Functional glutaredoxin activity in rat brain & liver mitochondria

Abstract

Glutathione (GSH) serves as a major anitoxidant in cells to protect from oxidative damage. One less studied role for GSH is in GSH‐protein‐mixed disulfide (PrSSG) formation where GSH is covalently linked to sulfhydryl groups on a protein. This reversible glutathionylation is catalyzed by glutaredoxin‐1 (Grx1). Grx1 was identified in brain cytosol (J. Neurochem. 1999, 72, 1170). A 2nd glutaredoxin (Grx2) was recently cloned & expressed in E. coli (JBC 2001 276, 26 269, 30 374). Grx2 contains a mitochondrial (mito) leader sequence. Recombinant Grx2 is also a thiol‐disulfide oxidoreductase. Functional endogenous Grx2 activity in mito has not been demonstrated. To study this, subfractionation of liver & brain was carried out to obtain mito & microsome fractions. Marker enzyme activities as well as EM monitored fraction purity. Intact mito were inactive, but intramitochondrial matrix from lysed mito were able to dethiolate mixed disulfides. The specific activity for dethiolation for liver & brain cytosol was similar & greater than that of liver or brain mito. Grx activity in brain mito was 2‐fold higher than in liver mito. The microsomal fraction also contained Grx activity, but was significantly less than that found in cytosolic or mito fractions. Like cytosolic Grx, the mito enzyme showed a preference for GSH containing mixed disulfides, had a pH optima of ∼ 8.0 & was inhibited by aurothioglucose. Consistent with findings from recombinant Grx2, antibody directed against Grx1 cross reacted with brain & liver cytosolic enzyme, but not the mito form. These findings show that brain & liver mito contain functional glutathione‐dethiolating activity. In mito, this activity may play an important role in protecting against free radical damage of mito proteins. Acknowledgements: Supported by NS36157 & M.J. Fox Foundation for Parkinson's Research.