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The modifier subunit of drosophila glutamate-cysteine
ligase regulates catalytic activity by covalent and non-
covalent interactions and influences glutathione Home-
ostasis in vivo.
Journal Article
How to cite:
Fraser, Jennifer A.; Kansagra, Pushpa; Kotecki, Claire; Saunders, Robert D.C. and McLellan, Lesley I.
(2003). The modifier subunit of drosophila glutamate-cysteine ligase regulates catalytic activity by covalent
and noncovalent interactions and influences glutathione Homeostasis in vivo. Journal of Biological Chemistry,
278(47), pp. 46369–46377.
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The Modifier Subunit of Drosophila Glutamate-Cysteine Ligase
Regulates Catalytic Activity by Covalent and Noncovalent
Interactions and Influences Glutathione Homeostasis in Vivo*
Received for publication, July 23, 2003, and in revised form, August 21, 2003
Published, JBC Papers in Press, September 3, 2003, DOI 10.1074/jbc.M308035200
Jennifer A. Fraser‡, Pushpa Kansagra§, Claire Kotecki§, Robert D. C. Saunders§,
and Lesley I. McLellan‡¶
From the ‡Biomedical Research Centre, University of Dundee, Ninewells Hospital and Medical School,
Dundee DD1 9SY, United Kingdom and the §Department of Biological Sciences, Open University, Walton Hall,
Milton Keynes MK7 6AA, United Kingdom
Glutamate-cysteine ligase (GCL) has a key influence
on glutathione homeostasis. It has been proposed that
mammalian GCL is regulated by the redox environment,
and we show here that cysteine residues in the Drosoph-
ila melanogaster GCL modifier subunit (DmGCLM) can
form covalent interactions with the catalytic subunit
(DmGCLC) and modify its activity. Candidate compo-
nents of intersubunit disulfides (Cys213, Cys214, and
Cys267) were identified using matrix-assisted laser des-
orption ionization time-of-flight spectroscopy of iodoac-
etamide-modified DmGCLM as well as examination of
the evolutionary conservation of cysteines. Mutation of
the 3 cysteine residues allowed DmGCLM to associate
with DmGCLC, but inhibited the formation of intersub-
unit disulfides. This caused a 2-fold reduction in the
catalytic efficiency of Drosophila GCL, although activity
remained significantly higher than the catalytic subunit
alone. The cysteine mutant was also more sensitive to
inhibition by glutathione than the unmodified holoen-
zyme. Notably, human GCLM could substitute for
DmGCLM in modification of DmGCLC activity. The role
of DmGCLM in vivo was examined by analysis of a Dro-
sophila mutant (l(3)L0580) containing a P-element inser-
tion in Gclm. We found that the P-element is not respon-
sible for the lethal phenotype and separated the
recessive lethal mutation from the P-element by recom-
bination. This yielded two fully viable and fertile recom-
binants bearing the P-element insertion, which Western
and Northern blotting indicated is a severely hypomor-
phic allele of Gclm. Glutathione levels were
2-fold
lower in the GclmL0580 mutants than in control strains,
demonstrating the importance of DmGCLM in the regu-
lation of glutathione homeostasis in vivo.
The intracellular redox environment is of critical importance
in cell physiology. It has a major influence on signaling path-
ways and cell fate in response to stress (1). Glutathione is one
of the key influences on the redox state of the cell; and accord-
ingly, intracellular glutathione levels are subject to multilat-
eral regulatory mechanisms (2, 3).
A major player in the regulation of glutathione homeostasis
is glutamate-cysteine ligase (GCL),1 which catalyzes the first
and rate-limiting step in de novo synthesis of glutathione from
precursor amino acids (4). GCL is located in the cytoplasm, and
its activity appears to be regulated at several levels by the
antioxidant status of the cell. In addition to being subject to
transcriptional activation by pro-oxidants (5–7), evidence sug-
gests that the catalytic activity is subject to redox regulation by
the reversible formation of disulfide bridges between its two
subunits (8, 9). Furthermore, GCL is subject to feedback inhi-
bition by glutathione at concentrations that are physiologically
relevant (10). The complex regulation of GCL activity high-
lights its pivotal role in controlling cellular glutathione
synthesis.
GCL is a heterodimer composed of a catalytic subunit
(GCLC) and a modifier subunit (GCLM). The presence of
GCLM modulates the catalytic properties of GCLC by lowering
its sensitivity to inhibition by glutathione and by increasing its
affinity for glutamate (8, 9). The sensitivity of GCLC to inhibi-
tion by glutathione is such that it has been proposed that
GCLC would function poorly in vivo without the presence of
GCLM. The pioneering biochemical studies of Meister and co-
workers (9) in the 1990s provided evidence to suggest that
GCLM could further enhance the function of GCLC by the
formation of intersubunit disulfide bonds. This proposition was
based on the observations that treatment of the GCL holoen-
zyme with dithiothreitol (DTT) lowered its affinity for gluta-
mate and increased its sensitivity to inhibition by the glutathi-
one analog ophthalmic acid. The effects of DTT were dependent
on the presence of GCLM. The results prompted the hypothesis
that intracellular GCL activity could be increased under con-
ditions that deplete glutathione, where the oxidizing environ-
ment within the cell would promote disulfide bond formation
within GCL.
More recently, the role of intermolecular disulfide linkages
in modifying GCL activity was investigated by mutagenesis of
cysteine residues in the catalytic subunit (11). 8 of the 14
cysteine residues in human GCLC were singly altered to gly-
cine, and the effects on activity and ability to form disulfide
linkages were examined. One of the 8 cysteines (Cys553) was
shown to be involved in influencing the ability of GCLM to
increase the activity of GCLC. The mutant holoenzyme was,
however, still able to form a 114-kDa complex when analyzed
by SDS-PAGE under non-reducing conditions. This suggests* This work was supported by Grants 94/G15091 and 108/G15090
from the Biotechnology and Biological Sciences Research Council. The
costs of publication of this article were defrayed in part by the payment
of page charges. This article must therefore be hereby marked “adver-
tisement” in accordance with 18 U.S.C. Section 1734 solely to indicate
this fact.
¶ To whom correspondence should be addressed. Tel.: 44-1382-
660111; Fax: 44-1382-669993; E-mail: lesley.mclellan@cancer.org.uk.
1 The abbreviations used are: GCL, glutamate-cysteine ligase; GCLC,
glutamate-cysteine ligase catalytic subunit; GCLM, glutamate-cysteine
ligase modifier subunit; Dm, Drosophila melanogaster; Hs, Homo sapi-
ens; DTT, dithiothreitol; MALDI-TOF, matrix-assisted laser desorption
ionization time-of-flight.
THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 278, No. 47, Issue of November 21, pp. 46369–46377, 2003
© 2003 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.
