Nitric Oxide-induced S-Glutathionylation and
Inactivation of Glyceraldehyde-3-phosphate Dehydrogenase*
Susanne
Mohr
,
Hazem
Hallak§,
Alexander
de Boitte¶,
Eduardo G.
Lapetina
, and
Bernhard
Brüne¶
From the
Molecular Cardiovascular Research Center,
Case Western Reserve University School of Medicine and the University
Hospitals of Cleveland, Cleveland, Ohio 44106-4958, the
§ Department of Cell Biology, Cleveland Clinic Foundation,
Cleveland, Ohio 44195, and the ¶ Faculty of Medicine, Department
of Medicine IV-Experimental Division, University of
Erlangen-Nürnberg, 91054 Erlangen, Germany
 |
ABSTRACT |
S-Nitrosylation of protein thiol
groups by nitric oxide (NO) is a widely recognized protein
modification. In this study we show that nitrosonium tetrafluoroborate
(BF4NO), a NO+ donor, modified the thiol groups
of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) by
S-nitrosylation and caused enzyme inhibition. The resultant
protein-S-nitrosothiol was found to be unstable and to
decompose spontaneously, thereby restoring enzyme activity. In
contrast, the NO-releasing compound S-nitrosoglutathione
(GSNO) promoted S-glutathionylation of a thiol group of
GAPDH both in vitro and under cellular conditions. The
GSH-mixed protein disulfide formed led to a permanent enzyme
inhibition, but upon dithiothreitol addition a functional active GAPDH
was recovered. This S-glutathionylation is specific for
GSNO because GSH itself was unable to produce protein-mixed disulfides.
During cellular nitrosative stress, the production of intracellular
GSNO might channel signaling responses to form protein-mixed disulfide
that can regulate intracellular function.
 |
INTRODUCTION |
Nitric oxide (NO)1 is an
important biological messenger that plays a role in physiological and
pathophysiological conditions such as endothelium-dependent
vasorelaxation, inflammation, and septic shock (1, 2). These multiple
effects are based on its redox chemistry. NO can react with oxygen
species and transition metals to form NOx, peroxynitrite
(ONOO
), and metal-NO adducts, respectively (3, 4). The
interactions of NO with sulfhydryl-containing molecules and enzymes has
gained considerable importance (5, 6). In many biological systems, nitrosation reactions transferring NO+ from a NO donor to a
protein S
group affect protein function. Targets for this
type of modification, among others, are bovine serum albumin (7),
tissue-type plasminogen activator (8), gyceraldehyde-3-phosphate
dehydrogenase (GAPDH) (9, 10), the
N-methyl-D-aspartate receptor (11), oncogenic p21ras (12), and transcriptional activators
(13).
The S-nitrosothiol of glutathione (GSNO) may be the most
relevant biological molecule to carry out nitrosation reactions under physiological conditions (14-16). It has been reestablished that the
actions of the endothelium-derived relaxing factor more closely resemble a low molecular weight nitrosothiol rather than the NO·
radical itself (17). However, S-nitrosothiols can decompose to form NO· and thiyl radicals (18), and the thiyl radical can
lead to the production of protein-mixed disulfides also known as
protein S-glutathionylation.
In this study, we investigated the influence of NO donors on the
glycolytic enzyme GAPDH which catalyzes the reversible oxidative phosphorylation of D-glyceraldehyde-3-phosphate by
NAD+ and inorganic phosphate. GAPDH is comprised of four
identical 37-kDa subunits. Each subunit contains four cysteines; two of them (Cys-149 and Cys-153) are located in the catalytic site of each
GAPDH subunit. The catalytically active cysteine 149 interacts with a
histidine to form a highly reactive thiolate group
(cys-S
) which is required for GAPDH activity. Recently,
it has been shown that NO inhibits GAPDH activity (15). In this study,
we examined the effect of BF4NO (a
NO+-releasing compound) and GSNO on the thiol groups of the
enzyme. Our results point to specific differences in thiol modification mechanisms that can be triggered by competing
S-nitrosylation and S-glutathionylation.
 |
EXPERIMENTAL PROCEDURES |
Materials--
[14C]N-ethylmaleimide
(40 mCi/mmol) and [35S]glutathione (83.1 Ci/mmol) were
purchased from NEN Life Science Products.
