PEROXIDATIVE DAMAGE TO CARDIAC MITOCHONDRIA .2. IMMUNOLOGICAL ANALYSIS OF MODIFIED ADENINE-NUCLEOTIDE TRANSLOCASE

We have previously reported that treatment of isolated rat heart mitoc hondria with the free radical-generating system Cu2+/tert-butylhydrope roxide produces striking changes in the adenine nucleotide translocase (ANT) of the inner membrane. These changes include a small increase i n apparent molecular weight as determined by sodium dodecyl sulfate-po lyacrylamide gel electrophoresis, followed by its disappearance from t he polypeptide profile upon further oxidant treatment (Zwizinski and S chmid (1992) Arch. Biochem. Biophys. 294, 178-183). In order to charac terize its peroxidative modification in more detail, we have purified rat heart ANT and prepared polyclonal antibody against it. Using this antibody, we have observed that increasing oxidant treatment results i n a gradual increase in the ANT protein's apparent molecular weight by up to 1 kDa. The progressive nature of the molecular weight shift, wh ich parallels the generation of thiobarbituric acid reactive substance s, supports the hypothesis that this phenomenon may be the result of c ovalent addition of increasing amounts of lipid peroxidation products. Strong oxidative treatment of cardiac mitochondria also causes fragme ntation and polymerization of the ANT protein. However, Western blot a nalysis showed that a major portion of the original ANT survives even extensive oxidation as a distinct, modified protein. Therefore, the al most complete disappearance of ANT from Coomassie-stained gels appears to be the result of cross-linking and fragmentation reactions, as wel l as a decreased efficiency of the Coomassie staining. Because a measu rable molecular weight shift of ANT occurs at the mildest oxidative tr eatment tested (resulting in the production of only 1.1 nmol malondial dehyde/mg protein), it may be relevant as a parameter of myocardial is chemia-reperfusion injury. (C) 1994 Academic Press, Inc.