This paper is available on line at http://www.jbc.org 46369

that covalent interactions could still occur between the mu-
tated catalytic subunit and GCLM, indicating that Cys553 may
not be the only cysteine involved in GCLC association with
GCLM. It is possible that more than one disulfide linkage may
form between the subunits.
We reasoned that the most straightforward method of inves-
tigating the importance of intermolecular disulfide linkages in
GCL catalysis would be to target the cysteine residues in the
modifier subunit; GCLM has fewer cysteines, and interpreta-
tion of data would not be confounded by mutation of cysteine
residues with a role in catalysis (12). We have shown previously
that GCL from Drosophila melanogaster contains an
31-kDa
modifier subunit (DmGCLM) that is capable of forming disul-
fide linkages with the
80-kDa catalytic subunit (DmGCLC) in
vitro (13). The disulfide status of specific cysteines in either
DmGCLC or DmGCLM is unknown. In this study, we have
investigated the importance of intermolecular disulfide link-
ages in DmGCL activity and show that the ability to form
disulfide bonds impinges on both activity and sensitivity to
feedback inhibition by glutathione. We also show that Drosoph-
ila Gclm mutants, although viable, have approximately half
the levels of glutathione compared with the control strains,
demonstrating the importance of DmGCLM in glutathione ho-
meostasis in vivo.
EXPERIMENTAL PROCEDURES
Construction of pET20bDmGCLC—To make recombinant DmGCLC
with a histidine tag at the C terminus, the open reading frame of
the Gclc gene was excised from pETDmGCLC (13) and subcloned into
the NdeI and XhoI sites of pET20b (Novagen), generating
pET20bDmGCLC. A 254-bp fragment from the 3-end of the open read-
ing frame was amplified from pDmGCS4.3.3 (14) by PCR using up-
stream (5-GGGAATTCGCCGGCGAGCTAATCACCACG-3) and down-
stream (5-CCGCTCGAGTTTCTCCTCGCAGCAGCC-3) oligonucleo-
tides designed to insert an EcoRI site into the 5-end and an XhoI site
into the 3-end of the fragment and to remove the terminal stop codon.
The 254-bp cDNA fragment was subcloned into the EcoRI and XhoI
sites of pBluescript SK and sequenced before it was digested with
SgrAI (underlined sequence) and XhoI and used to replace the corre-
sponding SgrAI/XhoI fragment in pET20bDmGCLC.
Site-directed Mutagenesis—Site-directed mutagenesis was used to
alter the cysteine residues within DmGCLM and was carried out using
the QuikChange site-directed mutagenesis kit (Stratagene). Mutations
were introduced via PCR-based site-directed mutagenesis using the
DmGCLM open reading frame cloned into pBluescript SK (13) as a
template and pairs of complementary oligonucleotides (Table I). Oligo-
nucleotides were designed to introduce a single nucleotide change re-
sulting in an amino acid substitution from cysteine to serine. Mutations
were introduced sequentially. Amplified plasmid DNA was isolated,
and the presence of point mutation(s) was determined by sequencing
before the mutated open reading frame was subcloned into the NdeI
and XhoI sites of pET15b (Novagen) to generate pETDmGCLM plas-
mids, which were used to express mutant recombinant DmGCLM
polypeptides.
Expression and Purification of Recombinant Proteins—pET20bDmGCLC
and mutant forms of pETDmGCLM were expressed separately in Esche-
richia coli strain BL21(DE3) and purified by nickel-agarose chromatography
as described previously (13), except that LB broth was used in place of
Terrific Broth for the culture medium. Recombinant human GCLM
(HsGCLM) cloned into pET15b was expressed in BL21(DE3) cells and
purified as described previously (15). Preparations of the GCL holoenzyme
were generated by mixing purified recombinant DmGCLC (with a C-termi-
nal histidine tag) and GCLM polypeptides (each tagged with histidine at the
N terminus) and purifying the protein complexes by gel filtration chroma-
tography under the conditions described previously (13).
Carboxymethylation of Cysteine Residues within DmGCLM—Sam-
ples of DmGCLM were diluted to a final concentration of 200
g/ml with
100 mM Tris-HCl (pH 8.0) and 1 mM EDTA, containing no additives, 5
mM DTT, or 5 mM DTT plus 6 M guanidine HCl. Samples were incubated
with 10 mM iodoacetamide for 5 h in the dark at room temperature and
were subsequently dialyzed overnight in 100 mM Tris-HCl (pH 8.0) and
1 mM EDTA in the dark (16). The dialyzed samples were digested with
trypsin and analyzed by matrix-assisted laser desorption ionization
time-of-flight (MALDI-TOF) mass spectroscopy as described (17) and
performed by Douglas J. Lamont at the “FingerPrints” Proteomics
Facility, Post-Genomics and Molecular Interactions Centre, School of
Life Sciences, University of Dundee.
Circular Dichroism Spectroscopy—Samples of mutant and unmodi-
fied DmGCLM polypeptides (1 mg/ml) for CD analyses were dialyzed
overnight in 20 mM Tris-HCl (pH 7.4) and degassed by bubbling N2
through the samples. The CD analyses were carried out using the
CONTIN procedure (18) by Dr. Sharon Kelly at the Circular Dichroism
Facility of the University of Glasgow (Glasgow, United Kingdom).
Analysis of GCL Activity—GCL activity was determined at 25 °C
spectrophotometrically (13) using L--aminobutyric acid as a substrate
instead of L-cysteine and adapted for use on 96-well plates. Reaction
mixtures (0.21 ml) contained 5 mM L-glutamate, 50 mM L--aminobu-
tyric acid, 2.4 mM phosphoenolpyruvate, 0.24 mM NADH, 1 unit of lactic
dehydrogenase, and 1 unit of pyruvate kinase. The reaction was initi-
ated by the addition of ATP to a final concentration of 5 mM. L-Gluta-
mate concentrations ranging from 0.25 to 16 mM were used to deter-
mine the Km and Vmax values from initial reaction rates. Michaelis-
Menten parameters were fitted via a Hanes plot using hyperbolic
regression analysis software (19). For inhibition studies with glutathi-
one, reduced glutathione was included in the standard reaction mixture
at concentrations between 0.25 and 16 mM.
Western Blot Analysis—Western blotting was performed using anti-
serum raised against recombinant DmGCLM, DmGCLC, or HsGCLM
as described previously (13, 15). For analysis of DmGCL content in flies,
10 male flies were homogenized in 150
l of sample loading buffer (100
mM Tris-HCl (pH 6.8), 4% (w/v) SDS, 20% (v/v) glycerol, 3.6 M 2-mer-
captoethanol, and 0.05% (w/v) bromphenol blue) and incubated at
100 °C for 5 min before being subjected to SDS-PAGE and Western
blotting.