[35S]L-cysteine (1075 Ci/mmol) was from ICN.
BF4NO was obtained from Aldrich. Mouse
anti-glyceraldehyde-3-phosphate dehydrogenase monoclonal antibody was
from Chemikon. Rabbit muscle GAPDH (80 units/mg) was bought from
Boehringer Mannheim. Other chemicals were of the highest grade of
purity from Sigma.
Synthesis of GSNO--
GSNO was prepared as described previously
(19). GSNO displayed absorption maxima at 335 and 545 nm with
extinction coefficients of 922 and 15.9 dl (2) mol
1
cm
1, respectively. GSNO solutions were prepared prior to
the experiment.
Synthesis of [35S]GSNO--
Commercial
[35S]GSH solutions (83.1 Ci/mmol) were stabilized with 10 mM DTT. DTT was removed according to a published procedure (20). Briefly, the [35S]GSH solution (89 µl) was
adjusted to pH 2 with 2 N HCl and then extracted twice with 900 µl of
ethyl acetate. The [35S]GSH containing phase was used for
[35S]GSNO-synthesis. An aliquot of 82 µl of
[35S]GSH and 25 mg of GSH were dissolved in 0.2 M HCl in a total volume of 100 µl at 4 °C followed by
the addition of 5.67 mg of NaNO2. The following procedure
was carried out as described for GSNO synthesis.
[14C]N-ethylmaleimide (NEM) Labeling of
GAPDH--
GAPDH (50 µg/assay) was incubated in 100 mM Tris
buffer (pH 8.0) and different NO donors in a total volume of 100 µl
at 30 °C for 5 min, or as otherwise indicated. Proteins were
precipitated with 200 µl of 20% ice-cold trichloroacetic acid (TCA),
left on ice for 15 min, and centrifuged for 10 min at 10,000 × g and 4 °C. The pellets were washed twice with 1 ml of
ice-cold water-saturated ether and resuspended in 100 mM
Tris buffer (pH 8.0) containing 0.1% Triton X-100 and 50 µM [14C]NEM (0.17 µCi). Samples were
incubated for 1 h at 30 °C. The reaction was stopped with 200 µl of 20% ice-cold TCA. Proteins were left on ice for 15 min and
then centrifuged for 10 min at 10,000 × g and 4 °C.
The pellets were washed twice with 1 ml of ice-cold water-saturated
ether and separated in a nonreducing 11% sodium dodecyl
sulfate-polyacrylamide gel. Radioactivity was quantified using the
PhosphorImager system (Molecular Dynamics). NO donor solutions were
freshly prepared prior to the experiments. A stock solution of
BF4NO was prepared under acidic conditions (pH 2.0; 0.2 M HCl) as described previously (9).
DTT Reversibility of NO-induced GAPDH Modification--
GAPDH
(50 µg/assay) was incubated with 100 mM Tris buffer (pH
8.0) and different NO donors in a total volume of 100 µl at 30 °C.
After 5-min incubation, proteins were precipitated with 200 µl of
20% ice-cold TCA, left on ice for 15 min, and then centrifuged for 10 min at 10,000 × g and 4 °C. The pellets were washed
twice with 1 ml of ice-cold water-saturated ether and resuspended in 100 mM Tris buffer (pH 8.0) containing 10 mM
DTT. Incubations went on for 30 min at 30 °C. Proteins were
precipitated again with 200 µl of 20% ice-cold TCA, left on ice for
15 min, and then centrifuged for 10 min at 10,000 × g
and 4 °C. The pellets were washed twice with 1 ml of ice-cold
water-saturated ether and resuspended in 100 mM Tris buffer
(pH 8.0) containing 0.1% Triton X-100 and 50 µM
[14C]NEM (0.17 µCi). Incubations were then processed as
described for the [14C]NEM-labeling.
