Information on EC 6.3.2.2 - glutamate-cysteine ligase and Organism(s) Homo sapiens

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The expected taxonomic range for this enzyme is: Bacteria, Eukaryota, Archaea


The taxonomic range for the selected organisms is: Homo sapiens

EC NUMBER
COMMENTARY hide
6.3.2.2
-
RECOMMENDED NAME
GeneOntology No.
glutamate-cysteine ligase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
ATP + L-glutamate + L-cysteine = ADP + phosphate + gamma-L-glutamyl-L-cysteine
show the reaction diagram
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
carboxamide formation
-
-
-
-
carboxylic acid amide formation
-
-
-
-
Ligation
-
-
PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
ergothioneine biosynthesis I (bacteria)
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glutathione biosynthesis
-
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homoglutathione biosynthesis
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glutathione metabolism
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Cysteine and methionine metabolism
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Glutathione metabolism
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Metabolic pathways
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SYSTEMATIC NAME
IUBMB Comments
L-glutamate:L-cysteine gamma-ligase (ADP-forming)
Can use L-aminohexanoate in place of glutamate.
CAS REGISTRY NUMBER
COMMENTARY hide
9023-64-7
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ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
physiological function
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
ATP + D-Glu + L-2-aminobutyrate
ADP + phosphate + gamma-D-Glu-L-alpha-aminobutyrate
show the reaction diagram
-
-
-
?
ATP + L-Glu + gamma-aminobutyrate
ADP + phosphate + L-Glu-gamma-aminobutyrate
show the reaction diagram
-
-
-
ir
ATP + L-Glu + L-2-aminobutanoate
ADP + phosphate + gamma-L-Glu-2-aminobutanoate
show the reaction diagram
-
-
-
-
-
ATP + L-Glu + L-2-aminobutyrate
ADP + phosphate + L-Glu-2-aminobutyrate
show the reaction diagram
ATP + L-Glu + L-alpha-aminobutyrate
ADP + phosphate + gamma-L-Glu-L-alpha-aminobutyrate
show the reaction diagram
ATP + L-Glu + L-Cys
?
show the reaction diagram
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key regulatory enzyme in glutathione biosynthesis
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-
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ATP + L-Glu + L-Cys
ADP + phosphate + gamma-L-Glu-L-Cys
show the reaction diagram
ATP + L-glutamate + L-cysteine
ADP + phosphate + gamma-L-glutamyl-L-cysteine
show the reaction diagram
ATP + L-glutamate + L-cysteine
ADP + phosphate + L-glutamyl-L-cysteine
show the reaction diagram
-
-
-
-
?
glutamate + ATP + L-cysteine
ADP + phosphate + gamma-L-glutamyl-L-cysteine
show the reaction diagram
-
assay at pH 8.2
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-
?
additional information
?
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NATURAL SUBSTRATES
NATURAL PRODUCTS
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
ATP + L-Glu + L-Cys
?
show the reaction diagram
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key regulatory enzyme in glutathione biosynthesis
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-
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ATP + L-Glu + L-Cys
ADP + phosphate + gamma-L-Glu-L-Cys
show the reaction diagram
ATP + L-glutamate + L-cysteine
ADP + phosphate + gamma-L-glutamyl-L-cysteine
show the reaction diagram
ATP + L-glutamate + L-cysteine
ADP + phosphate + L-glutamyl-L-cysteine
show the reaction diagram
-
-
-
-
?
additional information
?
