidation of the catalytic Cys149 followed by
reaction with GSH rather than GSSG. In vivo, such a
mechanism would be favored under conditions of oxi-
dative stress, leading to enhanced ROS production in
the chloroplast, such as exposure to high light under
unfavorable conditions for photosynthetic metabolism
(e.g. cold or water stress). Considering the high sensi-

tivity of A
4
-GAPDH to oxidation, the formation of a
mixed disulfide between GSH and the active site cys-
teine oxidized to sulfenic acid would prevent its irre-
versible oxidation. The results we obtained in vitro
indeed confirmed that GSH-mediated glutathionylation
of A
4
-GAPDH can effectively protect the enzyme from
irreversible oxidation.
However, besides protecting A
4
-GAPDH, glutath-
ionylation might play a more general role in the regu-
lation of photosynthesis under stress. TRX f plays a
major role in the regulation of Calvin cycle enzymes
that are mostly inactive in the dark and are activated
by TRXs under illumination [55]. Glutathionylation of
a conserved extra cysteine of TRX f, distinct from the
two active site cysteines, strongly decreases the ability
of TRX f to activate target enzymes, including
GAPDH isoforms containing B subunits [46]. This
suggests that, under conditions leading to protein glu-
tathionylation in the chloroplast (e.g. under conditions
of enhanced ROS production), the activity of TRX-
dependent Calvin cycle enzymes would be decreased.
In particular, A
n
B

n
-GAPDH would be down-regulated
A
B
C
Time (min)
0 2 4 6 8 10 12 14 16
A
8
B
8
)%()HPDAN(ytivitcAHPDAG-
0
20
40
60
80
100
Buffer
H
2
O
2
(0.1 mM)
H
2
O
2
(1 mM)
H

2
O
2
(0.1 mM)+GSH
Time (min)
0 2 4 6 8 10 12 14 16
A
2
B
2
)%()HPDAN(ytivitcAHPDAG-
0
20
40
60
80
100
Buffer
H
2
O
2
(0.1 mM)
H
2
O
2
(1 mM)
BPGA then H
2

O
2
(0.1 mM)+GSH
H
2
O
2
(0.1 mM)+GSH
)%()HPDAN(ytivitcAHPDAG
0
20
40
60
80
100
H
2
O
2
)
Mm
1.
0(
H
2
O
2
)Mm
1
.0(

HSG+
H
2
O
2
)Mm
1
(
A
2
B
2
A
8
B
8
H
2
O
2
)
Mm
1.
0(
H
2
O
2
)Mm
1

.0(
HSG
+
H
2
O
2
)Mm1(
+dithiothreitol
+dithiothreitol
+dithiothreitol
+dithiothreitol
+dithiothreitol
+dithiothreitol
Fig. 10. Inactivation of A
n
B
n
-glyceraldehyde-3-phosphate dehydroge-
nase (A
n
B
n
-GAPDH) and reversal by dithiothreitol. A time-dependent
inactivation of nonaggregated A
n
B
n
-GAPDH (tetrameric form) by
1m

M H
2
O
2
(open circles), 0.1 mM H
2
O
2
alone (open squares),
0.1 m
M H
2
O
2
and 0.5 mM GSH, in the absence (open triangles)
or presence of BPGA (open diamonds), or in tricine buffer as a con-
trol (closed circles). Aliquots of the incubation mixtures were
withdrawn at the indicated time points, and the remaining NADPH-
dependent activity was determined. Activity is given as a percent-
age of the initial activity. (B) Time-dependent inactivation of
aggregated A
n
B
n
-GAPDH (hexadecameric form) by 1 mM H
2
O
2
(open circles), 0.1 mM H
2

O
2
alone (open squares) or in the pres-
ence of 0.5 m
M GSH (open triangles), or in K-phosphate buffer as
the control (closed circles). Aliquots of the incubation mixtures
were withdrawn at the indicated time points and the remaining
NADPH-dependent activity was determined. Activity is given as a
percentage of the initial activity. Note that the specific activity of
A
8
B
8
-GAPDH with NADPH as the coenzyme is about fourfold lower
than that of A
2
B
2
-GAPDH. (C) Reversal of A
n
B
n
-GAPDH inactivation
by dithiothreitol. Nonaggregated and aggregated forms of GAPDH
were inactivated by incubation with either 1 m
M H
2
O
2
or 0.1 mM

