PEROXIDATIVE MODIFICATION OF A MEMBRANE-PROTEIN - CONFORMATION-DEPENDENT CHEMICAL MODIFICATION OF ADENINE-NUCLEOTIDE TRANSLOCASE IN CU2+ TERT-BUTYL HYDROPEROXIDE TREATED MITOCHONDRIA/

Peroxidative treatment of rat heart mitochondria results in a gradual increase of the apparent molecular weight of the adenine nucleotide tr anslocase (ANT) by up to 1.2 kDa. ANT isolated from mitochondria treat ed with 1 mM tert-butyl hydroperoxide and 5-40 mu M 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, dithiot hreitol, and the specific inhibitor of ANT, carboxyatractyloside (CAT) , inhibited the peroxidation-induced molecular weight shift without in hibiting lipid peroxidation, which is believed to be the primary cause of the observed ANT modification. Bongkrekic acid, which stabilizes A NT in a conformation different from that brought about by CAT, did not inhibit the ANT molecular weight shift. Dithiothreitol, as well as CA T, was found to protect ANT against most of the losses of amino acid r esidues, indicating that alteration of sulfhydryl residues is required for chemical modification of, not only cysteine, but also lysine, arg inine, and valine. We conclude that the peroxidative modification of A NT is conformation-dependent and involves chemical modification of cys teine as a critical step.