-
COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Cu2+
-
induces expression of heavy subunit
Mn2+
bound to the erythrocyte enzyme
Sodium arsenite
-
induces expression of heavy subunit
Zn2+
-
induces expression of heavy subunit
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
2-mercaptoethanol
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buthionine sulfoximine
cysteamine
dithiothreitol
glutathione
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feedback inhibition, subunit GCLM increases the Ki for GSH-mediated feedback inhibition of GCL, competitive to glutamate
L-buthionine sulfoximine
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95% inhibition at 0.001 mM
L-buthionine-R-sulfoximine
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L-buthionine-S-sulfoximine
strong inhibition
NF-kappaB
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inhibits induction of enzyme expression by other substances, e.g. buthionine sulfoximine or tert-butylhydroquinone
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additional information
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ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
1-(4-amino-2-methyl-5-pyridimidyl)-methyl-3-(2-chloroethyl)-3-nitrosurea
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induces expression of heavy subunit
2,3-dimethoxy-1,4-naphthoquinone
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induces expression of heavy and light subunit
4-hydroxy-2-nonenal
6-Hydroxydopamine
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induces expression of heavy subunit
AP-1
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apigenin
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nearly 2fold induction of the heavy subunit gene promotor and heavy subunit expression
apocynin
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induces expression of heavy subunit
beta-naphthoflavone
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induces expression of heavy subunit
buthionine sulfoximine
-
induces expression of heavy and light subunit
butylated hydroxyanisole
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induces expression of heavy and light subunit
butylated hydroxytoluene
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induces expression of heavy subunit
caffeic acid
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treatment of the cells with 100 and 500 microg/ml of caffeic acid increases gamma-GCS activities by 1.4- and 1.8fold compared to the control group, respectively. At the same doses of caffeic acid, the treated cells show increased levels of glutathione by 1.7- and 2.7fold compared to the control, respectively
cigarette smoke condensate
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induces expression of heavy subunit
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diethyl maleate
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induces expression of heavy and light subunit
erythropoietin
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induces expression of heavy subunit
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Ethacrynic acid
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induces expression of heavy subunit
ethoxyquin
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induces expression of heavy subunit
iodoacetamide
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induces expression of heavy subunit
kaempferol
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2fold induction of the heavy subunit gene promotor and heavy subunit expression
menadione
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induces expression of heavy and light subunit
oltipraz
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induces expression of heavy subunit
onion extract
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containing flavonoids, which increase the expression of both subunits of the enzyme in COS-1 cells
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oxidative stress
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activation of GCL occurrs within min of treatment and without any change in GCL protein levels, and coincides with an increase in the proportion of GCL catalytic subunit in the holoenzyme form. Likewise, GCL modifier subunit shifts from the monomeric form to holoenzyme and higher molecular weight species. Neither GCL activation, nor the formation of holoenzyme, requires a covalent intermolecular disulfide bridge between GCL catalytic subunit and GCL modifier subunit. In immunoprecipitation studies, a neutralizing epitope associated with enzymatic activity is protected following cellular oxidative stress. Thus, the N-terminal portion of GCL catalytic subunit may undergo a change that stabilizes the GCL holoenzyme. Results suggest a dynamic equilibrium between low- and high-activity forms of GCL, which is altered by transient oxidative stress
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oxidized low density lipoprotein
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induces expression of heavy subunit
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phorone
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induces expression of heavy subunit
Prostaglandin A2
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induces expression of heavy subunit
pyrrolidine dithiocarbamate
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time-, dose-, and Cu2+-dependent induction and increase in expression levels of the 2 subunits of the enzyme in HepG2 cells, mechanism, can be partially blocked by N-acetylcysteine and by copper chelator bathocuproine disulfonic acid
quercetin
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3fold induction of the heavy subunit gene promotor and heavy subunit expression, best at 0.05 mM, induction even of a distal part of the promotor sequence containing only 2 antioxidant-response/electrophile-response elements, i.e. ARE/EpRE
tert-butylhydroquinone
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induces expression of heavy and light subunit