H
2
O
2
, with or without 0.5 mM GSH, for 10 min at 25 °C, and sub-
sequently treated with 10 m
M dithiothreitol for 10 min at 25 °C.
The NADPH-dependent activity was determined before (black bars)
and after (white bars) treatment with dithiothreitol. Activities are
given as a percentage of the initial activity measured before the
inactivation treatment.
M. Zaffagnini et al. Glutathionylation of chloroplastic GAPDH
FEBS Journal 274 (2007) 212–226 ª 2006 The Authors Journal compilation ª 2006 FEBS 221
through glutathionylation of TRX f, whereas A
4
-
GAPDH would be directly inhibited through glutath-
ionylation of its catalytic cysteine. The activity of
another Calvin cycle enzyme, fructose-1,6-bisphosphate
aldolase, could also be inhibited by glutathionylation
[44]. Hence, all these considerations suggest that glu-
tathionylation could contribute to slowing down the
Calvin cycle under stress.
A general down-regulation of the Calvin cycle under
stress would allow redistribution of the reducing power
in the chloroplast by decreasing NADPH consumption
and thereby increasing electron availability at the redu-
cing side of photosystem I. As a consequence, more
reductants would be available for ROS detoxifying
enzymes, such as ascorbate peroxidase, monodehydro-

ascorbate reductase, glutathione peroxidase or pero-
xiredoxins [56–59]. Alternatively, over-reduction of
photosystem I could lead to enhanced ROS production
and thereby reinforce the initial oxidative signal.
Clearly, further work is required to identify the factors
that determine the distribution of reductants between
the various photosystem I electron acceptors and to
elucidate the complex interplay between the different
redox factors that regulate chloroplast metabolism.
In any case, reducing power is required for the
deglutathionylation of proteins and recovery of their
enzymatic activity once ROS accumulation has been
overcome. In nonphotosynthetic organisms, glutare-
doxins, small oxidoreductases of the thioredoxin super-
family using GSH as the electron donor, appear to be
key regulators of protein glutathionylation [60].
Indeed, glutaredoxins are very efficient catalysts of
protein deglutathionylation, although other enzymes
could also participate in these reactions [6,60,61]. In
addition, recent genomic analyses have suggested the
existence of chloroplast glutaredoxins in plants [62,63],
and proteomic studies have identified several potential
glutaredoxin targets, including the A-subunit of
GAPDH, fructose-bisphosphate aldolase and other
Calvin cycle enzymes [64]. Thus, glutaredoxins could
participate in the regulation of the Calvin cycle by
controlling deglutathionylation of A
4
-GAPDH and
other glutathionylated enzymes in the chloroplast.

Indeed we observed that glutathionylated A
4
-GAPDH
is stable in the presence of high levels of GSH (data
not shown), suggesting that an oxidoreductase, such as
a glutaredoxin, might be required for reduction of the
mixed disulfide. In this hypothesis, glutaredoxins
would be involved in a signaling pathway contributing
to the redox regulation of the Calvin cycle by control-
ling deglutathionylation of A
4
-GAPDH and other glu-
tathionylated enzymes in the chloroplast.
H
2
O
2
+ GSH
35000 36400 37800 39200 40600
42000
Mass (m/z)
35000 36400 37800
36136.2
36129.5
36347.1
36442.3
39369.2
36345.4
39357.3
39200 40600

42000
Mass (m/z)
+ dithiothreitol
% Intensity
0
20
40
60
80
100
% Intensity
0
20
40
60
80
100
Fig. 11. MALDI-TOF mass spectrometry of
A
n
B
n
-glyceraldehyde-3-phosphate dehydrog-
enase (A
n
B
n
-GAPDH). Mass spectra of A
n
B

n
GAPDH treated with 0.1 mM H
2
O
2
and
0.5 m
M GSH for 1 h at 25 °C were per-
formed before and after treatment with
10 m
M dithiothreitol (30 min at 25 °C). Accu-
racy of the measurement is ± 7 Da (0.02%).
Similar spectra were obtained with A
2
B
2
and A
8
B
8
isoforms.
Glutathionylation of chloroplastic GAPDH M. Zaffagnini et al.
222 FEBS Journal 274 (2007) 212–226 ª 2006 The Authors Journal compilation ª 2006 FEBS
Experimental procedures
Reagents
Glutathione was purchased from Roche Diagnostics Cor-
poration (Indianapolis, IN, USA). All other reagents were
obtained from Sigma-Aldrich (St Louis, MO, USA).
Expression and purification of Arabidopsis
A