GAPDH Activity--
GAPDH (1 µg/assay) and up to 200 µM individual NO donor were incubated in 50 mM triethylammonium buffer (pH 7.5) in a total volume of 50 µl at 37 °C for 5 min. Samples were diluted into 950 µl of 50 mM triethylammonium buffer (pH 7.5) containing 50 µM arsenate, 2.4 mM glutathione, and 100 µg/ml glyceraldehyde-3-phosphate, at 37 °C. The enzymatic
reduction of NAD+ to NADH was started with the addition of
250 µM NAD+. GAPDH activity was monitored by
recording the fluorescence emission above 430 nm after excitation at
313 and 366 nm, respectively. Samples without NO donors served as controls.
Modification of GAPDH by [35S]GSNO--
GAPDH (10 µg/assay) was incubated in 100 mM Hepes buffer (pH 7.5)
and 0.5, 1, or 2 mM [35S] GSNO (45000 cpm/assay), respectively. Incubations containing GAPDH (10 µg/assay),
100 mM Hepes buffer (pH 7.5) and 0.5, 1, or 2 mM [35S]GSH (45000 cpm/assay) served as
controls. After 5 or 10 min at 37 °C, proteins were precipitated
with 200 µl of 20% ice-cold TCA, left on ice for 15 min, followed by
centrifugation for 10 min at 10,000 × g and 4 °C.
The pellets were washed twice with 1 ml of ice-cold water-saturated
ether and separated in a nonreducing 11% sodium dodecyl
sulfate-polyacrylamide gel. For the detection of radioactivity, gels
were exposed (PhosphorImager exposing cassettes) for 8 up to 30 days.
Isolation of Endothelial Cells (EC)--
Bovine aortic EC were
isolated as described (21) and were passaged and maintained in
Dulbecco's modified Eagle's and F12 media, supplemented with 5%
fetal bovine serum in a humidified atmosphere containing 5%
CO2. Confluent cultures in 10-cm dishes were made quiescent
by replacing medium with serum-free medium containing 1% gelatin for
24 h before use.
Metabolic Labeling of EC--
For protein
S-thiolation studies, EC were prelabeled by incubation with
[35S]L-cysteine (50 µCi/ml) for 2 h in
the presence of 200 ng/ml cycloheximide to block protein synthesis.
Cells were then treated with GSNO (500 µM) or hydrogen
peroxide H2O2 (500 µM),
respectively, for times indicated.
Immunoprecipitation of GAPDH--
Total cell lysate of EC was
denatured by incubation with 0.5% SDS, 50 mM sodium
phosphate, pH 8.0, and 2 mM EDTA at 90 °C, to allow
immunoprecipitation. The samples were supplemented to obtain a
composition of 50 mM sodium phosphate, pH 7.2, 1% sodium deoxycholate, 1% Triton X-100, 0.5% SDS, 150 mM NaCl, 2 mM EDTA, 5 mM NaF, 2 mM
Na4P2O7, 2 mM
Na3VO4, 1% aprotinin, and 200 µg/ml leupeptin at 4 °C. Mouse anti-GAPDH monoclonal antibody (usually at
a 1:100 dilution) was added, followed 18 h later by protein A-Sepharose. Washed immunoprecipitates were analyzed by autoradiography (for 35S) after separation in a nonreducing or reducing
11% sodium dodecyl sulfate-polyacrylamide gel.
 |
RESULTS |
We have examined the effect of nitric oxide-releasing compounds on
sulfhydryl group modifications of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). To detect the thiol modifications of the GAPDH
subunits, we used NEM which binds to free, unmodified sulfhydryl groups. Nitrosonium tetrafluoroborate (BF4NO) is an
exclusively NO+ releasing NO donor that forces
protein-S-nitrosothiol generation via a transnitrosation
reaction. In the first set of experiments we incubated GAPDH with
BF4NO for 5 min. Following protein precipitation, the
enzyme was resuspended, exposed to [14C]NEM, and
electrophoretically separated on a nonreducing 11% SDS gel (Fig.
1). A concentration of 200 µM BF4NO completely modified all accessible
cysteines of GAPDH subunits as shown by the lack of subsequent
[14C]NEM binding to GAPDH thiol groups. GAPDH that was
not exposed to BF4NO showed the highest amount of
[14C]NEM labeling (Fig. 1).

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Fig. 1.