additional information
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
1.3
4-aminobutyrate
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pH 8.2, 37C
0.26 - 4.47
ATP
0.86 - 1.3
gamma-L-Glu-L-Cys
0.14
L-2-aminobutanoate
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1.4 - 5
L-2-aminobutyrate
0.25 - 2.39
L-alpha-aminobutyrate
0.05 - 0.17
L-Cys
0.1 - 0.8
L-cysteine
0.03 - 7.16
L-Glu
0.7 - 3.5
L-glutamate
additional information
additional information
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kinetics, recombinant enzyme
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Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.45 - 1000
GSH
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.001
native COS cells
0.014
transformed COS cells expressing both the recombinant subunits at equal amounts
0.038
transformed COS cells expressing the recombinant catalytic subunit
0.12
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purified recombinant mutant C249G catalytic subunit and mutant C295G catalytic subunit
0.17
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purified recombinant mutant C52G catalytic subunit and mutant C248G catalytic subunit
0.38
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purified recombinant mutant C605G catalytic subunit
0.44
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purified mutant R127C enzyme, substrates L-glutamate and L-alpha-aminobutyrate
0.48
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purified recombinant mutant C501G catalytic subunit
0.63
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purified recombinant mutant C491G catalytic subunit
0.92
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purified recombinant mutant C553G catalytic subunit
1.83
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purified recombinant mutant C553G holoenzyme
4.67
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purified recombinant mutant C249G holoenzyme
5.17
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purified recombinant mutant C605G holoenzyme
5.33
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purified recombinant mutant C295G holoenzyme
5.67
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purified recombinant mutant C248G holoenzyme
6.67
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purified recombinant mutant C52G holoenzyme
7
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purified recombinant mutants C491G holoenzyme and C501G holoenzyme
8.86
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purified wild-type enzyme, substrates L-glutamate and L-alpha-aminobutyrate
28.75
-
-
additional information
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7.5
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assay at
8.2
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assay at
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
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A2780/100 ovarian carcinoma cell exhibits resistance to DNA crosslinking agents, chlorambucil, cisplatin, melphalan, and ionizing radiation compared to the parental cell line, A2780. Drug-resistant cells have the inherent ability to maintain increased gamma-GCS activity
Manually annotated by BRENDA team
malignant cell line
Manually annotated by BRENDA team
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treatment of human breast cancer cells with 2-deoxy-D-glucose causes metabolic oxidative stress that is accompanied by increases in steady-state levels of glutamate cysteine ligase mRNA, glutamate cysteine ligase activity and glutathione content
Manually annotated by BRENDA team
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bronchial epithelial cells
Manually annotated by BRENDA team
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oligophosphopeptides derived from egg yolk phosvitin up-regulate gamma-glutamylcysteine synthetase and antioxidant enzymes against oxidative stress in Caco-2 cells
Manually annotated by BRENDA team
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from brain
Manually annotated by BRENDA team
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bronchial epithelial cells
Manually annotated by BRENDA team
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oncogene MYCN-amplified cells
Manually annotated by BRENDA team
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gamma-glutamylcysteine synthetase mediates the c-Myc-dependent response to antineoplastic agents in melanoma cells
Manually annotated by BRENDA team
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oncogene MYCN-amplified cells
Manually annotated by BRENDA team
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oncogene MYCN-non-amplified cells
Manually annotated by BRENDA team
additional information
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
additional information
-
while GCLC and GCLM are generally considered to be cytosolic proteins there is evidence that they may exhibit altered subcellular localization in certain circumstances
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Manually annotated by BRENDA team
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
30700
1 * 72800, heavy catalytic subunit, + 1 * 30700, light regulatory subunit, SDS-PAGE
32000
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x * 72000 + x * 32000, denaturing SDS-PAGE
72000
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x * 72000 + x * 32000, denaturing SDS-PAGE
72800
1 * 72800, heavy catalytic subunit, + 1 * 30700, light regulatory subunit, SDS-PAGE
75000
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2 * 75000, recombinant wild-type and mutant enzymes, SDS-PAGE
114000
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
?
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x * 72000 + x * 32000, denaturing SDS-PAGE
additional information
POSTTRANSLATIONAL MODIFICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
lipoprotein