4
-GAPDH
The sequence encoding the putative mature form of the
A. thaliana plastidial A
4
-GAPDH isoform (GapA-1 cDNA
At3g26650 provided by TAIR, Stanford, CA, USA) was
amplified by PCR using the following primers: forward
(NcoI site underlined), 5¢-TGTGA
CCATGGCCAAGC-3¢;
reverse (BamHI site underlined), 5¢-CAA
GGATCCCTCA
CTTC-3¢. The PCR product was digested by NcoI and
BamHI restriction enzymes and cloned into the expression
vector pET-28a(+) (Novagen, Barmstadt, Germany), diges-
ted by the same enzymes. Site-specific mutant C153S was
constructed by PCR. The following primers, with the muta-
tions indicated in bold, were used according to the proce-
dures of the QuickChange site-directed mutagenesis kit
(Stratagene, La Jolla, CA, USA): C153S-up, 5¢-TGCATC
TTGCACTACCAACTCTCTTGCTCCCTTTGTC-3¢; and
C153S-down, 5¢-TGACAAAGGGAG CAAGA GAGTTGG
TAGTGCAAGATGC-3¢. The recombinant plasmids were
introduced into Escherichia coli BL21 (DE3) for overexpres-
sion of the protein. Production and purification procedures
were performed as previously described [65]. The final prep-
arations were electrophoretically pure, as judged by
SDS ⁄ PAGE and Coomassie blue staining.
Purification and preparation of native
A

n
B
n
-GAPDH from spinach leaves
Native A
n
B
n
-GAPDH was purified from spinach chloro-
plasts, as previously described [34]. Native enzyme prepar-
ation were pure, as judged by SDS ⁄ PAGE. Purified protein
was desalted in 25 mm potassium phosphate, pH 7.5, stored
at 4 °C and used within the next 48–72 h. The protein con-
centration was determined by the Bradford method, using
bovine serum albumin as a standard. Nonaggregated and
aggregated A
n
B
n
-GAPDH were obtained by incubating the
enzyme on ice for 2 h in the presence of 0.2 mm NADP
(nonaggregating condition) or 0.2 mm NAD (aggregating
condition). Following incubation, the aggregation state of
A
n
B
n
-GAPDH samples was analyzed by size-exclusion
chromatography on a Superdex 200 HR 10 ⁄ 30 column con-
nected to an A

˚
KTA Purifier system (GE Healthcare
Bioscience AB, Uppsala, Sweden), as described previously
[65]. Fractions of 350 l L were collected and assayed for
NAD(P)H-GAPDH activity. Active fractions corresponding
to A
2
B
2
and A
8
B
8
forms were pooled separately and
desalted, before each treatment, in 100 mm Tricine, pH 7.9,
0.2 mm NADP, or 25 mm K-phosphate, pH 7.5, 0.2 mm
NAD, respectively.
Enzyme treatments and activity measurements
Before each experiment, the enzyme storage buffer was
exchanged for 100 mm Tricine-NaOH, pH 7.9, by a pas-
sage through a Sephadex G-25 HiTrap Fast Desalting
column (Amersham Bioscience AB, Uppsala, Sweden).
The protein concentration was determined spectrophoto-
metrically (e
280
¼ 149.2 mm
)1
Æcm
)1
). All incubations of

A
4
-GAPDH (2.5 lm final concentration) were performed
in 100 mm Tricine buffer (pH 7.9) at 25 °C in the pres-
ence of GSSG, GSH, diamide, H
2
O
2
or CuCl
2
at the
indicated concentrations. Aliquots were withdrawn for the
assay of enzyme activity, monitored as described previ-
ously [34]. BPGA was prepared using 5 unitsÆmL
)1
of
3-phosphoglycerate kinase, 2 mm ATP and 3 mm 3-phos-
phoglycerate.
MALDI-TOF mass spectrometry
All spectra were acquired in positive-ion mode on a Voy-
ager DE-STR MALDI-TOF mass spectrometer (Applied
Biosystems, Foster City, CA, USA) equipped with a 337-
nm nitrogen laser.
Determinations of the molecular masses of the A
4
-GAP-
DH, C153S A
4
-GAPDH and A
n

B
n
-GAPDH proteins were
performed in linear mode (accelerating voltage, 25 kV; grid
voltage, 93%; guide wire, 0.3%; delay, 600 ns) with exter-
nal calibration using sinapinic acid as matrix after proteins
were previously concentrated and desalted with C4 reverse-
phase Zip-Tip (Millipore, Bedford, MA, USA) following
the recommendations of the manufacturer.
Replicates
All the results reported are representative of at least three
independent experiments and expressed as mean ± stand-
ard deviation.
Acknowledgements
We thank Eliane Keryer for expert technical assistance.
This work was supported, in part, by Agence Nationale
de la Recherche Grant JC05-45751 (to SDL) and in
part by Ministero dell’Istruzione, Universita
`
e Ricerca
(PRIN 2005 to MZ, FS and PT).
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