title: Several glutathione S-transferase isozymes that protect against oxidative injury are expressed in human liver mitochondria.
abstract: The mitochondrial environment is rich in reactive oxygen species (ROS) that may ultimately peroxidize membrane proteins and generate unsaturated aldehydes such as 4-hydroxy-2-nonenal (4HNE). We had previously demonstrated the presence of hGSTA4-4, an efficient catalyst of 4HNE detoxification, in human liver mitochondria to the exclusion of the cytosol. In the present study, GSH-affinity chromatography was used in conjunction with biochemical and proteomic analysis to determine the presence of additional cytosolic glutathione S-transferases (GSTs) in human hepatic mitochondria. HPLC-subunit analysis of GSH affinity-purified liver mitochondrial proteins indicated the presence of several potential mitochondrial GST isoforms. Electrospray ionization-mass spectrometry analysis of eluted mitochondrial GST subunits yielded molecular masses similar to those of hGSTP1, hGSTA1 and hGSTA2. Octagonal matrix-assisted laser desorption/ionization time of flight mass spectrometry and proteomics analysis using MS-FIT confirmed the presence of these three GST subunits in mitochondria, and HPLC analysis indicated that the relative contents of the mitochondrial GST subunits were hGSTA1>hGSTA2>hGSTP1. The mitochondrial localization of the alpha and pi class GST subunits was consistent with immunoblotting analysis of purified mitochondrial GST. Enzymatic studies using GSH-purified mitochondrial GST fractions demonstrated the presence of significant GST activity using the nonspecific GST substrate 1-chloro-2,4-dinitrobenzene (CDNB), as well as 4HNE, delta(5)-androstene-3,17-dione (ADI), and cumene hydroperoxide (CuOOH). Interestingly, the specific mitochondrial GST activities toward 4HNE, a highly toxic alpha,beta-unsaturated aldehyde produced during the breakdown of membrane lipids, exceeded that observed in liver cytosol. These observations are suggestive of a role of GST in protecting against mitochondrial injury during the secondary phase of oxidative stress, or modulation of 4HNE-mediated mitochondrial signaling pathways. However, other properties of mitochondrial GST, such as conjugation of environmental chemicals and binding of lipophilic non-substrate xenobiotics and endogenous compounds, remain to be investigated.
authors.affiliation: Department of Environmental and Occupational Health Sciences, University of Washington, Seattle, USA. evang3@u.washington.edu
authors.forename: Evan P
authors.initials: EP
authors.lastname: Gallagher
authors.forename: James L
authors.initials: JL
authors.lastname: Gardner
authors.forename: David S
authors.initials: DS
authors.lastname: Barber
mesh_terms: D000328:Adult; D000447:Aldehydes / Q000378:metabolism; D015153:Blotting, Western; D002851:Chromatography, High Pressure Liquid; D005982:Glutathione Transferase / Q000737:chemistry / Q000302:isolation & purification / Q000502:physiology*; D006801:Humans; D007527:Isoenzymes / Q000502:physiology*; D008297:Male; D008930:Mitochondria, Liver / Q000201:enzymology*; D018384:Oxidative Stress; D021122:Protein Subunits
chemical_list: D000447:Aldehydes; D007527:Isoenzymes; D021122:Protein Subunits; D005982:Glutathione Transferase; C027576:4-hydroxy-2-nonenal