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myristoylation is responsible for regulation of GCL subunit subcellular localization to membranes and mitochondria, overview
phosphoprotein
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phosphorylation plays an important role in regulating GCL activity in vivo, phosphorylation of GCLC occurs on serine and threonine residues in vitro and the phosphorylation sites are likely identical for all three kinases protein kinase C, PKC, cAMP-dependent protein kinase, PKA, or Ca2+-calmodulin-dependent protein kinase II, CMKII
proteolytic modification
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caspase-mediated cleavage of GCLC, overview
additional information
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post-translational modifications of GCLC, e.g. phosphorylation, myristoylation, caspase-mediated cleavage, have modest effects on GCL activity
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
homology model of the catalytic subunit of human glutamate cysteine ligase. Examination of the model suggests that post-translational modifications of cysteine residues may be involved in the regulation of enzymatic activity
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GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
activity of the holoenzyme and of the catalytic subunit is reduced by 20% and 10%, respectively, after 1 cycle of freezing and thawing
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enzyme is inactivated by freezing
freezing of the purified recombinant enzyme in solution results in irreversible inactivation
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glycerol is required for enzyme stability during storage
glycerol stabilizes
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L-glutamate stabilizes the enzyme during purification,
Mn2+ destabilizes the enzyme during purification
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-20C, 25% glycerol, 20 mM imidazole HCl buffer, pH 7.4, 1 mM EDTA, without freezing, stable for at least 1 year
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-20C, purified enzyme, 25% glycerol, indefinitely stable
4C, purified holoenzyme, 10% loss of activity after 1 week
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4C, purified recombinant enzyme, 20 mM imidazole HCl buffer, pH 7.4, 1 mM EDTA, stable for at least 7 days
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
from erythrocyte, from malignant astrocytoma cell line, recombinant from Escherichia coli to homogeneity
recombinant His-tagged holoenzyme and individual subunits from Sf9 insect cells
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recombinant His-tagged wild-type and mutant enzymes from Sf9 insect cells
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recombinant His-tagged wild-type and mutant R127C enzyme from Rosetta cells
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recombinant holoenzyme from Escherichia coli, to homogeneity
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
both regulatory subunit GCLM and catalytic subunit GCLC
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catalytic subunit DNA sequence determination and analysis, expression as His-tagged wild-type enzyme and mutant R127C in Rosetta cells, functional expression of wild-type and mutant enzyme in enzyme-deficient cells
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coexpression of the catalytic and the regulatory subunit from 2 different plasmids in Escherichia coli BL21(DE3), intracellular assembly of the holoenzyme
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coexpression of the His-tagged catalytic and regulatory subunits in Spodoptera frugiperda Sf9 cells via baculovirus infection, formation of the holoenzyme in the cells
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DNA sequence determination and analysis, 2 genes encode the 2 subunits, chromosomal mapping to 6p12 and 1p21, constitutive expression, expression of the heavy subunit alone or in combination with the light subunit in mammalian cells reveals that the regulatory subunit improves the activity, genetic regulation involving AP-1, overview, expression of several constructs in HepG2 cells, signaling for enzyme expression
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DNA sequence determination and analysis, heterodimer of a catalytic subunit and a regulatory subunit, encoded by 2 genes: GLCLC for the catalytic subunit, and GLCLR for the regulatory subunit, GLCLC polymorphism and existence of 5 alleles as defined by the trinucleotide repeat, which exhibits a range of 4 to 10 uninterrupted repeat, genotyping for the repeat of 60 tumor cancer cell lines, overview
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DNA sequence determination and analysis, mapping to chromosome 6p12, heavy and light subunits, overexpression in Escherichia coli, individual or coexpression of the 2 subunits in COS cells, expression patterns, expression of several deletion mutants created fom the 5'-flanking region of the gene in human hepatoblastoma HepG2 cells, overexpression in human leukemia HL-60 cells; DNA sequence determination and analysis, mapping to chromosome 6p12, heavy and light subunits, overexpression in Escherichia coli, individual or coexpression of the 2 subunits in COS cells, expression patterns, expression of several deletion mutants created fom the 5'-flanking region of the gene in humen hepatoblastoma HepG2 cells, overexpression in human leukemia HL-60 cells
expression of a construct consisting of 3.8 kb of the enzyme's heavy subunit gene promotor in front of luciferase as well as a fragment of the 5'-flanking sequence, transient expression in COS-1 cells
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expression of the His-tagged wild-type enzyme and mutants in Spodoptera frugiperda Sf9 cells via baculovirus infection
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gene GCLC, DNA and amino acid sequence determination and analysis, genotyping
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gene GCLC, semiquantitative expression analysis in endothelial cells