Glutathione Determination—The total glutathione content of whole
flies was determined by a modification of the glutathione reductase/
5,5-dithiobis(2-nitrobenzoic acid) recycling assay described by Tietze
(20) and adapted for use on a 96-well plate. Routinely, 10 male flies
were manually homogenized in 100
l of ice-cold 10% (w/v) sulfosalicylic
acid. Reaction mixtures (0.15 ml) contained 1 mM 5,5-dithiobis(2-nitro-
benzoic acid) and 0.34 mM NADPH in 150 mM sodium phosphate buffer
(pH 7.5) containing 7.5 mM EDTA. The reaction was initiated by the
addition of 0.1 unit of glutathione reductase (Sigma). Glutathione con-
centrations were determined from a standard curve generated from
known concentrations of glutathione prepared in 10% (w/v) sulfosalicylic
acid and are expressed as picomoles of glutathione/fly. To determine
whether glutathione levels differed significantly by genotype, we used a
one-way analysis of variance with post hoc analyses carried out using
Scheffe’s test and the SPSS statistical software package. Values were
considered to be significantly different from each other when p  0.05.
Drosophila Procedures—Flies were maintained on a standard oat-
meal medium. We obtained the P-element insertion stock l(3)L0580
from the Bloomington Drosophila Stock Center. This stock is reported
to bear a recessive lethal mutation insertion of a P{lacW}-element (21)
within the 5-untranslated region of Gclm. The location of the P-ele-
ment was confirmed by PCR and sequencing. The recessive lethal
present on the l(3)L0580 chromosome was removed by recombination
with an e4 wo1 ro1 stock obtained from the Bloomington Drosophila
TABLE I
Sequences of oligonucleotides used for mutation of DmGCLM cysteine residues
Mutant Oligonucleotide sequencea Cysteine location
DmGCLM-A 5-CAACCTGTCTACGTcCTGCGTGGTGCCAC-3 Cys213
DmGCLM-B 5-CAACCTGTCTACGTGCTcCGTGGTGCCAC-3 Cys214
DmGCLM-C 5-GCAGGAGTTcTCCACCGCTCACC-3 Cys224
DmGCLM-D 5-GTGCATGTcCGCTCCGGGGCG-3 Cys267
DmGCLM-E 5-CCAAACCGCCCTCTTcCGAGGATTCC-3 Cys139
a The mutated nucleotides are in lowercase letters.
Drosophila Glutamate-Cysteine Ligase46370

Stock Center. Recombinant progeny bearing the L0580 P-element in-
sertion (marked with w) and either e4 or ro1 were selected and tested
for recessive lethality. Approximately 50% of the recombinants of each
class were homozygous viable. We did not map the location of the lethal
mutation. Viable stocks bearing the L0580 P-element and marked with
either e4 or ro1 were established. We have named the L0580 P-element
insertion GclmL0580 to indicate its identity as a mutant allele of Gclm.
Revertants were obtained from the e4 GclmL0580 stock by crossing to a
stock bearing a third chromosome insertion of the P{ry2-3}-element,
which produces constitutively active P-transposase. Revertants were
selected on the basis of loss of eye pigmentation, associated with loss of
the w-bearing P-element. We confirmed the loss of the element by
PCR. No apparent aberrations were seen. The level of DmGCLM in the
revertant stock was estimated by Western blotting.
Multiple Sequence Alignment—Multiple sequence alignments were
performed using the Genetics Computer Group Pileup software.
RESULTS
Mutation of All 5 Cysteines within DmGCLM Inhibits Inter-
action with DmGCLC—DmGCLM contains 5 cysteine residues
at positions 139, 213, 214, 224, and 269, which were named E,
A, B, C, and D, respectively (Table II). To investigate the role of
intersubunit disulfide linkages in the regulation of DmGCL
activity, we performed site-directed mutagenesis on the modi-
fier subunit to generate a polypeptide that entirely lacks cys-
teine (DmGCLM-FM). Purified recombinant DmGCLM-FM ap-
peared as a single polypeptide with an apparent molecular
mass of
35 kDa when analyzed by SDS-PAGE (Fig. 1). Its
mobility is slower than that of wild-type DmGCLM (Fig. 1),
which has an apparent molecular mass of 31 kDa (13). The
reason for the difference in electrophoretic motility is un-
known, but it suggests that DmGCLM-FM may have a less
compact structure than the wild-type protein. The secondary
structures of the polypeptides were examined by CD spectros-
copy over the absorbance range 260 to 320 nm. The CD anal-
yses did not, however, reveal any significant differences be-
tween DmGCLM-FM and the unmodified DmGCLM
polypeptide (data not shown). The predicted composition of
-helices and -sheets in DmGCLM is 6  1.3 and 61  1.45%,
respectively, similar to that of DmGCLM-FM (7  1.5 and 62 
1.6%, respectively).
Regulation of mammalian GCLC activity by GCLM is
thought to involve, at least in part, the transient formation of
reversible covalent interactions promoting conformational
change(s) around the active site of GCLC (9). To investigate the
role of these interactions in DmGCL regulation, we attempted
to generate a DmGCL holoenzyme using DmGCLC and
DmGCLM-FM.
DmGCLM-FM was mixed with DmGCLC, and the mixture
was subjected to gel filtration chromatography. When the wild-
type DmGCLC/DmGCLM mixture was resolved, two peaks
were identified corresponding to the holoenzyme complex
(
140 kDa) and uncomplexed DmGCLM (
30 kDa) (Fig. 2)
(13). However, when the DmGCLC/DmGCLM-FM mixture was
resolved, a major peak corresponding to the
140-kDa complex
was not observed, and the profile obtained was more akin to
that of uncomplexed DmGCLC (
80 kDa) and DmGCLM (
30
kDa) (Fig. 2). This was confirmed by SDS-PAGE analysis of the
peak fractions obtained from the DmGCLC/DmGCLM-FM pro-
file, which showed that DmGCLM did not co-elute with
DmGCLC in the
80-kDa peak (data not shown). A small
shoulder on the leading edge of the 80-kDa peak was observed
and found to contain a trace of DmGCLM. Analyses of these
fractions for GCL activity did not demonstrate an enhancement
of activity compared with the catalytic subunit alone (data not
shown). These findings suggest that mutation of all of the
cysteine residues in DmGCLM substantially impairs its ability
to interact with DmGCLC.