Effect of BF4NO on
NEM-labeling of GAPDH. GAPDH (50 µg/assay) was preincubated with
BF4NO for 5 min at 30 °C. Following protein
precipitation and resuspension in 100 mM Tris (pH 8), 0.1%
Triton X-100, and 50 µM [14C]NEM (0.2 µCi/assay), incubations were carried out for 1 h at 30 °C.
Experimental details are given under "Experimental Procedures."
Results are representative of three similar experiments.
|
|
Next, we investigated the effect of the NO donor
S-nitrosoglutathione (GSNO) that may act as a
NO+-transferring agent on GAPDH modification (Fig.
2). However, GSNO can also be degraded
homolytically to produce NO· and GS· radicals. In
contrast to BF4NO, a concentration of 500 µM
GSNO was not able to block GAPDH cysteine residues within a 5-min
incubation. Only after a period of 60 min with GSNO were all accessible
thiols modified as shown by the disappearance of [14C]NEM
binding (Fig. 2). These data show a clear difference between the
actions of BF4NO and GSNO. It might indicate that the
action of GSNO is not related to NO+.

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Fig. 2.
Effect of GSNO on NEM-labeling of GAPDH.
GAPDH (50 µg/assay) was incubated with 500 µM GSNO for
the indicated times. Following individual incubations, proteins were
precipitated and resuspended in 50 µM
[14C]NEM (0.2 µCi/assay). Incubations were for 1 h
at 30 °C. For other details, see legend to Fig. 1. Similar results
were obtained in three separate experiments.
|
|
It has been established that nitrosylation of Cys-149 in the active
site of GAPDH attenuates enzymatic activity. To further characterize
the effect of NO donors on this reactive cysteine, we assessed enzyme
activity. A concentration of 200 µM BF4NO
decreased GAPDH activity by about 60% within 1 min (Fig.
3). However, attenuation of enzyme
activity was only transient, and GAPDH activity was recovered over a
time period of 15 min (Fig. 3). This effect was paralleled by
[14C]NEM labeling of the cysteines, although with a
slower progression (Fig. 3). In contrast, the effect of GSNO on GAPDH
activity was much different. GSNO promoted enzyme inhibition that was
not spontaneously reversible (Table I).
As shown in Table I, GSNO inhibited enzyme activity by 60% within 1 min, and activity was only marginally recovered within 30 min.

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Fig. 3.
Effect of BF4NO on
GAPDH activity and NEM-labeling. GAPDH (50 µg/assay) was
incubated at 30 °C in the presence of 100 µM
BF4NO. GAPDH activity ( ) was measured at the indicated
times as described under "Experimental Procedures." Additional
assays were performed for NEM labeling of GAPDH. , image quants.
Proteins were precipitated, and pellets were resuspended in 100 mM Tris (pH 8), 0.1% Triton X-100, and 50 µM
[14C]NEM (0.2 µCi/assay). Incubations were for 1 h
at 30 °C. Results are representative of three other similar
experiments.
|
|
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Table I
Enzyme inhibition of GAPDH by BF4NO or GSNO
GAPDH (1 µg/assay) was incubated with 200 µM
BF4NO or GSNO, respectively, at 37 °C for times indicated.
GAPDH enzyme activity was measured as described under "Experimental
Procedures." Results represent the mean (±S.D.) of three different
experiments.
|
|
Both, BF4NO and GSNO can produce NO+ which in
turn leads to transnitrosylation. However, the differences of the
effects of BF4NO and GSNO on GAPDH can be explained by the
fact that GSNO can be homolytically degraded to NO· and
GS· radicals. The GS· radical can cause a cysteine
modification via S-glutathionylation.
To investigate whether the permanent enzyme inhibition caused by GSNO
is related to protein-mixed disulfide formation via GS·
radicals, we synthesized radioactive [35S]GSNO. A 10-min
treatment of GAPDH with 500 µM [35S]GSNO
was sufficient to label the enzyme, thus indicating
S-glutathionylation of GAPDH (Fig.
4A). This effect was absent
when we used [35S]GSH. These data indicate that the
formation of a nitrosothiol by nitric oxide facilitates the
S-glutathionylation of GAPDH. [35S]labeling of
GAPDH was reversed by DTT, which is consistent with the formation of a
protein-mixed disulfide (Fig. 4A).