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generation of C57Bl/6 mice that conditionally overexpress glutamate-cysteine ligase
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genes gclC and gclM, genotyping in healthy individuals and chronic obstructive pulmonary disease patients
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genes gclC and gclM, genotyping in healthy individuals and in schizophrenia patients, overview
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genes gclc and gclm, genotyping, analysis of correlation between genotype and smoking effects, overview
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transfection of COV-434 granulosa tumour cell with vectors designed for the constitutive expression of Gcl catalytic subunit, Gcl modifier subunit, or both Gcl catalytic subunit and Gcl modifier subunit
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transfecttion of embryonic fibroblast from GCLC null mice and expression in Saccharomyces cerevisiae
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EXPRESSION
ORGANISM
UNIPROT
LITERATURE
actinomycin D and cycloheximide suppress enzyme expression
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both the glutamate-cysteine ligase catalytic (GCLC) and modifier (GCLM) subunit mRNA levels are upregulated in response to a lack of cysteine or other essential amino acids, independent of GSH levels
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catalytic subunit GCLC protein levels do not increase, whereas regulatory subunit GCLM protein levels increase in the cells cultured in cysteine-deficient medium
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indomethacin inhibits the gamma-glutamylcysteine synthetase promoter activity. Co-treatment by indomethacin and doxorubicin increases the cytotoxicitiy of doxorubicin by decreasing the intracellular contents of glutathione and its conjugates with decreasing expression of gamma-glutamylcysteine synthetase
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oncogen MYCN directly binds to an E-box containing GCL catalytic subunit promoter and over-expression of MYCN in MYCN-non-amplified cells stimulates GCL catalytic subunit expression and provides resistance to oxidative damage. Knock-down of MYCN in MYCN-amplified cells decreases GCL catalytic subunit expression and sensitizes them to oxidative damage
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presence of an ethanol-responsive element in the human GCL catalytic subunit promoter, it spannes bases 1432 to 832 in hepatocytes and HepG2 cells transfected with cytochrome P450 2E1. The region lacks an ARE but has a putative nuclear factor-kappaB element
-
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
C248G
-
site-directed mutagenesis in the catalytic subunit, reduced activity of the catalytic subunit, activity of the holoenzyme is similar to the wild-type enzyme
C249G
-
site-directed mutagenesis in the catalytic subunit, reduced activity of the catalytic subunit, reduced activity of the holoenzyme compared to the wild-type enzyme
C295G
-
site-directed mutagenesis in the catalytic subunit, reduced activity of the catalytic subunit, activity of the holoenzyme is similar to the wild-type enzyme
C491G
-
site-directed mutagenesis in the catalytic subunit, reduced activity of the catalytic subunit, activity of the holoenzyme is similar to the wild-type enzyme
C501G
-
site-directed mutagenesis in the catalytic subunit, reduced activity of the catalytic subunit, activity of the holoenzyme is similar to the wild-type enzyme
C52G
-
site-directed mutagenesis in the catalytic subunit, reduced activity of the catalytic subunit, activity of the holoenzyme is similar to the wild-type enzyme
C553G
-
site-directed mutagenesis in the catalytic subunit, slightly reduced activity of the catalytic subunit, about 3.5fold reduced activity of the holoenzyme compared to the wild-type enzyme
C605G
-
site-directed mutagenesis in the catalytic subunit, reduced activity of the catalytic subunit, activity of the holoenzyme is similar to the wild-type enzyme
H370L
-
clinically relevant mutation in catalytic subunit GCLC. Significantly lower levels of glutathione relative to that of the wild type. Compromised enzymatic activity can largely be rescued by the addition of GCLM
P158L
-
clinically relevant mutation in catalytic subunit GCLC. Significantly lower levels of glutathione relative to that of the wild type, kinetic constants comparable to those of wild-type GCLC
P414L
-
clinically relevant mutation in catalytic subunit GCLC. Significantly lower levels of glutathione relative to that of the wild type, most compromised mutant among those studied. Compromised enzymatic activity can largely be rescued by the addition of GCLM
P462S
-
non-synonymous polymorphism in the gene encoding the catalytic subunit of glutamate-cysteine ligase. The polymorphism is present only in individuals of African descent and encodes an enzyme with significantly decreased in vitro activity when expressed by either a bacterial or mammalian cell expression system. Overexpression of the P462 wild-type GCLC enzyme results in higher intracellular glutathione concentrations than overexpression of the P462S isoform. Apoptotically stimulated mammalian cells overexpressing the P462S enzyme have increased caspase activation and increased DNA laddering compared to cells overexpressing the wild-type enzyme. The P462S polymorphism is in Hardy-Weinberg disequilibrium, with no individuals homozygous for the P462S polymorphism identified
additional information
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
medicine
pharmacology
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co-treatment by indomethacin and doxorubicin increases the cytotoxicitiy of doxorubicin by decreasing the intracellular contents of glutathione and its conjugates with decreasing expression of gamma-glutamylcysteine synthetase. Indomethacin inhibits the gamma-glutamylcysteine synthetase promoter activity.