Redox Status of Cysteine Residues in DmGCLM—The disul-
fide status of cysteine residues in DmGCLM was analyzed by
peptide mass fingerprinting. Recombinant DmGCLM was
treated with iodoacetamide under native, reducing, or reducing
and denaturing conditions. Tryptic digests of iodoacetamide-
treated DmGCLM were analyzed by MALDI-TOF mass spec-
trometry. The iodoacetamide-modified cysteine-containing
polypeptide fragments are shown in Table II. In native DmG-
CLM, the fragment containing Cys139 (residues 137–147) and
the fragment containing Cys213, Cys214, and Cys224 (residues
177–260) were both singly modified by iodoacetamide (data not
shown). In the reduced sample, a further iodoacetamide modi-
fication was identified in the fragment containing Cys213,
Cys214, and Cys224, whereas under denaturing and reducing
FIG. 1. SDS-PAGE analysis of DmGCLM-FM. Recombinant
DmGCLM polypeptides were purified from E. coli by nickel-agarose
chromatography and analyzed by SDS-PAGE. Protein (0.75
g) was
loaded as follows: lane 1, DmGCLM; lane 2, DmGCLM-FM.
TABLE II
Cysteine-containing tryptic fragments of DmGCLM
Drosophila Glutamate-Cysteine Ligase 46371

conditions, all 3 cysteine residues within this fragment were
modified. We were unable to identify the fragment containing
Cys267 (residues 267–268) due to its being small and poorly
retained on the high pressure liquid chromatography column.
Nevertheless, MALDI-TOF analysis implicated Cys267 as being
involved in disulfide formation, as peaks corresponding to
mixed disulfides between Cys267 and Cys139 and between
Cys267 and Cys213, Cys214, or Cys224 were identified when the
non-reduced iodoacetamide-treated samples were analyzed.
These peaks were absent in samples treated with DTT.
It is important to note that, in the absence of DmGCLC and
glutathione or DTT, purified recombinant DmGCLM was pre-
dominantly multimeric (data not shown). This suggests that
some of the disulfide bonds that become reduced upon DTT
treatment and subsequently modified by iodoacetamide are
likely to be intermolecular disulfides rather than intramolecu-
lar disulfides. It is unclear whether this has biological signifi-
cance, but it is possible that multimerization may occur as a
result of the artificial environment of the E. coli expression
system, in which reactive cysteines could readily form intermo-
lecular disulfide bonds giving rise to homodimers or trimers.
We reasoned that cysteine residues involved in forming inter-
FIG. 3. Alignment of GCLM amino
acid sequences from different phyla
and animal classes. Black boxes show
amino acid sequence identity. Gray boxes
show amino acid similarity. The con-
served cysteine residues potentially in-
volved in redox regulation of GCL activity
are indicated by asterisks. Xl, X. laevis;
Dr, D. rerio; Ce, C. elegans; Sp, S. pombe.
FIG. 2. Gel filtration of DmGCLC
combined with DmGCLM-FM. Puri-
fied recombinant DmGCLM-FM (10 mg)
was mixed with purified DmGCLC (10
mg). Uncomplexed DmGCLC (
), wild-
type (WT) DmGCL (), and the DmG-
CLC/DmGCLM-FM combination (Œ) were
separately subjected to gel filtration chro-
matography as described under “Experi-
mental Procedures.” Eluted fractions
were analyzed for protein concentration.
Drosophila Glutamate-Cysteine Ligase46372

molecular disulfide bonds in the absence of DmGCLC are po-
tential candidates for forming disulfide bridges with DmGCLC
in the holoenzyme.
Our findings from the proteomic analyses indicate that two
cysteines on the surface of DmGCLM are predominantly in a
reduced state and can be modified by iodoacetamide, two sur-
face cysteines are involved in formation of disulfide bonds, and
one cysteine is inaccessible to iodoacetamide without denatur-
ation of the protein. The MALDI-TOF analyses suggest that
Cys139 is principally (but not entirely) present as a free thiol on
the surface of DmGCLM and that Cys267 can participate in
disulfide bridge formation with other cysteines. Tryptic frag-
ment 177–260 appears to contain one free thiol, one cysteine as
part of a disulfide, and one cysteine that is buried within the
native protein. A caveat to our interpretation is that the vicinal
cysteines may not be amenable to simultaneous modification by
iodoacetamide in the nondenatured protein. Disulfide shuffling
may also occur. Due to the positions of the tryptic cleavage
sites, we were unable to dissect this further, and we used a
bioinformatics approach to gain additional insight into which of
the cysteine residues in tryptic fragment 177–260 could be
involved in disulfide formation with DmGCLC.
Comparison of the amino acid sequences of DmGCLM, Hs-
GCLM, and hypothetical GCLM polypeptides from Caenorhab-
ditis elegans (GenBankTM/EBI accession number NP_491305),
Xenopus laevis (accession number AAH44107), and Danio rerio
(accession number AAH44532) showed that both Cys213 and
Cys214 are conserved in representatives from mammals, am-
phibians, fish, arthropods, and nematodes (Fig. 3). A partial
sequence of an expressed sequence tag encoding a hypothetical
GCLM cDNA from Gallus gallus (accession number AJ447023)
also contained the conserved Cys213 and Cys214 residues (data
not shown). The only putative GCLM sequence that we found
that does not contain both of the conserved cysteines was from
Schizosaccharomyces pombe (accession number NP_588368), in
which only Cys214 is conserved (Fig. 3). Assuming that the sole
function of GCLM is to regulate GCLC activity and that inter-
molecular disulfide bridges are involved in the regulation of
activity in vivo, the evolutionary conservation of cysteine resi-
dues infers a possible role for Cys213 and/or Cys214 (rather than
Cys224) in the regulation of DmGCLC activity.
We went on to generate a mutant form of DmGCLM lacking
Cys213, Cys214, and Cys267 (named DmGCLM-ABD). We also
generated the converse mutant lacking Cys139 and Cys224
(named DmGCLM-CE) and investigated the ability of both
mutants to form the 140-kDa holoenzyme complex.
Mutation of Cys213, Cys214, and Cys267 Impairs Intermolecu-
lar Disulfide Bridge Formation within DmGCL—Purified re-
combinant DmGCLM-ABD appeared as single polypeptide
with an approximate molecular mass of 31 kDa when analyzed
by SDS-PAGE (Fig. 4). This is in agreement with the molecular
mass obtained for wild-type DmGCLM. Purified recombinant
DmGCLM-CE also appeared as a single polypeptide, although
the estimated molecular mass of
34 kDa (Fig. 4, lane 3) is
similar to that found for DmGCLM-FM. Interestingly,
DmGCLM containing a single mutation at Cys224 also exhib-
ited retarded motility (data not shown). This was not observed
in DmGCLM with a single mutation at Cys139, implicating
Cys224 in this effect.