S-Glutathionylation of GAPDH by [35S]GSNO
occurred within 5 min and only slightly increased after 10 min (Fig.
4B).

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Fig. 4.
S-Glutathionylation of GAPDH.
A, GAPDH (10 µg/assay) was incubated with different
concentrations of [35S]GSNO (45,000 cpm/assay) at
37 °C for 10 min in the presence or absence of DTT (10 mM). Incubations containing GAPDH (10 µg/assay) and
[35S]GSH (45,000 cpm/assay) were performed in parallel.
B, GAPDH (10 µg/assay) was incubated with 2 mM
[35S]GSNO (45,000 cpm/assay) at 37 °C for the
indicated times. Incubations with GAPDH (10 µg/assay) and 2 mM [35S]GSH (45,000 cpm/assay) were also
performed in parallel. The incorporated radioactivity was measured as
described under "Experimental Procedures." Results are
representative of four similar experiments.
|
|
To verify a homolytic breakdown of GSNO under our experimental
conditions, we measured the NO· release from GSNO incubated in
buffer. Under these specific conditions, GSNO was not homolytically
degraded as determined by an NO· electrode (9), which excludes
the formation of GS· radicals and, furthermore, the possibility
of GSSG generation as a likely S-glutathionylation agent.
The results indicate more a direct attack of the reactive cysteine of
GAPDH at the S
N bond leading to a homolytic breakdown of GSNO.
After we have shown that GSNO promoted S-glutathionylation
of GAPDH in vitro, we were interested to see if GSNO was
able to induce formation of GAPDH/GSH-mixed disulfides under cellular conditions. Therefore, freshly isolated bovine EC were preincubated with [35S]L-cysteine in the presence of the
protein synthase inhibitor cycloheximide to generate intracellular
[35S]GSH. After treatment of EC with GSNO, GAPDH was
immunoprecipitated and separated on an SDS-gel under nonreducing
conditions (Fig. 5A). GSNO
(500 µM) evoked significant [35S]labeling
of GAPDH within 15 min, indicating S-glutathionylation of
the enzyme similar to our in vitro studies. The
[35S]labeling of GAPDH was stable for at least 30 min
(data not shown). Hydrogen peroxide H2O2 (500 µM), known to produce intracellular protein-mixed
disulfides, also caused S-glutathionylation of GAPDH, albeit
to a lesser extent (Fig. 5A). The addition of DTT to the sample buffer (reducing SDS gel) removed the [35S]label
from GAPDH caused by GSNO or H2O2, respectively
(Fig. 5B). In contrast, alcohol dehydrogenase that contains
a non-histidine-activated cysteine could not be modified by GSNO via
S-glutathionylation under cellular conditions (data not
shown).

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Fig. 5.
GSNO-induced
S-glutathionylation of GAPDH in bovine endothelial
cells. Freshly isolated bovine EC were prelabeled with
[35S]L-cysteine (50 µCi/ml) for 2 h in
the presence of cycloheximide (200 ng/ml) to allow formation of
[35S]GSH. EC were treated with GSNO (500 µM) or H2O2 (500 µM), for times indicated. Following immunoprecipitation
of GAPDH with a mouse anti-GAPDH monoclonal antibody, protein was
separated in an 11% SDS-gel under nonreducing (A) or
reducing (B) conditions. Radioactivity was measured as
described under "Experimental Procedures." Results are
representative of three independent experiments.
|
|
 |
DISCUSSION |
Our study shows that GSNO causes the
S-glutathionylation of GAPDH. This effect is not seen when
GSH is used. Therefore, the homolytic degradation of GSNO is essential
for S-glutathionylation to occur. Because we excluded the
possibility of NO· formation as a result of homolytic GSNO
cleavage with resultant GSSG formation as a potential
S-glutathionylating agent, we conclude that the breakage of
GSNO is directly induced by a histidine-activated thiol group such as
cysteine 149 in the active site of GAPDH. A strong nucleophile like the
histidine-activated thiolate 149 can attack the S
NO bond of GSNO,
leading to the formation of GSH-mixed protein disulfides. Thiols that
are not activated by a histidine have a lower nucleophilicity and are
not strong enough to break the S
NO bond of GSNO. These thiols are
more likely to become nitrosylated by GSNO. This is exemplified for
alcohol dehydrogenase that contains a non-histidine-activated cysteine
in its active site that does not become S-glutathionylated
by GSNO in comparison to GAPDH. Therefore, GSNO can induce
S-nitrosylation or S-glutathionylation according
to the nucleophilicity of the cysteine residues. On the contrary,
BF4NO can only produce S-nitrosylation of
cysteines by spontaneous release of NO+. This also explains
why BF4NO reduces NEM labeling of GAPDH fast, whereas GSNO
affects the labeling at a much slower rate, despite rapid enzyme
inactivation which results from S-glutathionylation.
