Initial characterization of the glutamate-cysteine ligase modifier subunit Gclm(-/-) knockout mouse. Novel model system for a severely compromised oxidative stress response.
Glutamate-cysteine ligase (GCL) is the rate-limiting enzyme in the GSH biosynthesis pathway. In higher eukaryotes, this enzyme is a heterodimer comprising a catalytic subunit (GCLC) and a modifier subunit (GCLM), which change the catalytic characteristics of the holoenzyme. To define the cellular function of GCLM, we disrupted the mouse Gclm gene to create a null allele. Gclm(-/-) mice are viable and fertile and have no overt phenotype. In liver, lung, pancreas, erythrocytes, and plasma, however, GSH levels in Gclm(-/-) mice were 9-16% of that in Gclm(+/+) littermates. Cysteine levels in Gclm(-/-) mice were 9, 35, and 40% of that in Gclm(+/+) mice in kidney, pancreas, and plasma, respectively, but remained unchanged in the liver and erythrocytes. Comparing the hepatic GCL holoenzyme with GCLC in the genetic absence of GCLM, we found the latter had an approximately 2-fold increase in K(m) for glutamate and a dramatically enhanced sensitivity to GSH inhibition. The major decrease in GSH, combined with diminished GCL activity, rendered Gclm(-/-) fetal fibroblasts strikingly more sensitive to chemical oxidants such as H(2)O(2). We conclude that the Gclm(-/-) mouse represents a model of chronic GSH depletion that will be very useful in evaluating the role of the GCLM subunit and GSH in numerous pathophysiological conditions as well as in environmental toxicity associated with oxidant insult.
Yang
Yi
Y
Department of Environmental Health, University of Cincinnati Medical Center, Cincinnati, OH 45267-0056, USA.
Dieter
Matthew Z
MZ
Chen
Ying
Y
Shertzer
Howard G
HG
Nebert
Daniel W
DW
Dalton
Timothy P
TP
D000367:Age Factors; D000483:Alleles; D000818:Animals; D015152:Blotting, Northern; D015139:Blotting, Southern; D001835:Body Weight; D016923:Cell Death; D002850:Chromatography, Gel; D003545:Cysteine / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D005347:Fibroblasts / Q000378:metabolism; D005838:Genotype; D005721:Glutamate-Cysteine Ligase / Q000737:chemistry* / Q000235:genetics; D018698:Glutamic Acid / Q000737:chemistry; D005978:Glutathione / Q000378:metabolism; D006720:Homozygote; D006861:Hydrogen Peroxide / Q000494:pharmacology; D015151:Immunoblotting; D007668:Kidney / Q000201:enzymology; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D051379:Mice; D018345:Mice, Knockout; D008957:Models, Genetic; D016297:Mutagenesis, Site-Directed; D018384:Oxidative Stress; D010100:Oxygen / Q000378:metabolism; D010641:Phenotype; D016133:Polymerase Chain Reaction; D017434:Protein Structure, Tertiary; D013997:Time Factors; D014018:Tissue Distribution
D018698:Glutamic Acid; D006861:Hydrogen Peroxide; D005721:Glutamate-Cysteine Ligase; D005978:Glutathione; D003545:Cysteine; D010100:Oxygen