DmGCLM-ABD and DmGCLM-CE were each mixed sepa-
rately with DmGCLC, and the mixtures were resolved by gel
filtration chromatography. The profiles obtained following
separation of DmGCLC/DmGCLM-ABD and DmGCLC/
DmGCLM-CE were identical to that of the wild-type DmGCLC/
DmGCLM mixture (data not shown), suggesting that mutation
of Cys139 and Cys224 together or Cys267, Cys213, and Cys214
together has little or no impact on the ability of DmGCLM to
associate with DmGCLC to form an
140-kDa complex. This
was confirmed by SDS-PAGE analysis, which showed that the

140-kDa complexes obtained following gel filtration con-
tained both DmGCLC and the mutant forms of DmGCLM (data
not shown).
To explore the role of cysteine residues within DmGCLM in
disulfide interactions, we investigated the ability of mutant
DmGCLM to form the 140-kDa complex with DmGCLC under
non-reducing conditions. The peak fractions obtained from gel
filtration were subjected to SDS-PAGE and Western blotting
under non-reducing conditions (Fig. 5). In the control
DmGCLC/DmGCLM sample, a complex with an approximate
molecular mass of 140 kDa was apparent when the sample was
probed with antiserum raised against DmGCLC (Fig. 5). A
corresponding band was also identified in the DmGCL-CE sam-
ple analyzed separately in the same way, suggesting that mu-
tation of Cys139 and Cys224 has little impact on disulfide bond
FIG. 4. SDS-PAGE analysis of DmGCLM-ABD and DmGCLM-CE
mutants. Recombinant DmGCLM polypeptides were purified from E.
coli by nickel-agarose chromatography and analyzed by SDS-PAGE.
Protein (0.75
g) was loaded as follows: lane 1, DmGCLM; lane 2,
DmGCLM-ABD; lane 3, DmGCLM-CE.
FIG. 5. Analysis of intermolecular disulfide bridge formation
in the mutant DmGCL holoenzymes. After dialysis to remove DTT,
protein (200 ng) from the
140-kDa peak fractions obtained after gel
filtration chromatography of DmGCLC/DmGCLM (lane 1), DmGCLC/
DmGCLM-ABD (lane 2), or DmGCLC/DmGCLM-CE (lane 3) was re-
solved by SDS-PAGE in the absence of 2-mercaptoethanol and analyzed
by Western blotting using antiserum raised against recombinant
DmGCLC.
TABLE III
Kinetic constants for Drosophila GCL with respect to L-glutamate
Enzymea Km Vmax kcal/Km
mM
mol/min/mg min1 mM1
DmGCL 0.91  0.06 12.1  0.41 1096.1  85
DmGCLC/DmGCLM-ABD 0.97  0.02 5.85  0.40 490.46  33
DmGCLC 0.92  0.08 1.51  0.02 134.44  11
a Assays were performed in triplicate from one preparation each of
DmGCLC, DmGCLM, and DmGCLM-ABD. The catalytic subunit in
each preparation is from the same source, and all proteins were purified
on the same day under the same conditions. DmGCLC with a hexahis-
tidine tag at the C terminus was used rather than the N-terminal tag
employed previously (13).
Drosophila Glutamate-Cysteine Ligase 46373

formation within DmGCL. Conversely, the 140-kDa band was
not detectable in the lane containing DmGCLC/DmGCLM-
ABD, suggesting that mutation of these residues inhibits di-
sulfide bond formation between the DmGCL subunits under
non-reducing conditions. Taken together with the results from
the peptide fingerprinting experiments, these findings impli-
cate Cys267 and either Cys213 or Cys214 (or both) as the cysteine
residues within DmGCLM responsible for intermolecular disul-
fide linkages.
Cysteine Residues in DmGCLM Impact on GCL Activity—
To determine whether inhibition of intermolecular disulfide
bond formation affects the catalytic characteristics of DmGCL,
we compared the catalytic activities of the dialyzed DmGCL
and DmGCLC/DmGCLM-ABD samples. Kinetic analyses of
DmGCL activity were carried out on at least four separate
occasions on different preparations of DmGCL. We found that,
unlike the Km values, which were fairly consistent between
experiments, the specific activity and maximal velocity of
DmGCLC varied between preparations by as much as 2-fold.
Despite this interbatch variation, the magnitude of changes in
Vmax and kcat/Km between the unmodified and mutant DmGCL
polypeptides and the catalytic subunit alone was consistent
between preparations.
The Km of DmGCL for L-glutamate is 0.91 mM, whereas the
Km of DmGCLC/DmGCLM-ABD is 0.97 mM (Table III). This
observation implies that loss of disulfide bond formation has
little impact on the affinity of DmGCL for L-glutamate. In
contrast, the Vmax of DmGCL was found to be 12.1
mol/min/
mg, approximately twice that of DmGCLC/DmGCLM-ABD.
This suggests that intermolecular disulfide bridge formation
has a significant impact on the catalytic efficiency of the
DmGCL holoenzyme. The Vmax of DmGCLC is substantially
lower than that of either DmGCLC/DmGCLM-ABD (
4-fold)
or unmodified DmGCL (
8-fold) (Table III), emphasizing the
importance of non-covalent subunit interactions in regulating
DmGCL activity. In contrast, the Km obtained for DmGCLC
is not significantly different from that of DmGCL or
DmGCLC/DmGCLM-ABD.
Absence of Intermolecular Disulfide Linkages between
DmGCLM and DmGCLC Enhances Sensitivity to Feedback
Inhibition by Glutathione—The DmGCL holoenzyme is subject
to feedback inhibition by glutathione (13). Inhibition is mixed,
and reduction of intermolecular disulfide linkages by glutathi-
one may facilitate its access to the active site to inhibit activity.
We hypothesized that a DmGCL mutant unable to form disul-
fide linkages would be more susceptible to inhibition by
glutathione.
Wild-type DmGCL activity was lowered in the presence of
glutathione by a maximum of
40% (Fig. 6). By contrast,
DmGCLC/DmGCLM-ABD activity was more susceptible to in-
hibition by glutathione; activity was decreased by
60% at the
highest concentration of GSH (16 mM). The difference in the
extent of inhibition between DmGCL and DmGCLC/
DmGCLM-ABD was particularly evident at lower concentra-
tions of glutathione. These findings highlight the potential
importance of intermolecular disulfide bridges within DmGCL
in regulating the mechanism of feedback inhibition by gluta-
thione in the holoenzyme. The susceptibility of DmGCLC/
DmGCLM-ABD to inhibition by glutathione was not, however,
as marked as that of DmGCLC, where activity was almost
completely abolished at higher concentrations of GSH (Fig. 6).
Inhibition of the catalytic subunit by glutathione is competitive
(13), and the difference in the extent of inhibition between
DmGCLC and DmGCLC/DmGCLM-ABD underscores the im-
pact that non-covalent intersubunit interactions have upon the
susceptibility of DmGCLC to glutathione inhibition.
