Glutathionylation regulates cytosolic NADP+-dependent isocitrate dehydrogenase activity.
Cytosolic NADP+-dependent isocitrate dehydrogenase (IDPc) is susceptible to inactivation by numerous thiol-modifying reagents. This study now reports that Cys269 of IDPc is a target for S-glutathionylation and that this modification is reversed by dithiothreitol as well as enzymatically by cytosolic glutaredoxin in the presence of GSH. Glutathionylated IDPc was significantly less susceptible than native protein to peptide fragmentation by reactive oxygen species and proteolytic digestion. Glutathionylation may play a protective role in the degradation of protein through the structural alterations of IDPc. HEK293 cells treated with diamide displayed decreased IDPc activity and accumulated glutathionylated enzyme. Using immunoprecipitation with an anti-IDPc IgG and immunoblotting with an anti-GSH IgG, we purified and positively identified glutathionylated IDPc from the kidneys of mice subjected to ischemia/reperfusion injury and from the livers of ethanol-administered rats. These results suggest that IDPc activity is modulated through enzymatic glutathionylation and deglutathionylation during oxidative stress.
Shin
Seoung Woo
SW
School of Life Sciences and Biotechnology, College of Natural Sciences, Kyungpook National University, Taegu 702-701, Korea.
Oh
Chang Joo
CJ
Kil
In Sup
IS
Park
Jeen-Woo
JW
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D003600:Cytosol / Q000201:enzymology; D017931:DNA Primers / Q000235:genetics; D004229:Dithiothreitol / Q000494:pharmacology; D000431:Ethanol / Q000633:toxicity; D054477:Glutaredoxins / Q000378:metabolism; D005978:Glutathione / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D007521:Isocitrate Dehydrogenase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007668:Kidney / Q000201:enzymology / Q000293:injuries; D008099:Liver / Q000187:drug effects / Q000201:enzymology; D051379:Mice; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D018384:Oxidative Stress; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015427:Reperfusion Injury / Q000201:enzymology
D017931:DNA Primers; D054477:Glutaredoxins; D011994:Recombinant Proteins; D000431:Ethanol; D007521:Isocitrate Dehydrogenase; C039355:isocitrate dehydrogenase (NADP+); D005978:Glutathione; D004229:Dithiothreitol