The formation of GSH-mixed protein disulfides is mostly a consequence
of cellular oxidative stress. A recent report demonstrated that a small
pool of glutathione (less than 15% of the total) is able to produce a
significant increase in protein-mixed disulfides (22). GAPDH has been
identified as a major S-glutathionylated protein in
endothelial cells that were exposed to hydrogen peroxide (23) and in
activated monocytes (24). Other reports described inactivation of GAPDH
in the ischemic myocardium, most likely via
S-glutathionylation (25). Besides GAPDH, several other
proteins such as actin, creatine kinase, glycogen phosphorylase
b, and the homodimeric HIV-1 protease are targets for
S-glutathionylation in intact cells following exposure to
oxidative stress (for review, see Ref. 26). In line, it recently was
shown that treatment of endothelial cells with the combination of GSNO
and cysteine, a system that generates GSSG and nitric oxide, evoked
loss of intracellular glutathione probably because of the formation of protein-mixed disulfides (27).
Our study shows that GSNO induces S-glutathionylation of
GAPDH in endothelial cells. In comparison with hydrogen peroxide, the
formation of the GAPDH/GSH-protein-mixed disulfide caused by GSNO is
more efficient. Hydrogen peroxide probably oxidizes GSH to GSSG. In
turn, the reactive cysteine of GAPDH might be nucleophilic enough to
break the disulfide bond in GSSG, thus leading to the formation of a
protein-mixed disulfide. The S
N bond in GSNO is much weaker than the
S
S bond in GSSG. Therefore, S-glutathionylation induced by
GSNO is facilitated because it needs less energy to break a S
N rather
than a S
S bond.
The concentration of glutathione in cells could be as high as 10 mM. This glutathione can be S-nitrosylated, and
the S-nitrosothiol of glutathione has been implicated in the
storage and delivery of cellular NO. Our present study points to a new
cellular function of GSNO which is the covalent modification of
proteins by S-glutathionylation. This function of GSNO is
strengthened by the fact that it is GSNO, and not GSH, that facilitates
mixed disulfide formation. Therefore, cells under conditions of
nitrosative stress (formation of NO) could produce GSNO which in turn
promotes oxidative stress by formation of GSH-mixed-disulfides. The
homolytic degradation of GSNO facilitated by histidine-activated thiol
groups represents a unique mechanism of protein glutathionylation. It
would be important in the future to differentiate the effects of NO
donors on signal transduction events that could be mediated by
S-glutathionylation or S-nitrosylation.
 |
FOOTNOTES |
*
The work was supported by the Deutsche
Forschungsgemeinschaft (to B. B.), Mo 779/1-1 (to S. M.), the
European Community BMH4-CT96-0979 (to B. B.), and the American Heart
Association AHA 9730028N (to H. H.) and AHA 9750720N (to E. G. L.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: University of
Erlangen-Nürnberg, Faculty of Medicine, Department of Medicine IV-Experimental Division, Loschgestrasse 8, 91054 Erlangen,
Germany. Tel.: +49-9131 85-36311; Fax: +49-9131 85-39202; E-mail:
mfm423{at}rzmail.uni-erlangen.de.
 |
ABBREVIATIONS |
The abbreviations used are:
NO, nitric oxide;
GSNO, S-nitrosoglutathione;
BF4NO, nitrosonium
tetrafluoroborate;
DTT, dithiothreitol;
NEM, N-methylmaleimide;
GAPDH, glyceraldehyde-3-phosphate
dehydrogenase;
TCA, trichloroacetic acid;
EC, endothelial cells.
 |
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