HsGCLM Is Able to Interact with DmGCLC in Vitro via
Covalent Interactions—To gain a better understanding about
some of the regions on GCLM that may be involved in subunit
interactions, we investigated whether we could create a hybrid
GCL holoenzyme using HsGCLM and DmGCLC. Purified re-
FIG. 6. Inhibition of recombinant DmGCLC/DmGCLM-ABD activity by glutathione. The specific activities of the wild-type (WT) DmGCL
holoenzyme (f), the DmGCLC/DmGCLM-ABD holoenzyme (DmGCL ABD; E), and DmGCLC (Œ) were measured under standard assay conditions
in the presence of increasing concentrations of glutathione. Activity is expressed as a percentage of the control activity in the absence of glutathione
(mean  S.E.).
FIG. 7. Analysis of the subunit composition of the hybrid Dro-
sophila/human GCL holoenzyme. Protein (2
g) from the peak
fractions obtained after gel filtration of DmGCLC (lane 1), DmGCLC/
DmGCLM (lane 2), or DmGCLC/HsGCLM (lane 3) was resolved by
SDS-PAGE in the presence of 2-mercaptoethanol.
Drosophila Glutamate-Cysteine Ligase46374

combinant HsGCLM was mixed with purified DmGCLC and
resolved by gel filtration chromatography. A major peak with
an estimated molecular mass of
140 kDa and a minor peak
with an estimated molecular mass of
28 kDa, corresponding
to uncomplexed HsGCLM, were identified when the DmGCLC/
HsGCLM mixture was separated by gel filtration (data not
shown). This was identical to the profile obtained following
resolution of the DmGCLC/DmGCLM mixture. Fig. 7 shows
that polypeptides corresponding to DmGCLC (
80 kDa) and
HsGCLM (
28 kDa) are present in the 140-kDa peak. This
indicates that, despite having only 27% sequence identity to
DmGCLM, HsGCLM is able to interact with DmGCLC to gen-
erate a new higher molecular mass protein complex when they
are mixed together in vitro.
To determine whether HsGCLM can form covalent interac-
tions with DmGCLC, the
140-kDa sample was subjected to
SDS-PAGE under non-reducing conditions. Western blot anal-
yses using antiserum raised against DmGCLC showed the
presence of an
140-kDa band in the DmGCLC/HsGCLM sam-
ple (Fig. 8A), similar to the
140-kDa band observed when the
normal DmGCL holoenzyme was analyzed under the same
conditions. This band was absent in the sample containing
DmGCLC alone (Fig. 8A). The 140-kDa band was also detected
when the DmGCLC/HsGCLM sample was probed with anti-
serum raised against HsGCLM (Fig. 8C). These results indi-
cate that HsGCLM is capable of forming disulfide linkages with
DmGCLC. As only 2 cysteine residues (Cys213 and Cys214) are
conserved between the Drosophila and human modifier sub-
units (Fig. 3), is seems likely that either one or both residues
are involved in intermolecular disulfide bond formation.
HsGCLM Can Enhance the Activity of DmGCLC—The cata-
lytic activity of the wild-type DmGCL holoenzyme was com-
pared with that of the DmGCLC/HsGCLM hybrid. The Km for
the DmGCLC/HsGCLM hybrid enzyme is 1.01 mM, which is
similar to the value of 0.89 mM obtained for the Drosophila
GCL holoenzyme. The Vmax for the DmGCLC/HsGCLM hybrid
enzyme was found to be 8.39
mol/min/mg, similar to the Vmax
determined for the DmGCL holoenzyme (8.45
mol/min/mg)
generated from the same preparation of DmGCLC. These re-
sults indicate that HsGCLM is able to modify the activity of
DmGCLC in a similar way to DmGCLM.
Effect of DmGCLM Ablation on Glutathione Homeostasis in
Vivo—The gene encoding DmGCLM (Gclm) maps to 94C on the
third chromosome. We obtained the P-element-induced reces-
sive lethal mutant stock l(3)L0580, which contains a P{lacW}-
element inserted in the 5-noncoding region of the Gclm gene.
The P-element in this stock was not responsible for the lethal-
ity, as judged from failure to recover dysgenically induced
revertants, from failure to rescue lethality with a transgene,
and by deficiency mapping (data not shown). Accordingly, we
separated the recessive lethal mutation in this stock from the
P-element by recombination with an e4 wo1 ro1 chromosome.
This yielded two fully viable recombinants bearing the P-ele-
ment insertion, e4 GclmL0580 and GclmL0580 ro1. We have
named this P-element allele GclmL0580 and refer here to the
two marked recombinant derivatives as e GclmL0580 and
GclmL0580 ro. Levels of DmGCLM in homozygous e GclmL0580
and GclmL0580 ro flies were examined by Western blotting. Fig.
9A shows that DmGCLM was substantially diminished in e
GclmL0580 or GclmL0580 ro fly lysates. Fig. 9B shows that the
reduction in DmGCLM was not accompanied by any change in
DmGCLC protein levels. Northern blotting showed a dramatic
reduction in Gclm transcription in adult GclmL0580 homozy-
gotes (data not shown). Collectively, these data indicate that
GclmL0580 is a severely hypomorphic allele. GclmL0580 homozy-
gous flies are viable and fertile under normal laboratory con-
ditions and have no obvious phenotype. We obtained a rever-
tant (e Gclmrev4) by mobilizing the L0580 element from the e
GclmL0580 chromosome. The e Gclmrev4 homozygotes have wild-
type levels of DmGCLM, as judged by Western blotting (Fig. 9).
As DmGCLM enhances the catalytic efficiency of DmGCLC
and reduces its sensitivity to feedback inhibition by glutathi-
one, we hypothesized that e GclmL0580 and GclmL0580 ro flies
may have an impaired capacity to synthesis glutathione. We
analyzed the whole body glutathione contents of e GclmL0580
and GclmL0580 ro flies and compared them with those of Canton
S and w1118 flies, both of which are wild-type for Gclm, and
with the revertant, e Gclmrev4 (Fig. 10). Canton S and w1118
flies contained 348.15  20.9 and 305.5  27.3 pmol of gluta-
thione/fly, respectively. The revertant strain (e Gclmrev4) con-
tained slightly less glutathione than the wild-type strains
FIG. 8. Analysis of the subunit behavior of the hybrid Drosophila/human GCL holoenzyme under nonreducing conditions. After
dialysis to remove DTT, protein (200 ng) from the peak fractions obtained after gel filtration of DmGCLC (lane 1), DmGCLC/DmGCLM (lane 2),
or DmGCLC/HsGCLM (lane 3) was resolved by SDS-PAGE in the absence of 2-mercaptoethanol and analyzed by Western blotting using antiserum
raised against DmGCLC (A), DmGCLM (B), or HsGCLM (C).
