title: Multiple isoforms of mitochondrial glutathione S-transferases and their differential induction under oxidative stress.
abstract: The mitochondrial respiratory chain, which consumes approx. 85-90% of the oxygen utilized by cells, is a major source of reactive oxygen species (ROS). Mitochondrial genetic and biosynthetic systems are highly susceptible to ROS toxicity. Intramitochondrial glutathione (GSH) is a major defence against ROS. In the present study, we have investigated the nature of the glutathione S-transferase (GST) pool in mouse liver mitochondria, and have purified three distinct forms of GST: GSTA1-1 and GSTA4-4 of the Alpha family, and GSTM1-1 belonging to the Mu family. The mitochondrial localization of these multiple GSTs was confirmed using a combination of immunoblot analysis, protease protection assay, enzyme activity, N-terminal amino acid sequencing, peptide mapping and confocal immunofluorescence analysis. Additionally, exogenously added 4-hydroxynonenal (HNE), a reactive byproduct of lipid peroxidation, to COS cells differentially affected the cytosolic and mitochondrial GSH pools in a dose- and time-dependent manner. Our results show that HNE-mediated mitochondrial oxidative stress caused a decrease in the GSH pool, increased membrane lipid peroxidation, and increased levels of GSTs, glutathione peroxidase and Hsp70 (heat-shock protein 70). The HNE-induced oxidative stress persisted for longer in the mitochondrial compartment, where the recovery of GSH pool was slower than in the cytosolic compartment. Our study, for the first time, demonstrates the presence in mitochondria of multiple forms of GSTs that show molecular properties similar to those of their cytosolic counterparts. Our results suggest that mitochondrial GSTs may play an important role in defence against chemical and oxidative stress.
authors.affiliation: Department of Animal Biology and the Mari Lowe Center for Comparative Oncology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA 19104-6047, U.S.A.
authors.forename: Haider
authors.initials: H
authors.lastname: Raza
authors.forename: Marie-Anne
authors.initials: MA
authors.lastname: Robin
authors.forename: Ji-Kang
authors.initials: JK
authors.lastname: Fang
authors.forename: Narayan G
authors.initials: NG
authors.lastname: Avadhani
mesh_terms: D000447:Aldehydes / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D003600:Cytosol / Q000378:metabolism; D004789:Enzyme Activation; D005982:Glutathione Transferase / Q000096:biosynthesis* / Q000737:chemistry / Q000378:metabolism; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism; D015227:Lipid Peroxidation; D008297:Male; D051379:Mice; D008813:Mice, Inbred ICR; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D008928:Mitochondria / Q000201:enzymology*; D008930:Mitochondria, Liver / Q000201:enzymology; D008969:Molecular Sequence Data; D018384:Oxidative Stress; D020033:Protein Isoforms; D017434:Protein Structure, Tertiary; D017382:Reactive Oxygen Species; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013997:Time Factors
chemical_list: D000447:Aldehydes; D018840:HSP70 Heat-Shock Proteins; D020033:Protein Isoforms; D017382:Reactive Oxygen Species; D005982:Glutathione Transferase; C027576:4-hydroxy-2-nonenal