title: Peroxidative modification of a membrane protein. Conformation-dependent chemical modification of adenine nucleotide translocase in Cu2+/tert-butyl hydroperoxide treated mitochondria.
abstract: Peroxidative treatment of rat heart mitochondria results in a gradual increase of the apparent molecular weight of the adenine nucleotide translocase (ANT) by up to 1.2 kDa. ANT isolated from mitochondria treated with 1 mM tert-butyl hydroperoxide and 5-40 microM Cu2+ for 1 h at 37 degrees C exhibited a progressive loss of lysine, cysteine, arginine, and valine residues compared to native ANT. N-Ethylmaleimide, dithiothreitol, and the specific inhibitor of ANT, carboxyatractyloside (CAT), inhibited the peroxidation-induced molecular weight shift without inhibiting lipid peroxidation, which is believed to be the primary cause of the observed ANT modification. Bongkrekic acid, which stabilizes ANT in a conformation different from that brought about by CAT, did not inhibit the ANT molecular weight shift. Dithiothreitol, as well as CAT, was found to protect ANT against most of the losses of amino acid residues, indicating that alteration of sulfhydryl residues is required for chemical modification of, not only cysteine, but also lysine, arginine, and valine. We conclude that the peroxidative modification of ANT is conformation-dependent and involves chemical modification of cysteine as a critical step.
authors.affiliation: Hormel Institute, University of Minnesota, Austin 55912, USA.
authors.forename: J
authors.initials: J
authors.lastname: Girón-Calle
authors.forename: H H
authors.initials: HH
authors.lastname: Schmid
mesh_terms: D000818:Animals; D001278:Atractyloside / Q000031:analogs & derivatives / Q000494:pharmacology; D001865:Bongkrekic Acid / Q000494:pharmacology; D002084:Butylated Hydroxytoluene / Q000494:pharmacology; D003300:Copper; D004229:Dithiothreitol / Q000494:pharmacology; D005033:Ethylmaleimide / Q000494:pharmacology; D008297:Male; D008353:Mannitol / Q000494:pharmacology; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D008928:Mitochondria / Q000187:drug effects* / Q000201:enzymology; D000226:Mitochondrial ADP, ATP Translocases / Q000737:chemistry / Q000378:metabolism*; D009206:Myocardium / Q000201:enzymology / Q000648:ultrastructure; D010545:Peroxides / Q000494:pharmacology*; D011487:Protein Conformation; D051381:Rats; D017207:Rats, Sprague-Dawley; D017382:Reactive Oxygen Species; D020122:tert-Butylhydroperoxide
chemical_list: D008565:Membrane Proteins; D010545:Peroxides; D017382:Reactive Oxygen Species; D001865:Bongkrekic Acid; D001278:Atractyloside; D002084:Butylated Hydroxytoluene; D008353:Mannitol; D003300:Copper; D000226:Mitochondrial ADP, ATP Translocases; D020122:tert-Butylhydroperoxide; D005033:Ethylmaleimide; C003853:carboxyatractyloside; D004229:Dithiothreitol