FIG. 9. DmGCL protein levels in GclmL0580 flies. Portions (5
l for
analysis of DmGCLM or 10
l for analysis of DmGCLC) of fly homoge-
nates from wild-type, mutant, or revertant strains were analyzed by
Western blotting using antiserum raised against DmGCLM (A) or
DmGCLC (B). Lane 1, Canton S; lane 2, w1118; lane 3, e GclmL0580; lane
4, GclmL0580 ro; lane 5, e Gclmrev4.
Drosophila Glutamate-Cysteine Ligase 46375

(258.52  22 pmol of glutathione/fly). The concentrations of
glutathione in the e GclmL0580 and GclmL0580 ro flies were
substantially less (152.47  8.35 and 168.89  6.01 pmol of
glutathione/fly, respectively) than those in all of the control
strains, implicating DmGCLM as an important component of
the regulatory pathway for glutathione synthesis in vivo.
DISCUSSION
Glutamate-cysteine ligase has a profound influence on intra-
cellular redox status. Despite this, the complex regulatory
mechanisms that modify GCL activity to control glutathione
homeostasis remain poorly understood. In this study, we have
examined the role of intermolecular disulfide linkages in reg-
ulating Drosophila GCL activity and showed that abrogation of
the ability to form disulfide bridges between the catalytic and
modifier subunits has a significant impact on the catalytic
efficiency of the holoenzyme as well as sensitivity to feedback
inhibition by glutathione. The proposed importance of
DmGCLM in regulating glutathione homeostasis in vivo was
substantiated by the observation that Drosophila strains with
a mutation in Gclm have approximately half as much glutathi-
one as wild-type strains.
Using a combination of MALDI-TOF mass spectroscopy and
examination of evolutionary conservation of cysteines, we iden-
tified Cys213, Cys214, and Cys267 in DmGCLM as candidate
disulfide-forming cysteines important for the interactions with
DmGCLC. A mutant form of DmGCLM that lacks these 3
cysteine residues (DmGCLM-ABD) could form a stable holoen-
zyme complex with DmGCLC, but was unable to form intermo-
lecular disulfide bridges under non-reducing conditions. Our
kinetic analyses showed that the mutant DmGCLC/DmGCLM-
ABD holoenzyme was less active than the unmodified holoen-
zyme, but significantly more active than DmGCLC, supporting
the notion that both covalent and non-covalent interactions are
important in regulating DmGCL activity. We also found that
the DmGCLC/DmGCLM-ABD mutant was more sensitive to
inhibition by glutathione than the wild-type holoenzyme. This
observation is in keeping with the hypothesis that disulfide
linkages generate a conformational change that causes the
active site to be less susceptible to competitive inhibition by
glutathione (9); in the absence of disulfide bridges (or where
intermolecular disulfides are reduced by glutathione), the ac-
tive site would adopt a more open conformation, which would
allow access of glutathione and competition with glutamate.
Interestingly, the degree of inhibition observed for DmGCLC/
DmGCLM-ABD in the presence of very high concentrations of
glutathione was still greater than that observed for wild-type
DmGCL. It would be expected that, under such conditions, the
disulfide linkages between DmGCLC and DmGCLM would be
fully reduced, and the sensitivity of wild-type DmGCL to glu-
tathione inhibition would be identical to that of the DmGCLC/
DmGCLM-ABD mutant. It is possible, however, that a propor-
tion of the recombinant holoenzyme contains glutathione-
resistant disulfides. Pertinently, it was shown previously that
a proportion of native rat kidney GCL remained as undissoci-
ated holoenzyme when subjected to SDS-PAGE after incuba-
tion with the physiologically attainable concentration of 10 mM
glutathione (9).
At the outset of this study, we noticed that replacing the
N-terminal histidine tag with one at the C terminus caused
GCL activity to increase by 10-fold. This implies that the
N-terminal tag may interfere with the conformation of the
protein. Although we have not analyzed DmGCLC activity
without a histidine tag, previous studies have shown that an
N-terminal histidine tag has a modest inhibitory effect on
HsGCLC activity (11). The low activity of N-terminally tagged
DmGCLC made it less amenable to study than the more active
C-terminally tagged protein, and therefore, we carried out all of
our subunit interaction studies using the latter DmGCLC form.
Furthermore, to circumvent potential problems due to the
rapid oxidation of the cysteine substrate in vitro, we performed
our enzyme assays with -aminobutyrate rather than cysteine,
which we had used previously (13). We were surprised, how-
ever, to find that there was no significant difference in the Km
values for glutamate between DmGCLC and either the mutant
or wild-type DmGCL holoenzyme. In our earlier study (using
cysteine as substrate and DmGCLC with an N-terminal tag),
we found that the Km value for glutamate was nearly six times
higher when the modifier subunit was absent. This difference
in Km values is highly reproducible. We are uncertain why the
more active C-terminally tagged DmGCLC does not exhibit this
DmGCLM-dependent difference in Km for glutamate. It is pos-
sible that the cysteine residue that has been proposed to be at
or near the active site (12, 22) is more susceptible to oxidation
by the cysteine substrate in uncomplexed DmGCLC due to
FIG. 10. Effect of mutation of Gclm on glutathione levels in vivo. Glutathione levels in whole fly homogenates were determined as
described under “Experimental Procedures.” Glutathione concentrations are expressed as the mean  S.D. of three experiments. Glutathione levels
differed significantly by genotype (analysis of variance, F(4,40) 68.42, p  0.001). Post hoc comparisons indicated that the levels in Canton S and
w1118 flies did not differ significantly from each other, but did differ from the levels in the other three genotypes (p  0.001); the levels in the two
GclmL0580 stocks did not differ from each other, but did differ from the levels in the other three genotypes (p  0.001); and the levels of glutathione
in the Gclmrev4 stock were significantly different from those in each of the other genotypes in the analysis.
Drosophila Glutamate-Cysteine Ligase46376

DmGCLM-induced conformational alterations. Indeed, we
have shown previously that DmGCLC is more susceptible to
inactivation by cystamine than the DmGCL holoenzyme (13).
Alternatively, it is possible that oxidation of cysteine to cystine
in the assay mixture could cause differential inhibition, which
might be reflected in apparent differences in Km values. One
other possibility is that the C-terminal histidine tag on DmGCLC
actually generates a conformational change in the catalytic
subunit to increase activity and to decrease the Km for gluta-
mate. Despite this caveat, we have demonstrated convincingly
that interaction with DmGCLM increases the catalytic effi-
ciency of DmGCLC and that the potential to form intersubunit
disulfide linkages can further modulate activity and suscepti-
bility to inhibition by glutathione.
We found that HsGCLM was able to functionally substitute
for DmGCLM in the regulation of DmGCLC activity. The mod-
ifying effect of HsGCLM was very similar to that of unmodified
DmGCLM, suggesting that intersubunit disulfide bridges may
form between HsGCLM and DmGCLC to enhance activity.
Supporting this notion, HsGCLM was able to form disulfide
bonds with DmGCLC. These data strongly implicate Cys213
and/or Cys214 as the principal modulator of GCLC activity, as
Cys213 and Cys214 are the only cysteines that are conserved
between HsGCLM and DmGCLM. We should note, however,
that mutant DmGCLM polypeptides in which only Cys213 and
Cys214 were changed to serine could still form covalent linkages
with DmGCLC when analyzed by SDS-PAGE under non-reduc-
ing conditions (data not shown). In this study, it was therefore
important to perform the biochemical analyses on GCL protein
complexes in which the formation of disulfide linkages in vitro
was not apparent.
In view of the number of hypothetical GCLM proteins iden-
tified from representatives of a variety of eukaryotic classes, it
seems likely that many eukaryotes utilize a modifier subunit to
regulate glutathione synthesis. Nevertheless, it probable that
this mechanism is not common to all eukaryotes. Previous work
has shown that GCL from Trypanosoma brucei is highly un-
likely to be regulated by a modifier subunit (23). This hypoth-
esis is substantiated by the fact that T. brucei GCL lacks the
conserved cysteine residue (Cys553 in HsGCLC) that has been
proposed to be involved in disulfide interactions between
HsGCLC and HsGCLM (11). When we examined GCLC se-
quences from organisms in which we found a hypothetical
GCLM subunit (D. rerio, X. laevis, G. gallus, C. elegans,
Anopheles gambiae, S. pombe, and Neurospora crassa), as well
as the characterized mammalian forms, all were found to con-
tain the conserved Cys553, supporting the hypothesis that this
cysteine residue plays an important role in regulating GCL
activity by participating in intermolecular disulfide bridge for-
mation. It is of interest to note that, unlike S. pombe GCLC,
Saccharomyces cerevisiae GCLC does not contain the conserved
Cys553, and we were unable to find a GCLM ortholog in the
genome sequence.
Despite the wide distribution of GCLM, GclmL0580 flies are
fully viable and fertile. The P-element insertion in the 5-
untranslated region of Gclm almost entirely ablates expres-
sion, and the mutants have
50% less glutathione than control
strains. However, the lack of phenotype under standard labo-
ratory conditions is perhaps not surprising in light of a recent
study in which targeted disruption of mouse Gclm caused no
overt phenotype, despite causing significant decreases in glu-
tathione levels in all tissues examined (24). Gclm/ mouse
fetal fibroblasts were, however, substantially more susceptible
to H2O2 toxicity than those with the Gclm
/ or Gclm/
genotype.
The genetic experiments with fruit flies and mice have high-
lighted the importance of GCLM in glutathione homeostasis in
vivo. It is likely that an impaired capacity to synthesize gluta-
thione would sensitize mutant Gclm animals to cellular oxida-
tive damage. As oxidative stress has been proposed to play a
prominent role in aging (25), it will be interesting to study the
relationship between glutathione homeostasis, resistance to
various stresses, and aging in mutant Gclm flies. The use of
Drosophila as a model system will allow us to address the
importance of reversible disulfide bridges as a mechanism for
regulating glutathione synthesis in vivo. We should be able to
use gene replacement strategies to generate flies that express
the DmGCLM-ABD mutant instead of the wild-type protein,
furthering our understanding of the fundamental mechanisms
of regulation and the biological relevance of this essential thiol
antioxidant.
Acknowledgment—We thank Douglas J. Lamont for advice and help-
ful discussion on the MALDI-TOF analyses.
REFERENCES
1. Finkel, T. (2003) Curr. Opin. Cell Biol. 15, 247–254
2. Dickinson, D. A., and Forman, H. J. (2002) Biochem. Pharmacol. 64,
1019–1026
3. Lu, S. C. (1999) FASEB J. 13, 1169–1183
4. Griffith, O. W. (1999) Free Radic. Biol. Med. 27, 922–935
5. Griffith, O. W., and Mulcahy, R. T. (1999) Adv. Enzymol. Relat. Areas Mol.
Biol. 73, 209–267
6. Hayes, J. D., and McLellan, L. I. (1999) Free Radic. Res. 31, 273–300
7. Soltaninassab, S. R., Sekhar, K. R., Meredith, M. J., and Freeman, M. L. (2000)
J. Cell. Physiol. 182, 163–170
8. Huang, C. S., Anderson, M. E., and Meister, A. (1993) J. Biol. Chem. 268,
20578–20583
9. Huang, C. S., Chang, L. S., Anderson, M. E., and Meister, A. (1993) J. Biol.
Chem. 268, 19675–19680
10. Richman, P. G., and Meister, A. (1975) J. Biol. Chem. 250, 1422–1426
11. Tu, Z., and Anders, M. W. (1998) Biochem. J. 336, 675–680
12. Seelig, G., and Meister, A. (1984) J. Biol. Chem. 259, 3534–3538
13. Fraser, J. A., Saunders, R. D. C., and McLellan, L. I. (2002) J. Biol. Chem. 277,
1158–1165
14. Saunders, R. D. C., and McLellan, L. I. (2000) FEBS Lett. 467, 337–340
15. Tipnis, S. R., Blake, D. G., Shepherd, A. G., and McLellan, L. I. (1999)
Biochem. J. 337, 559–566
16. Kim, S. O., Merchant, K., Nudelman, R., Beyer, W. F., Jr., Keng, T., DeAngelo,
J., Hausladen, A., and Stamler, J. S. (2002) Cell 109, 383–396
17. Walsh, E. P., Lamont, D. J., Beattie, K. A., and Stark, M. J. R. (2002) Bio-
chemistry 41, 2409–2420
18. Provencher, S. W., and Glockner, J. (1981) Biochemistry 20, 33–37
19. Easterby, J. S. (1996) Hyper, Version 1.1s, University of Liverpool, Liverpool,
United Kingdom
20. Tietze, F. (1969) Anal. Biochem. 27, 502–522
21. Spradling, A. C., Stern, D., Beaton, A., Rhem, E. J., Laverty, T., Mozden, N.,
Misra, S., and Rubin, G. M. (1999) Genetics 153, 135–177
22. Brekken, D. L., and Phillips, M. A. (1998) J. Biol. Chem. 273, 26317–26322
23. Lueder, D. V., and Phillips, M. A. (1996) J. Biol. Chem. 271, 17485–17490
24. Yang, Y., Dieter, M. Z., Chen, Y., Shertzer, H. G., Nebert, D. W., and Dalton,
T. P. (2002) J. Biol. Chem. 277, 49446–49452
25. Finkel, T., and Holbrook, N. J. (2000) Nature 408, 239–247
Drosophila Glutamate-Cysteine Ligase 46377